new year, new name and new milestones scope journal of circulating biomarkers editorial shidong jia1 and winston patrick kuo2 1 genentech inc, south san francisco, usa 2 harvard medical school, boston, usa received 21 apr 2014; accepted 29 apr 2014 doi: 10.5772/58638 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this editorial article introduces a renaming of journal exosomes and microvesicles (exmv) to the journal of circulating biomarkers with a new editorial scope, mission and our approach for the upcoming year in relation to engaging at the international level, the translational art of the study of exosomes and microvesicles, and the interface between exosomes and microvesicles, circulating tumor cells, cell-free circulating dna and circulating protein markers in precision medicine and drug development. there is a slight change in the members of the editors in chief, editorial board and extending collaborations to international societies, such as the american society for exosomes and microvesicles (asemv). keywords exosomes, microvesicles, circulating tumor cells, cell-free circulating dna, editorial in the past year, we have witnessed many excitements, exemplified by our sharp focus on engaging with international key opinion leaders, translational researchers and diagnostic and therapeutic innovators from both academia and industry working in the field of exosomes and microvesicles. we had hoped to leverage exmv as a niche medium to showcase their latest research and advances, though we only received 13 manuscripts and 10 of which were published requiring the service of 19 individual referees, we have decided to make significant changes to the existing journal. in order to achieve this goal we have decided to implement the following; we have invited a new member to join the exmv editors in chief team, dr. shidong jia (bio available on the journal website). dr. jia is a scientist at genentech inc. where he spearheads the biomarker development efforts in support of oncology pipeline and drug-companion diagnostic co-development. dr. jia is an internationally recognized scientist in the extracellular vesicle field, among other circulating biomarkers and we look forward to working closely with him in bringing international exposure and success to the journal. and when considering the recent breakthrough in blood-based biomarker development, the new team of editors-inchiefs have decided to rename and re-index the journal name from exmv to “journal of circulating biomarkers” (abbreviated herein as jcb), and to expand the current 1shidong jia and winston patrick kuo: new year, new name and new milestones scope journal of circulating biomarkers article j circ biomark, 2014, 3:1 | doi: 10.5772/58638 journal of circulating biomarkers scope from exosomes and microvesicles to the interface between exosomes/microvesicles, circulating tumor cells, cell-free circulating dna and circulating protein markers in the context of precision medicine and clinical drug development. new discoveries utilizing blood-based biomarkers will potentially enhance our understanding of the pathology and etiology of disease, particularly originating from the genetic and molecular world. the use of exosomes/microvesicles, circulating tumor cells, cell-free circulating dna and circulating protein markers as a biomarker in clinical medicine for early diagnosis, prognosis and monitoring of therapy will be a significant advancement in the field. significant progress has been accomplished up to this point, yet we hope this this new renamed journal will be the source to publish the process to the best clinical use of these new discoveries. as our first task, the editors in chief have and will actively expand our editorial board and develop collaborations/partnerships with international societies through conferences, meetings, webinars we will be attending, which will include scientific experts who understand how discovery moves from lab to the market and to attract contributors and participation from a more global audience. these experts will serve as the central hub of the circulating biomarker community for the latest advances and provide us guidance. the jcb manuscript processing and peer-review is entirely online. the editorial manager facilitates the manuscript processing time, reducing costs and is a better experience for our authors and reviewers. we have received numerous positive comments on this evolving electronic system, and we are indebted to our publisher, intech, for managing this program for us. for more information, please visit our new manuscript submission system at http://www.editorialmanager.com/exo/default.asp. in addition, we are enforcing to expedite the acceptance to publishing time for our authors and will request informal opinions from our editorial board for borderline cases. jcb will continue to work on partnering with other scientific societies, academic and industry leaders to develop and advance the circulating biomarker field and to allow free access to knowledge. it’s our pleasure to thank the editorial team and publishers to make this journal possible. 2 j circ biomark, 2014, 3:1 | doi: 10.5772/58638 article journal of circulating biomarkers year of expanding into circulating biomarkers editorial shidong jia1 and winston patrick kuo2* 1 genentech inc., south san francisco, usa 2 ies diagnostics, inc., cambridge, usa *corresponding author(s) e-mail: wkuo@iesdiagnostics.com doi: 10.5772/60126 © 2015 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract this editorial article summarizes the achievements and current challenges for the journal of circulating biomarkers (jcb) regarding a more strategic approach to branding and attracting a high quality variety of articles. more emphasis is placed on fostering engagement with academic and industry sources operating at the cutting-edge of transla‐ tional technologies applied to the field of circulating biomarkers (interface between extracellular vesicles including exosomes and microvesicles, circulating tumour cells, cell-free circulating dna and circulating protein markers) and with those in the investment arena seeking and providing private funding for this area of research. keywords extracellular vesicles, exosomes, microvesicles, circulating tumour cells, cell-free circulating dna, edito‐ rial we are proud to announce that during the past year we have experienced several ongoing developments, one of which has been the expansion of our editorial board with key opinion leaders in the circulating biomarker field including yvon e. cayre (pierre and marie curie univer‐ sity, france), alain charest (tufts university school of medicine, usa), yong chen (ecole normale superieure, france), daniel haber (harvard medical school, usa), pavel laktionov (russian academy of sciences, russian federation) and david t. wong (university of california los angeles, usa), all of whom contribute as part of our associate editor’s team. additionally, aled clayton (cardiff university, united kingdom), aleksandra fučić (institute for medical research and occupational health, croatia), stefan holdenrieder (university hospital bonn, germany), hongjun kang (chinese pla general hospital, china), ming-lin liu (university of pennsylvania, usa), jan lotvall (göteborgs universitet, sweden), jie ma (fudan university, china), pierre-yves mantel (harvard school of public health, usa), dario marchetti (baylor college of medicine, usa), p. shannon pendergras (ymir genomics, usa), eva rohde (paracelsus medical university, austria), an song (genentech inc., usa), matthew j. wood (uni‐ versity of oxford, united kingdom), bruce r. zetter (harvard medical school, usa) and hanyu zhu (chinese pla general hospital, china) serve as members of our editorial board. in 2014, we published high quality papers covering research topics in the area of cell-cell communication via exosomal heat-shock proteins, the function of erythrocytederived extracellular vesicles in malaria as it pertains to understanding their role in immune regulation and cell-cell communication, the development of stem cell-derived exosomes as a cell-free regenerative medicine, as well as the detection of human c-myc and egfr amplifications in 1j circ biomark, 2015, 4:1 | doi: 10.5772/60126 circulating extracellular vesicles in mouse tumour models. we also published a paper on circulating free dna and the influence thereof following lung parenchyma surgical manipulation. in addition, we are pleased to have established a partner‐ ship/collaboration with the biopharma research council (http://www.biopharmaresearchcouncil.org/), spearhead‐ ed by its executive director, joanne gere. the biopharma research council has been developing a vibrant commun‐ ity for scientists as they navigate today's complex science environments. our unique resources – including close relationships with leading companies and suppliers – and agile approaches have established recurrent opportunities for breakthrough discussion and debate throughout the drug discovery process and delivery pipeline. this part‐ nership/collaboration resulted in the establishment of a short course titled, “extracellular vesicles: the transition from tissue to liquid biopsies”, which was conducted as a four-session webinar series in october and november 2014. the goal of the course was to provide, at an introduc‐ tory level, an exchange between researchers from within academia and industry pertaining to the characterization of the applications of evs in clinical and translational research, eventually to be implemented within clinical practice. we were able to utilize our editorial team to present topics that included: “overview of extracellular vesicles” by jan lötvall, md, phd; “clinical diagnostic applications of extracellular vesicles” by johan skog, phd; “commercialization aspects of extracellular vesicles” by alexander “sasha” vlassov, phd; “extracellular vesicles: therapeutic hurdles” by eva rohde, md; “funding opportunities for extracellular vesicle research” by angel ayuso sacido, phd. a meeting dispatch was published online and is available on the jcb website (http:// www.intechopen.com/journals/journal-of-circulatingbiomarkers/short-course-in-extracellular-vesicles-ndashthe-transition-from-tissue-to-liquid-biopsies). from this experience, we learned that coordinating the preparation process of such webinars required approximately six months. we plan to continue several webinar series on circulating tumour cells and cell-free circulating dna later this year. finally, we launched a special issue titled, “the biology and the clinical utility of circulating tumor cells”, with a focus on detecting the latest findings in ctc research and clinical value. more details are located in the news section of the journal of circulating biomarkers (http://www.inte‐ chopen.com/journals/journal-of-circulating-biomarkers). as we continue to strive to publish the best scientific research in all fields related to circulating biomarkers, the editorial team aims to focus on additional topics such as cell free dna and disruptive technologies, among others. jcb accepts original and review articles, as well as editori‐ als, perspectives, short research reports, protocols and methods, notes to the editor, letters to the editor and meeting dispatch reports. we would also like to mention that intech publishing have decided that jcb will not apply any article processing charges for authors whose research papers have been accepted for publication in volume 4/2015 of the journal. all manuscripts are peer-reviewed for scientific quality and the review process is conducted entirely online. the editorial manager facilitates the manuscript processing time, thereby reducing costs and providing a better experience to both our authors and reviewers. we have received numerous positive comments about this evolving electronic system and are indebted to intech for managing this programme for us. for more information, please review our new manuscript submis‐ sion system at http://www.editorialmanager.com/exo/ default.asp. in addition, we are expediting the acceptance to publishing time for our authors and will request informal opinions from our editorial board for borderline cases. jcb will continue to partner with scientific societies and academic and industry leaders in order to advance the field and to allow free access to knowledge in the area of circulating biomarkers. we gratefully acknowledge the editorial team and publish‐ ers for making the continued success of the journal possible. 2 j circ biomark, 2015, 4:1 | doi: 10.5772/60126 "i have a dream" perspective article stefano fais1,* 1 anti-tumour drug section, department of drug research and medicine evaluation, istituto superiore di sanità, italy * corresponding author e-mail: stefano.fais@iss.it received 22 apr 2014; accepted 21 may 2014 doi: 10.5772/58709 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. “i have a dream that one day every valley shall be exalted, every hill and mountain shall be made low, the rough places will be made plains, and the crooked places will be made straight, and the glory of the lord shall be revealed, and all the flesh shall see it together. this is our hope…” (martin luther king, washington d.c., august 28, 1963) keywords nanomedicine, teranostic, exosomes, biomarkers, cancer, diseases unfortunately, we are all witness to a dramatic failure in new, effective and possibly non-toxic therapies against major diseases. in fact, the diseases that were incurable seven decades ago remain incurable today, excluding of course infectious diseases. cancer, as well as neurodegenerative diseases (e.g. multiple sclerosis, alzheimer’s ) and autoimmune diseases (including systemicus lupus erythematosus, rheumatoid arthritis and scleroderma), all suffer from poorly effective and often extremely toxic therapies. however, while many of these diseases can be controlled as chronic diseases, cancer is becoming a sort of nightmare rather than a disease. this is for two main reasons: first, because the standard therapy did not show to be effective, largely under the expectancy, while extremely toxic and often destroying the patient’s body rather than helping it in defeating the disease. these two concepts are clearly expressed in two articles published in the last four years. the first one, by david shaywitz and nassim taleb, was published in the financial times in 2008 (1), and discusses the reasons for this dramatic failure in drug discovery. in the authors’ own words: “the molecular revolution was supposed to enable drug discovery to evolve from chance observation into rational design, yet dwindling pipelines threaten the survival of the pharmaceutical industry. what went wrong? the answer, we suggest, is the mismeasure of uncertainty, as academic researchers underestimated the fragility of their scientific knowledge while pharmaceuticals executives overestimated their ability to domesticate scientific research.” of course, there is no reason not to agree with these statements. there is a looseness between academic science and the pharmas in biomedical research. there appears to be something like an “unrealistic ambition”: we take the discoveries of scientists and we apply to the concept of research and development the potentially applicable findings coming from basic research. this approach has not achieved innovative or effective therapies for major diseases. shaywitz and taleb write further: “for all the breathless headlines proclaiming breakthrough discoveries, the truth is that we still do not understand what causes most disease. even when we can identify a responsible gene or implicate an important mutation, we have made only limited progress in turning these results into treatments.” unfortunately, this is the truth, and moreover it 1stefano fais: “i have a dream” article j circ biomark, 2014, 3:5 | doi: 10.5772/58709 journal of circulating biomarkers represents a dramatic truth. however, i fear we have to seriously regard for this truth, in order to find a way to overcome this “big, big problem.” another important point from the same article is: “medical research is particularly hampered by the scarcity of good animal models for most human disease, as well as by the tendency of academic science to focus on the “bits and pieces” of life – dna, proteins, cultured cells – rather than on the integrative analysis of entire organisms, which can be more difficult to study.” again, this is dramatically true. in fact, following the age of the big discoveries in medicine, where medical scientists often tested their ideas on their own bodies, today, biomedical research is mostly deprived of mds or physicians, with positions instead filled by basic scientists, with no medical culture and – even worse with no interest in discovery of “the causes of diseases.” the last point upon which i want to comment is: “nevertheless, real scientific progress has occurred, inviting the question: why do pharmaceutical companies, which spend billions of dollars each year trying to turn advances into treatments, have so little to show for their efforts? answer: spreadsheets are easy; science is hard.” i fear that the problem is that, during recent decades, “science” has become a sort of spreadsheet in its application. this is because the research projects in biomedicine were set up in a nasa-like way. something like: “we want to get to the moon.” yes; but discovery of the cause of diseases (in order to try to cure them) does not correspond with the will to get to the moon. the unforgotten genius and 1931 nobel prize for medicine, prof. otto h. warburg, suggested to all medical scientists at the beginning of the last century: “we can only cure what we can understand first.” i think that, really, we should reset our research in understanding diseases. the example of cancer is emblematic, inasmuch as we continue to ignore the prime aetiology of tumours. the result is that, after more than 60 years from the introduction of chemotherapy in human beings, the gold standard anti-tumour strategies offered to cancer patients are still based on chemotherapy, surgery and radiotherapy, which physically try to destroy cancer with brute force rather than by selectively interacting with cancer cells’ unique biological characteristics. actually, cancer represents an area with significant unmet medical need, with more than 20 million people worldwide being diagnosed annually and, despite the current available therapies, more than a million patients dying from this disease every year. there is an urgent need for safe and effective new treatments resulting in durable disease remissions and increased overall survival. at this point, i would like to introduce an article by robert gatenby (2), which proposes a change of strategy in the war against cancer. he begins by listing some facts: “the german nobel laureate paul ehrlich introduced the concept of ‘magic bullets’ more than 100 years ago: compounds that could be engineered to selectively target and kill tumour cells or disease-causing organisms without affecting the normal cells in the body. the success of antibiotics 50 years later seemed to be a strong validation of ehrlich’s idea. indeed, so influential and enduring was medicine’s triumph over bacteria that the ‘war on cancer’ continues to be driven by the implicit assumption that magic bullets will one day be found for the disease.” after so many years, we are still waiting for this magic bullet against malignant tumours and, of course, this is generating the idea that something went wrong along the way (or from the very beginning). gatenby concludes: “however, in battles against cancer, magic bullets may not exist and evolution dictates the rules of engagement.” actually, gatenby proposes that a reasonable approach may be to set up therapeutic strategies aimed at controlling cancer rather than trying to cure it, through highly toxic drug combinations that are seemingly more destructive to the patient’s body than to cancer. altogether, these considerations suggest that we should proceed along two different but parallel paths in trying to find a way to cure diseases while at the same time avoiding treatments that are needlessly aggressive for the patient’s body (being extremely toxic and poorly effective). for this purpose, i would like to come back to a concept which in the past had a pivotal role in the identification of drugs proven effective in different diseases, namely ‘serendipity’. today, the word ‘serendipity’ is used in everyday language, though with different definitions, such as “the faculty of making happy and unexpected discoveries by accident,” and “the faculty of finding valuable or agreeable things not sought for” and “an accidental discovery” (i.e., “finding one thing while looking for something else”). however, the word itself has a very ancient origin. in fact, serendip was the old arabic name for ceylon, now known as sri lanka. the real origin of the word ‘serendipity’ comes from a persian fairy tale telling of the three princes of serendip who, during their travels, accidentally discovered numerous things they were not in fact on a quest for. in the 16th century, the tale was translated from persian to italian, and from italian to french. horace walpole (1717-1797), an english man of letters, encountered it in a collection of oriental tales in french, and coined the english term ‘serendipity’ in a letter to his friend, horace mann, dated june 28, 1754. we should not forget that serendipity is one of the pivotal factors contributing to drug discovery. whether we want to keep the definition whereby serendipity implies the finding of one thing while looking for something else, we should recall the discovery of penicillin first. fleming was studying “staphylococcus influenza” when one of his culture plates had become contaminated, developing a mould that created a bacteria-free circle. later, he found within the mould a substance that has highly resistant 2 j circ biomark, 2014, 3:5 | doi: 10.5772/58709 against the vast majority of bacteria infecting human beings. equally, serendipity played a key role in the discovery of a wide range of psychotropic drugs, including aniline purple, lysergic acid diethylamide, meprobamate, chlorpromazine and imipramine (3). it appears quite clear that, at least in the past, serendipity played a pivotal role drug discoveries – or, more precisely, in the discovery of drugs that were effective against different diseases. thus, while we are confronted with an unbelievable failure in the discovery of effective drugs based on cross-communication between basic science and drug companies, we have to realize that this approach should be abandoned if we want to get to better results. probably, we should have another look at the thousands of drugs on the market with different eyes. probably, we would be better to think of the ‘offtargeting’ of drugs. in fact, there is an interesting approach using side-effects’ similarities for drug target identification, meaning that there is a trend in drug discovery based on the identification of common offtargets of different drugs through the evidence of common side effects (4). an example of the off-targeting approach comprises proton pump inhibitors, which in addition to having some off-targets in the central nervous system (4) have also been shown to have a potent antitumour effect, through the inhibition of proton pumps on tumour cells (which are similar, but not identical, to gastric proton pumps (5-10)). however, in addition to thinking about the careful identification of effective off-targeting drugs, we should also think about different systems of drug delivery. in the strategic platforms for nanomedicine, it is clearly stated that nanomedicine seeks to exploit the improved (and often novel) physical, chemical and biological properties of materials at the nanometric scale. however, these documents specify that there is an urgent need for biomimetism, namely the process of simulating what occurs in nature. exosomes are nanovesicles, naturally released from almost every cell in our body and which, whether in a normal or a diseased state, deliver a mess of molecules including proteins, lipids and nucleic acids. they are able to interact with the target cells within an organ or at distance using different mechanisms, including ligand-to-receptor interaction (11-12) and fusion with the cell plasma membrane via the transfer of their contents within the cell cytoplasm (13). thus, exosomes appear as a vectorized signalling system operating inside a donor cell by either binding to the membrane receptors or directly interacting with internal compartments of the target cell. these notions place the exosome at the centre of the real novelties in translational science, and mark it as a potential candidate autologous nanoshuttles for drugs potentially useful for the future strategies in nanomedicine. the future use of exosomes for new therapeutic and diagnostic approaches has to be discussed and given serious consideration. exosomes are becoming the real novelty in the identification of novel biomarkers. in fact, new tests offering the possibility of the contemporary characterization and quantification of exosomes in human body fluid have been set up recently (14). this dual potentiality of the exosome recommends the use of these nanovesicles as the ideal tool in ‘theranostics’. this new area of nanomedicine focuses on multi-disciplinary research to build new systems for various nanobiomedical applications, ranging from the medical use of nanoplatform-based diagnostic agents, to therapeutic agents and even possible future applications of diagnosis and therapy theranostics. theranostics is the medical application of nanobiotechnology and refers to highly specific medical intervention at the nanoscale in diagnosing, curing and preventing diseases. it includes the early detection of diseases, the monitoring of therapeutic responses and the targeted delivery of therapeutic agents. theranostics at the nanometric-scale encompasses, nanoprobes, nanocarriers and nanodiagnostics. however, the most important task of a theranostic strategy concerns theranostic nanoformulations, which deal with the development of new agents based on a ‘whole in one approach’ that should have its maximal application in the field of personalized medicine.. the exosome appears as the ideal nanovector for theranostics, with maximal potentiality for targeting the disease site with only minimal side effects. if successful, the proof-of-concept in the use of exosomes as the autologous nanovector for both the diagnosis and therapy of major diseases will allow for widespread preclinical and clinical applications. i have a dream, as well: “that serendipity, ideas and an open mind, will drive new research in biomedicine, acquiring the best results with the aim of freeing human beings from the nightmare of uncurable diseases” references [1] shaywitz d, taleb n. drug research needs serendipity. the financial times july 30, 2008. [2] gatenby ra. a change of strategy in the war on cancer. nature. 2009 may 28;459(7246):508-9. [3] ban ta. the role of serendipity in drug discovery. dialogues clin neurosci. 2006;8(3):335-44. [4] campillos m, kuhn m, gavin ac, jensen lj, bork p. drug target identification using side-effect similarity. science. 2008 jul. 11;321(5886):263-6. [5] luciani f, spada m, de milito a, molinari a, rivoltini l, montinaro a, marra m, lugini l, logozzi m, lozupone f, federici c, iessi e, parmiani g, arancia g, belardelli f, fais s. effect of proton pump inhibitor pretreatment on the resistance of solid tumors to cytotoxic drugs. j natl cancer inst. 2004 nov. 17;96(22):1702-1713. 3stefano fais: “i have a dream” [6] de milito a, iessi e, logozzi ma, lozupone f, spada m, marino ml, federici c, perdicchio m, matarrese p, lugini l, nilsson a, fais s. proton pump inhibitors induce apoptosis of human b cell tumors through a caspase-independent mechanism involving reactive oxygen species. cancer res. 2007 jun. 1;67(11):5408-17. [7] fais s, de milito a, you h, qin w. targeting vacuolar h+-atpases as a new strategy against cancer. cancer res. 2007 nov. 15;67(22):10627-30. [8] de milito a, canese r, marino ml, borghi m, iero m, villa a, venturi g, lozupone f, iessi e, logozzi m, della mina p, santinami m, rodolfo m, podo f, rivoltini l, fais s. ph-dependent antitumor activity of proton pump inhibitors against human melanoma is mediated by inhibition of tumor acidity. int j cancer. 2010 jul 1;127(1):207-19 [9] fais s. proton pump inhibitor-induced tumour cell death by inhibition of a detoxification mechanism. j intern med. 2010 may;267(5):515-25. [10] spugnini e, baldi a, buglioni s, carocci f, milesi de bazzichini g, betti g, pantaleo i, menicagli f, citro g, fais s. lansoprazole as a rescue agent in chemoresistant tumors: a phase i/ii study in companion animals with spontaneously occurring tumors. j transl med 2011 dec. 28; 9:221. [11] andreola, g., rivoltini, l., castelli, c., huber, v., perego, p., deho, p., squarcina, p., accornero, p., lozupone, f., lugini, l., stringaro, a., molinari, a., arancia, g., gentile, m., parmiani, g., fais, s. j. exp. med. 2002 195, 1303–1316. [12] huber, v., fais, s., iero, m., lugini, l., canese, p., squarcina, p., zaccheddu, a., colone, m., arancia, g., gentile, m., seregni, e., valenti, r., ballabio, g., belli, f., leo, e., parmiani, g., rivoltini, l. gastroenterology 2005 128, 1796–1804. [13] parolini i, federici c, raggi c, lugini l, palleschi s, de milito a, coscia c, iessi e, logozzi m, molinari a, colone m, tatti m, sargiacomo m, fais s. microenvironmental ph is a key factor for exosome traffic in tumor cells. j biol chem. 2009 dec. 4;284(49):34211-22. [14] logozzi m, de milito a, lugini l, borghi m, calabrò l, spada m, perdicchio m, marino ml, federici c, iessi e, brambilla d, venturi g, lozupone f, santinami m, huber v, maio m, rivoltini l, fais s. high levels of exosomes expressing cd63 and caveolin-1 in plasma of melanoma patients. plos one. 2009;4(4):e5219. 4 j circ biomark, 2014, 3:5 | doi: 10.5772/58709 article journal of circulating biomarkers influence of lung parenchyma surgical manipulation on circulating free dna original research article marco anile1*, caterina chiappetta2, daniele diso1, valeria liparulo1, martina leopizzi2, carlo della rocca2 and federico venuta1,3 1 university of rome sapienza, department of thoracic surgery, rome, italy 2 university of rome sapienza, department of pathology, rome, italy 3 fondazione eleonora lorillard spencer cenci, rome, italy *corresponding author(s) e-mail: marco.anile@uniroma1.it received 27 august 2014; accepted 20 october 2014 doi: 10.5772/59875 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract objectives: metastatic recurrence is the most frequent cause of death after surgical resection of lung cancer. manipulation during surgery has been advocated as one of the causes contributing to promotion of spreading. methods: we investigated if the detection of plasma circulating free dna (cfdna) is influenced by surgical manipulation in 25 lung cancer patients (17 males and eight females) undergoing complete resection; 20 health subjects formed the control group. bloodstream levels of cfdna were detected before surgery, one week and one month after surgery. results: cfdna levels measured preoperatively and in the control group were 23 07 ± 7 4 ng/ml and 7 5 ± 3 4 ng/ml respectively (p=0 0002); levels at one week and one month were 68 2 ± 36 2 ng/ml and 9 6 ± 3 1 ng/ml respectively. the difference between the three time points were statisti‐ cally significant (preop vs. one week p=0 0006; one week vs. one month p=0 0003) with an increase in the first week and a strong decrease after one month. cfdna levels at one month were not statistically different from those recorded in the control group. there was no correlation between preoperative cfdna levels, tumour stage, grading and histology and patient demographics. no correlation was found between postoperative cfdna, type of surgical procedure, histology and stage. after a median follow-up of 16 months no recurrence was detected. conclusions: surgical manipulation determines increased cfdna levels in the early postoperative period; however, after one month they decrease within the normal range, at levels that are statistically comparable with healthy subjects. keywords lung cancer, circulating free dna, real-time quantitative polymerase chain reaction, surgical resection, recurrence 1. introduction lung cancer is the leading cause of cancer related death worldwide in both genders [1]. despite the improved diagnostic tools and treatment strategy, overall survival is still poor; metastatic recurrence is also the most important cause of death after complete resection. the metastatic process involves a number of steps that unfortunately are not yet completely understood [2]. in particular, several authors postulated that manipulation of the tumour during 1j circ biomark, 2014, 3:7 | doi: 10.5772/59875 surgery might promote early spreading [3, 4]. release of tumour growing factors, surgical stress-related suppres‐ sion of the innate immunity, reduction of anti-angiogenesis factors and tumour cells shedding in the bloodstream were considered as possible causes; however, the reported results were often contradictory due to the heterogeneity of the study populations. in 1948 mandel and metais demonstrated the presence of circulating free dna (cfdna) in the bloodstream and subsequent studies showed a correlation between high cfdna levels and neoplastic diseases [5–8]. recently, with the improvement of extraction and quantification methods, the determination of cfdna is becoming a useful diagnos‐ tic and prognostic tool also in lung cancer patients [9, 10]. it has been demonstrated that high preoperative cfdna levels show a negative impact on long term survival, independently from a patient’s age and stage of the disease [11]. however, the explanation of the mechanism involved in cfdna release still remains uncertain. several hypothe‐ ses (tumour cells lysis, apoptosis, necrosis or active secretory effects of neoplastic cells) have been proposed, but none of them is exhaustive [12, 13]. furthermore, the relationship between cfdna levels, disease progression and metastatic spreading is still debated. based on previous experimental studies on cfdna plasma levels after surgical procedures, some authors focused on circulating cancer cells and nucleic acids, postulating in animal models that their postoperative increased levels might show an impact on the mechanism of metastatic spreading [14, 15]. how‐ ever, this hypothesis has not yet been confirmed since there are no clinical studies evaluating the levels of circulating nucleic acids during the early postoperative course; thus, we are not able to assess correctly the potential modifica‐ tions during follow-up and understand the role of surgical manipulation on their release within the bloodstream. 2. materials and methods twenty-five consecutive patients (17 men – eight women; mean age 62 4 ± 10 years) with lung cancer underwent complete surgical resection. none of them had a previ‐ ous history of cancer or received induction therapy. twenty voluntary healthy subjects (10 men – 10 wom‐ en; mean age 37 ± 15 years) were considered as the control group. after an exhaustive explanation of the objectives of the study, all patients and controls signed an in‐ formed consent. fifteen patients (60%) had squamous cell carcinoma and 10 (40%) had adenocarcinoma. none of them had chronic viral infections. we performed eight right upper lobectomies, six right lower lobectomies, five left upper lobectomies, five left lower lobectomies and one right intrapericardial pneumonectomy; in four lobectomy patients, because of neoplastic ribs invasion, a chest wall resection was required. in all cases a com‐ plete hilar and mediastinal lymph node dissection was performed at the end of the procedure. interlobar fissures were created in all cases with surgical staplers, with the obvious exception of pneumonectomy. no blood transfu‐ sions were required. fifteen patients (60%) were at stage i, seven (28%) at stage ii and three (12%) at stage iiia. a withdrawal of 6 ml peripheral blood was collected in tubes containing edta the day of surgery just before induction of anaesthesia, one week and one month postoperatively. the samples were processed to obtain plasma within an hour after the withdrawal of the blood sample. plasma was separated from the cellular fraction by centrifugation two times at 2500 rpm for 10 minutes at 4°c. the resulting supernatant (plasma) was frozen at-80°c. dna was extracted from plasma by using qiaamp dna mini kit (qiagen, italy) according to the supplier’s instructions; this provided an elution in a final volume of 50 ul. the dna was stored at-20°c until it was analysed. to quantify the plasma circulating dna we used a realtime quantitative polymerase chain reaction (pcr) ap‐ proach based on the 5’ nucleotide method. this methodology is based on continuous monitoring of a progressive fluorogenic pcr by an optical system [16]. the pcr system uses two amplification primers and an additional amplicon-specific and fluorogenic hybridiza‐ tion probe, the target sequence of which is located within the amplicon. the probe is labelled with two fluorescent dyes. one server was used as a reporter on the 5’ end (fam dye; applied biosystems, foster city, ca, usa). the emission spectrum of the dye is quenched by a second fluorescent dye at the 3’ end (tamra; applied biosystems, foster city, ca, usa). if amplification occurs, the 5’ to 3’ exonuclease activity of the amplitaq (biosystems, foster city, ca, usa) dna polymerase cleaves the reporter from the probe during the extension phase, thus releasing it from the quencher [17]. the resulting increase in fluorescent emission of the reporter dye is monitored during the pcr process. the real-time pcr was performed on an abi prism 7500 fast real time pcr system (applied biosystem, foster city, ca, usa). primers and probes were designed to specifically amplify the ubiquitous gene of interest, the actb2 single copy gene mapped on 5q11.2 (gene id: 345651, applied biosystem, foster city, ca, usa). the amplicon size of the actbl2 gene was 67 bp. pcr reaction was carried out on 96-well plate with 20 ul per well using 1x taqman master mix. after incubation for two minutes at 50°c and 10 minutes at 95°c, the reaction continued for 40 cycles at 95°c for 15 seconds and 60°c for one minute. each plate consisted of patient samples in triplicates and multiple water blanks as negative control. for construction of the calibration curve on each plate, we used a standard taqman control human genomic dna (applied biosys‐ tems, foster city, ca, usa) with appropriate serial dilutions at 100, 10, 1, 0 1, 0 01, and 0 001 ng. all of the data were analysed using the abi prism 7500 software (ap‐ plied biosystems foster city, ca, usa) to interpolate the 2 j circ biomark, 2014, 3:7 | doi: 10.5772/59875 standard amplification curve of dna at a known quantity with amplification cycle threshold of the unknown target sample, obtaining the relative amount of dna in the experimental sample. 3. statistical analysis quantitative variables were reported as mean ± standard deviation and the differences were analysed with the student’s t test. correlation analysis was performed between cfdna levels and quantitative variables (age and tumour dimension). qualitative variables as stage, patho‐ logical nodal status, histology, presence of tumour ne‐ crosis, grading, type of surgery (standard vs. extended), and history of smoking were reported as present or absent and used to identify two groups of patients at the three different steps. differences of cfdna levels between these groups were assessed by the student’s t test. statistical analysis was performed with spss software (version 17 0, spss inc., usa). 4. results the surgical procedure lasted a mean of 88 4 ± 21 3 minutes (range 62 – 115 minutes). after surgery, all patients were recovered in the surgical ward. no major postoperative complications occurred with an uneventful postoperative course in all patients. no adjuvant therapy has been administered before the third blood withdrawal at one month. in all patients the third blood sample was obtained; furthermore, in 14 patients (56%) we were able to collect blood samples at six and 12 months. the median follow-up was 16 months. in the study population and in the control group the mean preoperative level of cfdna was 23 07 ± 7 4 ng/ml and 7 5 ± 3 4 ng/ml respectively (p=0 0002). after one week the cfdna bloodstream level was 68 2 ± 36 2 ng/ ml (p=0 0006) and after one month the cfdna values significantly dropped below preoperative levels (9.6 ± 3.1 ng/ml) (p=0.0001). the difference between the cfdna levels measured one month after surgery and those obtained in healthy subjects did not reach statistical significance (9 6 ± 3 1 ng/ml vs. 7 5 ± 3 4 ng/ml p=0 1). no correlation was found between preoperative and one week cfdna levels, age (p=0 989 at both time points) and tumour diameter (p=0 925 and p=0 325 at each time point). no other variable considered for statistical analysis showed an impact on cfdna levels at the different time points of the study (table 1); the increase of cfdna values after one week seems absolutely independent from any assessed variable. no statistically significant differences on cfdna values were reported between pathological stages neither preoperatively nor during the follow-up. after a median follow-up of 16 months no patient showed recurrence; stage iii patients underwent adjuvant chemo-radiothera‐ py. eighteen patients (72%) were reassessed six months after surgery and 14 (56%) after 12 months showing stable cfdna levels (fig.1). t0 t1 t2 nodal status (n0-1vs n2] 0.63 0.7 0.67 histology (epi vs. adc) 0.57 0.68 0.56 necrosis (yes/no) 0.07 0.45 0.79 surgery (standard vs. extended) 0.43 0.54 0.41 smoking (yes/no) 0.98 0.1 0.87 t0: preoperative; t1: 1 week; t2: 1 month; epi: squamous cell carcinoma; adc: adenocarcinoma table 1. p values of cfdna levels at three steps according to qualitative variables assessed by independent student’s t test figure 1. trend of cfdna levels in patients undergoing surgery 5. discussion metastatic disease is still the leading cause of death after complete surgical resection of lung cancer. metastatic spreading involves several steps: the presence in the bloodstream of circulating cancer cells (with their nucleic acids), their passage from blood to distant organs and the growth in the new site. the mechanism of this process is not completely understood yet, although it has been postulated that a reciprocal transition of cancer cells between the epithelial and mesenchymal status should be involved [18]. several authors suggested that surgical manipulation could promote this cascade already during the immediate postoperative course; many different mechanisms have been advocated to elicit this process, in particular the decrease of immune-competency after surgery, the production of oxygen free radicals by leuko‐ cytes or tumour cells shedding into the bloodstream [19, 20]. cell-free tumour nucleic acids (cfdna) levels have been proposed as a diagnostic and prognostic tool in several solid cancers. furthermore, cfdna determination in lung cancer patients has gained importance during recent years. yoon and colleagues in 2009 compared cfdna levels between lung cancer patients and healthy subjects finding higher values in the former group [9]. paci and colleagues demonstrated that in 151 lung cancer patients cfdna levels were higher when compared to healthy individuals and that the risk of disease increases with higher cfdna levels; in particular with a cut-off of 2 ng/ml, higher concentrations are strongly connected with the presence of neoplastic disease [10]. van der drift and colleagues in 2010 reported that high cfdna levels at the time of diagnosis were a prognostic factor for poor survival, although no correlation was found with stage, histology and age [11]. our study confirms that in the lung cancer population cfdna levels are higher than in healthy subjects. these molecules might not be produced only from tumour cells degradation; they can also be released by an active secretory mechanism and they can play a role in the metastatic process. in fact, it has been demonstrated in an 3marco anile, caterina chiappetta, daniele diso, valeria liparulo, martina leopizzi, carlo della rocca and federico venuta: influence of lung parenchyma surgical manipulation on circulating free dna animal model that haematogenous dissemination of tumours due to surgery seems related to the presence of cfdna [15]. however, in a study on percutaneous liver biopsy in patients with hepatocarcinoma no relationship was found between this procedure and cfdna plasma dissemination [21]. our results show that one week after surgery, cfdna levels increase, probably due to manipu‐ lation, but after one month they drop below preoperative values in all patients and remain stable during the time period of the study. thus, since all patients show this pattern, if surgical manipulation related release should be involved in the metastatic process, it must be in combina‐ tion with other variables different from patient to patient (release of tumour growing factors, suppression of innate immunity, reduction of anti-angiogenesis factors, etc.). the absence of statistical significance between the study population one month after surgery and the control group allows us to conclude that the impact of lung manipulation is restricted to the very early postoperative course and cannot influence the metastatic process by itself. the surgical stress entails an increased inflammatory response with determination of high cfdna levels, but probably the causes and mechanisms of the metastatic process should be researched somewhere else. 6. conflict of interest all authors declare no conflict of interest. 7. references [1] jemal a, center mm, desantis c, ward em. global patterns of cancer incidence and mortality rates and trends cancer epidemiol biomarkers prev 2010; 19: 1893-907 [2] pantel k, brakenhoff rh. dissecting the metastatic cascade. nat rev cancer 2004; 4: 448-56 [3] ben-eliyahu s. does surgery promote metastasis? lancet oncology 2002; 3: 578-9 [4] da costa ml, redmond p, bouchier-hayes dj. the effect of laparotomy and laparoscopy on the establishment of spontaneous tumor metastases. surgery 1998; 124: 516-24 [5] mandel p, matais p. les acides nuclèiques du plasma sanguine chez l’homme. cr acad sci paris 1948; 142: 241-3 [6] silva jm, silva j, sanchez a. et al. tumor dna in plasma at diagnosis of breast cancer patients is a valuable predictor of disease-free survival. clin cancer res 2002; 8: 3761-6 [7] kamat aa, bischoff fz, dang d. et al. circulating cell-free dna: a novel biomarker for response to therapy in ovarian carcinoma. cancer biol ther 2006; 5: 1369-74 [8] hauser s, zahalka t, ellinger j. et al. cell-free circulating dna: diagnostic value in patients with renal cell cancer. anticancer res 2010; 30: 2785-9 [9] yoon ka, park s, lee sh, kim jh, lee js. compar‐ ison of circulatin plasma dna levels between lung cancer patients and healthy controls. j mol diagn 2009; 11: 182-5 [10] paci m, maramotti s, bellesia e. et al. circulating plasma dna as diagnostic biomarker in non-small cell lung cancer. lung cancer 2009; 64: 92-7 [11] van der drift ma, hol bea, klaassen chw et al. circulating dna is a non invasive prognostic factor for survival in non-small cell lung cancer. lung cancer 2010; 68: 283-7 [12] garcia-olmo d, garcia olmo dc. functionality of circulating dna. the hypothesis of genometasta‐ ses. ann n y acad sci 2001; 945: 265-75 [13] stroun m, lyautey j, lederrey c, olson-sand a, anker p. about the possible origin and mechanism of circulating dna apoptosis and active dna release. clin chim acta 2001; 313: 139-42 [14] garcia-olmo d, ontanon j, garcia-olmo dc, vallejo m, cifuentes j. experimental evidence does not support use of the “no-touch” isolation techni‐ que in colorectal cancer. dis colon rectum 1999; 42: 1449-56 [15] garcia-olmo dc, gutierrez-gonzalez l, samos j, picazo mg, atienzar m, garcia-olmo d. surgery and hematogenous dissemination: comparison between the detection of circulating tumor cells and of tumor dna in plasma before and after tumor resection in rats. ann surg oncol 2006; 13: 1136-44 [16] heid ca, stevens j, livak k, williams pm. real time quantitative pcr. genome res 1996; 6: 986-94 [17] holland pm, abramson rd, watson r, gelfand dh. detection of specific polymerase chain reaction product by utilizing the 5’-3’ exonuclease activity of thermus aquaticus dna polymerase. proc natl acad sci usa 1991; 88: 7276-80 [18] polyak k, weinberg ra. transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits. nat rev cancer 2009; 9: 265-73 [19] yamaguchi k, takagi y, aoki s, futamura m, saji s. significant detection of circulating cancer cells in the blood by reverse transcriptase-polymerase chain reaction during colorectal cancer resection. ann surg 2000; 232: 58-65 [20] zetter br. angiogenesis and tumor metastasis. ann rev med 1998; 49: 407-24 [21] yu sch, lo dym, ip cb, liew ct, leung twt, lau wj. does percutaneous liver biopsy of hepatocellu‐ lar carcinoma cause haematogenous dissemina‐ tion? ajr 2004; 183: 383-5 4 j circ biomark, 2014, 3:7 | doi: 10.5772/59875 article journal of circulating biomarkers short course in extracellular vesicles – the transition from tissue to liquid biopsies meeting dispatch jan lötvall1, johan skog2, alexander v. vlassov3, angel ayuso sacido4, eva rohde5,6, joanne gere7 and winston patrick kuo8* 1 department of internal medicine, gothenburg university, gothenburg, sweden 2 exosome diagnostics, cambridge, ma, usa 3 thermo fisher scientific, austin, tx, usa 4 thoracic and brain tumour laboratory, ciocc-imma, fundacion hospital de madrid, madrid, spain 5 spinal cord injury and tissue regeneration center, salzburg, austria 6 transfusion medicine department, paracelsus medical university, salzburg, austria 7 biopharma research council, tinton falls, nj, usa 8 ies diagnostics, inc., cambridge, ma, usa *corresponding author(s) e-mail: winston@iesdiagnostics.com doi: 10.5772/60053 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract extracellular vesicles (evs), including exosomes and microvesicles, carry a variety of bio-macromolecules, including mrna, microrna, other non-coding rnas, proteins and lipids. evs have emerged as a promising, minimally invasive (liquid biopsies) and novel source of material for molecular diagnostics, and may provide a surrogate to tissue biopsy-based biomarkers for a variety of diseases. although evs can be easily identified and collected from biological fluids using commercial kits, further research and proper validation is needed in order for them to be useful in the clinical setting. currently, several ev-based research and diagnostic companies have developed research-based kits and are in the process of working with clinical laboratories to develop and validate ev-based assays for a variety of diseases. the successful clinical application of ev-based diagnostic assays will require close collaboration between industry, academia, regulatory agencies and access to patient samples. we expect that international, integrative and interdisciplinary translational research teams, along with the emergence of fda-approved platforms, will set the framework for evbased diagnostics. we recognize that the ev field offers new promise for personalized/precision medicine and targeted treatment in a variety of diseases. a short course was held as a four-session webinar series in september and october 2014, presented by pioneers and experts in the ev domain, covering a broad range of topics from an overview of the field to its applications, and the current state and challenges of the commercialization of evs for research and an introduction to the clinic. it was concluded with a panel discussion on the regulatory aspects and funding opportunities in this field. a summary of the short course is presented as a meeting dispatch. keywords extracellular vesicles, exosomes, short course, liquid biopsies, therapeutics, funding, clinical trials 1j circ biomark, 2014, 3:8 | doi: 10.5772/60053 1. introduction evs are small vesicles, containing diverse “nucleic acids” and protein cargo that are spontaneously secreted by all cells and found in abundance in all human body fluids. depending upon the cell or tissue of origin, many different roles and functions have been attributed to evs, for example: the eradication of obsolete molecules, the facili‐ tation of the immune response, antigen presentation, programmed cell death, angiogenesis, inflammation, coagulation, the dissemination of oncogenes from tumour cells and the spread of pathogens such as prions and viruses from one cell to another. importantly, evs deliver macromolecular messages that enable cell-to-cell commu‐ nication and signalling. the short course, evs: the transi‐ tion from tissue to liquid biopsies was sponsored by the biopharma research council and was held as a foursession series in october and november 2014. the goal of the short course was to provide, at an introductory level, an exchange between researchers from academia and industry in the characterization of applications of evs in clinical and translational research and, eventually, clinical practice. the ev topics included an overview of its appli‐ cations, the commercial aspects, therapeutic hurdles and funding opportunities. 2. session 1 – september 11, 2014 2.1 overview of extracellular vesicles by jan lötvall, md, phd the first session presented by jan lötvall, provided an overview of evs (their function and role in medicine and therapy). jan started by describing and providing a history of evs. evs are membrane vesicles with a lipid bilayer that have surface molecules present on the surface containing cytoplasmic molecules, which can vary in size, with the smaller evs ranging from 40-150 nm, which have a regulatory function. evs were first described in 1983 as debris by two groups, harding et al. and pan et al., discussing the transferrin receptor sent out from the cells becoming erythrocytes. earlier studies, in 1967 and 1977, discussed platelet dust and prostasomes, respectively. jan continued to discuss where evs are found, what they contain and what can they do in disease. the three major groups of evs, even though there are many names used in the literature, are: 1) microvesicles budding off the surface of the cells which are released, containing surface receptors, and attaching to another cell (surface-to-surface interac‐ tion) or even being taken up by another cell; 2) exosomes, which are smaller evs, are produced by multivesicular endosomes, contain different types of cargo, and are taken up by another cell (recipient cell); and 3) larger vesicles produced by programmed cell death, the apoptotic bodies. these are important in cell-to-cell communication. jan further discussed how the production of evs is a conserved process, as seen in bacteria, plants and parasites, which is not specific in human cells. a recent paper in science reported bacterial vesicles found in marine ecosys‐ tems (the ocean) as they relate to ocean-depth. they demonstrated the transfer of carbon when giving these vesicles to another bacterium to sustain life. in another example, jan discussed a controversial issue, the presence of rice mirna in the circulation of chinese people, where intact mirna molecules from the rice are present in the evs, whereby harbouring the rice cargo. lastly, two abstracts presented at the isev meeting discussed the presence of evs in beer, especially in unfiltered beer, and how they are ingested by humans. what are the functions of evs? evs have several functions, including surface-to-surface interactions (antigen presen‐ tation), first described in 1996. another function is the shuttling of proteins, rna and lipids between cells. the presence of nucleotides in evs was described in the late 1990s, considering the packaging of dna and rna in apoptotic bodies during apoptosis. in 2006, a study demonstrated that vesicles greater than 100 nm provided evidence that mrna and protein can be delivered from one cell to another. in 2007, jan’s group showed the presence of mrna and mirna in smaller evs 40-50 nm shuttling materials between cells. to prove that the rna was functional, they took mouse exosomes with mrna to human mast cells, translating proteins in human cells. they found a number of proteins from exosomes, and a number of proteins that were present as mrna and not present at proteins in exosomes, suggesting that mouse rna can be translated to protein in the recipient cell. there was also a presence of functional mirna. how do you test whether the rna is functional? jan’s group exposed exosomes to oxidative stress and placed evs on top of cells, placing them under stress again – mediating a protective message under stress. where do you find extracellular rna? they are found in all the body fluids, like blood, plasma, saliva, breast milk and urine. the communication of rna containing exo‐ somes can be made by kissing or may be by breast milk (mother to child communication – uptake by macrophages – immune tolerance and t-regulatory cells). evs are present in fetal bovine serum (fbs). in cell culture experiments, fbs is often used to supplement the cell culture medium as a nutrient, but it is important to know that the fbs also contains significant quantities of evs. jan’s group showed how rna-containing evs in fbs can be removed to almost 95% of cases with an 18-hours centrifu‐ gation protocol, and this approach strongly reduces the functionality of fbs vesicles in relation to epithelial cell migration. 2 j circ biomark, 2014, 3:8 | doi: 10.5772/60053 what can evs do in medicine? they can promote and attenuate disease. jan described the presence of bacterial evs (outer membrane) released from pseudomonas aeruginosa, where his group isolated the evs from the cell to look to see whether they induce pseudomonas aerugi‐ nosa related pathologies/symptoms. in another example, outer membrane evs from pseudomonas aeruginosa showed strong inflammation (travel through the body quickly by evs) without any bacteria at all; clearly, evs are very potent, inducing symptoms. clearly, all bacterially (both pathogenic and non-pathogenic) released evs are distributed throughout the body, inducing an inflammato‐ ry process. evs have also been found in the peripheral blood of sepsis patients. in the cancer field, jan discussed a few examples of the role of evs in tumours and cancers. tumours contain cancer cells, fibroblasts, immune cells and inflammatory cells, but evs play a role in influencing these cells in the microenvir‐ onment. jan further discusses the role of evs secreted under hypoxia and how they enhance the invasiveness of prostate cancer cells cells secreting evs from the center of the tumour. another study discussed how evs from triplenegative breast cancer cells can transfer phenotypic traits. lastly, jan discussed a recent study which showed the reprogramming of patient-derived adipose stem cells by prostate cancer cell-associated evs that actually produce tumours. jan stated there are many subsets of evs. his group showed that apoptotic bodies, microvesicles and exosomes contain fundamentally different rna profiles, arguing that microvesicles isolated from cell cultures often do not contain considerable amounts of rna. the rrna was primarily found in apoptotic bodies, which should be considered when the functionality of rna in different vesicles is studied. exosomes as therapy? jan discussed a paper published by a group from tokyo who systemically injected exosomes targeted to egfr to deliver anti-tumour mirna to breast cancer cells, suggesting that you can target to tumours partly by functional rna. in summary, jan concluded the following: 1. exosomes and other ev contain rna 2. the rna can be shuttled between cells 3. mediate biological messages 4. circulating esrna can be biomarkers 5. exosomes and other evs can deliver therapy 6. possibilities for treating cancer and inflammatory diseases in addition, he invited everyone to the upcoming isev meeting held in washington dc that will take place during april 23-26, 2015, at the bethesda north marriot hotel. 3. session 2 – september 18, 2014 3.1 clinical diagnostic applications of extracellular vesicles by johan skog, phd the second session, by johan skog, discussed the clinical diagnostic applications of evs, including sample collection and processing approaches, and platform applications, including exosome rna mutation analysis and expression profiling. johan started off by describing an overview of evs. various imaging technologies like electron microsco‐ py (em) and scanning em can be used to characterize evs, and it has been shown that evs are heterogeneous in nature and that they vary in size (30 nm to greater than 1,000 nm). cryo-em show that evs are a double-lipid membrane, but interestingly evs are single-lipid bi-layers and one can see vesicles within vesicles. the contents of the evs are interesting they contain mrna, mirna, non-coding rna and a variety of proteins – a good platform for biomarkers. the package of evs is about 10 kb of rna, not the entire transcriptome. you will find one or a few transcripts per vesicle – carrying different messages. johan discussed the extraction of nucleic acids from biofluid such as plasma. using the exosome platform enables the sub-fractionation of rna from different cellular processes by way of total exosome extraction (ultracentrifugation, filter purification, etc.) and affinity purifi‐ cation (magnetic beads, microfluidics, etc.). in using wholeplasma extraction, there is a problem in that there is no selectivity from the sub-fractions (rna is acquired from all kinds of components) and there is a limit in the efficiency of the rna extraction with higher volumes due to rnase activity. johan showed that exosomal isolation demon‐ strates the good quality/yield of intact rna and 18s and 28s ribosomal rna peaks compared to direct extraction. johan stated that the pre-processing of one’s samples is a critical step, like avoiding cell carry-over. he emphasized that the need for standardized sops for the collection and pre-processing of your samples is important. when isolating nucleic acid isolation, you need to know whether you are extracting dna or rna, since small amounts of rna are easily be biased by dna. it is important to have controls in your extraction method. johan uses the exorna isolation method because it is scalable. most blood collec‐ tion tubes are compatible with extraction methods, but have different biases. johan emphasized using controls/ standards and the same collection protocol for all patients. exosome rna is stable once isolated, even under freezethaw cycles. johan mentioned that there are multiple sources of rna in biofluids and that exosome rna is not the same as the direct precipitation of rna. by conducting the lysis on the filter using the exorneasy kit, one can capture the rna, even the flow-through (mirna), directly. high volumes of samples are needed for highsensitivity applications, like looking for oncogenes and tumour mutations. 3jan lötvall, johan skog, alexander v. vlassov, angel ayuso sacido, eva rohde, joanne gere and winston patrick kuo: short course in extracellular vesicles – the transition from tissue to liquid biopsies exosome rna profiling can be used to monitor treatment responses johan found unique expression changes in the responder group when compared to the non-responder group and when validated by single-plex qpcr validation. johan concluded there are benefits in measuring tumour mutations in biofluids because there is less risk for the patient than invasive biopsy, it is cheaper than surgery, and it can obtain multiple samples over time to track changes longitudinally (although there are still challenges in isolations and in detecting rare tumour transcripts). 4. session 3 – september 25, 2014 4.1 commercialization aspects of extracellular vesicles by alexander “sasha” vlassov, phd the third session on the commercialization aspects of evs was presented by alexander “sasha” vlassov. his session discussed how the spectrum of current scientific interest in exosomes is wide, ranging from studying their functions and pathways, to utilizing them in the development of diagnostics and therapeutics. for example, exosome diagnostics developed a biofluid-based molecular diagnos‐ tic test for use in personalized medicine. the company's proprietary exosome technology makes use of this natural enrichment to achieve high sensitivity and specificity for rare gene transcripts, as well as the expression of genes responsible for cancers and other diseases. caris life sciences developed carisome – an innovative, proprietary and versatile testing technology that has the potential to reveal critical information about disease at its earliest stages, from a simple blood test. currently being developed for a number of types of cancers, including prostate, breast, lung and colorectal cancers, the technology has the poten‐ tial to provide diagnostic, prognostic and theranostic information for patients. exosome sciences developed exosome-based solutions to improve the identification and monitoring of acute and chronic conditions. candidate products are focused on diagnostic advancements in the fields of oncology, infectious disease and brain injury. exosomics sienna performs applied research and r&d activities in the field of exosome-associated biomarkers, with a specific focus on the development and validation of proprietary immunometric multiplex assays for noninvasive cancer diagnostics and monitoring and point-ofcare devices for cancer screening. in speaking about therapeutic applications, sasha men‐ tioned how exosomes have tremendous potential as vesicles for the in vivo delivery of various therapeutic cargos. for instance, one group reported the successful use of exosomes for the delivery of short interfering rna (sirna) to the brain in mice. targeting was achieved by engineering dendritic cells to express lamp2b, a membrane protein found in exosomes, fused to the neuron-specific rvg peptide3. loaded exosomes were purified from conditioned culture media of these cells with synthetic sirna using electroporation and intravenously injected using rvg-p3-tagged exosomes that delivered sirna specifically to neurons, microglia and oligodendrocytes in the brain, resulting in a specific gene's knockdown. a completely different therapeutic approach was proposed by aethlon medical. a few years ago, it was reported that tumour-secreted exosomes actually suppress the immune response to the cancer. aethlon medical developed the hemopurifier® medical device to selectively remove the tumour-secreted exosomes from the circulatory system. their postulate is that this will restore the immune system of the cancer patients. the technology uses a large format flow-through canister that can work in an apheresis mode. proprietary lectins attached to the canister-bed matrix act as unique affinity-capture moieties for exosomes, targeting mannose residues on their surface. taking into account that the number of applications and commercial opportunities is rapidly increasing, exosomes are very small and complex entities there is a growing need for quick and easy methods for both the isolation of exosomes and the analysis of the containing cargo. life technologies (now part of thermo fisher scientific) developed a complete exosome workflow solution: (i) the fast and efficient recovery of exosomes from serum, plasma, urine, cerebrospinal fluid, amniotic fluid, ascitic fluid, milk, saliva and cell media using total exosome isolation reagents; (ii) the extraction of their “cargo” with total exosome rna and a protein isolation kit; (iii) the characterization of exosomal rna content using the ion torrent proton sequencing and qrt-pcr with taqman® assays. a number of additional kits and reagents were released recently, allowing the versatile and advanced analysis of exosomes and evs (www.lifetechnologies.com/ exosomes). for example, dynabeads with conjugated anticd63, cd81, cd9, epcam antibodies enable the isolation of exosomal subpopulations. the continuous development of next-generation tools is crucial in furthering our understanding of exosomes and expanding the range of their practical applications. more‐ over, based on the recent reports, this goes beyond mam‐ malian systems to plants, bacteria and possibly other kingdoms, where evs have many undiscovered roles and many exciting practical uses including, for example, food and cosmetic industries. 5. session 4 part 1– october 2, 2014 5.1 extracellular vesicles: therapeutic hurdles by eva rohde, md part one of the fourth session was a presentation on the therapeutic hurdles in the ev ecosystem by eva rohde. eva expressed the increasing interest in the putative therapeutic potency of human cell-derived evs. ev-based therapeutics are biologicals that may be categorized as ‘high risk medicinal products’ due to currently unknown mecha‐ 4 j circ biomark, 2014, 3:8 | doi: 10.5772/60053 nisms of action (moas). she stressed the challenges in ev characterization and the design of preclinical studies as well as aspects of pharmaceutical engineering which have to be addressed to overcome the hurdles on the way to clinical application. a lack of safety data as well as partic‐ ular knowledge about the nature of the therapeutic target or further biomedical uncertainties (as, for example, the most representative animal models for ev-based therapies) may setback the translational process. unsurprisingly, data from clinical trials (cts) testing ev-based therapeutics are scarce. from this point of view, we are not yet perfectly prepared for the clinical testing of ev-based therapeutics. nevertheless, study protocols evaluating the therapeutic potency of evs will emerge and broad clinical testing will hopefully start in the foreseeable future. to accelerate the developmental process bringing ev-based therapies into clinics, a close collaboration between researchers and public health authorities is urgently required. 6. session 4 part 2– october 2, 2014 6.1 funding opportunities for extracellular vesicle research by angel ayuso sacido, phd the second part of the session was a discussion on the funding opportunities for ev research by angel ayuso sacido. angel primarily focused on the funding opportu‐ nities within the european union based on his experience. he provided an overview of the structure of the european parliament, the council and the commission. the europe‐ an union is based on a 10-year milestone process. the previous programme, entitled the “lisbon strategy”, from 2000-2010, was based on growth and jobs, whereas the horizon 2020 is very broad, whereby 3% of the eu’s gdp is focused on research and development. angel suggested three cores industrial leadership, societal challenges and excellent science in which ev research could fall under. angel also mentioned partnership opportunities, “looking for partners” using a matchmaking tool, “fit for health” and “funding for networks” by “eranets”, a networking mechanism of national and regional programmes, and “cost”, the european cooperation in science and tech‐ nology programme. 7. conclusion based on the session presentations, it was evident that the field of ev research is quickly evolving and that it continues to advance in all facets of science. overall, the short course fulfilled its goal of providing a balanced forum of relevant content from researchers from both academia and industry. the webinar presentations are available on the biopharma research council website (http://www.biopharmare‐ searchcouncil.org/webinar-short-course-in-the-exosome). 5jan lötvall, johan skog, alexander v. vlassov, angel ayuso sacido, eva rohde, joanne gere and winston patrick kuo: short course in extracellular vesicles – the transition from tissue to liquid biopsies exosomes and microvesicles editorial announcing exosomes and microvesicles, the official journal of the american society for exosomes and microvesicles stephen j. gould1, douglas taylor2, antonio chiesi3 and winston p. kuo4 1 department of biological chemistry, the johns hopkins university, baltimore, md, usa 2 department of obstetrics, gynecology & women’s health, university of louisville school of medicine, louisville, ky, usa 3 exosomics siena spa, siena, italy 4 harvard catalyst, laboratory for innovative translational technologies, harvard medical school, boston, ma, usa; department of developmental biology, harvard school of dental medicine, cambridge, ma, usa * corresponding e-mail: exmv@intechopen.com © 2013 gould et al.; licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract  this  editorial  article  introduces  the  new  scientific  journal exosomes and microvesicles  (exmv),  the  official journal of the american society for exosomes and  microvesicles  (asemv),  and  describes  its  editorial  line  and mission in relation to the role of the society, the state  of the art of the study of exosomes and microvesicles, and  the overall approach of the publication.    keywords exosomes, microvesicles, asemv, editorial                                            1. introduction    the  past  decade  has  witnessed  an  extraordinary  explosion  of  research  in  the  field  of  exosomes,  microvesicles,  and  other  extracellular  vesicles.  on  the  wave of the huge interest in this emerging field coming  from the academic, industrial and financial communities,  in 2012 the american society for exosomes and microvesicles  (asemv) was established by the initiative of a number of  stakeholders in the united states. asemv is a non‐profit  scientific  society  dedicated  to  advancing  exosome  and  microvesicle  research,  promoting  interactions  among  researchers,  funding  agencies,  and  educating  scientists  and policy makers about  the biological, biomedical and  biotechnological  importance of secreted vesicles  in both  basic and applied/clinical sciences. its mission, in short, is  to advance the field of extracellular vesicle research and  to support the scientists working in this domain, both in  the academic and industrial environments. the society is  already  active  in  sponsoring  scientific  meetings,  partnering  with  other  scientific  societies  for  interdisciplinary  developments  and  working  with  funding agencies to promote this exciting field of biology.  today, we expand these activities by launching exosomes  and  microvesicles  (exmv),  the  official  journal  of  our  society,  a  peer‐reviewed,  open  access  scientific  publication with an  innovative approach, being open to  both sides: for readers and for authors (no fee requested).  the  mission  of exosomes and microvesicles  is  to  publish  papers  that  provide  a  significant  contribution  to  our  understanding,  development  and  translation  of  knowledge  to  concrete  applications  in  this  field.  the  journal  is  addressed  to  experts  and  emerging  scientists  such as cell and molecular biologists, medical researchers,  clinicians  and  surgeons  specializing  in  exosome‐based  approaches  for  the  diagnosis  or  treatment  of  human  diseases,  but  also  to  researchers  coming  from  other  relevant  scientific  fields  such  as  biomedical  engineers,  biomaterials  scientists,  chemists  and  pharmacists  developing new technologies and applications in the field.  the goal of the journal is to connect all of them and thus  enhance the translation of knowledge in the exosome and  microvescicle  sciences  among  disciplines  and  among  1stephen j. gould, douglas taylor, antonio chiesi and winston p. kuo: announcing exosomes and microvesicles (exmv), the official journal of the american society for exosomes and microvesicles www.intechopen.com article www.intechopen.com exosomes microvesicles, vol. 1, 1:2013 academia and industry. no other existing journal focuses  on the application of exosomes and microvesicles in the  maintenance  of  human  health,  and  the  development  of  strategies to better diagnose and fight diseases.    2. the field of research    the  study  of  exosomes  (nano‐scaled  lipid‐based  membrane vesicles of endocytic origin that are shed from  most  living  cells)  and  other  secreted  microvesicles  (heterogeneously‐sized  membrane‐delimited  vesicles  shed  from  the  cell  membrane)  is  a  domain  of  biotechnology  and  biomedicine  that  crosses  multiple  disciplines,  including  the  application  of  molecular  and  cell  biology,  biochemistry,  genetics,  physiology,  immunology  and  biophysics  to  analytics,  to  help  elucidate  the  structure,  biogenesis,  function  and  trafficking,  providing  hints  on  distinctive  features  and  biomedical potential of different vesicle categories [1‐4].  discovered  over  30  years  ago,  exosomes  were  long  considered  an  alternative  secretion  pathway  for  unwanted  molecules  meant  to  be  discarded  from  the  parent  cells  [5].  over  last  decade,  important  roles  of  exosomes  have  emerged  as  mediators  of  intracellular  communication  and  immune  regulation,  both  in  physiological and pathological conditions [2, 5‐7]. today,  these  vesicles  are  often  recognized  for  their  specific  molecular  composition  dependent  on  parent  cell/tissue  type  and  condition,  and  their  involvement  in  the  transferring  of  proteins,  lipids  and  genetic  material  affecting the function of the recipient (target) cell [8,9].  the  recent  increased  interest  in  exosomes  and  microvesicles has been related to the discovery of their  role  in  intercellular  communication  vectors  in  parent  cell microenvironments or at a distance, by  trafficking  through the lymphatic and circulatory system, and can  thus be found  in a variety of human biofluids such as  blood and urine [10‐12]. these features make exosome  research  an  appealing  field  for  the  discovery  of  exosome‐associated biomarkers and the development of  targeted  diagnostic  applications  [8,  13‐15].  recent  papers  have  shown  the  diagnostic  value  of  exosomes  purified  from  blood  and  urine  in  pathological  conditions such as cancer [12,13,16]. moreover, the use  of  exosomes  as  therapeutic  tools  in  cancer  immunotherapy  [17],  regenerative  medicine  [19]  and  therapeutic  vaccine  [18]  approaches,  as  well  as  in  targeted drug delivery  [20‐22], has been  the subject of  many on‐going pioneering studies.    the field of investigation in the domain of exosomes and  secreted  microvescicles  is  therefore  an  exciting  and  rapidly  expanding  field  of  clinical  and  translational  science, with a pressing demand for further insights into  the roles of exosomes and their functions in physiologic  and pathologic conditions. further development is needed  in  the area of standardizing methodological approaches  for  the  exchange  and  validation  of  independently  obtained  data  from  an  increasing  number  of  on‐going  exosome  research  focused  groups  worldwide.  overall,  this  field  comprises  a  multidisciplinary  scientific  arena  with unprecedented potential to open new research paths  and  yield  novel  biotechnological  and  medical  applications.    3. publishing approach    as a response to the rapid growth of this research field  and to the wide range of topics and applications that  it  may have in the future, both in basic research and clinical  applications, the mission of exosomes and microvesicles is  to  facilitate  and  foster  communication  among  scientists  from  different  backgrounds  and  with  differing  provenances, and between academia and industry, for an  effective  translation  of  discoveries  and  applications.  several categories of papers will be published, including  original research articles, technical reports on methods  and protocols, review articles, hypotheses and opinions  on contradictory or visionary issues, meeting reports and  letters to the editor. submissions are encouraged from all  institutions  and  on  all  topics  related  to  extracellular  vesicles, including:     biogenesis of extracellular vesicles, both in eukaryotic  and prokaryotic cells   mechanisms of vesicle : cell interactions   intercellular signalling   intercellular traffic of proteins, nucleic acids and other  macromolecules   modulation of the extracellular matrix   roles of extracellular vesicles in normal physiological  processes,  including  differentiation,  development,  immunity, tissue repair, stem cell biology, etc.   roles  of  extracellular  vesicles  in  disease,  including  cancer,  neurodegeneration,  diabetes,  chronic  inflammatory  diseases,  cardiovascular  diseases,  infectious disease, ageing, etc.   virology and bacteriology   biomarkers   technical  advances  in  vesicle  purification  and  characterization   design and production of cell‐derived vesicles   design and production of synthetic vesicles   use  of  extracellular  vesicles  in  diagnostics  and  companion diagnostics   use of extracellular vesicles in therapeutics delivery   use  of  extracellular  vesicles  in  antigen  presentation  and vaccine development   use  of  extracellular  vesicles  in  cell  free  regenerative  medicine   use of extracellular vesicles in association with other  synthetic nanoparticle for different scopes  2 exosomes microvesicles, vol. 1, 1:2013 www.intechopen.com exosomes  and  microvesicles  aims  to  be  the  preeminent  journal for scientific advances in the field of extracellular  vesicle research. to achieve this goal, we have arranged for  papers  in  exmv  to  be  published  in  an  open  access,  electronic  format,  with  no  charge  to  its  readers.  open  access  will  make  all  published  material  available  to  everybody, without the barrier of affordability or copyright  ownership. therefore, all papers will be distributed under  the  terms  of  the  creative  commons  attribution  license  (http://creativecommons.org/licenses/by/3.0), which permits  unrestricted  use,  distribution  and  reproduction  in  any  medium, provided the original work is properly cited.    in  line  with  the  idea  of  making  knowledge  free  to  the  scientific community, the journal currently also offers free  of charge publication to all authors. the journal will take  it  upon  itself  to  finance  the  costs  of  production,  processing  and  publication  of  all  submitted,  peer‐ reviewed  and  accepted  articles  for  the  benefit  and  advancement of the exosomes and microvesicles field of  research, and the scientific community of interest in this  particular area of study.    further on the asemv and the journal aim to work on  partnering  with  other  scientific  societies  and  industry  leaders  to advance  the  field and  to allow  free access  to  knowledge.    an additional  feature of exmv  is  its plan  to expand  its  activity  to  ancillary  projects  such  as  special  issues  and  collections of papers, books and events. the first act will be  the publication of reports and abstracts from the exosomes  and microvesicles conference 2013 that will be held in late  september in orlando, florida, organized by asemv, with  the exmv journal as the official conference journal.    exmv’s promise of excellence  is certainly evident  in  its  associate  editors  and  members  of  its  editorial  board.  they  are  international  experts  with  diverse  expertise  mirroring the journal’s scope. on this occasion we would  like  to  thank  our  reviewers  whose  skill  and  rigour  contributed to the launch of the journal.    4. inaugural papers    the  first  papers  published  by  exmv  consist  of  four  articles that illustrate the journal’s scope.    the  paper  by  atay  et  al.  [23]  addresses  exosomes  as  mediators of cross talk between tumour cells and tumour  microenvironments, essential for creating the favourable  context for tumour progression and evasion. in this case,  the  authors  report  the  induction  of  proinflammatory  cytokines  production  by  activated  macrophages,  thus  providing hints for an explanation of increased levels of  inflammatory  mediators,  such  as  il‐1  observed  in  cancer  patients  and,  more  importantly,  identifying  possible therapeutic intervention targeting the exosome‐ related  mechanisms  leading  to  tumour  stroma  “education”.     hendrix  et  al.  [24]  address  another  phenomenon  characteristic of tumour microenvironments: intratumoural  acidification  caused  by  the  switch  from  oxidative  phosphorylation  towards  aerobil  glucolysis  that  is  a  fundamental  prerequisite  for  the  invasive  growth  and  chemoresistance  of  metastatic  cancers.  the  authors  identify  the  role  of  rab27b  in  promoting  the  metabolic  switch  and  so‐called  filopodia  phenotype  through  a  parakrine  activation  of  distinct  signalling  pathways  potentially mediated by exosomes. indeed rab27b is one  of  the  essential  regulators  of  exosome  biogenesis  and  release, and are themselves secreated by exosomes. this  paper reinforces already published evidence on the close  inter‐connection  between  microenvironmental  ph  and  exosome traffic in cancer.    romagnoli et al. [25] provide data on the incorporation of  mature  dendritic  cell‐derived  exosomes  by  diverse  tumour cells that thereby acquire the surface display of  co‐stimulatory  molecules  involved  in  antigen  presentation.  the  authors  suggest  this  mechanism  of  conversion  into  potentially  immunogenic  cells  as  an  avenue  to  be  explored  in  tumour  immunotherapy.  the  real potential of such an approach is that it fosters further  investigation.  this  paper  also  addresses  the  molecular  mechanisms  and  components  involved  in  the  selective  uptake of exosomes by target cells, correlating the rate of  uptake with the cell expression of cd9.    finally, a review article  from suntres et al.  [26] gives a  timely  comment  on  appealing  use  of  exosomes  in  a  variety  of  therapeutic  applications,  ranging  from  immunotherapy  in cancer, vaccines  in  infective diseases  such as parasitic and viral infections, through to their use  in  the  treatment  of  autoimmune  and  inflammatory  conditions  and  targeted  drug  delivery.  the  authors  highlight some unsolved issues that currently hamper full  lavaraging of enourmes exosome potential as therapeutic  effectors,  in  particular,  standardization  of  methodology  for their isolation and characterization.    therefore,  the  journal  will  act  as  a  means  of  tying  together and maintaining a scholarly community around  exosomes and microvesicles by staking out the intellectual  territory  of  this  new  field  to  serve  as  a  forum  to  inform/discuss issues around exosomes and microvesicles,  whether it is basic science or clinical and translational.    we hope that you will find our first papers and the ones  to  follow  inspiring  and  insightful.  we  invite  you  to  consider exosomes and microvesicles as a means of learning  about and contributing to the further development of this  evolving field.  3stephen j. gould, douglas taylor, antonio chiesi and winston p. kuo: announcing exosomes and microvesicles (exmv), the official journal of the american society for exosomes and microvesicles www.intechopen.com 5. references    [1]  keller s, sanderson mp, stoeck a, altevogt p. exosomes:  from biogenesis and  secretion  to  biological function.  immunol lett. 2006 nov 15;107(2):102‐8.   [2] théry c, ostrowski m, segura e. membrane vesicles  as  conveyors  of  immune  responses.  nat  rev  immunol. 2009 aug;9(8):581‐93.  [3]  györgy b, szabó tg, pásztói m, pál z, misják p, aradi  b, lászló v, pállinger e, pap e, kittel a, nagy g, falus  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uses  of  exosomes.  exosomes  and  mi‐ crovesicles.  4 exosomes microvesicles, vol. 1, 1:2013 www.intechopen.com article journal of circulating biomarkers size exclusion hplc detection of smallsize impurities as a complementary means for quality analysis of extracellular vesicles original research article tao huang1, anna b. banizs1, weibin shi1, alexander l. klibanov2 and jiang he1* 1 department of radiology and medical imaging, university of virginia, charlottesville, va, usa 2 department of medicine, university of virginia, charlottesville, va, usa *corresponding author(s) e-mail: jh6qv@virginia.edu received 23 march 2015; accepted 18 june 2015 doi: 10.5772/61148 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract for extracellular vesicle research, whether for biomarker discoveries or therapeutic applications, it is critical to have high-quality samples. both microscopy and nanosight tracking analysis (nta) for size distribution have been used to detect large vesicles. however, there is currently no well-established method that is convenient for routine quality analysis of small-size impurities in vesicle samples. in this paper we report a convenient method, called ‘sizeexclusion high-performance liquid chromatography’ (sehplc), alongside nta and microscopy analysis to guide and qualify the isolation and processing of vesicles. first, the se-hplc analysis was used to detect impurities of small-size proteins during the ultra-centrifugation process of vesicle isolation; it was then employed to test the changes of vesicles under different ph conditions or integrity after storage. as se-hplc is generally accessible in most institutions, it could be used as a routine means to assist researchers in examining the integrity and quality of extracellular vesicles along with other techniques either during isolation/preparation or for further engineering and storage. keywords extracellular vesicles, exosomes, hplc, quality analysis 1. introduction exosomes and extracellular vesicles are secreted by most cell types under both normal and pathological conditions [1, 2, 3]. these vesicles have been detected in a wide range of biological fluids, such as blood, urine, saliva, and breast milk [4, 5, 6, 7]. carrying abundant biomolecules like proteins, rnas and lipids, they are important messengers in intercellular communications and play a pivotal role in tumour progression and metastasis, as well as other diseases [8]. in addition to efforts to understand the biology of exosomes and extracellular vesicles in different diseases, extensive attention has recently also been focused on the study of these nanoparticles as biomarkers and the engi‐ neering of vehicles for drug and gene delivery. all such research, which has used different approaches including proteomics [9, 10, 11], transcriptomics [10, 11], lipidomics [10, 12], and vesicle engineering [13, 14], demands samples of high quality. however, current isolation and characterization methods leave much to be desired in terms of ensuring high-quality vesicles. various methods have been used for vesicle isolation, including ultracentrifugation [15], size exclusion (filtration or chromatography) [15, 16, 17, 18, 19], immu‐ 1j circ biomark, 2015, 4:6 | doi: 10.5772/61148 noaffinity isolation [15, 20], precipitation (exoquick or “salting-out”) [21, 22], or combinations of these. each method yields different results and standardization has not yet been established – though the vesicle research com‐ munity is working hard on this [23]. a few techniques have been commonly used to characterize isolated vesicles, such as transmission electron microscopy (tem) or cryo-em [24], nanoparticle tracking analysis (nta, nanosight) [25], and western blot for protein marker confirmation [15]. recently, webber et al. proposed the use of the ratio of vesicle counts to protein concentration as an indirect means to check the purity of vesicle preparation [26]. all these approaches are based on the presence of vesicular particles and present some limitations and challenges. for example, electron microscopy was employed to give informative visualization of vesicular morphology and identify the presence of larger-non-vesicular particulates. nta re‐ quires a lot of optimization of parameters and presents several challenges related for example to detection thresh‐ old, minimum expected particle size, blur, and minimum track length [25]. on the other hand, for impurity detection, while presence of protein biomarkers not expressed by vesicles [26] was proposed for quality assay, the selection of these markers is challenging and may not be accurate due to the limited understanding of how proteins are packaged into vesicles. to ensure high-quality vesicles, not only must the desired population of particles be confirmed to be present, con‐ taminants and impurities must also be demonstrated to be absent. unfortunately, there is no easy method available to rule out the presence of contaminants. thus, in this paper we report a simple se-hplc analysis to detect watersoluble small-size proteins in samples. se-hplc is widely used and accessible in most institutions and could be used alongside other current techniques to characterize these exosomes and extracellular vesicles. it could confer a routine and a convenient means to qualify the vesicle products. 2. materials and methods 2.1 materials chemicals or reagents were purchased from commercial sources and used without further processing unless otherwise stated. research-grade foetal bovine serum (fbs, sterile triple 100 nm filtered) and dmem cell-culture media were purchased from fisher scientific. mda (mdamb-231 human breast adenocarcinoma) and msto (msto-211h human biphasic mesothelioma) cell lines were from atcc (manassas, va). sephadex g-25 nap-5 columns were purchased from ge healthcare. the ultracentrifugation was performed on a beckman coulter optima le-80k ultra-centrifuge with an sw28 rotor. highperformance liquid chromatography (hplc) was per‐ formed on an agilent 1260 infinity system equipped with an auto-sampler and a variable wavelength uv detector. the hplc column was a size-exclusion column (superose 12 10/300 gl from ge healthcare) (9 µm-13 µm). the gel filtration hmw calibration kits were purchased from ge healthcare and the liposome labelled with dio (120 nm) for calibration was prepared in-house by dr alexander klibanov. a nanosight ns300 system (malvern instru‐ ments inc., westborough, ma) and a tecnai f20 twin transmission electron microscope (fei, hillsboro, or) were used to characterize the vesicle samples. 3. methods 3.1 cell culture cells, including mda and msto, were cultured at 37°c at 5% co2 in dmem media complemented with 4% vesicledepleted fbs. the fbs was depleted of vesicles by over‐ night ultra-centrifugation at 120,000 g followed by filtration through pvdf 0.22 µm vacuum-driven filters (millipore). the cell-culture supernatant was collected for vesicle isolation when cell confluence reached 90% (usually after days, from cell seeding to final supernatant collection). 3.2 isolation of vesicles vesicles were isolated from cell-culture supernatants or the commercially available fbs. the isolating procedure was adapted from a reported protocol [15] with minor modifi‐ cations. briefly, to remove large cell debris the collected cell-culture supernatant was first subjected to subsequent centrifugations at 400 g for 5 min and 3000 g for 30 min, followed by sequential filtration through 0.45 µm and 0.22 µm pvdf filters. fbs was used as purchased. the liquid was further ultra-centrifuged at 120,000 g at 4℃ for 90 minutes, followed by removal of the supernatant. the vesicle pellet was subjected to washing by re-suspending in a large volume of fresh dpbs (30 ml) and ultra-centri‐ fuging at 120,000 g at 4℃ for 90 minutes. this washing step was repeated several times and aliquots from each round of ultra-centrifugation were saved for hplc analysis and other assays, as shown in scheme 1. aliquots of final vesicle pellets were used for experiments immediately or stored at 4℃ for future use. 3.3 size-exclusion hplc analysis size-exclusion hplc (se-hplc) was performed with a superose 12 10/300 gl column with exclusion limit of 2× 106 daltons to detect the contaminants and vesicular nanoparticles. 50-100 µl of an aliquot was injected via the auto-sampler. the mobile phase was dpbs solution (137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4, 1.8 mm kh2po4) with a flow rate of 1.0 ml/min. uv absorbance was detected at a wavelength of 254 nm. 2 j circ biomark, 2015, 4:6 | doi: 10.5772/61148 3.4 cryogenic transmission electron microscopy (cryo-tem) analysis vesicles were vitrified by standard methods of cryo-tem [27]. in brief, an aliquot (~3.5 µl) was applied to a glowdischarged, perforated carbon-coated grid, manually blotted with filter paper, and rapidly plunged into liquid ethane. the grids were stored in liquid nitrogen, then transferred to a gatan 626 cryo-specimen holder and maintained at -180°c. low-dose images were collected at a nominal magnification of 29,000× on a tecnai f20 twin transmission electron microscope operating at 120 kv. the digital micrographs were recorded on a gatan us4000 ccd camera. cell culture supernatant pre-clearing centrifugation: 400 g 5min; 3000 g 30min filtrations: 0.45 um and 0.22um centrifugation: 120,000 g 90 min hplc: fig. 1. a pbs wash then centrifugation: 120,000 g 90 min pbs wash then centrifugation: 120,000 g 90 min hplc: fig. 1. b pbs wash then centrifugation: 120,000 g 90 min hplc: fig. 1. c supernatant supernatant or fbs pellets pellets pellets pellets hplc: fig. 4 tem, nanosight, elisa: fig. 2 hplc: fig. 1. d hplc: fig. 3 storage ph5, ph9 scheme 1. flow chart of vesicle isolation and purification based on differential ultracentrifugation combined with filtration. se-hplc was used to qualify the intermediates and final product as well. 3.5 nanosight particle analysis real-time nanoparticle detection, counting and sizing were performed on the ns-300 nanosight instrument following manufacturer protocols. the instrument settings were as follows: camera type – scmos; shutter length – varied; shutter setting – 1300; camera gain – 512; frame rate – varied; analysis – blur setting, minimal expected size, minimal track length, all set to automatic. the version of the software was nta 2.3. 3.6 proteins and elisa analyses total protein of isolated vesicles and fractions from hplc were measured using the micro bcatm protein assay kit (thermo scientific pierce, rockford, il) following the protocol provided by the manufacturer, while the two protein markers, cd63 and cd9, were confirmed by using an enzyme-linked immunoassay kit (elisa, system biosciences, mountain view, ca). all measurements were performed following the protocol provided by the manu‐ facturers. 3.7 se-hplc detection of small-size impurities in vesicles at different phs or in refrigerated storage the possible changes of vesicles under different ph conditions were evaluated by using se-hplc analysis. the buffer medium of purified vesicles was changed from pbs to sodium acetate (0.1m, ph5) or sodium bicarbonate (0.1m, ph9) by using nap-5 column. aliquots (about 5×108 particle/ml) were kept at room temperature and analysed by se-hplc over time. to test the possible changes of vesicles in refrigerated storage, an aliquot of vesicles in pbs buffer (ph7.4) was stored at 4°c for up to one month, during which vesicles were analysed weekly by se-hplc for possible appear‐ ance of small-size impurities, and by nanosight analysis for the changes of size distribution. 4. results and discussion for research seeking to understand the biology of exosomes and extracellular vesicles, to discover biomarkers, especial‐ ly proteomins, lipidomics and genomics, and to engineer these vesicles for drug and gene delivery, it is crucial to ensure high-quality samples by removing interfering contaminants such as non-vesicular proteins or other biological molecules of small size. to that end, a simple means for quality analysis of vesicle samples is highly desirable. although several techniques, such as microsco‐ py and nta, can give visual information on particle size and global size distribution, respectively, there is still no easy technique to detect impurities of small size. size chromotagraphy has been used for isolation or purification of extracellular vesicles with different gel mediums, but no real hpl analysis has been reported for quality assay (abstract p-iv-13 at isev 2015: journal of extracellular vesicles, 2015,4: 27783). in this paper, we demonstrate that size-exclusion hplc (se-hplc) could be a convenient method to detect proteins and other impurities of small size. the size-exclusion column used in this study was first calibrated using standard gel filtration hmw calibration kits (purchased from ge healthcare) and nano-sized 3tao huang, anna b. banizs, weibin shi, alexander l. klibanov and jiang he: hplc quality analysis of extracellular vesicles liposomes. the void volume of this column was deter‐ mined using blue dextran 2000 (molecular weight 2000 kda) to be at 8 min and 4 sec at a flow rate of 1 ml/min. further confirmed by dio-labelled liposomes (120 nm), components eluting before 8 min were not resolved since they were within the void volume. proteins as big as thyroglobumin and ferritin can be resolved on this column (see details in supporting materials), so it is selected to separate non-associated proteins and other smaller impur‐ ities from vesicles. several techniques have been used to isolate vesicles. in this study, sequential ultra-centrifugation was employed to prepare vesicles from msto, mda tumour cells and fbs, and the samples were analysed by se-hplc. according to the ultra-centrifugation protocol [15], three rounds of ultracentrifugations (70 min each run) were used to isolate and purify vesicles from cell-conditioned media. since the procedure is empirical, the reproducibility of high-quality vesicles remains an issue. by introducing se-hplc to separate and detect vesicles and free small-size proteins, we were able to analyse the aliquot at different centrifuging levels in the whole process. as demonstrated in fig. 1, after two rounds of ultra-centrifugation, the species eluting out of the column before 10 min were negligible in the mixture compared to those appearing after 10 min. after the third round of ultra-centrifugation, the peak with the retention time of 7.5 min (identified as vesicle peak as shown in fig 2) became more dominant while other peaks after 10 min were decreased. with one more round of washing and ultra-centrifugation, a single peak was observed at 7.5 min, which represented the final vesicle product. all three kinds of vesicles from different sources (fbs, msto, mda) showed similar patterns in hplc analysis for this ultracentrifugation process. the vesicle samples representing the single peak in sehplc were subjected to cryo-tem and nanosight analysis (fig. 2). in tem, these vesicles appeared as well-defined membrane-bound vesicles ranging in size from 30 to 150 nm in all three kinds of vesicle. nanosight analysis gave a more broad distribution of 40 to 200 nm. to further confirm the identity of the samples representing the different peaks in se-hplc, fractions at 7.5 min and 12.8 min were collected and examined by cryo-tem and elisa for the presence of extracellular protein markers cd63 and cd9. the results showed that the fraction at 7.5 min was extrac‐ ellular-vesicles positive in both cd63 and cd9, while the fraction sample at 12.8 min was not positive in either of these. in recent years, nano-sized extracellular vesicles have emerged as potential drugand gene-delivery carriers, where nano-sized vesicles may need to be subjected to ph conditions other than the physiological one (ph 7.2-7.4). to test for any changes in vesicles under different ph condi‐ tions, the buffer for vesicle samples was changed from pbs (ph7.4) to sodium acetate buffer (ph5) or sodium bicar‐ bonate buffer (ph9). the se-hplc analyses were per‐ formed at different times. as illustrated in fig. 3, fbs vesicles showed a response to ph changes, with intensity of vesicle peak continuously decreasing while that of newly generated peaks increased in the study period. although the details of these new peaks are not known, and further identification is challenging and beyond the focus of the figure 1. representative chromatograms of extracellular vesicles from msto cell-culture supernatant. aliquots after one (a), two (b), three (c) and four (d) ultra-centrifugations were analysed. the peak with a retention time of 7.5 min stands for the exosome particles while the other peaks are impurities of small size. 4 j circ biomark, 2015, 4:6 | doi: 10.5772/61148 current study, it is apparent that changes did happen in these vesicles. these new impurity peaks might be due to the vesicular particles disrupted or the proteins or other biomolecules of small size falling off from the vesicles, resulting the lower and lower signal from the vesicles. nevertheless, we have demonstrated that it would be useful to use se-hplc to detect impurities or changes of vesicles under different conditions. another question for vesicle research regards the appro‐ priate storage condition for vesicle samples. generally, -80oc has been recommended [23] for long-term storage. however, the cycle of “freeze and thaw”, if it happens frequently, may do harm to membrane vesicles. is it safe to keep the vesicles at 4°c, and how long will they remain good? as illustrated in fig. 4, aliquots of purified mda vesicles in pbs were kept at 4°c for up to one month and there was no obvious change in the se-hplc graph. the nta analysis also showed minimum changes of size in these samples. the other two vesicle samples from msto cells and fbs showed similar results, without any substan‐ tial changes in size distribution and small-size impurities. these results suggest that it may be safe to keep vesicle aliquots in a refrigerator for a short period of up to four weeks. however, our se-hplc evidence only implies physical stability as particulates at 4°c. to draw conclu‐ sions about the biological functions of these extracellular vesicles after storage, further evaluation is required. it is important to note that there are also several limitations related to this hplc detection technique. first, in our study, no detailed information on vesicle size is gained by figure 2. characterization of purified extracellular vesicles isolated from fbs (a), msto cells (b), and mda cells (c). representative cryogenic transmission electron images of vesicles show round-shape membrane-bound particles from all three sources (fbs, msto and mda cells; left column) with occasional irregularly distributed electron-dense content, both inside and on the surface of the membrane. the nanosight plots depict typical size distributions of extracellular vesicles from the above-described sources (right column). bars correspond to 100 nm. 5tao huang, anna b. banizs, weibin shi, alexander l. klibanov and jiang he: hplc quality analysis of extracellular vesicles hplc analysis alone, since this column cannot resolve nanoparticles of different size. all nanoparticles elute at the void volume as demonstrated by calibrations where blue dextran 2000 (2, 000 k daltons) and dio-labelled liposomes of size 120 nm were used to determine the void volume under the same condition used for vesicle samples (shown figure 3. chromatograms of fbs vesicles in (a) bicarbonate buffer (0.1m, ph9) and (b) acetate buffer (0.1m, ph5). from top to bottom in both a and b, the time point was 0h, 1h, 2h, 4h and overnight (14h), respectively. 6 j circ biomark, 2015, 4:6 | doi: 10.5772/61148 in supporting materials). the intention of this study was to test whether se-hplc can be used as a means of quality analysis of soluble proteins and other impurities of small size. for a detailed size measurement by hplc, more evaluations are needed. it would be best to use the simple and straightforward se-hplc to qualify the vesicle preparation followed by other characterizations such as tem, nta, western blot and flow cytometry. secondly, a high exclusion limit of 2x106 daltons was used in this study to give a wide detection range and better resolution of small impurities for the extracellular vesicles of size less than around 200 nm. other exclusion limit ranges may give different results, depending on the vesicles and the sources to prepare these vesicles. from our experience, sizeexclusion columns with higher exclusion limits may be able to resolve large particles but will give less desirable resolution of small-size molecules. therefore, it is impor‐ tant to choose an appropriate column and calibrate it with a series of molecules of different sizes. finally, these results were based on the ultra-centrifugation purification method using commercially available fbs and tumour cells. for biomarker discovery research dealing with real samples of blood, etc., different isolation techniques such as affinity selection, filtration, or others, as well as any combinations of these could be employed to generate particular types of vesicles. nonetheless, purity of samples would be the top priority to avoid false biomarkers due to impurities. therefore, in addition to quality-analysis techniques (nta as a global analysis of size distribution) to exclude large particles or aggregates, techniques such as the hplc analysis reported here represent a convenient complemen‐ tary means to detect impurities of small size. 5. conclusions in this study, we conclude that size-exclusion hplc analysis can be used to help determine the purity of extracellular vesicle samples by detecting proteins and other impurities of small size that can have a substantial impact on biomarker discovery. this method has been used to examine the purity of vesicular nanoparticles with changes in ph and under storage at 4°c. the results exemplify the potential of such hplc analysis to monitor vesicle disruption during storage, modification or other processes in extracellular vesicle research focusing on biomarker discovery or therapeutic application. 6. compliance with ethical research standards the authors declare no conflicts of interest. no part of this study was performed on any human or animal subjects. 7. acknowledgements we are grateful to dr janet cross for providing us with the mda-mb-231 breast-cancer cells, dr kimberly kelly for allowing us to use the nanosight instrument, and dr kelly dryden for assistance with cryo-tem analysis. the research reported in this publication was partially support‐ ed by the national cancer institute (ccsg p30 ca44579) and the national heart, lung, and blood institute (r21hl120003) of the national institutes of health. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. figure 4. representative chromatograms of mda vesicles stored at 4°c for one month. the top was fresh aliquot and the bottom was aged for one month in a 4°c refrigerator. 7tao huang, anna b. banizs, weibin shi, alexander l. klibanov and jiang he: hplc quality analysis of extracellular vesicles 8. references [1] thery c, zitvogel l, amigorena s. exosomes: composition, biogenesis and function. nat. rev. immunol. 2002; 2: 569-579. 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[27] baker ts, olson nh, fuller sd. adding the third dimension to virus life cycles: three-dimensional reconstruction of icosahedral viruses from cryoelectron micrographs. microbiol. mol. biol. rev. 1999; 63: 862-922. 8 j circ biomark, 2015, 4:6 | doi: 10.5772/61148 article journal of circulating biomarkers the most favourable procedure for the isolation of cell-free dna from the plasma of iso-immunized rhd-negative pregnant women original research article riyaz ahmad rather1,2*, subhas chandra saha1 and veena dhawan2 1 department of obstetrics and gynaecology, post graduate institute of medical education and research (pgimer), chandigarh, india 2 department of experimental medicine and biotechnology, post graduate institute of medical education and research (pgimer), chandigarh, india *corresponding author(s) e-mail: farhan.rizu@gmail.com received 08 september 2015; accepted 26 november 2015 doi: 10.5772/62113 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract background: the ability to achieve quality recovery of cellfree foetal dna is important for making non-invasive prenatal diagnoses. in this study, we performed quantita‐ tive and qualitative analyses of isolated dna from mater‐ nal plasma, using different dna-isolation methods. method: dna was isolated from 30 iso-immunized women via the qiaamp column-based method, using four differ‐ ent elution volumes and two conventionally based meth‐ ods. real-time polymerase chain-reaction quantification of rhd and β-globin genes was performed in order to determine foetal-specific sequences and total genome equivalents, respectively. results: the column-based method at a 3 µl elution volume yielded the highest quality and quantity of total dna (67.0±0.6 ng/µl). at a 3 µl elution volume, the β-globin and rhd-gene sequences were estimated to be the highest among all isolation procedures, with 2778.13±1.5 and 66.9±0.6 geq/ml, respectively, and a 100% sensitivity for rhd-gene sequence detection. among the two conven‐ tional manual methods, the boiling lysis method yielded a higher dna concentration (53.8±0.8 ng/µl) and purity (1.73±0.05). in addition, the method's sensitivity for foetaldetection sequences was only 80%, whereas the salting-out method's sensitivity was just 70%. conclusions: this study confirms the theory that the qiaamp method is a specific and sensitive approach for purifying and quantifying plasma dna, when used in the minimum elution volume. keywords cell-free foetal dna, plasma, iso-immuniza‐ tion, genome equivalents, elution volume 1. introduction maternal blood contains cell-free foetal dna (cffdna), which is detectable as early as the sixth week of gestation; its level rises during the advance of gestational age and disappears rapidly after delivery [1]. invasive procedures, 1j circ biomark, 2015, 4:12 | doi: 10.5772/62113 such as amniocentesis or chorionic villus sampling (cvs), carry the risk of miscarriage as they rely on the sampling of foetal tissues [2]. a consistent, convenient and reliable method has been sought for prenatal diagnosis in order to reduce this risk of miscarriage. the discovery of cffdna in plasma and serum had a wide application in non-invasive prenatal diagnosis (nipd), which ultimately reduced the risk of foetal loss by a miscarriage. the many reported applications off cffdna include rhd genotyping [3–5], foetal sexing [6], pregnancy-associated conditions such as pre-eclampsia [7] and aneuploid detection [8]. the quality recuperation of cffdna in maternal plasma is key for noninvasive prenatal diagnosis; however, limitations in exploring cffdna for nipd include the associated recov‐ ery of maternal dna and the failure to recover highly purified cffdna, both of which impede quantification strategies and assay sensitivity [9]. as cffdna represents just 3–6% of the total cell-free dna in maternal plasma, and poses some technical difficulties in dna extraction due to its extremely low concentrations, an efficient dnaextraction method or technology is required [10]. to improve the yield of foetal dna, various methods were designed and formulated for the recovery of plasma from maternal blood and the subsequent dna isolation, as plasma is considered to be a better source of foetal dna than maternal serum [11]. for efficient plasma-dna recovery, multiple methods of centrifugation and filtration of plasma to remove apoptotic bodies and protein impuri‐ ties have been explored [9]. commercial kits are used widely to isolate dna of mediumand large-molecular sizes from body fluids, but less work has been done to isolate and purify fragmented dna and dna with a low molecular weight (about 50–150 bp). to improve the quality and concentration of cffdna, earlier studies have compared manual methods to an automated dnaisolation system on the basis of sequence-specific copy numbers [12, 13]. differential elution volumes were used in manual and automated dna-isolation methods to augment the concentration and foetal-detection sensitivity [14]. moreover, it was recently proposed that comparison of manual, automated, or commercial dna-isolation methods increased the recognition, detection and predict‐ ability of foetal sequences, because observed concentra‐ tions of cffdna differ depending on the processing and analysis methods used [15, 12]. due to these challenges, the recovery of an optimal quantity of dna and foetaldetection markers remain both problematic and exigent. achieving this goal would pave a path for additional dnaisolation procedures that can enhance dna concentration, purity, throughput and efficacy in prenatal testing. in this study, we have evaluated the suitability of three different methods — one commercial method using different elution volumes and two low-cost conventional methods — by comparing the methods cffdna yields in terms of concentration, purity and the rate of detection of foetal-specific sequences. 2. materials and methods 2.1 subjects the study was conducted in collaboration between the department of obstetrics and gynaecology and the department of experimental medicine and biotechnology of the postgraduate institute of medical education and research (pgimer), chandigarh, india. gestational age was calculated based on menstrual-cycle dates and was confirmed by ultrasound. where gestational age was not certain, it was confirmed by ultrasound before 15 weeks of gestation had passed. a cohort of 30 rhd-negative isoimmunized women (length of gestation, 23–34 weeks), whose foetus and partner were both rhd-positive, were registered for the study. pregnancies that experienced intrauterine foetal death, foetal gross-congenital malfor‐ mations, multiple pregnancies and ones that had an rhdnegative partner were excluded from the study. full written, informed consent was obtained from all the study subjects prior to their participation. the institute ethics committee of pgimer approved the study's protocol. 2.2 dna extraction from each subject, 10 ml of fresh blood was collected from an antecubital vein via an edta vacutainer and was transported the same day to the laboratory, where it was kept at -4˚c and processed within 12 h. plasma was separated by centrifuging the samples at 3000 x g for 10 min. isolated plasma was centrifuged again at 2000 x g for 7 min in order to remove the residual cells and leukocyte carryover. the plasma samples were stored at -80°c before further use. from each sample, 200 µl of plasma was used in order to extract dna for use in each method. in the commercial column-based method, hereafter referred to as a qiaamp-column method, dna was extracted from 200 µl of plasma per column, using a qiaamp dna blood mini kit (qiagen; hilden, germany) according to the “blood and body fluid protocol” [16] suggested by the manufacturer, with a slight adjustment to accommodate the higher plasma volume. the protocol was further modified by incubating samples at 37°c for 2 h instead of 56°c for 10 min. dna extracted via the commercial column-based qiaamp method was finally eluted in four different elution volumes using an ae elution buffer (50, 30, 10 and 3 µl) in order to check whether or not a modified protocol combined with the use of different elution volumes affects dna concentration. this method is based on the ability of dna to bind to the silica surface of the spin-binding column in a chaotropic salt solution after proteinase k lysis has occurred. the second method, hereafter referred to as conventional method 1 (cm1), was performed according to the specifications of miller et al. [17] and involves proteinase k digestion, dehydration and precipitation of proteins using a saturated nacl solution, followed by phenol:chloroform:isoamyl alcohol extraction and ethanol precipitation. the dna extracted using this 2 j circ biomark, 2015, 4:12 | doi: 10.5772/62113 method was eluted in 50 µl of nuclease-free water. the third method involved the lysis of proteins in phenol-based chaotropic salt by boiling samples at 58°c for 10 min, followed by chloroform:isoamyl alcohol and isopropanol precipitation, and 50 µl of nuclease-free water was used to elute the extracted dna. this method is hereafter called conventional method 2 (cm2) [18]. one µl of the undilut‐ ed, extracted dna sample was analyzed using a nano‐ drop nd-1000 spectrophotometer (nanodrop technologies, wilmington, de) in order to check its purity and concentration. the purity of the extracted dna was based on the a260:a280 optical density (od) ratio, and elution buffers used in each protocol were used as controls for od measures. 2.3 real-time polymerase chain-reaction analysis quantitative real-time polymerase chain-reaction analysis (q-pcr) was performed as previously described [19, 20], with modifications to the pcr chemistry and target primers. eluted dna from each method was analyzed via a real-time q-pcr abi 7500 detection system (applied biosystems), using power sybr® green chemistry. the pcr assay targeted rhd loci, a cell-free foetal dna marker [21] and β-globin gene loci. β-globin was used as an invariant control and it differentiated between true-negative and false-negative results deriving from a deficient dnaextraction process. total dna and cffdna quantities were represented as genome equivalents per millilitre (geq/ml) of plasma, based on copies of β-globin and rhd sequences detected per microlitre of plasma. the rhd primer sequence consists of a forward primer, 5’-cct ctc act gtt gcc tgc att-3’, and a reverse primer, agt gcc tgc gcg aac att-3’, resulting in a 74 bp pcr product. the β-globin primer sequence consists of a forward primer, 5´-gtg cac ctg act cct gag gag a-3´, and a reverse primer, 5´-cct tga tac caa cct gcc cag-3´, resulting in a 102 bp pcr product. amplification reactions were set up in a 25 µl reaction volume consisting of a 10 µl power sybr® green pcr master mix (applied biosystems) and 5 µl of template dna, in addition to primers. primers were optimized in order to determine the minimum primer concentrations that give the maximum amount of normalized reporter. primers were used at the final concentration of 300 nm. dna amplification was carried out in 96 well plates (applied biosystems). pcr conditions for all reaction mixtures consisted of an initial denaturation step at 95°c for 10 min and, finally, 40 cycles of 95°c for 15 s, 60°c for 1 min and 72°c for 30 s. melting-curve analysis was carried out at the end of each pcr assay in order to verify the specificities of the amplified pcr products. an elution buffer was used as a non-template control (ntc) and dna from a healthy rhd-positive individual was used as a positive control in order to generate a standard curve using 10-fold dilutions (30000, 3000, 300, 30 and 3 geq/ml) [21]. this standard curve was run in parallel with each pcr reaction. all samples were analyzed in triplicate for both genes, and the mean of the values was determined using the 7700 software and the standard curve of known dna concentrations [9]. 2.4 statistical analysis a graph pad prism v5.0 (la jolla, ca, usa) was used for statistical analysis. for each parameter, the mean, the standard error of the mean and the range were calculated. differences were evaluated via t-tests and non-parametric mann-whitney tests, and values of p<0.05 were considered to be statistically significant. 3. results to determine whether a difference in elution volumes using the same method influences dna's concentration and purity, we checked four different elution volumes in eluting the extracted dna from plasma using the qiaamp column-based method. as expected, dna concentrations and purities varied considerably between different elution volumes (table 1, figure. 1, 2). qiaamp method (n=30) cm1 (n=30) cm2 (n=30) elution volume 50 µl 30 µl 10 µl 3 µl 50 µl 50 µl *mean dna concentration (ng/µl) 20.1±3.1 30.3±2 39.5±1.6 67.0±0.6 29.4±1.5 53.8±0.8 mean dna purity (a260:a280 ratio) 1.38±0.05 1.37±0.05 1.81±0.07 1.82±0.11 1.27±0.08 1.73±0.05 * values are shown as mean with ± standard deviation (sd). table 1. dna recovered from maternal plasma of rhd negative isoimmunized women using different dna isolation methods as measured by spectrophotometer figure 1. bar diagram showing a comparison of dna concentrations isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 µl qiaamp method) (#= 30 µl qiaamp method) (α= 10 µl qiaamp method) (β= 3 µl of qiaamp method) (ψ=50 µl cm1); **p<0.01; ***p<0.001; #p<0.05, ###p<0.001; βββp<0.001; αp<0.05, αααp<0.001. 3riyaz ahmad rather, subhas chandra saha and veena dhawan: the most favourable procedure for the isolation of cell-free dna from the plasma of iso-immunized rhd-negative pregnant women figure 2. bar diagram showing a comparison of the quality of dna isolated from the maternal plasma of iso-immunized women using different isolation procedures. (n=30). (*=50 µl qiaamp method) (#= 30 µl qiaamp method) (α= 10 µl qiaamp method) (β= 3 µl of qiaamp method) (ψ=50 µl cm1); **p<0.01; ***p<0.001; #p<0.05, ###p<0.001; βββp<0.001; αp<0.05, αααp<0.001. among the four different elution volumes, the 3 µl volume yielded a significantly higher concentration of dna (p<0.001) as compared to the three other elution volumes. the dna concentration derived using the 3 µl volume was 3.36 and 2.26-fold higher than concentra‐ tions of the 50 and 30 µl volumes, respectively. howev‐ er, there was only a 1.6-fold increase in dna concentration when compared to use of a 10 µl elution volume (p<0.001). these data reveal that there is a negative correlation between the elution volume used and dna concentra‐ tion, as an increase in elution volume inhibits high dna concentration. the overall dna yield was less while using a 3 µl volume when compared with other volumes using the same method (data not shown). this is probably due to dilution of the sample, which decreases the elution's efficiency. among the two conventional methods, cm1 yielded a 1.82-fold increase in dna concentration as compared to cm2 (p<0.001). cm1 proved useful only in obtaining a 1.4-fold increase in dna concentration when compared with the qiaamp method using a 50 µl elution volume. however, the qiaamp commercial method combined with 10 and 3 µl elution volumes increased the dna concentration by 1.2 and 2.27-fold, respectively, when compared to cm1, and by 1.3and 1.2-fold when compared to cm2. these results show that a larger elution volume decreases the overall dna concentration, and vice versa (table 1, figure 1). the purity of the extracted dna was counted as a measure of the a260:a280 od ratio using a nanodrop spectropho‐ tometer, and that value is shown in table 1, figure 2. the purified dna sample was expected to show an a260:a280 ratio of 1.7 to 2.0. in a commercially based qiaamp method, the best ratio was achieved using 10 µl (1.81±0.07) and 3 µl (1.79±0.11) elution volumes; however, this method did not produce optimal ratios when using 50 or 30 µl vol‐ umes. nor did cm1 produce an optimal ratio, suggesting a partial precipitation of proteins reflecting an impure dna sample. between the two conventional methods, cm2 showed a significantly improved (p<0.01) a260:a280 ratio (1.73±0.05) as compared to cm1. of all methods used, the cm1 procedure was the least efficient dna purityyielding method. the β-globin gene was quantified via q-pcr in order to determine the total geq/ml, representing the total cell-free dna (maternal plus foetal dna). the mean values are shown in table 2 with ±sd. among the four different elution volumes used in the qiaamp commercial method, a higher β-globin concentration in terms of geq/ml was achieved (2778.13±1.5) by using a 3 µl volume as compared to a 50 or 30 µl volume, which harvested mean concentra‐ tions of 2498.15±1.61 (p<0.001) and 2541.99±4.53 (p<0.001) geq/ml, respectively (table 2). this accounts for around 1.11-fold (p<0.01) and 1.09-fold (p<0.05) increases in the βglobin concentration compared to use of 50 or 30 µl volumes, respectively. however, no significant increase in the β-globin concentration for the 10 µl elution volume was observed when compared with that of the 3 µl volume (p>0.05). in the two conventional methods, cm1 slightly increased the β-globin concentration, though to a nonsignificant degree (p>0.05), which is contrary to the spectrophotometric results. this anomaly might be due to the generation of pcr inhibitors, which affects quantifica‐ tion of the β-globin. when a qiaamp commercial method was compared with both conventional methods, it proved to be more efficient and significant (p<0.001) at increasing β-globin genome equivalents, for all four elution volumes. the quantity of cffdna was determined by estimating the rhd gene sequence for each isolation method (table 2). comparison of the two conventional methods, cm1 and cm2, found that they yielded almost equal genome qiaamp method (n=30) cm1 (n=30) cm2 (n=30) elution volume 50 µl 30 µl 10 µl 3 µl 50 µl 50 µl β-globin (geq/ml) range 2498.15±1.61 (1124.2-3017.8) 2541.99±4.53 (1037.1-3209.1) 2771.8±2.73 (743.0-2817.7) 2778.13±1.5 (798.3-3798.5) 1709.4±1.22 (540.1-4194) 1697.5±1.82 (744.4-2435.8) rhd (geq/ml) range 49.87±1.4 (14.3-87.0) 47.1±0.4 (17.1-79.0) 55.2±0.9 (19.1-103.6) 66.9±0.6 (18.6-160) 65.7±1.09 (27.9-178.12) 65.1±0.2 (17-172.2) table 2. dna recovered from maternal plasma of rhd negative iso-immunized women using different dna isolation methods as measured by real-time polymerase chain reaction 4 j circ biomark, 2015, 4:12 | doi: 10.5772/62113 equivalents of rhd, with mean values of 65.7±1.09 and 65.1±0.2, respectively (p>0.05). in addition, the rhd sequence detection-sensitivity rates were 70% (seven out of 10) and 80% (eight out of 10) for cm1 and cm2, respec‐ tively. among the different elution volumes used in the qiaamp commercial kit, the 3 µl volume yielded the highest concentration (66.9±0.6) of the rhd sequences, with 1.3-, 1.4and 1.2-fold increases in the 50, 30 and 10 µl volumes, respectively. however, the foetal-sequence detection rate was 100% (10 out of 10) for all elution volumes used in the qiaamp column-based method. the concentration of the rhd gene sequences, determined in terms of their genome equivalents, differed consistently when compared with the β-globin genome equivalents. when all methods are compared, the qiaamp commercial kit method, used with a 3 µl elution volume, proved the most efficient method for the detection of both β-globin concentrations and rhd sequences. 4. discussion the recovery of quality dna is necessary for proficient molecular diagnostic and clinical investigation [22]. due to the presence of a small amount of foetal dna that is associated with complex proteins, the dna-extraction method needs to be optimized in such a way that the maximum quantity of plasma dna can be extracted with the fewest impurities [1]. hence, it is imperative to deter‐ mine which plasma dna isolation and recovery methods increase the efficiency, reliability and reproducibility of foetal dna extraction. to address this quality and quantity issue, we tested three different methods for plasma dna isolation: the qiaamp commercial method and two conventional methods. the qiaamp commercial method was further tested for the issue of quality dna recovery, using four different elution volumes. the qiaamp com‐ mercial method, when used at a 3 µl elution volume, proved to be the most efficient in terms of overall yield. using the qiaamp commercial method, we concluded that dna purity and concentration were at their maximum when using the 3 µl elution volume; however, the detec‐ tion rates of total and foetal sequences were the same (100%) for all elution volumes, although they differed in achieving optimal dna concentrations and purities. we did find that using a small elution volume increases the dna concentration considerably, but also that it correlated negatively with the dna yield. in the qiaamp method, our study demonstrated a negative correlation between the elution volume, the purity and the quality of dna and a direct positive correlation between the purity of the samples and the amount of total and foetal sequences detected. these results are in accordance with early studies carried out by carolina et al. [9]. we achieved overall 3.3and 1.3-fold increases in dna concentration and purity, respectively, by using a 3 µl elution volume (table 1). in addition, total and foetal dna sequences, measured using real-time pcr, concomitantly increased by 1.1and 1.3fold, respectively. these findings suggest that using a smaller elution volume enhances the enrichment of total dna and small fragments of free foetal dna. we also observed a 4.8% reduction in ct value in the q-pcr signal for dna isolated in a 3 µl elution volume as compared to the three other elution volumes from the same method, which indicates an increased quantity of dna. this result is supported by other findings showing that dna eluted while using a smaller amount of elution buffer gives an early ct value, indicating the presence of concentrated dna in the sample [23]. elution carried out with the smallest volume increased the final dna concentration in the elute, which is very important for any down-streaming process. this result accords with other studies using columns, indicating the ability of the column matrix to enhance dna concentrations while using a smaller elution volume [24, 25]. however, using smaller elution volumes led to a significantly lower dna yield. the probable cause for this result may be that the qiaamp column matrix in the presence of the smallest elution volume might have increased the ionic strength, which promoted the highest dna adsorption with the column matrix. the yield can be improved significantly by eluting the dna twice using the same buffer; however, dilution renders lower cffdna quality/concentration, leading to false-negative pcr results. none of the four elution volumes in the qiaamp commercial method gave false-positive results, suggesting the high specificity of this method for cell-free foetal dna isolation. to determine the efficiency and reproducibility of the qiaamp commercial method, we compared it with two other conventional dna-isolation methods, i.e., cm1 and cm2, which are based on either the salting-out and precipitation of proteins or the lysis of proteins by boiling, respectively. cm2 produced an overall higher dna yield and quality (table 1) than cm1. however, the total amount of dna measured by real-time pcr using the β-globin loci in cm2 was lower than that of cm1. partial protein lysis and nacl contamination (used for the precipitation of proteins) could be the possible reason for the low purity and small quantity of dna in cm1; however, a nonsignificant total dna and foetal dna sequence were detected in cm2 than in cm1 (table 2). a possible pro‐ posed reason might be the loss of smaller-size cffdna fragments, which might have developed due to an incre‐ mental temperature increase that was set up as 58°c for 10 min. in summary, six different methods for the isolation of cffdna from the maternal plasma of rhd-negative isoimmunized women were evaluated. a commercially available qiaamp kit at a lower elution volume provides optimal results for dna purification with high sensitivity and specificity. the resulting high-quality dna should facilitate precise quantification and sequence analysis, and enable more efficient examination into the molecular nature of the cffdna in maternal plasma. 5riyaz ahmad rather, subhas chandra saha and veena dhawan: the most favourable procedure for the isolation of cell-free dna from the plasma of iso-immunized rhd-negative pregnant women 5. conflict of interest the authors declare no conflict of interest. 6. acknowledgements we thank the indian council of medical research (icmr), new delhi, india for providing financial support for this study. 7. references [1] lo ymd, corbetta n, chamberlain pf, rai v, sargent il, redman cw: presence of fetal dna in maternal plasma and serum. lancet. 1997; 350:485– 7. 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[25] hagiwara n, mechanic le, trivers ge, cawley hl, taga m, elise d. bowman ed, kumamoto k, peijun h, bernard m, doja s, miyashita m, tajiri t, sasajima k, nomura t, makino h, takahashi k, hussain sp, harris cc: quantitative detection of p53 mutations in plasma dna from tobacco smokers. cancer res. 2006; 66:8309–17. 7riyaz ahmad rather, subhas chandra saha and veena dhawan: the most favourable procedure for the isolation of cell-free dna from the plasma of iso-immunized rhd-negative pregnant women cbx663648 1..5 report addition of thrombin reduces the recovery of extracellular vesicles from blood plasma anush arakelyan 1 , wendy fitzgerald 1 , murad vagida 2 , elena vasilieva 2 , leonid margolis 1 , and jean-charles grivel 1,3 abstract extracellular vesicles (evs) are widely studied as a system of intercellular communication, as markers of various diseases, as well as a vehicle for delivery of various bioactive molecules to various cells. investigation of evs’ structure and function requires their isolation and precise quantification. however, in the current literature, there are significant discrepancies in the estimated numbers of evs in different body fluids. in part, this discrepancy is due to the difference in evs isolation protocols used by different investigators. a common protocol that includes exoquicktm is often used to isolate evs from body fluids and culture medium. here, we show that in the case of isolation of evs from blood, thrombin should be omitted from the protocol as clots formed due to the thrombin-triggered coagulation may entrap many evs thus leading to the underestimation of their numbers. keywords extracellular vesicles, quantification, thrombin, nanoparticles date received: 25 august 2015; accepted: 26 may 2016 introduction small extracellular vesicles (evs) are released by cells of almost all types both in vivo and in vitro. 1 it is currently understood that evs are important biological factors constituting a third system of intercellular communication after contact interaction and communication via soluble factors. populations of evs are heterogeneous, carrying large varieties of proteins and different types of small rnas that both may alter the physiology of the interacting cells. 2 over the last several years, there is a growing interest in studying evs as biological messengers and exploring their role as promising candidates for new potential therapeutic and diagnostic tools. 3 in particular, blood evs are now investigated as a factor in cardiovascular diseases. 2 detailed analysis of evs and their relevance to various pathologies requires evs isolation and precise quantification. however, in the current literature, there are significant discrepancies in the estimated numbers of evs in different body fluids. in part, this discrepancy is due to the difference in evs isolation protocols used by different investigators. various methods of evs isolation have been described. 2,4 the demonstration of the presence of exosomes in plasma by caby et al. in 2005 5 already relied on two major methods of isolation: differential centrifugation followed by sucrose gradient floatation and immunoaffinity purification on large latex beads coated with anti-cd63 antibodies. in this early work, aside from a longer initial centrifugation to accommodate the high viscosity of plasma, no other modifications were introduced in the sample treatment. later, new methods were introduced, including volume excluding polymers 1 section on intercellular interactions, eunice kennedy shriver national institute of child health and human development, national institutes of health, bethesda, md, usa 2 laboratory of atherothrombosis, moscow state university of medicine and dentistry, moscow, russia 3 sidra medical and research center, doha, qatar corresponding author: leonid margolis, section on intercellular interactions, eunice kennedy shriver national institute of child health and human development, national institutes of health, 10 center drive, nih, bldg 10, rm 9d58, bethesda, md 20892, usa. email: margolil@helix.nih.gov journal of circulating biomarkers volume 5: 1–5 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416663648 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:margolil@helix.nih.gov https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454416663648 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage used for viral preparations, 6–8 which are the basis for commercial products such as exoquick. size exclusion chromatography (sec) has also become a method of choice for ev isolation from plasma. 9,10 when compared to other methods, sec seems to provide a preparation devoid of protein contamination as determined by mass spectrophotometry 11 and results in the identification of new exosome markers in plasma. more recently, a new method based on plasma protein organic solvent precipitation has been developed for the purification of plasma microvesicles. 12 the considerations linked to the choice of the source, blood versus plasma, and the method of purification have been discussed in a position article from the international society of extracellular vesicles, 4 which concludes that because of clot-induced platelet vesiculation, the number of evs is higher in serum than in plasma, making plasma the most physiological milieu to study blood evs. the direct purification of microvesicles using nano-sized magnetic particles has been reported for the detection of cd34 þ exosomes in plasma of acute myeloid leukemia patient 13 or epithelial cell adhesion molecule (epcam) þ evs, 14 although such studies did not compare the effect of coagulation on the isolation of vesicles. in particular, fractions of small (<300 nm) evs carrying specific surface antigens can be isolated by capturing them with magnetic particles coupled to antibodies against ev surface proteins. many of the ev isolation methods are used in combination with a commercial product, exoquick tm , a volume excluding polymer that precipitates evs. exoquick is widely and successfully used to purify evs from various biological fluids such as ascites, urine as well as from cell culture media. 15,16 however, for various aims, in particular, those related to the study of cardiovascular diseases, evs need to be isolated from blood plasma. for blood plasma, thrombin is added to the exoquick isolation protocol. here, we report on the comparison of individual ev isolation from blood plasma by ev capturing with magnetic particles through major histocompatibility complex class-i (mhc-i) or cd31, a technique we recently developed that allows analysis of antigenic composition of individual evs. 17 we compared direct evs isolation from blood plasma with: (i) a commercially available kit ‘‘exoquick 5 tm ’’ which includes thrombin, (ii) same protocol but without thrombin treatment, and (iii) without exoquick but with thrombin treatment. we found that in the case of thrombin, the yield of evs was significantly lower compared to direct isolation or isolation with exoquick but without thrombin. thrombin-induced clotting may entrap a significant number of vesicles thus leading to the underestimation of evs quantities. materials and methods normal blood plasma was obtained from the nih blood bank. blood was collected in several 8-ml tubes with sodium citrate (3.2%); the first tube was discarded to avoid collecting evs released by platelets activated by venipuncture. collection tubes were centrifuged at 3000 � g for 15 min to obtain platelet-poor plasma (ppp) that was aliquoted and frozen at �80�c. from one of the pppcontaining tubes, after thawing, we isolated evs directly using carboxyl-terminated 15-nm iron oxide magnetic nanoparticles (mnps; 1 mg; ocean nanotech, springdale, arkansas, usa) coupled to mouse–anti-human monoclonal antibodies (either against mhc-i or against cd31); (biolegend, san diego, california, usa) or with isotype control antibodies (figure 1). we stained captured vesicles with fluorescent anti-cd9-phycoerythrin (pe) (biolegend) and anti-cd41-allophycocyanin (apc) (bd, pharmingen, san diego, california, usa) antibodies or with isotype control antibodies 17 (figure 1). alternatively, in combination with the mnp capture, we used exoquick either with thrombin according to the manufacturer instruction or without thrombin. the mnp–ev-detection antibodies complexes were separated in a strong magnetic field from free antibodies and evs that do not carry the capture antigen and their numbers were evaluated with a flow cytometer. evs captured by nanoparticles were analyzed with an lsrii (bd biosciences, san jose, california, usa) flow cytometer equipped with 355-, 407-, 488-, 532-, and 638-nm laser lines. for volumetric control, 123count ebeads™ (ebioscience, san diego, california, usa) were used. a known number of beads were added to the sample to be analyzed and by taking into account the dilution factor of the original bead solution, the volume of sample analyzed was estimated based on the number of 123 count ebeads acquired in each sample. on the basis of this volumetric measurement, the numbers of recorded events were recalculated into ev concentrations. also, to estimate evs’ size, a megamix-plus ssc (biocytex, marseille, france) was used. compensation beads (bd) were used to perform compensation controls. alexa-fluor 488 5c maleimide (carlsbad, california, usa)-labeled evs were a generous gift of dr lifson (nci-frederick, frederick, maryland, usa). the arbitrary fluorescent units (afu) of the spiked samples were measured with a safire 2 microplate reader (tecan, männedorf, switzerland) with the following settings: excitation at 490 nm (5 nm bandwidth) and emission at 540 nm (20 nm bandwidth, 10 flashes). ev concentration measurements were performed on nanosight ns 300 (salisbury, uk) according to the manufacturer instruction. results and discussion in this study, we compared protocols for evs isolation from blood ppp using as a test system mhc-i þ /cd9 þ / cd41 þ evs. after purification of plasma with exoquick or without such purification, we captured evs from the ppp with mnps coupled to anti-mhc-i antibodies. 17 we stained these vesicles for cd41 and cd9 and quantified them with flow cytometry. 17 also, we isolated evs with mnps coupled to cd31 and stained these vesicles for 2 journal of circulating biomarkers cd9 and for cd41, an antigen that attributes these vesicles to platelets. the vesicles we isolated were not necessarily exosomes but may be membrane-derived vesicles as well. as we showed earlier and confirmed here, this can be used either directly or after purification of the ev-containing fluid with exoquick. 17 capturing with mnps coupled to specific antibodies against evs surface antigens and isolation of these evs on a magnetic column resulted in obtaining an antigen-specific fraction of individual evs 17 (figure 1). specifically, to compare different protocols of evs isolation, ppp sample from each donor was divided into several aliquots. one aliquot was subjected to the exoquick purification according to the manufacturer’s protocol with thrombin, another aliquot was purified with exoquick but the thrombin step was omitted, the third aliquot of evs was treated with thrombin only, and the fourth aliquot was left untreated. then, all four samples were subjected to ev isolation by capturing with anti-mhc-i mnps coupled to specific antibodies, stained with fluorescent anti-cd9-pe and anti-cd41-apc antibodies, and separated the mnp-captured evs on a magnetic column. the majority of mhc-i captured evs from untreated ppp were double positive for cd41 and cd9 and therefore we compared their numbers isolated with three other protocols. on average, upon direct isolation, there were 3276 + 1875 (n ¼ 4) of cd41þ cd9þ mhc-i mnpcaptured evs per microliter of plasma. as expected, the number of evs directly isolated from ppp varied from donor to donor. to exclude the effects of this variability, for plasma from each donor, we normalized the number of isolated evs with the exoquick and/or thrombin by the number of evs isolated directly from untreated ppp. also, we characterized the size of the vesicles using megamix sizing beads. the size of evs isolated directly from untreated ppp and with exoquick without thrombin was similar (figure 2). however, when we used the full exoquick protocol (with thrombin), we isolated with mnps fewer evs by about one order of magnitude compared to direct isolation (figure 3). a similar decrease of mnp-isolated vesicles was observed when ppp was treated with thrombin only (figure 3). sizing of evs with flow cytometry indicated that the number of larger vesicles decreased predominantly (figure 2). unlike ascites, urine, or cell culture media, addition of thrombin to blood induces coagulation and formation of clot that is stabilized with fibrin. 18 in order to test whether the decrease of ev counts was due to formation of the clot that may trap evs, we omitted the thrombin step from our protocol and added only the exoquick polymer alone. in this case, the numbers (2757 + 1608, n ¼ 4) of evs captured with anti-mhc-i antibodies and positive for cd41 and cd9 were not significantly different from the corresponding numbers of evs isolated from the untreated ppp. to confirm these results with other antigens, from a sample figure 1. specificity of ev capture and staining. (a) evs were captured from untreated ppp either with mhc-i-mnps (red) or with isotype control mouse immunoglobulin g (msigg)-mnps (gray) and stained with anti-cd41-apc and anti-cd9-pe antibodies (left panel); evs were captured from untreated ppp with mhci-mnps and stained for isotype control antibodies mouseigg1kapc and mouseigg1k-pe (right panel). (b) evs were captured from ppp treated with exoquick either with mhc-i-mnps (red) or with isotype control msigg-mnps (gray) and stained with anti-cd41apc and anti-cd9-pe (left panel); evs were captured from ppp treated with exoquick with mhc-i-mnps and stained for isotype control antibodies mouseigg1k-apc and mouseigg1k-pe (right panel). (c) evs were captured from ppp treated with thrombin and exoquick, either with mhc-i-mnps (red) or with isotype control msigg-mnps (gray) and stained with anti-cd41-apc and anticd9-pe antibodies (left panel); evs were captured from ppp treated with thrombin and exoquick with mhc-i-mnps and stained for isotype control antibodies mouseigg1k-apc and mouseigg1k-pe (right panel). (d) evs were captured from ppp treated with thrombin either with mhc-i-mnps (red) or with isotype control msigg-mnps (gray) and stained with anti-cd41apc and anti-cd9-pe (left panel); evs captured from ppp treated with thrombin with mhc-i-mnps and stained for isotype control antibodies mouseigg1k-apc and mouseigg1k-pe (right panel). ev: extracellular vesicle; ppp: platelet-poor plasma; mhc-i: major histocompatibility complex class-i; mnp: magnetic iron oxide nanoparticle; cd: cluster of differentiation. arakelyan et al. 3 of plasma, we isolated evs with anti-cd31 mnps and again stained them with fluorescent antibodies against cd41 and cd9. in agreement with the above-described results with the anti-mhc-i mnps, the number of evs in the preparation subjected to exoquick (with the thrombin step) was close to an order of magnitude lower than that isolated from the untreated ppp obtained from the same donor (4.7 � 102 vs. 3.3 � 103/ml). if the thrombin was omitted from the exoquick protocol, the number of evs was similar to that obtained from untreated ppp (3.47 � 103/ml). these results were confirmed with two other techniques: to monitor the distribution of evs between different fractions in the course of their isolation, we spiked plasma with alexa-fluor 488 5c maleimide-labeled evs. 17 the initial fluorescence of this spiked plasma was on average 5954 afu. we divided this preparation of ev-spiked plasma into two parts: to one part, we added exoquick and to the second part, we added thrombin and then exoquick as described in the original protocol. measurements of fluorescence showed that with exoquick only, we recovered on average 4268 afu (72% of the spiked evs). in contrast, when we added thrombin resulting in clot formation, only 1620 afu of the fluorescent evs remain in the solution (about 25%) for further isolation with exoquick, while the rest was entrapped in the clot. furthermore, we evaluated the loss of evs in these spiked preparations with nanosight (salisbury, uk). according to this evaluation, due to thrombin-induced clotting, we lost about 65% of particles (1.4 � 108/ml with thrombin þ exoquick vs. 4 � 108/ml with exoquick only). in summary, we captured evs with magnetic nanoparticles coupled to mhc-i antibodies as this antigen is presented on most of the cells and by this way we captured evs generated by a broad spectra of cells. also, in this article, we captured evs through cd31 and stained them for cd9 and for cd41, which links them to platelets. whatever the biogenesis of the captured evs, when isolating them from blood samples either a direct capture with magnetic particles should be applied or if the exoquick protocol is to be used, the thrombin step should be omitted since it seems that the formed clot traps a significant number of evs leading to the underestimation of the amount of evs in the original plasma. in addition to this quantitative effect, induction of clotting by thrombin may selectively trap those vesicles that express receptors for clotting proteins such as tissue factor. 19 this may explain the numerical difference in the amount of antigen-specific versus non-specific figure 2. size evaluation of captured evs. evs were captured with anti-mhc-i-mnps from: (a) untreated ppp, (b) ppp treated with exoquick, (c) ppp treated with thrombin, and (d) ppp treated with thrombin and exoquick and stained for cd41 (purple). size of evs was estimated by megamix beads of different sizes (160 nm, 200 nm, 240 nm, and 500 nm) shown on each dot plot in red. ev: extracellular vesicle; mhc-i: major histocompatibility complex class-i; mnp: magnetic iron oxide nanoparticle; ppp: platelet-poor plasma; cd: cluster of differentiation. figure 3. comparison of different protocols for ev isolation. cd9þ/cd41þ evs were isolated from ppp by capture with mnps coupled to anti-mhc-i antibodies and the numbers of captured vesicles were evaluated. for each sample, isolation was performed under four conditions: evs were captured with mnps (i) from untreated ppp, (ii) from ppp treated with thrombin, (iii) from ppp treated with thrombin and exoquick, and (iv) from ppp treated with exoquick. to exclude the effects of the donor-to-donor variability, for plasma from each donor, the number of captured evs was normalized by the number of evs captured from untreated ppp. presented are means + sem. ev: extracellular vesicle; cd: cluster of differentiation; ppp: platelet-poor plasma; mnp: magnetic iron oxide nanoparticle; mhc-i: major histocompatibility complex class-i; sem: standard error of the mean. 4 journal of circulating biomarkers evs trapped in the clot in the above-described experiment. isolation of evs from plasma is different from evs isolation from other body fluids or from culture medium when thrombin is not recommended to be used with exoquick. even if thrombin is added into these media, there will be no coagulation and therefore the original exoquick protocol results in an adequate isolation of evs. differences in the protocols for evs isolation may contribute to various discrepancies in the reported estimation of the number of different vesicles in body fluids. acknowledgement author jean-charles grivel is currently affiliated with sidra medical and research center, doha, qatar. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study of aa, wf, j-c.g, and lm was supported by the nichd intramural program. the work of mv and ev was supported by the russian federation government grant #14.b25.31.0016. references 1. robbins pd and morelli ae. regulation of immune responses by extracellular vesicles. nat rev immunol 2014; 14(3): 195–208. 2. yanez-mo m, siljander pr, andreu z, et al. biological properties of 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of factor xiii in fibrin clot formation and effects of genetic polymorphisms. blood 2002; 100(3): 743–754. 19. bastida e, ordinas a, escolar g, et al. tissue factor in microvesicles shed from u87mg human glioblastoma cells induces coagulation, platelet aggregation, and thrombogenesis. blood 1984; 64(1): 177–184. arakelyan et al. 5 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice article journal of circulating biomarkers short course in the microbiome meeting dispatch kimberly falana1, rob knight2, camilia r. martin3, romina goldszmid4, k. leigh greathouse5, joanne gere1, howard young6 and winston patrick kuo7* 1 biopharma research council, tinton falls, nj, usa 2 department of pediatrics, computer science and engineering, university of california san diego, san diego, ca, usa 3 beth israel deaconess medical center, harvard medical school, usa 4 laboratory of experimental immunology cancer and inflammation program, national cancer institute, nih, usa 5 laboratory of human carcinogenesis, national cancer institute, nih, usa 6 laboratory of experimental immunology, national cancer institute, nih, usa 7 ies diagnostics, inc., cambridge, ma, usa *corresponding author(s) e-mail: winston@iesdiagnostics.com doi: 10.5772/61257 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract over the past decade, it has become evident that the microbiome is an important environmental factor that affects many physiological processes, such as cell prolifer‐ ation and differentiation, behaviour, immune function and metabolism. more importantly, it may contribute to a wide variety of diseases, including cancer, inflammatory diseases, metabolic diseases and responses to pathogens. we expect that international, integrative and interdiscipli‐ nary translational research teams, along with the emer‐ gence of fda-approved platforms, will set the framework for microbiome-based therapeutics and diagnostics. we recognize that the microbiome ecosystem offers new promise for personalized/precision medicine and targeted treatment for a variety of diseases. the short course was held as a four-session webinar series in april 2015, taught by pioneers and experts in the microbiome ecosystem, covering a broad range of topics from the healthy microbiome to the effects of an altered microbiome from neonates to adults and the long term effects as it is related to disease, from asthma to cancer. we have learned to appreciate how beneficial our microbes are in breaking down our food, fighting off infections and nurturing our immune system, and this information provides us with ideas as to how we can manipulate our microbiome to prevent certain diseases. however, given the variety of applications, there are scientific challenges, though there are very promising areas in reference to the clinical benefits of understanding more about our micro‐ biome, whether in our gut or on our skin: the outlook is bright. a summary of the short course is presented as a meeting dispatch. keywords microbiome, bacteria, gut, cancer, therapy, antibiotics, prenatal, personalized medicine, short course 1. introduction the human microbiome is an array of microorganisms, commonly referred to as the microbiota, which resides in our body and on our skin. our body is home to about 100 trillion bacteria/microbial cells (in and on our body) and two million microbial genes, but each of us only has about 40 trillion human cells and approximately 20, 000 human genes. bacterial cells alone outnumber our own by a factor of 20. therefore, understanding the microbial side and their interactions with us (the host) is of critical importance for our understanding of human biology, the variety of 1j circ biomark, 2015, 4:8 | doi: 10.5772/61257 diseases as mentioned, susceptibility to infectious and chronic disease, and even behaviour and drug responses. the short course on the microbiome organized by the biopharma research council and supported by intech open access publisher was held as a four-session webinar series in april 2015. the goal of the short course was to provide, at an introductory and in some cases a deeper glance, an exchange between researchers from academia in the current stage of the microbiome in clinical and transla‐ tional research and, eventually, clinical practice. the microbiome topics included a systems biology approach to understanding our microbiome, a spatially explicit map, how a mother’s biota affect physiology, and responses to certain stresses in babies to the role of the microbiome in cancer therapy. 2. session 1: april 9, 2015 2.1 the systems biology microbiome approach by rob knight, phd the first session was presented by rob knight, who provided a systems biology approach/overview to under‐ standing the microbiome. he introduced the current diversity measures used to study the microbiome including alpha diversity, beta diversity, phylogenetic diversity, and taxonomy diversity. for the past decade, dr. knight’s laboratory focused on developing computational methods/ tools for mapping the microbiome data. he discussed the unifrac method that exploits evolutionary relationships to compare different communities by using sequences to build phylogenetic trees, and then uses the trees to cluster the samples [1, 2]. dr. knight highlighted in his talk that the key is to identify connections between microbes and different conditions which we never thought of as being involved, including links between obesity and colon cancer, rheumatoid arthritis and (in mouse models) even things like autism, depression and multiple sclerosis, and finding out which of these conditions microbes cause — and which we can either predict or modify with improved knowledge about the microbial world. as an example of the potential utility of measuring microbial diversity, dr. knight described research from his group in which they sequenced gut microbiomes and revealed whether or not someone was obese with an accuracy greater than 90%, apparently more accurate than dna sequencing [3]. dr. knight further explained that faecal microbial samples are a good representation of the gut microbiome and are easy to collect. he presented figures displaying faecal microbial samples from a wide selection of animals clustered together based on a similar diet, gut type and lineage using the unifrac method [4, 5]. dr. knight also explained how researchers are able to distinguish the cause-effect relationship between the microbiome and many related diseases and conditions using koch’s postulates. as an example, he described research performed using gnotobiotics and model organ‐ isms. through these experiments, scientists discovered that transmissible gut microbiota can determine food intake [6, 7]. dr. knight described how, in a malawian twin study, children with kwashiorkor, or malnutrition, had different microbes to their healthy twins despite having an identical diet. upon treatment with ready-to-use therapeutic foods, the malnourished twins recovered while their microbial gut population changed, but these effects were only shortterm. the microbes from both children in three twin pairs were transferred to germ-free organisms to see if they lost or gained weight. in two cases, those that received microbes from malnourished children lost weight, indicating a causal relationship. dr. knight described a new comprehensive approach involving culturing, gnotobiotics and metagenomics that can help in scientifically determining the impact of our microbiome. in this method, he discussed the methods in which one will isolate the many strains of microbes from a single faecal sample and grow them separately, then mixing them to see if a specific disease phenotype emerges from the mixture. this technique can be utilized to disprove any hypotheses as to whether the cause of the disease phenotype is a virus, cytokine or metabolite as opposed to microbiota [8]. 3. session 2: april 16, 2015 3.1 part i: “roundup of the microbiome news” by winston patrick kuo, dds, dmsc, ms dr. kuo gave an insightful overview of recent and exciting discoveries in microbiome research. these discoveries included studies on the relationship of the microbiome to the immune system, obesity, cancer, mental health, and drug metabolism. he discussed how the microbiome affects us directly from birth (whether via traditional or csection) until we get old, and most of these microbes come from the mother’s skin, birth canal and gut. he highlighted in a recent publication how a microbe called bifidobacteri‐ um has potentially beneficial effects for babies, as they are among the first microbes to show up in a baby's intestinal tract after birth. studies suggest a particular type of bifidobacteria can prevent infections and help establish the newborn's immune system. a single gene in the mother called fut2 controls the behaviour of bifidobacterium, and this gene works through breast milk [9]. dr. kuo mentions how we carry up to 2 kg of microbes in our gut, and two thirds of our gut microbiome is unique to each individual. but the question is: how could your gut microbiota be influencing your health and risk of disease? he describes how the microbes in our gut play an important role in digestion: although sometimes the stomach and small intestine are unable to digest certain foods the gut 2 j circ biomark, 2015, 4:8 | doi: 10.5772/61257 microbes assist in ensuring we get the nutrients we need. for example, the gut bacteria help in the production of vitamins b and k that play a major role in immune function. dr. kuo highlighted the role of the gut microbiota and an individual's risk of obesity and other metabolic conditions. he discussed the research conducted at cornell and king's college where they identified a certain strain of bacteria christensenellaceae minuta that was more common in people with a low body weight, and that the presence of this particular strain is highly influenced by genes. he mentioned the study was confirmed by introducing this bacteria to the guts of mice, which then caused the animals to gain less weight, indicating the bacteria may reduce or prevent obesity [10, 11]. dr. kuo highlighted studies where the gut bacteria were linked to cancers, where researchers discovered specific bacteria in the intestines, lactobacillus johnsonii, that may play a role in the development of lymphoma [12]. he discussed a study conducted by uk researchers that found that a common gut bacteria called helicobacter pylori may cause stomach cancer and duodenal ulcers by deactivating a part of the immune system involved in regulating inflammation. late last year, investigators from mount sinai associated a specific combination of gut bacteria with the development of colorectal cancer [13-16]. dr. kuo’s next topic was the relationship between the microbiome and mental health. can gut microbes alter the metabolites associated with communication between the gut and the brain, which interferes with brain function? he discussed how bacteria has been shown to play a role in producing neurotransmitters, such as norepinephrine, serotonin, or dopamine, as well as how certain probiotic bacteria can actually modulate the effects of neurotrans‐ mitters. he further discussed a microbiome-gut-brain study in germ-free mice, where it was demonstrated that the lack of gut microbes affects sociability, decreases memory, and increases stress responses. the study showed how specific strains of lactobacillus rhamnosus modulate stress, and this effect appears to be mediated through the vagus nerve in mice [17]. dr. kuo discussed other recent topics related to stress and anxiety, autoimmune and cardiovascular diseases and drug metabolism. dr. kuo concluded his brief overview by stating how we need a better understanding of microbiome and drug interactions so that someday this will allow us to devise strategies to improve drug efficacy and reduce side effects. not too far ahead, we can start manipulating and explore potential therapeutic uses for the microbiome. 4. session 2: april 16, 2015 4.1 part ii: “the establishment of the microbiome in newborn infants: challenges and new opportunities” by camilia martin, md, ms dr. camilia martin discussed the influences of the early establishment of the microbiome in the maternal-foetal environment and ex-utero determinants. dr. martin discussed recent studies that have challenged the dogma that the maternal-foetal unit is sterile; however the placenta has been determined to harbour its own unique microbiome, and analysis of the first passage of stool in the infant reveals microorganisms [18, 19]. in addition, she mentioned the analysis of tracheal aspirates after early postnatal intubation reveals organisms, presumably from amniotic fluid swallowed when the infant was in utero [20]. dr. martin highlighted how the principle perinatal and postnatal determinants of microbial colonization patterns in the newborn include delivery mode, diet, hospitaliza‐ tion, and medications. of these factors, a vaginal delivery versus caesarean section and breast milk versus formula lead to a more favourable microbiome profile that contains known commensal organisms and fewer organisms considered to be pathogenic. in the preterm infant, the microbial pattern is dominated by pathogenic rather than commensal organisms due to factors unique to critically ill populations such as exposure to indigenous hospital organisms and to medications that are known to alter the enteric flora, including antibiotics and h2 blockers [21]. dr. martin went on to discuss the medical consequences of an altered microbiome where the establishment of an intestinal microbiome is critical for immune ontogeny and ongoing development of the intestinal tract. she observed how the microbiome profile of a preterm infant appears to have significant health consequences. unfavourable profiles have been linked to an increased risk of necrotizing enterocolitis, lung disease, and sepsis [20, 22, 23]. the microbiome patterns of older infants and children have also been linked to atopic disease (allergies, asthma), type i diabetes mellitus, celiac disease and obesity [24]. martin also mentioned how an increasing awareness of the influence of the microbiome on health and risk of disease has already begun to change perinatal maternal and neonatal medical practices. there is a concerted effort to decrease the national caesarean section rate, increase maternal–infant skin-to-skin colonization after birth, promote early and continued exposure to breast milk, and reduce antibiotic and other medication exposures. dr. martin went ahead to discuss strategies to protect and restore microbial diversity. she emphasized the need to optimize the influence of the microbiome on health, which is critical to protect and restore microbial diversi‐ ty. for the preterm infant, protecting microbial diversity would entail an understanding of how current medical practices alter the microbiome and a re-evaluation of the implementation of these practices. restoration of microbi‐ al diversity may include promoting dietary strategies that are known to optimize the intestinal microbiome, minimizing the use of medications known to disturb the microbial balance, delivering probiotics (although this 3kimberly falana, rob knight, camilia r. martin, romina goldszmid, k. leigh greathouse, joanne gere, howard young and winston patrick kuo: short course in the microbiome remains controversial due to limited well-designed studies), and changing medical practices to those that attempt to emulate natural patterns of colonization, such as strategies to expose the infant to vaginal flora even if delivered by caesarean section [25]. dr. martin concluded by discussing the challenges in bridging the gap between the microbiome and personal‐ ized medicine. she mentioned how patterns in the intestinal microbiome have been linked to specific diseases in various populations, overlap is often ob‐ served between cases and controls in many of the measures used to define the microbiome. as a result, it is difficult to clinically apply these observations on an individual basis. in parallel, an individual’s microbiome profile can be distinctly unique from other individuals such that it serves as a fingerprint to that person’s identity. thus, the precise nature of the influence of the microbiome in an individual’s health can be difficult to determine, and one’s microbiome-host relationship can be quite distinct from another’s. dr. martin concluded that non-invasive omics strategies have the potential to increase our understanding of the unique microbial-host interactions bridging the path to both personalized medicine and applied populational health [26]. 5. session 3: april 23, 2015 5.1 spatially explicit maps by rob knight, phd in this session, dr. knight gave examples of how computational methods can be utilized to map micro‐ biome genomic data. as an example, dr. knight present‐ ed a map of several microbiome clusters representing oral, vaginal, skin and faecal microbiomes from adults in the same cultural community. through the sequence of maps, he demonstrated how the faecal microbiome of an infant begins with a composition identical to their mother’s vaginal or skin microbiome, and after 2.5 years ends with a composition similar to that of an adult faecal microbiome [27]. as another example, dr. knight discussed a study involv‐ ing spatially explicit maps that illustrated the distribution and diversity of the bacteria on kitchen surfaces. bacterial samples from four kitchens were tested and averaged. the resulting maps demonstrated relatively high abundances of campylobacter on the stove exhaust fans, salmonella on stoves and sinks, clostridium on the cabinets and e. coli on the refrigerator draws. the researchers also differentiated bacteria based on particular sources including skin, produce and faucet water. in relatively high abundance, bacteria derived from the skin were found on trashcans, produce bacteria was found on the stove and counter, and faucet water bacteria was found on faucets. this study emphasizes implications for bacterial survivability, growth and transmission within the kitchen [28]. dr. knight believes there is a better way to illustrate the data from such studies than is currently available, and suggests a more spatially explicit approach. researchers in his lab were able to map the microbial communities of one’s face on a map of their face and correlated the different colours to each area of the face based on the composition/ make-up of its microbiome. dr. knight highlighted how another emerging and intri‐ guing area of research is establishing and validating the relationships between the metabolome and the micro‐ biome. he discussed how researchers have created micro‐ bial and metabolite maps of the whole human body using mass spectroscopy data: by mapping the metabolites and microbiomes onto the human body, researchers can hypothesize which microbes are performing the biotrans‐ formations that use or produce those metabolites. dr. knight mentioned that infection by microbial patho‐ gens, such as mrsa or clostridium difficile, is highest in a hospital environment. the ultimate goal of the human microbiome project is to reduce the nosocomial infection rate of hospitals through improved disinfection measures and in order to create and support such measures; several factors were studied to determine if they had significant influence on the microbial community and rate of microbial succession within the hospital. these factors included human demographics, physical conditions such as temper‐ ature and humidity, building materials, patient microbiota, duration of patient occupancy, patient room and nurse station usage, and the composition and diversity of an existing microbial community derived from previous occupants. in total, 84 different variables were considered. the results will be published in the near future. 6. session 4: april 30, 2015 6.1 part i: microbiome and cancer therapy by romina goldszmid, phd dr. romina goldszmid discussed how mammals live in partnership with a rich commensal microbiota on their bodies’ epithelial surfaces. this partnership is critical for tissue formation, metabolism and the development and function of the innate and adaptive resistance. the micro‐ biota are also closely linked to cancer development both locally (e.g., colorectal carcinoma) and at distant sites (mammary carcinoma, lymphoma). she discussed how recent studies demonstrated that disruption of the com‐ mensal gut microbiota impairs the response of subcutane‐ ous cancers to cpg odn-immunotherapy and platinum chemotherapy, and in both cases innate myeloid cells are responsible for the impaired response, albeit through distinct mechanisms [29]. the failure to respond to immu‐ notherapy was due to the inability of monocyte-derived cells in the tumour microenvironment to produce proinflammatory cytokines (e.g., tnf and il-12) in response to cpg and the subsequent necrosis needed to induce tumour regression. 4 j circ biomark, 2015, 4:8 | doi: 10.5772/61257 dr. goldszmid further discussed how the composition of the faecal microbiota was distinct among mice displaying high and low tnf responses to cpg treatment, and several bacterial species were found to either positively or nega‐ tively correlate with the tnf response. the impaired response to platinum chemotherapy correlated with a lack of an early genotoxic effect of the drug and reduced ros production by tumour infiltrating myeloid cells. these data point to a role of microbiota in priming tumour-associated myeloid cells to respond to immunoand chemotherapy. she highlights how the gut microbiota can also exert an adjuvant effect. for example, certain chemotherapeutics (e.g., cyclophosphamide) or the total body irradiationconditioning regime performed prior to adoptive t cell transfer therapies cause damage of the gut mucosa allow‐ ing bacteria translocation into the draining lymph nodes and increased levels of bacterial products in circulation [30]. the translocated bacteria and their products induce activation of antigen-presenting cells and subsequently the priming of t cells needed for an effective anti-tumour response [31]. together, these findings suggest that the composition of the commensal microbiota modulates the response to cancer therapy, thus providing new targets and possibilities for therapeutic intervention [32]. 7. session 4: april 30, 2015 7.1 part ii: microbiome and cancer therapy by k. leigh greathouse, phd, mph, ms, rd dr. leigh greathouse discussed that lung cancer is the leading cancer diagnosis worldwide (1.8 million/year) and a major health and financial burden to our healthcare system (us$ 12.1 billion/year). it has a mortality rate higher than that of the top three cancers combined. epidemiolog‐ ical evidence suggests that alterations in microbial com‐ munities due to repeated antibiotic exposure are associated with increased lung cancer risk. the microbiome consists of bacteria, archaea, fungi, eukaryotes and viruses which outnumber host cells ten to one and host genes >100 times. she elaborated that several bacteria are associated with chronic inflammation and a subsequent increased risk of lung and colon cancer, including mycobacterium tuberculo‐ sis (lung cancer) and fusobacterium nucleatum (colon cancer), a bacterium commonly isolated from inflammato‐ ry bowel disease patients and a risk factor for colon cancer. the more virulent strains of f. nucleatum affect colon cancer progression and increase tumour multiplicity by various mechanisms including favouring the infiltration of tu‐ mour-promoting myeloid cells to create a pro-inflammato‐ ry environment. colorectal carcinomas associated with a high abundance of faecal f. nucleatum were found to have the highest number of somatic mutations, suggesting that these mutations create a pathogen-friendly environment. furthermore, the loss of p53 in enterocytes impairs the epithelial barrier and allows infiltration of bacteria, resulting in nf-κb signalling, which was required for tumour progression. dr. greathouse emphasized that the microbiome of lung cancer is largely unknown. exposure to cigarette smoke reduces epithelial barrier function and increases suscepti‐ bility to infections. we hypothesized that somatic muta‐ tions together with cigarette smoke create a dysbiotic microbiota that is associated with lung carcinogenesis. to explore this hypothesis, we sequenced 16s rrna in tissue from lung cancer cases and controls, and lung cancer samples in the cancer genome atlas (tcga) as validation. lung cancer cases could be classified by the relative abundance of two taxa, variovorax and streptococcus, with an increase in variovorax abundance in tumours as compared to non-tumour adjacent lung tissue. a group of taxa were significantly associated with squamous cell carcinoma (scc), of which acidovorax spp. were enriched in smokers. further, we observed that these taxa, including acidovorax, exhibit higher abundance among the subset of scc cases with tp53 mutations. therefore, scc-associated taxa are enriched in tumours with tp53 mutations, estab‐ lishing a microbiome-gene interaction in lung cancer tissue. 8. conclusion based on the session presentations, it was evident that the microbiome translational research is quickly evolving and that it continues to advance in all facets of science. overall, the short course fulfilled its goal of providing a balanced forum of relevant content from academic researchers. the webinar presentations are available on the biopharma research council website (http://www.biopharmare‐ searchcouncil.org). due to the interest level in this space, the biopharma research council and the nci will cosponsor a one day event at the national cancer institute on september 24, 2015, entitled, “altering the microbiome: can it impact health?” the purpose of the meeting is to discuss how the host microbiome can be altered and whether such approaches can result in a positive impact on the host. 9. references [1] lozupone c, hamady m, knight r. unifrac--an online tool for 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[32] goldszmid rs, dzutsev a, viaud s, zitvogel l, restifo np, trinchieri g. microbiota modulation of myeloid cells in cancer therapy. cancer immunolo‐ gy research, 3(2), 103-109 (2015). 6 j circ biomark, 2015, 4:8 | doi: 10.5772/61257 article journal of circulating biomarkers detection and characterization of circulating tumour cells from frozen peripheral blood mononuclear cells original research article david lu1, ryon p. graf1, melissa harvey1, ravi a. madan2, christopher heery2, jennifer marte2, sharon beasley1, kwong y. tsang2, rachel krupa2, jessica louw1, justin wahl1, natalee bales1, mark landers1, dena marrinucci1, jeffrey schlom2, james l. gulley2 and ryan dittamore1* 1 epic sciences, inc., san diego, ca, usa 2 national cancer institute, national institutes of health, bethesda, md, usa *corresponding author(s) e-mail: ryan.dittamore@epicsciences.com received 29 january 2015; accepted 21 april 2015 doi: 10.5772/60745 licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http:// creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract retrospective analysis of patient tumour samples is a cornerstone of clinical research. ctc biomarker character‐ ization offers a non-invasive method to analyse patient samples. however, current ctc technologies require prospective blood collection, thereby reducing the ability to utilize archived clinical cohorts with long-term outcome data. we sought to investigate ctc recovery from frozen, archived patient pbmc pellets. matched samples from both mcrpc patients and mock samples, which were prepared by spiking healthy donor blood with cultured prostate cancer cell line cells, were processed “fresh” via epic ctc platform or from “frozen” pbmc pellets. samples were analysed for ctc enumeration and biomark‐ er characterization via immunofluorescent (if) biomarkers, fluorescence in-situ hybridization (fish) and ctc mor‐ phology. in the frozen patient pmbc samples, the median ctc recovery was 18%, compared to the freshly processed blood. however, abundance and localization of cytokeratin (ck) and androgen receptor (ar) protein, as measured by if, were largely concordant between the fresh and frozen ctcs. furthermore, a fish analysis of pten loss showed high concordance in fresh vs. frozen. the observed data indicate that ctc biomarker characterization from frozen archival samples is feasible and representative of prospec‐ tively collected samples. keywords circulating tumour cells, peripheral blood mononuclear cells, metastatic castrate resistant prostate cancer, androgen receptor, biorepository 1. introduction the molecular characterization of circulating tumour cells (ctcs) in the blood of patients with cancer has garnered great interest for its potential to longitudinally monitor an evolving disease, response to therapy and/or define prognosis [1-5]. while numerous ctc technologies are in development, a recognized unmet need is the ability to retrospectively analyse ctc samples from previously archived (frozen) clinical samples with associated long clinical histories [6]. the epic ctc platform utilizes a non-enrichment-based method and slide-based immunofluorescence (if), 1j circ biomark, 2015, 4:4 | doi: 10.5772/60745 coupled with digital pathology and genomic techniques, to detect and molecularly characterize ctcs. as part of the epic sciences standard operating procedures (sops), blood tubes are shipped to epic sciences and processed within 96 hours from blood draw. following red blood cell (rbc) lysis, the nucleated fraction is plated onto microscope slides and frozen at -80°c for storage and subsequent analysis. in contrast, many enrichmentbased strategies are unable to store morphologically intact ctcs for future analysis [7-9]. in order to augment the epic sop for sample processing, we sought to deter‐ mine if the epic platform might be compatible with previously banked (frozen) patient material to enable retrospective analysis and expand the patient sample pool amenable to epic ctc characterization. the collection of patient tissue, blood, saliva and urine is common in clinical trials, often with peripheral blood mononuclear cells (pbmcs) subjected to isolation and cryopreservation [10]. often, samples are banked until retrospective analyses are initiated. to enable the retro‐ spective analysis of existing archived samples, we sought to test whether ctcs could be detected and molecularly characterized from pelleted, frozen pbmc fractions using the epic ctc platform. we then compared the ctcs recovered from these “frozen” samples with matched material, which was prepared “fresh” per epic sop. in this study, we report the enumeration and biomarker characterization of spiked controls and patient samples, which were processed fresh at epic sciences. these are compared to matched samples from frozen/archived pbmcs, which were prepared by ficoll separation. we also compare the morphological characteristics, protein expres‐ sion and genetic alterations of ctcs that were processed using the epic platform with ctcs from frozen pbmcs, which had been stored up to 7.5 years prior to analysis. 2. material and methods 2.1 preparation of control cell line cell (clc) slides healthy donor (hd) blood was collected in sodium heparin vacutainer® tubes (bd, franklin lakes, nj) and whole blood white blood cell (wbc) counts were recorded. known amounts of vcap or pc3 (atcc, manassas, va) prostate cancer cell line cells (clcs) were spiked into the hd samples and nucleated cells were isolated by either the epic sop or ficoll density separation (ficoll-paque; ge healthcare, buckinghamshire, uk), as per the manufactur‐ er’s protocol. for a description of the epic sop, please see “analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumor cell detec‐ tion characterization” [11]. wbc/pbmc counts were taken after purification and were used to calculate the per cent recovery. the recovered cell line-spiked pbmc pellets were either: 1) used to create control clc slides for if staining per epic sop via cell deposition on microscope slides and storage in the epic biorepository, or 2) cryopreserved in 90/10 human ab serum/dmso (sigma-aldrich, st. louis, mo) and stored in liquid nitrogen for at least 48 hours. the cryopreserved pbmc pellets were then thawed and deposited onto glass slides for staining and analysis or storage in the epic biorepository. 2.2 patient blood sample processing blood samples were collected using cell free dna bct® tubes (streck, omaha, ne) from metastatic castrate resist‐ ant prostate cancer (mcrpc) patients at the national cancer institute and shipped to epic sciences for process‐ ing within 96 hours. all patients gave informed consent and enrolled on a biospecimen collection protocol ap‐ proved by the nci irb (nct00034216). nucleated cells were deposited onto glass slides and subsequently stored at -80°c, as per epic sop. for matched patient blood samples, two identical blood tubes were drawn. one tube per patient was shipped immediately to epic sciences and processed via sop, while trained operators processed the second tube at the clinical site via ficoll separation. ficoll purified pbmcs were cryopreserved in 90/10 human ab serum/dmso and frozen in liquid nitrogen for up to 90 months prior to ctc analysis. archived samples were shipped frozen to epic sciences and processed with a modified protocol for pbmc and ctc thaw and recovery, followed by immediate cell deposition onto slides and subsequent epic biorepository storage. for concordance testing, matched samples were stained and processed identically for morphology and biomarker assessment. 2.3 cryopreserved clc/ctc thaw for cryopreserved samples, the clcs and patient samples, which were stored in liquid nitrogen, were thawed in a 37°c water bath, followed by an immediate dilution with rpmi 1640 medium supplemented with 10% foetal bovine serum (life tech, carlsbad, ca). the pbmcs were pelleted and washed with pbs. this was followed by cell counting, resuspension in plating medium and deposition onto glass slides for -80°c storage and ctc analysis. 2.4 immunofluorescent staining and analysis epic sciences’ ctc identification and characterization technology has been described in previous publications [11, 12]. in brief, slides created from clc-spiked hd samples or mcrpc patient samples were subjected to automated immunofluorescent (if) staining utilizing commercially available monoclonal primary antibodies against pan-cytokeratin (ck), cd45 and androgen receptor (ar). fluorescent-labelled alexa fluor secon‐ dary antibodies (life tech, carlsbad, ca) were used to allow for fluorescent detection and semi-quantitative characterization of protein biomarkers. the stained slides were analysed with automated fluorescent scanners and morphology algorithms for the identification of tradition‐ al (ck+) ctcs, ctc clusters, small ctcs, apoptotic cells 2 j circ biomark, 2015, 4:4 | doi: 10.5772/60745 and ck-negative (ck-) ctcs. a more thorough descrip‐ tion of ctc types and their stage of validation is described in “analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumor cell detection characterization” [11]. trained classifiers conduct a final classification of ctc subpopu‐ lations and record subcellular biomarker localization, where applicable. 2.5 fluorescence in situ hybridization following ctc enumeration, if and morphological characterizations, select slides with sufficient ctcs were further tested for genetic alterations by fluorescence in situ hybridization (fish). coverslips were removed, immuno‐ fluorescence staining was attenuated and cells were fixed and dehydrated with formaldehyde and ethanol, respec‐ tively. after dehydration, a probe solution targeting the dna sequences of interest (i.e., pten) was applied to each slide, denatured for 10 minutes at 83°c and hybridized for 14-24 hours at 37°c. the slides were washed in a series of saline sodium citrate (ssc)/detergent (igepal) solutions, counterstained with dapi and mounted with an anti-fade mounting medium. as the exact coordinates of every ctc were recorded during the enumeration process, each ctc can be relocated and analysed by fish for specific genetic alterations. patient wbcs were used as internal controls, representing the normal genetic status for each respective sample. 2.6 statistical analyses statistical analyses were conducted using graphpad prism software (la jolla, ca). kruskal-wallis anova was used to compare the measures with >2 groups. a mann-whitney u test was used to compare the differences between the matched sample pairs from each individual patient. p<0.05 was considered to be significant. 3. results 3.1 androgen receptor (ar)+ and arcell lines demonstrate feasibility of ctc recovery from cryopreservation to initially assess the capabilities of frozen ctc recovery on the epic platform, ctc surrogate (vcap (ar+) and pc3 (ar-)) clcs were spiked into hd blood and subjected to the same cell isolation, cryopreservation and sample deposition protocols to be used for the processing of archived patient samples (figure 1a). matched slides containing clcs that were deposited fresh or after freez‐ ing, were stained for ck, cd45 and ar. the concordance of pbmc and clc morphology, as well as protein expres‐ sion, were compared (figure 2a). no statistically signifi‐ cant differences in recovery rates were observed between clcs that were processed with or without cryopreserva‐ tion (not shown). no significant changes in the mean ar expression were seen in either vcap or pc3 cells in frozen samples, indicating the preservation of ar protein integri‐ ty. vcap ck expression also remained unchanged, while pc3 ck expression decreased by 35% in the frozen samples (p<0.01). however, in the latter case, relative ck staining intensity was still observed at 180-fold over wbc back‐ ground. 3.2 concordance of ctc recovery and protein expression from archived mcrpc patient samples to determine concordance of patient ctc recovery and characterization, two matched blood tubes were drawn from 10 mcrpc patients. one tube was shipped to epic sciences and processed within 96 hours using standard cell deposition protocols (fresh), while the second tube was subject to ficoll pbmc isolation and archived in liquid nitrogen (frozen; figure 1b). compared to the fresh samples that were undergoing rbc lysis at epic sciences, the ficoll separation resulted in a lower net yield of figure 1. schematic of sample preparation and experimental protocol for fresh and frozen sample processing. (a) to create control slides, cell line cells (clcs) were spiked into healthy donor blood and processed via ficoll separation. clc-containing pbmc pellets were split into identical fractions: one fraction was cryopreserved in liquid nitrogen prior to cell deposition (blue arrow), and the other fraction was deposited fresh onto slides (grey arrow). (b) matched mcrpc patient samples were drawn: one sample was sent immediately to epic sciences for fresh sample deposition (grey arrow), and one sample was processed via ficoll and frozen at the clinical site. the frozen pbmc sample was cryopreserved for up to 7.5 years prior to shipment and subsequent processing at epic sciences (blue arrow). 3david lu, ryon p. graf, melissa harvey, ravi a. madan, christopher heery, jennifer marte, sharon beasley, kwong y. tsang, rachel krupa, jessica louw, justin wahl, natalee bales, mark landers, dena marrinucci, jeffrey schlom, james l. gulley and ryan dittamore: detection and characterization of circulating tumour cells from frozen peripheral blood mononuclear cells nucleated cells due to the preferential enrichment for pbmc over the whole wbc (median recovery: 67% and 20%, respectively; figure 2b). notably, the subsequent freeze/thaw of the ficoll isolated samples had no significant effect on the net pbmc recovery (p>0.05), indicating that the freeze/thaw process lost very little patient material. the total ctcs and ar+ ctcs, as detected by if staining, were normalized to 7.5 ml blood volume and com‐ pared between the freshly processed and archived samples (figure 2c, d). ctcs were found in 8/10 of the fresh samples and 8/10 of the frozen archived samples. typical ctc recovery in frozen samples was significant‐ ly lower as median ctc recovery in frozen samples was 18% that observed from freshly processed blood. howev‐ er, the relative order of ctc burden in patients re‐ mained fairly consistent, with the exception of two patients, ne1-8, -9. furthermore, ctcs were found in the fresh sample from patient ne1-15, which were undetect‐ ed in the matched frozen sample. meanwhile, ctcs were found in the frozen ne1-14 sample, which went undetect‐ ed in the corresponding fresh material. ar+ ctcs were found in both preparations of ne1-13, -6 and -10, and the median recovery of ar+ ctcs from the frozen samples was 16% of that observed in freshly processed blood. notably, the ctc count correlates linearly (though sample size is limited) between the fresh and frozen samples when normalized to blood volume or wbc count (r2>0.90, n=10; figure 3a, b). additionally, the per cent of ar+ ctc/clc subpopulations also correlate between the matched samples (r2=0.90, n=10; figure 3c). 3.3 cytokeratin and androgen receptor expression is retained in cryopreserved ctcs heterogeneous ck and ar protein expression, as quantified by if staining, was observed in all of the ctc + patients (figure 3d, e). moreover, traditional ck+ ctcs, ctc clusters and ckctc subpopulations were found in both the fresh and frozen material. with the exception of ne1-6, in which the median ck expression was higher in the cryopreserved sample (p<0.01), no other significant differences in ck or ar protein levels were observed between the matched material. furthermore, within the ar+ ctcs, subpopulations with predominately nuclearlocalized ar as well as ctcs with diffuse cytoplasmic ar were detected (representative images, figure 4a, b). no notable differences in the distribution of ar localization were observed in the ar+ ctc subpopulations. (figure 4c). thus, the process of cryopreservation did not disrupt the ar or ck protein localization in these samples. figure 2. assessment of cell line and mcrpc patient ctc recovery. (a) the mean clc ck and ar protein expression levels were compared between cells that were processed fresh and after cryopreservation and thaw. the cryopreserved vcap and pc3 samples showed a mean decrease in ck expression of 15% (p>0.05) and 35% (p<0.001), respectively, compared to freshly processed cells. mean vcap and pc3 ar expression remained unchanged. (b) compared to whole blood wbc counts, the median wbc recovery in the patient samples that were processed fresh per epic sop was 67% (range: 32-79), while ficoll purification resulted in a median pbmc yield of 20% (range: 3.9-49). ficoll-processed frozen sample recovery was not significantly changed post-thaw (p>0.05). the total ctcs (c) and ar+ ctcs detected (d) from matched mcrpc patient blood draws, which were normalized to 7.5 ml blood, are plotted and tabulated alongside control pc3 and vcap clc control slides. patient ctcs were found in 8/10 of the fresh samples and 8/10 of the frozen samples. compared to the fresh samples, the median total and ar+ ctc recovery in the frozen samples were 18% and 16%, respectively. notable differences: ctcs were found in the fresh sample from patient ne1-15 but were undetected in the matched frozen sample, while ctcs were found in the frozen ne1-14 sample, which went undetected in the fresh material. **p<0.01, ***p<0.001, n=10 patient samples. 4 j circ biomark, 2015, 4:4 | doi: 10.5772/60745 figure 3. comparison of ctc enumeration and biomarker expression. the ctcs that were recovered from the matched samples (or vcap control) show a linear correlation between the fresh and cryopreserved preparations when normalized to either 7.5 ml blood (a), 3m wbc (b), or (c) per cent ar+ ctcs (r2≥0.90 for each comparison). ctcs with heterogeneous ck (d) and ar (e) expression levels were detected in the majority of patients from both the fresh (f) and cryopreserved (c) samples. the dot plot depicts the presence of individual ctcs found in each patient sample. ck and ar positivity was defined for any ctc with a relative protein expression of greater than 2.8or 3-fold over background, respectively (dotted lines). with the exception of ne1-6 ck expression (d; p<0.01), no other matched patient samples expressed significantly different levels of mean ck or ar protein between the fresh and frozen material (p>0.05; mann-whitney u test). figure 4. representative images of patient ctcs demonstrate the preservation of wbc and ctc morphology and retention of ar localization. of the eight patients with detectable ctcs, three patients harboured ar+ ctcs in both fresh and frozen samples. representative images of arctcs, nuclear ar+ ctcs and diffuse cytoplasmic ar+ ctcs, which were recovered from both the fresh (a) and frozen (b) samples, are shown depicting preserved wbc and ctc morphology and consistent biomarker localization. (c) numerical assessment of ar localization in ar+ patients confirms the retention of subcellular protein localization after ctc cryopreservation. the percentages of ctcs with nuclear or cytoplasmic localized ar are similar between the matched ar+ samples, with no significant bias of localization between the fresh and frozen samples. (*>1300 ctcs present per slide; only 100 ctcs were assessed for ar localization.) 5david lu, ryon p. graf, melissa harvey, ravi a. madan, christopher heery, jennifer marte, sharon beasley, kwong y. tsang, rachel krupa, jessica louw, justin wahl, natalee bales, mark landers, dena marrinucci, jeffrey schlom, james l. gulley and ryan dittamore: detection and characterization of circulating tumour cells from frozen peripheral blood mononuclear cells 3.4 concordance of genetic assessment by fish to test the viability of downstream genetic analyses on cryopreserved ctcs, pten fish was conducted on patient ctcs detected from freshly processed blood (epic sop) and ficoll-processed cryopreserved samples. in each sample from patient ne1-13, at least 20 ctcs were assessed for pten alterations. the assessment of homozygous (pten = 0, cep10 ≥ 1) and hemizygous (0 < pten < cep10) pten loss found near-equivalent populations of genetic alterations between the fresh and frozen ctcs (figure 5). these findings demonstrate the preservation of dna and the feasibility of genetic analyses in cryopreserved ctcs. 3.5 ctc recovery from additional banked mcrpc patient samples cryopreserved mcrpc patient pbmc samples from a previously completed clinical trial were shipped to epic sciences for ctc recovery and characterization (5-92 months old). ctcs were detected in 16 of 20 (80%) samples, with 11 patients also harbouring ar+ ctcs (figure 6). akin to previously analysed samples, ctcs of varying morphol‐ ogy (i.e., traditional ck+, ctc clusters, apoptotic ctcs and ckctcs) were found. furthermore, heterogeneous ck and ar protein levels and ar localization patterns were observed in ctcs from these samples. 4. discussion in this study, we report enumeration and characterization of morphology, biomarker expression, and genetic altera‐ tions of ctcs isolated from frozen pbmc fractions stored up to 7.5 years prior to analysis. we found that patient blood samples processed by ficoll separation and cryopre‐ served in liquid nitrogen are amenable to ctc recovery and characterization using the epic platform. the ficoll gradient method enriches for pbmcs, removing granulo‐ cytes and thus accounting for the decreased apparent yield of nucleated cells after processing. compared to the epic sciences standard rbc lysis, this in part may account for the decreased ctc recovery in samples processed by ficoll. however, the procedure of cryopreservation and sample thaw alone does not substantially alter cell recovery, indicating that the pre-analytic phase of nucleated cell isolation may play a large role in influencing wbc/pbmc (and presumably, ctc) recovery rates. on average, approximately one quarter of the total ctc counts were detected when the pbmcs were isolated via ficoll and frozen, as compared to the matched blood tubes, figure 5. cryopreserved ctcs from pbmc pellets are viable for genetic analysis. after if staining and protein characterization, patient ctcs recovered from cryopreservation were assessed via pten fish. ctc homozygous pten loss is defined as the complete absence of pten (10q23.31; green) signals with the presence of ≥1 centromeric cep10 (10p11.1-q11.1; red) signal(s) within the same cell. ctcs with present pten signals < cep10 signals are indicative of hemizygous pten loss. preservation of wbc and ctc morphology was observed in frozen samples after fluorescent probe hybridization as well as a consistent assessment of pten hemizygous loss in all ctcs assessed (95% & 90% pten loss in fresh and frozen material, respectively). neighboring wbcs were used as internal controls for normal genetic status and to establish proper assay specificity. 6 j circ biomark, 2015, 4:4 | doi: 10.5772/60745 which were sent immediately to epic and banked fresh per the epic sop. however, though absolute ctc recovery diminishes in the archived samples, the relative abundance of patient ctc burden, as well as the protein biomarker (ck, ar) expression of those ctcs, remains largely consistent with the matched fresh samples. in the case of the archived samples, a decreased net ctc recovery can potentially be overcome by testing a larger volume of blood. importantly, the archived ctcs demonstrate preserved morphology, genetic alterations and biomarker localization as compared to the fresh samples that were received and processed within 96 hours. in our initial feasibility study with matched mcrpc patient samples, we were able to assess ctc ck and ar protein expression, ar localization and pten status, all of which were found to be largely concordant between the fresh and frozen material. taken together, all of the ctcs across all of the frozen samples had a mean magnitude of ck and ar protein expression 90% and 107% of matched freshly processed material. correlating these metrics between the matched samples shows that the process described does not intro‐ duce a significant bias towards any given cell population. furthermore, no particular ctc subpopulation appears to be favoured by cryopreservation. these findings suggest that while precise longitudinal studies focused on ctc enumeration may not be accurate given pre-analytic variability (though relative abundance may be feasible), genetic and protein characterization of archived ctcs is possible on the epic ctc platform, enabling retrospective biomarker validation studies in large cohorts of patients with detailed clinical history. in addition to these concordance studies, ctcs were detected in an additional 16 of 20 advanced mcrpc samples that were banked for up to 7.5 years prior to analysis. notably, no correlation was seen between sample age and ctc recovery, and ctcs were found in both baseline and follow-up draws. the data presented here focus on the feasibility of studies based on archived samples and do not themselves constitute a comprehensive validation. further work is underway to relate our findings with clinical data and assess the ability to prognosticate patient outcome based on data garnered from cryopre‐ served ctc analysis. additionally, while we have ob‐ served close concordance of genetic pten alterations between fresh and frozen ctcs from the same patient, ongoing studies are working to relate ctc genetic altera‐ tions with incidence of mutations in both primary and metastatic tumour tissue. a manuscript describing close concordance of pten status between mcrpc ctcs characterized on the epic platform and solid tumour biopsy figure 6. additional cryopreserved patient samples were processed via the epic ctc platform. twenty additional cryopreserved mcrpc patient samples of up to 7.5 years of age were processed. similar to previously characterized matched samples, if staining detected ck+ ctcs, ctc clusters, ckctcs and apoptotic cells with heterogeneous ck (a) and ar (b) expression. the threshold for ck and ar positivity is 2.8and 3-fold over background signals, respectively (dotted lines). (c) out of 20 samples, 16 (80%) had detectible ctcs (range: 2-40 ctcs/3m wbcs) and 11/16 (69%) ctc+ samples included ar+ ctcs (range: 1-10 ctcs/3m wbcs). 7david lu, ryon p. graf, melissa harvey, ravi a. madan, christopher heery, jennifer marte, sharon beasley, kwong y. tsang, rachel krupa, jessica louw, justin wahl, natalee bales, mark landers, dena marrinucci, jeffrey schlom, james l. gulley and ryan dittamore: detection and characterization of circulating tumour cells from frozen peripheral blood mononuclear cells tissue is currently under review for publication. further‐ more, the feasibility of single cell picking on the epic ctc platform coupled to next generation sequencing (ngs) has been demonstrated as a proof of concept (manuscript in preparation), and in-depth ngs characterization of archived patient ctcs is underway. the ability to retrospectively analyse samples from completed prospective studies might yield additional ctc biomarkers or insights into drug response or resistance when more time-related clinical information, like pfs and os, are available. retrospective genomic analyses have been reported on “exceptional responder” patient samples to define the likely mechanism of response to targeted therapies [13, 14]. the ability to detect and characterize material from archived patient blood samples offers the potential for an additional window to augment archived solid tissue biopsies for such analyses. 5. compliance with ethical research standards all of the patients gave informed consent and enrolled on a biospecimen collection protocol, approved by the nci irb (nct00034216). 6. disclosures dl, rpg, mh, sb, rk, jl, jw, nb, ml, dm and rd are employees of epic sciences, inc., san diego, ca. all other authors declare no conflicts of interest. 7. references [1] mateo j, gerlinger m, rodrigues d and de bono j s (2014) the promise of circulating tumor cell analysis in cancer management. genome biol. 15: 448 [2] yap t a, lorente d, omlin a, olmos d 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choueiri t k, gandhi l, cleary j m, elfiky a a, taplin m e, stack e c, signoretti s, loda m, shapiro g i, sabatini d m, lander e s, gabriel s b, kantoff p w, garraway l a and rosenberg j e (2014) activating mtor mutations in a patient with an extraordinary response on a phase i trial of everolimus and pazopanib. cancer discov. 4: 546-53 [15] peng g, woodman s e and mills g b (2014) radical response puts an exceptional responder in chkmate: a synthetic lethal curative response to dna-damaging chemotherapy? cancer discov. 4: 988-90 9david lu, ryon p. graf, melissa harvey, ravi a. madan, christopher heery, jennifer marte, sharon beasley, kwong y. tsang, rachel krupa, jessica louw, justin wahl, natalee bales, mark landers, dena marrinucci, jeffrey schlom, james l. gulley and ryan dittamore: detection and characterization of circulating tumour cells from frozen peripheral blood mononuclear cells detection of human c-myc and egfr amplifications in circulating extracellular vesicles in mouse tumour models original research article leonora balaj1,2, #,*, fatemeh momen-heravi3,#, weilin chen1, sarada sivaraman1, xuan zhang1, nicole ludwig4, eckart meese4, thomas wurdinger2, david noske2, alain charest5, fred h. hochberg1, peter vandertop2, johan skog6, winston patrick kuo3,7,8 1 departments of neurology and radiology, massachusetts general hospital, and program in neuroscience, harvard medical school, boston, ma, usa 2 department of neurosurgery, amsterdam cancer center, amsterdam, netherlands 3 formerly at harvard catalyst laboratory for innovative translational technologies, harvard medical school, boston ma, usa 4 department of human genetics, medical school, saarland university, homburg-saar, germany 5 molecular oncology research institute, tufts university, boston ma, usa 6 exosome diagnostics inc., cambridge, ma, usa 7 formerly at department of developmental biology, harvard school of dental medicine, boston, ma, usa 8 ies diagnostics, cambridge, ma, usa # these authors contributed equally * corresponding author e-mail: balaj.leonora@mgh.harvard.edu received 27 may 2014; accepted 22 aug 2014 doi: 10.5772/59174 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract essentially, all cells release extracellular vesicles (evs) that end up in biofluids, including blood, and the contents of these evs can provide a window into the status of the cells from which they are released. this is particularly interesting in cancer, since these evs allow for ‘ex-vivo’ analysis of the properties of the tumours without the need for biopsy. gene mutations, rearrangements, amplifications, and epigenetic changes in the transcriptome can be monitored in circulating evs. in this study, we used two human tumour cell lines derived from an epidermoid carcinoma and a medulloblastoma, which had amplification for the epidermal growth factor receptor (egfr) and c-myc genes, respectively. cells were implanted subcutaneously into immunocompromised mice, and levels of gene amplification in both groups of subcutaneous tumours were quantified. we then determined if elevated levels of transcripts for the human egfr and c-myc were represented in circulating evs in tumour-bearing mice. the expression levels of both human egfr (h-egfr) and human c-myc (h-c-myc) mrnas in circulating evs correlated well with their amplified status in the tumours. this data provides further support to the idea that circulating evs are a potential platform for tumour biomarkers. keywords cancer, epidermoid carcinoma, extracellular vesicles, medulloblastoma, biomarkers, microvesicles, gene amplification 1leonora balaj, fatemeh momen-heravi, weilin chen, sarada sivaraman, xuan zhang, nicole ludwig, eckart meese, thomas wurdinger, david noske, alain charest,fred h. hochberg, peter vandertop, johan skog, winston patrick kuo: detection of human c-myc and egfr amplifications in circulating extracellular vesicles in mouse tumour models article j circ biomark, 2014, 3:6 | doi: 10.5772/59174 journal of circulating biomarkers 1. introduction brain tumours comprise a variety of phenotypic and genotypic subtypes. currently, genetic analysis is performed on biopsies obtained during surgical tumour resection or by surgical biopsy at a later date for suspected recurrences. as many newly developed drugs target specific pathways affected by the genetic status of the tumour [1], understanding specific transcriptome signatures that characterize them is critical for personalized clinical care. extracellular vesicles are nano-sized membrane bound particles released by essentially all cell types, including tumour cells, in vivo as well as in vitro, and comprise a mixed population of exosomes, shed microvesicles, and apoptotic blebs [2,3,4], collectively termed extracellular vesicles (evs). they are found in all body fluids, including serum, plasma, cerebrospinal fluid (csf), urine, and saliva [3], and can provide a window into the genotype and consequent phenotype of the tumour from which they are derived. the nucleic acid content of evs provides a promising platform for companion diagnostics for monitoring tumour responses and changes during treatment [5, 6]. in the case of glioblastomas (gbms), mutant oncogenic egfrviii mrna and protein have been successfully detected in brain tumour-derived evs found in the blood of gbm patients [6,7,8]. in addition, tumours are very dynamic entities and tend to change their genetic profile over time, especially after drug treatment and during recurrence. for example, lung cancer patients treated with egfr tyrosine kinase inhibitors can relapse with a tumour harbouring a resistance mutation in egfr [9]. this and other findings stress the importance of longitudinal tumour profiling to select and modify proper clinical treatment. amplification of the epidermal growth factor receptor (egfr) gene is a common event in gbms and in about 40% of cases these amplified genomic fragments appear as double minute chromosomes [10]. medulloblastoma is the most common and most malignant tumour of the central nervous system in children [11]. one histopathological feature that correlates with poor prognosis is large cell/anaplastic changes (20-25% of cases), which has been reported to be associated with amplifications in the c-myc gene, with these amplifications being highly heterogeneous in the tumour tissue [11]. an ev-based assay for gene amplification would analyse nucleic acid derived from all cells in the tumour, thus having a better chance of representing different heterogeneous tumour characteristics. in this study, the rna content of circulating evs released by human tumours in xenograft mouse models was examined. the following two cell lines were used: brain tumour medulloblastoma cell line amplified for c-myc, but not egfr [6], and a peripheral tumour epidermoid carcinoma cell line amplified for egfr, but not c-myc [12]. tumour cells were injected subcutaneously into both flanks of immunocompromised mice and tumours allowed to grow for one month. rna was extracted from serum-derived evs, as well as from the subcutaneous (s.c.) tumour tissues. genomic dna (gdna) was also extracted from the s.c. tumours. levels of human egfr, c-myc and glyceraldehyde 3-phosphate dehydrogenase (gapdh) mrnas and gdna were assessed by qrt-pcr and qpcr, respectively. amplification of egfr and cmyc were found to be well reflected in the corresponding mrna extracted from circulating evs in mice bearing epidermoid carcinomas and medulloblastoma tumours, respectively. this data supports the idea that serum evs can provide an important platform for cancer biomarkers, in this case tumour gene amplification, and can provide useful information to tailor treatment strategies. 2. materials and methods 2.1 cell lines epidermoid carcinoma cell line a431 was obtained from atcc and cultured in dulbecco’s modified essential medium (dmem; invitrogen, carlsbad, ca usa) containing 10% foetal bovine serum (fbs; jrh biosciences, lenexa, ks) and penicillin/streptomycin (10 iu/ml and 10 μg/ml, respectively; cellgro, manassas, va usa). medulloblastoma cell lines d384 were obtained from dr. scott pomeroy at children’s hospital, boston, ma and were originally generated at duke university. medulloblastoma cell lines were cultured in suspension in dmem containing 10% fbs, 1×glutamax (invitrogen) and penicillin/streptomycin (as above). 2.2 xenograft tumour models a431 cells were cultured as monolayers, and on the day of injection they were rinsed in pbs twice, resuspended using trypsin, and then diluted in fbs-free dmem and centrifuged at 300 x g for 5 min to pellet the cells. d384 cells were grown in suspension, and after being dispersed they were centrifuged for 5 min at 300 x g. the cell pellets (5.0 x 107 cells of each cell line) were resuspended in 500 μl of pbs and mixed with 500 μl matrigel (bd biosciences, san jose, ca usa). five million cells per flank were injected into each flank of all animals (total of 1.0 x 107 cells/mouse). two groups of five adult mice (nu/nu, nci) were each injected s.c. in both flanks with 100 μl/5 x 106 epidermoid carcinoma (a431; five mice) or medulloblastoma (d384; five mice) cells per injection. tumours were allowed to grow for a maximum of one month. blood was drawn by cardiac puncture while mice 2 j circ biomark, 2014, 3:6 | doi: 10.5772/59174 were under deep anaesthesia prior to sacrifice. approximately 1 ml blood was collected into eppendorf tubes and allowed to clot at room temperature for 1 hr. samples were then centrifuged at 1,300 x g for 10 min. approximately 600 μl serum from 1 ml of blood was separated, filtered through a 0.8 μm filter and stored at 80c until further processing. tumours were also resected, weighed and snap frozen in liquid nitrogen. all animal procedures were performed according to the guidelines issued by the committee of animal care of massachusetts general hospital. 2.3 isolation of evs and rna extraction from serum and tumours serum samples were thawed on ice and centrifuged at 100,000 x g for 90 min using an optima max-xp, fixed angle mla-55 rotor (k factor = 116; beckman coulter, miami, fl, usa). the evs pellets were lysed in qiazol lysis buffer and rna was extracted using the mirneasy kit (qiagen, valencia, ca usa) according to the manufacturers’ recommendations. tumour tissue was homogenized and rna was extracted using the mirneasy kit (qiagen). then, 50 ng of rna from tumour tissue or evs was used for qrt-pcr. rna yield and purity was assessed using nanodrop 2000 (thermo scientific, wilmington, de usa). 2.4 genomic dna extraction around 20 μg of tumour tissue was cut from the frozen samples of each animal (epidermoid carcinomas and medulloblastomas) and lysed overnight at room temperature using the blood and tissue dna extraction kit (qiagen, valencia ca) as suggested by the manufacturer’s recommendations. the dna was rnase treated using rnase a (fermentas inc., glen burnie, md usa; 50 mg/ml) and the quality was assessed by nanodrop 2000. then, 10 ng of genomic dna was used as input for each qpcr reaction. 2.5 reverse transcription and qpcr twelve μl of the rna from tumour tissue or evs were reverse transcribed into cdna using superscript® vilo™ cdna synthesis kit (invitrogen), according to the manufacturer’s recommendations. samples were then preamplified using the taqman® preamp master mix (applied biosystems, carlsbad, ca, usa). briefly, 12.5μl of the cdna was added to the preamp master mix together with human egfr, c-myc, and gapdh primers and pre amplified for 14 cycles, according to the manufacturer’s recommendations. primers for the preamplification step are the same as those used in the taqman pcr (see below). the samples were then diluted 1:10 and taqman qrt-pcr was performed on all samples for the corresponding cdna. the amplification was performed using abi prism 7500 with the following program: 50°c, 2 min; 95°c, 10 min; 40 cycles of 95°c, 15 s; 60°c, 1 min on standard mode. primers and probes were as follows: egfr forward: tatgtcctcattgccctcaaca; egfr reverse: ctgatgatctgcaggttttcca; fam labelled probe aaggaattcgctccactg; c-myc forward caacccttgccgcatccac ; c-myc reverse agtcgcgtccttgctcgg; fam labelled probe: agcagcgggcgggcactttgcact; gapdh applied biosystems kit, catalogue number hs03929097_g1. all assays were specific for the human genes and did not detect any mouse sequences. 2.6 nanoparticle tracking analysis (nta) serum samples were diluted 1:3000 in pbs and particles were counted using the nanosight lm-10 system (nanosight, amesbury, uk) supplemented with a fast video capture and nanoparticle tracking analysis (nta) software. particles were recorded for 30 secs per run and analysed using the following settings: gain set at 10; detection threshold set between three and seven depending on sample concentration. each sample was measured three times and the data expressed as mean ± standard deviation. 2.7 statistics statistical analyses were performed using the student’s ttest (two tailed). a value of p < 0.05 was considered significant. all data is represented as mean ± standard deviation (sd). 3. results 3.1 primary tumour gene amplification is reflected in circulating evs human epidermoid carcinoma (a431) and medulloblastoma (d384) tumour cells were injected subcutaneously into nude mice, and animals were monitored for one month. at the time of sacrifice, the mice were deeply anaesthetized and blood was collected by cardiac puncture, tumours were resected, and evs were isolated from serum by high-speed ultracentrifugation for rna qrt-pcr analysis. all data is normalized to gapdh and represented as fold change compared to the tumour with the lowest levels of the gene of interest (i.e., nonamplified for that gene and has the lowest level within the group); a431 tumours are represented as fold change compared to the d384 tumour with the lowest levels of human egfr (h-egfr); and d384 tumours are represented as fold change compared to the a431 tumour with the lowest levels of human c-myc (h-c-myc). human egfr mrna in tumour tissues from epidermoid 3leonora balaj, fatemeh momen-heravi, weilin chen, sarada sivaraman, xuan zhang, nicole ludwig, eckart meese, thomas wurdinger, david noske, alain charest,fred h. hochberg, peter vandertop, johan skog, winston patrick kuo: detection of human c-myc and egfr amplifications in circulating extracellular vesicles in mouse tumour models carcinoma (a431) xenografts was elevated between 1.2 x 105 and 3.5 x 105-fold compared to the medulloblastoma tumour d384 (not amplified for egfr), while genomic dna (gdna) for h-egfr was between 22 to 29-fold higher (table 1). as expected, medulloblastoma xenograft tumour tissues (d384) had lower levels of h-egfr mrna in the range of 1.0-24-fold, with no elevation in h-egfr gdna levels (table 1). evs isolated from the serum of mice injected with the epidermoid carcinoma cell line had elevated levels of hegfr mrna in the range of 46—1.3 x 106-fold, while rna from serum-evs of medulloblastoma (d384) tumours had on average lower amounts of h-egfr mrna, ranging from 1.0-73-fold (figure 1 and table 1). three animals injected with the medulloblastoma cell line (d384) had high levels of circulating h-egfr rna (31-73fold), although the tumour tissue itself did not show any h-egfr amplification, suggesting an increase in transcription rather than amplification (table 1). on average there was about 100,000 times more h-egfr mrna in epidermoid carcinomas tumour tissue when compared to medulloblastoma tumour tissue (p≤0.001). the difference in h-egfr mrna levels in evs from mice with epidermoid carcinoma tumours, as compared to medulloblastoma tumours, was also highly significant (p≤0.001; figure 1a). evs from the epidermoid carcinoma tumour in mouse a431–1 had the lowest levels of circulating h-egfr rna, which was more comparable to the levels of h-egfr in three of the medulloblastoma injected animals. figure 1. egfr and c-myc amplifications in the primary tumour are reflected in circulating evs. human egfr and c-myc mrna levels were quantified in circulating evs from xenograft models injected with either an epidermoid carcinoma cell line (a431; n=5) or a medulloblastoma cell line (d384; n=5), respectively. box plot graph represents levels of h-egfr (a) and h-c-myc (b) mrna in ct values (lower ct corresponds to higher levels of the message). the data is normalized to h-gapdh mrna levels. values are represented as average ± s.d. (h-egfr p≤0.001, as compared to d384 which is not amplified for egfr; n=5 per group; n.d. = not detected). tumour tissue circulating evs h-c-myc h-egfr h-c-myc h-egfr gdna ±s.d. mrna fold ±s.d. gdna ±s.d. mrna fold ±s.d. mrna fold ±s.d. mrna fold ±s.d. a431 1 1.0 ±0.8 2.2 ±0.1 28 ±1.1 257,905 ±17,203 n.d. n.d. 46 ±0.4 a431 2 0.9 ±0.1 1.0 ±0.1 22 ±1.3 122,516 ±4,069 n.d. n.d. 1,042 ±148 a431 3 1.0 ±0.2 2.5 ±0.6 26 ±1.4 219,609 ±9,660 n.d. n.d. 1,230 ±89 a431 4 1.1 ±0.1 3.7 ±0.1 24 ±1.0 354,072 ±4,831 n.d. n.d. 908 ±26 a431 5 0.8 ±0.1 3.9 ±0.1 29 ±1.3 154,010 ±6,906 n.d. n.d. 1,327,610 ±64,262 d384 1 20 ±1.2 45 ±2.9 1.2 ±0.1 1.1 ±0.1 2,910 ±356 73 ±2.4 d384 2 21 ±1.3 30 ±0.6 1.2 ±0.1 24 ±0.3 1.962 ±36 67 ±0.4 d384 3 20 ±1.1 39 ±3.9 1.1 ±0.2 1.4 ±0.1 976 ±30 31 ±0.7 d384 – 4 19 ±1.3 27 ±1.1 1.0 ±0.1 17 ±0.2 126 ±14 1.0 ±0.1 d384 5 22 ±1.1 51 ±4.0 1.2 ±0.2 1.0 ±0.2 1.5 ±0.6 2.1 ±0.1 table 1. human c-myc and egfr mrna fold change normalized to gapdh in xenograft tumour tissue and circulating evs as well as level of genomic dna (gdna) 4 j circ biomark, 2014, 3:6 | doi: 10.5772/59174 figure 2. particle counts and tumour sizes in mouse xenografts. five animals were used in each group and injected in both flanks with either epidermoid carcinomas (a; a431) or medulloblastoma (b; d384) tumour cells. after one month tumours were resected, masses recorded and snap frozen in liquid nitrogen; serum was collected and stored at -80°c for gene amplification analysis. for nanosight analysis, serum was diluted 1:3000 and particles were counted. particle counts are represented as mean ± s.d. the y-axis represents evs counts and the x-axis represents tumour masses. human c-myc mrna in tumour tissues from medulloblastoma tumours (d384) was elevated between 27-51-fold compared to the c-myc non-amplified epidermoid tumour (a431—1 set to 1), while gdna for h-c-myc was 19-22-fold higher (table 1). the levels of circulating h-c-myc at evs reflected this amplification, ranging from a 1.5-2.910-fold increase as compared to the c-myc non-amplified epidermoid carcinoma group (a431 set to ct = 37/non-detectable). human c-myc rna was not detected in evs from any of the epidermoid carcinoma xenografts (figure 1b and table 1). 3.2 xenograft tumour sizes and circulating ev (particle) counts medulloblastoma (d384) and epidermoid carcinoma (a431) tumours were resected after one month of in vivo growth. tumours were weighed and recorded as follows: a431-1 – 1.14g, a431-2 – 0.96g, a431-3 – 1.06g, a431-4 – 1.06g, a431-5 – 0.6g, d384-1 – 1.01g, d384-2 – 0.43g, d384-3 – 0.54g, d384-4 – 0.54g, d384-5 – 1.65g. epidermoid carcinoma (a431) and medulloblastoma (d384) tumour masses were on average 0.9 ± 0.2g (figure 2a) and 0.8 ± 0.5g (figure 2b), respectively. serum samples from xenografts were diluted 1:3000 with pbs and counted using nanoparticle tracking analysis (nta). pearson correlation between particle number and tumour size revealed there to be no correlation in epidermoid carcinoma tumours (a431; r2=0.2). medulloblastoma tumours (d384) showed an initial correlation (4/5 mice) but the largest tumour had a low number of circulating evs, rendering the correlation negative (r2=-0.4; figure 2b). 4. discussion tumour-derived evs provide a promising platform for cancer biomarkers. they provide a window into the genetic status of the tumour by allowing ‘sampling’ of the tumour from a distant, accessible site. once released from tumour cells, evs can end up in the bloodstream where they can be isolated and analysed for tumour markers and expression signatures. one of the major challenges of this field is the high background of evs released from normal cells that contain a ‘wild-type’ transcriptome. several approaches have been used to enrich for tumourspecific evs and the field is currently expanding to include new and more advanced techniques, such as microfluidics [13], magnetic resonance imaging signals [8], antibody specific capturing [13], and streptavidin beads [14]. currently, a widely used technique for ev isolation is ultracentrifugation, which sediments particles based on their size so that both normal and tumour cellderived evs are collected, although variations in recovery vary with fluid viscosities [15]. we have previously reported that human c-myc rna is found in circulating evs from xenograft models injected with a medulloblastoma cell line [6]. in this study, we show that levels of gene amplification of c-myc and egfr in human tumour lines are reflected in circulating evs. xenograft mouse models were used and were injected with human cancer cell lines amplified for a specific oncogene, either egfr (a431) or c-myc (d384). mice were then sacrificed and tumours removed and serum collected for rna analysis of evs. evs were isolated by filtration and differential centrifugation, and rna was analysed for quantity of human egfr, c-myc and gapdh mrnas. epidermoid carcinoma egfr amplification was wellrepresented in circulating evs from a431 mouse models. overall, ct values for h-egfr mrna in circulating evs from mice bearing epidermoid carcinomas (a431) amplified at the genomic level for this oncogene were lower (corresponding to higher amounts; p≤0.001), than h-egfr levels from medulloblastoma (d384)-derived evs not amplified for this oncogene (figure 1a). human 5leonora balaj, fatemeh momen-heravi, weilin chen, sarada sivaraman, xuan zhang, nicole ludwig, eckart meese, thomas wurdinger, david noske, alain charest,fred h. hochberg, peter vandertop, johan skog, winston patrick kuo: detection of human c-myc and egfr amplifications in circulating extracellular vesicles in mouse tumour models egfr gene amplification levels in the primary tumours were in concordance with what has been previously reported for cultured a431 cells [16]. human egfr mrna levels in circulating evs varied greatly among different tumour-bearing mice with one of them having 1.6 x 106-fold more egfr message (compared to gapdh message). this mouse did not have the highest levels of egfr mrna in the primary tumour or the highest number of circulating evs (figure 2a). in fact, this mouse had the smallest tumour size (0.6 g) in that group, but it appeared to be the most malignant tumour, as the mouse was cachexic (data not shown). this difference in the levels of egfr mrna in the primary tumour and circulating evs may be due to increased transcription rates of mrna and the subsequent more aggressive behaviour of the tumour. in addition, different copies of mrna could exist at different locations, stabilities, and translations, and thus this kind of variation might be expected. high vascularization and invasion of the tumour may be another reason for such high malignancy, which in turn results in a lower body mass. furthermore, only a small portion of circulating evs are thought to be of tumour origin (5-10%), and this may indicate that these evs exerted their immune-suppressive properties on the host immune cells, thus decreasing the total number of circulating normal evs. this inhibition may have led to enrichment of the tumour-derived evs and thus explains the high levels of h-egfr mrna in evs detected by qpcr. another epidermoid carcinoma in one mouse (a431-1) had very low levels of h-egfr mrna in circulating evs (table 1). the size of the tumour was similar to those in a431-2, a431-3 and a431-4 mice, but the number of vesicles was around 70% less compared to these three animals, which may account for the lower level of h-egfr mrna in circulating evs (figure 2). human c-myc mrna was detected in circulating evs from all five xenografts injected with the medulloblastoma cell line (d384) and was not detected in circulating evs isolated from mice injected with the epidermoid carcinoma (a431) cell line. worth noting is that human c-myc mrna was not as high in the medulloblastoma tumour as egfr mrna was in the epidermoid carcinoma tumour (average mrna increase was 29 vs. 1.1 x 105, respectively). the data suggests that, most likely, at least an 18-fold amplification for h-c-myc in the tumour is required for detection in circulating evs, while h-egfr can be detected even when not amplified (data normalized to gapdh and expressed as fold difference compared to h-egfr non-amplified tumour [17]). one reason for this could be the difference in the half-life for these mrna species, which is relatively short for c-myc and therefore may be degraded before packaging inside evs [18], or different localization of h-cmyc and h-egfr mrnas inside the cells. according to the proposed kinetic model of rna, genes encoding mrnas with short half-lives are more likely to respond quickly to transcriptional activation or suppression [19]. thus, the intrinsic stability of the mrna would be expected to affect the rate of induction as well as the suppression of mrna levels, because transcripts with short half-lives can respond more rapidly than stable transcripts to changes in transcription rates. the difference in h-c-myc and h-egfr rna detection in evs may also suggest specific packaging mechanisms for human egfr mrna into evs, possibly exacerbated by the fact that this is occurring in a mouse background— especially because in 4/5 mice, h-c-myc mrna levels in evs were higher than in the tumour itself. there seems to be a slight increase in ev numbers as tumour size increases, but this is not reflected in all animals. this may be due to the small sample size as well as the malignant nature of the tumour; in fact, epidermoid carcinomas (a431) cannot grow very large because the mouse quickly becomes heavily sick (data not shown), which may not allow enough time to detect an increase in circulating evs. medulloblastoma tumours, on the other hand, showed an initial increase in circulating ev number, but the animal with the largest tumour had a low circulating evs count (figure 2b), suggesting a higher malignancy, which may have inhibited the number of normal hostderived circulating evs. another variability might be the degree of vascularization of the tumour, which could lead to a different number of circulating evs. within the limitations of an animal study, this study confirms the notion that evs provide a potential platform for tumour biomarkers. genetic mutations, rearrangements, and amplifications are reflected in rna in evs released by the tumour cells [20]. these evs can be isolated from biofluids and provide a window into studying the primary tumour, either at diagnosis or later on for treatment response or follow-up studies. 5. acknowledgements this work was supported by nih/nci grants ca069246 (f.h..), ca141226 (x.o.b.); ca156009 (x.o.b.) and ca141150 (x.o.b.); brain tumour funders' collaborative (x.o.b.); american brain tumour association (abta; j.s.); l.b. is supported by mgh ecor fmd fellowship and richard floor biorepository fund. this work was conducted, at least in part, through the harvard catalyst laboratory for innovative translational technologies (hclitt) with support from harvard catalyst—the harvard clinical and translational science center (nih award #ul1 rr 025758 and financial contributions from harvard university and its affiliated academic health care centres). the content is solely the responsibility of the authors and does not necessarily represent the official views of harvard catalyst, harvard university and its affiliated academic health care centres, the national center for research resources, or the national institutes of health. 6 j circ biomark, 2014, 3:6 | doi: 10.5772/59174 this work was performed in compliance with ethical research standards 6. conflict of interest johan skog is an employee of exosome diagnostics. thomas wurdinger is an employee of thrombodx. all other authors declare no conflicts of interest. 7. references [1] roychowdhury s, iyer mk, robinson dr, lonigro rj, wu ym, cao x, kalyana-sundaram s, sam l, balbin oa, quist mj, et al: personalized oncology through integrative high-throughput sequencing: a pilot study. sci transl med 2011, 3: 111–21. 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[19] keene, jd: the global dynamics of rna stability orchestrates responses to cellular activation. bmc biol 2010, 8: 95. [20] al-nedawi, k, meehan, b, rak, j: microvesicles: messengers and mediators of tumor progression. cell cycle 2009, 8: 2014–18. 7leonora balaj, fatemeh momen-heravi, weilin chen, sarada sivaraman, xuan zhang, nicole ludwig, eckart meese, thomas wurdinger, david noske, alain charest,fred h. hochberg, peter vandertop, johan skog, winston patrick kuo: detection of human c-myc and egfr amplifications in circulating extracellular vesicles in mouse tumour models article journal of circulating biomarkers influence of pre-analytical factors on thymusand activation-regulated chemokine quantitation in plasma original research article xuemei zhao1*, liliana delgado1, russell weiner1,2 and omar f. laterza1 1 translational molecular biomarkers, merck research laboratories, rahway, nj, usa 2 translational sciences, biomarkers & companion diagnostics, daiichi sankyo, inc., edison, nj, usa *corresponding author(s) e-mail: xuemei_zhao@merck.com received 09 april 2015; accepted 01 october 2015 doi: 10.5772/61749 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract thymusand activation-regulated chemokine (tarc) in serum/plasma associates with the disease activity of atopic dermatitis (ad), and is a promising tool for assessing the response to the treatment of the disease. tarc also exists within platelets, with elevated levels detectable in ad patients. we examined the effects of pre-analytical factors on the quantitation of tarc in human edta plasma. tarc levels in platelet-free plasma were significantly lower than those in platelet-containing plasma. after freeze-thaw, tarc levels increased in platelet-containing plasma, but remained unchanged in platelet-free plasma, suggesting tarc was released from the platelets during the freezethaw process. in contrast, tarc levels were stable in serum independent of freeze-thaw. these findings underscore the importance of pre-analytical factors to tarc quantitation. plasma tarc levels should be measured in platelet-free plasma for accurate quantitation. pre-analytical factors influence the quantitation, interpretation, and implementa‐ tion of circulating tarc as a biomarker for the develop‐ ment of ad therapeutics. keywords pharmacodynamic biomarker, pre-analytical factors, platelet-free plasma, platelet-containing plasma, freeze-thaw, thymus, activation-regulated chemokine, atopic dermatitis 1. introduction the hallmark of inflammation is the infiltration of specific leukocyte subsets from the blood into the affected tissue [1]. chemokines, which are small (8–10 kda) secreted proteins and their receptors expressed on the cell surface, orches‐ trate the guided migration of leukocytes to inflammatory sites. thymusand activation-regulated chemokine (tarc/ccl17) is a high-affinity ligand for cc chemokine receptor 4 (ccr4), as well as being a th2 type, pro-allergic secreted chemokine. tarc serves in the recruitment and migration of ccr4-expressing cells, such as th2 cells, cutaneous leukocyte-associated antigen (cla)+ skinhoming t cells, and cd25+ t suppressor cells [2]. it is closely related to the pathogenesis of allergic diseases with the accumulation of th2 lymphocytes in the inflammatory regions. in inflamed skin lesions, tarc acts on ccr4 at the endothelial surface to promote vascular recognition for subsequent cellular localization [3]. tarc is constitutively expressed in the thymus [4], and is also present in mono‐ cyte-derived dendritic cells [5–7], endothelial cells [8], and epidermal keratinocytes [9]. circulating levels of tarc have been associated with allergic skin diseases such as atopic dermatitis (ad), contact dermatitis, and psoriasis, in addition to asthma [10] 1j circ biomark, 2015, 4:10 | doi: 10.5772/61749 and epilepsy [11], but not allergic respiratory diseases such as allergic rhinitis and allergic asthma [12–14]. elevated levels of tarc have been reported in the serum of patients with ad, compared to that of healthy control subjects [12– 16]. similar observations have also been reported in the plasma of ad patients, compared to that of healthy control subjects [15, 16]. moreover, the plasma or serum levels of tarc from ad patients correlated significantly with the severity of the disease, as classified by the objective scorad score [17, 18]. thus, plasma/serum tarc is a disease activity marker for ad. furthermore, plasma or serum levels of tarc from ad patients decreased after treatment, in accordance with the improvement of clinical symptoms [12–15, 19]. therefore, plasma/serum tarc can also be utilized as a pharmacodynamic (pd) marker to assess disease pathway engagement in the clinical devel‐ opment of ad therapeutics. in blood, tarc also exists within platelets. platelets from ad patients contain higher levels of tarc than those found in healthy control subjects [15]. consistent with the finding of the presence of tarc in platelets, serum tarc levels were much higher than those in plasma, owing to the release of tarc from the platelets during coagulation in serum preparation [15, 16]. platelets may represent the predominant source of tarc in circulation, and platelet rupture, aggregation, or activation may affect levels of noncell-associated tarc in blood. thus, the alteration of platelets post-phlebotomy would artificially increase the levels of non-cell-associated tarc in blood. although preanalytical conditions may influence circulating tarc levels significantly, they have not been investigated thoroughly for the measurement of this marker. in this article, we describe the effects of pre-analytical factors on tarc quantitation in human edta plasma, including the method for edta plasma preparation and plasma sample handling prior to sample analysis. our observations underscore the importance of the influence of pre-analytical factors in clinical biomarker quantitation, interpretation, and implementation in drug development. 2. material and methods 2.1 ethical conduct of research the authors state that they have obtained appropriate institutional review board approval, or have followed the principles outlined in the declaration of helsinki, for all human or animal experimental investigations. in addition, for investigations involving human subjects, informed consent has been obtained from the participants involved. 2.2 reagents recombinant human tarc protein was purchased from r&d systems, inc. (minneapolis, mn, usa; catalogue number: 364-dn). the human tarc ultra-sensitive kit was purchased from meso scale discovery (msd) (rock‐ ville, usa; catalogue number: k151bgc-2). the kit was supplied with cytokine panel 2 ultra-sensitive plate (catalogue number: n45016a-1); human tarc detection antibody (catalogue number: d21bg-3); cytokine panel 2 calibrator blend (catalogue number: c0016-2); diluent 2 (catalogue number: r51bb-3); diluent 3 (catalogue number: r51ba-5); and msd read buffer t (4x) (cata‐ logue number: r92tc-3). dpbs (dulbecco’s phosphatebuffered saline) (1x) was purchased from mediatech, inc. (manassas, va, usa; catalogue number: 21-031-cv). surfact-amps 20 (10% tween 20) was purchased from thermo fisher scientific (rockford, il, usa; catalogue number: cas 9005-64-5). blocker casein in pbs (1% (w/v) casein) was also purchased from thermo fisher scientific (catalogue number: 37528). hbr1 (heterophilic blocking reagent 1) was purchased from scantibodies laboratory, inc. (santee, ca, usa; catalogue number: 3kc534-075). stabilzyme select stabilizer was purchased from surmodics, inc. (eden prairie, mn, usa; catalogue number: sz03-1000). distilled water was purchased from life technologies (grand island, ny, usa; catalogue number: 15230-147). edta plasma samples of healthy control subjects were purchased from bioreclamation, inc. (hicksville, ny, usa). 2.3 sample collection human plasma and serum samples from apparently healthy subjects were obtained with informed consent. for plasma preparation, whole blood was drawn into lavender top vacutainer tubes (6 ml, k2edta (spray dried), bd #367863). the blood tubes were mixed by gentle inversion 10x, incubated at room temperature (rt) for 60 min, and subsequently centrifuged at 1500 g for 15 min at rt, or prepared as described in figure 3. edta plasma samples were collected, aliquoted, and stored at -80°c. for serum preparation, whole blood was drawn into red top vacutain‐ er tubes (6 ml, clot-activatorand silicone-coated interior, bd #367815). the blood tubes were incubated at rt for 30 min, and then centrifuged at 1500 g for 15 min at rt. serum samples were collected, aliquoted, and stored at -80°c. 2.4 freeze-thaw treatment of plasma and serum samples a freeze-thaw treatment of the plasma or serum samples was executed using the following procedure. samples (100 μl per aliquot) were stored at -80°c for at least 4 h. they were then removed from the -80°c freezer and thawed at rt for 2 h. after one complete freeze-thaw cycle, samples were either analysed in the tarc assay or stored at -80°c. 2.5 preparation of standards and qc samples recombinant human tarc protein (one vial, 25 μg) was reconstituted in 2.5 ml of stabilzyme select stabilizer to a final concentration of 10 μg/ml. then 200 μl of 10 μg/ml tarc was mixed with 199.8 ml of casein-t (1% casein 2 j circ biomark, 2015, 4:10 | doi: 10.5772/61749 and 0.05% tween 20 in pbs) to obtain the tarc standard stock at 10, 000 pg/ml. the tarc standard stock was aliquoted and stored at -80°c. prior to each analysis, standard samples were prepared by serial dilution of the tarc standard stock with casein-t, resulting in a ninepoint standard curve with a range of 1.5–10, 000 pg/ml. four sets of quality control (qc) samples (extra high, high, medium, and low) were prepared by spiking recombinant human tarc protein into a human plasma sample purchased from bioreclamation, inc. the qc samples were aliquoted and stored at -80°c. 2.6 tarc ecl assay procedure and sample analysis all samples (plasma, serum, standards, qc, and blank) were analysed manually in duplicate on an msd-kitsupplied 96-well plate. all reagents were brought to room temperature prior to analysis. plasma/serum samples and qc samples were diluted 1:4 in a [casein-t + hbr] buffer, containing 0.4 mg/ml hbr1 in casein-t (1.2 μg of hbr1 per μl of plasma or serum). a plate with 150 μl casein per well was incubated for 1.5 h with vigorous shaking (1000 rpm) at rt. the plate was then washed three times with 200 μl per well of pbs-t (0.05% tween 20 in pbs). after the addition of 50 μl casein-t per well, and 50 μl of standards, diluted samples, or qc samples, the plate was incubated at rt for 2 h with vigorous shaking (1000 rpm). upon completion of the sample incubation, the plate was washed with pbs-t buffer three times, and 25 μl of human tarc detection antibody were added to each well. the plate was incubated for 2 h with vigorous shaking (1000 rpm) at rt. the plate was again washed with pbs-t buffer three times. after the addition of 150 μl of 2x read buffer t (1:2 dilution of 4x read buffer t in distilled water) to each well, the plate was read on the msd sector imager 6000. 2.7 software and statistical analysis the msd discovery workbench® analysis software was used for data acquisition and data analysis. the standard curve was modelled using a least-squares fitting algorithm, meaning that signals from standards with known levels of tarc could be used to calculate the concentration of tarc in the sample. the software utilized a four-parameter logistic model and included a 1/y2 weighting function to determine the mean, standard deviation (stdev), and % coefficient of variance (%cv). back-calculated concentrations and % difference of the back-calculated concentrations were calculated using microsoft excel. 3. results and discussion 3.1 differential effects of freeze-thaw on tarc quantitation in human edta plasma and serum during the development of an ultra-sensitive electroche‐ miluminescence (ecl) assay for tarc quantitation in human edta plasma, we observed that increased levels of tarc were detected after repeated freeze-thaw cycles [20]. we decided to further examine the effects of freeze-thaw on tarc quantitation in edta plasma, and in serum as a comparison, including freshly prepared plasma and serum samples without any freeze-thaw. edta plasma and subject-matched serum samples were prepared simultane‐ ously from six apparently healthy subjects following standard procedures, with low-speed centrifugation at 1500 g to separate blood cells from the plasma or serum. aliquots of the freshly prepared plasma or serum samples were either analysed in the tarc assay immediately after sample preparation, or frozen and stored at -80°c. after the freeze-thaw treatment, the fresh frozen samples and fresh frozen samples following additional freeze-thaw cycles were also analysed in the tarc assay. a fresh frozen plasma/serum sample was defined as a plasma/serum sample that was frozen immediately after sample prepara‐ tion. therefore, every fresh frozen plasma/serum sample had already gone through one freeze-thaw cycle prior to the sample analysis for tarc measurement. figure 1 illustrates the effects of freeze-thaw cycles on tarc quantitation in plasma samples. in general, the tarc levels in the fresh frozen plasma that had gone through one freeze-thaw cycle were higher than those in the fresh plasma without freeze-thaw. exceptions were observed in certain subject samples, such as k362 and p287. in addition, the difference in tarc levels between the fresh and the fresh frozen plasma was particularly large in some subjects, such as subject a376. consistent with our previ‐ ous observations, compared to the fresh frozen plasma, tarc levels increased in plasma that had gone through one additional freeze-thaw cycle for all subjects. further‐ more, increased levels of tarc were detected in plasma with repeated freeze-thaw cycles, up to at least three additional cycles. in contrast to the observations in plasma, in subjectmatched serum, tarc levels remained unchanged between the fresh and the fresh frozen samples for all subjects, even after three additional freeze-thaw cycles (figure 2). the blood draws for serum and subject-matched edta plasma were performed during the same visit, and the serum and edta plasma samples were prepared at the same time. consistent with previous observations, tarc levels in serum were much higher than those in plasma, probably owing to the release of tarc from platelets during coagulation (30 min whole blood incubation at rt) in serum preparation (compare figure 1 and figure 2) [15, 16]. in addition, the comparable levels of tarc detected in fresh serum and serum that had gone through multiple freeze-thaw cycles suggested that almost all tarc in the platelets was released into serum during coagulation; therefore, tarc levels in fresh serum were much higher than those in fresh plasma. the large ratio of tarc levels in serum versus plasma — 4–12 fold in six subjects — suggested that platelets are the predominant source of circulating tarc. 3xuemei zhao, liliana delgado, russell weiner and omar f. laterza: influence of pre-analytical factors on thymusand activation-regulated chemokine quantitation in plasma 3.2 tarc levels in platelet-free, but not platelet-containing, edta plasma were independent of freeze-thaw we were intrigued by the differential effects of freeze-thaw on tarc quantitation in matched edta plasma and serum samples from six individual donors (figure 1 and figure 2). the fact that freeze-thaw induced increased tarc levels in plasma, but not in serum, led us to hy‐ pothesize that plasma samples may have contained residual platelets, and that these platelets released tarc during freeze-thaw and contributed to the increase of tarc in plasma. the edta plasma samples used in these experiments were prepared following the guidelines of a standard plasma preparation method (national cancer institute, the early detection research network (edrn) standard operating procedure (sop) for collection of edta plasma, http://edrn.nci.nih.gov/resources/standardoperating-procedures/standard-operating-procedures/ biological-specimens), with low-speed centrifugation of whole blood at 1500 g for 15 min. these plasma samples most likely contained residual platelet cells from the whole blood. we then investigated the effect of the whole blood centrifugation speed on tarc levels in plasma. in partic‐ ular, we examined tarc levels in platelet-containing and platelet-free edta plasma samples. varo et al. performed a thorough analysis of the prepara‐ tion of platelet-rich and platelet-free plasma, by increasing the speed of whole blood centrifugation (which gradually depletes platelets and leucocytes from plasma), and discovered that at least 2000 g centrifugal force is required to produce platelet-free plasma samples [21]. thus, plasma samples prepared through centrifugation at 1500 g most likely contain residual platelet cells. in addition, ivandic et al. [22] and halldorsdottir et al. [23] have described two platelet-free plasma preparation methods, involving whole blood centrifugation greater than 2000 g. centrifugation at high speed, as described in these two methods, did not shear platelet cells. here, we prepared edta plasma using three different methods (figure 3): the standard low-speed centrifugation method (1500 g), which produces plateletcontaining edta plasma (method a), as well as two additional methods described in the literature, which allow the preparation of platelet-free edta plasma (methods b and c) [22, 23]. the major difference between these three methods is centrifugation speed, which determines whether platelet cells in whole blood are completely separated from edta plasma or not. additional variables in edta plasma preparation (such as the use of glass versus plastic collection tubes) could potentially impact the success of an assay [23, 24]. nevertheless, the scope of this 0 25 50 75 100 fresh fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t ta r c ( pg /m l) p287m003k362k224k005a376 fresh fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t ta r c ( pg /m l) p287m003k362k224k005a376 0 50 100 150 200 250 300 350 figure 1. effects of freeze-thaw (f-t) cycles on tarc levels in plasma. tarc levels were quantitated in edta plasma samples from six subjects, prepared by low-speed centrifugation (1500 g): fresh plasma, fresh frozen plasma, and fresh frozen plasma samples following one, two, or three additional f-t cycles. 0 25 50 75 100 fresh fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t ta r c ( pg /m l) p287m003k362k224k005a376 fresh fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t ta r c ( pg /m l) p287m003k362k224k005a376 0 50 100 150 200 250 300 350 figure 2. effects of freeze-thaw (f-t) cycles on tarc levels in serum. tarc levels were quantitated in serum samples from six subjects, prepared by lowspeed centrifugation (1500 g): fresh serum, fresh frozen serum, and fresh frozen serum samples following one, two, or three additional f-t cycles. the blood draws for the serum and the edta plasma (figure 1) were performed during the same visit. 4 j circ biomark, 2015, 4:10 | doi: 10.5772/61749 http://edrn.nci.nih.gov/resources/standard-operating-procedures/standard-operating-procedures/biological-specimens http://edrn.nci.nih.gov/resources/standard-operating-procedures/standard-operating-procedures/biological-specimens http://edrn.nci.nih.gov/resources/standard-operating-procedures/standard-operating-procedures/biological-specimens study is to compare the effects of platelet-containing and platelet-free edta plasma in tarc quantitation. invert 10 times to mix. incubate at rt for 60 min. 1500 g for 15 min at 4ºc (plasma_1500 g) 2500 g for 15 min at 4ºc (plasma_2500 g) 2790 g for 5 min at rt (plasma_2790 g) 16,000 g for 3 min at rt (plasma_2790 g_16,000 g) a b c figure 3. preparation of platelet-containing and platelet-free edta plasma samples. venous blood was collected into three purple/lavender top edta vacutainer tubes. after the blood tubes were inverted 10 times to mix thoroughly, and incubated at room temperature (rt) for 60 min, edta plasma samples were prepared following three methods: (a) centrifugation of the blood tube at 1500 g for 15 min at 4°c, to generate platelet-containing edta plasma; (b) centrifugation of the blood tube at 2500 g for 15 min at 4°c, to generate platelet-free edta plasma [22]; and (c) centrifugation of the blood tube at 2790 g for 5 min at rt to separate cells from the plasma, then re-centrifugation of the plasma at 16, 000 g for 3 min at rt to remove any residual platelets, for platelet-free edta plasma [23]. from each of the six apparently healthy subjects, we collected three tubes of venous blood and prepared three different types of edta plasma samples, following the methods illustrated in figure 3: (a) plasma_1500 g, (b) plasma_2500 g, and (c) plasma_2700 g_16, 000 g. the fresh frozen plasma samples and the fresh frozen plasma samples following additional freeze-thaw cycles were analysed in the tarc assay. three subjects — a376, k362, and m003 — participated in this study in addition to the study of freeze-thaw effects on tarc levels in plasma and matched serum samples described above. however, the blood draws for these two studies were five weeks apart. thus, the plasma tarc levels of the same subject in these two studies cannot be compared directly, owing to intrasubject biological variability. figure 4 illustrates the differential effects of three different edta plasma preparation methods on tarc levels from six apparently healthy subjects. for each plasma prepara‐ tion method, tarc levels in the fresh frozen plasma and the plasma samples following three additional freeze-thaw cycles were quantified. again, in the platelet-containing edta plasma samples (plasma_1500 g), compared to the fresh frozen plasma, elevated levels of tarc were ob‐ served in the plasma following repeated freeze-thaw cycles, for all six subjects. in contrast, tarc levels re‐ mained unchanged (<20% difference) even after at least three additional freeze-thaw cycles, in platelet-free edta plasma prepared following method c (plasma_2700 g_16, 000 g). after one additional freeze-thaw cycle, the differ‐ ence between tarc levels in method a (plasma_1500 g) and method c (plasma_2700 g_16, 000 g) was significant between the six subjects, with a p value of 0.008 determined by a paired t test. furthermore, this significant difference was maintained with an additional second and third freezethaw cycle, with p values of 0.009 and 0.011, respectively. in the platelet-free edta plasma prepared following method b (plasma_2500 g), tarc levels increased after one additional freeze-thaw cycle. these observations suggest that there was sufficient platelet contamination in the edta plasma prepared by low-speed centrifugation, and that these platelets released tarc during freeze-thaw, leading to elevated tarc levels in the plasma after repeated freeze-thaw cycles. in addition, it is possible that platelet activation could also contributed to the increased tarc levels in the edta plasma prepared by low-speed centrifugation although it is unlikely. therefore, it was platelet contamination (and perhaps activation) that caused the tarc accumulation in the stored frozen plasma. furthermore, tarc levels were consistently lower in samples that underwent high-speed centrifugation in all six subjects (figure 4: compare the tarc levels in plasma prepared by method a and method c). we hypothesize that this was owing to the elimination of residual platelets achieved with the 16, 000 g centrifugal force. we recognize that it is also possible that a higher centrifugal force may minimize the activation of platelets, and thus the release of tarc. similar findings have previously been reported; for instance, morita et al. reported that, in edta plasma samples prepared by whole blood centrifugation at 2000 g, tarc levels were not altered after at least five freeze-thaw cycles [16], suggesting that there was no or minimal platelet contamination in these plasma samples. data from the freeze-thaw study, however, was not shown in the report [16], making it difficult to perform a careful comparison with the results that we observed (figure 4). fujisawa et al. reported tarc levels in edta plasma samples prepared by whole blood centrifugation at 1800 g [15], but unfortu‐ nately the effect of freeze-thaw cycles on the tarc levels in these plasma samples was not examined. the use of tarc in clinical studies requires reliable sample preparation and analytical measurement. our present results underscore the importance of the careful removal of platelets to determine free tarc in blood and to avoid interference with the ligand derived from the cell-associat‐ ed pools. plasma prepared in this manner can undergo at least three freeze-thaw cycles without perturbing the analysis. it is very important to avoid platelet contamina‐ tion in plasma preparation because variabilities in platelet abundance, tarc expression in platelet, level of residual platelets in plasma, and degree of ruptured platelets in plasma after freeze-thaw can lead to incorrect interpreta‐ 5xuemei zhao, liliana delgado, russell weiner and omar f. laterza: influence of pre-analytical factors on thymusand activation-regulated chemokine quantitation in plasma tion of tarc in plasma, the biomarker data in a clinical study. the assessment of citrated plasma or plasma collected in ctad (citrate, theophylline, adenosine, and dipyridamole) tubes for tarc quantitation is warranted. both of these tube types are known to help prevent ex-vivo platelet activation [22]. platelets produce a number of proinflammatory mediators during activation; as with tarc, circulating levels of these mediators are complicated in platelet-containing plasma. these mediators include vascular endothelial growth factor (vgef), which is a platelet-derived growth factor involved in both angiogen‐ esis and pulmonary capillary permeability [25]; platelet factor 4 (pf4), which is a cytokine stored in platelets and released during activation [25]; and soluble cd40 ligand (scd40l), which is a trans-membrane protein released from the platelet in its soluble form during platelet activa‐ tion, and is involved in inflammation and thrombosis [21– 23, 25]. thus, investigators should be cautious of the choice of specimen and specimen-processing method when measuring circulating biomarkers that also exists in platelets. 4. conclusions in this article, we have presented the influence of preanalytical factors in the quantitation of circulating tarc levels. owing to its presence in platelets, the circulating levels of this pro-inflammatory chemokine were dramati‐ cally altered in platelet-containing edta plasma samples and were further complicated after repeated freeze-thaw cycles. therefore, the choice of specimen type and speci‐ men-handling procedures should be carefully considered for the measurement of circulating non-cell-associated tarc. when using tarc as a biomarker to aid the clinical development of atopic dermatitis therapeutics, the opti‐ mum strategy would be to measure tarc in platelet-free plasma or serum. 6. conflict of interest x. zhao and o. f. laterza are employees of merck sharp & dohme. l. delgado and r. weiner were employees of merck sharp & dohme during the course of this work. x. fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t fresh frozen 1 additional f-t 2 additional f-t 3 additional f-t 40 30 20 10 0 t r a c ( pg /m l) method a method b subject k362 method c 70 60 50 40 30 20 10 0 t r a c ( pg /m l) method a method b subject a376 method c 70 60 50 40 30 20 10 0 t r a c ( pg /m l) method a method b subject a376 method c method a method b method c method a method b method c t r a c ( pg /m l) 20 10 0 t r a c ( pg /m l) subject k777 30 20 10 0 t r a c ( pg /m l) method a method b subject l005 method c t r a c ( pg /m l) 90 10 20 30 40 50 60 70 80 0 t r a c ( pg /m l) subject m001 50 30 40 20 10 0 t r a c ( pg /m l) method a method b subject m003 method c figure 4. differential effects of three different edta plasma preparation methods on tarc quantitation in plasma. plasma samples were prepared by method a, b, or c, as illustrated in figure 3, from six apparently healthy subjects: a376, k362, k777, l005, m001, and m003. the tarc levels in the fresh frozen plasma and in the plasma samples that went through an additional one, two, or three freeze-thaw cycles were quantified. 6 j circ biomark, 2015, 4:10 | doi: 10.5772/61749 zhao and o. f. laterza own company stock/stock options. the authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. no writing assistance was utilized in the production of this manuscript. 7. acknowledgements we thank derek l. chappell for suggestions during the course of this work, thank leonidas n. carayannopoulos for comments on the manuscript. 8. references [1] ben-baruch a, michiel df, oppenheim jj. signals and receptors involved in recruitment of inflamma‐ tory cells. j biol chem. 1995 may 19;270:11703–6. 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[25] bercovitz rs, kelher mr, khan sy, land kj, berry th, silliman cc. the pro-inflammatory effects of platelet contamination in plasma and mitigation strategies for avoidance. vox sang. 2012 may; 102:345–53. 8 j circ biomark, 2015, 4:10 | doi: 10.5772/61749 exosomes and microvesicles rab27b-mediated metabolic reprogramming induces secretome acidification and chemoresistance in breast cancer cells original research article an hendrix1,2,*, carla ciccone3, christian gespach4, marc bracke1, olivier de wever1 and wendy westbroek3 1 laboratory of experimental cancer research, department of radiation oncology and experimental cancer research, ghent university hospital, belgium 2 department of medical oncology, ghent university hospital, belgium 3 medical genetics branch, national human genome research institute, usa 4 inserm u673 molecular and clinical oncology of solid tumors and inserm u938, université pierre et marie curie-paris, faculté de médecine, france * corresponding author e-mail: an.hendrix@ugent.be   accepted 18 mar 2013 © 2013 hendrix et al.; licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract  the  secretory  rab27b  small  gtpase  promotes  invasive  growth,  tumourigenicity  and  metastasis  in  oestrogen  receptor  (er)‐positive  human  breast  cancer  cells. coherently,  increased rab27b expression  in breast  cancer patients is associated with a poor prognosis. in the  present  study,  bio‐energetic  profiling  revealed  that  oxidative phosphorylation is significantly reduced in er‐ positive  breast  cancer  cells  engineered  to  overexpress  rab27b  levels  as  observed  in  invasive  clinical  primary  breast cancer. rab27b‐induced metabolic reprogramming  to aerobic glycolysis was further evidenced by increased  extracellular  acidification  followed  by  cathepsin  b  activation and doxorubicin resistance. transient silencing  of rab27b and stable transfection of rab27a, and rab27b  mutants in er‐positive breast cancer cells confirmed that  this response was rab27b‐specific and dependent upon  rab27b‐gtp activation and vesicle membrane attachment   through  the  c‐terminal  geranylgeranyl  group  of  this  small gtpase. rab27b‐driven extracellular acidification is  required  and  is  sufficient  to  induce  filopodia‐like  morphological changes, primarily involved in the process  of  cancer  cell  invasion.  our  data  demonstrate  that  a  rab27b‐dependent switch from oxidative phosphorylation  towards  aerobic  glycolysis  in  er‐positive  breast  cancer  cells  is  accompanied  by  acidification  of  the  tumour  environment.    keywords rab gtpase, aerobic glycolysis, filopodia    1. introduction  metastatic  cancer  is  strongly  dependent  on  an  adapted  metabolism  to  fulfil  the  bioenergetic  requirements  for  uncontrolled invasive growth [1]. proliferating cells are in  1an hendrix, carla ciccone, christian gespach, marc bracke, olivier de wever and wendy westbroek: rab27b-mediated metabolic reprogramming induces secretome acidification and chemoresistance in breast cancer cells www.intechopen.com article www.intechopen.com exosomes microvesicles, vol. 1, 3:2013   much  greater  need  of  reduced  carbon  and  nitrogen,  as  well  as  cytosolic  nadph  for  reductive  biosynthetic  reactions [2]. this metabolic phenotype is characterized by  a shift from oxidative phosphorylation (oxphos) towards  aerobic  glycolysis  supplying  nucleotides,  proteins  and  lipids  for  intense  and  constitutive  macromolecular  synthesis.  glycolysis  first  requires  the  conversion  of  glucose to pyruvate and eventually to the waste product  lactic acid. glycolysis is inhibited by oxygen consumption,  which allows mitochondria to oxidize pyruvate to co2 and  h2o. conversion of glucose to lactic acid in the presence of  oxygen,  but  without  its  consumption,  is  known  as  the  warburg  effect.  aerobic  glycolysis  is  detected  clinically  with  positron  emission  tomography  to  measure  the  increased  uptake  of  the  glucose  analogue  tracer  18fluorodeoxyglucose in many cancers [3].    altered metabolism in cancer cells is characterized by the  production  and  excretion  of  lactate,  resulting  in  intratumoural acidification [4]. typically, the extracellular  ph  (phe)  in  tumours  is  heterogeneous  with  a  range  of  6.44<phe<6.79  [5].  low  phe  promotes  excretion  of  numerous proteolytic enzymes that are involved in tissue  remodelling.  cathepsins  are  well  described  as  ph‐ regulated membrane remodelling enzymes with increased  secretion  and  activity  in  tumours  [6].  acidic  phe  contributes  to  increased  exosome  release  and  paracrine  exosome  uptake  [7].  these  small  membrane  vesicles  contain numerous proteins,  lipids and even nucleic acids  and  can  travel  to  distant  tissues,  which  allows  them  to  influence multiple aspects of tumour behaviour, including  local  invasive  growth  and  distant  metastasis  [8].  acidity  has been shown to play an important role during resistance  to chemotherapy by impairing the uptake of weakly basic  anti‐cancer drugs. for example, doxorubicin (dox), a well‐ established drug for breast cancer treatment, is a weak base  (pka between 7.5 and 9.5) that is protonated in the acidic  tumour environment and consequently displays decreased  cellular uptake [9].     the  small  gtpase  rab27b  regulates  the  transport  and  exocytosis  of  multiple  organelles,  including  multivesicular  endosomes  (mves),  resulting  in  the  release  of  exosomes  [10,  11].  rab27b‐mediated  derailed  exocytosis  releases  pro‐invasive  growth  regulators  into  the  tumour  environment  for  extracellular  matrix  degradation. in addition to the reorganization of the actin  cytoskeleton and invasion, rab27b also induces cell cycle  progression and proliferation [8, 12]. these observations  prompted  us  to  investigate  the  effect  of  rab27b  small  gtpase on the bio‐energetic profile of breast cancer cells.  2. materials and methods  2.1 cell culture  we  used  previously  described  cell  lines  that  stably  express  gfp  or  gfp‐rab  fusion  proteins:  mcf‐7  (or  t47d) gfp (gfp control protein), mcf‐7 (or t47d) gfp‐ rab27b (rab27b wild type protein), mcf‐7 gfp‐rab27a  (rab27a  wild  type  protein),  mcf‐7  gfp‐rab27b  q78l  (constitutively  active  mutant  protein),  mcf‐7  gfp‐ rab27b  t23n  or  n133i  (inactive  mutant  proteins  defective  in  gtp  binding),  mcf‐7  gfp‐rab27b  ger  (geranylgeranylation  mutant  protein  defective  in  membrane anchoring) [12]. the er‐positive human breast  cancer  cell  lines  were  maintained  in  a  culture  medium  (dmem supplemented  with 10%  fbs  (serum),  100u/ml  penicillin  and  100μg/ml  streptomycin)  (invitrogen,  merelbeke,  belgium).  to  prepare  a  serum‐free  conditioned medium (cm), 2 × 107 cells per flask of each  cell type were washed three times and incubated for 24  hours at 37°c with 15ml serum‐free culture medium. the  medium  was  harvested,  centrifuged  at  1250g  for  five  minutes at 4°c and passed through a 0.22μm filter. cm  was 30× concentrated at 4°c  in centriprep tubes ym‐10  (millipore,  billerica,  ma).  we  transiently  expressed  rab27b‐specific hp guaranteed sirna’s and a scrambled  rnai  negative  control  into  mcf‐7  gfp‐rab27b  cells  according to the manufacturer’s guidelines. sirnas were  purchased from qiagen (venlo, netherlands): sirab27b‐1  target  5’  aaa  cgt  gtg  gtt  tat  aat  gca  3’  and  sirab27b‐2  target  5’  tag  gaa  tag  act  ttc  ggg  aaa 3’. cell morphology and intracellular gfp‐rab27b  vesicle  distribution  were  studied  using  phase  contrast  and laser scanning confocal microscopy (zeiss 510 meta,  carl  zeiss,  micro‐imaging  inc.,  heidelberg,  germany)  respectively.     2.2 bioenergetic profiling  the xf extracellular flux analyser (seahorse bioscience,  billerica, ma)  is a 24‐well  instrument  that continuously  measures  the  uptake  and  excretion  of  metabolic  end  products.  the  xf  analyser  contains  a  sensor  cartridge,  embedded with 24 pairs of fluorescent biosensors (o2 and  ph), which monitor substrate concentration changes by a  fibre  optic  waveguide.  the  specialized  plates  allow  for  the  measure  of  oxygen  consumption  over  time.  cells  were seeded at a density of  15,000  in xf24 cell culture  microplates  (seahorse  bioscience)  in  a  culture  medium  and incubated at 37°c in 10% co2 for 24 hours. the assay  is initiated by aspirating the culture medium, replacing it  with 1ml of pre‐warmed  (37°c) assay medium  (dmem  without  bicarbonate  but  containing  sodium  pyruvate,  glutamax‐1 and 10mm glucose, ph=7.4) (invitrogen), to  wash the cells. after washing, almost all of the media is  removed  and  an  additional  625μl  of  assay  media  are  added.  the  plate  is  incubated  at  37°c  in  a  non‐co2  incubator for 30 minutes. the calibration plate (seahorse  bioscience)  is prepared one day  in advance. the probes  on the plate are immersed in calibrate solution (seahorse  bioscience)  for  a  minimum  of  24  hours  prior  to  the  experiment and placed at 37°c  in a non‐co2  incubator.  on the day of the experiment, the treatments are placed  2 exosomes microvesicles, vol. 1, 3:2013 www.intechopen.com   in the ports of the calibration plate. the three treatments,  all from sigma‐aldrich, used in this experiment are fccp  (200nm),  oligomycin  (1μm)  and  rotenone  (1  μm).  two  rows of the plate were injected with all three drugs, while  the remaining two rows were injected with assay media  only to serve as control wells. the three treatments and  media were loaded into the appropriate ports in a volume  of 75μl. three measurements were taken as  initial basal  measurements  followed  by  three  measurements  after  each injection. the wells are mixed for two minutes, there  is  a  three  minute  wait  time  and  then  the  wells  are  measured  over  a  two  minute  time  frame.  oxygen  consumption rates (ocr) are reported in pmol/min over  the time course of the experiment.    2.3 measurement of extracellular medium ph (phe)  the  measurement  of  phe  is  initiated  by  aspirating  the  culture  medium  of  exponential  growing  cells  and  washing  with  a  pre‐warmed  (37°c)  assay  medium  (dmem  without  bicarbonate  but  containing  sodium  pyruvate,  glutamax‐1  and  10mm  glucose,  ph=7.4)  (invitrogen).  after  washing,  additional  assay  media  is  added and the cultures are  incubated at 37°c  in a non‐ co2  incubator.  24  hours  later  the  medium  is  analysed  using a calibrated ph meter.    2.4 western blot analysis  for western blot analysis, mcf‐7 cells (1–10 × 106) were  harvested in laemmli lysis buffer (0.125 m tris–hcl [ph  6.8], 10% glycerol, 2.3% sodium dodecyl sulphate [sds]).  cell  lysates  (25μg)  and  cm  (20μl)  were  suspended  in  10μl reducing sample buffer (1 m tris–hcl [ph 6.8], 30%  glycerol,  6%  sds,  3%  β‐mercaptoethanol,  0.005%  bromophenol blue) and boiled for five minutes at 95°c.  samples were run on nupage4%–20% bis–tris gradient  gels  (invitrogen),  transferred  to  polyvinylidene  fluoride  membranes,  blocked  in  5%  non‐fat  milk  in  phosphate‐ buffered  saline  (pbs)  with  0.5%  tween‐20  and  stained  using  rabbit  polyclonal  anti‐cathepsin  b  (santa  cruz  biotechnology, santa cruz, ca), mouse monoclonal anti‐ parp (bd pharmigen, belgium) and mouse monoclonal  anti‐tubulin (sigma–aldrich, st louis, mo) antibodies.  3. results  3.1 rab27b attenuates oxphos capacity  to  investigate  the  impact  of  rab27b  on  energy  metabolism  pathways  we  tested  pharmacologic  modulators  interfering  with  the  mitochondrial  respiratory  pathway  in  the  seahorse  xf24  extracellular  flux analyser [13]. we engineered mcf‐7 breast cancer  cells  stably  overexpressing  rab27b  similarly  to  levels  found  in  invasive breast tumours [12]. treatments were     performed with the uncoupler fccp (carbonyl cyanide p‐ (trifluoromethoxy)  phenylhydrazone)  (30,  40  and  50  minutes), the complex v inhibitor oligomycin (60, 70 and  80 minutes) and the complex i inhibitor rotenone (90 and  100  minutes).  after  each  drug  treatment,  we  observed  that  the  oxygen  consumption  rate  (ocr)  was  significantly  lower  in  gfp‐rab27b  transfected  mcf‐7  cells  compared  to  mcf‐7  gfp  cells,  suggesting  that  rab27b  decreased  oxphos  activity  and  increased  dependency  on  aerobic  glycolysis  (figure  1).  this  is  consistent  with  our  previous  finding  that  rab27b  stimulates invasive tumour growth in a xenograft mouse  model, a high‐energy dependent process [12].      figure 1. reduction of oxidative phosphorylation by rab27b in  human  breast  cancer  cells  mcf‐7.  real‐time  analysis  of  the  oxygen  consumption  rate  (ocr)  by  xf  extracellular  flux  analyzer  in  mcf‐7  gfp  and  gfp‐rab27b  transfected  cells.  timepoints  (minutes)  one,  10  and  20  represent  baseline  measurements  before  drug  treatment;  30,  40  and  50:  ocr  measurements  after  carbonyl  cyanide  p‐(trifluoromethoxy)  phenylhydrazone  (fccp)  treatment;  60,  70  and  80:  ocr  after  oligomycin addition; 90 and 100: measurements after rotenone  treatment.  ocr  measurements  are  the  mean  of  three  independent experiments.    3.2 rab27b promotes acidic extracellular medium phe  in  exponentially  growing  cell  cultures,  rab27b  reduced  the  extracellular  medium  phe  of  mcf‐7  cells  by  0.64  units  after  24  hours,  from  7.40  to  6.76  (figure  2a).  in  contrast,  phe  of  control  vector  transfected  mcf‐7  cells  was  7.14.  importantly,  rab27a  overexpression  had  no  significant  effect  on  phe.  in  addition,  mcf‐7  cells  overexpressing  rab27b  defective  in  gtp‐binding  (i.e.,  inactive rab27b protein, n133i) or membrane anchoring  (i.e.,  no  geranylgeranylation  of  the  protein,  ger)  were  both ineffective in regulating phe. transient targeting by  a single sirna depleted rab27b protein by 70‐80% and  resulted in loss of extracellular acidification (figure 2b).  similar  observations  were  made  when  phe  was  measured in another model, the t47d breast cancer cells  overexpressing rab27b (data not shown).  3an hendrix, carla ciccone, christian gespach, marc bracke, olivier de wever and wendy westbroek: rab27b-mediated metabolic reprogramming induces secretome acidification and chemoresistance in breast cancer cells www.intechopen.com       figure  2.  role  of  rab27b  in  the  extracellular  acidification  process. (a) phe measurement of the culture medium prepared  from  mcf‐7  cells  stably  transfected  with  gfp‐rab27b  (wt),  gfp‐rab27b  mutants  defective  in  gtp‐binding  (n133i)  or  vesicle  membrane  anchoring  (ger)  n133i  and  ger  or  gfp‐ rab27a  (wt) were compared with phe values  recorded  from  mcf‐7 gfp cells. cells were cultured  for 24 hours during  the  exponential growth after assay medium refreshment. (b) control  mcf‐7 gfp‐rab27b cells  (con) and mcf‐7 gfp‐rab27b cells   transfected  with  scrambled  sirna  (sicon)  or  rab27b  sirna,  were  cultured  under  exponential  growth  conditions.  after  72  hours,  the  culture  medium  was  refreshed  and  phe  was  measured  24  hours  later.  inset:  silencing  of  rab27b  after  96  hours of rab27b sirna or sicon transfection  in mcf‐7 gfp‐ rab27b  cells  assessed  by  western  blot.  all  phe  data  are  expressed as the mean ± s.e.m. of three independent experiments  performed  in  triplicate. p‐values are calculated using  the  two‐ sided student’s  t‐test  following d’agostino‐pearson  testing  for  normal  distribution.  statistically  significant  p‐values  are  indicated.    3.3 functional consequences of rab27b‐induced acidification  mcf‐7 cells secrete cathepsin b, a cysteine protease that  is  activated  by  acidic  phe.  we  therefore  analysed  cathepsin b activation in the conditioned media of mcf‐ 7  gfp  and  gfp‐rab27b  cells.  western  blot  analysis  showed  that  the  cathepsin  b  protein  is  exclusively  activated  in  the  extracellular  milieu  of  mcf‐7  gfp‐ rab27b cells  (figure 3a). however, cathepsin b  is not  trafficked  by  secretory  rab27b  vesicles:  1)  mass  spectrometry analysis of the rab27b vesicle content did  not  reveal  cathepsin  b  protein  [12]  and  2)  confocal  microscopy revealed that cathepsin b did not co‐localize     with  rab27b‐positive  vesicles  (data  not  shown).  in  addition,  overexpression  of  rab27b  in  mcf‐7  cells  decreased  3.3‐fold  the  potency  of  the  cytotoxic  agent  doxorubicin (dox) (ic50= 1.56 μm), compared to mcf‐ 7  gfp  cells  (ic50=0.47 μm,  p<0.001).  this  was  further  evidenced  by  absence  of  the  85kda  cleaved  active  poly(adp‐ribose)polymerase‐1  (parp)  fragment  involved in cell death pathways (figure 3b).      figure 3. impact of rab27b‐mediated extracellular acidification  on cathepsin b activation and parp cleavage. (a) western blot  analysis of cathepsin b in the conditioned culture media of mcf‐ 7  gfp  and  mcf‐7  gfp‐rab27b  cells.  pro‐cathepsin  b  has  a  molecular  weight  of  40kda,  while  maturated  cathepsin  b  is  characterized by protein bands at 25 and 30kda. (b) expression  and  cleavage  of  the  apoptosis‐related  protein  parp  (85kda)  analyzed  by  western  blot  after  a  doxorubucin  treatment   (0.63 μm)  for  24  hours.  tubulin  (55kda)  was  used  as  loading  control.    3.4 rab27b induced acidic phe promotes filopodia formation  next,  we  analysed  the  impact  of  induced and  reversed  acidification  on  the  morphology  of  mcf‐7  cells  under  control and rab27b conditions. phe was experimentally  adjusted to physiological and acidic phe (respectively 7.2  and 6.5) for 24 hours in mcf‐7 gfp and gfp‐rab27b cell  cultures.  in  this  phe  range,  we  observed  that  experimental acidification of the culture medium of mcf‐ 7  gfp  cells  for  24  hours  did  not  induce  any  morphological  changes  (figure  4a).  in  contrast,  mcf‐7  gfp‐rab27b cells displayed more filopodia extrusions at  acidic  phe  as  compared  to  cells  maintained  at  physiological  phe  (figure  4a).  these  filopodia  often  contained  rab27b‐positive  vesicles  at  their  distal  ends  (figure  4c).  this  was  not  the  case  in  mcf‐7  gfp  cells  cultured  at  these  two  experimentally  adjusted  phe  conditions.  in  a  next  step  we  compared  the  morphological  impact  of  acidified  culture  medium  induced by rab27b‐dependent mechanisms (figure 4b)  with experimental and direct acidification of the culture  medium  prepared  from  mcf‐7  gfp  cells  (figure  4a,  lower left panel). only transfer of culture medium from  mcf‐7  gfp‐rab27b  cells  induced  the  formation  of  filopodia  protrusions  in  mcf‐7  gfp  acceptor  cells  (figure 4b).       4 exosomes microvesicles, vol. 1, 3:2013 www.intechopen.com     figure 4. influence of manipulating directly extracellular phe at  physiological  and  acidic  values  (phe  7.2  versus  6.5)  on  the  morphology  by  phase‐contrast  microscopy  of  control  and  rab27b‐transfected mcf‐7 cells. (a) mcf‐7 gfp (left) and gfp‐ rab27b cells (right) were cultured at phe 7.2 (upper panels) or  phe 6.5 (lower panels). filopodia formation was induced only in  rab27b‐transfected  cells  maintained  at  acidic  phe.  scale  bar:  100μm.  (b)  control  mcf‐7  gfp  cells  treated  with  the  conditioned  media  prepared  from  mcf‐7  gfp‐rab27b  cell  cultures  maintained  at  acidic  phe  induced  the  production  filopodia extensions. scale bar: 100μm. (c) representative laser  scanning  confocal  image  of  gfp‐rab27b  positive  vesicle  distribution.  arrowheads  indicate  gfp‐rab27b  vesicle  accumulation in filopodia protrusions. scale bar: 20μm.  4. discussion  our  data  suggest  that  rab27b  up‐regulation  in  er‐ positive  breast  cancer  cells  induces  a  switch  from  oxidative phosphorylation towards aerobic glycolysis, to  sustain the energy demands required for invasive tumour  growth,  which  is  accompanied  by  acidification  of  the  tumour  environment.  this  metabolic  reprogramming  is  rab27b‐specific  and  exclusively  requires  gtp‐binding  and geranylgeranyl‐dependent membrane targeting.    the metabolism of cancer cells is reprogrammed to meet  the  challenges  of  macromolecular  synthesis  associated  with  cell  growth  and  proliferation  [2].  this  altered  metabolism  can  be  related  to  growth  factor  signalling  since  activated  pi3k/akt  signalling  downstream  of  receptor  tyrosine  kinase  activation  leads  to  enhanced  glucose uptake and glycolysis [14, 15]. how can altered  expression  of  the  small  gtpase  rab27b  contribute  to  a  metabolic  shift?  rab27b  transports  mves  towards  the  plasma  membrane  and  fusion  results  in  the  release  of  exosomes [11]. we previously demonstrated that rab27b‐ associated vesicles release hsp90, recently identified as  an exosome surface protein, in the tumour environment  [12,  16].  extracellular  hsp90  activates  a  cassette  of  proteins that function collectively in invasive growth [8].  extracellular  hsp90  interacts  with  the  extracellular  domain  of  her‐2  tyrosine  kinase  receptor  in  breast  cancer  cells,  which  is  necessary  for  receptor  activation  and  heterodimerization  with  ligand‐activated  her‐3.  this  will  in  turn  mediate signal  transduction  pathways  via  mapk  and  pi3k‐akt,  known  to  play  key  roles  in  cellular  proliferation,  differentiation,  migration  and  metabolic reprogramming [14, 17]. thus, rab27b‐induced  hsp90  release  can  contribute  to  metabolic  reprogramming  required  to  sustain  invasive  growth.  other  members  of  the  ras  superfamily,  such  as  rho  gtpases,  have  been  linked  to  cellular  metabolism  through glutaminase activity and glutamine dependence.  sirna  knockdown  or  pharmacological  inhibition  of  glutaminase inhibits rho gtpase induced transformation  and proliferation [18]. rab25, a small gtpase involved in  endosomal recycling, which  is genomically amplified  in  multiple  cancers,  is  a  key  regulator  of  cellular  bioenergetics. rab25 induced an akt‐dependent increase  in  glucose  uptake  and  glycogen  storage  which  allows  maintenance of atp levels during bioenergetic stress [19].    in  addition  to  acidic  phe,  a  side  effect  of  aerobic  glycolysis,  we  have  shown  here  that  the  secretome  of  mcf‐7 gfp‐rab27b cells is required to induce filopodia  formation.  this  suggests  that  the  rab27b  secretome  contains putative positive regulators of the small gtpase  rho  involved  in  filopodia  formation  [20].  these  results  also  indicate  a  paracrine  diffusion  of  the  filopodia  phenotype from rab27b transfected breast cancer cells to  the control cells, potentially by the means of exosomes. it  has  been  demonstrated  that  addition  of  exosomes  containing notch  ligand delta‐like  four confer a  tip cell  phenotype on endothelial cells characterized by filopdia  formation  [21].  the  relocalization  of  rab27b‐associated  vesicles  to  the  filopodia  protrusions  may  facilitate  exocytosis  at  sites  of  active  migration  and  matrix  remodelling.  similar  observations  were  published  by  glunde  et  al.  demonstrating  that  secretory  lysosome  distribution in breast cancer cells alters at acidic phe [22].  human  melanoma  cells  exhibit  more  lamellipodia  and  stronger adhesion at lower phe, and displayed enhanced  invasion  and  increased  secretion  of  proteases  and  proangiogenic  factors  in  response  to  extracellular  acidification.  also,  in  human  breast  cancer  cells,  the  number  and  length  of  filopodia  has  been  shown  to  increase  at  acidic  phe  [22].  in  conclusion,  our  data  demonstrate a rab27b‐dependent switch from oxidative  phosphorylation  towards  aerobic  glycolysis  in  er‐ positive breast cancer cells to sustain invasive growth in  acidified environments.  5an hendrix, carla ciccone, christian gespach, marc bracke, olivier de wever and wendy westbroek: rab27b-mediated metabolic reprogramming induces secretome acidification and chemoresistance in breast cancer cells www.intechopen.com   5. conflict of interest    the authors declare no conflict of interest.    6. acknowledgements    this  work  was  in  part  supported  by  the  intramural  research  program  of  the  national  human  genome  research  institute  (nih,  bethesda,  maryland),  inserm  research  center  u938  and  paris  vi  university  upmc,  spearhead  oncology  of  ghent  university  hospital,    a  postdoctoral  grant  (odw  and  ah)  from  fund  for  scientific  research‐flanders  and  special  research  fund  ghent university.    7. references    [1] vander  heiden  mg,  cantley  lc,  thompson  cb:  understanding  the  warburg  effect:  the  metabolic  requirements  of  cell  proliferation.  science  2009,  324(5930):1029‐1033.  [2] ward ps, thompson cb: metabolic reprogramming: a  cancer  hallmark  even  warburg  did  not  anticipate.  cancer cell 2012, 21(3):297‐308.  [3] gatenby  ra,  gillies  rj:  why  do  cancers  have  high  aerobic  glycolysis?  nat  rev  cancer  2004,  4(11):891‐ 899.  [4] fais s, de milito a, you h, qin w: targeting vacuolar  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cells. neoplasia 2003, 5(6):533‐545.        6 exosomes microvesicles, vol. 1, 3:2013 www.intechopen.com protocol standardization reveals mv correlation to healthy donor bmi original research article philip hexley1,*, kyle p. rismiller2, chad t. robinson3 and george f. babcock3,4 1 university of nebraska medical center, omaha, ne 2 university of dayton. dayton, oh 3 university of cincinnati. cincinnati, oh 4 shriners hospitals for children, cincinnati, oh * corresponding author e-mail: phil.hexley@unmc.edu received 22 aug 2013; accepted 12 mar 2014 doi: 10.5772/58527 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract microvesicles (mvs) are cell-derived vesicles which are of interest in a clinical setting, as they may be predictive of early signs of disease and/or of treatment progression. however, there are growing concerns about using conventional flow cytometry (cfmc) for the detection and quantification of microvesicles. these concerns range from error-sources in collection through to the physical limitations of detection. here we present a standardized method for collection and analysis which shows that the mv numbers detected by cfcm correlate to donor body mass index (bmi). although unlikely to be comprehensive, we also demonstrate how cfcm is a useful and valid tool in the analysis of mvs. keywords quantification, bmi, flow cytometry 1. introduction extracellular vesicles (evs) are small membranous vesicles ranging from 0.05 to three microns in diameter, released from cells during apoptosis and cell activation (1). they have been subcategorized and defined in multiple ways by numerous groups (2, 3, 4). although the nomenclature is used somewhat ambiguously, an excellent review article by györgy et al. (2011) recently outlined the broad classification we are following. one category of evs is that of microvesicles (mvs), which are formed by activation or apoptosis (4). mvs range from 0.1 to one micron in diameter. in blood, circulating mvs have been found to range more specifically from 0.1 to 0.5 microns in diameter (5). it is these circulating plasma mvs which are the focus of this article. for decades, fcm has been used as a cellular analysis technique due to its rapid, multiparametric capabilities at the single cell level (6, 7, 8). the development of a greater understanding of the importance of microvesicle biology is a growing issue, both in basic research and in a clinical setting (9, 10, 11). as a result of the cellular origin of mvs, as well as the fact that the requirements for analysing mvs are the same as those for cells, fcm appears to be a logical extension of cellular analysis for investigating mvs. a conceptual concern is the fact that cells and mvs are not one and the same thing; mainly due to the small size of mvs, it is slowly becoming accepted that fcm is not a straightforward technique for the analysis of mvs (12, 13, 1philip hexley, kyle p. rismiller, chad t. robinson, and george f. babcock: protocol standardization reveals mv correlation to healthy donor bmi article exosomes microvesicles, 2014, 2:2 | doi: 10.5772/58527 exosomes and microvesicles 14, 15). there are many theoretical concerns in fcm which make this a suboptimal technique for the analysis of mvs. the use of polymer beads as a standardization measure is one of these concerns, as it is understood that a bead may scatter up to 100 times more light than an mv (16). consequently, if we can detect a 100 nm diameter polymer bead by light scatter, this does not mean that we can detect a 100 nm diameter mv by the same means. this leads to the conclusion that conventional fcm (cfcm) is not a comprehensive technique for mv analysis; it in fact only analyses a proportion of the larger mvs as well as, potentially, the coincidence of smaller mvs. another concern relates to coincidence event detection, where no individual mv would be detected above the background but, due to their close proximity when passing the detector, multiple mvs are registered as a single event (17, 18). finally, in fcm we conventionally identify an event when light scatter is detected above the threshold in the forward scatter parameter. in small particle flow cytometry and microbial flow cytometry, there are physical reasons for identifying events when light scatter is detected above the threshold in the 90o angle (side scatter) parameter (19, 20). however, if such small particles do not create light scatter that can be detected above the threshold, then the option to identify events by fluorescence is also available. some literature suggests that fluorescence is actually a better parameter for identification (21). however, it is yet to be determined whether fluorescence or light scatter is a better method for detecting mv events by fcm, as there is now evidence in the literature that not all mvs are annexin v positive (4, 22). currently, there is no allencompassing mv-stain by which to identify events by fluorescence, although there are several potential stains that appear to be suitable (23). as yet, however, this has not been confirmed. therefore, while some mv analysis would be better suited to fluorescence detection, there would be sub-populations which may not be detected using this method. it may be that, depending on the application, this factor will determine whether fluorescence or scatter detection is more appropriate. currently, there are no well-developed answers to these analysis issues. some suggestions have, however, been made, such as coincidence event determination by monitoring fluorescence and by serial dilutions of a sample. in the latter, a sample with high coincidence would be expected to show a reduced fluorescence signal in the dilutions. there still remains, however, the issue that only sub-populations of mvs can be identified and the fact that to date there is no agreed standard in mv analysis. in addition to the concerns over analysis, there are mounting concerns over protocol standardization and over how to define the pre-analytical and analytical variables which impact the analysis. these variables include those relating to sample collection: phlebotomy technique, needle gauge and position of tourniquet (24); those relating to sample handling: vortexing and centrifugation conditions (25); those relating to detection protocols including instrument set-up (26); and those relating to potential false positives caused by confounding reagent particulates and antibody aggregates (27). these concerns are of intense importance as the manner in which these protocol steps, which may appear trivial, are carried out can lead to a significant variation in mv numbers. in addition to the need for standardization of sample collection, handling and detection, there are also patient factors which need to be taken into account. these factors need to be considered so that analytical variables are understood and so that mvs can be reliably quantified in patients and other clinical settings. while there many studies which investigate the relationship between pathological states and physiological variables, to date few studies have examined variation among healthy individuals in relation to mv quantification. the possibility that donor parameters such as body mass index (bmi) could function as a confounding factor on absolute baseline mv quantification has not been extensively examined. to further complicate the already challenging area of mv study, it has recently been shown there is a cellular inflammatory response to fatty meals (28) which has been linked to an increase in mv numbers (29, 30). another recent study suggests that strenuous exercise increases relative mv counts (31). in this study, we examine baseline mv numbers, as mv numbers are affected by many factors. here, we have defined baseline mv numbers, after minimal disruption during collection and optimization of protocol. this allows us to measure a healthy individual’s circulating plasma mv numbers from normal cellular turnover. 2. materials and methods 2.1 prerequisites this study was performed under an approved institutional review board protocol. the inclusion criteria for the study included a minimum of four hours fasting as well as no meal over 50 g in fat (30) and no strenuous physical exercise (31) for 12 hours prior to sample collection. 2.2 donor demographics all donors gave self-declarations of good health and were caucasian males and females except for one east asian female. the mean age was ± sd, 40.2 ± 15.7 years; age range 21 – 64 years. 2 exosomes microvesicles, 2014, 2:2 | doi: 10.5772/58527 2.3 sample collection a stock of anticoagulant citrate dextrose (acd) solution was made by dissolving 22.0 g trisodium citrate (sigmaaldrich, mo), 8.0 g citric acid (fisher scientific, pa) and 24.5 g dextrose (sigma-aldrich) in one l deionized water (millipore, ma), which was then passed through a 0.22 micron filter (corning, ny). two ml of the adc solution was filtered with a 0.2 micron filter (pall, mi) immediately prior to use. two hundred µl of acd solution was pre-loaded into a 10 ml syringe (bd biosciences, nj) to prevent clotting when drawing blood. all peripheral blood was drawn by a standard procedure from the arm, where the tourniquet was placed approximately four inches above the selected puncture site, using a 21 gauge butterfly needle (bd biosciences). at this stage, a 100 µl aliquot was taken for analysis on a hemavet 1700fs (drew scientific, ct), following the manufacturer’s recommendations. immediately after, the blood was put into a 15 ml falcon tube (bd biosciences) containing 1.8 ml of acd solution, giving a total volume of 12 ml with a 5:1 ratio of blood to acd solution. 2.4 sample handling the 100 µl aliquot for acquisition on the hemavet 1700fs (drew scientific) was used to analyse the total white blood cell counts, neutrophil counts, lymphocyte count, monocyte count, eosinophil count, basophil count, platelet count, red blood cell count and percent haematocrit blood parameters, all of which were recorded (table 1). the 15 ml conical centrifuge tube (bd biosciences) containing the acd-blood solution (5:1 blood/adc) was gently inverted to mix the sample. the sample was then centrifuged at 1500 x g for 30 minutes at room temperature. the plasma supernatant was removed and transferred to an ultra-centrifuge tube (beckman coulter, in), leaving at least 300 µl supernatant undisturbed above the cell phase (3). the plasma was then centrifuged at 11,000 g for 30 minutes at room temperature to remove platelets. the platelet-free plasma supernatant was removed and placed into a 15 ml conical centrifuge tube, leaving at least 300 µl supernatant undisturbed above the pellet. from 10 ml of blood we obtained approximately four ml of platelet-free plasma. 2.5 flow cytometry sample data were acquired on an epics xl (beckman coulter) flow cytometer, equipped with a 15 mw, 488 nm argon-ion laser and using a nano fluorescent particle size standard kit, nfpps-52-4k (spherotech, il), for instrument standardization. using the epics xl volumetric acquisition setting, 20 µl of plasma was acquired by identifying events above threshold on the side scatter parameter, displayed in logarithmic scale. side scatter was set to 556 volts with a gain of five and a threshold value of three, as identified by a dashed line across figure 1, plots a to d. gates were set on a forward angle and a side scatter angle dual parameter plot, based on a sample population falling below the 1.33 micron bead population on side scatter. all reagents were diluted in 0.2 micron filtered facsflow (bd biosciences) and used as background controls. post-confirmation of mv presence in the sample was carried out via systematic triton lysis (26, 32) using 0.1% triton x-100™ (mallinckrodt, mo), where the mv population would dissolve from the mv gate, as seen in figure 1. 2.6 dynamic light scatter sample data were acquired on a coulter n4 plus instrument, acquiring the data at a 90o angle. experiments were carried out at 22oc to determine the size distribution of the microvesicles. 2.7 data analysis hemavet blood parameters were recorded. the flow cytometric circulating plasma mv numbers were recorded within the mv gate, counts within the mv gate from posttriton x100™ lysis were then subtracted and this value was corrected for the 5:1 blood-to-acd dilution to give absolute counts in 20 µl. this number was used to calculate the circulating plasma mv baseline number per ml of plasma. the baseline number was compared to the total bmi, age, sex and cellular blood parameters of the donor, using statistical analysis software (sas institute, inc., cary, nc 27511) by means of a curve-linear coefficient of correlation where a p>0.05* was considered significant and graphically displayed using microsoft excel 2010. 3. results volumetric fcm analysis of plasma samples quantified all subjects’ absolute baseline circulating plasma mv numbers. a background sample was acquired for each patient immediately prior to the plasma sample being taken (figure 1a). twenty µl was acquired for each sample and the mv population was determined from a bead standard defining the upper limit of the mv population (figure 1b). gating was confirmed post-acquisition by systematic triton x-100 lysis of the mv population (figure 1c). the absolute baseline circulating plasma mv number was derived by subtracting the gated background number from the plasma sample gated number and then converting the result to numbers of mvs per ml of plasma. donor experimental data from a hemavet 1700sf was acquired immediately after the sample was drawn. the haematocrit, white blood cell, red blood cell and platelet count were in the normal range, as shown in table 1.there was found to be no correlation between the baseline circulating plasma mv number and any of the cellular blood parameters measured. 3philip hexley, kyle p. rismiller, chad t. robinson, and george f. babcock: protocol standardization reveals mv correlation to healthy donor bmi figure 1. typical epics xl fcm plot showing contour plots fs/ss for (a) 1.33µ diameter bead standard, (b) triton control background, (c) mvs, (d) immediately after 0.1% triton x-100 lysis and (e) dls size distribution data of representative circulating plasma mvs. the baseline circulating plasma mv numbers were plotted against donor bmi using sas by means of a curve-linear coefficient of correlation, as seen in figure 2. there is significance (p<0.05) and a strong coefficient of correlation (r2=0.8867) in the relation between baseline circulating plasma mv number and donor bmi. figure 2. chart showing the relationship between bmi and baseline circulating plasma mv numbers per ml, with derived equation and correlation values. there are many areas for discussion, but as to the biological relevance of this correlation between quantitative mv count and bmi, we show that, when strict protocol standardization and rigorous control steps are taken, there is a strong correlation (r2=0.8867) between baseline circulating plasma mv numbers and healthy donor bmi when analysed by fcm. the relevance of this is amplified by the lack of correlation in other measured cell-derived blood parameters and bmi from the hemavet results (table 1). no significant differences between circulating plasma mv numbers between male and female samples were observed, nor was there any relationship between baseline mv number and the age of the donor (table 1). d on or i d a ge (y ea rs ) s ex w ei gh t ( lb s) w b c c ou n t (1 06 /m l) n eu tr op h il c ou n t (1 06 /m l) ly m p h oc yt e co u n t (1 06 /m l) m on oc yt e co u n t (1 06 /m l) e os in op h il c ou n t (1 06 /m l) b as op h il c ou n t (1 06 /m l) p la te le t c ou n t (1 06 /m l) r b c c ou n t ( 10 9 /m l) h ae m at oc ri t % b as el in e m v /m l 1* 28 m 265 6.32 3.10 1.69 0.65 0.62 0.25 223 5.64 55.17 499,960 2* 41 m 190 5.09 2.27 1.87 0.26 0.54 0.15 147 4.57 47.83 257,748 3* 56 m 125 3.44 2.04 0.79 0.18 0.33 0.10 166 4.48 46.80 103,020 4 31 f 145 6.68 4.72 0.85 0.79 0.16 0.16 184 5.13 49.90 128,460 5 52 f 155 4.28 2.35 0.73 0.54 0.53 0.13 153 4.93 50.40 180,000 6 56 f 250 6.62 3.78 1.90 0.50 0.27 0.18 111 5.07 51.10 756,000 7 64 m 202 3.14 1.68 0.76 0.26 0.41 0.03 96 4.30 43.70 368,520 8 31 f 115 5.38 2.89 1.79 0.39 0.19 0.12 140 4.60 42.10 104,400 9 22 f 120 5.80 3.13 1.73 0.29 0.55 0.09 293 6.20 39.20 89,520 10 21 m 185 6.18 2.47 2.04 0.95 0.57 0.15 60 5.27 45.90 164,640 table 1. table showing donor age, sex, bmi, circulating plasma mv numbers and hemavet 1700fs (drew scientific) cellular blood parameters. each sample was analysed by a curve linear coefficient of correlation (p>0.05). * where available, averaged patient data from replicated samples taken on different days with standard error of the mean (sem) are shown. 4 exosomes microvesicles, 2014, 2:2 | doi: 10.5772/58527 4. discussion previous studies investigating similar relationships between mv quantification and either patient bmi (33) or waist-to-hip ratio (34) did not report as strict a standardization of patient variables, nor acknowledge the rigorous protocol standardization necessary to minimize the confounding factors when quantifying mvs. furthermore, these studies did not report the rationale for the analysis. therefore, in light of issues raised recently, both here and in numerous previous publications (4, 10, 12, 15, 25, 29, 31), any further interpretation of the data in these respective publications is hard to justify. it is known that mvs are released from cells during apoptosis and upon cell activation (2, 3, 4). increased weight is intrinsically related to a state of chronic oxidative and inflammatory stress (35); it has also recently been implicated as a cause of endothelial damage and increased c-reactive protein levels, leading to apoptosis (36, 37). as mvs have been shown to increase oxidative activity (38) and been implicated in inflammatory stress (39), it is evident that there is significant overlap of associative effects between increased weight and increased mv activity. most importantly, it has been demonstrated that increased weight increases cell activation (40), which we know to be a process of mv formation. this strongly supports our findings that bmi and mv number are inherently related, indicating that weight-related factors may be mechanistic in mv formation. irrespective of the concerns, there is a distinct correlation between baseline circulating plasma mv numbers and bmi (figure 2). these findings suggest that circulating plasma mv numbers may have important and unique implications as a clinical cell-derived blood parameter and therefore warrant continued investigation. it should be noted, however, that we acknowledge that we did not consider racial difference, which has recently been recognized as a factor affecting mv numbers (1). as there only limited donors of different racial backgrounds were used in this study, intra-variance due to this factor is cannot be determined by it. although cfcm has limitations in mv analysis, including in relation to detection and polymer beads for standardization (16) and coincidence events as a source of error (17, 18), as well as whether scatter or fluorescence is the best parameter to identify an event (41), we are able to show here that, with strict protocol standardization, cfcm does have a place in mvs research. even with the acknowledged potential pitfalls of this study, we have seen a strong correlation of mv number and bmi. if the global mv population could be analysed and coincidence events reduced, then the results would be more accurate. the accuracy error cannot currently be defined by either cfcm or dls alone, but the techniques used in tandem give greater assurance in the interpretation of data. it has been suggested that dielectric measurements or modified fcm and dls would be more accurate in determining the count and size distribution of mvs than conventional light scatter measurements alone (42). there are a number of potential instruments which would overcome these sources of error: the qnano by izod (17, 43); the invitrox surface antigen detection and enumeration (isade) by invitrox (44); the delsamax pro by beckman coulter; and the nanoparticle tracking analysis (nta) by nanosight (45, 46). these are just a few which appear very promising in their ability to compliment cfcm analysis of mvs. although there are many concerns remaining with regard to best practices in fcm analysis of mvs [47], using fcm to analyse mvs to address biologically relevant questions should and no doubt will continue. at the same time, however, there is a need 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healthy donor bmi article journal of circulating biomarkers quantification of cell-free her-2 dna in plasma from breast cancer patients: sensitivity for detection of metastatic recurrence and gene amplification original research article patricia diana sørensen1,2*, rikke fredslund andersen1, niels pallisgaard1, jonna skov madsen1,2, erik hugger jakobsen3 and ivan brandslund1,2 1 department of clinical immunology and biochemistry, vejle, lillebaelt hospital, denmark 2 institute of regional health research, faculty of health sciences, university of southern denmark, odense, denmark 3 department of clinical oncology vejle, lillebaelt hospital, denmark *corresponding author(s) e-mail: diana_2000b@hotmail.com received 12 may 2015; accepted 19 august 2015 doi: 10.5772/61320 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly abstract the purpose of this study was to quantify the free-circu‐ lating plasma her-2 dna (cfher-2 dna) and to assess the ability of analysis to discriminate between patients with primary breast cancer and healthy controls in order to detect metastatic recurrence in comparison with serum her-2 protein and also her-2 gene amplification. the study population consisted of 100 patients with primary breast cancer and 50 healthy female donors. an additional 22 patients with metastases were subsequently included. cfher-2 dna was quantified with a quantitative pcr method together with a reference gene. results: using a cut-off of 2.5 for the ratio of the cfher-2 dna/reference gene, three (of 15) tissue her-2-positive patients had a ratio >2.5 prior to the detection of metastatic disease. in the post-metastatic/pre-chemotherapy setting, 11 (of 23) tissue her-2-positive patients with metastases had a ratio >2.5. there was no difference between absolute preoperative cfher-2 dna values for patients with primary breast cancer and those for healthy controls. there was no difference between cfher-2 dna values taken within nine months of development of the metastatic disease and the levels in patients without metastases, but there was a significant difference in the corresponding serum her-2 protein levels in the tissue her-2-positive patient group. conclusion: amplified her-2 dna can be detected in plasma when using a ratio between cfher-2 dna and a reference gene. cfher-2 dna could not be used to dis‐ criminate between patients with primary breast cancer and healthy controls, and could not predict the development of metastatic disease. keywords breast cancer, cell-free dna, gene amplifica‐ tion, her-2, metastatic recurrence, sensitivity 1j circ biomark, 2015, 4:9 | doi: 10.5772/61320 1. introduction breast cancer is the most common cancer in women in the western world. although significant progress has been made in the adjuvant treatment of primary breast cancer, more than 20% of patients initially diagnosed with regional disease develop systemic relapse later in life [1]. this implies the need for biomarkers for evaluating the effect of treatment after primary therapy and also for early recur‐ rence detection. the presence of increased free-circulating dna in serum from cancer patients was described more than 30 years ago [2]. subsequent studies reported increased levels of freecirculating dna in plasma/serum from patients affected by colon and lung cancer [3]. the source of the free-circulating dna is still a debated subject. in healthy people it has been stated to originate from the apoptosis of nucleate cells [4], but some consider it to also originate from active release [5;6]. in cancer patients, an increased level is hypothesized to originate from the necrosis of cancer cells, and perhaps also from active release from cancer cells [7]. another hypothesis is a possible perturbation of the mechanism of dna clearance from plasma, which is also unconfirmed [5]. quantification of her-2 gene-copy number in the circulat‐ ing cell-free dna is a novel suggested biomarker to detect possible amplification. recent studies have investigated its potential for detecting her-2 amplification in plasma [8] and also for monitoring response to trastuzumab treat‐ ment [9]. the purpose of this study is to quantify the free-circulating plasma her-2 dna. for this purpose a quantitative method for detecting plasma her-2 gene-copy number in the total free-circulating dna was developed and validat‐ ed. the study focuses on the ability of the analysis to discriminate between patients with primary breast cancer and healthy controls, as well as to detect metastatic recurrence and establish correlation to tissue her-2 status and serum her-2 protein. 2. methods 2.1 study design and population this study was performed on plasma samples from a previous prospective project, which was carried out on breast cancer patients in the period 2004–2009. the moni‐ toring time had a range of 1.1–6.3 years (mean 3.6). the patients were primarily and prospectively monitored with serum her-2 protein; the total population is described in a previous article [10]. both plasma and serum samples were available. serum samples were used to analyse serum her-2 protein, and plasma samples were used for plasma cell-free dna. of the 841 patients included in the study, 100 consecutive patients who fulfilled the following inclusion and exclusion criteria were selected for the circulating plasma cell-free her-2 dna analyses. inclusion criteria: 1. twenty-five tissue her-2-positive patients known to have elevated serum her-2 protein levels; 2. twenty-five tissue her-2-positive patients known to have normal serum her-2 protein levels; 3. twenty-five tissue her-2-negative patients known to have elevated serum her-2 protein levels; 4. twenty-five tissue her-2-negative patients known to have normal serum her-2 protein levels. exclusion criteria: patients without available preoperative blood sample and patients with fewer than five blood samples available in the follow-up. baseline samples were taken prior to surgery and/or chemotherapy. an additional 22 patients with metastases (14 tissue her-2positive and eight tissue her-2-negative), who had had a blood sample taken after being diagnosed with metastases and before beginning chemotherapy, were analysed with the purpose of strengthening the results. the results from the additional patients were evaluated together with samples from patients with metastases from the study population. fifty healthy female donors (10 aged 25–44 years, 20 aged 45–59 years, and 20 aged 60–75 years) from the control group at the diabetes research bank, vejle hospital, served as healthy controls. the work was approved by the regional science ethics committee (reg. nr. s-vf-20040101). 2.2 clinical follow-up in brief, following surgery for primary breast cancer the patients underwent routine clinical controls at the oncolo‐ gy department. patients with inoperable disease at the time of diagnosis received neoadjuvant treatment prior to surgery. after surgery, the patients were treated with adjuvant therapy according to the current guidelines from the danish breast cancer group. patients with tissue her-2positive breast cancer determined by ihc/fish received trastuzumab and/or tykerb. patients with tissue her-2negative breast cancer received adjuvant fec or ec (fluorouracil, epirubicin, cyclophosphamide or epirubi‐ cin, cyclophosphamide) and/or docetaxel. the patients with oestrogen-positive primary tumour received antihor‐ monal treatment. the patients who presented symptoms of clinical recur‐ rence were further investigated. metastases were identified by imaging techniques with computed tomography/ ultrasound/magnetic resonance (ct/ul/mr) and verified by pathological examination, if possible. the levels of freecirculating dna and serum her-2 did not contribute to diagnostic decision-making. 2 j circ biomark, 2015, 4:9 | doi: 10.5772/61320 2.3 laboratory methods 2.3.1 quantification and validation of cell-free plasma her-2 dna 1. samples: the patients had blood tests taken in routine clinical controls at the oncology department during the “serum her-2 protein project” [10]. besides serum samples for that project, 3 ml of peripheral blood was also collected in an edta tube at each visit. the blood was centrifuged at 2000 x g for 10 minutes within two hours of sampling, and the plasma was divided into two cryotubes and frozen at -80°c until analysis could take place. we used 0.6 ml plasma/patient/visit; a few samples had less volume, between 50 and 550 μl. 2. purification: dna was purified with a qiasymphony robot using the qiagen virus/bacteria midi kit (qiagen, hilden, germany). the robot required 1.2 ml volume of each plasma sample; the plasma was therefore supplemented with water up to 1.2 ml, which was taken into account when the results were calcu‐ lated. 3. quantitative pcr analyses: plasma her-2 gene-copy number and a reference gene-copy number were detected by quantitative real-time pcr using in-house assays. at the same time, we analysed cpp1 (cysteinerich polycomb-like protein 1), a plant gene [11] that was added to all plasma samples with the purpose of monitoring for loss during the purification process, and b-cell lysis, using a peripheral-blood (pb) assay that detects the rearranged immunoglobulin genes in b-lymphocytes [12]. results were excluded if they showed signs of b-cell-lysis contamination. the her-2 gene-copy number was calculated as 10 [(y,intercept(gher2) − meanct (gher2)) / slope(gher2)] and the b2m gene-copy number as 10 [(y,intercept(gb2m) − meanct (gb2m)) / slope(gb2m)]". the plasma volume was taken into account in the calculation of the results. primers and probes for both assays were designed using the oligo primer analysis software version 7 (molecular biology insights, colorado, usa). the following concentrations were used: for the b2m gene – 3 microm forward (f) plus reverse (r) primer and 2 microm probe; for the her-2 gene – 9 microm forward (f) plus reverse (r) primer and 2 microm probe. primers were purified using a reverse-phase fast-centrifuge purification method, while the probes were purified using an hplc method (dna technology, aarhus, denmark). primers and probe for the her-2 gene: f-primer: her2ex26:64u20, 5’-gcggtggggacctga‐ cacta-3’ r-primer: her2ex26:122l18, 5’-ccttcggagggtgc‐ cagt-3’ probe: her2ex26:93f26, fam-ccctctgaagag‐ gaggcccccaggtc-tamra primers and probe for the b2m gene: f-primer: b2mex4:972u29, 5’-taaaact‐ taatgtcttccttttttttctc-3’ r-primer: b2mex4:1047l27, 5’-aaacattttct‐ caaggtcaaaaactta-3’ probe: b2mex4:387l26, fam-cctccatgatgctgctta‐ catgtctc-tamra the reactions were performed on abi7900ht using 20 μl taqman universal mastermix (life technologies, ca, usa) with primers and probe and 5 μl of purified dna solution, as follows: initial step 50°c for two minutes and activation step 95°c for 10 minutes, followed by 50 cycles at 95°c for 15 seconds and 60°c for one minute. the results are expressed in copies/ml. all the analyses were performed in triplicate. the her-2 gene amplification in the cell-free dna from plasma was determined by dividing the number of cfher-2 dna copies by the number of cfb2m dna copies. 2.4 validation process according to the miqe guidelines 2.4.1 standard curves the levels of plasma cfher-2 dna and the reference gene b2m were determined using standard curves calculated with digital pcr, which is the most accurate technique for absolute quantification. current articles about digital pcr analyses for her-2 expression in breast-cancer tissue showed good concordance with ihc/fish [13, 14]. dna was purified from blood from 50 healthy control subjects and was subsequently pooled. five microliters of pooled dna and subsequent five-, 25-, 125-, 625-, 3125-, and 15,625-fold dilutions were all analysed with both digital pcr and quantitative pcr. the results from the digital pcr, counting a colour signal/one copy, were given in copies/microliter. the results from the analysis on the abi7900ht real-time pcr system were given in cq. standard curves were drawn by matching the cq values with the copy number from the digital pcr. the slope for the her-2 standard curve was -3.43, which corresponds to a pcr efficiency (e) of 96% where e = (10 ^ (-1/slope) -1) * 100. the slope for the b2m standard curve was -3.49, corresponding to an efficiency of 93%. 2.4.2 recovery test 1.2 ml of plasma from a patient with metastatic breast cancer was analysed on abi7900ht. the mean values from the triple determination were 20544 (19979, 22674, 18978) her-2 copies/ml and 5864 (6067, 5231, 6295) b2m copies/ml. half the portion from the initial plasma samples (600 microliters) was mixed with water and analysed with the following results for the mean value: 8362 her-2 copies/ml and 2513 b2m copies/ml. 1.2 ml of plasma from the same patient was subsequently diluted 10-fold to produce 10 samples. each sample was 3patricia diana sørensen, rikke fredslund andersen, niels pallisgaard, jonna skov madsen, erik hugger jakobsen and ivan brandslund: plasma cfher-2 dna in breast cancer analysed in triplicate and the average was taken as the result of the sample. the mean values were 2153 her-2 copies/ml (cv=7%) and 824 b2m copies/ml (cv=9%). each of the 10-fold dilutions was again diluted 10 times to produce 10 samples with the following mean values: 230 her-2 copies/ml (cv=9%) and 74 b2m copies/ml (cv=9%). each of the 100-fold dilutions was again diluted 10 times to produce 10 samples with the following mean values: 21 her-2 copies/ml (cv=19%) and seven b2m copies/ml (cv=25%). 2.4.3 quality control, reproducibility genomic dna from healthy donors was used as control material and was analysed with each master-mix. cv over an approx. six-month period was 15% for her-2 copy detection and 14% for b2m copy detection. 2.4.4 repeatability repeatability in the normal area was determined by performing repeated measurements on the same sample. 20 ml of whole blood was collected from two healthy donors. the plasma obtained from each donor was divided into 10 portions and the dna purified and analysed on abi7900ht. the first donor had a mean of 1901 her-2 copies/ml (cv=20%) and a mean of 1621 b2m copies/ml (cv=23%). the results from the second donor showed a mean of 2490 her-2 copies/ml (cv=22%) and a mean of b2m copies/ml of 2280 (cv=17%). 2.4.5 carry-over dna purification was done using the qiasymphony robot, which placed water samples between plasma samples in order to detect possible cross-contamination. no cross-contamination was detected. water samples were also used as a negative control for each master-mix – non template control (ntc). no positive ntcs were detected. 2.4.6 reference range reference range was determined using values from healthy controls. plasma from 50 healthy controls was analysed for her-2, b2m, cpp1 and pb. seven controls were excluded because of b-cell contamination. the remaining 43 healthy controls had values between 1591 and 6252 her-2 copies/ml (mean 3556) and 97.5 percentile 6244 her-2 copies/ml (95%ci 5637 –6252). the values for the reference gene b2m were between 1377 and 6019 b2m copies/ml (mean 2923) and 97.5 percentile 5975 b2m copies/ml (95%ci 4626–6019). 2.4.7 analytical sensitivity the detection limit was determined according to a dilution experiment where 100,000 her-2/b2m copies from a normal cell-line mcf10a were added to 100 microliters of water. after analyses, repeated dilutions were performed (first two-fold, then four-fold then 10-fold, 100-fold, 1000fold and 10,000-fold). the mean value for the lowest detection limit was 40 her-2 copies/ml (range 20–80 copies/ml; cv=42%) and 20 b2m copies/ml (range 0–20 copies/ml; cv=42%). 2.4.8 analytical specificity the primers and probe ensured analytical specificity. a blast search was performed and there were no other predicted amplified domains. her-2 and b2m gene amplification products were both run in agarose gel and each showed one band corresponding to the predicted amplicon length (fig. 1). figure 1. in gel: her-2 gene pcr product corresponding to 76 bp and b2m gene pcr product corresponding to 102 bp 2.4.9 method comparison page et al. used a similar method in their article about the detection of her-2 amplification in cfdna from breast cancer patients. however, they used other primers for targeting the her-2 gene, another reference gene (gapdh), and a delta cq for determining the her-2 gene amplification. 2.5 serum her-2 protein serum her-2 was previously analysed using the advia centaur assay, which is an automated sandwich immuno‐ assay that uses direct chemiluminescent technology and two monoclonal antibodies specific to unique epitopes on the extracellular domain of the her-2 receptor [15]. serum her-2 values above 15 μg/l were considered positive. analytical cv% in the period was determined by commer‐ cial and in-house controls at 8, 14 and 113 μg/l; the values were 6.6, 4.6 and 4.8, respectively. 2.6 pathological analyses with ihc/fish tissue her-2 was routinely assessed with ihc and fish. ihc 3+ or ihc 2+ and fish>2.0 were considered positive. ihc and fish were performed on formalin-fixed paraffinembedded breast-cancer tissue. ihc was analysed using the hercep test (dakocytomation, glostrup, denmark) and fish was analysed with the her-2 fish pharmdx kit (dakocytomation). 4 j circ biomark, 2015, 4:9 | doi: 10.5772/61320 2.6.1 statistical analysis statistical analyses were performed on stata ic 11. a mann-whitney test was used to compare two patient groups. a fisher-exact or chi2 test were used to compare data from 2x2 tables. 3. results the characteristics of the follow-up patients are presented in table 1. in total, 1,142 analyses were performed on plasma from the followed patients, with each patient having a minimum of five samples. eighty-five patient samples and seven samples from the healthy controls that were positive for pb (a sign of b-cell-lysis contamination) were excluded from the data evaluation. thirteen patient samples with haemolysis were also excluded. age, years <=40 1 41-50 19 51-60 31 61-70 33 >=70 16 tissue her-2 status positive 50 negative 50 tumour size at diagnosis (mm) t1 40 t2 52 t3 3 t4 2 tx 3 no. of positive nodes at diagnosis n1 83 n2 12 n3 0 nx 5 metastases status at diagnosis m0 100 histological type invasive ductal carcinoma 88 invasive lobular carcinoma 6 other 5 unknown 1 oestrogen receptor status positive 69 negative 31 progesterone receptor status positive 53 negative 44 unknown 3 table 1. patient population characteristics: 100 follow-up patients twenty-five follow-up patients with initial primary breast cancer developed metastatic disease in the monitoring period. the results from the follow-up patients who developed metastatic disease were analysed together with the results from the additional patients with metastases. 3.1 preoperative plasma/serum samples from tissue her-2positive patients vs. healthy controls of the 50 preoperative plasma samples from patients with tissue her-2-positive primary tumour, seven samples were excluded because of b-cell-lysis contamination. there was no significant difference between preoperative plasma cfher-2 dna or cfb2m dna from tissue her-2-positive patients and healthy controls (suppl. table 1, figure 2a). there was no correlation between preoperative plasma cfher-2 dna and the corresponding serum her-2 protein (range 6–52, mean 14.8, median 13 μg/l) in the tissue her-2-positive patient group (p=0.14). 3.2 preoperative plasma/serum samples from tissue her-2negative patients vs. healthy controls of the 50 preoperative plasma samples from patients with tissue her-2-negative primary tumour, four samples were excluded because of b-cell-lysis contamination. there was no significant difference between preoperative plasma cfher-2 dna from tissue her-2-negative patients and healthy controls (suppl. table 1). the preoperative plasma cfb2m dna values from tissue her-2-negative patients were lower than the cfb2m dna values from healthy controls (p=0.02) (figure 2b). there was no correlation between preoperative plasma cfher-2 dna and the corresponding serum her-2 protein (range 6–21, mean 12, median 11.75 μg/l) in the tissue her-2-negative patient group (p=0.96). 3.3 ability of cfher-2 dna to detect metastatic recurrence compared with serum her-2 protein 3.3.1 samples taken before detection of metastatic disease compared to patients without metastasis (tissue-positive patients) of the 50 tissue her-2-positive patients, 18 patients developed metastatic disease in the monitoring period, while 32 remained clinically relapse-free. the plasma/ serum sample from patients with metastases was taken within nine months of detection of symptomatic metasta‐ ses. for the patients without metastatic disease, the highest value taken in a period without chemotherapy was chosen. three plasma samples from patients with metastasis were excluded because of b-cell contamination. there was no significant difference between the plasma cfher-2 dna or cfb2m dna values in patients before the development of metastatic disease and those in patients without metastasis (suppl. table 2). there was a significant difference between the corresponding serum her-2 levels 5patricia diana sørensen, rikke fredslund andersen, niels pallisgaard, jonna skov madsen, erik hugger jakobsen and ivan brandslund: plasma cfher-2 dna in breast cancer taken before the development of metastatic disease (range 4–1729, mean 162, median 22.3 μg/l) and serum her-2 levels in patients without metastasis (range 5–18, mean 10, median 10.1 μg/l) (p=0.0003). in our previous work [10], we found a good correlation between increased serum her-2 values and the development of metastatic disease in tissue-positive patients. serum her-2 prior to metastases detection (tissue-positive) with metastases without metastases total elevated 8 3 11 normal 7 29 36 total 15 32 47 cfher-2 dna prior to metastases detection (tissue-positive) with metastases without metastases total elevated 8 9 17 normal 7 23 30 total 15 32 47 cfb2m dna prior to metastases detection (tissue-positive) with metastases without metastases total elevated 6 9 15 normal 9 23 32 total 15 32 47 table 2. number of tissue her-2-positive patients with elevated or normal serum her-2, cfher-2 dna and cfb2m dna levels prior to metastases detection we have also examined the ability of cfher-2 dna and cfb2m dna to detect metastatic recurrence compared to serum her-2 protein using cut-offs obtained from healthy controls, which were 6244 her-2 copies/ml for cfher-2 dna and 5975 b2m copies/ml for the reference gene b2m. for the serum her-2 protein, we used the known cut-off of 15 microgram/l. the results in the tissue her-2-positive group showed a significant difference between the meta‐ static and non-metastatic group in the serum her-2 protein values (p=0.002 with a sensitivity of 53% [95%ci 27–77] and a specificity of 90% [95%ci 73–97]) but no significant difference for cfher-2 dna (p= 0.09 with a sensitivity of 53% [95%ci 27–77] and a specificity of 71% [95%ci 53–85]) or for cfb2m dna (p=0.41 with a sensitivity of 40% [95%ci 17–67] and a specificity of 71% [95%ci 53–85]) (table 2). 3.3.2 samples taken before detection of metastatic disease compared with patients without metastasis (tissue-negative patients) of the 50 tissue her-2-negative patients, 10 patients developed metastatic disease in the monitoring period, while 40 remained clinical relapse-free. the plasma/serum sample from patients with metastasis was taken within nine months of the detection of symptomatic metastasis. there was no significant difference between plasma cfher-2 dna or cfb2m cna in patients before the development of metastatic disease and patients without metastasis (suppl. table 3). there was no significant difference between the corresponding serum her-2 levels taken before the development of metastatic disease (range 10.3–36.7, mean 17, median 14 μg/l) and serum her-2 from patients without metastasis (range 7.3–23.4, mean 13, median 12 μg/l) (p=0.13). in our previous article [10], we found no correlation between increased serum her-2 values and the development of metastatic disease in tissuenegative patients. using the previously specified cut-offs, the results in the tissue her-2-negative group showed no difference between the metastatic and non-metastatic group for either serum her-2 protein (p=0.3 sensitivity of 40% [95%ci 13–72], specificity 72% [95%ci 55–84]), cfher-2 dna (p= 0.1, sensitivity of 40% [95%ci 13–72], 0 1 0 ,0 0 0 2 0 ,0 0 0 3 0 ,0 0 0 plasma healthy controls preoperative plasma tissue her-2 positive cfher-2 dna (copies/ml) cfb2m dna (copies/ml) 0 2 ,0 0 0 4 ,0 0 0 6 ,0 0 0 8 ,0 0 0 1 0 ,0 0 0 plasma healthy controls preoperative plasma tissue her-2 negative cfher-2 dna (copies/ml) cfb2m dna (copies/ml) (a) (b) figure 3 figure 2. (a) preoperative plasma cfher-2 dna and cfb2m dna levels from tissue her-2-positive patients compared with levels from healthy controls. (b) preoperative plasma cfher-2 dna and cfb2m dna from tissue her-2-negative patients compared with levels from healthy controls. 6 j circ biomark, 2015, 4:9 | doi: 10.5772/61320 specificity of 85% [95%ci 69–93]) or cfb2m dna (p=0.1 with a sensitivity of 30% [95%ci 8–64], and a specificity of 90% [95%ci 75–96]) (table 3). serum her-2 prior to metastases detection (tissue-negative) with metastases without metastases total elevated 4 11 15 normal 6 29 35 total 10 40 50 cfher-2 dna prior to metastases detection (tissue-negative) with metastases without metastases total elevated 4 6 10 normal 6 34 40 total 10 40 50 cfb2m dna prior to metastases detection (tissue-negative) with metastases without metastases total elevated 3 4 7 normal 7 36 43 total 10 40 50 table 3. number of tissue her-2-negative patients with elevated or normal serum her-2, cfher-2 dna and cfb2m dna levels prior to metastases detection 3.4 detection of her-2 gene amplification in plasma cfdna cfher-2 dna/cfb2m dna ratio levels had a range of 0.69– 31.88 (mean 1.95, median 1.38) in 590 follow-up samples from 50 tissue her-2-positive patients. samples from the tissue her-2-negative patients (454) had a ratio-level range of 0.75–2.76 (mean 1.38, median 1.34). there was a signifi‐ cant difference between the ratio levels of the tissuepositive patients compared with those of the tissuenegative patients (p=0.03) (figure 3). if a cut-off of 2.5 (which is 99 percentiles from the tissue her-2-negative values) was chosen, 46 (8%) samples from the tissue her-2-positive group had a ratio >2.5 and four (0.9%) samples from the tissue-negative group had a ratio >2.5. 3.5 ratio levels from preoperative samples in the preoperative setting, there was no significant difference between the ratio levels of the tissue her-2positive patients compared with those of the tissue her-2negative patients or the healthy controls (suppl. table 4). one preoperative sample from the tissue her-2-positive group had ratio >2.5 and the same was observed for the tissue-negative group. 3.6 ratio levels before detection of metastatic disease ratio levels from samples taken within nine months before the detection of metastatic disease in 15 tissue-positive patients were compared with ratio levels from 10 tissuenegative patients, but the results showed no statistically significant difference between the two groups (table 4). when the results were compared with the ratio levels from healthy controls, there was a statistically significant difference between the ratio levels of healthy controls and the ratio levels of tissue-positive patients (p=0.0001) but also between the ratio levels of healthy controls and the ratio levels of tissue-negative patients (p=0.001). three (of 15) tissue her-2-positive patients had ratio >2.5 prior to metastases detection. regarding the tissue-negative group, none of the patients (out of 10) had ratio >2.5 prior to metastases detection. 0 1 0 2 0 3 0 t is su e h e r 2 ne gat ive w ith ih c / f i s h t is su e h e r 2 pos it iv e w ith i h c / f i s h r a ti o c fh e r 2 d n a / c fb 2 m d n a figure 3. ratio levels of cfher-2 dna/cfb2m dna in tissue her-2-negative patients (n=454) compared to tissue her-2-positive patients (n=590) 7patricia diana sørensen, rikke fredslund andersen, niels pallisgaard, jonna skov madsen, erik hugger jakobsen and ivan brandslund: plasma cfher-2 dna in breast cancer ratio her-2/b2m ratio p-value p-value tissue her-2positive patients (n=15) range 0.92–6.77 0.34 0.0001 mean 2.26 median 1.524 tissue her-2negative patients (n=10) range 0.98–2.23 1.0 0.001 mean 1.55 median 1.520 healthy controls (n=47) range 0.08–1.59 1.0 mean 1.23 median 1.25 table 4. ratio between cfher-2 dna and cfb2m dna prior to metastases detection 3.7 ratio levels after detection of metastatic disease we also examined the ratio levels after the diagnosis of metastatic disease prior to the start of palliative chemo‐ therapy both in the available samples from the study population (nine tissue her-2-positive and eight tissue her-2-negative patients) and in the extra samples (14 tissue her-2-positive and eight tissue her-2-negative patients), in total 39 patients: 23 tissue her-2-positive and 16 tissue her-2-negative. in the post-metastatic/pre-chemotherapy setting, there was a significant difference between the ratio levels in the tissue her-2-positive group and those in the tissue-negative group (p=0.01) (table 5). in the tissue her-2-positive group, 11 patients had ratio >2.5 while in the tissuenegative group none had ratio >2.5. ratio her-2/b2m ratio p-value tissue her-2-positive patients (n=23) range 0.9–10.7 0.01 mean 3.55 median 2.4 tissue her-2-negative patients (n=16) range 0.9–2.23 1.0 mean 1.42 median 1.4 table 5. ratio between cfher-2 dna and cfb2m dna after metastases detection 4. discussion the results gained in our study showed no difference between the absolute preoperative cfher-2 dna values for patients with primary breast cancer, regardless of tissue her-2 status, and absolute cfher-2 dna values for healthy controls. the explanation here could be that the tumour is localised, and that very little tumour dna is released into circulation. the same was observed for the levels of the reference gene b2m, which was used to express the total level of cfdna in the tissue her-2-positive group. the results published in the literature on the levels of cfdna in patients with primary breast cancer are contra‐ dictory. most studies show a higher level of total cfdna in plasma or serum from patients with primary breast cancer compared with healthy controls [16-19], but others find no difference between the two groups [20, 21]. holdenrieder et al. [21] found no difference in plasma cfdna concentra‐ tions between patients with benign diseases and patients with various types of primary cancer including breast cancer. one recent study [20] has investigated her-2 gene ampli‐ fication in plasma cfdna and the total cfdna in patients with primary breast cancer during neoadjuvant chemo‐ therapy. the authors found no difference in the baseline total plasma cell-free dna or her-2 gene-copy number compared to healthy controls, and no difference in baseline plasma her-2 gene-copy number between tissue her-2positive and -negative patients; nor were they able to detect gene amplification in plasma in the tissue her-2-positive patient group with primary breast cancer. the discrepancies between cfdna analysis results could be explained by different sample material (serum or plasma), different assays, or b-cell-lysis contamination. a recent study performed at our department by andersen et al. [22] found plasma more suitable than serum for cfdna analysis. b-cell-lysis contamination may cause false positive values. in the present study, a control assay was used which detects rearranged genes that are only present in lymphocytes (pb assay), and in this way it was possible to exclude contaminated samples. furthermore, not all the performed studies are reported according to the miqe guidelines. regarding the patients who developed metastatic disease in the present study, no difference was found between cfher-2 dna or cfb2m dna levels taken before (within nine months of) the development of metastatic disease and those taken for patients without metastases. neither specific cfher-2 dna nor the total cfdna (reference gene b2m) could predict the development of metastatic disease. on the other hand, in the tissue her-2-positive patient group, there was a significant difference between the corresponding serum her-2 protein levels before recur‐ rence developed and those for patients without recurrence. the difference in the tissue-negative group was not significant. this agrees with the previous work on serum her-2 protein [10], where a high correlation was found between the increase in serum her-2 and metastatic recurrence for tissue-positive patients, though the correla‐ 8 j circ biomark, 2015, 4:9 | doi: 10.5772/61320 tion was not significant for the tissue-negative group after adjustment for confounders with a logistic regression analysis. regarding the her-2 gene amplification in plasma, the ratio between cfher-2 dna and the reference gene cfb2m dna was significantly higher in the tissue her-2-positive group compared with the tissue-negative group when all the patient values were used. the values after the devel‐ opment of metastasis could also be useful in a clinical setting in order to differentiate between her-2-positive and -negative disease, since it is known that amplification status is not always the same in metastatic tissue as in the primary tumour. after the development of metastatic disease and prior to start of treatment, there was a signifi‐ cant difference between the ratio levels in the tissue her-2positive group and those in the tissue-negative group. others have examined the same issue. page et al. [8] found amplification in cfdna in eight out of 68 patients following treatment for primary breast cancer and also in five out 30 patients with metastatic disease, but not in 22 patients with primary breast cancer. bechmann et al. [20] included 15 patients with diagnosed metastatic disease (nine tissue her-2-positive and six tissue her-2-negative) in their study and found significantly higher plasma her-2 amplification in the tissue-positive group compared to healthy controls, but not for the tissue-negative group. sorensen et al. [9] found plasma her-2 amplification in 50% (14 of 28) of patients with tissue her-2-positive metastatic breast cancer using a cut-off value of 2xsd above the mean of the controls. 5. conclusion we conclude that in our population, neither absolute values of specific cfher-2 dna nor the total cfdna (reference gene b2m) could discriminate between patients with primary breast cancer and healthy controls, or predict the development of metastatic disease. amplified her-2 dna can be detected in plasma, mostly in the tissuepositive group and after the diagnosis of metastatic disease. more studies are needed in order to validate the usefulness of detection of amplified cfher-2 dna in plasma from breast cancer patients. improvement of the method of detection of cell-free dna could facilitate interpretation of results and increase both the sensitivity and the specificity of the analysis. in our population, with the current meth‐ ods, serum her-2 protein seems more sensitive and reliable in detecting metastatic recurrence than cell-free dna. 6. abbreviations ca 15-3: cancer antigen 15-3; cea: carcinoembryonic antigen; her-2: human epidermal growth factor receptor 2; ihc: immunohistochemistry; fish: fluorescence in-situ hybridization; fec: fluorouracil, epirubicin, cyclophos‐ phamide; cpp1: cysteine-rich polycomb-like protein 1; pb: peripheral blood; b2m: beta-2 microglobulin; hplc: highperformance liquid chromatography; cq: cycle threshold; gapdh: glyceraldehyde 3-phosphate dehydrogenase; ntc: non template control 7. conflict of interests ivan brandslund has received honoraria from siemens denmark for two lectures about serum her-2 protein. 8. references [1] dbcg danish breast cancer cooperative group guidelines. 2011 jun 15. [2] leon sa, shapiro b, sklaroff dm, yaros mj. free dna in the serum of cancer patients and the effect of therapy. cancer res 1977;37:646-50. [3] anker p, mulcahy h, chen xq, stroun m. detection of circulating tumour dna in the blood (plasma/ serum) of cancer patients. cancer metastasis rev 1999;18:65-73. [4] pathak ak, bhutani m, kumar s, mohan a, guleria r. circulating cell-free dna in plasma/serum of lung cancer patients as a potential screening and prognostic tool. clin chem 2006;52:1833-42. [5] van der vaart m and pretorius pj. the origin of circulating free dna. clin chem 2007;53:2215. [6] van der vaart m and pretorius pj. characterization of circulating dna in healthy human plasma. clin chim acta 2008;395:186. [7] pinzani p, salvianti f, pazzagli m, orlando c. circulating nucleic acids in cancer and pregnancy. methods 2010;50:302-7. [8] page k, hava n, ward b, brown j, guttery ds, ruangpratheep c, blighe k, sharma a, walker ra, coombes rc, shaw ja. detection of her2 amplifi‐ cation in circulating free dna in patients with breast cancer. br j cancer 2011;104:1342-8. [9] sorensen bs, mortensen ls, andersen j, nexo e. circulating her2 dna after trastuzumab treat‐ ment predicts survival and response in breast cancer. anticancer res 2010;30:2463-8. [10] sorensen pd, jakobsen eh, madsen js, petersen eb, andersen rf, ostergaard b, brandslund i. serum her-2: sensitivity, specificity, and predictive values for detecting metastatic recurrence in breast cancer patients. j cancer res clin oncol 2013;139:1005-13. [11] cvitanich c, pallisgaard n, nielsen ka, hansen ac, larsen k, pihakaski-maunsbach k, marcker ka, jensen eo. cpp1, a dna-binding protein involved in the expression of a soybean leghemo‐ globin c3 gene. proc natl acad sci usa 2000;97:8163-8. [12] pallisgaard n, spindler kl, andersen rf, brand‐ slund i, jakobsen a. controls to validate plasma 9patricia diana sørensen, rikke fredslund andersen, niels pallisgaard, jonna skov madsen, erik hugger jakobsen and ivan brandslund: plasma cfher-2 dna in breast cancer samples for cell free dna quantification. clin chim acta 2015;446:141-6. [13] belgrader p, tanner sc, regan jf, koehler r, hindson bj, brown as. droplet digital pcr measurement of her2 copy number alteration in formalin-fixed paraffin-embedded breast carcino‐ ma tissue. clin chem 2013;59:991-4. [14] heredia nj, belgrader p, wang s, koehler r, regan j, cosman am, saxonov s, hindson b, tanner sc, brown as, karlin-neumann g. droplet digital pcr quantitation of her2 expression in ffpe breast cancer samples. methods 2013;59:s20-s23. [15] luftner d, cheli c, mickelson k, sampson e, possinger k. advia centaur her-2/neu shows value in monitoring patients with metastatic breast cancer. int j biol markers 2004;19:175-82. [16] gal s, fidler c, lo ym, taylor m, han c, moore j, harris al, wainscoat js. quantitation of circulating dna in the serum of breast cancer patients by realtime pcr. br j cancer 2004;90:1211-5. [17] kohler c, radpour r, barekati z, asadollahi r, bitzer j, wight e, burki n, diesch c, holzgreve w, zhong xy. levels of plasma circulating cell free nuclear and mitochondrial dna as potential biomarkers for breast tumors. mol cancer 2009;8:105. [18] zanetti-dallenbach r, wight e, fan ax, lapaire o, hahn s, holzgreve w, zhong xy. positive correla‐ tion of cell-free dna in plasma/serum in patients with malignant and benign breast disease. anti‐ cancer res 2008;28:921-5. [19] zhong xy, ladewig a, schmid s, wight e, hahn s, holzgreve w. elevated level of cell-free plasma dna is associated with breast cancer. arch gynecol obstet 2007;276:327-31. [20] bechmann t, andersen rf, pallisgaard n, madsen js, maae e, jakobsen eh, bak jylling am, steffensen kd, jakobsen a. plasma her2 amplification in cellfree dna during neoadjuvant chemotherapy in breast cancer. j cancer res clin oncol 2013;139:995-1003. [21] holdenrieder s, burges a, reich o, spelsberg fw, stieber p. dna integrity in plasma and serum of patients with malignant and benign diseases. ann n y acad sci 2008;1137:162-70. [22] pallisgaard n, spindler kl, andersen rf, brand‐ slund i, jakobsen a. controls to validate plasma samples for cell free dna quantification. clin chim acta, 446 (2015), p. 141-146). 10 j circ biomark, 2015, 4:9 | doi: 10.5772/61320 cbx766966 1..10 report strategies for enumeration of circulating microvesicles on a conventional flow cytometer: counting beads and scatter parameters mohammad j alkhatatbeh1, anoop k enjeti2,3,4,5, sara baqar6,7, elif i ekinci6,7, dorothy liu6,7, rick f thorne3,4, and lisa f lincz2,3,4 abstract enumeration of circulating microvesicles (mvs) by conventional flow cytometry is accomplished by the addition of a known amount of counting beads and calculated from the formula: mv/ml ¼ (mv count/bead count) � final bead concentration. we sought to optimize each variable in the equation by determining the best parameters for detecting ‘mv count’ and examining the effects of different bead preparations and concentrations on the final calculation. three commercially available bead preparations (trucount, flow-count and countbright) were tested, and mv detection on a bd facscanto was optimized for gating by either forward scatter (fsc) or side scatter (ssc); the results were compared by calculating different subsets of mv on a series of 74 typical patient plasma samples. the relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (r2 � 0.997). however, trucount beads produced the most consistent (concentration variation ¼ 3.8%) calculated numbers of plasma cd41 þ /annexin v þ mv, which were significantly higher from that calculated using either flow-count or countbright (p < 0.001). the facscanto was able to resolve 0.5 mm beads by fsc and 0.16 mm beads by ssc, but there were significantly more background events using ssc compared with fsc (3113 vs. 470; p ¼ 0.008). in general, sample analysis by ssc resulted in significantly higher numbers of mv (p < 0.0001) but was well correlated with enumeration by fsc for all mv subtypes (r¼ 0.62–0.89, p < 0.0001). we conclude that all counting beads provided linear results at concentrations ranging from 6 beads/ml to 100 beads/ml, but trucount was the most consistent. using ssc to gate mv events produced high background which negatively affected counting bead enumeration and overall mv calculations. strategies to reduce ssc background should be employed in order to reliably use this technique. keywords flow cytometry, absolute counting, microvesicle, microparticles, extracellular vesicles, submicron particles, scatter date received: 21 november 2017; accepted: 21 february 2018 1 department of clinical pharmacy, faculty of pharmacy, jordan university of science and technology, irbid, jordan 2 haematology unit, calvary mater newcastle, new south wales, australia 3 hunter medical research institute, new lambton, new south wales, australia 4 faculty of health and medicine, university of newcastle, new south wales, australia 5 pathology north hunter, nsw health pathology, new south wales, australia 6 department of endocrinology, austin health, victoria, australia 7 department of medicine, austin health, the university of melbourne, victoria, australia corresponding author: lisa f lincz, haematology unit, level 4, new med building, calvary mater newcastle, edith street, waratah, new south wales 2298, australia. email: lisa.lincz@calvarymater.org.au journal of circulating biomarkers volume 7: 1–10 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418766966 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). http://orcid.org/0000-0002-1612-2382 http://orcid.org/0000-0002-1612-2382 mailto:lisa.lincz@calvarymater.org.au https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418766966 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage introduction microvesicles (mvs) are a type of extracellular vesicle (ev) that bud off directly from the plasma membrane of activated or dying cells. 1 they are small (<1 mm) membrane-bound particles that can be identified by their cell surface markers, and they circulate throughout the body carrying biological remnants of their cells of origin. 1 mvs are regularly found in body fluids, including human plasma. 2 their numbers and constitution have been documented to change in times of pathological conditions, and as such, they are considered ideal biomarkers for diagnosis and prognosis of various disorders. 3 however, absolute mv counts vary widely between studies, suggesting that their detection and accurate quantification remains a challenge. despite many new ‘nano’ technologies emerging in recent years, enumeration and analysis of circulating mv by flow cytometry continues to be the method of choice. 4 in spite of its sizing limitations, flow cytometry offers one of the few means to simultaneously detect multiple subsets of mvs and is also the most likely method to be easily adopted for clinical purposes owing to its widespread use in diagnostic pathology. standardization remains a priority, and there have been numerous reports on how different variables can affect mv measurements. these variables include pre-analytical variables, such as blood collection and timing, needle gauge, anticoagulants, sample transport, centrifugation and storage; testing variables, such as choice of antibody and fluorochrome; and analytical variables, such as brand of flow cytometer, fluorescence compensation approaches, threshold settings and gating strategies. 4–9 to this end, the international society on thrombosis and haemostasis (isth) has been instrumental in developing consensus guidelines detailing best practice recommendations for evaluation of circulating mvs by flow cytometry. 7,10,11 more recently, isth has collaborated with other key organizations, the international society of extracellular vesicles (isev) and the international society on advancement of cytometry, to develop a comprehensive set of methodological guidelines for collection, isolation and measurement of ev using a range of common techniques. 12 in addition, isev published ‘minimal information for studies of ev’ in 2014, providing advice on methods and reporting of ev isolation, characterization and functional studies. 13,14 such standardization is imperative to reduce the variability within and between methods in order to allow comparison between studies as well as develop diagnostic parameters for routine testing of circulating mvs. although the newer generation of flow cytometers can measure volume and thus provide absolute particle counts, more conventional instruments without this capability can still be used successfully for quantitative mv detection. 15–17 an often overlooked aspect of mv enumeration using such classical flow cytometry methods involves the spiking of samples with a known amount of commercially available counting beads to enable calculation of the concentration of mv in the starting material. the general formula for this is: mv=ml ¼ðmv count= bead countþ �ð total number of beads= test volumeþ hence, more variables are introduced, which can ultimately affect the final mv result. while all of the variables in the above formula can be manipulated in each experiment, there is limited information on changes of bead concentration and its impact on total mv enumeration. counting beads are distinct from the small (<1.0 mm) sizing beads available to establish mv gating parameters, and unlike the latter, there are no commercially available counting beads specific for mv enumerations; hence, much larger beads, meant for lymphocyte enumeration, are generally employed. few manufacturers specify the final analysis volume, leaving the bead concentration up to the individual researcher. hence, it is unknown whether the relationship remains linear over a wide range of bead concentrations or whether this differs between bead types and sizes. as for the parameters used to determine the ‘mv count’, the main testing variable is the actual flow cytometer and gating strategies employed. enumeration and analysis of such small particles can be performed using forward scatter (fsc) or side scatter (ssc) as the main sizing parameter. the choice is usually determined by the type of instrument being used; with wide angle fsc (1–19�) machines such as beckman coulter (brea, california, usa) generally performing better using fsc, compared with low angle fsc (1–8�) machines such as bd biosciences (san jose, california, usa), which typically perform better using ssc. a thorough cross-instrument evaluation has been performed to standardize gating parameters between the different types. 7 however, for those cytometers that perform equally well on either parameter, it is difficult to know which provides more reliable results. thus, the aims of this study were to (i) compare different concentrations of commercially available counting beads to establish their limitations and optimal parameters and (ii) determine whether fsc or ssc is a better sizing parameter for enumeration of circulating mvs by flow cytometry on a single bd facscanto (bd biosciences). materials and methods subjects a series of 74 platelet-free plasma (pfp) samples from patients with type ii diabetes were chosen as representative of a typical patient cohort for analysis. these samples were collected from patients (aged 50–75 years, body mass index (bmi) 25–35 kg/m 2 ) recruited between 2014 and 2016 from diabetes clinics at the department of endocrinology in austin health, melbourne, victoria, as part of a separate 2 journal of circulating biomarkers study on circulating mv levels in type ii diabetes. the study followed the guidelines set out within the australian national statement on ethical conduct in human research (2007; updated may 2015) and was approved by the austin health (hrec/12/austin/63) and hunter new england area human research ethics and governance committees (ssa/15/hne/141). all procedures were conducted in accordance with the helsinki declaration of 1975, as revised in 2008, and written informed consent was obtained from all participants. blood processing peripheral blood was collected into 3.2% sodium citrate and processed at room temperature within 2 h of collection. whole blood was centrifuged at 400�g for 15 min to separate the cellular fraction from the plasma. the latter was carefully removed, transferred to a fresh tube and further centrifuged at 2100�g for 15 min. all but the bottom 500 ml was transferred to a fresh tube and centrifuged again at 2100�g for 15 min to produce pfp. this was aliquoted, stored at �80�c and then thawed at 37�c immediately prior to analysis. antibody staining of pfp for mv analysis staining of mv was performed as previously described. 11,18 a 10 ml aliquot of pfp was incubated at room temperature for 30 min with various combinations of antibodies conjugated to phycoerythrin (pe), fluorescein isothiocyanate (fitc), allophycocyanin (apc) or pe-cyanine (pe-cy5): cd41-pe (clone pl2-49, biocytex, marseille, france; platelet marker), cd42b-fitc (clone hip1, bd pharmingen, san diego, california, usa; platelet marker), cd235aapc (clone ga-r2 (hir2), bd pharmingen; erythrocyte marker), cd105-pe (clone 1g2, beckman coulter; endothelial marker), cd31-pe (clone wm59, bd pharmingen; against endothelial marker pecam-1), cd62e pe-cy5 (clone 68-5h11, bd pharmingen, against activated endothelial marker e-selectin) and annexin v-apc (ebioscience, san diego, california, usa; phosphatidylserine). all assays were diluted to a final volume of 500 ml in phosphate-buffered saline (without ca 2þ and mg 2þ ) or calcium-rich binding buffer (for those stained with annexin v), with the addition of a known quantity of counting beads and 15 mm d-phe-pro-arg-chloromethylketone (ppak) to inhibit clumping. counting beads three different popular brands of counting beads were compared: trucount (bd biosciences; size not specified), flow-count (beckman coulter; 10 mm diameter) and countbright (molecular probes, eugene, oregon, usa; 7 mm diameter). each bead preparation was diluted into the final test volume at the indicated concentration by adding exact quantities based on the individual lot concentration provided by the manufacturer. the plasma source, concentration and acquiring time on the flow cytometer were kept constant. for experiments comparing fsc with ssc, countbright beads were used at a final concentration of 50 beads/ml. analysis of mv by flow cytometry all flow cytometry analyses were performed on a standard configuration bd facscanto (bd biosciences) equipped with two lasers (488 nm and 640 nm). the facs flow pressure was set to 3.0 lbf/in 2 and the low flow rate adjusted to a factor of 0.61 (decreased from the original factory settings of 4.5 lbf/in 2 and 0.75, respectively) to improve resolution at smaller sizes. analysis of mv was performed as previously described 11,18 and according to guidelines established by the isth vascular biology scientific standardization committee on the standardization of platelet microparticle enumeration by flow cytometry incorporating modifications suggested for the bd facscanto (bd biosciences). 10 the cytometer was calibrated for fsc resolution using megamix sizing beads (a blend of 2:1:1 of 0.5, 0.9 and 3 mm diameter fluorescent beads) or for ssc resolution using megamix-plus ssc (a mixture of 0.16 mm, 0.20 mm, 0.24 mm and 0.5 mm beads) both purchased from biocytex. voltages were set at fsc ¼ 570 v and ssc ¼ 390 v for fsc detection or fsc ¼ 350 v and ssc ¼ 631 v for ssc detection. the lower mv detection limits were set according to the manufacturer’s instructions, with thresholds of fsc ¼ 200/ssc ¼ 200 employed for fsc enumeration and ssc ¼ 3200 for ssc gating. fluorescent voltages were set to 654 v for fitc, 485 v for pe, 544 v for pecy5 and 400 v for apc (with the exception of annexin vapc detected at 500 v). counting beads were detected on percp-cy5.5 at 290 v using fsc gating and 549 v using ssc gating. events were collected for 60 s (bead experiments) or 120 s (patient samples; to enable adequate number of counting bead events 19 ) at low flow rate prior to analysis using facs diva software (bd biosciences). the absolute number of mv in each plasma sample was calculated using the formula: mv/ml ¼ (mv count/bead count) � (total # beads/test volume). statistical analysis data for continuous variables are expressed as mean + standard deviation or median (interquartile range) where appropriate. variables that were not normally distributed were analysed using non-parametric tests. differences in mean levels of multiple normally distributed continuous variables were assessed using one-way analysis of variance (anova) with post hoc scheffe test for multiple comparisons. mann–whitney u test and wilcoxon matched-pair tests were used to detect differences between medians for individual and paired data, respectively. correlations alkhatatbeh et al. 3 between continuous variables were assessed by pearson’s product-moment or spearman’s rho (r) where appropriate. all calculations were performed with statistica v10.0 (statsoft, tulsa, oklahoma, usa) or stata v11 (statacorp llc, college station, texas, usa) using two-tailed tests, and p values <0.05 were considered statistically significant. results comparison of counting beads the manufacturer’s method was followed as closely as possible to prepare a series of identical plasma samples containing varying concentrations of three different brands of counting beads: trucount (bd biosciences), flowcount (beckman coulter) and countbright (molecular probes), respectively. all tests were performed in triplicate. figure 1 shows that for all three brands of counting beads tested, the relationship between the number of beads added to each test and the number of beads counted by flow cytometry remained linear over a wide range of bead concentrations (up to 200 beads/ml), as indicated by all correlation coefficients (r 2 ¼ 0.999, 0.997, 0.997, respectively), which were close to 1.0. importantly, these relationships were maintained even at the lowest bead concentrations of 6.26 beads/ml (equivalent to adding just 3125 beads to a 500 ml test). however, the slope (m) of the lines fitted for the countbright (m ¼ 1.54) and flow-count (m ¼ 1.51) beads was slightly higher than that of the trucount beads (m¼1.22). thus, the absolute number of trucount bead events became significantly different from that of the countbright and flow-count at concentrations greater than 50 beads/ml (p < 0.05). we next sought to determine whether the number of raw mv events counted was stable in the presence of different levels of counting beads. as indicated by the dotted lines in figure 1, the number of cd41 þ /annexin v þ raw events was on average 428 + 42 and was not significantly different in tubes with added counting beads versus tubes without counting beads (data not shown). this was with the exception of tubes containing 200 beads/ml of flow-count beads that derived significantly less mv events than expected (p < 0.001), indicating that spiking with high amounts of flow-count beads interferes with mv detection. the numbers of bead and cd41 þ /annexin v þ events were then used to calculate the final concentration of cd41 þ /annexin v þ mvs in each sample for the three brands of counting beads at different concentrations (table 1). with the exception of the highest concentration figure 1. correlation between the number of bead events counted versus added to each test and mv events counted using different brands of absolute counting beads. solid lines and filled icons represent bead events, whereas dotted lines and outlined icons represent corresponding raw cd41þ/annexin vþ mv events detected by flow cytometry for each bead dilution. *p � 0.05 for trucount versus countbright and/or flow-count bead events; **p � 0.001 for flow-count versus trucount and/or countbright mv events. mv: microvesicle. 4 journal of circulating biomarkers of flow-count beads (this data point was omitted from the overall analysis), the results were consistent within each bead manufacturer, showing no significant difference between the calculated values at all bead concentrations (within manufacturer anova p values ¼ 0.685, 0.417, 0.479, respectively). trucount tubes gave the highest overall consistency, with a concentration variation of 3.80% compared to 8.86% and 7.50% for flow-count and countbright, respectively. the number of calculated mv events was highest using trucount beads (3768 + 143 mv/ml), and this was significantly different from that calculated using flow-count (3058 + 271 mv/ml, p < 0.001) and countbright (2886 + 217 mv/ml; p < 0.001). comparison of fsc versus ssc for gating mv by flow cytometry in an attempt to optimize the number of raw mv events detected by flow cytometry, we compared two alternate gating strategies on our facscanto, one using fsc and the other using ssc as the main sizing parameter. fluorescent beads of known diameters selected to cover a major part of the theoretical mv size range (0.1–1.0 mm) were used to determine resolution aptitude and establish appropriate mv gates. as the relative position of biological mvs and beads in ssc is different from that in fsc, reference beads of sizes specifically designed for each parameter were used. as shown in figure 2, the facscanto was equally capable of adequately resolving the respective bead mixtures by either fsc or ssc. threshold parameters were set to exclude as much background as possible, leaving the mv gates set to capture all events below the 0.5 mm bead cloud using fsc and all events between the 0.2 and 0.5 mm bead limits detected using ssc. these mv gates have been shown to be equivalent in order to allow inter-platform comparisons of mv counts. 19 the respective mv gates were used to detect six different mv subsets in a series of 74 patient plasma samples. the number and type of bead were kept constant (countbright beads were added to all samples at a final concentration of 50 beads/ml), and events were collected from the same tube for 120 s on each gating parameter. table 2 presents the number of raw events detected using the respective mv gates as well as the number of countbright beads counted. these amounts were determined in the absence of added patient plasma at the beginning of each run (n ¼ 9) in order to establish the amount of background electronic noise detected using either parameter. this consistently showed significantly more background events in the mv gate using ssc compared to fsc (3113, 2098–23,860 vs. 470, 404–3994; p ¼ 0.008), but the number of bead events detected remained equivalent (786, 733–804 vs. 822, 730–865; p ¼ 0.374). with the addition of individual plasma samples, events detected in the mv gate were significantly higher using ssc compared to fsc (119,640, 84,320– 180,233 vs. 12,476, 7530–27,211; p < 0.00001) with an increase that was disproportionate and could not be explained by the initial higher background noise events. in addition, the number of beads counted was significantly reduced in the presence of plasma using ssc (730, 697–771 vs. 822, 730–865 without plasma; p ¼ 0.008) and when plasma containing samples were measured on ssc compared to fsc (730, 697–771 vs. 766, 738–793; p < 0.0001). in contrast, the number of beads enumerated remained stable when fsc was used to count beads in samples with or without additional plasma (766, 738–793 vs. 786, 733–804; p ¼ 0.520). this is further illustrated in figure 3, which shows a strong negative correlation between the number of countbright beads counted and the number of events in the mv gate when ssc but not fsc is used as the main gating parameter. table 1. calculated number of cd41 þ /annexin v þ mvs using different manufacturer’s brands of counting beads at different concentrations. trucounttm flow-count countbrighttm final bead concentration per ml mean + stdev %cv mean + stdev %cv mean + stdev %cv 200 3397 + 211 6.2 32 + 16 50.1 2621 +49 1.9 100 3912 + 449 11.5 2591 + 613 23.7 2841 + 159 5.6 50 3581 + 203 5.7 3086 + 249 8.1 2653 + 153 5.8 25 3745 + 204 5.4 3154 + 244 7.7 3001 + 171 5.7 12.5 3910 + 638 16.3 3290 + 452 13.7 3147 + 870 27.6 6.26 3693 + 632 17.1 3167 + 565 17.8 3051 + 217 7.1 mean + stdev 3768 + 143 10.4 3058 + 271a 14.21a 2886 + 217 9.0 %cv 3.80 8.86a 7.50 p values 0.685 0.417 a 0.479 <0.001 0.724 between brands <0.001 cv: coefficient of variation; mv: microvesicle; stdev: standard deviation. a omitting 200 beads/ml results. alkhatatbeh et al. 5 not surprisingly, calculation of plasma mv concentrations using data derived from ssc analysis resulted in significantly higher absolute amounts of most mv subsets compared to analysis using fsc (p < 0.0001; figure 4(a)). however, the results for each mv subset were individually well correlated between the two methodologies (r¼ 0.619–0.992; p < 0.0001; figure 4(b)). discussion absolute mv counts vary widely between studies, with circulating platelet mv levels ranging from hundreds to thousands even in control populations. 20–22 although much attention has been given to the many pre-analytical and methodical variables that can result in such discrepancies, 23 few have addressed the addition of the all-important table 2. comparison of raw mv events and beads counted in the presence or absence of plasma using fsc or ssc as the flow cytometry sizing parameter. number of events in mv gate a number of beads counted a without plasma (n ¼ 9) with plasma (n ¼ 74) without plasma (n ¼ 9) with plasma (n ¼ 74) p value fsc 470 (444–656) 12,476 (7530–27,211) 786 (733–804) 766 (738–793) 0.520 ssc 3113 (2497–4727) 119,640 (84,320–180,233) 822 (730–865) 730 (697–771) 0.008 p value 0.008 <0.00001 0.374 <0.00001 mv: microvesicle. avalues are presented as median (interquartile range). figure 2. flow cytometry resolution of sizing beads and mv gate settings using fsc versus ssc as the main size parameter. (a) the histogram in the top panel shows resolution of 0.5 mm (orange) and 0.9 mm (yellow) megamix beads by fsc. the same beads are depicted in the dot plot below where the 0.9 mm bead cloud is used to set the mv gate. (b) the histogram in the top panel shows resolution of 0.16 mm (pink), 0.20 mm (blue), 0.24 mm (green) and 0.5 mm (red) megamix-plus ssc beads using fitc as the main parameter. the same beads are depicted in the dot plot below using ssc as the threshold to eliminate the 0.16 mm (pink) beads and use the 0.5 and 0.2 mm bead clouds to set the mv gate. the gate for capturing the counting beads is depicted in blue on both dot plots. 6 journal of circulating biomarkers counting beads that enable such calculations. herein, we compared three different popular brands of fluorescent absolute counting beads: trucount by bd biosciences, which is supplied as a lyophilized pellet in individual flow cytometry tubes; flow-count (beckman coulter) and countbright (molecular probes), both of which are supplied as slurries. all are brightly fluorescent and intended to be used for determining absolute counts of leucocytes in blood. our results show that although the number of beads counted by the flow cytometer remained linear over a wide range of concentrations for all three brands tested, the trucount beads gave the most accurate enumerations with the least variation in mv calculated levels. this is perhaps due to the lyophilized format of the beads that may deter clumping and thus minimize the potential for pipetting error that would be more common with slurries. however, the single-use tubes are the most expensive of the three preparations, contain a set amount of beads and require the presence of proteins (such as from plasma or serum) for proper performance, which may limit the utility of such tubes for other, such as purified, mv preparations. although all manufacturers specify a certain amount of beads to use per test (equating to 50,000–100,000 beads), only bd provides a recommended final test volume of 520 ml (100 beads/ml) of their trucount beads, while countbright manufacturers warn to maintain a final volume of at least 300 ml per test (143 beads/ml). despite this, both brands performed well at concentrations of up to 200 beads/ml. of concern was the finding that high levels of flow-count beads (� 200 beads/ml) significantly interfered with the detection of mv particles. the underlying reasons for inhibition are unclear, and we cannot discount factors other than the beads themselves, such as proprietary stabilizers added by the manufacturer. nevertheless, our findings are instructive to delineate the concentration parameters where flow-count beads can be used to measure mv. we did not count 1000 bead events as suggested by the manufacturers of flow-count and countbright because our mv enumeration protocol has always been based on that recommended by the isth standardization papers. the first of these employed 30 ml of flow-count beads in a final volume of 580 ml (approximately 50 beads/ml) and a timed collection of events for 60 s at low flow rate. 10 on our instrument this allows for counts of 350–400 beads, but obviously this varies between laboratories and will be highly dependent on the fluidics pressure of individual machines. we, therefore, have adopted the latest recommendation to increase the collection time to 120 s if the number of beads counted is <500. 19 however, the results presented herein suggest that this will not make any significant difference to the calculated mv results for major populations that are readily detectable (i.e. approximately 400 positive events detected in the mv gate). perhaps a better guide would be to collect a minimum number of mv events of interest. original attempts to standardize flow cytometry analysis of mv using fsc as the main sizing parameter proved that reproducible platelet-derived (cd41 þ ) mv counts could be obtained across many different laboratories worldwide. 10 however, this success was not always shared by laboratories using bd instruments, which demonstrated discrepancies between location of sizing beads and that of biological particles on the fsc parameter, thought to be due to the relatively lower solid angle used to collect fsc signals on these instruments. this could be ameliorated by removing the upper gate limit set by the megamix beads, a recommendation adopted for the current study. 10 however, the same authors found a more reproducible solution in using ssc as the main sizing parameter, with the use of different sized reference beads for fsc versus ssc being the most critical element for standardization across the different platforms. 7,19 our bd instrument produced less variability in mv subset calculations using fsc. although we found higher figure 3. correlation between the number of beads counted and the number of mv events detected by ssc. scatterplots show relationship between the number of beads counted and the number of mv events detected in the mv gate when using (a) fsc or (b) ssc as the main detection parameter. alkhatatbeh et al. 7 background events detected by the ssc channel compared to fsc, these were not above the recommended background noise threshold ratio of 1.0 (calculated as the number of events per second/maximal number of events per second acceptable by the instrument ¼ [3113/60]/4000 ¼ 0.012 for ssc on our facscanto) and consistent, if not better, then most instruments surveyed by cointe et al. 19 however, the significantly increased number of mv events detected in plasma suggests that ssc may be a more sensitive parameter for small particles, resulting in much higher absolute counts for the majority of mv subsets. this phenomenon was not observed across different instruments in the isth multicentre workshop, with similar counts recorded between instruments using ssc or fsc as the preferred sizing parameter. 19 however, the results were highly variable, with standard deviations of up to 50% of the mean for identical samples measured on different instruments. hence, only a paired study design would be able to address the difference between results measured by different scatter parameters on individual flow cytometers. the utility of employing polystyrene beads to establish sizing gates for biological material has been the source of much contention. it is well known that polystyrene has a much higher refractive index, resulting in light scattering properties much different from plasma membranes. 24,25 a 400 nm polystyrene microsphere has been shown to produce the same forward light scatter as a 1 mm lipid or cellular vesicle. 24 the megamix gating strategy originally figure 4. results of mv subsets calculated from data using fsc or ssc as the main detection parameter. (a) bar graph illustrating differences in concentrations of mv subsets when calculated from data obtained using fsc versus ssc as the main detection parameter. bars represent median and interquartile ranges. (b) correlation of individual mv subset concentrations when calculated from data obtained using fsc versus ssc as the main detection parameter. scatter graphs are presented on log scales. *p < 0.0001. mv: microvesicle; fsc: forward scatter; ssc: side scatter. 8 journal of circulating biomarkers established by the scientific standardization committee set the upper size detection limit using 900 nm beads, but this has been estimated in actuality to gate biological vesicles measuring 800–2400 nm in diameter. 25 such discrepancies have led to proposals by us and others that triggering on fluorescence may provide a much more useful approach. 6,26,27 however, plasma contains many different sizes and shapes of particles, some as small as 30 nm, 28,29 and current technology in flow cytometry remains biased towards detection of only the largest and brightest particles, with many events destined to be lost in the instrument ‘noise’. 23 much more sensitive detection and sizing methods of nanoparticle tracking analysis and/or resistive pulse sensing can provide more accurate measurement of ev concentrations and have confirmed that total plasma ev is highly underestimated by flow cytometry. 28,30,31 the current study is limited by the few different types of counting beads assayed and the use of a single flow cytometer. smaller sized counting beads such as the 5.2 mm cytocount (dako, agilent pathology solutions, santa clara, california, usa) are becoming more popular and would have made a welcome comparison to the larger flow-count and countbright beads used here. similarly, all the analyses were done on a single flow cytometer, rendering it the equivalent of a technological ‘case study’. it would be of interest to compare our results to other facscanto machines as well as other newer instruments with integrated cell counting. nonetheless, we have highlighted the importance of bead selection, concentration and background minimization for mv analysis by flow cytometry. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: this study was funded in part by a hunter medical research institute (hmri) special project grant, supported by lions district 201 n3 diabetes foundation, awarded to rft and lfl. maintenance for the bd facscanto was supplied by the hunter cancer research alliance. mja was supported by jordan university of science and technology. orcid id lisa f lincz http://orcid.org/0000-0002-1612-2382 references 1. raposo g and stoorvogel w. extracellular vesicles: exosomes, microvesicles, and friends. j cell biol 2013; 200: 373–383. 2. george j, thoi l, mcmanus l, et al. isolation of human platelet membrane microparticles from plasma and serum. blood 1982; 60: 834–840. 3. enjeti ak, lincz lf 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momen-heravi3,8, jie hu4, xin zhang4, ying wu5, hongguang zhu5, jiping wang6, jian zhou4 and winston patrick kuo3,7 1 northern ontario school of medicine, thunder bay, ontario, canada 2 borgess hospital, kalamazoo, mi, usa 3 harvard catalyst laboratory for innovative translational technologies, harvard medical school, boston, ma, usa 4 liver cancer institute, zhongshan hospital, fudan university, shanghai, china 5 department of pathology, shanghai medical college, fudan university, shanghai, china 6 division of surgical oncology, brigham and women’s hospital, harvard medical school, usa 7 department of developmental biology, harvard school of dental medicine, cambridge, ma, usa 8 department of medicine, university of massachusetts medical school, worcester, ma, usa *corresponding author(s) e-mail: zsuntres@nosm.ca received ; accepted 26 march 2013 doi: 0000 © 2013 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract exosomes are membrane vesicles with a diameter of 40-100 nm that are secreted by many cell types into the extracel‐ lular milieu. exosomes are found in cell culture superna‐ tants and in different biological fluids and are known to be secreted by most cell types under normal and pathological conditions. considerable research is focusing on the exploitation of exosomes in biological fluids for biomarkers in the diagnosis of disease. more recently, exosomes are being exploited for their therapeutic potential. exosomes derived from dendritic cells, tumor cells, and malignant effusions demonstrate immunomodulatory functions and are able to present antigens to t-cells and stimulate antigenspecific t-cell responses. exosomes have also been exam‐ ined for their therapeutic potential in the treatment of infections such as toxoplasmosis, diphtheria, tuberculosis and atypical severe acute respiratory syndrome as well as autoimmune diseases. attempts to find practical applica‐ tions for exosomes continue to expand with the role of exosomes as a drug delivery system for the treatment of autoimmune/inflammatory diseases and cancers. keywords exosomes, therapy, delivery 1. introduction exosomes are membrane vesicles with a diameter of 40-100 nm, a sub-fraction of extracellular vesicles that are secreted by many cell types into the extracellular milieu [1, 2]. they are equivalent to cytoplasm enclosed in a lipid bilayer with the external domains of transmembrane proteins exposed to the extracellular environment. exosomes form in a particular population of endosomes, called multivesicular bodies (mvbs), by inward budding into the lumen of the compartment. upon fusion of mvbs with the plasma membrane, these internal vesicles are secreted. while the biological function of exosomes is still under investigation, they can mediate communication between cells, provide a protective effect against or induce intra-and extracellular stress and are involved in the exchange of functional genetic information [1-3]. exosomes are found in cell culture supernatants and in different biological fluids and are known to be secreted by most cell types under normal and pathological conditions. so far, exosomes have been found to be 1j circ biomark, 2013, 1:0 | doi: 0000 released by all cells examined today such as b-cells, dendritic cells (dcs), t-cells, mast cells, epithelial cells, and platelets and have been found to be present in physiological fluids, such as bronchoalveolar lavage (bal) fluid, serum, urine, breast milk, cerebrospinal fluid, saliva, and malignant effusions [4-14]. the presence of exosomes in biological fluids could be exploited for biomarkers in the diagnosis of disease [12, 13, 15-20]. the protein composition of exosomes has been character‐ ized using immunoblotting [21], peptide mass spectrosco‐ py mapping [22], and affinity extraction into magnetic beads, followed by phenotyping by flow cytometry [23]. exosomes derived from dendritic cells [22, 24], b lympho‐ cytes [21], intestinal epithelial cells [25] and other cell types [26-33] revealed the presence of common as well as cell type specific proteins. exosomes from different cellular origins sequester a common set of molecules that are essential for their biogenesis, structure and trafficking – as well as celltype specific components which, presumably, reflect the biological function of the parent cell. ubiquitous proteins in exosomes include cytoplasmic proteins, such as tubulin, actin and actin-binding proteins, the heat shock proteins hsp70 and hsp90, and trimeric g proteins, as well as membrane proteins, such as members of the tetraspanin family (cd9, cd63, cd81, cd82) [37], which have been suggested to be involved in cell adhesion, activation, proliferation and antigen presentation. in addition to the conserved set of proteins, exosome functionality seems to be determined by cell-type specific proteins that reflect the specialized function of the original cells. for example, exosomes originated from dendritic cells are enriched with major histocompatibility complex (mhc) class i and ii and express co-stimulatory molecules like cd54, cd80 and cd86 (also known as icam-1, b7-1 and b7-2, respectively) suggesting a t-cell stimulatory capacity [15, 22, 24, 32-34]. exosomes from synovial fluid contain citrullinated pro‐ teins (eg. fibrin α-chain fragment, fibrin β-chain, fibrinogen d fragment and spα receptor) which might play an important role in converting nonimmunogenic proteins into autoimmunogenic proteins [35]. exosomes collected in the urine contain aquaporin-2 which might be used as a biomarker for renal diseases [36]. lipids found on exosomes are characteristic of the cell origin, with most of the lipid analytical work being performed on exosomes derived from cancer cells, reticu‐ locytes, mast cells, b lymphocyte cell lines and human dcs [37-40]. the typical lipid composition of mast cell-derived exosomes includes lysophosphatidylcholine, sphingomye‐ lin, phosphatidylcholine, phosphatidylserine, phosphati‐ dylethanolamine, cholesterol and diglyceride [40]. although most of these lipids are also present on exosomes isolated from other cell types, the ratios of these lipids vary. for example, the ratio of cholesterol/phospholipid is lower in exosomes derived from mast cells and reticulocytes when compared with b-cell-derived exosomes [39]. 2. isolation techniques exosomes represent only a small fraction of all components present in a culture medium, cytosol or body fluids. recognizing the fact that the exosomes have a size that range between 40 to 100 nm, with density ranging between 1.13 and 1.21 g/ml, and contain cell type specific proteins, isolation procedures have focused on techniques based on size and density and biochemical properties. usually, exosomes have been isolated by serial centrifugation of culture supernatant and body fluids to eliminate cells and debris which consists of multiple steps: first, a low speed spin (300 x g for 10 minutes) which eliminates dead cells and bulky apoptotic debris, followed by higher speed spins, which varies among different protocols, from 1000 x g to 20,000 x g and eliminates larger vesicles and debris. a final high speed spin at 60,000-100,000 x g for a period of more than 1 hour precipitates a pellet, which consists of extracellular vesicles including exosomes. some protocols integrate the usage of filtration steps (like 0.8 µm and 0.22 µm filters) and spinning to eliminate cell debris and other contaminants. however, ultracentrifugation results in the formation of a pellet that could aggregate exosomes with other vesicles, particles, apoptotic bodies or other cell debris and interfere with purification. moreover, applying excessive centrifugation force and time may lead to rupturing the exosomes. also, taking advantage the density properties of exosomes, they can be purified from protein aggregates, apoptotic bodies and nucleosomal fragments by floatation into a sucrose density gradient. this procedure can eliminate impurity with composition different from that of exosomes. however, with these procedures, we can only obtain exosomes heterogeneous with microvesicles, because current methods could not distinguish a 50-100nm “exosome” from a 50-200nm “microvesicle”. these processes result in variable recovery of the starting amount of exosomes [13, 41-44]. although this branch of science is growing so fast, the quality and purity of these methods for exosomes preparation do not fulfill the common good manufacturing practice (gmp) criteria. because exosomes prepared in this way are easily contaminated with media proteins and contain only 5-25% of starting concentration. instead of differential centrifuga‐ tion, a newer method for purifying clinical grade (cgmp) exosomes derived from antigen presenting cells employs ultrafiltration cartridges and pumps and is especially useful for purifying exosomes from large volumes (>1 liter) of conditioned medium. more specifically, the ultrafiltra‐ tion process were incorporated through a 500-kda nm wco hollow fiber cartridge that allowed the passage of unaggregated media proteins through the pores of the membrane, while retaining aggregate proteins in the retentate without significant changes on the composition and performance of the media [44]. as it is believed, the protein aggregates are much more immunogenic than the soluble form because of preferential capture by antigen presenting cells [45]. thus, the removal of co-purifying 2 j circ biomark, 2013, 1:0 | doi: 0000 proteins such as human haptoglobin and albumin aggre‐ gates prevents their undesirable immune responses to serum components [45-48]. furthermore, during previous co-purifying with exosomes, these proteins can reach a higher concentration in the final product making it an essential aspect for the purification of cgmp exosomes. another isolation method, which is based on biochemical composition of exosomes utilizes magnetic beads coated with monoclonal antibody specific for a protein known to be present on the exosome membrane. for example, with the use of antibody-coated magnetic beads, using antibod‐ ies against tumor-specific proteins, it has been possible to collect her2-expressing tumor exosomes from the culture supernatant of breast adenocarcinoma cell lines and ascites of an ovarian cancer patient [41, 43, 49]. 3. therapeutic use of exosomes in light of the fact that exosomes secreted by neoplastic cells are close copies of the originating cells in terms of their antigenicity, the use of exosomes in cancer immunotherapy holds promise. for example, melanoma-derived exosomes contain the highly immunogenic antigens melana/mart-1 and gp100 and those released by colon carcinoma cells express cea and her2. this antigenic content is not only a feature of in vitro-released exosomes, but also can be found in microvesicles (or fragments of plasma membrane ranging from 50 nm to 1000 nm shed from almost all cell types) isolated from plasma of cancer patients as well, evidence that demonstrates the tumor origin of these organelles [14, 50]. exosomes containing tumor antigens have been shown to stimulate cd4+and cd8+t cells and exosomes from in vitro cultured antigen presenting cells (apcs) administered in vivo can induce t-cell responses resulting in inhibition of tumor growth [51-53]. also, dendritic cell-derived exosomes pulsed with tumorderived antigens elicit potent antitumor t-cell responses and tumor regression in experimental animals [14]. however, while translating findings from mouse to human, we should be cautious about the difference between human and mouse immune systems. despite many features conserved between human and mouse systems, there are substantial differences between them. although extensive conservation exists when comparing activated immune t-cells, the pro-inflammatory response of mice is distinct from humans. importantly, canonical th17 differentiation signature (il17a, f, il23r, rorc, batf, and ccl20) is different in human either because of an inherently higher responsiveness of the th17 module in human or presence of fast reactive memory t-cells in human cells. in contrast, activation of cd24 or lag3 seemed exclusive to mouse cells. moreover, after pre-stimulation in similar conditions, mouse cd4 t cells activated slightly weaker than humans. phase i clinical trials in human cancer evaluated the effectiveness of patient-specific exosomes released by dendritic cells and loaded with tumor antigen-derived peptides (dexosomes [dex]) for melanoma and non-small cell lung cancer and showed that dexosome immunother‐ apy was feasible, safe and led to the induction of both innate and adaptive immune responses, disease stabilization and long-term survival for several patients [54, 55]. also, ascites-derived exosomes derived from colorectal cancer patients were shown to be safe, nontoxic, and tolerable when used as a cancer vaccine, and in combination with gm-csf can efficiently induce potent carcinoembryonic antigen (cea)-specific antitumor immunity in advanced colorectal cancer patients [56]. it should be noted however that the potential antitumor effects of tumor-derived exosomes is still unclear as evidenced by the fact that in cancer patients with advanced disease, tumor-derived exosomes do not exert any effective immune-stimulatory or antitumor effects despite the abundant production of tumor-derived exosomes [53]. tumor-derived exosomes have also been shown to be immunosuppressive with direct administration of tumor-derived exosomes actually resulting in promoted tumor growth [53, 54]. tumorderived exosomes were shown to directly suppress the activity of effector t cells or target myeloid cells to modu‐ late their differentiation and function such as in the case where exosomes derived from human melanoma cell lines and colorectal carcinoma cell lines were demonstrated to skew monocyte differentiation into dcs toward the generation of myeloid-derived suppressor cells (mdscs) and exert tgf-β1 mediated suppressive activity on t cells in vitro [53-55]. a better characterization of tumor-derived exosomes and understanding of their effects on cancer pathogenesis are warranted to further improve their use in cancer chemotherapy. exosomes are favorable as vaccine candidates in infections such as toxoplasmosis, diphtheria, tuberculosis and atypical severe acute respiratory syndrome. toxoplasmo‐ sis is induced by the obligate intracellular parasite toxo‐ plamsa gondii. it has been reported that transfer of dcs pulsed with t. gondii antigens (tag) to healthy mice induced protection against a virulent oral challenge of t. gondii but this approach is limited due to difficulty to obtain high quantity of dcs suitable for vaccination [57-59]. an alternative to dc-based vaccines being investigated is the ability of exosomes, especially those derived from dcs, to induce protective immune responses. exosomes secreted by srdc (cd8α+cd4− dc cell line) pulsed in vitro with toxoplasma gondii-derived antigens (exo-tag) induced protective responses against infection with the parasite in both syngeneic and allogeneic mice. after oral infection, syngeneic cba/j mice exhibited significantly fewer cysts in their brains and allogeneic c57bl/6 mice survived immune protection is associated with the induction of humoral and cellular tag-specific responses[60]. exosomes have been examined for their therapeutic potential in the treatment of other infectious diseases as well. it has been shown that murine bone marrow-derived dcs (bmdcs) pulsed in vitro with intact diphtheria toxin 3zacharias e. suntres, milton g. smith, fatemeh momen-heravi, jie hu, xin zhang, ying wu, hongguang zhu, jiping wang, jian zhou and winston patrick kuo: therapeutic uses of exosomes (dt)-released exosomes, which upon injection into mice induce immunoglobulin g (igg)2b and igg2a responses specific for dt [61]. infection with m. tuberculosis primes macrophages for the increased release of exosomes and microvesicles bearing m. tuberculosis peptide-mhc-ii complexes that may generate antimicrobial t-cell respons‐ es [62, 63]. exosomes as a vaccine has also been explored in infection with the sars-associated coronavirus (sarscov) known to induce an atypical pulmonary disease with a high lethality rate. studies by kuate et al. demonstrated that exosomes containing spike s protein of sars-cov induced neutralizing antibody titres and this immune response was further enhanced by priming with the sarss exosomal vaccine and then boosting with the currently used adenoviral vector vaccine [64]. exosomes may be potential candidates as vaccines for allergic diseases. exosome-like vesicles isolated from the bronchoalveolar lavage fluid of tolerized mice by respira‐ tory exposure to the olive pollen allergen ole e 1 or found to induce tolerance and protection against allergic sensiti‐ zation in mice [65]. serum containing exosomes from ovafed experimental animals can induce tolerance to ova when injected into naïve recipients [66, 67]. exosomes found in breast milk [68] contain molecules such as muc-1, mhc class i and ii, cd86 and heat-shock proteins (hsps) and have an immune regulatory role as they inhibit il-2 and ifn-γ production and induce treg cells (foxp3+cd4+cd25+cells); however, the biological role of exosomes in milk and their impact on allergy development remains under investigation [15]. exosomes have also proved useful in treatment of autoimmune diseases in animal models. kim et al. showed that administration of exosomes derived from dcs-expressing recombinant il-4 was able to modulate the activity of apc and t cells in vivo, partly through a fasl/fas-dependent mechanism, resulting in effective treatment against collagen-induced arthritis through suppression of the delayed-type hypersensitivity inflam‐ matory response [69]. also, vaccination of mice with exosomes from il-10, fasl, and indoleamine 2,3-dioxyge‐ nase-modified dc reduced the clinical manifestation of mice with rheumatoid arthritis [70-75]. exosomes from tgf-β1-modified dcs reduced disease activity and incidence of intestinal bleeding in a murine model of inflammatory bowel disease (ibd)[75, 76]. more recently, exosomes have been seen as an alternative to liposomes in the delivery of therapeutic agents [77-79]. exosomes are comprised of natural non-synthetic compo‐ nents, and their small size and flexibility enables them to cross major biological membranes, while their bi-lipid structure protects the cargo from degradation, facilitating delivery to its target [80, 81]. in addition, these naturallyoccurring secreted membrane vesicles are less toxic, and better tolerated in the body as evidenced by their ubiqui‐ tous presence in biological fluids [80]. for example, exosomes have been used to deliver anti-inflammatory agents, such as curcumin, to activated myeloid cells in vivo. immune dysfunction is properly investigated during various tumor growth and progression. cd8+cytotoxic t lympocytes play a substantial role in antigen-specific tumor destruction and cd4+t cells assists cd8+t-cells in this scenario. tumors frequently target and inhibit t-cell function to evade from immune response. curcumin has been shown to inhibit the suppressive activity of t-cells via down-regulation of the production of tgf-β and il-10 in t-cells as well as increasing the ability of effectors t-cell to destroy cancer cells. curcumin delivered by exosomes is more stable and more highly concentrated in the blood [82]. exosomes can be used therapeutically to target egfrexpressing cancerous tissues with nucleic acid drugs [83]. in this situation, targeting can be achieved by engineering the donor cells to express the transmembrane domain of platelet-derived growth factor receptor fused to the ge11 peptide. intravenously injected exosomes delivered let-7a mirna to egfr-expressing xenograft breast cancer tissue in rag2(-/-) mice [83]. exosomes are natural carriers of rna making them a valuable tool for the delivery of rna interference (sirna) and microrna (mirna) regulatory molecules in addition to other single-stranded oligonucleotides [84]. it has been demonstrated that exosomes can be used as vehicles for delivering sirna to suppress the growth of cancer cells [85]. in addition, tumor-suppressive mirnas delivered via exosomes confer a gene silencing effect on recipient cells, inhibiting cancer proliferation [86]. dendritic cell (dc)derived exosomes have been exploited for targeted rnai delivery to the brain after systemic injection [87]. similarly, encapsulation of bace (a therapeutic target in alzheimer’s disease) sirna in exosomes derived from dendritic cells expressing lamp2b, an exosomal membrane protein that reduces immunogenicity, fused to the neuron-specific rvg peptide resulted in delivery of bace sirna to the brain and decreased gene expression in neurons, microglia and oligodendrocytes in the brain [88]. exosomes are being considered as a potential therapeu‐ tic tool in modulating neovascularization. activation of neovascularization can lead to healing of wounds and reconstruction of hypoxic injury while hampering neovascularization delays tumor development [89]. exosomes secreted from human cd34(+) cells have angiogenic activity in isolated endothelial cells and in murine models of vessel growth and have been postulat‐ ed to represent a significant component of the paracrine effect of progenitor cell transplantation for therapeutic angiogenesis enhancing recovery from ischemic disease or injury [90]. endothelial-derived exosomes carrying proteins such as delta-like 4 (a transmembrane ligand for notch receptors that is expressed in arterial blood vessels and sprouting endothelial cells) and matrix metalloprotei‐ nases lead to angiogenesis [66, 67, 89]. 4 j circ biomark, 2013, 1:0 | doi: 0000 in conclusion, investigation in exosome biology has been a relatively new area of research and much work remains to be done to ensure the safe and effective use of exosomes for therapeutic applications. exosomes appear to be noncytotoxic and well tolerated. as our understanding of the biology of exosomes intensifies, so will the range of principles for the design of exosomes and exosomal conjugates used in the development of immunotherapeu‐ tics, vaccines, and angiogenesis modulators. the role of exosomes as a next generation drug delivery system appears to be advantageous over existing drug delivery systems because of their small size, lack of toxicity and target specificity although loading of exosomes without compromising their biological properties remains a challenge. 4. acknowledgements this work was conducted with support from harvard 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[90] sahoo s, klychko e, thorne t, misener s, schultz km, millay m, ito a, liu t, kamide c, agrawal h et al: exosomes from human cd34(+) stem cells mediate their proangiogenic paracrine activity. circ res 2011, 109(7):724-728. 9zacharias e. suntres, milton g. smith, fatemeh momen-heravi, jie hu, xin zhang, ying wu, hongguang zhu, jiping wang, jian zhou and winston patrick kuo: therapeutic uses of exosomes cbx782617 1..10 research article improved ovarian cancer emt-ctc isolation by immunomagnetic targeting of epithelial epcam and mesenchymal n-cadherin joseph w po1,2, aflah roohullah1,2,3, david lynch1,2, anna defazio4,5,6, michelle harrison3,7, paul r harnett4,5,6, catherine kennedy4,5,6, paul de souza1,2,3,8, and therese m becker1,2,4,8 abstract epithelial cell adhesion molecule (epcam)-targeted capture remains the most common isolation strategy for circulating tumor cells (ctcs). however, epithelial-to-mesenchymal transition (emt) leads to decreased epithelial epcam expression affecting the optimal ctc capture. in this study, we tested a cohort of ovarian cancer cell lines using flow cytometry to identify n-cadherin as the additional immunomagnetic cell surface target for ovarian cancer cell isolation. combined immunomagnetic targeting of mesenchymal n-cadherin and epithelial epcam enriched ctcs from advanced ovarian cancer patient blood approximately three times more efficiently than targeting of epcam alone. we also show that more emt-phenotype ctcs are captured by including n-cadherin targeting into ctc isolation protocols. however, after n-cadherin-based ctc isolation, in some blood samples of healthy individuals, we also observed the presence of cells expressing markers common to ctcs. our data show that these “false positives” can be largely distinguished from ctcs as circulating endothelial cells (cecs) by vascular endothelial–cadherin co-staining. cec counts are highly variable in patients and healthy controls. our data demonstrate that a combination of epcam with n-cadherin-targeted isolation can improve ctc detection and widen the emt-phenotype spectrum of captured ctcs. keywords ctc, emt, epcam, n-cadherin, ve-cadherin, vimentin date received: 1 january 2018; accepted: 11 april 2018 1 centre for circulating tumour cell diagnostics and research, ingham institute for applied medical research, liverpool, new south wales, australia 2 school of medicine, western sydney university, campbelltown, new south wales, australia 3 department of medical oncology, liverpool hospital, liverpool, new south wales, australia 4 centre for cancer research, westmead institute for medical research, westmead, new south wales, australia 5 the university of sydney, new south wales, australia 6 the crown princess mary cancer centre westmead, westmead hospital, new south wales, australia 7 department of medical oncology, chris o’brien lifehouse, camperdown, new south wales, australia 8 south western clinical school, university of new south wales, liverpool, new south wales, australia corresponding author: therese becker, ingham institute for applied medical research, 1 campbell st., liverpool, new south wales 2170, australia. email: t.becker@unsw.edu.au journal of circulating biomarkers volume 7: 1–10 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418782617 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:t.becker@unsw.edu.au https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418782617 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage introduction ovarian cancer is the fifth leading cause of cancer in women and the leading cause of gynecological cancer death worldwide. while 70–80% of patients initially respond to first-line platinum-based chemotherapy, others have intrinsically resistant tumors. further, the majority (70%) of advanced-stage patients will eventually develop treatment resistance. 1,2 the analysis of circulating tumor cells (ctcs) is emerging as a promising way to monitor cancer progression and the effectiveness of therapy. ctcs are cells that have shed from the primary or metastatic tumor and intravasated into the blood stream. ctc isolation and analysis can give an insight into the disease biology and its behavior. expectedly, high ctc counts have been correlated with disease progression and poorer prognosis in colorectal, breast, and prostate cancers (reviewed by caixeiro et al. 3 ). a comprehensive study in ovarian cancer patients (n ¼ 216) showed that ctc counts above two at therapy commencement correlated with poorer progression-free and overall survival. 4 another study found that enhanced ctc counts predicted relapse or progression in 31 epithelial ovarian cancer patients. 5 although some smaller studies found no correlation of ctcs with disease progression, four recent metaanalyses showed that ctc positivity in ovarian cancer patients was significantly associated with shorter overall, disease-free, and progression-free survival as well as advanced stage in ovarian cancer. 6–9 potential diagnostic application of ovarian cancer patient ctcs include ctc ercc1 transcript detection associated with platinum resistance, detection of ctc clusters associated with platinum resistance, and in vitro assaying of platinum sensitivity in cultured ctcs which correlated with patient response. 10–12 thus, although ctcs are often considered of minor relevance in ovarian cancer because it metastasizes mainly throughout the peritoneum, a view that was challenged by data using an elegant parabiosis mouse model, ovarian cancer ctcs appear to have value as biomarkers. 13,14 currently, the most common method of ctc isolation relies on immunomagnetic cell capture by targeting the epithelial cell adhesion molecule (epcam). however, with epcam expression lost or reduced, ctc detection may be difficult, 15,16 and there is evidence for epcam heterogeneity in ovarian cancer cells. 17 moreover, epcam is downregulated during epithelial-to-mesenchymal transition (emt), a process that is implicated in the metastatic spread of cancer and especially the egress of ctcs into the circulation. 18 a recent study evaluating epithelial and mesenchymal gene expression of ovarian cancer patient ctcs before and after chemotherapy suggested that platinum-based therapy enriches emt-like ctcs. 19 similarly, emt-phenotype changes may be a marker of resistance to platinum therapy as shown for ovarian cancer cell lines, 20 and gradual change towards emt gene expression signatures in ovarian cancer tissue during progression to platinum resistance was correlated with poor prognosis. 21,22 quite contrary, another study indicates that the epithelial cell phenotype combined with high nuclear factor kb activity is associated with ovarian cancer platinum resistance. 23 taking the evidence together, liquid biopsies and ctc analysis may provide important predictive and prognostic information, and heterogeneity in resistance mechanisms suggest that both epithelial and mesenchymal cells need to be investigated to follow changes of disease progression biomarkers in a representative population of ctcs. a well-characterized central step during emt is the expression switch of the epithelial cell–cell adhesion molecule e-cadherin to the mesenchymal cell–cell adhesion molecule n-cadherin (reviewed by lamouille et al. 24 ), and an e-cadherin-to-n-cadherin switch was shown in ovarian cancer tissue at progression from stage ii to stage iii. 25 therefore, in this study, we assessed e-cadherin, epcam, and n-cadherin expression on the surface of ovarian cancer cell lines to identify n-cadherin, in addition to epcam, as a useful target for immunomagnetic ctc isolation. we demonstrate that additional ctcs are isolated by combining epcam with n-cadherin-targeted ctc isolation by establishing a method to identify emt-phenotype ctcs. materials and methods patients patients were recruited from liverpool cancer therapy centre and the crown princess mary cancer centre westmead. clinical information was sourced from patient medical records. information at the time of blood sampling was collected including age and primary cancer site. treatment information was collected including chemotherapy regimen, previous lines of therapy prior to ctc isolation, serum ca-125 levels, and radiological assessments (online supplementary table s1). blood samples from healthy individuals were analyzed as controls. cell culture ovarian cancer cell lines a2780, caov3, colo316, es2, ovcar3, peo1, peo4, peo14, skov3, and the wme099 ebv-transformed human b-lymphocyte cell line were maintained in rpmi 1640 media (lonza, basel, switzerland) supplemented with 10% fetal bovine serum (fbs) (interpath, melbourne, australia) in a humidified incubator with 5% atmospheric carbon dioxide at 37�c. all cell lines were authenticated by short tandem repeat (str) profiling (australian genome research facility, melbourne, australia) and tested negative for mycoplasma. cells were seeded at 15–20% confluency and cultured for 3 days. adhered cells were harvested with 0.2 mm ethylenediaminetetraacetic acid (edta) in phosphate-buffered saline 2 journal of circulating biomarkers (pbs) at 37�c to maintain cell surface protein integrity. cell scrapers were used to help detach any cell adhering beyond 5–10 min of pbs/edta incubation. flow cytometry detached cells were pelleted, resuspended, and aliquoted at 1–5 � 105 cells. cells were blocked with 10% fbs in pbs and probed sequentially with primary and secondary antibodies for 30 and 20 min, respectively (online supplementary table s2) and resuspended in 300 ml of pbs for fluorescence-activated cell sorting (facs) analysis (facs canto ii cell analyzer, bd biosciences, north ryde, australia). flowing software 2.5.1 was used for analysis (turku centre for biotechnology, turku, finland). immunocytostaining cells were seeded on sterile 18-mm diameter coverslips in 12-well plates at 2.5 �104 cells/well and grown for 3 days. non-fixed cells were blocked with 10% fbs in pbs for 10 min and sequentially incubated with primary and secondary antibodies for 45 and 30 min, respectively (online supplementary. table s2). prolong gold antifade reagent with 40,6-diamidino-2-phenylindole (dapi) (life technologies, melbourne, australia) was used for mounting. fluorescent microscopy cell images were taken with a bx53 microscope (olympus, notting hill, australia) with 20� objective using the cellsens dimension imaging software. ctc capture immunomagnetic beads, rare cell isolation kit (fluxion, san francisco, california, usa), were incubated with antiepcam or anti-n-cadherin antibodies for conjugation according to the distributer’s protocol (online supplementary table s2). conjugated beads were stored at 4�c and used within 4 weeks. at each blood collection, three 9 ml peripheral blood tubes were drawn per patient into edta vacutubes (greiner bio-one, frickenhausen, germany) and processed within 24 h. a total of 26 blood collections from 22 patients were analyzed. lymphoprep and sepmate tubes (stemcell technologies, vancouver, canada) were used to separate the peripheral blood mononuclear cells (pbmcs), containing ctcs, according to the manufacturer’s instructions. pbmcs derived from 9 ml blood each were washed once in pbs and resuspended in 800 ml binding buffer; then, 40 ml fc buffer (fluxion) and either 30 ml anti-epcam antibodycoupled beads or 30 ml anti-n-cadherin antibody-coupled beads or 30 ml of each were added. cells were incubated for 90 min at 4�c on a rotating platform and then loaded into primed isoflux cartridges for ctc enrichment using the isoflux standard isolation protocol (fluxion). enriched ctcs samples were fixed with 3.7% formaldehyde in pbs before immunocytostaining. ctc immunocytostaining enriched ctc samples were washed with binding buffer and blocked with 25 ml of 10% fbs in binding buffer, followed by 15-min incubation with 25 ml of anti-cd45 antibody (fluxion), 1:100 in 10% fbs/binding buffer. after a binding buffer wash, cells were incubated with 25 ml cy3-conjugated donkey anti-rabbit immunoglobulin g (igg) antibody (fluxion), 1:200 dilution in 10% fetal calf serum (fcs)/binding buffer for 15 min, washed again, then permeabilized with 25 ml 0.2% triton x-100, and incubated with 25 ml of fluorescein isothiocyanate (fitc) conjugated anti-cytokeratin antibody diluted in 10% fcs/binding buffer (online supplementary table s2) for 30 min. after final wash steps, samples were transferred to glass slides and mounted with prolong gold antifade reagent with dapi (life technologies, melbourne, australia). cells were imaged as outlined above. initially, ctcs from the blood samples of 10 patients and 10 healthy blood donors were detected and enumerated in this standard way by establishing nuclear dapi (nucþ), cytokeratin (ckþ), and cd45� cells. ve-cadherin quadruple immunocytostaining for quadruple staining, enriched ctc samples were preblocked with mouse immunoglobulins as above. after fixing in 3.7% formaldehyde, the ctc immunocytostaining protocol was followed by the cy3-conjugated donkey antirabbit igg antibody probing. sample was then washed in binding buffer and incubated with vioblue-conjugated antihuman vascular endothelial (ve) -cadherin (cad) antibody for 30 min (online supplementary table s2), followed by cytokeratin probing. sample mounting media included draq5 nuclear dye (abcam, melbourne, australia) instead of dapi. imaging was performed as outlined above. vioblue ve-cad staining was scored immediately in nucþ/ckþ/cd45� cells to avoid background associated with slide storage. blood samples from six patients and nine healthy donors were analyzed this way. finally, blood samples from six patients were compared using quadruple staining with either antihuman vimentin or ve-cad antibodies (as described above) analyzing ctc isolations based on targeting either the cell surface marker epcam or the combination of epcam and n-cadherin. vimentin quadruple immunocytostaining for quadruple ctc and vimentin staining, enriched ctc samples were preblocked in 25 ml mouse immunoglobulins (abacus, brisbane, australia) at a final concentration of 1.2 mg/ml in binding buffer for 20 min to saturate any po et al. 3 remaining free anti-mouse antibody present on magnetic beads. samples were then washed briefly in binding buffer and fixed in 3.7% formaldehyde for 10 min. initially, the ctc immunocytostaining protocol was followed. after the cytokeratin probing, samples were washed in binding buffer and incubated with alexa fluor 647-conjugated mouse antihuman vimentin antibody for 1 h at room temperature (online supplementary table s2). after three washes (2� pbs, 1� h2o) mounting media included hoechst nuclear dye, and cells were observed and images were captured as mentioned earlier. nucþ/ckþ/cd45� cells were scored as emt-ctcs when showing strong vimentin reactivity (vimþ). n-cadherin quadruple immunocytostaining for n-cadherin quadruple ctc staining, enriched ctc samples or pbmcs were preblocked with mouse immunoglobulins and then washed, fixed, and blocked as mentioned earlier. samples were then incubated with fitc-conjugated anti-cd45 (biolegend, san diego, ca, usa) and fitc-conjugated anti-cd144 (miltenyi biotec, nsw, australia) for 30 min. samples were washed in binding buffer and probed with rabbit anti-n-cadherin (novus, biologicals, litleton co, usa) for 60 min and then its alexafluor647-conjugated secondary donkey anti-rabbit igg antibody (jackson immunoresearch, west grove, pa, usa) for 30 min. after permeabilization with 0.2% triton-x in pbs, samples were probed with alexafluor555-conjugated pan-cytokeratin (cell signaling technology, beverly, ma, usa) for 30 min (see online supplementary table s2, for antibody dilutions). after three washes (2� pbs, 1� h2o), cells were observed and images captured as mentioned earlier. compliance with ethical research standards the study was conducted in accordance with the declaration of helsinki and approved by the south western sydney local health district ethics committee (ref: hrec/ 13/lpool/158). all patients and healthy controls included in this study gave informed written consent for blood collection and subsequent ctc analysis. results epithelial and mesenchymal cell surface proteins on ovarian cancer cells to identify suitable ovarian cancer cell surface proteins for evaluating immunomagnetic ctc isolation, a heterogeneity representative cohort of nine different ovarian cancer cell lines was analyzed by facs and immunocytostaining for the expression of e-cadherin, epcam, and n-cadherin. a human b-lymphocyte cell line (wme-099) was included in the analysis to rule out antibody interaction with lymphocytes. as expected, epcam was heterogeneously expressed and was never found on the entire cell population of any ovarian cancer cell line. in all, 60–85% of cells expressed detectable epcam levels, while in two cell lines, es-2 and a2780, epcam was undetectable. e-cadherin expression was found in the same cell lines that expressed epcam, although the proportion of cells expressing detectable e-cadherin tended to be slightly lower, ranging from 30% to 80%, again es-2 and a2780 lacked e-cadherin. n-cadherin expression, on the other hand, tended to be inversely related to epcam expression, with epcam-negative es-2 cells expressing detectable n-cadherin in approximately 80% of cells. exceptions were epcam and e-cadherin-positive peo14 cells that also expressed high n-cadherin levels in approximately 75% of cells, while epcam/e-cadherin-negative a2780 cells also lacked detectable n-cadherin in the majority (>60%) of cells (figure 1). overall, n-cadherin emerged as a possible target for emt-ctc isolation. n-cadherin-targeted ctc isolation from advanced ovarian cancer patients ctc isolation was performed using the isoflux microfluidic ctc isolation instrument. we confirmed in initial experiments that the gc-4 anti-n-cadherin antibody is suitable for immunomagnetic cell capture of es-2 cells (data not shown). to validate that n-cadherin in addition to epcam targeting improves ctc isolation, we compared ctc isolation in 20-patient blood collections from 18 advanced ovarian cancer patients when analyzed according to standard ctc identification (nucþ/ckþ/cd45�). ctcs were captured in 90% (18 of 20) of the patient samples when epcam and n-cadherin were targeted together, and slightly fewer (80%, 16 of 20) by directing isolation at epcam alone (figure 2(a), table 1). ctc counts showed high intra-patient variability in all antibody groups (0–376 for epcam�, 0–853 for n-cadherin�, and 0–1300 for combined targeted isolation). n-cadherin-directed ctc isolation outperformed epcam-based isolation by 2.1fold, based on median fold change of ctc capture, while combined targeting of n-cadherin and epcam increased ctc capture 3.0-fold (table 1). in 10 healthy blood donor control samples, we also observed a background of cells that met the staining criteria for ctcs when n-cadherin alone or in combination with epcam was targeted for isolation. importantly, these “false-positive ctcs” were highly variable in number, and most healthy individuals had only moderate counts. howver, 40% (4 of 10) of healthy individuals had >10 cells in 9 ml blood meeting the ctc definition of nucþ/ckþ/cd45� (figure 2(b)). distinguishing cecs from ctcs circulating endothelial cells (cecs) are rare cells in the circulation, expressing n-cadherin and cytokeratin 28 and thus are likely to be co-enriched with our assay as “false4 journal of circulating biomarkers figure 1. cell surface proteins on ovarian cancer cells and lymphocytes. the nine indicated ovarian cancer cell lines and the lymphocyte line wmm-099 were tested for the expression of epcam, e-cadherin (e-cad), and n-cadherin (n-cad) by facs analysis and immunocytostaining. (a) representative facs histograms and immunocytostaining for mesenchymal es-226 and epithelial peo1 cells27 are depicted. (b) the proportion (gray columns) of cells from the indicated cell lines expressing the designated proteins with mean expression level (red bars) is graphed (mean + sem. n � 2). facs: fluorescence-activated cell sorting; sem: standard error of mean. table 1. comparative ctc isolation approaches with indicated antibodies presented by common ctc identification (nucþ/ckþ/ cd45�). patient epcam n-cadherin fold change combination fold change pt 1 20 38 1.9 24 1.2 pt 2 0 17 — 2 — pt 3 0 0 — 0 — pt 4 19 25 1.3 58 3.1 pt 5* 8 288 36.0 82 10.3 pt 6 48 82 1.7 125 2.6 pt 7 376 853 2.3 1300 3.5 pt 7a,* 236 307 1.3 697 3.0 pt 8 8 24 3.0 42 5.3 pt 9* 16 162 10.1 68 4.3 pt 10 4 7 1.8 9 2.3 pt 11 0 0 — 0 — pt 12 51 530 10.4 465 9.1 pt 13 7 47 6.7 29 4.1 pt 13 a 20 38 1.9 74 3.7 pt 14 6 46 7.7 8 1.3 pt 15 0 2 — 1 — pt 16 5 21 4.2 8 1.6 pt 17 4 6 1.5 9 2.3 pt 18 4 3 0.8 1 0.3 median fold change 2.1 median fold change 3.0 ctc counts per 9 ml blood. data from 20 blood collections (18 patients) with ctcs stained by the common identification stain (nucþ/ckþ/cd45�) (n ¼ 10) or relevant data from common stain plus vim (n ¼ 4) or ve-cadherin (n ¼ 6) are combined. pt: patient; epcam: epithelial cell adhesion molecule; ctcs: circulating tumor cells. arecollection post >3 months. * <9ml blood available, data normalised to 9ml. po et al. 5 positive ctcs” when isolation is directed at emt markers. to identify cecs, a quadruple-staining identification strategy, including probing for the endothelial marker ve-cad, revealed that isolated cecs (nucþ/ckþ/ve-cadþ/ cd45�) were enriched in blood collections from our patients and healthy controls with comparable distribution to the cells we identified as “false-positive ctcs” (figure 2(b) and (c)). to help estimate how much of an issue false positive (non-ctcs) are for this assay, we spiked skov3 cells into healthy donor blood and compared a small proportion of pre-enrichment pbmcs with the major proportion of blood after ctc enrichment using the same staining. mimicked ctcs (skov3) were found in the pre-enrichment sample, consistent with the minor proportion of pbmcs analyzed. importantly, more cells met the cec criteria in the pre-enriched sample (5 from 1 ml blood vs. 1 after n-cadherin-based ctc enrichment from 8.7 ml blood). together with the fact that no cecs were detected after epcam or combined targeted ctc enrichment, this indicates that the healthy donor had relatively low overall cec counts (online supplementary figure s1). the prevalence of cecs in ovarian cancer patients and healthy controls confirmed that cec co-isolation by epcam targeting is rare, while n-cadherin targeting is associated with increased identification of co-isolated cecs. cec counts are highly variable between individuals and low (<5) in approximately 60% of assayed healthy controls and patient samples. strikingly though, in patient 14, cecs (44 of 46) outnumbered ctcs (2 of 46) (figure 3(a)), highlighting the value of cec co-staining during ctc identification. emt-ctc detection and discrimination from cecs to confirm that we can detect emt-ctcs, we developed another quadruple stain for nucþ/ckþ/cd45� ctcs by including the emt marker vimentin (vim) into our immunomagnetic ctc isolation method. due to limitations of our microscope (four-color detection only), we were unable to include vim and ve-cad detection in the same assay. to determine whether contaminating cecs will affect emt-ctc detection, we decided to focus on ctcs isolated by either epcam alone or in combination with n-cadherin targeting. we performed a quadruple stain for ve-cad or for vim in ctcs of parallel samples from six patients, five positive for ctcs (figure 3(b)). emt-ctcs were observed at higher counts than cecs in the majority of patient samples regardless of isolation method. only patient 22 displayed higher cec counts than emt-ctc counts associated with ctc isolation by combination targeting. importantly, regardless of varying cec co-purification, true emt-ctc counts were always higher in combination-based ctc isolates in comparison to epcam-alone–based isolation (figure 3(b)). to estimate how prevalent rare blood cell expression of n-cadherin is and how well we are able to distinguish emt-ctcs from potential false positives, we took 27ml blood from one patient. we then kept a small proportion of the pbmcs pre-ctc enrichment to compare immunostaining with staining after ctc enrichment using our different figure 2. cell isolation from advanced ovarian cancer patients. (a) data from 20 blood collections (18 patients) are presented to compare epcam, n-cadherin (n-cad), or the combination (combi) targeted ctc isolation efficiencies when applying the standard ctc identification (nucþ, ckþ, and cd45�). (b) false-positive “ctcs” in 10 healthy donor blood samples, when using the standard ctc identification (nucþ, ckþ, and cd45�). (c) cecs in healthy individuals and patients: the same cell isolation method using a ve-cadherin (ve-cad) cec staining protocol identified co-isolated cecs in patients (n ¼ 6) and healthy controls (n ¼ 9). red symbols: patient-derived ctcs (a) or cecs (c); black symbols: healthy control-derived false positive “ctcs” (b) or cecs (c). all counts are presented as cells per 9 ml blood. epcam: epithelial cell adhesion molecule; ctcs: circulating tumor cells; cecs: circulating endothelial cells. 6 journal of circulating biomarkers isolation strategies. to also evaluate n-cadherin expression in that setting, we combined cd45 with ve-cad probing in the green fluorescent channel. the patient evidently had very low ctc numbers (online supplementary table s3). thus, not surprisingly, we did not detect any ctc in the pre-enriched sample from 1 ml blood but detected four non-ctcs (nucþ/(cd45, ve-cad)þ/ckþ). in total, more non-ctcs were detected in the enriched ctc samples, indicating that the patient had high cec counts which could be appropriately distinguished from ctcs (online supplementary table s3). however, normalized on the blood volume (8.7 ml) only n-cadherin-alone isolation produced similarly high non-ctc numbers, further evidencing that this enrichment strategy may also enrich potential false positives most effectually (online supplementary table s3). the detected non-ctcs might be cecs, although with some uncertainty, due to cd45 and ve-cad detection in the same fluorescent channel in these experiments. of note, ncadherin was detected in some but not all of these cells (online supplementary figure s2). we set out to demonstrate increased isolation efficiency with our method, and blood collections were not restricted to specific time points throughout treatment. thus, not unexpectedly, no significant correlation between ctc numbers and disease parameters was observed (data not shown). discussion the aim of this study was to establish a method to improve immunomagnetic ctc isolation from ovarian cancer patients by capturing both ctcs with epithelial and mesenchymal phenotype. the emt-phenotype change is characterized by an e-cadherin-to-n-cadherin switch (reviewed by lamouille et al. 24 ), while expectedly the two epithelial markers epcam and e-cadherin are closely coexpressed in our large ovarian cancer cell line cohort. thus, well-established epcam-based ctcs isolation is likely adequate to isolate epithelial cells. as expected, we confirmed increased levels of n-cadherin in our ovarian cancer cell lines with low epcam levels, and these flow cytometry data informed our decision to add n-cadherin to our immunomagnetic targeting assay using the isoflux platform. however, it is worth highlighting that n-cadherin was not expressed on approximately 75% of epcamnegative a2780 cells, implying that a2780-like ctcs are likely to remain as poorly detectable with our method as with established epcam-based ctc isolation methods. figure 3. cec and emt-ctc capture by immunomagnetic isolation (a) top: representative quadruple staining of a ctc (ve-cad�, ckþ, cd45�, and nucþ) and cec staining (ve-cadþ, ckþ, cd45�, and nucþ). bottom: ovarian cancer patient cells were isolated by epcam, n-cadherin (n-cad), or combined targeting as indicated. the proportion of total ctcs and cecs captured with each isolation strategy is displayed. all counts are presented as cells per 9 ml blood. (b) top: representative quadruple staining of an epithelial (epi) and an emt-phenotype ctcs. bottom: comparison of emt-ctc and cecs isolated from advanced ovarian cancer patients by epcam or combined epcam plus n-cadherin (combi) targeting. all counts are presented as cells per 9 ml blood. cecs: circulating endothelial cells; emt: epithelial-to-mesenchymal transition; ctcs: circulating tumor cells; cecs: circulating endothelial cells; epcam: epithelial cell adhesion molecule. po et al. 7 notably, a2780 ovarian origin has been questioned previously as genetic clustering puts these cells closer to intestinal or lung cancer cells. 25,29 our data show that targeting the emt marker n-cadherin with a commercially available anti-ncadherin antibody isolates 2.1-fold more ctcs from advanced ovarian cancer patients than epcam-alonebased isolation and 3.0-fold more when used together with epcam targeting. our ctc isolation efficiencies from 80% to 90% of patients are comparable to other ovarian cancer studies that detected ctcs from 14% to 85% of patients dependent on disease stage and ctc detection methods. 4,5 notably, ctc counts when targeting epcam alone were similar to those reported in a recent ovarian cancer study using the isoflux platform (range 0–1208; median 55). 30 in comparison to epcam-alone–targeted ctc isolation, ctc counts were always higher when n-cadherin was targeted alone or in combination with epcam, although some co-purification of cecs was observed when including n-cadherin targeting into isolation strategies. in our study, n-cadherin-alone–based isolation of ctcs always improved on epcam-alone–based isolation. also, the combined targeting always improved on epcam-alone targeting; however, the combination strategy was only superior to n-cadherin-alone–based ctc isolation in about half of the samples. we speculate that immunomagnetic beads with different antibodies in the same admixture can cause interference with cell capture if only one of the targeted antigens is predominantly expressed. thus, an emtctc population predominantly expressing n-cadherin would be expected to be more efficiently isolated with n-cadherin-only targeting than by adding potentially interfering beads coupled to, in the context less relevant, antiepcam antibodies. we, therefore, propose that antibody cocktails used in ctc isolation should be considered with caution, as antibodies that might only aid in exceptional cases to isolate relatively rare cells may interfere in the appropriate isolation of the intended common cell population. nevertheless, we propose that combining epcam with n-cadherin-based ovarian cancer ctc isolation will ultimately be a more successful strategy, particularly if also investigating early-stage patients, who would be expected to have more epithelial ctc phenotypes. our data confirm that, similar to size filtration-based ctc enrichment methods, ctc isolation strategies focusing not only on epcam expressing ctcs lead to higher total ctc counts. 31,15 however, we confirm that epcamonly–based ctc isolation yields purer ctc populations as contamination with false positives is rare in comparison to n-cadherin-based ctc isolation. our data also confirm, importantly, that cecs are an important cause of falsepositive ctc identification and are not distinguished by the common ctc identification (nucþ/ckþ/cd45�). moreover, cecs not only express n-cadherin but also a number of other markers that have been previously proposed for ctc isolation, such as epidermal growth factor receptor (egfr), vimentin, and fibronectin. with a cell size ranging up to around 10 mm, they may also not be filtered out sufficiently by size exclusion methods of ctc isolation. our data agree with studies that specifically targeted cecs for isolation, where cec counts were highly variable in healthy individuals (0–29/ml blood) and tended to be increased in some disease states including cancer (reviewed by po et al. 28 ). therefore, it is important to account for cecs when using any non-epcam-based ctc isolation method. we developed quadruple-stain ctc identification (nucþ/ckþ/cd45�/ve-cad�) that largely diminishes the identification of false-positive ctcs by distinguishing them from co-isolated cecs. importantly, regardless of whether cecs might make up a varying predominantly smaller proportion of cells in a ctc isolate, parallel samples confirmed that immunomagnetic ctc isolation based on epcam plus n-cadherin does isolate more emt-phenotype ctcs than epcam targeting and any cec can be readily distinguished. this indicates that although cecs can be considerable for a few individuals, when isolating ctcs by other than epcam-based methods, they play mostly a minor role especially if a study only analyses ctc numbers. our study was aimed to improve immunomagnetic ctc isolation by also targeting emt-ctcs, and we confirmed our isolation strategy in predominantly advanced disease ovarian cancer patients. our patient recruitment did not require liquid biopsies at specific times during a patient’s disease progression or with regard to timing before and after treatment cycles. in our small heterogeneous cohort used for method validation, we, therefore, did not observe any obvious correlation of ctc counts with disease stage or outcomes. we did, however, capture a larger number of ctcs in a higher proportion of advanced ovarian cancer patients by combined targeting of epcam and n-cadherin for immunomagnetic isolation. therefore, our data also support the notion that ovarian cancer ctcs are more heterogeneous with regard to emt status, which agrees with the findings that emerging emt-phenotype ctcs might be associated with response to therapy. 19 . in conclusion, we established a combined epcam and n-cadherin immunomagnetic targeting strategy to improve on the overall ctc isolation. we suggest that nonepcam-based ctc isolation methods should employ a quadruple staining method to avoid false positives. the significance of detecting more and a wider range of ctc phenotypes is that they are more likely to accurately represent the biology of a patient’s ovarian cancer at that point in time, thus improving their value as potential tumor biomarkers. finally, our combined antibody targeting approach may also be useful in improving ctc capture in other cancer types. acknowledgements we thank the members of the gynaecological oncology biobank westmead (member of the australasian biospecimen network8 journal of circulating biomarkers oncology group, funded by the australian national health and medical research council, enabling grants id 310670 & id 628903 and the cancer institute nsw grants id 12/rig/1-17 & 15/rig/1-16) for patient recruitment. wme-099 cells were kindly provided by professor graham mann. human ethics approval, hrec/13/lpool/158, was obtained and managed by the concert biobank. declaration of conflicting interests the author/s declared no conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: jp and dl are recipients of rotary health australia and ingham research director’s phd scholarships, respectively. pds 1 , tmb 1 , adef 2 , and prh 2 are supported by the cancer institute new south wales through; 1 the centre for oncology education and research translation (concert), and 2 the sydney-west translational cancer research centre. adef was also funded by the university of sydney. supplemental material supplementary material for this article is available online. references 1. kim a, ueda y, naka t, et al. therapeutic strategies in epithelial ovarian cancer. j exp clin cancer res 2012; 31: 14. 2. matsuo k, lin yg, roman ld, et al. overcoming platinum resistance in ovarian carcinoma. expert opin investig drugs 2010; 19(11): 1339–1354. 3. caixeiro nj, kienzle n, lim sh, et al. 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plos one 2015; 10(6): e0130873. 9. zeng l, liang x, liu q, et al. the predictive value of circulating tumor cells in ovarian cancer: a meta analysis. int j gynecol cancer 2017; 27(6): 1109–1117. 10. chebouti i, kuhlmann jd, buderath p, et al. ercc1expressing circulating tumor cells as a potential diagnostic tool for monitoring response to platinum-based chemotherapy and for predicting post-therapeutic outcome of ovarian cancer. oncotarget 2017; 8(15): 24303–24313. 11. pearl ml, dong h, zhao q, et al. ictc drug resistance (cdr) testing ex vivo for evaluation of available therapies to treat patients with epithelial ovarian cancer. gynecol oncol 2017; 147: 426–432. 12. lee m, kim ej, cho y, et al. predictive value of circulating tumor cells (ctcs) captured by microfluidic device in patients with epithelial ovarian cancer. gynecol oncol 2017; 145(2): 361–365. 13. pradeep s, kim sw, wu sy, et al. hematogenous metastasis of ovarian cancer: rethinking mode of spread. cancer cell 2014; 26(1): 77–91. 14. van berckelaer c, brouwers aj, peeters dj, et al. current and future role of circulating tumor cells in patients with epithelial ovarian cancer. eur j surg oncol 2016; 42(12): 1772–1779. 15. punnoose ea, atwal sk, spoerke jm, et al. molecular biomarker analyses using circulating tumor cells. plos one 2010; 5(9): e12517. 16. gorges tm, tinhofer i, drosch m, et al. circulating tumour cells escape from epcam-based detection due to epithelialto-mesenchymal transition. bmc cancer 2012; 12: 178. 17. stimpfl m, schmid bc, schiebel i, et al. expression of mucins and cytokeratins in ovarian cancer cell lines. cancer lett 1999; 145(1–2): 133–141. 18. tsai jh and yang j. epithelial-mesenchymal plasticity in carcinoma metastasis. genes dev 2013; 27(20): 2192–2206. 19. chebouti i, kasimir-bauer s, buderath p, et al. emt-like circulating tumor cells in ovarian cancer patients are enriched by platinum-based chemotherapy. oncotarget 2017; 8(30): 48820–48831. 20. haslehurst am, koti m, dharsee m, et al. emt transcription factors snail and slug directly contribute to cisplatin resistance in ovarian cancer. bmc cancer 2012; 12: 91. 21. marchini s, fruscio r, clivio l, et al. resistance to platinumbased chemotherapy is associated with epithelial to mesenchymal transition in epithelial ovarian cancer. eur j cancer 2013; 49(2): 520–530. 22. tothill rw, tinker av, george j, et al. novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. clin cancer res 2008; 14(16): 5198–5208. 23. miow qh, tan tz, ye j, et al. epithelial-mesenchymal status renders differential responses to cisplatin in ovarian cancer. oncogene 2015; 34(15): 1899–1907. 24. lamouille s, xu j, and derynck r. molecular mechanisms of epithelial-mesenchymal transition. nat rev mol cell biol 2014; 15(3): 178–196. 25. quattrocchi l, green ar, martin s, et al. the cadherin switch in ovarian high-grade serous carcinoma is associated with disease progression. virch arch 2011; 459(1): 21–29. po et al. 9 26. davidson b, trope cg, and reich r. epithelialmesenchymal transition in ovarian carcinoma. front oncol 2012; 2: 33. 27. chung vy, tan tz, tan m, et al. grhl2-mir-200-zeb1 maintains the epithelial status of ovarian cancer through transcriptional regulation and histone modification. sci rep 2016; 6: 19943. 28. po jw, lynch d, de souza p, et al. importance and detection of epithelial-to-mesenchymal transition (emt) phenotype in ctcs. in: xu k (ed) tumor metastasis. nigeria: intech, 2016, pp. 241–256. 29. domcke s, sinha r, levine da, et al. evaluating cell lines as tumour models by comparison of genomic profiles. nat commun 2013; 4: 2126. 30. rao q, zhang q, zheng c, et al. detection of circulating tumour cells in patients with epithelial ovarian cancer by a microfluidic system. int j clin exp pathol 2017; 10: 9699–9706. 31. krebs mg, hou jm, sloane r, et al. analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial 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kn shashidhar 1 , v lakshmaiah 2 , and muninarayana 3 abstract liver is the vital organ for synthesis of proteins whose concentration in blood reflects liver dysfunction. variations in protein domain can generate clinically significant biomarkers. biomarker pipeline includes discovery of candidates, qualification, verification, assay optimization, and validation. advances in proteomic approach can discover protein biomarker candidates based on “up-or-down” regulation or fold change in expression which is correlated with disease state. despite numerous biomarker candidates been discovered, only few are useful in clinical practice which indicates the need for well-established validation regimen. hence, the main purpose of this review is to understand the protein biomarker development and pitfalls. companion diagnostics provide insights into potential cost-effective diagnosis for chronic liver disease. keywords chronic liver disease, proteomics, biomarker discovery, assay optimization, specificity and sensitivity, validation strategies, pitfalls, companion diagnostics date received: 22 december 2017; accepted: 18 april 2018 introduction human liver is a vital organ for health and survival, performing biochemical functions, namely, protein synthesis, production of digestive enzymes, and detoxification. 1 liver fibrosis/cirrhosis is a pathological condition in which functions of liver are impaired by chronic liver insult. cirrhosis of liver is the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury which leads to portal hypertension and endstage liver disease. fibrosis is a reversible natural wound healing response to chronic liver injury resulting in accumulation of extra cellular matrix (ecm); precursor of cirrhosis. despite varied etiology, the pathological characteristics which include degeneration, necrosis of hepatocytes, and replacement of liver parenchyma by fibrotic tissues and regenerative nodules are common and ultimately result in liver dysfunction. 2 after an acute injury, there will be regeneration and replacement of liver parenchymal cells to necrotic and apoptotic cells. if injury persists, there will be substitution of hepatocytes with abundant ecm having contractile, inflammatory, and fibrogenic properties. 3 activation of hepatic stellate cells (hscs) is a crucial step of tissue injury and regeneration. 4 quiescent hscs present in space of disse will be activated and trans-differentiate into myofibroblasts like cells which are responsible for ecm production and accumulation in injured liver. 5 accumulation of 1 department of biochemistry, sri devaraj urs medical college, sduaher, karnataka, india 2 department of medicine, sri devaraj urs medical college, sduaher, karnataka, india 3 department of community medicine, sri devaraj urs medical college, sduaher, karnataka, india corresponding author: kn shashidhar, department of biochemistry, sri devaraj urs medical college, kolar, karnataka 563103, india. email: drshashikn1971@yahoo.co.in journal of circulating biomarkers volume 7: 1–9 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418777186 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:drshashikn1971@yahoo.co.in https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418777186 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage ecm is due to increased synthesis and decreased degradation by over expression of tissue inhibitors of metallo proteinases which inhibits matrix metallo proteinases. fibrotic liver contains 3–10 times more ecm compared to normal liver which includes collagen types, glycoproteins, proteoglycans, and glycosaminoglycans. 2 accurate assessment of degree of hepatotoxicity especially at early stage is crucial for clinical management to predict prognosis and therapeutic decision even to reverse liver fibrosis/cirrhosis to normal architecture of liver. despite development of potential diagnostic tests, for the past 50 years, liver biopsy is still considered as gold standard for diagnosis of chronic liver diseases (clds), which is associated with pain and complications. biomarkers are used to diagnose or monitor the activity of disease and to assess therapeutic response for cld. 2 a biomarker is a molecule that is analytically measured with well-established performance characteristics in an established scientific frame work of evidence that elucidates physiological, toxicological, pharmacological, or clinical outcome. 6 biomarkers can be gene variants, single nucleotide polymorphisms, gene expression products, metabolites, polysaccharides, circulating nucleotides, and proteins. 7 validation of a biomarker includes assessing the biomarker, measuring the performance characteristics, and determining the range of conditions for reproducibility and accuracy. biomarker validation relates biomarker with biological process and clinical end point and is necessary for fit-for-purpose. validation helps research data for better patient care. an ideal biomarker for cld should be specific, sensitive to indicate active damage, prior to histological changes, easily accessible in peripheral tissue, and cost-effective. 2,6 a biological marker objectively measures and evaluates normal biological, pathogenic process, or pharmacological response to a drug. surrogate markers serve as a substitute for a clinically meaningful end point. prognostic biomarker indicates likely outcome of a disease irrespective of treatment. predictive biomarker helps to assess response to a particular treatment. pharmacodynamic biomarker gives interaction between drug and target. 6,7 in this review, an attempt has been made to understand the process of proteomic biomarker development which includes protein biomarker discovery, validation, and pitfalls in biomarker pipeline for cld. biomarker development by proteomic approach establishment of correlation between disease state and biomarker alterations will help clinician for diagnosis and tailored therapy. 8 in cld, protein domain will have alterations where the amount of protein from liver enters into circulation and serves as an indicator for degree of liver dysfunction, which holds good for discovery of novel protein biomarkers using proteomics. 9 proteins have more structural diversity and stability than dna and rna and carry more information than nucleic acids which are dynamic and reflection of cellular physiology. 10 advances in proteomic approach help discover and identify clinically significant protein biomarker candidates for cld. protein biomarker pipeline includes a series of essential components such as discovery, research assay optimization, analytical and clinical validation, and clinical utility (figure 1). 11,12 protein biomarker candidate discovery protein biomarker discovery is a simplified, semiquantitative, unbiased binary comparison between diseased and figure 1. biomarker discovery and validation; advances in omics will generate clinically significant biomarker candidates which need validation through verification studies and clinical assay development. source: adapted from the study by nies et al.12 2 journal of circulating biomarkers normal using biological samples for maximal detection of significant protein expression differences. it needs avoiding contamination of other diseases and confounding factors. 2,13 proteins that are differentially expressed between cld and normal are due to changes in translation, posttranslational modifications, and degradation or that are involved in pathophysiological changes which are good sources of biomarker candidates. 10 comparative analysis between diseased and healthy generates hundreds of protein biomarker candidates that are differentially expressed. there is an inverse relationship between number of samples analyzed and number of proteins quantified (figure 2). 13,14 discovery of protein biomarker candidates for cld, plasma/serum is the best choice among other body fluids, represents physiological and pathological process. 15 during discovery phase, the variables (study design, preanalytical, and analytical) which affect precision should be minimized. study population should be selected from a well-defined study design with definite inclusion and exclusion criteria to minimize bias. case control study or cohort study usually considered as a better study design for discovery phase of biomarker. multiple sources of bias could be seen in retrospective and observational studies. 16 preanalytical variables such as type and manufacture of collection tubes, phlebotomy device, patient’s posture, time of sample collection, type of sample to be collected, storage conditions, and sample preparation should be controlled in order to get significant observation. analytical variables such as mass resolution and collision energy need to be controlled to minimize the source of variations. 13,14 in candidate discovery for cld, to obtain significant protein expressional difference, use of gold standard sample is recommended. plasma is the biofluid of choice (human proteomic organization), and contains proteins that reflect a variety of human diseases. 17 anticoagulants (edta or citrate) cause osmotic shifting of fluid from cell to plasma, which gives 10% less values when compared to serum and are known to chelate cations, and give negative results in case protein of interest has cations in its structure. antigenic epitope mask might happen which reduces immunoreactivity because of heparin. 13,18 compared to plasma, proteins are more stable in serum. for large studies, serum is the preferred sample by clinicians since it is the most simple matrix. 14 although individual sample analysis is recommended, pooling strategy with definite criteria from multiple individuals reduces sample number and cost. 19 protein biomarker discovery in plasma/serum is complicated. around 99% of protein content is comprised of 20 abundant proteins which interfere in identification and characterization of low abundant proteins by mass spectral and electrophoretic analysis. 20–23 depletion of abundant proteins allows detection of low abundant proteins. but there is a chance for further removal of low abundant proteins that are bound to high abundant proteins. 14 for figure 2. discovery of protein biomarker; protocol for the development of protein biomarker candidate. source: adapted from the study by rifai et al. 13 nallagangula et al. 3 accurate protein biomarker candidate discovery for cld, depletion of albumin (55% of total protein) and immunoglobulins may be achieved using high affinity columns. 21,22 in two-dimensional gel electrophoresis (2-de), depletion dilemma can be rectified using narrow ph (3–5.6) range which avoids interference of abundant proteins (albumin, transferrin, and immunoglobulins). 9,14 after depletion, discovery may be carried out by fractionation and purification using different analytical methods: 2-de for separation of proteins followed by identification of significant protein spots using software tools. identified spots are subjected for in-gel digestion to identify peptides and proteins either by surfaceenhanced laser desorption-ionization (seldi) or matrix-assisted laser desorption-ionization (maldi) or liquid chromatography-mass spectrometry (lc-ms) and proteomics search engines, that is, mascot or sequest. 24–26 unlike gel-based discovery, lc-ms carried out before or after enrichment of proteins by trypsin digestion, splitting long proteins into short peptides followed by chromatographic separation in addition to mass to charge ratio. 14,27,28 2-de has limited sensitivity and reproducibility compared to lc-ms. the main disadvantage of seldi/ maldi is difficulty in detection of differential pattern and identification of peaks. automated lc-ms is suitable for protein biomarker discovery. secondary ions collected from chromatographic profiles from ms spectra are subjected to proteomic search engines. 29,30 identified peptides are used to determine differential expression between cld and normal. use of parametric statistical tools prior to peptide identification is recommended. biomarker candidates reported and identified for cld by one group of researchers are not identified by another group (table 1) because of lack of standardization of multistep procedures. selection of specific criteria during lc-ms gives complexity and errors for reproducibility between laboratories. biomarker discovery and validation should be performed in a blinded fashion, free from bias, and performed in a similar fashion that remove all confounding factors and generate significant biomarker candidates. 30 biomarker validation biomarker validation is necessary to deliver high-quality research data for effective use of biomarker for better patient care. great interest and technological advancement in biomarker discovery results in identification of protein biomarker candidates for cld. biomarker candidates require verification that demonstrates the differential expression which remains detectable by assay to be used for validation. 13 despite numerous biomarker candidates identification, verification may be done only for few qualified candidates in terms of marker performance and reagent availability. 10 proteins that act in cellular pathways and deregulated in cld should be considered for further validation. 19 validation of biomarker and clinical assay optimization requires measurement of thousands of patient samples with narrow measurement coefficient of variation values. 13 assay optimization as ms is unable to achieve high measurement accuracy and precision, it is necessary to develop antibodies for quantification of biomarker candidates. concentration of protein in serum or plasma ranges from picograms to nanograms per milliliter; highly sensitive immunotechniques are required for quantification. enzyme-linked immunosorbent assay (elisa) is the best alternative for quantification of these proteins compared to sophisticated nonimmunebased techniques. 35 capture and detection antibodies (monoclonal or polyclonal), which detect distinct epitope of the protein, are needed to form sandwich reaction. specificity of antibodies is established using western blot or immunostaining. during development of elisa, care should be taken to minimize the effect of variables such as avidity, concentration of antibodies (monoclonal capture/detection 0.5–4/0.25–2 mg/ml and polyclonal capture/ detection 0.2–0.8/0.05–0.4 mg/ml), incubation time and temperature, sample volume, dilution of sample, ph, composition and concentration of diluents, enzyme, substrate, and quality of detector which affect performance characteristics. fluorescent or chemiluminescent are other alternatives for better sensitivity. 13,36 analytical evaluation newly developed assay requires analytical validation before evaluating clinical utility in terms of performance characteristics such as outcome studies, clinical requirement, proficiency testing, and goals set by regulatory agencies. 35 preanalytical variables should be characterized and controlled in various physiological and pathological conditions. time of collection of sample (fasting or fed state) should be defined. in fed state, chylomicrons do not affect elisa. selection of appropriate sample (plasma or serum) and use of anticoagulants should be determined. storage conditions and duration of storage should be examined. physiological factors such as age, gender, and ethnicity significantly affect protein concentrations along with lifestyle factors. pathological conditions and drugs which influence protein concentrations should be examined before estimation. 13 indicators of accuracy, precision, analytical measurement range, and reference intervals should be defined. trueness is the closeness of agreement between average measured values of different samples which reflect bias (systemic error). accuracy is the closeness of agreement between the values measured and true concentration of analyte. 37 newly discovered methods usually do not have reference materials and methods and should use alternative 4 journal of circulating biomarkers protocols such as spike, recovery, and linearity. use of specific antibodies should be necessary to have no cross reactivity with other proteins. care should be taken during elisa development to minimize the errors because of exogenous and endogenous substances. the factors (buffer components, sample matrix, compliment, and rheumatoid factor) can impact antibody binding in natural samples and therefore influence the accuracy of results should be ruled out. 38 repeatability is the measurement performed in the same condition, and reproducibility is the measurement performed in different conditions. to assess precision, two replicates per sample per run, and two runs per day for least 20 days are recommended. 39 reference intervals must be defined for protein of interest and new methodology by comparing healthy individual values similar to those of patient values. 39 reference values should be subdivided into groups based on age, gender, race, and physiological states. normal distribution of reference intervals for protein of interest for parametric analysis is presented as mean + 2sd and for nonparametric analysis will be presented as percentiles. limits of detection and quantification must be defined with acceptable accuracy and precision. limit of table 1. protein biomarker candidates identified by proteomic analysis for liver fibrosis. authors etiology of liver fibrosis type of sample proteomic techniques protein biomarker candidates identified white et al.31 hcv serum 2-de and lc-ms a2 macroglobulin haptoglobin complement c4 serum retinol binding protein apolipoprotein a1 apolipoprotein a-iv gangadharan et al.32 hcv serum 2-de and lc-ms a2 macroglobulin inter-a-trypsin inhibitor heavy chain h4 a1 antichymotrypsin apolipoprotein l1 paraoxonase/aryleserase 1 zinc-a2-glycoprotein cd5 antigen-like protein b2 glycoprotein i gangadharan et al.9 hcv serum 2-de, lc-ms, and in-solution isoelectric focusing beta chains of c3 and c4 gangadharan et al.33 hcv serum 2-de and lc-ms adiponectin sex hormone binding protein 14-3-3 protein zeta/delta complement c3dg immunoglobulin j chain apolipoprotein ciii corticosteroid binding globulin a2 hs glycoprotein lipid transfer inhibitor protein haptoglobin-related protein katrinli et al. 34 hbv liver tissue 2-de and lc-ms apolipoprotein a1 pyruvate kinase glyceraldehyde 3-phosphate dehydrogenase glutamate dehydrogenase alcohol dehydrogenase transferrin, peroxiredoxin 3 keratin 5, annexin nallagangula et al. (2017) (unpublished data) ald serum 2-de and lc-ms serotransferrin keratin isoforms vitamin d binding protein isoform 3 angiotensinogen preproprotein cd5 antigen-like protein hemopexin precursor a1 antichymotrypsin glycerol kinase isoform x1 sex hormone binding protein hcv: hepatitis c virus; hbv: hepatitis b virus; ald: alcoholic liver disease; 2-de: two-dimensional electrophoresis; lc-ms: liquid chromatography-mass spectrometry. nallagangula et al. 5 detection is the lowest value that exceeds the measurand value against blank sample which does not have protein of interest. linearity gives the relation between observed value and expected value which is above the range of measurand values. 13,39 clinical validation after analytical validation of new methodology for protein of interest, biomarker candidate should confirm the performance characteristics in terms of consistency and accuracy in clinical evaluation to diagnose or predict the clinical outcome of cld. the newly identified biomarker candidate should satisfy the following criteria. 40 sensitivity of biomarker the ability of a biomarker or change in magnitude of a biomarker with precision which is sensitive enough to reflect a meaningful change in clinical end point of cld. specificity of biomarker the ability of a biomarker or change in magnitude of a biomarker which distinguish patients who are responders and nonresponders in terms of change in clinical end point of cld. probability of false positive desired change of biomarker is not reflected by positive change in clinical end point or even worse is associated with negative change in a clinical end point of cld. probability of false negative no change or small change is observed in magnitude of biomarker which fails to signal positive and meaningful change in a clinical end point of cld. pharmacokinetic/pharmacodynamic model correlation between changes in biomarker and drug exposure, to predict future outcome or standardization of dose adjustments based on biomarker measurements. likelihood ratio of biomarker indicates certainty of the diagnosis of disease prevalence and calculates posttest odds of having a disease as the prevalence changes. receiver operating characteristic (roc) curve is the comparison of diagnostic accuracy of two or more tests and to define appropriate cutoff for clinical utility of test. likelihood ratio and roc curve are derived from sensitivity and specificity values. 2,41 clinical utility clinical utility predicts positive outcome of drug in selected and unselected groups. novel biomarker candidate needs to be evaluated in a series of human population (sub and stratified). in phase i (exploratory phase), test results should be different from patients with confirmed cld and those of control population without cld. area under roc curve should be >0.5 for newly identified biomarker candidate to proceed further. in phase ii (challenge phase), different cutoff values for sensitivity and specificity should be defined with diagnostic accuracy to predict the presence or the absence of cld. phase iii (advanced clinical phase) is to establish diagnostic accuracy of biomarker in target population in different geographical regions independently. phase iv (outcome phase) gives the positive influence of test to get healthy outcome of cld by evaluating both tested and untested patients with respect to diagnostic and therapeutic intervention. 13,42,43 pitfalls and limitations in current medical research, novel biomarker should have the ability to improve treatment which is cost-effective. 7 newly identified biomarkers for cld are unable to replace the existing conventional markers in clinical practice due to errors in study design or experimental execution. despite numerous biomarker candidates identified for cld, few biomarkers only validated successfully. pitfalls in biomarker pipeline are because of no proper relation between discovery, verification, and clinical validation (due to lack of definite selection criteria in discovery phase, biomarker verification (sensitivity and specificity), and less robustness in analytical validation) as well as less structure and scientific factors which fail to give strong evidence for better patient care (figure 3). 7,19 during discovery phase, appropriate and well characterized clinical specimen has impact on outcome of identification process. 44 selection of patients for biomarker studies should be done by specialist to ensure the presence or absence of the disease. randomization and optimal selection of patients with single etiology are necessary and are well matched with age and gender with the same ethnicity. other lifestyle factors play an important role in selection criteria, namely, body mass index, habits, physical activity as well as metabolic syndromes, and use of drugs. 7 small sample size and lack of information about history may give false negative values in discovery phase. 13,45 proper procedures for samples collection, handling, and storage to avoid denaturation of proteins should be followed. systematic monitoring of quality of sample over a time period is necessary. 7 suitable semiquantitative methods and sophisticated technologies like lc-ms along with proper analysis and data interpretation can improve biomarker candidate quality and yield. validation of biomarker is expensive and timeconsuming. protein biomarker quantification is essential to have specific detection and capture antibodies with high sensitivity to form sandwich method with low concentrated protein in biological specimens. analysis of protein of 6 journal of circulating biomarkers interest should be carried in triplicates and reported in mean and standard deviation. 7,13 in research laboratory, quality control procedures are less compared to clinical laboratories, and basic steps should be implemented to get accuracy and precision. 46 difficulties in validation strategies, which need well-defined sub and stratified population matched with pathological and physiological factors of early disease state, are main reasons for pitfalls. lack of sensitivity, specificity of biomarker for disease progression and regression with roc � 75%, and likelihood ratio � 5 with odd ratio � 1 are limitations in biomarker pipeline.7 transition from research to routine newly identified biomarker needs to satisfy and fulfill the need of application which can be able to separate patients into groups that clinicians would treat differently and could be able to give reliable outcome of the treatment and it should be evidence based. 46 new test should add or replace the information provided by existing biomarkers for cld and cost-effective for better patient care. 47 introduction of new biomarker from research laboratory into clinical laboratory is three-way collaboration involving research laboratory, diagnostic industry, and clinical laboratory. 48 care should be taken in research laboratory about selection of novel biomarker evaluation at early stage to minimize the methodological bias (preanalytical, analytical, and postanalytical) which may affect results. 49,50 validated biomarker in research laboratory will be transferred to specialist referral laboratory to confirm the assessment in clinical setting. 47 once new test has beneficial effect on patient outcome which is evidence based and cost-effective, it will be introduced into funded health care system. biomarker has to meet analytical validation, quality control, external validation, personal qualification, training, and documentation for approval (510(k)/premarket approval (pma)/in vitro diagnostic (ivd) directive 98/97/ec). 13,47 the test developed should be suitable for clinical laboratory and capable to meet basic requirements (robustness, stability of reagents, acceptable turnaround time, adaptability for automation, and low cost). if biomarker is ideally measured in serum which does not require special handling, storage with robust analytical procedure, rejection of samples will be minimized which decreases turnaround time. internal quality control should be robust as external quality control may not be available for newly discovered and validated biomarkers. interlaboratory comparison can provide information about accuracy and precision at early stages. definite reference intervals and linearity range will help for best practice of biomarker. 47 regulatory requirements ivd device to enter into market must meet the rules and regulations of 510(k) premarketing clearance or pma by the food and drug administration (fda) in the united states, the pharmaceutical affairs law (pal) and market authorization holder by pharmaceutical and medical devices agency of japan, and ivd directive 98/97/ec by member states of european union. according to fda, 510(k) process is that new test should measure existing fda classified analyte i or ii where there is predicate cleared test which are commercially available. information about new test should include classification, performance characteristics, and analytical capability (accuracy, precision, linearity, specificity, and sensitivity) comparison with that of existing predicate test. class iii, which is associated with high risk or clinical utility of biomarker or novel technological measurements or no predicate device, needs pma process. 13,51,52 companion diagnostics companion diagnostics (cdx) is the central part of personalized medicine. cdx is simultaneous development of drug and diagnostic test: in vitro diagnostic device which provides information about safe and effective use of corresponding therapeutic product. cdx includes screening and detection, prognosis, monitoring, and theranostics. the key indicator for cdx is robustness of financing environment for drug and diagnostic companies which minimizes costs from selection of patient population till clinical trials. cdx improves chances for approval and increases market uptake. there is a need for cdx which can be able to provide diagnostic test specific for therapeutic drug for cost-effective and successful management of cld. 7,53 conclusion early diagnosis of cld is essential for disease management and even reversibility of liver fibrosis/cirrhosis. concentration of proteins expressed from liver into circulation serves as an indicator for liver dysfunction and good source of biomarker development based on proteomic approach. technological advancement generates biomarker candidates which is a prerequisite for validation in terms figure 3. pitfalls and limitations in biomarker development. nallagangula et al. 7 of performance characteristics, analytical validation, accuracy, precision, and clinical utility. factors that affect discovery and validation should be controlled to overcome pitfalls in biomarker pipeline. evidence-based biomarker which fulfills regulatory requirements should be introduced into clinical practice by collaboration with research laboratory, diagnostic 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title¼21&part¼814&section¼1&subpart¼& type¼text (accessed august 2017). 53. agarwal a, ressler d, and snyder g. the current and future state of companion diagnostics. pharm personal med 2015; 8: 99–110. nallagangula et al. 9 http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text http://frwebgate.access.gpo.gov/cgi-bin/get-cfr.cgi?year=current&title=21&part=807&section=81&subpart=&type=text 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/hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice 404 not found article journal of circulating biomarkers perspectives on the potential therapeutic uses of vesicles review article giovanni camussi1* and peter j. quesenberry1 1 department medical sciences, university of torino, torino, italy department of medicine, the warren alpert medical school of brown university, providence, ri, usa *corresponding author(s) e-mail: giovanni.camussi@unito.it received ; accepted 8 november 2013 © 2013 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the role of extracellular vesicles as an important mediator of cell-to-cell communication has been well established by many studies that have shown their capability for exchang‐ ing proteins, bioactive lipids and nucleic acids. extracellu‐ lar vesicles have been implicated in several physiological and pathological processes according to the cell of origin. identification of the innate properties of extracellular vesicles derived from stem cells and from immune cells has led to the possibility of their exploitation in regenerative medicine and immune therapies. as extracellular vesicles are able to cross biological barriers, express surface receptors and contain defined cargoes able to target specific cells/tissues, they may represent a biocompatible and effective tool for drug delivery. herein, we review and discuss the perspectives related to the therapeutic oppor‐ tunities of extracellular vesicles. keywords exosomes, microvesicles, therapy 1. introduction liposomes, which are synthetic phospholipid vesicles, have been used in the delivery of anticancer agents for the treatment of different solid tumours [1]. anticancer agentcarrying liposomes are currently being investigated in several clinical trials [2-7]. in particular, polyethylene (peg)-coated liposomes, which are long-lasting circulating liposomes, passively accumulate within tumours as a consequence of increased micro-vascular permeability and defective lymphatic drainage [8, 9]. to reduce the side effects of liposomes, targeting strategies have been devel‐ oped using peptides, monoclonal antibodies and small organic molecules to achieve efficient internalization into the tumour vasculature and tumour cells [10]. neverthe‐ less, an ideal liposome, which specifically incorporates into target cells whilst avoiding the potential toxicity of its lipid membrane and the immunogenicity of targeting molecules, remains evasive. naturally-occurring secreted vesicles, which are present in large amounts within biological fluids and therefore physiological constituents, could represent a valid alternative for overcoming some of the limitations posed by liposomes. secreted vesicles are heterogeneous populations of small vesicles released by eukaryotic cells. they have been classified on the basis of the cell of origin, specific function, or of their biogenesis and are known in the literature by different names such as prostatosomes, cardiosomes, tolerosomes, microparticles, ectosomes, microvesicles and exosomes. moreover, the cell-released vesicles also include apoptotic bodies, generated by 1 blebbing of apoptotic cell membranes. virtually all cells can secrete vesicles in basal conditions; however, this event is particularly evident for certain cell types and it may increase during cell proliferation and cell activation or after expo‐ sure to stress conditions [11]. the two major groups of nonapoptotic vesicles defined by their biogenesis are microvesicles and exosomes. microvesicles have been defined as small vesicles generated by direct budding of the cell membrane, with a size ranging from 50 to 1000 nm. microvesicles express surface receptors that vary accord‐ ing to the membrane composition of the cells of origin and may include molecules such as integrins, selectins and the cd40 ligand [12]. exosomes have been defined as originating from inward budding of membranes of multivesicular bodies, followed by their fusion with the cell plasma membrane and release into the extra-cellular space [11, 13]. exosomes, which are thought to be smaller than microvesicles (30-120 nm), express cell type-specific proteins and molecules that are considered specific markers of exosomes of different origin, such as cd63, cd9, cd81 tetraspanin family members, flotillin, cd82, tsg101, alix and other components of the endosomal sorting complex required for transport (escrt). moreover, some exosomes may contain the heat shock 70kda protein 8 and rab-gtpases [13, 14]. this distinction, based on biogenesis, size, sedimentation on sucrose gradients, protein and lipid composition, remains confusing because the markers used for defining vesicles are frequently not exclusive, and may vary depending on the cell of origin. in addition, small vesicles have recently been reported to have a broad range and size [15, 16]. for this reason, the use of the generic term “ex‐ tracellular vesicles (ev)” has been suggested for all secreted vesicles [17]. recent studies have suggested that ev may act as vehicles for horizontal exchange of information between cells, independently from their biogenesis and characteristics [11-13, 18-20]. ev may either activate target cells by means of surface receptors or bioactive lipids, or by delivering their cargo, which may include transcription factors or nucleic acids, in particular, extra-cellular secreted rna (exrna) [21-24]. the exrna that may convey paracrine/endocrine signals are present in all human biological fluids in degradative enzyme-protected forms, and are associated with protein carriers such as ago2 and hdl or encapsulated within ev [25-29]. both microvesicles and exosomes are exrna enriched and include mrna, microrna (mirna) and long non-coding rna, and may enable transfer of genetic information between cells, which infers important physiological and pathological implica‐ tions. the mrna can be translated in the recipient cells, ensuing in the activation of intracellular pathways [22]. the mirnas, which are known to regulate more than 80% of all protein-encoding genes and the long non-coding rna (implicated in the regulation of the epigenome) may induce changes in the cell phenotype [30]. it is therefore conceiv‐ able that, under physiologic conditions, ev may play a critical role in signalling mechanisms for essential cellular and biological functions. naturally-occurring vesicles, given their properties of selectively targeting certain cell types or tissues in order to deliver their cargo, are potential candidates for therapeutic applications. on one hand, the innate therapeutic potential of ev derived from certain cell types can be exploited, for example stem cells; on the other hand, ev may represent a biocompatible and effective tool for drug delivery, as they are able to cross biological barriers [31]. 2. innate therapeutic potential of ev the possibility of exploiting the innate therapeutic poten‐ tial of ev is based on the observation that, by delivering their bioactive cargo, ev plays a critical role in cell-to-cell cross talk [32]. ev derived from certain cell types may deliver information that reprogram target cells. this is the case, for example, of ev produced by stem/progenitor cells, which may convey information required for tissue regen‐ eration or from immune modulatory cells that could potentially inhibit or promote specific immune responses. 2.1 role of ev in stem/progenitor cell biology ratajczak et al. [21] demonstrated that ev released by murine embryonic stem cells may modulate hematopoietic progenitor phenotypes by transfer of proteins and mrna, including nanog, rex-1, oct-4 and hoxb4 early-transcrip‐ tion factors. ev from embryonic stem cells were also found to carry abundant mirna, which can be transferred in vitro to mouse embryonic fibroblasts; this suggests that evderived stem cells can modulate the gene expression in recipient cells, as mirnas regulate protein translation [33]. we found that ev released by endothelial progenitor cells (epcs) activated angiogenesis in quiescent endothelial cells by transferring pro-angiogenic mrna [22] and mirna [34] fromepcs to endothelial cells. adult human stem cells, such as bone marrow-derived multipotent stromal cells (msc) and human liver stem cells (hlsc), secrete ev that contain specific subsets of func‐ tional mrna [35, 36] and mirna [24] associated with the mesenchymal phenotype, and control transcription, proliferation and immune regulation. ev released by msc and hlsc contain the ribonucleoproteins tia, tiar, hur and staufen, which are responsible for rna transport, stability and storage of mrna, along with the argonaute 2 (ago2) protein, a critical component of the rna-induced silencing complex (risc), involved in the transport and maturation of mirna [24]. by comparing the mirna content of ev with that of parental cells, an enrichment of certain subsets of mirna within ev was observed, suggesting non-random mirna compartmentalization during ev formation [24]. this compartmentalization may be modulated by certain stimuli, as illustrated by epc, where hypoxia was found to enhance ev expression of the angiomir mir-126 and mir-296 [34]. 2 direct ev-mediated delivery of mrna into bone marrow cells and induction of transcription was demonstrated by aliotta et al. [37]. several other studies using reporter mrna have shown its translation into proteins following ev-mediated delivery, both in vitro and in vivo, indicating that transferred mrna is functional [22, 24, 37]. likewise, mirnas transferred by ev were shown to block translation of target mrna, suggesting that they may influence the phenotype of recipient cells [23, 24, 33]. based on the properties of ev, quesenberry et al. [38, 39] recently revised the hierarchical vision of the stem cell niche [40] by proposing an alternative model of stem cell biology defined as “continuum” and characterized by reversible change of stem cell phenotype during the cell cycle [41]. the terminal-differentiating stimulus is provid‐ ed by environmental factors, including ev that may modulate the stem cell plasticity by means of genetic information exchange in a defined microenvironment. the genetic information exchange between tissue resident cells and stem cells is bi-directional and may account for stem cell phenotypic changes and activation of tissue regenera‐ tive programs. transfer of lung-specific mrnas, such as those coding for surfactant b and c and clara cell-specific protein, to bone marrow cells via ev released from the injured lung cells was shown by aliotta et al. [37]. further work indicated that immediate expansion of lung-specific mrna in marrow cells was due to transfer of both lung mrna and lung-derived transcriptional regulation, but long-term genetic change was due to transcriptional modulation on epigenetic change in target marrow cells. [42]. conversely, ev derived from stem/progenitor cells may modulate the phenotype of injured tissue and promote regeneration and cell differentiation. therefore, the observation that the phenotype of target cells can be modified by ev-mediated transfer of exrna provides a new perspective for the paracrine/endocrine hypothesis of stem cell action. 2.1.1 ev derived from stem/progenitor cells in tissue regeneration the beneficial effects of stem cell-based therapies are not supported by any tangible indication that in vivo stem cells are able to permanently engraft the injured tissues and substitute parenchymal cells, despite their in vitro plastici‐ ty. instead, the current view is that stem cells induce regeneration by paracrine/endocrine mechanisms [43, 44]. this hypothesis is supported by a number of studies showing that stem cell-conditioned media hold the same beneficial properties of the stem cell of origin [45-47]. moreover, a major contribution of resident cells in tissue regeneration after injury has been demonstrated in many organs such as the liver [48], heart [49] and kidneys [50]. in this context, if ev were able to reproduce the regenera‐ tive action of stem cells, they may provide an important new therapeutic perspective. we observed that ev from human bone marrow-derived mscs were able to promote the recovery of acute kidney injury (aki) in a manner similar to the cell of origin [35]. moreover, ev administered with a therapeutic regimen in a lethal model of aki induced significantly improved survival and tissue regeneration [51]. we have shown that msc-derived ev obtained by differential centrifugations express several mesenchymal markers, including cd105, cd73, cd44 and cd29, as well as a lamp-1 exosomal marker [51]. this healing effect of ev was associated with the transfer of human msc-specific mrna and its transient translation into proteins within the injured kidneys of scid mice [35, 51]. studies on bio-distribution indicated a selective accumulation within the injured kidney but not in the normal kidney. this tropism of ev for the injured tissue exploited the same adhesion molecules expressed by msc. within the kidney, ev are incorporated by injured endo‐ thelial and tubular cells, resulting in the prevention of cell apoptosis and the induction of proliferation of tubular epithelial cells, with a reconstitution of parenchymal integrity. in vitro, ev derived from human msc were shown to induce a stem cell-like phenotype of renal tubular epithelial cells, with subsequent activation of regenerative programs [35]. the transcription regulators delivered by ev caused modification of gene expression in tubular epithelial cells, with a consequent up-regulation of the anti-apoptotic genes birc8, bcl-xl and bcl2 and down-regulation of the pro-apoptotic genes casp1, casp8 and lta [51]. ev were also shown to transfer human igf-1r mrna from msc to cisplatin-injured murine proximal tubular cells, thus enhancing tubular cell sensitivity to igf-1, which is involved in renal regeneration [52]. this observation may provide an explanation for the powerful renoprotection provided by just a few msc that are engrafted-onto the kidney in msc-based therapy. a renoprotective action has been described for ev derived from epc in a model of ischemia-reperfusion injury (iri), characterized by diffuse endothelial and tubular cell damage [34]. epc-derived ev display pro-angiogenic properties, as they can transfer mrna associated with the pi3k/akt signalling pathway [22] and pro-angiogenic mir 126 and mir 296 [34]. once injected into diseased animals, ev localize within peritubular capillaries and tubular cells, restraining tissue damage and favouring a rapid recovery from aki, and preventing capillary rare‐ faction, glomerulosclerosis and tubulo-interstitial fibrosis, which causes chronic kidney damage [34]. non-specific mirna depletion of ev by dicer knockdown in epcs, specific depletion of mir-126 and mir-296 by sirna transfection in epc, or inhibition by the use of antagomirrs prevented the renoprotective effect of ev [3]. by means of a similar mechanism, epc-derived ev were found to improve vascularization and favour muscle regeneration in a model of hind limb ischemia made by ligation and resection of the left femoral artery in scid mice [53]. 3giovanni camussi and peter j. quesenberry: perspectives on the potential therapeutic uses of vesicles ev have also been shown to display a therapeutic effect in other organs. for example, in a murine model of myocar‐ dial ischemia/reperfusion injury, ev derived from the conditioned medium of msc were shown to decrease infarction size [54]. moreover, it was found that foetal tissue-derived msc was able to produce elevated amounts of ev with cardio-protective activity suitable for therapeu‐ tic use [55]. in addition, ev released from cardiac progen‐ itor cells may be exploited as a potential therapeutic resource for myocardial pathology [56]. our group also showed that ev derived from hlsc were able to stimulate liver repair in 70% hepatectomized rats [36]. in a recent study, li et al. [57] demonstrated that ev obtained from mesenchymal stem cells derived from the human umbilical cord alleviates liver fibrosis and protect hepatocytes in a carbon tetrachloride model of chronic liver injury. a neuro-regenerative potential of msc-derived ev has also recently been proposed [58]. ischemic brain extracts enhance the expression of mir 133b in msc-derived ev, which has been shown to play a critical role in functional recovery after spinal cord injury in zebra fish [59]. iglesias et al. [60] suggested a possible application of ev derived from msc of normal human subjects in the correction of a genetic disorder known as cystinosis. ev were found to transfer cystinosin protein and mrna to human cystinotic cells and to reduce in vitro cystine accumulation. 2.1.2 ev-mediated reprogramming of tumour cells although the physiological functions of ev in different tissues are mostly uncharacterized, it is emerging that they not only depend on the ev-carried molecules, but are also influenced by the functional and metabolic state of target cells. indeed, the same ev may exhibit contrary effects depending on the different states of activation or inhibi‐ tion of particular metabolic pathways in recipient cells. for instance, ev released from hlsc are able to accelerate liver regeneration [36] whilst inhibiting hepatoma growth, as hlsc-derived ev carry antitumor mirna, which pro‐ mote tumour regression [61]. these mirna lacking in tumour cells are delivered by ev and then reprogram tumour cells to a more benign phenotype. similarly, ev derived from bone marrow-mscs are able to induce regression of different tumours by inhibiting cell cycle progression and inducing apoptosis [62]. however, as previously shown for msc, the time of ev delivery is critical and msc-derived ev may in fact enhance tumour engraft‐ ment by promoting neoangiogenesis [63], or may induce regression of an established tumour by favouring tumour cell apoptosis [62], depending on when they are adminis‐ tered. recent studies conducted by the quesenberry and chatterjee groups indicate that vesicles derived from normal prostate cells can reverse the chemoresistance and anchorage-independent growth of malignant prostate cancer cells in vitro [64]. 2.1.3 ev-mediated modulation of the immune response raposo et al. [65] first demonstrated that b lymphocytes secrete antigen-presenting vesicles, which were shown to express peptide-bound class ii major histocompatibility complex (mhc) able to induce a t cell response. since then, a number of studies recently reviewed by gutierrezvasquez et al. [66] confirmed the potential involvement of ev modulation in the immune response. a critical role has been suggested for ev-mediated exchange of information at the level of immune synapses, leading to the initiation of the immune response. this innate ability of ev to potentiate an immune response can be exploit‐ ed for cancer immune therapy. indeed, zitvogel et al. [67] demonstrated the possibility of eradicating murine tumours using exosomes expressing class i and class ii mhc molecules derived from dendritic cells pulsed with tumour peptides. subsequent studies have demonstrat‐ ed the enhancement of the t cell response, the protec‐ tion of t cells from apoptosis, the enhancement of the production of pro-inflammatory cytokines and natural killer activity [68]. clinical trials based on vaccination with tumour antigen-loaded dendritic cell-derived exosomes are presently underway (clinicaltrials.gov). on the other hand, vesicles derived from cells that possess immune modulatory properties, such as msc [69] or il10-treated dendritic cells [70] may display antiinflammatory and immune inhibitory properties, which could be exploited in the treatment of immune-mediat‐ ed diseases. it has been proposed that ev released from msc may produce tolerogenic signals by stimulating production of il10 and tgf beta anti-inflammatory cytokine, and by expansion of cd4+, cd25+and foxp3+regulatory t cells [71]. 3. therapeutic potential of ev for drug delivery based on the knowledge that ev express surface receptors and contain selected patterns of proteins and exrna, we can potentially generate ev expressing or containing desired molecules by engineering the cells of origin. the feasibility of this approach has been demonstrated by the gould group through protein targeting ev by plasmamembrane anchors [72]. moreover, mirna or sirna transfected within the cell of origin have been shown to result in their incorporation in secreted ev [24, 61]. alvar‐ ez-erviti et al. [73] generated ev capable of specifically delivering sirna to oligodendrocytes, microglia and neurons in the brain of intravenously injected mice, by inducing dendritic cells to express the exosomal membrane protein lamp2b fused to the neuron-specific rvg peptide. the therapeutic potential of this strategy was shown by the knockdown of bace1 mrna and protein, a therapeutic target in alzheimer's disease [73]. this study also demon‐ strates that targeted ev can cross the blood-brain barrier and target neurons without significant immunogenicity or toxicity. it has also been shown by zhuang et al. [74] that ev loaded with anti-inflammatory drugs, unlike lipo‐ somes, were able to cross the blood-brain barrier after 4 intranasal administration. the therapeutic potential of this strategy was investigated in neuro-inflammation induced by lipopolysaccharide and in experimental encephalomye‐ litis with curcumin-containing ev. in addition, in a mouse model of glioblastoma, ev complexed with a stat3 inhibitor were used. to improve neuron targeting of ev, andaloussi et al. [75] generated exosomes expressing the rvg peptide. another example of the use of ev in therapy was demon‐ strated by ohno et al. [76], who engineered cells to obtain ev expressing the ge11 peptide fused with the transmem‐ brane domain of platelet-derived growth factor receptor and which showed an efficient in vivo delivery of mirna to breast cancer cells bearing the epidermal growth factor receptor (egfr). moreover, akao et al. [77] demonstrated that after transfection of mir-143bps in human monocytic leukaemia thp-1 cells, rna was secreted within ev. after intravenous injection of shed ev containing mir-143bps, the level of this mirna was significantly increased in the serum, tumour and kidneys of the host animals [77]. these studies indicate that the ex vivo manipulation of ev donor cells may modify the mirna content of ev; this could be an efficient strategy for delivering specific subsets of mirna to target cells. similarly, van den boorn et al. [78] used loaded exosomes to efficiently deliver sirnas to target cells in vivo in mice. taken together, these experiments provide proof of the concept that ev represent a potential biocompatible vehicle for different therapeutic molecules, enhancing their stability, limiting their potential toxicity and immunoge‐ nicity, targeting specific cells/tissues and enabling them to cross biological barriers. 4. clinical translation of ev-mediated therapies despite the promising results demonstrated in experimen‐ tal animals and the preliminary clinical trials that have tested exosomes in the field of cancer (clinicaltrials.gov), several points should be elucidated before envisaging the use of either the innate therapeutic potential or engineered ev in clinics. firstly, there is an urgent requirement to develop largescale methods for ev preparation. this implies identifica‐ tion of the best suitable cellular sources and culture conditions for gmp production of ev. culture conditions are critical, as it is known that they may influence the yield and the content of ev and thus their bioactivity. the use of ev for regenerative medicine and for drug delivery needs a source of cells that are expandable in gmp conditions and that generate non-immunogenic ev. msc derived from bone marrow, fat or the umbilical cord are potential candidates, as they retain the immune-modulatory prop‐ erties of msc. however, their senescence after repeated culture passages may limit their use. to overcome this limitation, chen et al. [79] immortalized human embryonic stem cell-derived mscs, enabling large-scale production of exosomes. these msc remained unchanged in quantity and quality, and the immortalizing oncogene was not detected within exosomes. however, cell immortalization may present safety concerns and cannot be accepted by regulatory agencies. another potential source of ev are hlsc [36]. hlcs can be expanded on a large scale in gmp conditions; they do not undergo senescence and they maintain a stable karyotype up to the 24th passage. we have found that these cells do not require immortalization and the derived ev share several properties with those of msc. another critical point is to develop an easy, reproducible and efficient gmp purification protocol. the gold standard protocol for ev purification against which all other techniques should be evaluated is based on differential ultracentrifugation to remove cell debris and large vesicles, and to collect the small vesicles. as the ultracentrifugation technique is time-consuming with a low yield and poses concerns about the loss of the biological activities of ev, some alternative techniques have been under evaluation, including immunoaffinity [80, 81] and ultrafiltration using membranes with different-sized pores, combined with gel filtration by liquid chromatography [55]. so far, no ideal scalable purification technique applicable to a gmp condition is available. ev purified by all these techniques contain heterogeneous populations and a more precise purification, by combining different methods and using a sucrose gradient, is not feasible for a gmp production. therefore, it is critical to define the healing ev population and to understand how far purifi‐ cation should be taken. questions that need to be answered are: would non-healing populations interfere with the desired biological activity? what is the purity level that is required? moreover, a test of potency should be developed for comparison of different ev batches. ideally, the test should be in vitro, easily reproducible and straightforward, and should be appropriately designed for each field of application. finally, it is important to define identity marker/s that correlate with the ev potency. pre-clinical studies are also needed to determine in vivo bio-distribu‐ tion of labelled ev to evaluate whether ev localize in the required organs. finally, the biosafety of acute and chronic administration of ev requires further studies. 5. conclusions ev have emerged as an important vehicle of information between cells, as they can transfer bioactive proteins, lipids and nucleic acids. this property can be exploited for therapy in different fields, as ev retain several biological activities of the cell of origin. ev released in physiological conditions have innate therapeutic potential. stem cellderived ev mimic the favourable effects of the cell of origin, thus promoting repair and limiting injury in several organs, as they are able to activate regenerative programs and coordinate tissue self-repair. due to the expression of membrane receptors derived from the stem cell of origin, ev can localize at the site of injury and deliver their cargo to damaged cells. the complex constituent array of ev 5giovanni camussi and peter j. quesenberry: perspectives on the potential therapeutic uses of vesicles allow them to influence multiple cellular pathways involved in different pathological conditions. ev derived from immune cells may potentiate the immune response and can therefore be used in cancer therapy. the identifi‐ cation of molecules responsible for the biological effect of ev may provide critical information for engineering ev for therapeutic purposes. the possibility of developing specifically targeted and drug-loaded ev may allow for the development of new therapeutic strategies. additional investigations into the pathological conditions that may benefit from an ev-based therapy, as well as a definition of suitable, scalable gmp protocols of ev production are needed. moreover, the biosafety and pharmacokinetics of ev require further studies. 6. acknowledgements research reported in this publication was supported by the national center for advancing translational sciences of the national institutes of health under award number uh2tr000880. the content is solely the responsibility of the authors and does not necessarily represent the official views of the national institutes of health. 7. references [1] schiffelers rm, storm g: liposomal nanomedicines as anticancer therapeutics: beyond targeting tumor cells. int j pharm 2008, 364:258-264. 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[81] mathivanan s, lim jw, tauro bj, ji h, moritz rl, simpson rj: proteomics analysis of a33 immunoaf‐ finity-purified exosomes released from the human 9giovanni camussi and peter j. quesenberry: perspectives on the potential therapeutic uses of vesicles colon tumor cell line lim1215 reveals a tissuespecific protein signature. mol cell proteomics 2010, 9:197-208. 10 article journal of circulating biomarkers exosomes: mechanisms of uptake review article kelly j. mckelvey1*, katie l. powell1,2, anthony w. ashton1, jonathan m. morris1,3 and sharon a. mccracken1 1 division of perinatal medicine, kolling institute of medical research, university of sydney at royal north shore hospital, st leonards, nsw, australia 2 pathology north, nsw health pathology at royal north shore hospital, st leonards, nsw, australia 3 department of obstetrics and gynaecology, royal north shore hospital, st leonards, nsw, australia *corresponding author(s) e-mail: kelly.mckelvey@sydney.edu.au received 25 march 2015; accepted 30 june 2015 doi: 10.5772/61186 © 2015 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract exosomes are 30–100 nm microvesicles which contain complex cellular signals of rna, protein and lipids. because of this, exosomes are implicated as having limitless therapeutic potential for the treatment of cancer, pregnancy complications, infections, and autoimmune diseases. to date we know a considerable amount about exosome biogenesis and secretion, but there is a paucity of data regarding the uptake of exosomes by immune and nonimmune cell types (e.g., cancer cells) and the internal signalling pathways by which these exosomes elicit a cellular response. answering these questions is of para‐ mount importance. keywords exosome, immunology, endocytosis 1. introduction in the 1960s, robert feynman implored researchers to try to develop nanotechnology with the ability to interact with the human body at the cellular level [1]. since the 1990s, nanomedicines generated from polymeric or liposomal nanoparticles—encapsulating or adsorbing one or more drugs—have been assessed in clinical trials and imple‐ mented in the clinic, for the treatment of cancer, hiv/aids, malaria, and tuberculosis. biologically, exosomes offer a viable ”natural” therapeutic avenue for immune modula‐ tion. exosomes are nanoscale (30–100 nm) vesicles contain‐ ing lipid, protein and rna species in a single biological unit. they are present in the intracellular space and in body fluids (including plasma, saliva, urine, pleural ascites, amniotic fluid, cerebrospinal fluid, colostrum, breast milk and semen), and may act locally or from a distance, through the secretion of soluble factors or cell-cell contact. previous reviews have detailed exosome biogenesis, composition, and the cellular response of t cells to tumour[2], dendritic cell (dc)[3–5], and placenta-derived exosomes [6]. this review highlights recent advances in these research areas of exosome function, but focuses on what is currently known about exosome targeting, inter‐ nalization and elimination, with specific reference to t cells. 2. exosome biogenesis and secretion exosome biogenesis starts with the invagination of the late endosomal limiting membrane into the lumen to form multi-vesicular bodies (mvbs). consistent with the formation of other cell-derived vesicles, the formation of exosomes requires the evolutionarily conserved soluble nethylmaleimide-sensitive factor attachment protein receptor (snare), rab, coat complex subunit and sec 1 1j circ biomark, 2015, 4:7 | doi: 10.5772/61186 proteins [7]. at this stage, mvbs contain “recycled” cellsurface proteins, and rna and proteins derived from the cytoplasm. “de novo” proteins from the endoplasmic reticulum and golgi complex may be directly sorted into the mvbs, guided by either (i) sequential action of the endosomal-sorting complex required for transport (escrt) machinery [8] (specifically aip1/alix/vps31 and tsg101/vps23 [9]), or (ii) by a ceramide/tetraspanindependent pathway [10]. most cells, including those grown in culture, are constantly releasing exosomes. in immune cells, this response can be enhanced by potent activation signals, such as antigenic, cytokine or mitogen stimulation [11]. upon stimulation of the cell, mvbs polarize to the immunological synapse within 10 min of engagement [12], and are released into the extracellular environment by fusion with the plasma membrane. the process of mvb docking at the plasma membrane and subsequent fusion is regulated by rab, snap and snare proteins. the rab27b and rab27 effector molecules, synaptotagmin-like 4 and exophilin 5, regulate docking of the mvbs at the plasma membrane [13]. meanwhile, rab11 and rab31 [13, 14], the r-snare protein ykt6 [15], and the v-snare protein vamp7/tivamp [16] are implicated in the fusion event between mvbs and the plasma membrane. rab27a has been shown to play a role in both docking and fusion [13, 14]. notably, it is plausible that some exosomes are released by cells via direct outward budding and fission of the plasma membrane, analogous to shedding microvesicles and apoptotic blebs. 3. exosome cellular recognition while there is a large body of evidence relating to exosome biogenesis, our understanding of exosome internalization is in its infancy. newer techniques such as fluorescent (dil)labelling and the polyparticletracker programme are being used to reveal the dynamics of exosomes during cellular internalization [17]. below is a discussion of the current data on exosome internalization. 3.1 free floating exosomes released from cells circulate in body fluids at least for a short period of time. intravenous administration of fluorescently labelled b cell-derived exosomes, via the lateral tail vein of mice, revealed that the half-life of exosomes in plasma was ~2 min [18]. however, exosomes were detectable in the spleen up to 2 hrs later. following intranasal administration, exosomes have been found within the brain and intestine after 3 hrs [19]. in both studies, exosomes were found to co-localize with macro‐ phages, and did so within 15 min of administration [19]. the data suggest that exosomes are rapidly sequestered by circulating monocyte/ macrophages in the liver and spleen, though whether this is for clearance or cellular signalling is unclear. from intravital video microscopy of capillaries in the ears of mice, in combination with computer modelling, it has been estimated that nanoparticles of the exosome size range (30–100 nm) randomly distribute within the blood vessel, drifting laterally from the red blood cell core toward the vessel wall where leukocytes localize [20]. this may partially explain the systemic diffusion of exosomes to sites distant from the secreting cells, and how exosomes may come into contact with monocytes/macrophages, as well as t cells. similarly to liposomes, this could be mediated by the opsonization of exosomes during circulation [21]. phosphatidylcholine—hydrolysed to lysophatidylcholine by calcium-independent phospholipase a2 (ipla2) on the surface of exosomes from rat reticulocytes—binds natural igm antibodies and complement component c3, promot‐ ing phagocytosis [22]. other opsonins present on the surface of exosomes, including phosphatidylserine and lactoadherin (mfg-e8) [23], may explain the co-localiza‐ tion of exosomes with macrophages noted in abovemen‐ tioned studies [18, 19]. while t cells are not considered ”professional” phagocytes, γδ t cells can phagocytose bacteria and synthetic beads via antibody opsonization and fcγriii receptors [24]. an alternative explanation could be that exosomes use chemokines to attract leukocytes to their location. exo‐ somes express an impressive array of chemokines, which may attract t cells and other cell types [25]. these include ccl2, ccl3, ccl4, ccl5, ccl7, ccl20, ccl28, cxcl1, cxcl2, and cxcl16. of these, ccl2, ccl5, ccl20, and cxcl16 are potent t cell chemoattractants. 3.2 adhesion a fundamental step for exosome-t cell communication is adhesion. lymphocyte adhesion by exosomes requires the conformational change of integrins from a low to a high affinity status. this enables oligomerization of integrins and coupling with cytoskeletal elements, to facilitate the high avidity binding of the lymphocyte to the integrin-bound exosome [26]. the presence of icam-1 (cd54) on mature dc-derived exosomes is critical for efficient naive t cell priming, mediated by the leukocyte integrin lfa-1 (cd11a/ cd18 or αlβ2) [27]. other integrins that have demonstrat‐ ed roles in exosomes-leukocyte adhesion are integrins β1 (cd29), α3 (cd49c) and αv (cd51), and lactadherin and vitronectin, which are ligands for αvβ3/β5 integrins [9, 28, 29]. while the initial binding/docking of t cells to exo‐ somes is regulated by icam-1/lfa-1 [30], firm adhesion is further facilitated via αl (cd11a), α4 (cd49d), cd44 and icam-1 expression on leukocytes, and the expression of tetraspanins cd9 and cd81 on exosomes [29, 31]. tetraspanins are highly conserved through evolution and have a role in adhesion, motility, signal transduction and cell activation. a number of tetraspanins have been reported on the surface of exosomes, including cd9, cd53, cd63, cd81 and cd82, although the combina‐ tion and proportion of exosomes expressing a particular 2 j circ biomark, 2015, 4:7 | doi: 10.5772/61186 tetraspanin differs depending on the cell of origin. one example is the abovementioned observation that only ~50% of exosomes from activated platelets express cd63 [32]. tetraspanins form heterobimolecular complexes with integrins (e.g., α3β1) [33], ig superfamily members (e.g., icam-1, mica, micb) and co-receptor molecules (e.g., cd4, cd8, cd19, cd21) [26, 34, 35]. it is thought that the expression of tetraspanins on exosomes contrib‐ utes to the spatial assembly for antigen recognition and may partially dictate the signal induced by the exo‐ some. for example, tcr co-stimulation via cd9 result‐ ed in only partial t cell activation before the t cell underwent apoptosis, while traditional co-stimulation via cd28 leads to the proliferation of t cells [36]. other adhesion proteins are demonstrated to play a role in the capture of exosomes, including cd169 (sialoadhesin) on macrophages [18], and heparin sulfate proteoglycans on both u-87 mg glioblastoma cells [37] and 293t human embryonic kidney cells [38], while the latter may also bind cytotoxic and/or helper t cells. 3.3 antigen recognition to date, molecular profiling and proteomic analysis has demonstrated that target cell specificity for exosomes appears to be dictated solely by a combination of antigen and mhc class i (tc cells) and ii molecules (cd4+ t cells) [39]. the expression of mhc molecules on exosomes is dependent on the expression of the molecule on the parent cell. while the micrornas contained in some exosomes differ greatly to those of the parent cell [12, 40], the expres‐ sion of mhc class i and ii molecules is similar to that of the originating cell [41]. the expression of mhc class i molecules on exosomes induces a negative signal via inhibitory receptors on the recipient cell, such as ilt2, ilt4 and kir2dl4, which promote the inhibition of cd8+ tc cells and nk cell responses. mhc class i molecules are often found on exosomes in conjunction with other immune modulating molecules, such as the b7 molecules cd274 (b7h-1) and cd276 (b7-h3) [42], and the mhc class i-related molecules mica and micb, which down-regulate the nk cellactivating molecule nkg2d on immune cells [43]. exo‐ somes expressing hla-g1 are secreted by melanoma cells [44], and may play a role in immune evasion, which enables escape from the immune response. mhc class ii molecules evoke a stimulatory signal and promote the proliferation and differentiation of cd4+ t cells. exosomes from antigen-presenting cells such as dcs and b lymphocytes express mhc class ii. exosomes bearing hla-dr1–haemagglutinin (306–318) complexes weakly activate ha/dr1-specific t cells; however, the incubation of hla-dr1+ exosomes with dcs resulted in highly efficient stimulation of antigen-specific t cells [45]. it remains to be seen whether there is another as-yetunidentified mechanism of cell targeting by exosomes. 4. exosome internalization it is still unclear as to whether exosomes must be internal‐ ized by immune and non-immune cells in order to elicit cellular responses. for example, cellular responses elicited by rna species rely on internalization. by comparison, cellular responses induced by membrane-bound or soluble fasl and trail from exosomes do not require internali‐ zation, but are dependent on location and temporary adhesion for juxtacrine or soluble signalling. equally, differing opinions as to the method by which exosome internalization occurs—either by fusion, receptor-mediat‐ ed endocytosis, macropinocytosis or phagocytosis—exists within the literature. however, the latter two methods may represent mechanisms for the clearance of exosomes, rather than the elicitation of a cellular response. a summary of the internalization mechanisms for exosomes, and some of the key proteins involved, is provided in figure 1. 4.1 soluble and juxtacrine signalling soluble signalling involves the proteolytic cleavage of ligands from the exosomal surface or alternative splicing, while juxtacrine signalling requires the juxtaposition of ligands and receptors on the surfaces of the exosome and target cell. membrane-bound fasl, trail and tnf can be cleaved by metalloproteinases to form soluble cytokines. nevertheless, it should be noted that the death ligands, soluble fasl and trail, have a reduced pro-apoptotic activity when compared to that of the membrane-bound form [46, 47]. exosomes from cultured placental explants or plasma from pregnant women have fasl and trail on their membrane, and induce apoptosis in jurkat t cells, via nf-κb, cd3ζ and jak3 down-regulation [48–50] [sharon mccracken, unpublished findings]. the same mechanism has been demonstrated for exosomes derived from tu‐ mours [51]. 4.2 fusion vesicle-cell fusion is the process by which a vesicle merges with the plasma membrane of a cell. using a fluorescent lipid-mixing assay and membrane fusion assay, monocytederived microvesicles were demonstrated to bind and fuse with the plasma membrane of activated platelets, and to transfer proteins to the recipient cell, such as tissue factor and p-selectin glycoprotein ligand-1 (psgl-1) [52]. in a similar experiment, exosomes from metastatic melanoma cells fused with the plasma membrane, which could have been inhibited by filipin [53]. proteins were found to have a minor, possibly structural, role during fusion. colocalization of exosomes with rab53 or lamp-1 suggested that exosomes are internalized and interact with cytoplas‐ mic vesicles [53]. unlike endocytosis, in which multiple mechanistic path‐ ways have been detailed, the mechanism of cell-cell fusion is incompletely understood. the phenomenon may be regulated by tetraspanin complexes on target cells. tetra‐ spanins have a role in t cell activation and membrane 3kelly j. mckelvey, katie l. powell, anthony w. ashton, jonathan m. morris and sharon a. mccracken: exosomes: mechanisms of uptake fusion between sperm-oocyte [54, 55], myoblasts [56], mononuclear phagocytes [57] and mammalian viral-cell fusion [58]. while tetraspanin cd81, which co-localizes with cd4, has been shown to be involved in exosome release from hivinfected t cells [59, 60], it has not yet been demonstrated whether tetraspanins are involved in exosome-t cell fusion. in the context of viral-cell fusion, tetraspanins inhibit cell fusion; cd9, cd63, cd81, cd82, cd151 and cd231 reduce hiv infection [61, 62] by inhibiting cell fusion [63, 64] and cell-cell transmission of viral particles [65]. the inhibition of cell-cell fusion of mononuclear phagocytes by cd9 and cd81 has also been reported [57]. whether this is also true in the context of exosome-cell fusion remains to be seen. it also seems plausible that, in a similar manner to leuko‐ cyte transendothelial migration, integrins are involved in exosome adhesion/attachment to the target cell, and following this, tetraspanin-enriched microdomains facilitate exosome fusion, which is alluded to in previous reports [66, 67]. 4.3 phagocytosis phagocytosis is an actin-mediated mechanism which requires the presence of specific opsonin receptors (i.e., fcr and complement receptors), scavenger receptors or toll-like receptors (figure 1). while phagocytosis is typically performed by “professional phagocytes” such as macro‐ phages and dcs, it can also be performed by “nonprofessional” cells, including γδ t cells [24]. phagocytosis has been proposed as a means of exosome internalization. not surprisingly, monocytic/macrophagic cell lines were able to internalize exosomes derived from erythroleukae‐ mia (k562) and t cell leukaemia (mt4) cells more efficient‐ ly than ”non-professional” phagocytic cells, including jurkat t cells and 293t human embryonic kidney cells [68]. the phagocytosis of exosomes was shown to be dependent on the actin cytoskeleton, phosphatidylinositol 3-kinase (pi3k), and dynamin2 [68]. notably, actin, pi3k and dynamin2 have all been implicated in both clathrinmediated endocytosis [69], and phagocytosis [70]. internal‐ ized exosomes co-localized with lamp-1, lysobisphosphatidic acid and rab7 [68] in late autophago‐ figure 1. schematic of exosome biogenesis, internalization and cellular response. the adhesion of exosomes to the recipient cell utilizes the interaction of various exosomal surface proteins and cellular receptors. once bound, the exosome may (i) elicit transduction of the signal via intracellular signalling pathways and be released (juxtacrine signalling); (ii) fuse with the cellular membrane transferring protein and genetic contents, into the cytoplasm of the recipient cell (fusion); or (iii) be endocytosed via phagocytosis, macropinocytosis or receptor-mediated endocytosis. this figure was produced using servier medical art, available from www.servier.com/powerpoint-image-bank. 4 j circ biomark, 2015, 4:7 | doi: 10.5772/61186 somes and/or endosomal and lysosomal vesicles [71]. determining whether phagocytosis represents a true method of exosome internalization for the purpose of intercellular communication, or is merely a means of elimination, requires further research. 4.4 macropinocytosis during macropinocytosis, plasma membrane protrusions driven by actin filaments form an invagination which nonspecifically endocytoses extracellular fluid and small particles. reportedly, phosphatidylserine (ptdser; figure 1) on the surface of oligodendrocyte-derived exosomes activated macropinocytosis in a subset of microglia/ macrophages without antigen-presenting capability [72]. macropinocytosis of exosomes is dependent on na+ and pi3k, with the inhibition of na+-h+ ion exchange and pi3k activity by the pharmacological inhibitors eipa and ly294002, respectively, reducing exosome uptake [73]. 4.5 receptorand raft-mediated endocytosis as the names suggest, receptor-mediated endocytosis and raft-mediated endocytosis require either a ligand on the exosomal surface to engage specific receptors on the cellular plasma membrane, or the presence of cholesteroland sphingolipid-rich microdomains in the plasma mem‐ brane, respectively. the former, also called clathrinmediated endocytosis, utilizes clathrin and adaptor protein 2 complexes which coat the membrane and induce the invagination of the membrane into a vesicle. the latter includes caveolae-mediated endocytosis, as well as the clathrinand caveolae-independent endocytosis mecha‐ nisms rhoa-, cdc42-, and arf6regulated endocytosis, which utilize distinct combinations of dynamin, flotillin and/or rab proteins [74]. the endocytosis of particles from the external environment or plasma membrane may be sent to lysosomes for degradation, or recycled back to the plasma membrane. exosomes released from cultured rat adrenal gland medulla (pc12) tumours are partially internalized by clathrin-mediated endocytosis, as demonstrated using pharmacological reduction (cpz) and sirna knockdown of clathrin [73]. in another report, the internalization of glioblastoma-derived exosomes involved non-classical, lipid raft-dependent endocytosis, and required erk1/2hsp27 signalling [75]. in this pathway, the negative regulation of erk1/2 by caveolin-1 inhibited exosome endocytosis. 5. exosome intercellular trafficking and cellular response a wide range of stimulatory or inhibitory functional outcomes are shown to be induced following cellular interactions with exosomes, including proliferation, angiogenesis, apoptosis, cytokine production, immune system modulation, and invasion or metastasis. whether or not a cellular response is elicited by the target cell likely depends on the mechanism of internalization. soluble, juxtacrine and fusion are most likely to end with a cellular response, as they do not directly engage the endosomal-lysosomal degradative pathway. phagocytosis inevitably results in the fusion of the phagosome with lysosomes and the degradation of its contents. the endocytosis of exosomes, whether by macropinocyto‐ sis, or receptoror raft-mediated mechanisms, always results in the delivery of the vesicular cargo to the endoso‐ mal pathway. early endosomes act as the sorting compart‐ ment. those sent to late endosomes are then unidirectionally sent for degradation by fusion with lysosomes; meanwhile some proteins and fluids may be redirected back to the plasma membrane via recycling endosomes. however, even those sent to the late endosome may escape degradation by the trans-golgi network. it is suggested that the macropinocytotic clearance of exosomes by cells lacking an antigen-presenting capability may represent a mechanism for the degradation of the cellular membrane by immune cells in an immunologically 'silent' manner [72]. this implicates macropinocytosis as a mechanism of exosomal clearance rather than cell signal‐ ling. thus, exactly which of these mechanisms represent bona fide mechanisms for exosome signalling and elicit a cellular response, and which are mechanisms of exosome clearance, needs to be delineated. the internal transport network of phagosomes, macropi‐ nosomes, clathrin-coated vesicles and caveosomes requires a spectrum of cellular components. as an example, exosomes from chronic myeloid leukaemia cells were shown to require v-snare protein vamp3/cellubrevin for the fusion of endosomes with autophagosomes [16], while vamp7/ti-vamp was required for fusion between the amphisome and lysosome. similar dynamics of diffusion on the plasma membrane and internalization have been observed for natural and synthetic viruses [76, 77]. the internalization of exosomes from glioblastoma cells was dependent on erk1/2 and hsp27 signalling [75]. not surprisingly, intracellular filamentous actin [78] and microtubules [79] are also critical for the transport of exosome-containing vesicles within the cell following internalization. whether other cellular proteins known to be involved in endosomal transport—such as p38α, pkcδ, arrestins and syntaxins—are utilized when exosomes are internalized remains to be demonstrated. 6. exosomes, t cells and therapy since exosomes are naturally present in the circulation of all individuals, and elicit effects that alter cellular function‐ ing, they present a possible therapy in many disease settings. molecular profiling and the functional analysis of tumour-derived exosomes show that they express death 5kelly j. mckelvey, katie l. powell, anthony w. ashton, jonathan m. morris and sharon a. mccracken: exosomes: mechanisms of uptake ligands and mediate apoptosis of cd8+ t cells (for a recent review, see [2]), while dc-derived exosomes promote cd4+ t cell proliferation and regulate t cell responses [80]. placenta-derived exosomes regulate both functional characteristics, including mediating apoptosis, and induc‐ ing the proliferation and generation of regulatory t cells (tregs) [48]. yet the question remains as to what signals are required to elicit the desired therapeutic effect. manipulating the signals on the surface of exosomes or the mrna/microrna content of exosomes seems most plausible and has precedents. il-10-treated exosomes from bone marrow-derived dcs prevented the onset of murine collagen-induced arthritis, and dampened the established disease [81], in a model shown to be associated with a robust and sustained t cell response to type ii collagen [82]. using this same model, exosomes derived from dcs genetically engineered to produce il-4 suppressed the activity of t cells in vivo, via a mhc class ii and fasl/fasdependent mechanism [83]. the reproducibility of using cell culture-derived exo‐ somes for therapy remains an issue. the proteomic profil‐ ing of three independent batches of exosomes, from cultured human embryonic stem cell-derived mesenchymal stem cells (hues9.e1), identified ~400 unique proteins; howev‐ er, only 154 proteins (~20%) were common to all three preparations [84]. thus, the use of manipulated cultures to generate exosomes may result in a vast diversity of exo‐ some protein and rna content. with the relative success of lipid and polymer-based nanomedicines, the extrapola‐ tion of the technology utilized for the extrusion, purifica‐ tion, analysis and labelling of these vesicles would most likely be required to generate therapeutic exosomes [21]. while this review updates what is currently known about the proteins involved in the interaction of (primarily) t cells with exosomes, and their internalization, further elucida‐ tion of these mechanisms is required, focusing on what governs the internalization mechanism used, and the signal transduction pathways leading to cellular responses. once this is known, the therapeutic capability of exosomes is potentially limitless! 7. compliance with ethical research standards the authors declare no conflicts of interest. 8. acknowledgements kelly j. mckelvey’s work is supported by the national medical research council (nhmrc), australia (cia jonathan m. morris, gnt1066606); katie l. powell’s by pathology north, nsw health pathology; and sharon a. mccracken’s by ramsay health care, australia. 9. references [1] feynman r (1960). there's plenty of room at the bottom. eng. sci. 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properly cited. abstract extracellular vesicles (ev), including exosomes, microvesicles and apoptotic bodies, are released from numerous cell types and are involved in intercellular communication, physiological functions and the pathology of disease. they have been shown to carry and transfer a wide range of cargo including proteins, lipids and nucleic acids. the role of evs in cardiac physiology and heart disease is an emerging field that has produced intriguing findings in recent years. this review will outline what is currently known about evs in the cardiovascular system, including cellular origins, functional roles and utility as biomarkers and potential therapeutics. keywords exosome, microvesicle, apoptotic bodies, cardiovascular disease 1. introduction cardiovascular disease (cvd) is a significant contributor to morbidity, mortality and health-care expenditures in the united states. with an improvement in earlier detection of disease, increased focus on primary prevention and better treatment of cardiovascular diseases, there has been a steady decline in mortality attributable to cvd over the past two decades. nonetheless, the burden of cvd remains extremely high, with 32.3% of all u.s deaths directly attributable to cvd [1]. additionally, with the decrease in acute mortality after diseases like acute coronary syndrome, treatment of cardiovascular conditions including heart failure (the late sequelae of myocardial infarctions) now accounts for an ever-increasing fraction of health-care expenditure, with an estimated 183,000,000,000 dollars spent in 2009 [1]. unfortunately, the development of novel therapies to treat cvd has stagnated in the past decade. in light of this, there is an urgent need for an improved mechanistic understanding of heart disease pathogenesis, the identification of novel targets and innovative strategies to deliver therapies. while cardiomyocytes (cms) comprise the majority of the volume of the heart, multiple other cell types, including fibroblasts (which comprise 90% of the non-myocyte cells that make up 5-10% of cardiac tissue), endothelial cells and vascular smooth muscle cells make up the remainder of the heart and play an important role in cardiac function, both in health and in disease [2]. recently, investigators have turned their attention to elucidating mechanisms of communication between these diverse cell-types, particularly as it relates to disease 1kirsty m. danielson and saumya das: extracellular vesicles in heart disease: excitement for the future ? article exosomes microvesicles, 2014, 2:1 | doi: 10.5772/58390 exosomes and microvesicles pathogenesis. extracellular lipid-bilayer enclosed particles have garnered recent attention as mediators of signalling between cell types [3, 4], particularly in the field of immunology and oncology, where their role in various processes including intercellular communication [5], immune induction [6], neoangiogenesis [7] and antigen presentation [8] have been described. investigations into the function of these particles, which include exosomes as well as microvesicles (collectively referred to as extracellular vesicles or evs, see definitions below), are in their infancy in cardiovascular biology. however, several recent studies suggest that these particles may not only play an important role in intercellular communication in the cardiovascular system, but may also have clinical utility as biomarkers in cvd and could potentially be utilized as vehicles for targeting therapies. this review will focus on the current state of knowledge about the role of evs in the cardiovascular system, including characteristics of cardiac evs and their mechanistic function, in addition to their potential use in the clinical arena. 2. characterization of cardiac extracellular vesicles extracellular vesicles (evs) consist of several distinct entities, namely exosomes, microvesicles and apoptotic bodies, which are differentiated by their mechanisms of biogenesis and secretion (figure 1). exosomes are derived from the endolysosomal pathway, stored in multivesicular bodies and are released from cells through fusion of these multivesicular bodies to the cell membrane [9]. they are approximately 40-100 nm in diameter and investigators have proposed several specific surface markers, including tumour susceptibility gene 101 and flotillin, which identify exosomes [9]. microvesicles, in contrast, are generally larger in size, ranging from 1001000 nm in diameter, are formed through budding of the cell membrane and thus are heavily enriched in phosphatidylserine with a membrane composition that resembles that of the parent cell membrane [10]. exosomes and microvesicles contain many cellular components capable of effecting intercellular communication on neighbouring and distant cells. these include cytoplasmic and membrane proteins [11], mrna and microrna (mirna) [3], other non-coding rna [12] and lipids [13]. finally, apoptotic bodies (50-2000 nm) result from blebbing of the surface of the apoptotic cells and contain cell organelles and nuclear fractions, including non-coding rnas that are specific to the nucleus or nucleolus [14]. investigators have described detailed characterizations of the contents of evs released by tumour cells [15], cells of the immune [16] and neural systems [17] and reviewed elsewhere. much of the recent work has focused on the non-coding, particularly the mirna, content of evs. of particular interest is work that notes that the mirna profile of evs is not representative of the parent cell mirna profile, raising the possibility of selective export of certain mirnas into evs [3]. this observation was supported by a subsequent study demonstrating that certain mirnas present in evs were not detectable in the parent tumour cell, thereby suggesting that these mirnas may have been transcribed specifically for transport in evs [18]. at the current time it is not known if a similar process of selective transport of mirnas into evs occurs in the cells that comprise the cardiovascular system. figure 1. extracellular vesicle secretion a significant obstacle in this rapidly developing field is the lack of standardized protocols for isolating each of these components of evs. detailed characterization of evs using proteomics [19], lipidomics [13] and rna-seq [20] has also been hindered by the diversity of the cellular origins of evs in biospecimens, coupled with the complexity of their contents. hence published studies have shown considerable divergence in their findings, with a lack of agreement about standardized surface or content markers for each of these components of evs. current isolation methods for evs include immunoaffinity capture, differential centrifugation, the use of gradients and specialized kits produced by vendors. these isolation methods rely on different characteristics of evs and therefore it is possible that different subpopulations of evs will be isolated, depending on the exact technique deployed. additionally, significant differences in ev viability have been found between different preparations [21]. evs produced using exoquick (system biosciences) were extremely robust and withstood changes in temperature and ph for up to 18 h. by contrast, evs produced using previously published methods for clinical utility 2 exosomes microvesicles, 2014, 2:1 | doi: 10.5772/58390 (ultrafiltration followed by ultracentrifugation through a sucrose/deuterium oxide column [22]) began degrading and releasing their contents within 2 h [21]. therefore, differences in methods used for ev isolation need to be addressed when comparing the findings from different studies. differences in evs from different cell types and species should also be taken into consideration. it is likely that evs of different origins express different surface markers; therefore, selection of evs based on specific markers, obtained through methods such as immunoaffinity, could result in the study of ev subpopulations that exclude some biologically relevant carriers. future detailed characterization of ev subpopulations could result in the ability to specifically isolate subpopulations of interest, which could prove to be a valuable research tool. the study of evs in the cardiovascular field is beset by these same complexities. most studies have relied on ultracentrifugation/ultrafiltration techniques to isolate evs; however, some studies call the isolated entities exosomes, while others refer to them as extracellular membrane vesicles (emvs). most of these studies do not investigate the biogenesis of the isolated evs or characterize their size and composition in detail; hence, in our opinion, it would be better to classify to them as evs. in this review, we will therefore refer to these membranebound extracellular vesicles as evs, regardless of how they were classified in the initial publication. 3. origin of evs in the cardiovascular system cms and endothelial cells have both been shown to secrete evs [23, 24]. like evs from other cell types, they contain a variety of proteins and rnas from the parent cell. proteomic analysis of evs (which were classified as exosomes in this study) from adult rat cms revealed some common proteins with previous studies from different cell origins, including glyceraldehyde-3phosphate dehydrogenase (gapdh) and actin [21]. however, many other proteins previously found in evs in other studies, including hsp90 and the tetraspanins, were not seen in the cm-derived evs [21]. in contrast, another study did identify previously reported tetraspanins in evs derived from human microvascular endothelial cells in culture [24]. it should be noted that many of the earlier proteomics studies focused on evs secreted from human tumour cells. the differences in protein expression between the evs of cardiovascular origin may reflect differences in both cell typeand speciesspecificity of ev characteristics. currently, no studies have directly demonstrated the release of evs from cardiac fibroblasts. however, in neonatal rat cell culture, paracrine factors from cardiac fibroblast-cultured media produced changes in cm electrophysiology [25] and improved cm viability in hypoxic conditions [26]. investigating whether evs are implicated in mediating these paracrine factors would be an interesting area for future research. another interesting finding from proteomics studies is that stimulation of cell cultures with different stressors produces evs with differing contents. this was observed in both adult rat cms in response to hypoxia/reoxygenation or ethanol treatment [21] and in human microvascular endothelial cells in response to hypoxia and tnf-α treatment [24]. in endothelial cell-derived evs, several of these findings, which were confirmed with immunblot analysis, included an increase in lysyl oxidase like-2 in evs from hypoxia-treated cells and an increase in icam1 in evs derived from tnf-α treated cells. in addition, differences in mrna expression in endothelial cells in response to these stressors were observed [24]. these studies raised the possibility that evs may play a role in inter-cellular transfer of stress signals and therefore play an important role in disease pathogenesis. 4. functional role of evs in cardiovascular biology and disease a summary of reported ev release from different cell types in vitro and their functional effects is represented in figure 2. 4.1 cardiomyocyte-derived evs ev release from cms (classified as exosomes in this study) was first reported by gupta and knowlton in primary cultures of adult rat cms, in which evs were isolated using classical differential centrifugation and ultracentrifugation techniques [27]. in this study, evs were released from cms at baseline, but their release was tripled (as measured by acetylcholine esterase activity [28]) in response to brief hypoxia. hsp60, which had not been previously found in evs from other cell types, was determined to be a component of these evs, in addition to the previously described markers hsc70 and hsp90 [27]. the release of evs containing tnf-α in response to hypoxia has also been reported [29]. in neonatal rat cms, hypoxia induced increases in tnf-α mrna within the cell and caused the release of the 26 kda transmembrane form of tnf-α in cd63+ evs. exposure of healthy cms to these evs induced apoptosis. the authors suggested that although tnf-α is not normally produced in the heart, cms can produce it under stress, package it into evs and thereby affect apoptosis in neighbouring cells [29]. transfer of tnf-α via evs may therefore provide a mechanism by which stressed cells participate in the propagation of an inflammatory response. 3kirsty m. danielson and saumya das: extracellular vesicles in heart disease: excitement for the future ? a subsequent study also showed the release of hsp60containing evs from cms in response to ethanol exposure [21]. the authors argued that this mechanism may be reactive oxygen species-mediated, as supported by the finding of increased cell-rox red staining in the ethanol treated cells compared to controls. the implications of the hsp60 in the evs derived from these cells remains unclear. hsp60 is found in both the mitochondria and cytosol of cms and has a complex role in apoptosis and cell signalling. increases in hsp60 have been reported to prevent apoptosis [30]; however, translocation of hsp60 to the plasma membrane has also been reported to increase apoptosis [31] and extracellular hsp60 (which has been detected in rodent and human plasma) appears to contribute to myocardial injury during ischemia [32]. in the current study, hsp60 was mono-ubiquitinated associated with lipid raft structures and tightly bound to the membrane surface with the evs. the authors suggested that ev-bound hsp60 functions as an intercellular signal without causing toxicity due to its encapsulation within the membrane, although these assumptions were not directly proven [21]. uptake of these exosomes by other cell types and the subsequent functional effects were not investigated and could be the subject of future studies. in contrast to the studies already described, where the fate of the secreted exosomes was not directly investigated, a recent study demonstrated the transfer of dna and rna from cms to fibroblasts [23]. evs released by hl-1 cells (an immortalized atrial myxoma cell line) were found to contain dna and rna, as labelled with acridine orange. when added to nih-3t3 cultures, the acridine orange-labelled evs were shown to transfer the acridine orange staining into nih-3t3 fibroblasts, often into their nuclei, thereby demonstrating ev-mediated transfer of genetic information from cms to fibroblasts. cargo present within the evs included ribosomal rna and mrna coding for proteins and transfer of this produced changes in gene expression within the fibroblasts [23]. however, the exact mrnas or mirnas that affected the changes in gene transcription in the recipient cells were not identified. furthermore, since nih-3t3 cells are not a cardiacspecific fibroblast cell line, the intriguing idea that there may be differences between uptake of cm evs in cardiac fibroblasts and in fibroblasts from other organs was not explored. 4.2 stem cell-derived evs in contrast to the limited data on ev secretion by mature cms, considerably more data exist for the release of evs by cardiac progenitor cells (cpcs) and other types of stem cells. spurred by the observation that beneficial effects of transplanted cpcs and other types of stem cells are often out of proportion to the number of surviving engrafted donor cells, investigators have proposed the ‘paracrine hypothesis’. this hypothesis postulates that in addition to possible direct differentiation of the engrafted donor stem cells to the appropriate cell lineages, paracrine factors released by the donor stem cells may contribute to improvements in heart function [33, 34]. this has been confirmed by numerous studies that have shown that conditioned media from stem cells can enhance cm survival after hypoxic injury [35], induce angiogenesis in infarcted myocardium [36] and reduce infarct size in both mouse [37, 38] and porcine [36, 39] models of myocardial ischemia reperfusion injury. in many cases, these paracrine factors are packaged within evs. conditioned media from mesenchymal stem cells (msc) has been shown to reduce infarct size and improve cardiac remodelling in a pig model of myocardial ischemia/reperfusion and some of this activity was contained within evs isolated by traditional ultracentrifugation methods [36]. similarly, msc-derived evs inhibited vascular remodelling and attenuated hypoxia-induced pulmonary hypertension, probably by inhibiting stat-3 signalling in endothelial cells [40]. at least some of the paracrine effects of stem cells in recruiting endothelial cells and mediating angiogenesis appear to be contained in flotillin-positive 100 nm extracellular vesicles (isolated by differential ultracentrifugation) [33]. while an extensive characterization of the contents of evs released from the different types of stem cell described in these studies has not been conducted, much of the recent attention has focused on the role of mirnas packaged in these entities [41]. following in the footsteps of cancer biology, future studies will no doubt pinpoint exact mirnas that are packaged and released in evs and may affect surrounding and distant cells. 4.3 endothelial cell-derived evs endothelial cells have been found to be a potent source of evs and several recent studies have shed light on the role of evs in mediating communication between vascular endothelial cells, the underlying smooth muscle cells and the circulating immune cells in the pathogenesis of atherosclerosis. at high levels, ox-ldl and homocysteine are risk factors for atherosclerosis and coronary artery disease [42, 43]. in contrast, high levels of circulating hsp70 have been suggested to be concurrent with a lower risk of vascular disease [44]. both ox-ldl and homocysteine induced the release of exosomes containing hsp70 from rat aortic endothelial cells [45]. interestingly, hsp70 activation of monocytes preceded their adhesion to endothelial cells. heat shock proteins are generally accepted to be protective within the cell; however, extracellular hsps 4 exosomes microvesicles, 2014, 2:1 | doi: 10.5772/58390 may be toxic or trigger inflammatory responses. whether monocyte activation by ev-derived hsp70 fulfils a protective or deleterious role remains unclear: it could act as an early immune response to vascular damage, or conversely play a role in early atherosclerotic lesion formation via monocyte infiltration of the endothelium. communication between monocytes and endothelial cells via evs occurs in both directions. evs released from the monocytic cell line thp-1 were capable of transferring mir-150 to human microvascular endothelial cells (hmec-1) [46]. similarly, mir-150 enriched evs isolated from human plasma were transferred to hmec-1 cells; however, it was not determined whether they originated specifically from monocytes, from another cell type, or from multiple cell types. treating thp-1 cells with lipopolysaccharide, hydrogen peroxide, advanced glycation end products and oleic acid/palmitic acid caused the release of evs with markedly different expression patterns and levels of mirnas, indicating specificity in the packaging and release of mirnas depending on the exact stimulus. the transfer of mir-150 to hmec-1 cells via evs induced migration of these cells, presumably through the knock-down of c-myb [46]. in addition to communication between circulating monocytes and endothelial cells, the transfer of information between endothelial cells and smooth muscle cells may play an important role in atherosclerotic plaque formation. disorganization and dysfunction of the smooth muscle cells underlying endothelium in the vasculature occurs during plaque formation and contributes to pathogenesis [47]. kruppel-like factor 2 (klf2) is a shear-responsive transcription factor believed to be atheroprotective by preventing the formation of atherosclerosis [48]. increased expression of klf2 in human umbilical vein endothelial cells (huvecs) via lentiviral transduction or exposure to shear stress causes the release of mir-143/145 in evs [49]. interestingly, mir-143/145 was increased approximately 30-fold in evs compared to only 10-fold in the cells, suggesting targeted transcription of the mirnas specifically for release. co-culture of huvecs with human aortic smooth muscle cells (hasmcs) resulted in the transfer of mir-143/145 to the smooth muscle cells and the reduction of multiple target mrnas. most importantly, transfer of mir-143/145 from endothelial cells to smooth muscle cells appears to be atheroprotective in vivo. injection of evs from klf2-transduced mouse endothelial cells into apoe-/mice produced a twofold reduction in aortic lesion area compared to controls [49]. figure 2. summary of reported ev release from cardiac cells in culture and their functional effects. ev release has been reported in numerous cell lines and primary cultures, both at baseline and in response to stimuli. transfer of evs between cell types has been demonstrated as well as delivery of cargo. huvec: human aortic endothelial cell; hmec-1: human microvascular endothelial cell; hasmc: human aortic smooth muscle cell; nrvm: neonatal rat ventricular myocyte. 5kirsty m. danielson and saumya das: extracellular vesicles in heart disease: excitement for the future ? finally, evidence of ev-mediated transfer of information between endothelial cells and cms was recently demonstrated [50]. while investigating the mechanistic basis of peripartum cardiomyopathy, it was found that a fragment of prolactin (16k prl) could induce the expression of mir-146a in endothelial cells, leading to inhibition of angiogenesis. furthermore, 16k prl could also induce the release of mir-146a-loaded evs from endothelial cells. these evs were found to be absorbed into cms where they decreased metabolic activity and down-regulated target molecules erbb4, notch1 and irak1. signal transduction by mir-146a packaged in evs appeared to be important in disease pathogenesis, as evidenced by amelioration of the disease phenotype by antagonism of mir-146a [50]. the majority of studies on the function of informational transfer via emvs have focused on evs; however, there is evidence that apoptotic bodies also play an important role in intercellular communication [51]. endothelial and smooth muscle cell apoptosis have been implicated in the pathology of atherosclerosis and apoptotic bodies are known to carry an array of dna fragments, rna and proteins. indeed, significant enrichment of mir-126 in apoptotic bodies released from serum-starved huvecs and hamscs has been demonstrated [14]. application of these apoptotic bodies to healthy huvecs induced increased expression of the chemokine cxcl12, which is known to recruit progenitor cells and counteract apoptosis in response to arterial injury. in addition, huvec-derived apoptotic bodies induced increased cxcl12 expression in hasmcs and mouse aortic endothelial cells. the increase in cxcl12 expression was attributed to suppression of rgs16 by mir-126. this indicates that the release of mir126 enriched apoptotic bodies may non-specifically signal to neighbouring vascular cells to promote cell survival. most importantly, mir-126-enriched apoptotic bodies derived from atherosclerotic plaques of human patients and administered to apoe-/mice reduced plaque area and macrophage content in the aortic root of mice [14]. in summary, the studies described above provide examples of the functional role of evs in mediating transfer of information and signal transduction between endothelial cell types and other important cell types in the cardiovascular system. while considerable literature exists documenting the important role played by evs in communication between tumour cells and endothelial cells, the last two years have seen some progress in describing this important mechanism in the cardiovascular system and may spur the development of novel biomarkers as well as therapeutic targets. 5. evs as biomarkers in the cardiovascular system as previously described, mirnas and signalling proteins specific to the parent cell may be selectively packaged into evs and released into biofluids including plasma, urine, saliva and cerebrospinal fluids. in the case of tumours, these evs may contain markers specific for these tumours. apart from their possible functional role in altering the tumour microenvironment, the sequestration of the rnas and proteins within the evs protect them from degradation by rnases and proteinases. these characteristics make evs attractive candidates for studying novel biomarkers. this has been extensively investigated in the fields of oncology and neurology [52, 53]. in cardiovascular biology, there has been considerable excitement in studying plasma mirnas as novel diagnostic and prognostic biomarkers. while several of the biomarkers described so far coincidentally reside within evs, the use and characterization of evs as biomarkers has not been systematically investigated. significantly increased serum levels of mir-1 and mir133a has been found in patients with unstable angina pectoris, takotsubo cardiomyopathy and acute myocardial infarction [54]. stimulation of a rat myoblast cell line (h9c2) with the calcium ionophore a23187 induced the release of evs containing mir-133a. interestingly, mir-133a levels were decreased in infarcted heart tissue in a mouse model of myocardial infarction and release of evs from h9c2 cells was associated with cell death. this led the authors to conclude that circulating mir-133a is released in response to cardiac injury and could be used as an indicator of cm death [54]. it is not known, however, whether these mirnas contribute to pathology and cell death, or whether they may play some compensatory role to prevent the death of surrounding cells. sera mir-192, mir-194 and mir-34a were identified as prognostic biomarkers in patients with acute myocardial infarction. they appeared to predict the development of clinical heart failure and ventricular remodelling within one year of acute myocardial infarction onset [55]. interestingly, all these mirnas were regulated by p53 and packaged in cd63+ evs. they appear to have a deleterious role as transfer of these secreted mirnas to cultured myoblasts from embryonic rat heart accelerated cell death, while cell viability was increased by knockdown of the three mirnas [55]. endothelial cells have been described as an important source of mirnas packaged into evs. mir-146a, as described above may play an important role in the pathogenesis of peripartum cardiomyopathy by mediating signalling between endothelial cells and cms [50]. as noted in the previous section, mir-146a is packaged in evs and released by endothelial cells and in human subjects was specifically diagnostic for peripartum cardiomyopathy, compared with idiopathic dilated cardiomyopathy [50]. 6 exosomes microvesicles, 2014, 2:1 | doi: 10.5772/58390 there have been numerous studies in recent years on putative mirna biomarkers in cardiovascular disease, as summarized in [56]. while they have not all been identified as membrane-enclosed entities, the demonstration of ev release from cardiac cells justifies additional investigation into this specific subclass of mirnas. further evidence supporting the origin of some of these biomarker plasma mirnas in the heart came from work that showed a concentration gradient of some of the mirnas recognized as biomarkers of myocardial injury, suggesting secretion from myocardial cells [57]. nonetheless, the exact cellular source of these mirnas, the mechanisms of packaging and secretion and their functional roles remain a matter of active investigation. thus, there is a rapidly expanding field in identifying and validating novel diagnostic and prognostic biomarkers in cardiovascular biology. a large majority of these appear to be packaged in evs and therefore protected from degradation by circulating rnases. however, the detailed characterization of evs released into the circulation by myocardial cells has lagged behind and now appears poised for further fruitful investigation. 6. evs as therapeutic modalities in cardiovascular diseases in addition to their use as biomarkers, evs could potentially be used in novel therapeutics for cardiovascular disease. as discussed in the previous section, several cellular pathways that are targeted by evs originating from cardiac cells have now been identified. possible therapeutic options in cardiovascular disease involve targeting the evs containing rna, dna or proteins as mediators of signalling pathways involved in disease pathogenesis. blocking the delivery of ev cargo to cells could be achieved in several ways, including the inhibition of vesicle release, uptake, or formation [58]. while no studies have yet described the use of such strategies in cardiovascular disease, they have been studied to some extent in cancer therapy. for example, amiloride-induced inhibition of ceramide, a lipid component of sphingomyelin, can reduce vesicle yields and it enhanced the efficacy of the chemotherapeutic drug cyclophosphamide in mice [59]. a problem with this strategy, however, is its potential off-target effects. it is now known that many different cell types produce evs and these contain a number of biologically active molecules. therefore a blanket disruption in vesicle biology could produce many unwanted effects. alternatively, evs could be used as therapeutic delivery tools. since evs are capable of carrying a wide range of cargo and transferring exogenous nucleic acids, they are an attractive option for gene therapy and drug treatments. in addition, some evs have innate therapeutic activity. as discussed previously, transfer of evs from stem cells has been shown to have beneficial effects in animal models of cardiovascular disease [36, 37, 39]. this suggests two possible strategies for using evs as a therapeutic: deliberate packaging of specific components into vesicles for targeted delivery and use of endogenouslyproduced stem cell evs without designed contents. proof of concept for the specific packaging and targeting of evs has been provided by recent studies including [60]. in this study, dendritic cells were engineered to express the exosomal membrane protein lamp2b fused to neural specific rabies virus glycoprotein peptide. evs derived from these cells were shown to deliver gapdh sirna specifically to the brain of mice following intravenous injection [60]. although a similar study is yet to be conducted on the cardiovascular system, this does provide encouraging evidence for the viability of evs as a therapeutic modality. furthermore, methods for the large scale production of clinical grade evs from dendritic cells [22] and mesenchymal stem cells [61,62] are currently being developed and offer considerable promise. with continuing research on the role and regulation of evs in cardiovascular disease, they could offer a novel therapeutic tool in the future. 7. conclusion in summary, evs released from cardiovascular cells have been shown to transfer to different cell types and cause changes in rna and protein expression that have significant downstream functional effects. functional changes reported to date appear be both protective and deleterious and may play an important role in normal physiology or disease pathogenesis. regardless of whether the observed effects of ev transfer produce positive or negative effects, evs provide an attractive target for potential therapies and use as novel biomarkers. there is considerable uncharted territory that is yet to be explored by investigators. evs have been shown to express surface ligands such as integrins that can interact with proteins of the extracellular matrix, where they may be stored as repositories of signalling. alterations in the extracellular matrix, as may occur with infarction or haemodynamic changes resulting from heart failure may result in the ‘uncovering’ of these evs and their signalling capabilities. this hypothesis has been explored in cancer biology, but remains to be examined in heart disease. detailed characterization of evs released by the components of the cardiovascular system both at baseline and with stress may provide additional insight into novel intercellular signalling mechanisms. as attempts to undertake these ventures gather pace, we may be on the threshold of a new era of mechanistic understanding of heart disease. 8. acknowledgements sd is funded by the klarman scholars foundation. 7kirsty m. danielson and saumya das: extracellular 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[62] yeo rwy, lai rc, zhang b, tan ss, yin yj, teh bj, lim sk: mesenchymal stem cell (2013) an efficient mass producer of exosomes for drug delivery. advanced drug delivery reviews 65: 336-341. 10 exosomes microvesicles, 2014, 2:1 | doi: 10.5772/58390 exosomal heat shock proteins as new players in tumour cell-to-cell communication review article claudia campanella1,2,*, celeste caruso bavisotto1,2, antonella marino gammazza1,2, dragana nikolic1, francesca rappa1,2, sabrina david1,2, francesco cappello1,2, fabio bucchieri1,2 and stefano fais3 1 department of experimental biomedicine and clinical neurosciences, section of human anatomy ‘‘emerico luna’’, university of palermo, palermo, italy 2 euro-mediterranean institute of science and technology, palermo, italy 3 department of therapeutic research and medicine evaluation, istituto superiore di sanità, rome, italy * corresponding author e-mail: claudia.campanella@unipa.it received 19 mar 2014; accepted 23 may 2014 doi: 10.5772/58721 © 2014 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract exosomes have recently been proposed as novel elements in the study of intercellular communication in normal and pathological conditions. the biomolecular composition of exosomes reflects the specialized functions of the original cells. heat shock proteins (hsps) are a group of chaperone proteins with diverse biological roles. in recent years, many studies have focused on the extracellular roles played by hsps that appear to be involved in cancer development and immune system stimulation. hsps localized on the surface of exosomes, secreted by normal and tumour cells, could be key players in intercellular cross-talk, particularly during the course of different diseases, such as cancer. exosomal hsps offer significant opportunities for clinical applications, including their use as potential novel biomarkers for the diagnoses or prognoses of different diseases, or for therapeutic applications and drug delivery. keywords extracellular vesicles, heat shock proteins, cell communication 1. introduction 1.1 cell-to-cell communication cellular communication is used by multicellular organisms to organize and coordinate the activities and the development of various organs and tissues [1]. in order to maintain cellular homeostasis or to respond to pathogens in the extracellular milieu, cells often exchange information through direct cell-to-cell contact or by secretion of soluble factors, either via ligand-receptor interactions or cellular ‘bridges’, such as nanotubes [2]. the cells interact with other cells through membrane surface molecules or by secreting several types of molecules such as soluble proteins, amino acids, fats, 1claudia campanella, celeste caruso bavisotto, antonella marino gammazza, dragana nikolic, francesca rappa, sabrina david, francesco cappello, fabio bucchieri and stefano fais: exosomal heat shock proteins as new players in tumour cell-to-cell communication article j circ biomark, 2014, 3:4 | doi: 10.5772/58721 journal of circulating biomarkers steroids and gas. these molecules can activate the target cells by interacting with the cell surface receptors [1]. recently, it has been shown that cells can also communicate through the direct exchange of nucleic acids. new evidence has shown that circulating mirnas may be important in intercellular communication; in particular, they may induce gene silencing in the target cells [3, 4]. in addition to soluble molecules, cells can also send information through cell junctions and adhesion contacts, which can act within the same cell in which they are produced, in neighbouring cells, or even over long distances in an endocrine manner [5]. in the past two decades, another mechanism for intercellular communication has emerged involving the intercellular transfer of extracellular vesicles (evs) [1]. abundant evidence has validated a newly identified mechanism of intercellular interaction through lipid vesicles, in which phospholipid-enclosed vesicles are released into the extracellular environment that can bind the specific receptor to the target cells, and vescicles are be internalized by recipient cells [6]. the release of evs is a well-conserved evolutionarily mechanism that cells use to exchange bioactive proteins, lipids and nucleic acids. evs released from cells are heterogeneous in origin and size, and include those derived from the endosomal membrane cell compartment, released by exocytosis after the fusion of multivesicular bodies with the plasma membrane, as well as those formed by direct budding of the plasma membrane [2, 7]. evs were first characterized in hematopoietic cells. in 1967, wolf observed these subcellular fractions (“microparticles”), which he described as “platelet dust” using electron microscopy [8]. initially, it was thought that evs were a mechanism of the depletion of the cytoplasm and a specific function of membrane reticulocytes. indeed, evs are rich in reticulocyte-specific proteins, transferrin receptor and devoid of some key plasma membrane proteins [9]. increasing evidence supports the notion that each cytotype produces evs (including t cells, b cells, dendritic cells, platelets, epithelial cells and cancer cells), which are essential players in intercellular communication and that they establish the ability of a cell to sense and adapt to environmental alterations [10, 13]. evs are composed of a lipid bi-layer and contain multiple functional molecules derived from the cytosol of the donor cell, such as proteins (both transmembrane and luminal), lipids, rnas, non-coding rnas, micrornas and retrotransposon elements [14]. evs constitute a heterogeneous population that differs in cellular origin, size, morphology, antigenic composition and functional properties. they are classified into various categories based on their size and composition, for example, exosomes (40–100 nm), apoptotic bodies (>800 nm) [15], microparticles (0.1–1 µm), prostasomes (50–500 nm) and tolerosomes (~40 nm), factors that create confusion in the nomenclature [16]. in addition, isolating them is extremely difficult. in fact, in recent years, researchers have tried to improve the various ev isolation protocols [1]. in many past studies, evs were isolated by differential ultracentrifugation, depending on their size and density, but isolation protocols have not been definitively standardized. following differential ultracentrifugation, a complementary characterization procedure using biochemical markers and electron microscopy imaging techniques is essential [13]. among the large group of evs, exosomes have been most studied to date, because of their involvement in both pathological and physiological events as mediators of cell-to cell communication [16]. 2. exosomes as described by pan in 1983, it was initially thought that exosomes could be a mechanism for shedding the cytoplasm in maturing sheep reticulocytes [17]. in fact, exosomes are cell-derived vesicles that are secreted by all cell types and are also present in many body fluids such as blood, urine, cerebrospinal fluid, breast milk, saliva, bronchoalveolar lavage fluid, ascitic fluid and amniotic fluid [18]. exosomes are released into the extracellular space after the merging of late endosomes with the cell membrane. early endosomes become part of multivesicular bodies (mvbs), which undergo a maturing process that provides a gradual change in protein composition of the vesicles (intraluminal vesicles (ilvs). during this maturation process, the vesicles accumulated in the mvbs, can have three potential outcomes: 1) they may merge with the lysosomes, causing protein content degradation (e.g., in the case of receptors); 2) they may constitute a temporary storage compartment; 3) they may blend with the plasma membrane, releasing exosomes. therefore, exosomes correspond to the intraluminal vesicles of mvbs. mvbs merge with the plasma membrane, resulting in exocytosis of the vesicles contained in mvbs; as such, vesicles maintain the same topological orientation as the plasma membrane [1, 19]. the endosomal sorting complexes required for transport machinery (escrt) are involved in exosome biogenesis and in their loading. different evidence sources support the idea that escrt could assist in the sorting of ubiquitinated cargo proteins at the endosome membranes. the escrt-associated protein alix (apoptosis-linked gene 2-interacting protein x) can regulate this function [20]. likewise, some evidence assumes that the sorting of proteolipid molecules to intraluminal vesicles functions independently of escrt. for example, in dendritic cells, during cognately antigenspecific cd4+ t cell interaction, the sorting of mhc ii to 2 j circ biomark, 2014, 3:4 | doi: 10.5772/58721 exosomes occurs independently from mhc ii ubiquitination and mhcii can be incorporated into detergent-resistant protein complexes of intraluminal vesicles, which are secreted as exosomes and transferred to the interacting t-cells [21]. another alternative cargo selection, independent from the escrt mechanism, occurs through lipid affinity, which requires sphingolipid ceramide and depends on raft-based microdomains [2224]. exosomes exhibit specific cell-type dependent content. it has been reported that their protein composition is similar to that of proteins found in plasma membranes, as well as in the endocytic or subcellular compartments of source cells and includes membrane proteins such as annexins [25]; cytoskeletal proteins (tubulin, actin) [26]; lysosomal markers (cd63, lamp-1/2); enzymes [27]; death receptors (fasl, trail) [28]; cytokines [29-31]; hla class i/ii [32] and some heat shock proteins (hsps) [33, 34]. it has been demonstrated that exosomes derived from various cell types contain a wide variety of rna, including mrna, mirna, rrna and trna [35-41]. the rna present in exosomes has been termed exosomal shuttle rna (esrna) [39] and can be transferred to recipient cells where they modulate cells' genetic expression [41]. exosomes might constitute an exquisite mechanism for local and systemic intercellular transfer, not only of proteins, but also of genetic information in the form of rna [39]. depending on the source cell, many different functions have been attributed to exosomes. they are involved in cell-to-cell information transfer [42], immune response [43], inflammation [44], coagulation [45], stem cell activation [46] and programmed cell death [47]. exosomes can participate in cellular responses against stress. clayton and colleagues [48] showed that exposing b-cell lines to heat stress results in a marked increase of hsps expression by exosomes and in an increase in the quantity of exosomes produced. given that exosomes can mediate the transfer of specific molecules, they may play a role in intercellular transmission in disease pathogenesis, including tumour development, viral infections and neurological diseases. for example, exosomes might carry viral proteins from the infected cells from which they are released, thereby playing a part in the intercellular dissemination of viral vectors, or in clearing viral proteins from infected cells [49, 50]. most studies have that many tumour cells release a large amount of evs and those tumour-derived vesicles can carry proteins, lipids and nucleic acid that contribute to cancer progression [51]. in the case of many tumours, such as ovarian carcinoma, prostate cancer and pancreatic cancer, high levels of exosomes have been reported and these data suggest that exosomes could be important diagnostic and therapeutic tools [52-55]. furthermore, neuronal exosomes can mediate the transfer of misfolded proteins, causing a transmission mechanism of systemic amyloidoses in neurodegenerative diseases [56, 57]. all these data show that exosomes are important players in various physiological and pathological processes, and could be useful for both diagnostic and therapeutic applications. some evidence obtained with exosomes released by human cancer cells support the existence of at least two entirely different mechanisms through which exosomes may interact with target cells. one is mediated by interaction of a ligand (often expressed on the exosome membrane) and its receptor (often expressed on the cell plasma membrane). this was clearly demonstrated for death-receptors/ligands interaction, which always led to the triggering of cell death [58-60]. however, exosomes may fuse with the plasma membrane of the target cells, in turn transferring their content to the cell cytoplasm and possibly fusing with internal vesicles, too [61]. the exosomes taken up by target cells may well have an effect, as it has been shown for nk cell-derived exosomes [60], suggesting that exosomes can be used as real effectors of the natural immune response against either tumours or foreign agents [62]. 3. hsps and exosomes molecular chaperones are a group of proteins conserved during evolution and are involved in the maintenance of other "client" proteins in folded and active conformations in all cellular organisms [63-65]. these chaperones protect the proteome from the dangers of misfolding and aggregation by facilitating protein folding, complex assembly and refolding of partially denatured proteins; additionally, they also drive protein translocation across membranes and in the case of protein damage, toward degradation [58, 59]. chaperonology is the science that studies molecular chaperones and pathological conditions in which chaperones become pathological factors, known as chaperonopathies. chaperone therapy involves the use of chaperones in the treatment of chaperonopathies [63, 64]. most hsps are molecular chaperones with crucial functions in the biosynthesis, folding/unfolding, transport and assembly of other proteins [63-66]. they are classified into families by their molecular weights: hsp100, 90, 70, 60, 40 and the ‘small hsps’, which includes hsp27 [65]. hsps were initially described as a group of proteins that are induced by heat shock, as well as by other stressors [67]. the expression of hsps is induced in response to a wide variety of stress conditions, such as hypoxia, ischemia, heavy metal or ethanol exposure and infections [68]. interest in these molecules has increased in recent years due to many studies having indicated that these proteins are involved in many physiological mechanisms in normal cells, such as dna replication and gene expression regulation [63]. several mechanisms are responsible for the cytoprotective 3claudia campanella, celeste caruso bavisotto, antonella marino gammazza, dragana nikolic, francesca rappa, sabrina david, francesco cappello, fabio bucchieri and stefano fais: exosomal heat shock proteins as new players in tumour cell-to-cell communication effect of hsps [69]. additionally, it has been demonstrated that hsps have other roles, such as participation in immune system regulation [70, 71], cell differentiation [72], apoptosis and carcinogenesis [73-75]. the levels of many hsps are elevated in various types of cancer and hsp overexpression suggests a poor prognosis in terms of survival and response to therapy in some types of cancer [65]. numerous studies have shown that hsps are involved in cell transformation, metastasis formation and multidrug-resistance development [65, 74, 75]. furthermore, several studies have reported that elevated levels of hsps can protect malignant cells against therapy-induced apoptosis [65,69]. hsps are traditionally considered intracellular molecules, but many studies have shown that they can also appear in extracellular locations or in the blood [7681. extracellular or membrane-bound hsps can mediate immunological functions and may act as a potent danger signal, activating the immune system response [82]. several years ago, some researchers hypothesized that exosomes may provide a secretory pathway, allowing cells to actively release specific hsps [48, 83]. exosomes are important in cell-to-cell communication; on the other hand, they are also considered to be key players in intercellular cross-talk [82]. recent studies have validated this hypothesis by demonstrating that specific members of the hsp family, such as hsp70, hsp90 and hsp60, can be secreted by cancerous cells via the exocytotic pathway (table 1) [13, 33, 84, 85]. hsp70 and hsp90 are classic cytosolic chaperonins and normally fulfil a cytoprotective role inside cells [89]. hsp70 is actively secreted by different types of cells through non-classical protein secretory routes, including exosome pathways [84]. extracellular hsp70 exert immunomodulatory effects and play a key role in the immune response to cancer cells [85]. for example, microvesicles containing hsp70 on their surface activate macrophages [90], or natural killer cells [86, 87] and play an important role in the regulation of vascular homeostasis [84]. hsp90α is released by invasive cancer cells via exosomes and its release enhances cancer cell migration [88]. in recent years, new data has revealed new extracellular roles for hsp60. hsp60 is considered a mitochondrial protein that is, together with its cochaperone hsp10, essential for mitochondrial protein folding [74]. there is increasing evidence localizing hsp60 outside of the cells, where it mediates interaction between immune cells and other body tissues [91]. in addition, much recent experimental evidence has demonstrated that hsp60 can be localized in extramitochondrial sites [65]. in particular, it has been detected in the cytosol [92], intracellular vesicles [88], on the surface of normal and tumour cells [33, 93] and in tumoural exosomal hsps tumoural cells origin functions ref. hsp70 human erythroleukemia cells breast carcinoma cells immunomodulatory functions [85] [85] human pancreas carcinoma cells human colon carcinoma cells human hepatocellular carcinoma cells stimulation of migration and cytolytic activity of natural killer cells [86] [86] [87] hsp90 human hepatocellular carcinoma cells glioblastoma cells fibrosarcoma cells breast cancer cells stimulation of migration and cytolytic activity of natural killer cells cancer cells motility [87] [88] [88] [88] hsp60 human erythroleukemia cells human lung adenocarcinoma human mucoepidermoid bronchial carcinoma human hepatocellular carcinoma cells immunomodulatory functions (?) stimulation of migration and cytolytic activity of natural killer cells [13] [13] [13, 33] [87] table 1. origin and hypothetic functions of tumoural exosomal hsps 4 j circ biomark, 2014, 3:4 | doi: 10.5772/58721 blood [76]. in the cytosol, hsps may play two distinct roles, given the numerous evidence implying that hsps have pro-survival effects, while under different conditions they have been shown to have pro-apoptotic effects [92]. it is known that circulating hsp can have immunosuppressing or immunostimulating effects, depending on the interaction between hsps and cells or immune system components. for example, hsp60 has been found in the blood of patients with hashimoto’s thyroiditis (ht) and its presence may be involved in ht pathogenesis via an antibody mediated immune mechanism [94]. moreover, extracellular hsp60 can interact with a variety of receptors present on the surface of plasma cells, such as tlr, cd14, cd40 and cd91 [59]. furthermore, hsp60 appears to be involved in the activation of macrophages and neutrophils in patients with chronic lung diseases [95]. levels of hsp60 are increased in many types of tumours and it has been hypothesized that hsp60 overexpression has an important role in cancer development and progression [65]. the use of hsp60 as a biomarker for disease has recently been proposed and some researchers are studying the use of potential hsp60 inhibitor agents in the treatment of certain diseases, including cancer [64, 96]. in heart failure, hsp60 is released by cardiomyocytes and its presence in the serum may be correlated with the severity of the disease and cardiovascular risk [77, 97]. moreover, gupta and knowlton [98] demonstrated that hsp60 is released by adult cardiomyocytes through an exosome-mediated process in both the basal state and following mild stress. another group of researchers demonstrated that fibrosarcoma cells release hsp60 through the conventional endoplasmic reticulum golgi protein transport pathway [99]. more recently, our research group showed that hsp60 is released by tumour cells and not by normal cells, and that the mechanism of release is mediated by an unconventional secretion mechanism, i.e., the lipid raft exosome pathway [13]. these findings suggest a new role for extracellular hsp60 in the cross-talk between tumour cells and the immune system [33, 99]. in fact, the expression of hsp60 on the surface of exosomes released by tumour cells may be considered as a danger signal for the immune system [33]. further studies are certainly needed to explain the unusual exosome membrane localization of hsps [33, 100]. experimental data show that exosomal hsps may have opposing roles, that is, immunosuppressing or immunostimulating effects. these different effects depend on the interaction between exosomal hsps and cells or immune system components. for example, it has been demonstrated that the histone deacetylase inhibitor, ms-275, can significantly alter the immune molecule content and categories in exosomes of hepatocarcinoma cells; in particular, treatment with ms-275 increased the expression of hsp70. exosome modification by ms-275 can significantly increase the cytotoxicity of nk cells and the proliferation of pbmc, determining a reduction in tumour growth [101-104]. on the other hand, designing inhibitors of hsp-associated exosomes may be useful to hindering the dissemination of metastases [88]. moreover, the presence of hsps associated with circulating exosomes can be evaluated and monitored quantitatively in the blood of patients with tumours associated with over-expression of one or more hsps. figure 1. pathways of secretion of heat shock proteins (hsps) by tumour cells. cytosolic hsps can be released in free, soluble forms by golgi transport vesicles or can be bound to exosomes. the latter is produced by multivesicular bodies (mvb) fusing with the plasma membrane of cells. lipid rafts participate in hsps release by exosomes, as they are internalized by endocytosis, which for various reasons that remain unknown reach the plasma membrane of tumour cells, and enter into mvb. secreted hsps may interact with other cells in the peritumoural environment or be released into the bloodstream. 4. conclusions exosomes are currently considered to be bioactive vesicles that are useful in the study of normal biological functions, but also for understanding pathological conditions. the molecular composition of exosomes reflects the specialized functions of the original cells. through exosomal ability to bind target cells and/or exchange molecules, they can modulate the activity of other cells. hsps were originally described as intracellular molecular chaperones with a cytoprotective role. however, more novel functions have now been attributed to the hsp proteins, depending on their localization. in particular, circulating hsps (free or associated to exosomes) have immunological functions and may be involved in tumour progression [see figure 1]. 5claudia campanella, celeste caruso bavisotto, antonella marino gammazza, dragana nikolic, francesca rappa, sabrina david, francesco cappello, fabio bucchieri and stefano fais: exosomal heat shock proteins as new players in tumour cell-to-cell communication hsps found on the surface of exosomes secreted by normal and tumour cells might be key players in intercellular cross-talk. there could be a novel and interesting link between hsps and the immune system. elsner et al. [101] demonstrated that hsp70-positive exosomes released by tumour cells increase the nk cell activity against cell targets, resulting in reduced tumour growth. therefore, exosomes can function as independent cell-to-cell carriers. effectively, the immune response can be facilitated and enhanced by exosomes and their immunomodulatory molecules (such as hsp70, 80, 90 and mhc class i molecules), released by the cell source into the blood [102]. exosomes secreted by tumour cells and engineered to express specific hsp molecules could improve antitumor immunity [105]. therefore, engineered exosomes could be used as potential tumour vaccines or immunotherapeutic vesicles. indeed, exosomes and their molecular cargo, including hsps, are essential players in cell-to-cell communication and immunoregulation. there is significant potential for future clinical applications, including the use of hsps as potential novel biomarkers for the diagnoses, prognoses and follow-up of different diseases, or for therapeutic applications and drug delivery. in particular, in light of new technical approaches, the levels of exosomes in human body fluids can be detected and quantified [106], and it is clear that exosomes have the potential to become important circulating biomarkers in the vast majority of human diseases [107]. moreover, their intriguing capacity to shuttle molecules of various origins may well become one of the most important available drug delivery systems; they may therefore be of paramount importance for the future of nanomedicine [108]. 5. compliance with ethical research standards conflict of interest the authors declare no conflicts of interest 6. references [1] raposo g, stoorvogel w (2013) extracellular vesicles: exosomes, microvesicles, and friends. j. cell biol. 18;200(4):373-383. 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[108] fais s, logozzi m, lugini l, federici c, azzarito t, zarovni n, chiesi a (2013) exosomes: the ideal nanovectors for biodelivery. biol. chem. 394(1):1-15. 10 j circ biomark, 2014, 3:4 | doi: 10.5772/58721 article journal of circulating biomarkers identification of immunoreactive tumour antigens using free and exosomeassociated humoral responses original research article carolyn d. roberson1, cicek gercel-taylor2, ying qi3, kevin l. schey3 and douglas d. taylor2* 1 department of microbiology and immunology, university of louisville school of medicine, louisville, ky , usa 2 exosome sciences, monmouth junction, nj, usa 3 department of biochemistry, mass spectrometry research center, vanderbilt university, nashville, tn, usa *corresponding author(s) e-mail: douglastaylor@exosomes.org received ; accepted 21 november 2013 © 2013 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract altered tumour antigens can initiate cellular and humoral immune responses; however, they often fail to eliminate tumours. in humans, the presence of cancer is generally associated with the suppression of t cell activation and effector responses, characterized as a th1 to th2 biased response. this th2 response leads to the production of tumour-reactive antibodies. further, neoplastic lesions and biological fluids of cancer patients contain an abundance of tumour-derived exosomes (tde) expressing tumour antigens. expression of tumour antigens on tde may represent an antibody target and serve to block antibody binding to the tumour, implicating a role for these nano‐ vesicles in tumour survival. in this study, ovarian tumour cell proteins were separated by two-dimensional electro‐ phoresis (2-de) and patient-derived antibodies were used to analyse immunoreactivity. common immunoreactive proteins among ovarian cancer patients were identified by mass spectrometry and six proteins were selected based on recognition and correlation with cancer pathogenesis. the identity of these proteins were confirmed by immunoreac‐ tivity of patient-derived antibodies with recombinant proteins and their presence on in vivo and in vitro-derived ovarian tumour exosomes was defined. analysis of the tde demonstrated bound tumour-reactive immunoglobu‐ lins, exhibiting immunoreactivity with specific antigens, suggesting that patient-derived antibodies recognize tumour antigens on circulating exosomes. keywords autoantibodies, ovarian , cancer, exosomes 1. introduction in the tumour microenvironment, antigens are functional‐ ly and/or structurally altered by overexpression, muta‐ tion, aberrant degradation or post-translational modification that makes them immunogenic [1]. in an effective immune response, these immunogenic proteins are detected and an antitumour response is promoted to eliminate the transformed precursors before they establish malignancy. effective elimination is characterized by the simultaneous collaboration of innate and adaptive cellmediated and humoral responses. in the adaptive antitu‐ mour response, t cells (with cognate tcr) recognize tumour-associated antigens processed/presented on the mhc of antigen-presenting cells (apc), along with subsequent costimulation and cytokine expression for facilitation and maintenance of the response [2]. elimina‐ 1 tion of the tumour is accomplished through the activation of cytotoxic t (ctl) cells to induce tumour cell apoptosis, activation of cd4+t cells to promote both cellular and humoral responses through stimulation of apc presenta‐ tion of antigens to ctl and activation of b cells to produce antigen-specific antibodies that enhance tumour cell uptake by apcs [3,4]. the primary antitumour response is facilitat‐ ed by the cellular arm of the adaptive immune system; however, humoral responses to tumour antigens are clearly demonstrated through the production of antitumour antibodies [5,6]. this production of antibodies is present‐ ed as elevated igg in the blood [7]. in ovarian cancer sera, levels of tumour reactive-igg are elevated [8], suggesting intact humoral immunity in ovarian cancer patients and an effective humoral antitumour response. however, in the midst of this igg-laden environment, ovarian tumours continue to thrive. a key factor in the progression of transformed cells to malignancy is the tumour microenvironment. the tumour microenvironment consists of a number of cellular partici‐ pants, including immune cells, which are critical for the suppression of tumour growth [9]. however, the function‐ al activities of these immune cells are often counterregulated by tumour cell expression and the release of a number of biologic components, which act to promote the growth and metastatic progression of the tumour [10]. one essential biologic component in growth and progression is the tumour-derived exosome (tde). studies indicate that increased release of exosomes facilitate communication between the tumour's microenvironment and the tumour cell [11]. tde express tumour-derived antigens; however, they are not molecular duplicates of the plasma mem‐ brane of their parental tumour cells; rather, they represent a ‘micromap’ that displays increased expression of anti‐ gens associated with the tumour [12,13]. tde are abundant‐ ly found in plasma and malignant effusions derived from cancer patients [14] and their presence and expression of tumour-related antigens has been documented to contrib‐ ute to tumour progression. progressive effects mediated by tde have been found to range from regulation of tumour growth to invasion, angiogenesis and metastasis [14,15] through expression of molecules such as matrix metallopro‐ teinases (mmp-2, mmp-9) and horizontal transfer of growth factor receptors (egfrviii)[16,17]. additionally, tde have been shown to directly and indirectly modulate the evasion of anti-tumour responses provided by effector t cells for assisting in progression. melanoma-derived exosomes have been shown to promote monocyte production of myeloidderived suppressor cells [14], which can act to suppress tcell responses. ovarian tde demonstrate induced apoptosis of t cells by enhanced expression of fas l on the exosomes and cd3zeta suppression on the t cell [18], while nasopharyngeal tde have been shown to express in‐ creased galectin-9 to induce t cell apoptosis via tim-3 [19]. since tde can mediate the progression of the tumour and its evasion of cellular antitumour responses, they may also intervene in the humoral antitumour response, thereby permitting further development of the tumour. utilizing these circulating antitumour antibodies to identify immu‐ noreactive antigens may define proteins that are essential to carcinogenesis [20]. likewise, analyses of antigens associat‐ ed with tde may unlock the mysteries of the essential, but inadequate, humoral response needed for efficient eradica‐ tion of the tumour and the role of the tde in tumour progression and the humoral response. we hypothesize that the circulating free and tde-associated humoral immune response can be used to identify specific tumour-associat‐ ed antigens, which in the context of tde may be a mecha‐ nism for diverting the immune response from the tumour. using human ovarian cancer as our model system, we investigated immunoreactivity of tumour-associated antigen of ovarian patient cancer cells, patient in vivo and in vitro-derived tumour exosome antigens and patientderived free and exosome-associated antibodies. we established that ovarian tumour-derived exosomes expressed tumour-associated antigens, which are recog‐ nized by tumour-reactive antibodies, thereby indicating a role for tde in the ovarian tumour humoral response. 2. materials and methods 2.1 patient sera, ascites and cell lines biofluids (sera or ascites) used in this study were derived from patients diagnosed with stage iii (t3c) serous adenocarcinoma (designated ul-124, ul-167, ul-190, ul-207, ul-351, ul-398), mucinous adenocarcinoma (ul-324), carcinosarcoma (designated ul-224), teratoma (designated ul-184) and serous/endometrioid mixed carcinoma (designated ul-472). samples were obtained from the gynecologic oncology repository at the univer‐ sity of louisville, under an approved irb protocol. addi‐ tional information about ascites sample ul-309 was unavailable. control sera were derived from age-matched women with no evidence of ovarian disease. primary ovarian tumour cell cultures were established from ul-124 ascites. primary cultures were grown in 75 cm2 tissue culture flasks, initially in hyclone rpmi 1640 medium (thermoscientific, pittsburgh, pa) supplemented with 2mm l-glutamine, 10% foetal bovine serum (fbs, biowest), 1mm sodium pyruvate (cellgro, mediatech, manassas, va), 0.1 mm nonessential amino acids (cellgro, medi‐ atech) and 100 units/ml penicillin-streptomycin (gibco, life technologies, carlsbad, ca) in a humidified incubator at 37ºc with 5% co2. cells were transferred into ultracul‐ ture general purpose serum-free media without lglutamine, but supplemented with the same amount of sodium pyruvate, non-essential amino acids and penicillinstreptomycin. cell viability was evaluated by trypan blue exclusion and all cultures utilized were >95% viable. 2.2 preparation of tumour cell lysates prior to the harvesting of cells, culture media was removed and saved for subsequent culture exosome isolation. cells were rinsed twice with cold hyclone dpbs/modified 2 (thermoscientific) and were solubilized with ripa buffer (1x, pierce chemical co., rockford il) supplemented with protease inhibitor cocktail iii (100x, rpi) and phosphatase inhibitor ii (100x, sigma chemical co., st. louis, mo). ripa-solubilized cells were kept on ice for 5-10 minutes and then cells were collected using a cell scraper. solubi‐ lized cell lysate was collected into 1.5 ml microcentrifuge tubes (eppendorf) and centrifuged at 14,000 x g for 15 minutes at 4ºc to pellet the cell debris. the resulting supernatant was collected and assayed for total protein by the bradford assay (bio-rad laboratories, herceles, ca) using bovine serum albumin (bsa) as a standard. 2.3 isolation of cellular protein by rotofor fractionation cellular lysate was desalted by column chromatography using p6 desalting gel (biorad laboratories) and the resulting protein was assayed by the bradford method. desalted protein concentration was used to determine the amount of ampholyte to combine with the protein sample. desalted protein was combined with 0.5% bio-lyte 3/10 ampholyte (bio-rad laboratories) and ddh2o and loaded onto the rotofor cell (biorad). protein was focused for 4 h using the powerpachv under recirculated refrigeration. focused proteins were collected into 20 fractions. the ph and protein concentrations were determined for each fraction. each fraction (50µg) was loaded onto a criterion tris hcl 4-15% gel, electrophoretically separated and silver stained or analysed by western immunoblotting. mem‐ branes were probed overnight at 4ºc with patient sera (1:250) followed by peroxidase-conjugated anti-human iggam (1:4000, sigma chemical co). bound immune complexes were visualized by immunostar hrp chemilu‐ minescence (biorad) and the resulting film was analysed for similarity in reactivity between different sera. thirteen bands corresponding to shared immunoreactivity were cut from of the rotofor fractionated-silver stained gel and analysed by mass spectrometry (vanderbilt university, nashville, tn). 2.4 protein identification by mass spectrometry silver-stained protein gel bands were thoroughly rinsed with water to remove residual acetic acid. fresh reducing reagents (30 mm of potassium ferricyanide, 100 mm of sodium thiosulfate) in a 1:1 ratio were mixed and immedi‐ ately added in sufficient volume to cover the gel piece. the gel slice was washed with hplc-grade h2o and 100 mm of nh4hco3, alternatively, until the gel piece was clear. each gel slice was diced into small pieces (1 mm2) and placed into a clean tube, then washed twice with 100 µl (or enough to cover the gel pieces) of 25 mm nh4hco3/50% acn with vortexing for 10 minutes. the gel pieces were completely dried by speedvac. dried gel pieces were partially rehydrat‐ ed with 5 µl of 100 mm nh4hco3 and the proteins re‐ duced in 25 µl of 10 mm dtt in 25 mm nh4hco3 for 1 hr at 56ºc. supernatant was removed and discarded and proteins were alkylated with 25 µl of 55 mm iodoaceta‐ mide in 25 mm nh4hco3 in the dark for 45 minutes at room temperature. gel pieces were washed twice in 100 µl of 100 mm nh4hco3 (with vortexing for 10 minutes), dehydrat‐ ed with 100 µl of 25mm nh4hco3 in 50% acn (with vortexing for 5 minutes, repeated once) and were complete‐ ly dried by speedvac for 20 minutes. dried gel pieces were rehydrated with 5 µl of trypsin solution (0.1 µg/µl trypsin in 100 mm nh4hco3) and 50-70 µl of 100 mm nh4hco3 was added until the gel pieces returned to their original size. digestion was carried out overnight at 37ºc and then stopped by the addition of 1.5 µl of trifluoroacetic acid. the resulting peptides were recovered by two-25 minute extractions using 30 µl of 50% acn/5% formic acid at room temperature with 20 minute vortex/5 minute sonication cycles. the extracts were combined and dried in a speed‐ vac. the tryptic peptides were reconstituted in 25 µl of hplc water with 0.1% formic acid. digested peptides were separated and mass analysed using an eksigent nano-lc system connected to a ltq velos (thermo fisher scientific) ion trap mass spectrometer. briefly, tryptic peptides were loaded onto a 150 µm id microcapillary fused silica pre-column, which was inhouse packed with 4 cm ξ 5 µm c18 resin (jupiter c18, 5µm particle size, 300å pore size). the c18 trap column was coupled to a nanoflow analytical column packed with 10 cm of 3 µm c18 reverse-phase resin (jupiter c18, 3µm particle size, 300 å pore size) constructed with an integrat‐ ed electrospray emitter tip. peptides were eluted with a 90 minute gradient (from 2%acn with 0.1% fa to 35% acn with 0.1% fa) over 35 minutes, followed by 35% acn to 90% acn over 15 minutes at a flow rate of 0.5 µl per minute. the ltq velos mass spectrometer was operated in datadependent mode in which an initial ms scan recorded the mass-to-charge range as 300-2000u and the five most abundant ions were selected for subsequent collisioninduced dissociation. dynamic exclusion (repeat count 1, exclusion list size 150 and exclusion duration 60s) was enabled to allow for the detection of less abundant ions. lc-ms/ms raw files were converted into.dta files by a custom scansifter algorithm. spectra that contained fewer than 25 peaks or that had less than 2e1 measured total ion current were removed. dta files for singly charged precursor ions were created if 90% of the total ion current occurred below the precursor ion m/z ratio and all other spectra were processed for doubly and triply charged ions. dta files were searched against the human protein database uniprot-human 155_200907_rev with 20,914 total protein entries using the sequest algorithm. the search parameters used allowed for the following differential modifications:+57 on cysteine and+16 on methionine. sequest-searched files (pepxml) were imported into id picker software for protein assembly. results were filtered using the following criteria: a minimum peptide length of five amino acids, a minimum of one unique peptide per protein (modifications to cysteines or methionines were not considered distinct from the unmodified peptides), overall maximum false positive rate (fdr) of 5%, a minimum of 3carolyn d. roberson, cicek gercel-taylor, ying qi, kevin l. schey and douglas d. taylor: identification of immunoreactive tumour antigens using free and exosome-associated humoral responses two additional peptides to establish a unique protein group, one protein reported per protein group and single protein groups indicated by proteins that shared the same set of peptides (indiscernible from each other based on available data). 2.5 verification of mass spectrometry-identified ovarian cancer antigen by immunoprecipitation and immunoblot to verify the presence of the ovarian cancer antigen identified by mass spectrometry, immunoprecipitation was performed using protein g hp spintrap columns (ge healthcare, piscataway, nj) and true blot anti-rabbit or anti-mouse ig ip beads (ebioscience, san diego, ca). ul-124 cell lysates were preclarified by combining 500µg lysate proteins with anti-rabbit/anti-mouse ig ip beads. lysate and beads were incubated at 4ºc for 1 hour, centri‐ fuged to obtain the precleared lysate and protein concen‐ trations of clarified lysate determined by the bradford method (bio-rad laboratories). for spintrap columnbased immunoprecipitation, commercial antibodies corresponding to the ms-identified antigens were conju‐ gated to the protein g spintrap matrix (20µg protein/ column) for 3-4 hours at room temperature and the manufacturer’s cross-link protocol was followed. the antibodies used included grp-78 (rabbit polyclonal), annexin 2 (mouse monoclonal), cathepsin d (mouse monoclonal), alpha-enolase (mouse monoclonal), hsc 70 (mouse monoclonal) and pdi (mouse monoclonal). all precipitating antibodies were obtained from santa cruz biotechnology (scbt, santa cruz, ca). the unbound antibody was removed and precleared lysate in trisbuffered saline (tbs) was added to the immunoaffinity beads and incubated overnight at 4ºc. bound antigens were eluted using 0.1m glycine/urea as an elution buffer and the fractions were neutralized by addition of 1m tris base. to determine the quantity of eluted protein, the bradford protein assay was performed. for dot blot analysis, 5µl (0.5µg) of each antigen sample (in duplicate) were spotted onto individual nitrocellulose membranes (bio-rad laboratories) with bsa (1 mg/ml) and hela or a431 (epidermoid carcinoma cell line) lysates added as negative and positive controls, respectively. membranes were blocked in 5% nonfat milk/tbst, washed with 1xtbst (tbs +0.1% tween-20) and incubated at 4ºc overnight with corresponding commercial antibodies (diluted 1:1000) and control or ovarian patient sera (diluted 1:250). secondary hrp goat anti-mouse/anti-rabbit (1:5000, bio-rad labora‐ tories) or peroxidase-conjugated anti-human iggam (1:4000, sigma chemical co) was added and immunoreac‐ tivity analysed by immune star hrp chemiluminescence (bio-rad laboratories). for immunoprecipitation with the trueblot immunopreci‐ pitation system (pierce chemical co.), 5 µg of each of the antibodies listed above were added to the precleared lysate and incubated for three hours at 4ºc. the incubated grp-78/lysate sample was added to the true blot antirabbit beads, with the remaining ab/lysate samples added to the true blot anti-mouse beads for overnight incubation at 4ºc. following this incubation, supernatants were collected by centrifugation at 10,000xg; beads were washed 3x in a cold np-40 lysis buffer (50 mm tris hcl, ph 8, 150 mm nacl, 1% np-40) and supernatant aspirated, and beads were combined with 50 µl of 1x laemmli. the bead complexes were boiled and centrifuged to collect superna‐ tant containing the immunoprecipitated antigen. the immunoprecipitated antigen samples were loaded onto mini-protean tgx precast gels (4-15%, bio-rad laboratories), electrophoretically separated and analysed by western immunoblotting. membranes were blocked and probed overnight at 4ºc with corresponding commercial antibodies, as previously mentioned. secondary rabbit and mouse trueblot anti-rabbit/anti-mouse igg hrp (ebio‐ science) were added, followed by analysis of immunoreac‐ tivity using immune star hrp chemiluminescence (biorad laboratories). 2.6 recognition of recombinant proteins by ovarian cancer sera antibodies to verify recognition of the mass spectrometry-identified proteins by ovarian cancer sera, 0.2 µg human recombinant protein for grp-78 (prospec bio, east brunswick, nj), annexin 2 (prospec bio), cathepsin d (prospec bio), pdi (prospec bio), alpha-enolase (american research prod‐ ucts, waltham, ma) and hsc70 (enzo life sciences, farmingdale, ny) were combined with 4x lds nonreduc‐ ing buffer (thermoscientific), loaded onto a 10% sdspage gel and analysed by western immunoblotting using serum from a non-cancer control (ctrl, 1:250) and ovarian cancer patients (samples ul167, ul190, ul207, ul324, ul351, ul398). all ovarian sera were used at 1:250, with the exception of the mucinous sample (1:100), based on a lack of detection at the greater dilution. in addition, as a positive control, the recombinant proteins were probed with a mixture of commercial antibodies against the identified proteins (1:1000) to further ensure detection of the recombinant antigen. 2.7 isolation of ovarian tumour exosomes and analysis of tumourexosome protein immunoreactivity exosomes were isolated from ovarian tumour-patient (ul-124) ascites and conditioned ovarian tumour culture media by a two-step chromatographic procedure devel‐ oped in our laboratory [18]. ascites and media, either serum-containing or serum-free, (100 ml), were concen‐ trated by ultrafiltration and applied to a 2% agarose (agarose bead technologies, tampa, fl) chromatography column (1.5 x 20 cm) and equilibrated with ddh2o. fractions (1 ml) were collected and the elution was monitored by absorbance at 280 nm. fractions containing material greater than 50 million daltons were obtained and ultrafiltrated to a concentrate, and the quantity of protein 4 was assayed using the bradford method. the biofluidderived vesicular proteins (15µg) were mixed with a 1x laemmli sample buffer, loaded on a 10% sds-page gel and analysed using western immunoblotting. immunor‐ eactivity of tumour-exosome-derived protein was assayed by commercial antibodies corresponding to mass spec‐ trometry-identified proteins. 2.8 isolation of igg from tde and analysisof immunoreactivity 1 ml of the ul-124 ascites bound exosome fraction was combined with 50µl increments of the igg elution buffer until a ph of ~3.0 was obtained. the sample was allowed to incubate with end-over-end rotation for 30 minutes at room temperature. post incubation, the sample was added to a pre-rinsed (1x pbs) vivaspin 2 centrifugal concentrator with a membrane mwco of 1 x 106 (sartorius stedim biotech, bohemia, ny). igg was eluted from the exosomes by centrifugation (4000 x g, 10-20 min.) and collected from the filtrate tube. igg was neutralized using 1m tris-hcl, ph 9.0 and protein quantitated using the bradford method. 0.5µg human recombinant protein corresponding to the antigens of interest was loaded onto a mini protean tgx 4-15% gel and western immunoblot performed using the isolated igg at 1:50 to detect immunoreactivity. 3. results 3.1 recognition of ovarian tumour antigens by patient serum antibodies previously, traditional isoelectric focusing of ovarian tumour antigenic proteins evaluated by silver staining of ipg sds-page gels containing focused protein followed by immunoblotting with serum-derived antibodies from ovarian carcinosarcoma patient (ul-224) and an ovarian teratoma patient (ul-184) revealed distinct patterns of immunoreactivity associated with cancer patients. subse‐ quently, rotofor fractionation has been used to fractionate cellular proteins based on isoelectric focusing. ovarian tumour antigenic proteins were initially rotofor fractio‐ nated, this material was then separated by sds-page gels and subsequently immunoblotted with sera from patient ul-224 (figure 1a) and ul-184 (figure 1b) as primary antibodies. bands corresponding to shared recognition (n=13) between sera were subjected to mass spectrometry. similar reactivity between patient-derived antibodies to antigenic proteins was noted with some variation in intensity (for example, stronger recognition with ul-224: fraction 12; stronger recognition with ul-184: fraction 9, lower band). figure 1. shared immunoreactivity of rotofor-fractionated ul-124 serumfree lysate with serum of ovarian tumour-patient a) ul-224 (1:250) and b) ul-184 (1:250). boxes indicate randomly chosen bands' shared reactivity. molecular marker (mm) and rotofor fraction numbers (1-20) are indicated above each image. increasing isoelectric point (pi) is indicated by the arrow beneath images. 3.2 identification of ovarian tumour antigens by patient antibodies from the 13 selected bands, mass spectrometry analysis identified six proteins with high confidence: grp 78 (glucose-related protein 78), annexin 2, cathepsin d, alphaenolase, hsc70 and pdi (protein disulfide isomerase). these were selected for focus due to their protein scores, their association with tumour survival and their status as a potential biomarker. in order to verify the presence of these antigenic proteins in the ovarian cancer lysate sample, these proteins were isolated from tumour cell lysates by immunoprecipitation. the proteins of interest were subjected to western and dot immunoblotting and probing with commercial antibodies corresponding to the antigens of interest. for the dot blot assays, captured antigens were applied to the membrane in duplicate. immunoreactivity of precipitated antigen for all antigens of interest was detected by dot blot (figure 2a). in some cases, the presence of specific proteins was verified by western immunoblotting (figure 2b); however, some proteins failed to demonstrate reactivity in western immunoblot‐ ting. this was potentially due to the loss of specific epitopes through protein reduction and heating. 5carolyn d. roberson, cicek gercel-taylor, ying qi, kevin l. schey and douglas d. taylor: identification of immunoreactive tumour antigens using free and exosome-associated humoral responses figure 2. representative immunoblots validate the presence of mass spectrometry antigens of interest using commercial antibodies. ul-124 cellular antigen was immunoprecipitated by a) protein g hp spintrap columns, applied in duplicate and subjected to dot blot; and b) true blot ip beads subjected to western blot (representative immunoreactivity shown). 3.3 validation of serum antibody detection of mass spectrometry antigens of interest using corresponding recombinant proteins humoral responses detecting grp 78, annexin 2, cathepsin d, alpha-enolase, hsc70 and pdi were evaluated by western immunoblot using recombinant proteins and sera from patients with serous adenocarcinoma/carcinoma and mucinous adenocarcinoma. using patient-derived anti‐ bodies (figure 3a-e), western immunoblotting identified the presence of annexin 2, alpha-enolase and hsc70 with variations in intensities and isoforms between samples. preliminary assays for antigen detection by serum revealed minimal detection of cathepsin d by the serous samples tested, with the exception of patient ul-351. out of the serous carcinoma samples assayed, patient 351 displayed lone recognition of the intermediate cathepsin d isoform (~46 kda). additionally, reactivity to isoforms of hsc70 (~30 kda) and annexin 2 (~75-90 kda) were detected to varying degrees by the serous samples. mucinous adeno‐ carcinoma patient antibodies (figure 3f) detected grp78 and alpha-enolase, and faintly detected the intermediate form of cathepsin d (~46 kda). additionally, the higher molecular weight isoform was detected with annexin 2, as seen in some of the serous samples. a mixture of commer‐ cial antibodies (figure 3g) corresponding to recombinant proteins of interest was utilized to detect viability of the antibodies and their ability to recognize the antigens at their standard molecular weights. of the six recombinant proteins assayed, the commercial antibodies recognized grp78, annexin 2 (both isoforms), alpha-enolase and pdi. identification of recombinant antigens using serum antibodies from normal, non-cancer presenting controls (figure 3h) similarly recognized alpha-enolase and hsc70 at its standard molecular weight and the lower molecular weight form. figure 3. representative immunoblot validating ovarian cancer patient antibody recognition of antigens of interest. immunoreactivity of human recombinant protein with human ovarian serum from patients with: a-b) serous adenocarcinoma; c-d) serous carcinoma; d) mucinous adenocarcino‐ ma; f) commercial ab mix; g) non-cancer control (ctrl). recombinant proteins indicated as: g78 (grp78), a2 (annexin 2), a-eno (alpha-enolase), h70 (hsc70), pdi (protein disulfide isomerase) and cd (cathepsin d), were shown with serous sample 351, mucinous sample and control. ovals indicate recognition of cd. boxes indicate recognition of other antigens. molecular marker indicated by mm. initial assays showed no reactivity of cd with other sera samples. 3.4 enrichment of ovarian tumour mass spectrometry antigens of interest in in vivo and in vitro-derived ovarian tumour exosomes previous work conducted by our group demonstrated the presence of bound immunoglobulins on circulating exosomes. these exosome-bound antibodies appear to recognize specific proteins. to determine whether ovarian tumour-derived exosomes are enriched with the same antigens identified by mass spectrometry of ovarian tumour cells, exosomes from ul-124 ascites or cell culture were probed in western blots with commercial antibodies against the antigens of interest from the mass spectrometry analysis. western immunoblot analysis of ascites-derived and culture-derived exosomes demonstrated the presence of grp 78, alpha-enolase, pdi and varying isoforms of cathepsin d (figure 4). 6 figure 4. analysis of the association of mass spectrometry antigens of interest with in vivo and in vitro-derived exosomes. western immunoblots of ul-124 ascites-derived (asc), serum-containing (sc) and serum-free (sf) culture-derived exosomes demonstrating immunoreactivity to commercial antibodies corresponding to the antigens of interest. cathepsin d isoforms: immature (imm), intermediate (intm), mature (mat). the presence of the cathepsin d active intermediate enzyme (~46 kda) was detected in all samples, while the immature proenzyme (52-60 kda) was detected in ascites and culture media. the active mature form (~33 kda) was detected in ascites only. variation in recognition was seen between the culture-derived samples, particularly in the identification of annexin 2 and hsc70 in fbs-containing vesicles and whose expression was shared in the ascites sample (figure 4). since cells grown in serum-free media are more sensitive to factors such as ph, temperature, mechanical stresses, etc., the molecular expression of specific proteins may have been altered. 3.5 exosome-bound immunoglobulin reactivity to mass spectrometry antigens of interest to examine the ability of exosome-associated igg to recognize the antigens of interest, bound-immunoglobu‐ lins were eluted from exosomes isolated from the ascites fluid of patient ul-124. recombinant proteins correspond‐ ing to the antigens of interest were subjected to electropho‐ resis and their immunoreactivity was examined with the isolated exosome-associated antibodies (figure 5). exo‐ some-associated immunoglobulins exhibited recognition of both isoforms of annexin 2, alpha-enolase and hsc70, with minor recognition of grp 78. figure 5. recognition of antigens of interest by patient ascites exosomeassociated igg. western immunoblot of recombinant proteins’ immunor‐ eactivity to ul-124s ascites exosome-associated igg. molecular marker indicated by mm. recombinant proteins indicated as in previous figures. 4. discussion cellular proteins present in the tumour microenvironment can be functionally and/or structurally altered, resulting in them becoming immunogenic and provoking autoanti‐ body responses. these autoantibodies reflect responses to tumour-associated (differentially expressed on normal and cancer cells) antigens and can be detected in the circulation regardless of the magnitude of tumour-associated antigen expression or tumour size [21]. as a result of the long-lived humoral response, the antigen presence is “amplified,” which facilitates ease of detection. since autoantibodies act as sentinels of aberrant cellular activity and their presence reflects the execution of a humoral immune response to the tumour, detailed analysis of their immunoreactivity can provide a clearer understanding of the tumour-associated antigens that they recognize [22]. progress in the characterization of human cancers has been attained through proteomic analyses of cancer-associated serum proteins. proteomic analysis involves extensive characterization of proteins, including identification of any modifications and interactions, and structural determina‐ tion of isoforms and related functional components. traditional approaches to quantitative analysis of pro‐ teomes involve high resolution separation of the proteins (by size and charge) using 2de, accompanied by identifi‐ 7carolyn d. roberson, cicek gercel-taylor, ying qi, kevin l. schey and douglas d. taylor: identification of immunoreactive tumour antigens using free and exosome-associated humoral responses cation of the resolved proteins using mass spectrometry (ms) [23]. protein profiles from such analyses can reveal differential protein expression between samples, which may denote key molecules that are critical to protein function. in this investigation of ovarian cancer patients' humoral antitumour responses, we used patient serum autoanti‐ bodies for the detection of ovarian-cancer-specific-antigen‐ ic proteins. proteomic analysis allowed for the identification of numerous proteins with shared recogni‐ tion: six of particular interest (grp78, annexin 2, cathepsin d, alpha-enolase, hsc70 and pdi), due to their roles in tumour survival and as potential biomarkers for different cancers. in this instance, the identities of these antigenic proteins were confirmed by western immunoblotting and dot immunoblotting of immunoprecipitated ovarian tumour cell-derived proteins, using commercial antibodies against these proteins. one concern in this study was the effects of denaturation on the ability of the antigenic protein to be recognized by our commercial antibodies. hence, we utilized both western and dot immunoblot analysis to reduce the likelihood of “overlooking” the detection of the antigens. as expected, there was variability in detection of annexin 2, hsc70, and pdi between western and dot blot analysis. dot blot analysis revealed detection of all anti‐ gens, while western blot resulted in definitive detection of grp 78, cathepsin d and alpha-enolase. grp78, a member of the heat shock protein 70 family, is a chaperone protein commonly housed in the lumen of the endoplasmic reticulum, but is also expressed on the cell surface in various cancers [24]. cell-surface expression of grp78 in cancer cells has been shown to activate pathways that induce cellular survival and proliferation [24], and to correlate with the expression of circulating autoantibodies in prostate [25] and ovarian [26] cancers. previous studies of circulating ovarian cancer sera autoantibodies by this lab demonstrated recognition of grp78 and cathepsin d (immature proenzyme form and mature form) in ovarian cancer patients. in this study, immunoprecipitated antigen probed with commercial antibodies revealed bands ~75 kda (corresponding to grp78), ~48 kda (corresponding to the intermediate form of cathepsin d) and ~47 kda (corresponding to alpha-enolase). cathepsin d is an aspartic lysosomal peptidase present in normal cells, but its overexpression has been reported in a number of cancers [27]. functionally, it is believed to act on tumour growth and metastasis by assisting with stromal remodelling. processing of the single polypeptide immature (pro) form (52 kda) of cathepsin d involves removal of the propeptide to form the active intermediate isoform (48 kda) and eventual formation of the double-chained mature isoform (34 kda heavy chain, 14 kda light chain). alphaenolase is a glycolytic enzyme important in the catalysis of 2-phospho-d-glycerate (pga) to phosphoenolpyruvate [28], and is upregulated under stressful conditions, like hypoxia, in order to mediate enhanced anaerobic metabo‐ lism. increased autoantibodies against alpha-enolase have been recorded in some cases of organ-specific autoimmun‐ ity [29] and in a number of metastatic cell lines. this study indicates strong recognition of immunoprecipitated ovarian cancer antigens by commercial anti-cathepsin d and alpha-enolase antibodies. consistent recognition of human recombinant alpha-enolase by serous adenocarci‐ noma/carcinoma and mucinous adenocarcinoma serum antibodies was seen, with variation in cathepsin d and grp78 recognition between sera types. additionally, recombinant alpha-enolase also appeared to be weakly recognized by non-cancer controls. grp78 and alphaenolase were also detected in all 3 exosome samples, with detection of all 3 isotypes of cathepsin d among the exosome samples. exosome-associated antibodies also recognized alpha-enolase with faint detection of grp78. our data suggests that grp78, cathepsin d and alphaenolase are tumour-associated antigens that are transport‐ ed to the released exosome and elicit a humoral response, indicated by the presence of antibodies to these molecules. annexins are calcium-binding proteins with functions ranging from trafficking of vesicles, to apoptosis and regulation of cellular growth [29]. annexin 2, a key mediator in the plasminogen activator system, has been shown to be important in clot dissolution and wound healing, and integral to cancer progression. located on the surface of endothelial cells and various tumour cells, annexin 2 can exist as a monomer (~36 kda) or a heterote‐ tramer (~90 kda), which acts a binding protein for proca‐ thepsin b on the surface of tumour cells to facilitate invasion and metastasis. expression of annexin 2 is varied in different types of cancers with increased expression in pancreatic [30] and breast [31] cancers, but variable expression in prostate cancer [32]. in our studies, the presence of annexin 2 was validated by dot blot and ovarian cancer patient serum antibodies primarily recognized the heterotetramer form of the antigen, while both isoforms were detected in the ascites tde. furthermore, igg isolated from tde detected both isoforms. chaperone proteins hsc70 and pdi were detected by dot blot assay and were also found to be associated with tde. hsc70 is a constitutively expressed cytoplasmic protein that binds to new peptides exiting the ribosomes to protect the hydrophobic residues from inefficient interactions. additionally, it can catalyse the disassembly of clathrin cages and has been implicated in the regulation of tumouri‐ genesis and apoptosis. with the assistance of co-chaper‐ ones, it can be recruited to the intracellular membrane, but can be released from cells as a result of active secretion [33]. studies of a cell line transfected with cathepsin d showed that overexpression of cathepsin d resulted in an increase in the malignant phenotype of the transformed cells [34]. subsequent studies of these cells have revealed that this overexpression prevented the release of hsc70, which led to the malignant phenotype. in our study, the expression of cathepsin d was seen in the ovarian cancer cells and all 8 isoforms on the exosomes. however, the ovarian patient serum antibodies failed to detect cathepsin d in all cases. the antigen expression of cathepsin d, the lack of anti‐ bodies to cathepsin d, and the reduced hsc70 expression on the cells may indicate a “protective” mechanism used by the tumour in order to maintain high cathepsin d expression, so that hsc70 release can be minimized and the tumour growth can be maintained. furthermore, the exosome expression of all three isoforms may act to “block” recognition of antigens on the tumour cell, thereby protect‐ ing the tumour from immunosurveillance. pdi is a key endoplasmic reticulum chaperone that catalyses the breakage of disulfide bonds within a protein and rearrang‐ es them to form the native protein. because accumulation of misfolded proteins in the cell can lead to enhanced cellular stress and death [35], upregulation of pdi can act to reduce stress-associated apoptosis. human pdi-family member erp5 has been shown to promote shedding of mica from epithelial tumours [36]. mica is the ligand for the nkg2d (nk group member 2d) receptor on nk, cd8+t, nkt and t cells, and upon ligation, activates cytolysis of the target cell. tumour-derived exosomes have been shown to express mica/b, which downregulates nkg2d expression to reduce nkg2d-mediated killing [37]. in our studies, pdi was strongly expressed in tde. expression of pdi in tde may participate in the facilitation of mica expression, resulting in interference of innate and adaptive immune effector cell function against the tumour. 5. conclusions studies of tde have demonstrated their critical role in cancer pathogenesis; however, much remains to be discov‐ ered about their roles in the humoral antitumour response. the vast amount of autoantibodies present in the circula‐ tion clearly indicates that a humoral response is mounted against the tumour, but that its efficacy in mediating tumour elimination is compromised [38]. tde cannot only express tumour antigens, but are also able to elicit a humoral response against the antigen, as indicated in this study. we propose that the tumour cell expresses certain antigens in their exosomes in order to divert the humoral immune response away from the tumour, thereby prevent‐ ing its effective detection and 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[38] taylor dd, homesley hd, doellgast gj (1984) identification of antigenic components recognized by "membrane bound" antibodies from ovarian cancer patients. am. j. reprod. immunol. 6: 179-184. 11carolyn d. roberson, cicek gercel-taylor, ying qi, kevin l. schey and douglas d. taylor: identification of immunoreactive tumour antigens using free and exosome-associated humoral responses cbx779310 1..10 research article associations between smoking and lipid/lipoprotein concentrations among us adults aged �20 years ram b jain 1 and alan ducatman 2 abstract cross-sectional data from national health and nutrition examination survey for the years 1999–2012 for those aged �20 years, fasting for at least 8 h, and classified as smokers and nonsmokers on the basis of observed serum cotinine levels were used to evaluate the impact of smoking on the adjusted and unadjusted concentrations of low-density lipoprotein (ldl) cholesterol, high-density lipoprotein (hdl) cholesterol, total cholesterol (tc), and triglycerides (tg). adjustments were made for the effects of gender; race/ethnicity; survey year; dietary intake of alcohol; caffeine; cholesterol; saturated, unsaturated, and total fatty acids; fasting time; body mass index; and poverty income ratio. adjusted levels of ldl and tc did not vary among smokers and nonsmokers. smokers had lower adjusted levels of hdl than nonsmokers (48.8 vs. 51.4 mg/dl, p < 0.01) and higher adjusted levels of tg (124.4 vs. 111.9 mg/dl, p < 0.01) than nonsmokers. adjusted odds of smokers having abnormal levels were 1.6 (95% confidence interval (ci) 1.4–1.8) for hdl, 1.2 (95% ci 1.1–1.4) for tc, and 1.3 (95% ci 1.2–1.5) for tg. males had lower adjusted levels than females for hdl (45.2 vs. 55.4 mg/dl, p < 0.01) and tc (191.3 vs. 196.6 mg/dl, p < 0.01) but higher adjusted levels than females for tg (126.3 vs. 110.1 mg/dl, p < 0.01) and ldl (114.4 vs. 112.6 mg/dl, p ¼ 0.02). a unit increase in body mass index was associated with 1.4% decrease in the adjusted levels of hdl, 0.18% increase in the adjusted levels of ldl, and a 2.3% increase in the adjusted levels of tg. keywords smoking, triglyceride, cholesterol, lipids, lipoproteins, cotinine date received: 15 january 2018; accepted: 27 april 2018 introduction smoking has been shown to alter lipid/lipoprotein levels. komiya et al. 1 reported smokers with brinkman index � 554 (defined as the number of cigarettes smoked per day multiplied by duration of smoking in years) to have 1.657 times the odds of having abnormal triglyceride (tg) levels among japanese males aged 24–68 years. kuzuya et al. 2 also reported smokers to have lower levels of high-density lipoprotein (hdl), lower levels of low-density lipoprotein (ldl), lower levels of total cholesterol (tc), and higher levels of tg than nonsmokers. based on a review of 54 published studies, when compared with nonsmokers, smokers were found to have higher levels of tc by 3%, tg levels by 9.1%, vldl levels by 10%, ldl levels by 1.7%, and lower levels of hdl by 5.7%.3 furthermore, clear dose response relationships have been reported for tc, tg, and ldl findings. 4–6 the presence of obesity, diabetes, and metabolic syndrome is of obvious importance to the studies of lipids. among japanese males aged 42–81 years, those who had visceral area �100 cm2, also had higher proportion of abnormal tg (�150 mg/dl), and this risk factor was 1 independent researcher, dacula, ga, usa 2 department of occupational and environmental health, west virginia university school of public health, morgantown, wv, usa corresponding author: ram b jain, independent researcher, 2959 estate view ct, dacula, ga 30019, usa. email: jain.ram.b@gmail.com journal of circulating biomarkers volume 7: 1–10 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418779310 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). http://orcid.org/0000-0002-3278-7106 http://orcid.org/0000-0002-3278-7106 mailto:jain.ram.b@gmail.com https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418779310 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage further modified in relationship to their smoking status, with 18.8%, 17.3%, and 36.4% among nonsmokers, former smokers, and current smokers, respectively, but tg levels were not found to differ among these groups when visceral area was <100 cm 2 . 7 thus, an interaction between smoking and body fat distribution was found to affect tg levels. håglin et al. 8 reported female smokers who never had type 2 diabetes and male smokers who currently had type 2 diabetes to have higher levels of tg than nonsmokers. the presence of the number of metabolic syndrome components was not reported to be affected by smoking. 9 age may also be important, as de souza et al. 10 reported an association between increased tg levels and smoking among elderly people aged �60 years. a possible mechanism of how cigarette smoking may alter lipid levels in serum has been suggested. 6 absorption of nicotine leads to secretion of catecholamines, cortisol, and growth hormones, activating adenyl cyclase in adipose tissue. this results in lipolysis of stored tg and release of free fatty acids. this, in turn, results in increased hepatic synthesis of tg and vldl. recently, jain 11 reported data from national health and nutrition examination survey (nhanes, https://www. cdc.gov/nchs/nhanes/index.htm) for the period 1999–2012 to investigate the effect of smoking on the levels of hdl, ldl, tc, and tg for us population aged �20 years, based upon self-reported use of tobacco products during the last 5 days to determine the smoking status of participants. however, jain 12 reported on the possibility of bias in smoking classification based on self-reports. using a cutoff of 10 ng/ ml for serum cotinine to distinguish smokers from nonsmokers, 7.5% self-reported nonsmokers were found to be smokers and 2% self-reported smokers were found to be nonsmokers. in addition, for participants with missing selfreported smoking data, 20.8% were found to be smokers. thus, there is a possibility that measured associations between smoking and lipid/lipoprotein levels may differ depending upon how smoking is classified. the current study was undertaken to evaluate associations between smoking and lipid/lipoprotein levels when smoking classification is based on the observed levels of serum cotinine, instead of self-reports. the hypothesis is that self-report introduces bias, and that cotinine measures will improve the evaluation. smoking status will be determined by observed serum cotinine levels. in addition to evaluation of lipid profile differentials among smokers and nonsmokers, based on serum cotinine levels, an extended objective of this study compares odds of having abnormal values (as defined in the next section) of hdl, ldl, tc, and tg among smokers when compared with nonsmokers. this has potential implications for surveillance and treatment of lipid abnormalities in smokers. data from nhanes 1999–2012 were selected because nhanes provides data for a nationally representative sample of the non-institutionalized us population. methods data source and description data were obtained from nhanes (http://www.cdc.gov/ nchs/nhanes/index.htm) for the years 1999–2012 for those �20 years old who have fasted for at least 8 h prior to blood draw. data on demographics, body measures, physical activity, serum cotinine, total nutrient intake, hdl, ldl, tc, and tg levels were downloaded and match merged. in nhanes, sampling weights are created for each sampling domain. each sampling domain represents a specific combination of race/ethnicity, gender, age, and income. for nhanes survey 2011–2014, there were 87 sampling domains. 13 examples of age groups for nhanes sampling domains included 1–2 years, 3–5 years, 6–11 years, . . . , 50–59 years, and �60 years. each person within a sampling domain is assigned the domain weight whose value is based on sampling rate, response rate, and estimated us population for that sampling domain. for the purpose of analysis, sampling weight assigned to each person is taken into consideration. in addition, all analyses completed for this study used age as a continuous variable. sample sizes unweighted sample size for those aged �20 years and who have fasted for at least 8 h prior to blood draw was 15,267. of the 15,267 participants for whom data were available, self-reported smoking status was unknown for 1031 participants while smoking status based on serum cotinine (<10 ng/ml classified as nonsmokers, �10 ng/ml classified as smokers) levels were unknown for 196 participants. of the 3497 self-reported smokers, 281 or 8% were classified to be nonsmokers by the serum cotinine based status. of 10,739 self-reported nonsmokers, 215 or 2% were classified as smokers based upon serum cotinine status. it was determined that there was enough discrepancy between selfreported and cotinine-based smoking status to possibly lead to discrepancy between the estimated unadjusted geometric means (ugm) and adjusted geometric means (agm). in addition, a measured versus reported result decreases the percent of those with unknown smoking, thus providing a larger sample size. weighted and unweighted sample sizes for non-missing values of hdl, ldl, tc, and tg by gender, race/ethnicity, and smoking status are given in table 1. for some of the analyses conducted, the sample sizes were somewhat smaller because of missing values for physical activity levels and other variables. detailed age groupings among nonsmokers were as follows: nage: 20–29 years ¼ 1891, nage: 30–39 years ¼ 1868, nage: 40–49 years ¼ 1805, nage: 50–59 years ¼ 1595, nage: 60–69 years ¼ 1907, nage: 70–79 years ¼ 1425, and nage: �80 years ¼ 938. detailed age groupings among smokers were as follows: nage: 20–29 years ¼ 760, nage: 30–39 years ¼ 710, nage: 40–49 years ¼ 792, nage: 50–59 years ¼ 579, nage: 60–69 years ¼ 481, nage: 70–79 years ¼ 238, and nage: �80 years ¼ 82. 2 journal of circulating biomarkers https://www.cdc.gov/nchs/nhanes/index.htm https://www.cdc.gov/nchs/nhanes/index.htm http://www.cdc.gov/nchs/nhanes/index.htm http://www.cdc.gov/nchs/nhanes/index.htm derived variables self-reported levels of recreational physical activity were categorized as vigorous, moderate, none, or minimal. for the nhanes years 2007–2012, activity status was enquired during a typical week and during the last 30 days for nhanes 1999–2006. those who self-reported being engaged in vigorous activity with or without being engaged in moderate activity were classified as being engaged in vigorous activity. those who self-reported being engaged in moderate activity without being engaged in vigorous activity were classified as being engaged in moderate activity. those who did not answer the question about their recreational physical activity were considered to be engaged in minimal or no physical activity. abnormal values of hdl and tg were defined consistent with wildman et al. 14 : abnormal fasting triglyceride levels were �150 mg/dl; abnormal hdl levels were <40 mg/dl for males or <50 mg/dl for females. while variable risk-based abnormal values for ldl and tc have been suggested (https://www.nhlbi.nih.gov/files/docs/guide lines/atp3xsum.pdf), for the purpose of this communication, ldl values were considered to be abnormal ldl � 130 mg/dl. tc values were considered to be abnormal for tc � 240 mg/dl. software sas university edition software (www.sas.com) was used to analyze data for this study. statistical analyses ugms with 95% confidence intervals for hdl, ldl, tc, and tg levels by gender, race/ethnicity, and smoking status were computed by sas proc surveyreg. ugms by gender, race/ethnicity, and smoking status are given in table 2. for the adjusted analysis, log 10-transformed values of hdl, ldl, tc, and tg were used as dependent variables in regression analyses done by sas proc surveyreg. categorical independent variables used in regression models were gender (males and females), race/ethnicity (non-hispanic white (nhw), non-hispanic black (nhb), mexican americans (ma), and other unclassified race/ ethnicities (oth)), smoking status (nonsmoker and smoker), and physical activity level (vigorous, moderate, none, or minimal). continuous independent variables used in regression models were age, age 2 , body mass index, fasting time in hours, poverty income ratio (pir), total daily dietary intake of total cholesterol, alcohol, caffeine, carbohydrate, fiber, monounsaturated fatty acids, polyunsaturated fatty acids, saturated fatty acids, total fat, and survey year. however, because of very high correlations between monounsaturated fatty acids, polyunsaturated fatty acids, saturated fatty acids, and total fat may have led to multicollinearity, one variable at a time was entered in the model, and as such, there were four fitted models for each dependent variable. agms with 95% confidence intervals are given in table 3. table 4 provides data on associations (slopes) of continuous variables such as pir with hdl, ldl, tc, and tg. in order to compute adjusted odds of having abnormal levels of hdl, ldl, tc, and tg, sas proc surveylogistic was used with abnormal values (yes, no) of lipids/lipoproteins as dependent variables and independent variables, similar to the linear regression mentioned above except that dietary variables were not used. while the objectives of this study intended to evaluate the impact of smoking on the adjusted and unadjusted levels of ldl, hdl, tc, and tg as well as the adjusted odds of having abnormal levels of ldl, hdl, tc, and tg among smokers, the adjusted analyses did make table 1. unweighted and weighted sample sizes by gender, race/ ethnicity, and smoking status for non-missing values of hdl, ldl, tc, and tg for nhanes 1999–2012. n weighted n % hdl total 15,092 1,441,274,994 100.0 male 7261 693,906,283 48.1 female 7831 747,368,711 51.9 non-hispanic white 7245 1,009,503,646 70.0 non-hispanic black 2859 158,639,515 11.0 mexican american 2953 113,347,185 7.9 other race/ethnicities 2035 159,784,649 11.1 nonsmoker 11,401 1,057,412,610 73.7 smoker 3626 377,377,356 26.3 ldl total 14,717 1,406,279,496 100.0 male 7003 668,584,457 47.5 female 7714 737,695,039 52.5 non-hispanic white 7053 983,805,516 70.0 non-hispanic black 2824 156,746,995 11.1 mexican american 2846 109,670,666 7.8 other race/ethnicities 1994 156,056,319 11.1 nonsmoker 11,140 1,034,353,625 73.9 smoker 3514 365,674,864 26.1 tc total 15,091 1,441,201,566 100.0 male 7261 693,906,283 48.1 female 7830 747,295,283 51.9 non-hispanic white 7245 1,009,503,646 70.0 non-hispanic black 2858 158,566,086 11.0 mexican american 2953 113,347,185 7.9 other race/ethnicities 2035 159,784,649 11.1 nonsmoker 11,400 1,057,339,182 73.7 smoker 3626 377,377,356 26.3 tg total 15,073 1,439,979,380 100.0 male 7250 693,096,765 48.1 female 7823 746,882,615 51.9 non-hispanic white 7241 1,008,921,734 70.1 non-hispanic black 2850 158,238,706 11.0 mexican american 2948 113,067,635 7.9 other race/ethnicities 2034 159,751,305 11.1 nonsmoker 11,388 1,056,627,236 73.7 smoker 3620 376,867,116 26.3 hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; nhanes: national health and nutrition examination survey. jain and ducatman 3 https://www.nhlbi.nih.gov/files/docs/guidelines/atp3xsum.pdf https://www.nhlbi.nih.gov/files/docs/guidelines/atp3xsum.pdf www.sas.com adjustments for the simultaneous and interacting effects of age, gender, race/ethnicity, and other variables. this statistical adjustment mechanism resulted in impact of age, gender, race/ethnicity, and other variables also being evaluated on the adjusted levels of ldl, hdl, tc, and tg as well as the odds of having abnormal levels of ldl, hdl, tc, and tg. results univariate analysis unadjusted geometric means. males had lower ugms than females for hdl (46.1 vs. 56.2 mg/dl, p < 0.01, table 2) and tc (191.2 vs. 196.3 mg/dl, p < 0.01, table 2) but higher ugms than females for tg (124.2 vs. 108.0 mg/ table 2. unadjusted geometric means with 95% confidence intervals for hdl, ldl, tc, and tg by gender, race/ethnicity, and smoking status for those aged �20 years. hdl ldl tc tg total 51.1 (50.7–51.5) 112.4 (111.6–113.2) 193.8 (192.8–194.8) 115.5 (113.8–117.2) m 46.1 (45.7–46.5) 113.2 (112.1–114.2) 191.2 (189.9–192.5) 124.2 (121.6–126.8) f 56.2 (55.6–56.7) 111.8 (110.7–112.8) 196.3 (195–197.5) 108 (106–109.9) nhw 51.1 (50.6–51.6) 113.1 (112.1–114.1) 195.2 (193.9–196.5) 118.6 (116.6–120.7) nhb 53.8 (53.2–54.5) 109.3 (108–110.7) 187.8 (186.4–189.2) 90 (87.6–92.6) ma 48.3 (47.7–48.9) 112 (110.3–113.7) 192 (190.1–193.9) 125.2 (121.2–129.2) oth 50 (49–51) 111.7 (109.5–113.9) 192.4 (190.1–194.7) 117.8 (113.8–121.9) nsm 52.1 (51.7–52.5) 112.4 (111.5–113.3) 194.1 (193–195.2) 113.2 (111.3–115.2) sm 48.3 (47.7–49) 112.2 (110.8–113.7) 192.7 (191–194.4) 121.8 (119.1–124.6) statistically significant differences m < f (p < 0.01), nhw < nhb (p < 0.01), nhw > ma (p < 0.01), nhb > ma (p < 0.01), nhb > oth (p < 0.01), nsm > sm (p < 0.01) nhw > nhb (p < 0.01), nhb < ma (p ¼ 0.02) m < f (p < 0.01), nhw > nhb (p < 0.01), nhw > oth (p ¼ 0.01), nhw < oth (p ¼ 0.04), nhb < ma (p < 0.01), nhb < oth (p < 0.01) m > f (p < 0.01), nhw > nhb (p < 0.01), nhw < ma (p < 0.01), nhb < ma (p < 0.01), nhb < oth (p < 0.01), ma > oth (p ¼ 0.01), nsm < sm (p < 0.01) source: data from national health and nutrition examination survey 1999–2012. hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; m: male; f: female; nhw: non-hispanic white; nhb: non-hispanic black; ma: mexican american; oth: other race/ethnicities; nsm: nonsmoker; sm: smoker. table 3. adjusted geometric means with 95% confidence interval for fasting levels of hdl, ldl, tc, and tg in mg/dl for participants aged �20 years by gender, race/ethnicity, smoking, and physical activity status. hdl ldl tc tg gender m 45.2 (42.3–48.3) 114.4 (107.7–121.5) 191.3 (183–200) 126.3 (111.4–143.3) f 55.4 (51.9–59.2) 112.6 (105.9–119.7) 196.6 (188.2–205.3) 110.1 (97.5–124.5) race/ethnicity nhw 49.2 (45.9–52.6) 114.4 (107.5–121.7) 195.5 (186.9–204.5) 125.5 (111–141.8) nhb 54.2 (50.7–57.9) 111 (104.5–118) 189.3 (181.4–197.7) 89.8 (79.3–101.7) mexican american (ma) 48.5 (45.4–51.8) 115.1 (108.1–122.5) 196.7 (188.1–205.6) 134.2 (118.3–152.2) oth 48.6 (45.4–52.1) 113.5 (106.6–120.7) 194.3 (185.7–203.2) 128.1 (112.4–146) smoking nsm 51.4 (48.1–54.9) 113.6 (106.9–120.7) 193.9 (185.5–202.7) 111.9 (98.9–126.6) sm 48.8 (45.6–52.1) 113.3 (106.6–120.4) 193.9 (185.6–202.6) 124.4 (109.7–141) physical activity vig 50.4 (47.1–53.9) 115.5 (108.4–123) 195.2 (186.6–204.1) 114.6 (101.5–129.5) mod 49.2 (45.9–52.6) 114.5 (107.5–121.9) 194.4 (185.7–203.5) 120.4 (105.6–137.3) min 50.7 (47.4–54.1) 110.5 (104.2–117.2) 192.2 (184.2–200.6) 119 (105.1–134.6) statistically significant differences m < f (p < 0.01), nhw < nhb (p < 0.01), nhb > ma (p < 0.01), nhb > oth (p < 0.01), nsm > sm (p < 0.01), vig < mod (p < 0.01), mod < min (p ¼ 0.01) m > f (p ¼ 0.02), nhw > nhb (p < 0.01), nhb < ma (p < 0.01), vig > min (p ¼ 0.02), mod > min (p < 0.01) m < f (p < 0.01), nhw > nhb (p < 0.01), nhb < ma (p < 0.01), nhb < oth (p ¼ 0.01) m > f (p < 0.01), nhw > nhb (p < 0.01), nhw < ma (p < 0.01), nhb < ma (p < 0.01), nhb < oth (p ¼ 0.01), nsm < sm (p < 0.01), vig < mod (p < 0.01) source: data from national health and nutrition examination survey 1999–2012. hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; m: male; f: female; nhw: non-hispanic white; nhb: non-hispanic black; ma: mexican american; oth: other race/ethnicities; nsm: nonsmoker; sm: smoker; vig: vigorous; mod: moderate; min: none or minimal. 4 journal of circulating biomarkers dl, p < 0.01, table 2). among the three major race/ethnic categories, ugms for hdl were nhb (53.8 mg/dl) > nhw (51.1 mg/dl) > ma (48.3 mg/dl), and all three pairwise differences were statistically significant (p < 0.01, table 2). ugms for ldl were nhw > nhb (113.1 vs. 109.3 mg/dl, p < 0.01) and nhb < ma (109.3 vs. 112.0 mg/dl, p ¼ 0.02, table 2). ugms for tc were nhw (195.2 mg/dl) > ma (192.0 mg/dl) > nhb (187.8 mg/dl), and all three pairwise differences were statistically significant (p < 0.01, table 2). ugms for tg were ma (125.2 mg/dl) > nhw (118.6 mg/dl) > nhb (90.0 mg/ dl), and all three pairwise differences were statistically significant (p < 0.01, table 2). smokers had lower ugms than nonsmokers for hdl (48.3 vs. 52.1 mg/l, p < 0.01) but higher ugms than nonsmokers for tg (121.8 vs. 113.2 mg/l, p < 0.01). jain 11 also found smokers to have lower ugms for hdl than nonsmokers (48.2 vs. 52.1 mg/dl, p < 0.01) but higher tg than nonsmokers (123.2 vs. 113.5 mg/ dl, p < 0.01). ugms among smokers and nonsmokers were not found to differ for ldl and tc for the study conducted by jain. 11 multivariate analysis adjusted geometric means. males had lower agms than females for hdl (45.2 vs. 55.4 mg/dl, p < 0.01, table 3) and tc (191.3 vs. 196.6 mg/dl, p < 0.01, table 3) but higher agms than females for tg (126.3 vs. 110.1 mg/dl, p < 0.01, table 3) and ldl (114.4 vs. 112.6 mg/dl, p ¼ 0.02, table 3). among three major race/ethnic categories for hdl, agms were in the following order: nhb (54.2 mg/dl) > nhw (49.2 mg/dl) > ma (48.5 mg/dl) and nhw > nhb (p < 0.01) and nhb > ma (p < 0.01). agms for ldl were nhw > nhb (114.4 vs. 111.0 mg/dl, p < 0.01) and nhb < ma (111.0 vs. 115.1 mg/dl, p < 0.01). agms for tc were nhw > nhb (195.5 vs. 189.3 mg/dl, p < 0.01) and nhb < ma (189.3 vs. 196.7 mg/dl, p < 0.01). among three major race/ethnic categories for tg, agms were in the following order: ma (134.2 mg/dl) > nhw (125.5 mg/dl) > nhb (89.8 mg/dl), and all three pairwise differences were statistically significant (p < 0.01). as expected, nonsmokers had higher hdl agms than smokers (51.4 vs. 48.8 mg/l, p < 0.01) and lower agms for tg (111.9 vs. 124.4 mg/l, p < 0.01) than nonsmokers. contrary to this, jain 11 found smokers to have higher hdl (52.2 vs. 50.1 mg/dl, p ¼ 0.02) than nonsmokers using self-reported data. vigorous physical activity was associated with higher hdl than moderate physical activity (50.4 vs. 49.2 mg/dl, p < 0.01, table 3), but other than this, there were no clear dose-response relationships for exercise and outcomes with the exception of a paradoxical relationship for ldl for which vigorous and moderate physical activity were associated with higher ldl (115.5 and 114.5 vs. 110.5 mg/dl, p � 0.02, table 3) than none or minimal physical activity. associations between dependent and continuous independent variables. as expected, positive association (p < 0.01, table 4) was found between age and ldl (b ¼ 0.00937), tc (b ¼ 0.0072), and tg (b ¼ 0.00913) but age2 had a negative association (p < 0.01, table 4) with the adjusted levels of ldl (b¼�0.00009), tc (b¼�0.00006), and tg (b¼ �0.00007). the increase in adjusted levels of ldl, tc, and tg with age is therefore shown to attenuate as people age. depending upon the relative slopes associated with age and age 2 , the direction of change in ldl, tc, and table 4. regression slopes with significance probabilities for independent variables when log10-transformed values of hdl, ldl, tc, and tg in mg/dl were fitted as dependent variables.a dependent variables as log 10 of independent variables hdl ldl tc tg age 0.00051 (0.19) 0.00937 (<0.01) 0.0072 (<0.01) 0.00913 (<0.01) age2 0 (0.81) �0.00009 (<0.01) �0.00006 (<0.01) �0.00007 (<0.01) body mass index �0.0059 (<0.01) 0.00078 (<0.01) 0.0001 (0.49) 0.01006 (<0.01) pir 0.00591 (<0.01) �0.00287 (0.01) �0.00147 (0.03) �0.00878 (<0.01) survey year 0.00611 (<0.01) �0.008 (<0.01) �0.00436 (<0.01) �0.01032 (<0.01) fasting time (h) 0.00075 (0.19) 0.00167 (0.03) 0.00117 (0.01) 0.00018 (0.89) alcohol intake (g) 0.00059 (<0.01) �0.00009 (0.11) 0.00015 (<0.01) 0.00008 (0.44) caffeine intake (mg) 0 (0.6) 0.00002 (<0.01) 0 (0.42) �0.00005 (<0.01) cholesterol intake (mg) 0 (0.53) 0.00001 (0.38) 0 (0.37) 0.00001 (0.63) dietary fiber (g) 0.00091 (<0.01) �0.00033 (0.07) �0.00009 (0.4) �0.00142 (<0.01) total fat (g) 0.00022 (<0.01) 0.00001 (0.81) 0.00002 (0.55) �0.00044 (<0.01) monounsaturated fatty acids (g) 0.00043 (<0.01) 0 (0.97) 0.00003 (0.72) �0.0009 (<0.01) polyunsaturated fatty acids (g) 0.00063 (<0.01) �0.00025 (0.08) �0.00022 (0.03) �0.00175 (<0.01) saturated fatty acids (g) 0.0005 (0.01) 0.00024 (0.06) 0.00022 (0.01) �0.00059 (0.01) r 2 in % 27.8 6.1 8.9 17.2 source: data from national health and nutrition examination survey 1999–2012. hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; pir: poverty income ratio. a statistically significant slopes are shown in bold letters. jain and ducatman 5 tg may reverse at a specific age. as expected, bmi was negatively associated (b ¼ �0.0059, p < 0.01) with the levels of hdl but positively associated (p < 0.01) with the levels of ldl (b ¼ 0.00078) and tg (b ¼ 0.0106). a measure of income above poverty level (pir) was positively associated (b ¼ 0.00591, p < 0.01) with the levels of hdl and negatively associated (p � 0.03) with the levels of ldl (b ¼ �0.00287), tc (b ¼ �0.00147), and tg (b ¼ �0.00878). this is an expected finding. alcohol intake was positively associated (p < 0.01) with hdl (b ¼ 0.00059) and tc (b¼ 0.00015). caffeine intake was positively associated with ldl (b¼ 0.00002, p < 0.01, table 4) but negatively associated with tg (b¼�0.00005, p < 0.01). intakes of dietary fiber (b¼ 0.00091), total fat (b ¼ 0.00022), monounsaturated fatty acids (b ¼ 0.00043), polyunsaturated fatty acids (b ¼ 0.00063), and saturated fatty acids (b¼ 0.0005) were positively associated with the levels of hdl (p � 0.01). intakes of dietary fiber (b ¼ �0.00142), total fat (b ¼ �0.00044), monounsaturated fatty acids (b ¼ �0.0009), polyunsaturated fatty acids (b ¼ �0.00175), and saturated fatty acids (b ¼ �0.00059) were negatively associated with the levels of tg (p � 0.01). intake of polyunsaturated fatty acids (b ¼ �0.00022, p ¼ 0.03) was negatively associated with tc and dietary intake of saturated fatty acids (b ¼ 0.00022, p ¼ 0.01, table 4) was positively associated with tc. prevalence and odds of having abnormal levels as compared to females, males had lower prevalence of abnormal (low) levels of hdl (27.1% vs. 32%, p < 0.01, table 5) and lower prevalence of abnormal (high) tc (13.1% vs. 16%, p < 0.01). it should be recalled that the defined low abnormal hdl is a higher value for females than for males, so the comparison can be confusing. males had higher prevalence of the abnormal (high) levels of ldl (35.0% vs. 33.1%, p ¼ 0.04, table 5) and tg (33.4% vs. 25.9%, p < 0.01). the prevalence of abnormal levels of hdl by race/ethnicity was ma (35.1%) > nhw (29.2%) > nhb (24.2%), and all three pairwise differences were statistically significant (p < 0.01). the prevalence of abnormal levels of ldl by race/ethnicity was nhw (34.5%) > ma (34.3%) > nhb (31.8%) and nhw > nhb and nhb < ma (p ¼ 0.03). for tc, the prevalence of the abnormal levels by race/ethnicity was nhw (15.5%) > ma (12.7%) and nhb (11.5%). the prevalence of abnormal levels of tg by race/ethnicity was ma (34.3%) > nhw (31.1%) > nhb (15.3%), and all three pairwise differences were statistically significant (p � 0.02, table 5). as compared to females, adjusted odds of males having abnormal levels of hdl was 0.737 (0.662–0.820, table 6) and for abnormal levels of tc, it was 0.819 (0.785–0.914, table 6). as compared to females, adjusted odds of males having abnormal levels of ldl was 1.113 (1.021–1.212, table 6) and for abnormal levels of tg, it was 1.48 (1.335– 1.639, table 6). as compared to nhw, adjusted odds of abnormal levels of hdl, ldl, tc, and tg for nhb were 0.512, 0.871, 0.713, and 0.330, respectively (table 6) and adjusted odds for ma were 1.109, 1.082, 0.940, and 1.282, respectively. smokers had higher odds of having abnormal levels of hdl (1.596, 1.141–1.802) and tg (1.308, 1.155– 1.481). a 10-year change in age was associated with markedly higher odds of abnormal levels of ldl (3.585, 3.004– 4.278), tc (4.12, 3.763–5.154), and tg (1.887, 1.553– 2.92). for one unit change in bmi, odds of having abnormal levels of hdl, ldl, tc, and tg were 1.559, 1.061, 1.007, and 1.442. for a unit increase in pir, odds of having abnormal levels of hdl was 0.888 (0.853–0.924) and for tg, it was 0.936 (0.906–0.968). for each survey period, table 5. percent prevalence with 95% confidence intervals for abnormal values of hdl (<40 mg/dl for males, <50 mg/dl for females), ldl (>130 mg/dl), tc (>240 mg/dl), and tg (>150 mg/dl). hdl ldl tc tg total 29.6 (28.5–30.8) 34 (32.9–35.1) 14.6 (13.8–15.3) 29.5 (28.4–30.6) m 27.1 (25.6–28.6) 35 (33.6–36.5) 13.1 (12.1–14.1) 33.4 (31.8–35) f 32 (30.5–33.5) 33.1 (31.7–34.4) 16 (15–16.9) 25.9 (24.6–27.2) nhw 29.5 (27.9–31) 34.5 (33.1–35.9) 15.5 (14.5–16.5) 31.1 (29.8–32.3) nhb 24.2 (22.2–26.2) 31.8 (29.9–33.7) 11.5 (10.3–12.6) 15.3 (13.7–16.9) ma 35.1 (32.7–37.4) 34.3 (32.1–36.6) 12.7 (11.3–14) 34.3 (31.9–36.6) oth 32.2 (28.8–35.6) 32.6 (29.8–35.4) 13 (11.3–14.8) 30.3 (27.4–33.2) nsm 27.4 (26.3–28.5) 33.7 (32.5–34.9) 14.2 (13.4–15.1) 28.5 (27.3–29.8) sm 35.7 (33.4–38) 34.5 (32.8–36.3) 15.3 (14–16.7) 32.2 (30.4–34) statistically significant differences m < f (p < 0.01), nhw > nhb (p < 0.01), nhw < ma (p < 0.01), nhb < ma (p < 0.01), nhb < oth (p < 0.01), nsm < sm (p < 0.01) m > f (p ¼ 0.04), nhw > nhb (p < 0.01), nhb < ma (p ¼ 0.03) m < f (p < 0.01), nhw > nhb (p < 0.01), nhw > ma (p < 0.01), nhw < oth (p ¼ 0.02) m > f (p < 0.01), nhw < nhb (p < 0.01), nhw < ma (p ¼ 0.02), nhb < ma (p < 0.01), nhb < oth (p < 0.01), ma > oth (p ¼ 0.04), nsm < sm (p < 0.01) source: data from national health and nutrition examination survey 1999–2012. hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; m: male; f: female; nhw: non-hispanic white; nhb: non-hispanic black; ma: mexican american; oth: other race/ethnicities; nsm: nonsmoker; sm: smoker. 6 journal of circulating biomarkers odds of having abnormal levels of hdl, ldl, tc, and tg decreased by 0.879, 0.918, 0.935, and 0.911, respectively (table 6). discussion as previously mentioned, there are two ways to assess smoking status. study participants may be asked to selfreport their smoking status. but, at times, the accuracy of self-reports may be questionable. the accuracy of selfreports may depend on the specificity of the questions asked. in addition, there may be intentional misreporting. the respondents may report what they think is a socially acceptable smoking status. as discussed elsewhere, 12 in the current social setting, smokers are more likely to report themselves as nonsmokers than nonsmokers reporting themselves as smokers. thus, if the accuracy of smoking status is needed, use of one or the other biomarkers of tobacco smoke becomes a desirable alternative. proposed biomarkers of tobacco smoke include serum cotinine, urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (nnal), 15 volatile organic compounds like benzene and toluene measured in blood, 16 and urinary thiocyanate. 17 however, probably because of the ease of measurement using easily available assays, serum cotinine continues to remain a biomarker of choice. the half-life of serum cotinine is estimated to be 16–19 h, 18 and as such, serum cotinine can only be used as a biomarker of exposure to tobacco smoke during the last 5 days. on the other hand, half-life of urinary nnal has been estimated to be as much as 10–18 days, 19 and as such, urinary nnal can be used as the exposure to tobacco smoke during the last 6–12 weeks. 19 these data might suggest urinary nnal as a preferred biomarker of exposure to tobacco smoke. however, the assay used to measure nnal is complicated and expensive and nhanes data for nnal did not become available until nhanes 2007–2008. since the study period for this communication was between 1999 and 2012, serum cotinine was selected as a biomarker of tobacco smoke. based on a study among 215 patients, jarvis et al. 20 proposed a serum cotinine cut off of 14 ng/ml to distinguish smokers from nonsmokers. as far as we could determine, first mention of nonsmokers having <10 ng/ml of serum cotinine was also by jarvis et al. 18 however, since serum cotinine reflects exposure to tobacco smoke from all sources including main stream and second hand smoke, benowitz et al. 21 proposed a serum cotinine cut off of 2.99 ng/ml for adolescents aged 12–19 years and 3.08 ng/ml for adults aged �20 years to distinguish smokers from nonsmokers. these substantially lower cutoffs reflected reduction in exposure to secondhand smoke over 1988–2002 in us as documented by pirkle et al. 22 however, benowitz et al. 21 used self-reported smoking status as the gold standard to develop the proposed serum cotinine cutoffs as presented above. in other words, proposed cutoffs by benowitz et al. 21 depended upon the accuracy of self-reported smoking status. the use of cutoffs that depended upon the accuracy of self-reported smoking status would have been contrary to the objectives of this study. as such, we used the serum cotinine cutoff of 10 ng/ml for this study. many, if not all, of the studies that have previously investigated the association between smoking and lipid/ table 6. adjusted odds of having abnormal levels of hdl (<40 mg/dl for males, < 50 mg/dl for females), ldl (>130 mg/dl), tc (>240 mg/dl), and tg (>150 mg/dl) by gender, race/ethnicity, smoking status.a effect hdl ldl tc tg males versus females 0.737 (0.662–0.82) 1.113 (1.021–1.212) 0.819 (0.735–0.914) 1.48 (1.335–1.639) non-hispanic blacks versus nonhispanic whites 0.512 (0.443–0.591) 0.871 (0.768–0.988) 0.713 (0.616–0.825) 0.33 (0.285–0.382) mexican americans versus non-hispanic whites 1.109 (0.959–1.282) 1.082 (0.957–1.223) 0.94 (0.798–1.107) 1.282 (1.131–1.453) other race/ethnicities versus nonhispanic whites 1.098 (0.914–1.319) 0.943 (0.81–1.099) 0.861 (0.721–1.028) 1.086 (0.913–1.291) smoker versus non-smoker 1.596 (1.414–1.802) 1.073 (0.979–1.176) 1.228 (1.07–1.409) 1.308 (1.155–1.481) vigorous versus none/minimum physical activity 1.065 (0.884–1.283) 1.137 (0.928–1.394) 0.983 (0.745–1.296) 0.896 (0.756–1.061) moderate versus none/minimum physical activity 1.287 (1.078–1.537) 1.152 (0.99–1.341) 1.174 (0.919–1.5) 1.084 (0.916–1.283) age for a 10-year change 0.914 (0.766–1.091) 3.585 (3.004–4.278) 4.12 (3.235–5.246) 1.887 (1.553–2.292) age2 for a change of 100 1.001 (0.983–1.019) 0.891 (0.876–0.907) 0.887 (0.868–0.907) 0.954 (0.937–0.972) body mass index for a unit change 1.559 (1.491–1.63) 1.061 (1.022–1.101) 1.007 (0.963–1.052) 1.442 (1.38–1.507) pir for a unit change 0.888 (0.853–0.924) 0.956 (0.922–0.991) 0.944 (0.905–0.984) 0.936 (0.906–0.968) fasting for a 1-h change 0.992 (0.968–1.016) 1.039 (1.014–1.064) 1.033 (1.002–1.065) 0.991 (0.966–1.017) survey year for a 2-year change 0.879 (0.848–0.912) 0.918 (0.886–0.951) 0.935 (0.889–0.984) 0.911 (0.879–0.945) source: data from national health and nutrition examination survey 1999–2012. hdl: high-density lipoprotein; ldl: low-density lipoprotein, tc: total cholesterol; tg: triglyceride; pir: poverty income ratio. aphysical activity level, age, body mass index, pir, and survey year. statistically significant odds ratios are shown in bold letters. jain and ducatman 7 lipoprotein levels were in specific communities, not necessarily for a representative sample of the entire national population under investigation. this nationally representative sample of the us population aged �20 years used cotinine-based classification to distinguish smokers from nonsmokers and, therefore, is different from both community-based studies and previous results reported by jain. 11 the use of the biomarker reclassified about 10% of participants by smoking class and increased the sample size by 5.9%. advantages of the biomarker cotinine have been reported extensively, 23 and the accuracy of adult self-report has also been reported. 24 our findings support the inference that use of the biomarker is particularly pertinent for the analysis of prevalence data, such as found in nhanes. effect of smoking nhanes data reveal that the levels of both hdl and tg are adversely affected among smokers, a finding that is in the literature but which could be further emphasized in risk factor and tobacco control literature. compared to nonsmokers, adjusted levels among smokers were about 6% lower for hdl and 11% higher for tg, both undesirable associations. prevalence of abnormal or low levels of hdl among smokers was 8.3% higher than nonsmokers. the prevalence of abnormal or elevated levels of tg was 3.7% higher among smokers than nonsmokers. odds of having abnormal levels for smokers was about 60% higher for hdl and about 31% higher for tg than nonsmokers. in spite of the differences in study design and populations covered, some of the results of this study are consistent with the results reported by komiya et al., 1 koda et al., 7 devaranavadgi et al., 6 meenakshisundaram et al., 4 craig et al., 3 and gossett et al. 5 effect of age, race/ethnicity, and gender consistent with the literature, increase in age was found to be associated with increases in the adjusted levels of ldl, tc, and tg. however, the associations are not monotonic and attenuate with increasing age. despite the attenuation, a 10-year increase in age was associated with higher odds of abnormal levels of ldl (or: 3.6), tc (or: 4.1), and tg (or: 1.9) as per definitions used in this study. similar results using commercial clinical laboratory data have been reported by kaufman et al. 25 in a study conducted in anniston, alabama, united states, aminov et al. 26 reported african americans to have lower levels of total lipids and triglycerides and higher levels of hdl among those who were not on any lipid lowering medications. santos et al. 27 compared results in a brazilian population by race: blacks were reported to have a favorable profile, higher concentrations of hdl but lower concentrations of ldl and tg. similar to these findings, we report that nhb did have higher levels of hdl and lower levels of tc and tg (table 3) and, in addition, as compared to nhw, nhb had lower odds (table 6) of having abnormal values of hdl (or: 0.512), ldl (or: 0.871), tc (or: 0.713), and tg (or: 0.33). in this study, males were found to have higher adjusted levels of ldl and tg than females and lower adjusted levels of hdl and tc than females (table 3). gender differences in lipid/lipoprotein metabolism have been reported by russo et al., 28 habib et al., 29 kolovou et al., 30 duvernoy et al., 31 wang et al., 32 and others. males having higher levels of ldl than females have been reported by kaufman et al. 25 and russo et al. 28 females having higher levels of hdl than males have been reported by habib et al., 29 russo et al., 28 kolovou et al., 30 and duvernoy et al. 31 lower levels of tg among females as compared to males have been reported by habib et al., 29 russo et al., 28 kolovou et al., 30 and duvernoy et al. 31 kolovou et al. 30 reported females to have higher levels of tc than males. as compared to females, males had lower odds (table 6) of clinically “abnormal” hdl (or: 0.737) and tc (0.819) but higher odds of abnormal ldl (or: 1.113) and tg (or: 1.48). the hdl comparison for abnormal values requires understanding of the different cut-offs used. since we normally think of females as having favorable cardiovascular risk profiles until the age of menopause, the population data may provide additional detail to inform clinical risk factor considerations. conclusion results of this study do indicate that smoking is associated with adverse lipid/lipoprotein profiles among adult population of the united states. smokers were shown to have lower adjusted levels of hdl and higher adjusted levels of tg, as well as higher adjusted odds of having abnormal levels of hdl, ldl, and tg when compared with nonsmokers. use of serum cotinine rather than self-reports improves understanding of the relationship between smoking and unfavorable lipid profile. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) received no financial support for the research, authorship, and/or publication of this article. orcid id ram b jain http://orcid.org/0000-0002-3278-7106 8 journal of circulating biomarkers http://orcid.org/0000-0002-3278-7106 http://orcid.org/0000-0002-3278-7106 http://orcid.org/0000-0002-3278-7106 references 1. komiya h, mori y, yokose t, et al. smoking as a risk factor for visceral fat accumulation in japanese men. tohoku j exp med 2006; 208(2): 123–132. 2. kuzuya m, ando f and iguchi a, et al. effect of smoking habit on age-related changes in serum lipids: a cross-sectional and longitudinal analysis in a large japanese cohort. atherosclerosis 2006; 185(1): 183–190. 3. craig wy, palomaki ge, and haddow je. cigarette smoking and serum lipid and lipoprotein concentrations: an analysis of published data. bmj 1989; 298(6676): 784–788. 4. meenakshisundaram r, rajendiran c, and thirumalaikolundusubramanian p. lipid and 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/downsample16bitimages true /flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice article journal of circulating biomarkers exosome: a novel and safer therapeutic refinement of mesenchymal stem cell ronne wee yeh yeo1, ruenn chai lai1, kok hian tan2 and sai kiang lim1,3* 1 institute of medical biology, agency for science, technology and research, singapore 2 department of maternal fetal medicine, kk women’s and children’s hospital, singapore 3 department of surgery, yong loo lin school of medicine, national university of singapore, singapore *corresponding author(s) e-mail: saikiang.lim@imb.a-star.edu.sg received 14 november 2013; accepted © 2013 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract mesenchymal stem cell (msc) has just been approved as the first “off-the-shelf” stem cell pharmaceutical drug with an anticipation of more approvals following completion of numerous rigorous clinical trials. despite this progress, the rationale for msc therapeutic efficacy remains tenuous and is increasingly rationalized on a secretion rather than differentiation mechanism. recent studies identifying exosome as the secreted agent mediating msc therapeutic efficacy could potentially reduce a cell-based drug to a safer biologic-based alternative. here we review the develop‐ ment of msc exosome as a potential first-in-class thera‐ peutic, and the unique challenges in the manufacture and regulatory oversight of this new class of therapeutics. keywords exosomes, mesenchymal stem cell, myocardial ischemia / reperfusion injury, purification 1. introduction 1.1 mesenchymal stem cells as a therapeutic agent mscs are currently the most clinically evaluated stem cells. the clinicaltrials.gov database lists more than 300 msc trials to treat a wide range of pathological conditions (http://www.clinicaltrials.gov/, accessed march 2013). many of these trials were predicated on the efficacy of msc against a wide range of diseases in animal models. the therapeutic efficacy of transplanted mscs was initially attributed to their homing and engraftment in injured tissues, and subsequent differentiation to repair and replace damaged tissues. however, studies in animal models and patients indicated that <1% of transplanted mscs localize to the target tissue with most becoming trapped in the liver, spleen and lungs [1]. furthermore, evidence for differentiation of transplanted mscs at the site of injury could not rule out the possibility of fusion with endogenous cells [2-4]. in fact, the efficacy of msc trans‐ plantation in treating diseases in animal models and patients has been increasingly observed to be independent of engraftment or differentiation [5-9]. consequently, msc secretion is now implicated as the primary mediator of msc-based therapy. the refinement of msc therapy from a cell-to secretionbased therapy offers several advantages as it essentially translates the therapeutic agent from a living to non-living agent and obliterates the arduous task of preserving cell viability and function during manufacture, storage and delivery to patient. as such, cellular secretions are more amenable to development as an “off-the-shelf” therapeutic that can be delivered to patients in a timely manner. they also mitigate the safety risks inherent in administering 1 large, viable cells such as the risk of occlusion in microvas‐ culature or unregulated growth. for example, intra-arterial administration of mscs causes a high 25-40% incidence of pulmonary embolism and death [10]. msc transplantation has also been associated with adverse cellular growth such as cardiac sympathetic nerve sprouting and consequently hyper-innervation [11], or ossification and/or calcification in cryo-infarcted hearts [12]. therefore, the benefits of secretion-based therapy coupled with the risks of cell transplantations have spurred recent research interest in msc secretion. much of the initial attention on msc secretion has centered on small molecules such as growth factors, chemokines and cytokines [13]. although many candidates for mediating the efficacy of msc have been proposed, none could sufficiently account for the efficacy of msc against the diverse range of pathological conditions. for example, several cytokines, chemokines and enzymes shown to be secreted by mscs have been proposed as the secreted factor mediating immunotherapeutic efficacy of mscs [14]. they include interferon-γ (ifnγ) [15-17], a downstream ifnγinducible gene indoleamine 2,3-dioxygenase (ido) [18] and inducible nitric-oxide synthase (inos) [19]. both ifnγ and ido were eliminated as candidates when the immunomodulatory activity of mscs was subsequently found not to be ameliorated by a lack of ido production caused by either defective ifnγ receptor 1 or ido inhibi‐ tors [20]. also, murine mscs unlike human mscs do not have ido activity but could exert similar immunomodula‐ tory effects [21, 22]. in the case of inducible nitric-oxide synthase (inos) which appear to be important for the inhibition of t-cell proliferation by mouse mscs, it was deemed not important in human mscs as they expressed low level of inos [14]. mscs are also known to secrete many generic immuno-modulating factors such as trans‐ forming growth factor (tgf)-β1, hepatocyte growth factor (hgf), heme oxygenase 1, il-6, prostaglandin e2 (pge2), and hla-g5 [23-34] and it is unlikely that any one of these factors could sufficiently mediate the immunotherapeutic efficacy of mscs. efforts to identify the therapeutic agent in msc secretion have recently shifted from the small molecules to relatively large secreted lipid membrane vesicles known collectively as extracellular vesicles (evs) and these evs were shown to be as therapeutically efficacious as mscs [35-37]. 2. exosome, an extracellular vesicle (ev) evs consist of several classes of secreted vesicles such as exosomes, microvesicles, ectosomes, membrane particles, exosome-like vesicles or apoptotic bodies [38]. however, these different classes remain poorly defined and terms “exosomes”, “microvesicles”, “microparticles” are being used interchangeably. generally, evs could be formed by either inward budding of endolysosomal vesicles followed by exocyto‐ sis e.g. exosomes, or shedding from the plasma mem‐ brane e.g. microvesicles. of the evs characterized to date, exosomes remain the best characterized and are the only evs known to be formed by the invagination of endoly‐ sosomal vesicles. unlike other evs, exosomes are defined as having a diameter of 40-100 nm, a flotation density in sucrose of 1.1-1.18 g/ml, and bilipid membrane enriched in lipid rafts of cholesterol, sphingomyelin, and ceram‐ ide [39, 40]. the presence of exposed phosphatidylser‐ ine on exosome membrane is controversial and remains to be determined [41-44]. many cell types such as b cells [45], dendritic cells [46], mast cells [47], t cells [48], platelets [44], schwann cells [49], tumor cells [50], mesenchymal stem cells [35], human embryonic kidney cells [51], various cancer cell lines [52] and sperms [53] are known to secrete exosomes. consistent with this diverse range of cell types, exosomes are found in as many physiological fluids including bronchial lavage fluid [54], human urine [55, 56] and human blood [56]. together, these indicate that exosome secretion is a conserved cellular function. exosomes from different cellular sources share an evolu‐ tionary conserved set of protein molecules including tetraspanins (cd81, cd63, cd9), alix and tsg101 but they also carry proteins and rnas unique to their cell source and the pathophysiological states of the cell source [57]. for example, exosomes from reticulocytes, unlike those from lymphocytes and dendritic cells, are rich in transferrin receptors that had to be disposed of when reticulocytes mature [45, 58, 59] while exosomes from tumor cells carry tumor antigens [50, 60-62] or tumor-specific micrornas [63]. the cargo composition of exosomes is also modulated by its microenvironment. exosomes secreted by tumor cells during hypoxia carry proteins that facilitate angiogenesis and metastasis [64]. the list of proteins and rnas reported to be present in exosomes are freely accessible at exocarta (http://www.exocarta.org) or vesiclepedia (http:// www.microvesicles.org). the biogenesis of exosomes in the endosomal pathway involves the invagination of the endosomal membrane to form intraluminal vesicles (ilvs) resulting in the formation of a multivesicular body (mvb). the ilvs will either be degraded if the mvb fuses with a lysosome or released as exosomes if the mvb fuses with the plasma membrane [65]. the process by which cellular proteins and rnas is targeted to endosomes and subsequently exosomes has not been elucidated. although escrt (endosomal sorting complex required for transport) has been implicated [66], some proteins have been shown to be targeted to exosomes in an escrt-independent manner via higher-order oligomerization or a ceramide-dependent but escrtindependent process [67, 68]. notwithstanding this, it is obvious that the biogenesis of exosomes requires cellular expenditure of energy and resources, implying a functional importance for exosomes. 2 despite its discovery about four decades ago [69], research interest in exosomes was fueled only recently when exosomes were found to be secreted by different cell types and participate in many important cellular activities. for example, exosomes are secreted by dendritic cells pulsed with tumor peptides for tumor suppression [70], by neurons for neurotransmission [71, 72], by oligodendrocytes for myelin membrane biogene‐ sis [73], and by cardiomyocyte progenitor cells for migration of endothelial cells [74]. exosomes from mesenchymal stem cells were even found to be therapeu‐ tic and could reduce myocardial ischemia/reperfusion injury [35] or acute tubular injury [36]. unfortunately, exosomes have also been implicated in a wide spectrum of diseases from cancers and degenera‐ tive diseases to infectious diseases. exosomes secreted by tumor cells have been shown to be capable of enhanc‐ ing tumor or metastatic phenotype [75-77]. they are also implicated in the formation of disease-associated pro‐ tein aggregates such as alpha-synuclein aggregates in parkinson's disease [2, 78] and amyloid beta protein aggregates (aβ) in alzheimer disease [79]. it was also reported that during infection with diverse infectious agents such as scrapie, viruses and bacteria, cells secrete exosomes containing the infectious agent or its products and thus providing potential route for disseminating the infection [49, 70, 80-84]. as a consequence of the close association between the composition of exosomes and physiological or pathological states of the secreting cells, exosomes are good sentinels of cellular health and pathology, and have become an attractive source of disease biomarkers (reviewed [85]). 3. exosomes as a therapeutic agent the therapeutic potential of exosomes was first described in msc exosomes when it was observed that the exosomes were cardioprotective in a murine model of acute myocar‐ dial ischemia/reperfusion injury [35]. this observation provided a rationale for an increasing msc enigma. the cardioprotective potential of mscs have been widely reported; they reduce infarct size, improve left ventricular ejection fraction, and increase vascular density and myo‐ cardial perfusion in animal models of ami [86-90]. al‐ though this protection has always been rationalized on the potential of mscs to differentiate and replace damaged cardiac tissues, significant engraftment and differentiation of mscs into cardiomyocytes were seldom observed even when there were functional improvements [1-4]. in general, it was observed that the therapeutic efficacy of mscs was not dependent on their physical proximity to the tissue [37-41] leading to the proposal that this therapeutic efficacy was mediated by secreted factors [42]. timmers et al. first reported in 2008 that msc secretion mediated the cardioprotection of msc. they observed that after sterile filtration with a 0.2 µm filter, culture medium conditioned by human esc-derived mscs could reduce infarct size in pig and mouse models of acute myocardial ischemia and reperfusion injury [91]. this conditioned medium (cm) could also improve cardiac function in a pig model of myocardial ischemia by stimulating angiogenesis and preventing adverse myocardium [92]. size fractiona‐ tion of the cm subsequently revealed that the therapeutic activity resided in the 100 to 220 nm fraction. this fraction when purified by size-exclusion hplc was found to be enriched in lipid membrane vesicles that fulfilled the defining criteria of exosomes i.e. 40-100 nm, a flotation density in sucrose of 1.1-1.18 g/ml, lipid membrane enriched in lipid rafts of cholesterol, sphingomyelin, and ceramide, and a conserved set of protein molecules, namely the tetraspanins (cd81, cd63, cd9), alix and tsg101 [39, 40]. this fraction of exosomes was able to reduce the infarct size in a mouse model of acute myocardial ischemia and reperfusion injury [35]. although our lab was the first to describe the secretion and therapeutic potential of exo‐ somes from mscs, bruno et al. preceded us in describing msc secretion of lipid membrane vesicles which they termed “microvesicles” and the amelioration of glycerolinduced acute kidney injury by these vesicles [36]. as these microvesicles were not evaluated for exosome-associated properties and had an estimated diameter of 180 nm versus the 55-65 nm hydrodynamic radius [35] for the exosomes that we isolated, we postulated that these two populations of evs are different. in the last two years, msc exosomes have been found to be efficacious in an increasing number of animal models for human diseases such as cerebral ischemia [93], liver fibrosis [94], hypoxic pulmonary hypertension [95], and acute kidney injury [96, 97]. recently, msc exosomes were administered to a treatment-resistant grade iv acute gvhd patient. her symptoms were dramatically alleviat‐ ed and she remained stable for five months [98]. 4. translating msc exosome into a cardiac pharmaceutical drug acute myocardial infarction (ami) or heart attack causes ~7.2 million deaths globally each year and inflicts a heavy socioeconomic burden [99]. while advances in reperfusion therapy to restore blood flow and oxygenation to ischemic heart tissues have significantly improved ami survival rate [100], 65% of ami survivors go on to develop fatal heart failure within 5 years [101]. this progression to heart failure is complex but is largely determined by the final size of infarcted myocardium in surviving ami patients. al‐ though early reperfusion of ischemic myocardium salvages much of the tissue at risk, reperfusion of severely ischemic tissue paradoxically causes lethal injury, known as ische‐ mia/reperfusion (i/r) injury and is estimated to contribute up to 50% of the final infarct size in reperfused ami patients [102]. to date, i/r injury has been intractable to pharma‐ ceutical interventions [103]. the efficacy of msc exosomes in reducing reperfusion injury and long term preservation 3ronne wee yeh yeo, ruenn chai lai, kok hian tan and sai kiang lim: exosome: a novel and safer therapeutic refinement of mesenchymal stem cell of cardiac function and geometry in animal models of ami [35, 91, 92, 104, 105] therefore provides a compelling rationale for its translation into a pharmaceutical drug to treat i/r injury. if successful, exosome will be a “first-inclass”. this, in itself will present unique challenges in the manufacture and regulatory oversight. 5. msc exosome-mediated efficacy against reperfusion injury as the terms “exosomes”, “microvesicles”, “microparti‐ cles” are presently being used interchangeably in the literature, the term “msc exosome” in this section shall be used in a similar context to include all extracellular vesicles produced by mscs. msc exosomes, like their prodigious cell source, are reportedly efficacious in an equally wide range of diseases. however, it remains to be determined if a single class of exosomes or a single molecular mechanism is responsible for the therapeutic efficacy against these different diseases. we have previously proposed that msc exosomes ameliorate reperfusion injury through a proteo‐ mic complementation [106]. during acute myocardial ischemia, rapid reperfusion to reoxygenate the tissues halts ischemia-induced cellular damages and restores cardiac contractibility but delayed reperfusion after 40-60 minutes of ischemia cannot restore cardiac contractibility [107-111]. this failure to restore cardiac contractibility in severely ischemic myocardial tissues has been partially attributed to i/r injury. during ischemia, the lack of oxygen inhibits oxidative phosphorylation and causes atp production to collapse. this is particularly detrimental to contractile function of cardiomyocytes which have the highest basal atp con‐ sumption rate in a resting body. cardiomyocytes derive 95% of its atp from the highly efficient oxygen-dependent coupling of fatty acid β-oxidation and oxidative phosphor‐ ylation [112]. during ischemia, the lack of oxygen disrupts this atp production and aerobic glycolysis, leaving anaerobic glycolysis as the major source of atp produc‐ tion. consequently, the cells become atp deficient and atp-dependent cellular activities such as protein synthesis and turnover, gene transcription, ion pumps or transport‐ ers have to be curtailed. over time, this escalates to a loss of cellular homeostasis such as intracellular acidification, imbalance of ions such as sodium, potassium and calcium, and increased intracellular osmotic pressure through the accumulation of metabolic products such as adp, inorgan‐ ic phosphates, reduced coenzymes (e.g. nadh and fadh) and lactic acid, and eventually initiation of apoptosis [108-113]. therefore, restoring atp production and delaying initiation of apoptosis are critical therapeutic targets in reperfusion therapy. in less ischemic tissues, reperfusion re-oxygenates and reenergizes the mitochondria to generate atp and restores cellular activities. however, in the severely ischemic tissues where the cellular biochemistry is highly deranged, reoxygenation exacerbates mitochondrial damage and the perturbed ion homeostasis to cause i/r injury. reperfusion of severely ischemic cells not only fails to restore oxidative phosphorylation but causes further damages such as opening of the mitochondrial permeability transition pore (mptp) to generate and release reactive oxygen species (ros), and aggravation of ionic imbalances leading to sarcolemmal disruption and hypercontracture [112, 114-116]. these ultimately result in initiation of apoptosis and an acute inflammatory response, both of which contribute to infarct size [87-90]. our recent analysis of the cardiac proteome after ischemia and then reperfusion revealed that the dominant determi‐ nants of i/r injury, atp production and apoptosis, are underpinned by proteomic changes that persist for at least 60 minutes post-reperfusion [117]. therefore, therapeutic strategies targeting these determinants of i/r injury will have to rapidly overcome these proteomic alterations in order to alleviate the atp deficit and vulnerability to apoptosis. we had proposed that protein complementation by msc exosome could provide a direct intervention to overcome these proteomic alterations and elicit a timely cellular response that is critical for the survival of the atpdeficient and pro-apoptotic reperfused cells [106]. msc exosomes carry a full enzyme complement for the atp-generating phase of glycolysis and also cd73, an adenosine-generating extracellular enzyme [118]. we had demonstrated that these enzymes were active, and msc exosomes could increase atp production and adenosinemediated survival kinase signaling in cells [118] and a mouse model of i/r [105] (figure 1). as i/r injury is complex and causes extensive biochemical derangements as discussed earlier, ameliorating atp deficit and circum‐ venting initiation of apoptosis essentially provide a short time window of opportunity for the cells to rectify other molecular derangements. the remarkable efficacy of msc exosome in reducing i/r injury in animal models and its large diverse proteome suggest that msc exosome must have biochemical activi‐ ties beyond the glycolytic enzymes and cd73 to rectify the other molecular derangements in i/r injury. some of these activities include functionally active 20s proteasomes capable of reducing denatured protein aggregates in reperfused hearts of mouse models [119] and the inhibition of complement-mediated lysis by exosome-associated cd59 [118]. we hypothesize that by rapidly correcting a critical atp deficit and delaying initiation of apoptosis, the reperfused cells would have sufficient atp to support the diverse biochemical potential of msc exosomes and/or the endogenous repair mechanisms in the cells to rectify the othermolecular derangements caused by i/r injury [106]. 4 figure 1. msc exosomes ameliorate ischemia/ reperfusion injury by correcting atp deficit and circumventing a pro-apoptotic proteome. in a severely ischemic myocardium, atp-generating processes such as aerobic glycolysis, β-oxidation and citric acid cycle are curtailed, leading to a loss of cellular homeostasis. reperfusion further exacerbates this injury by opening the mitochondrial permeability transition pore (mptp) to release reactive oxygen species (ros), resulting in the initiation of apoptosis. exosomes ameliorate the atp deficit by provide a full complement of glycolytic enzymes to enhance glycolytic flux. in addition, amp derived from the degradation of atp and adp from injured or dying cells is hydrolyzed into adenosine by cd73 present on exosome surfaces. adenosine in turn binds to adenosine receptors to activate reperfusion injury salvage kinase (risk) pathways that mediate cell survival. the effectiveness of msc exosomes in ameliorating i/r injury could be partially attributed to the use of enzymes as therapeutic agents. by using enzymes, msc exosomes could exert an amplified catalytic effect that is calibrated to their microenvironment (e.g., substrate concentration or ph). in healthy tissues where the microenvironment for most enzymes is likely to be in homeostastic equilibrium, activity of administered enzymes will be modulated by regulatory feedback mechanisms to maintain this equili‐ brium. during injury when homeostasis is disrupted, enzyme activity will be modulated in proportion to the magnitude of loss in homeostasis which is, in turn propor‐ tional to injury severity until equilibrium is restored or injury is resolved. this potential to sense and respond accordingly to restore equilibrium mitigates the risk of over-or under-dosing. the use of enzymes as therapeutics is traditionally limited by the susceptibility to degradation and by the lack of a vehicle to transfer proteins intracellu‐ larly. their encapsulation in exosomes circumvents many of these limitations by protecting them against degradation to enhance their bioavailability, homing and enabling internalization by membrane fusion or endocytosis (reviewed [120]). in fact, the integrity of the exosome membrane is key to its efficacy in reducing i/r injury. when membrane integrity is compromised, exosomes lose their efficacy [105]. interestingly, the efficiency of exosome uptake has been correlated to intracellular and microen‐ vironmental acidity [121]. this provides a mechanism for exosome homing to ischemic tissues whereby msc exosomes are preferentially endocytosed by ischemic cardiomyocytes which reportedly have low intracellular ph [122]. 6. msc exosome-mediated efficacy against other pathologies unlike reperfusion injury, the proposed mechanism of msc exosome-mediated efficacy against other diseases have centered on either the rna components of msc exosomes or the rna machinery of the target cells. in glycerol-, gentamicin-, and cisplatin-induced acute kidney injury, msc exosomes reportedly shuttle mrna to tubular epithelial cells to promote proliferation and survival [36, 96, 97]. in a rat model of middle cerebral artery occlusion, it was demonstrated that msc exosomes transferred mir-133b to neurons and astrocytes to promote neurite outgrowth and functional recovery after stroke [93]. on the other hand, msc exosomes instead of delivering rna were recently shown to suppress induction of the proliferative mir-17 microrna superfamily but induce expression of antiproliferative mir-204, and prevent pulmonary vascular remodeling in a murine model of hypoxic pulmonary hypertension [95]. together, the studies of msc exosomes in animal models of diseases have implicated both proteins and rnas in mediating their therapeutic efficacy against different diseases. such versatility in the mode of action and disease targets is possible only because of the large payload that exosomes could carry and this clearly differentiates exosomes from traditional biologics. 7. msc exosomes as a pharmaceutical drug the main considerations in developing msc exosomes as a pharmaceutical drug are the manufacturing path and regulatory oversight. the key factor in the manufacture of msc exosomes is the cell source. the ideal cell source would be a reprodu‐ cibly high exosome-yielding cell with an infinite expan‐ sion capacity. we have observed that exosome production was inversely correlated with the age of the donor tissue with the most prolific producer being mscs derived from hescs followed by fetal tissues, umbilical cord and adult bone marrow (chen et al, 2013). hesc-mscs are also superior to those derived from other tissues in having a rapid doubling time of about 72 hours and being proliferative for at least 20 passages at a 1:4 split [123]. however, unlike other mscs, hesc-mscs are encum‐ bered by the same social, legislative and ethical controver‐ sies surrounding hescs. although these controversies have abated, complex patent protection and legislation have become increasingly intractable barriers to transla‐ tion [124, 125]. also, the large proliferative capacity of hesc-mscs is finite and would require costly and timeconsuming repeated derivation and validation to sustain a manufacturing process. to overcome this issue, we immortalized hesc-mscs by over-expressing myc and demonstrated that despite a compromised differentia‐ tion potential, the immortalized cells continued to secrete 5ronne wee yeh yeo, ruenn chai lai, kok hian tan and sai kiang lim: exosome: a novel and safer therapeutic refinement of mesenchymal stem cell cardioprotective exosomes [126]. notably, myc protein was not detectable in the exosomes. also, myc rna is not likely to be present in msc exosomes as the rnas in msc exosome are primarily less than 300 nt [127]. another key manufacturing consideration is the purifica‐ tion of exosomes. exosomes are conventionally purified by ultracentrifugation, ultra-filtration and gel filtration [128-131]. while these techniques result in exosome enrichment, the preparation is usually contaminated with protein aggregates and other cell debris. additionally, this method is time-consuming, requires expensive specialized equipment or has poor scalability. we have previously reported that the purity of the exosome preparation could be greatly enhanced by size exclusion high performance liquid chromatography (hplc) [35, 104]. however, this method requires expensive equipment, and has poor scalability and product yield. while immuno-affinity chromatography [132, 133] is highly scalable and could potentially enhance the purity of the exosome prepara‐ tions, none of the known exosome-associated antigens are exclusive to exosomes and immuno-affinity-based isola‐ tion protocols could still purify protein complexes and other evs. in addition, the non-physiological salt or ph concentration needed to extract exosomes from the immu‐ no-affinity column could affect the biological activity of the exosomes. in short, to date there is no ideal, scalable and cost effective method for the purification of exosomes. the lack of defining markers for exosomes also poses a challenge to the development of an identity for exosomes as therapeutic agents. exosomes are typically identified as particles of 100-200 ηm in diameter, a density of 1.10-1.18 g/ml in sucrose and marked by tetraspanins cd9, cd81 or cd63) on their surfaces. these parameters are either too generic or too cumbersome for routine identification assay. another challenge is a standardized unit of quantity to measure exosomes. the current use of protein concentra‐ tion is ambiguous as it is highly dependent on the purity of the preparation. this challenge has been somewhat mitigated by the recent advent of particle analysis technol‐ ogies such as nanosight’s particle tracking analysis and izon qnano’s tunable resistive pulse sensing which quantify exosomes as particle numbers. additionally, these technologies could profile size distribution of the particles which is useful in assessing of the level of contaminating ev populations (e.g. exosomes, microvesicles, apoptotic bodies) in the exosome preparation. however, particle count and size do not differentiate ev from non-ev particulates such as protein aggregates. a more specific quantification of exosome would be to assay for exosomeassociated membrane-bound antigen (e.g. tetraspanins cd9, cd81 or cd63) by elisa however, there are no definitive exosome markers, and most exosome-associated markers are also found on other evs. essentially, there is an urgent need for an unambiguous assay to identify and quantify exosomes. a third and probably most important challenge in devel‐ oping exosomes as a pharmaceutical drug is the develop‐ ment of an in vitro surrogate assay for in vivo therapeutic potency. in the development of msc exosome for the treatment of i/r injury, we envisage that such an assay is likely to involve the measurement of enzyme activity in intact exosomes as our observations thus far have indicated that therapeutic efficacy of msc exosomes is dependent on enzyme activity and intact exosome membrane. 8. regulatory oversight the use of exosomes as a pharmaceutical product presently has no precedent and therefore has no defini‐ tive product “class” guidance within regulatory agen‐ cies such as the fda and emea to instruct compliance. nonetheless, the over-arching guidance could be easily surmised from the universal principle of safety, quality and efficacy. msc exosome derives from a cell source that has been extensively tested in clinical trials for numer‐ ous indications. in general, administration of autolo‐ gous and allogeneic mscs has been found to be safe in humans and allogeneic mscs are well tolerated without overt immune rejection. by extrapolation, msc exosome which is essentially a minor constituent of msc would be expected to be equally safe and immunologically tolerated. consistent with the immune tolerance of msc exosomes, proteomic analysis of msc exosome revealed the absence of mhc and co-stimulatory molecules e.g. cd80 and cd86 [118, 119]. finally, msc exosomes have been demonstrated to be efficacious in clinically rele‐ vant animal models [35, 91, 92, 104, 105]. together, experimental and pre-clinical data support msc exo‐ some as a safe and efficacious therapeutic agent that could be manufactured reproducibly, and provides a compelling rationale for further clinical development. 9. conclusion the efficacy of msc against diverse disease indications is increasingly being attributed to its exosomes. this efficacy, which is prodigious for a single therapeutic entity, is not unreasonable particularly in view of its diverse cargo of proteins and rna. in fact, this diverse cargo load provides a distinct advantage over a single molecule drug in enabling a simultaneous targeting of multiple disease processes through a multitude of mechanisms of action, an essential therapeutic strategy for complex injury or disease. interestingly, many of the mechanisms of action are mediated by proteins and rnas that are generally ex‐ pressed in a wide variety of cell types, and that target generic housekeeping processes. these suggest that msc exosomes exert their efficacy by restoring basic housekeep‐ ing activities in the injured cell and its microenvironment to facilitate endogenous repair and regeneration, and this therapeutic strategy resonates well with the role of msc as a stromal support cell. 6 the identification of exosomes as the main agent mediating the therapeutic efficacy of mscs provides a rationale for refining msc-based therapy from a cellular to a noncellular one. although an exosome-based therapy could potentially reduce the complexities 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is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract investigation of the involvement of extracellular vesicles (evs) in parasite biology has burgeoned in recent years. human infecting protozoan parasites, such as trypanosoma cruzi, lesihmania sp. and trichomonas vaginalis, have all demonstrated the utilization of evs as virulence factors in order to activate or hamper host immunity. novel findings have provided evidence that the deployment of evs by plasmodium sp. has a major impact in disease outcomes and serves as an integral part in controlling stage switching in its life cycle. clinical studies have highlighted elevated levels of evs in patients with severe malaria disease and evs have been linked to increased sequestration of infected red blood cells to the endothelium, causing obstruction of blood flow. it has also been found that evs produced during malaria disease activate innate immunity. intriguingly, recent discoveries indicate that plasmodium sp. “highjack” the erythrocyte microvesiculation system in order to cross-communicate. both the transfer of dna and parasite density regulation has been suggested as key mechanisms of evs in malaria biology. keywords extracellular vesicles, malaria, cell communication, inflammation, plasmodium falciparum 1. introduction 'quorum sensing' refers to the regulation of gene expression in an organism as a response to variations in cell density in a population of cells. this phenomenon has been well studied in bacteria where certain species produce and secrete molecules called auto-inducers. the concentration of these molecules increases with increased cell density, while differential gene expression in a cell occurs once a minimal threshold stimulatory concentration has been reached [1]. more recently, it has been found that similar phenomena occur among disease 1johan ankarklev, daisy hjelmqvist and pierre-yves mantel: uncovering the role of erythrocytederived extracellular vesicles in malaria: from immune regulation to cell communication article j circ biomark, 2014, 3:3 | doi: 10.5772/58596 journal of circulating biomarkers causing protozoan parasites such as leishmania sp., which causes leishmaniasis, trypanosoma cruzi, the causative agent of chagas disease, trichomonas vaginalis, which induces vaginitis and plasmodium sp., the malefactor of malaria disease. malaria currently affects upwards of half a billion people each year and mortality rates have been estimated to exceed 600,000. the majority of deaths occur in the sub-saharan regions of africa, where young children, pregnant women and the elderly are at elevated risk of morbidity and mortality. several species of plasmodium can infect humans, namely p. ovale, p. malariae, p. knowlesi, p. vivax and p. falciparum, where the latter accounts for the majority of deaths. malaria disease includes symptoms such as fever and headaches, which in approximately 1% of p. falciparum infections can progress to severe cases and ultimately result in coma and death. the most common form of severe malaria is anaemia, but cerebral malaria, which comprises neurological disorder, is among the deadliest [2]. plasmodium species are obligate intracellular protozoan parasites that belong to the phylum apicomplexa. the complex life cycle of the human infecting plasmodium species entails switching between a mosquito vector and a primate host. in the host, the majority of parasites reproduce asexually; this stage gives rise to the severe symptoms and the mortality of the disease. however, differentiation into gametocytes (the sexual stage) also occurs inside the human host (figure 1). gametocytogenesis is the destiny of a minority of the total parasite population and the mechanisms underlying sexual differentiation are largely unknown. the gametocyte is the sole causative agent of transmission from the human to the mosquito, as this is the life cycle stage that is actively transmitted during a mosquito blood meal. in the mosquito midgut, male and female gametocytes reproduce sexually through meiosis, giving rise to millions of sporozoites ready to infect new human hosts upon subsequent mosquito feedings. several factors are hypothesized for controlling the rate of malarial sexual stage commitment. these include host immunity, chemotherapeutical agents and anaemia [3], however, the exact mechanisms remain largely unknown [4]. it has been proposed that unidentified factors secreted or released by parasites trigger differentiation to gametocytes [5]. recently however, extracellular vesicles (evs) derived from irbcs have been proposed to be a key factor in the regulation of sexual stage commitment. evs are small vesicles derived from intact cells. different types of vesicles have been described and are named according to their origin and size, such as exosomes, microvesicles, apoptotic bodies and oncosomes [6]. exosomes, which are a type of evs, originate from late endosomes and are formed in multi-vesicular bodies (mvb). once the mvb fuse with the cell membrane, the exosomes are released in the extracellular space. although exosomes were first described in reticulocytes in 1987 by johnstone and colleagues, they can be secreted by a large variety of cells, in particular, upon cell activation [7]. there are currently several markers used to identify exosomes, such as alix, tsg101, heat shock proteins and the tetraspanins (cd9, cd63 and cd81) [8,9]. exosomes mediate cell to cell transfer of information [10,11] and regulate the immune response during infections and tumourogenesis [12]. microvesicles (mvs) (syn. ectosomes, microparticles) are secreted after budding or shedding from the plasma membrane [6] and they display similar physiological features as the cells from which they originate. for example, mvs express receptors from the cell of origin on their surface, allowing their identification by antibody labelling in complex biological samples [13,14]. although it is not always possible to distinguish between exosomes and mvs, mvs are generally larger in size (0.1 1 μm), whereas exosomes range from 50-150 nm. in several published works, it is impossible to clearly distinguish between exosomes and mvs; thus, we will use the abbreviation ev as a general term for both types of vesicles. in this review, we discuss the involvement of evs in malaria pathology and disease, as well as the recent discoveries of evs as quorum sensinglike auto-inducers of parasite density regulation, including their suggested functional mechanisms. figure 1. parasite life-cycle and pathogenesis: the blood stage is responsible for the pathology and symptoms observed in malaria infection. within the 48 hours of the life cycle, the parasite develops into the rbc from a ring form to trophozoites and finally schizonts; after rupture of the irbcs, the merozoites invade uninfected rbcs to repeat a new cycle. during the asexual cycle irbc produce evs that interact with immune cells and mediate stage conversion to gametocytes. mature stages of parasites sequester in tissues to avoid clearance by the spleen, triggering local inflammation and activation of the endothelium and accumulation of immune cells, particularly in the brain. vascular cells release evs, contributing to the pathology. 2 j circ biomark, 2014, 3:3 | doi: 10.5772/58596 2. extracellular vesicles are elevated in the plasma of malaria patients evs can be detected in the different body fluids of healthy donors such as urine [15], plasma [16], breast milk [17] and saliva [18]. circulating evs are mostly derived from platelets, but rbcs, blood leukocytes and endothelial cells are also an important source of evs. ev production is elevated in several diseases, such as cancer [19], inflammation [20], cardiovascular disease [21], idiopathic thrombocytopenic purpura [22], thalassaemia [23] [14] in human immunodeficiency virus (hiv) patients [24] and ebola virus-infected macaques [25]. thus, evs are promising biomarkers for the diagnosis of pathologies, as well as in monitoring treatment response in patients. furthermore, evs are important mediators and regulators of physiological responses. they contribute to coagulation, inflammation, vascular homeostasis and the development of tumours and metastases [26]. several studies have highlighted elevated levels of evs during malaria episodes, especially in patients experiencing a severe form of the disease. in a study performed in malawi with children aged six months to 12 years infected with p. falciparum, the plasmatic concentration of evs-derived from endothelial cells was increased at the time of admission in patients with fatal malaria, as measured with the endothelial cellspecific marker cd51 by flow cytometry [27], whereas in uncomplicated cases, the level of evs remained at the same level as in healthy controls. the ev release may be the result of endothelium activation or a direct mechanical result of cytoadherence of irbc to the endothelium [28]. it has been shown that activation of the endothelial cells by tnf-alpha in lupus leads to the production of evs [29]. similarly to lupus, plasma tnf concentration is increased in patients with malaria [30] and tnf can induce the release of procoagulant and proadhesive evs from cultured cells in vitro [29]. therefore, tnf is likely to play a role in local inflammation and the release of endothelial evs. while the endothelium in the capillaries is activated, it interacts with leukocytes, platelets and irbcs; therefore, other cell types are likely to produce evs during malaria. a study performed in douala, cameroon, found that platelet, rbc, endothelial and leukocytic ev levels were elevated and to a lesser extent lymphocyte evs in malaria infected patients with cerebral dysfunctions; however, these had returned to normal at the time of discharge. interestingly, ev levels were found to correlate with a number of clinical and biological parameters. in cm patients, the platelet-ev increase was associated with the depth and duration of the coma, as indicated by the negative correlations between ev levels and coma resolution time (crt). thus, platelet-evs have the potential to be implemented as candidate markers for follow up diagnosis of patients post treatment, a topic of tremendous clinical importance. while the microvascular sequestration primarily involves irbcs, there was no correlation between parasitaemia and rbc-evs cd235a [31]. another study composed of 36 patients suffering from acute p. falciparum, p. vivax or p. malariae infection based in thailand, found that patients with p. falciparum had the highest concentration of rbcderived evs compared to healthy controls. in this study, a specific marker for phospholipid ps (annexin v) and anti-glycophorin a were used for the identification of rbc evs. in vitro, the evs were released mostly by mature stage parasites. infected rbcs release 10-15 times more evs than uninfected. about 40% of the evs were positive for resa, a parasite protein, which is exported by the parasite to the rbc cell membrane [32]. the concentration of rbc-derived evs quickly decreases after treatment with standard courses of artemisinin derivatives. evs are likely to be removed by the spleen, since splenectomised malaria patients have increased levels and prolonged circulation of evs [23]. the splenic reticular-endothelial system removes cells and particles that express ps on their surface. the liver and the lungs can also contribute to the clearance of evs, as has been shown in a murine model [33]. furthermore, it has been found that the concentration of evs was elevated in p. vivax and p. malariae infection, although to a lesser extent than in p. falciparum infection [34]. in a fourth study based in brazil, the evs derived from rbc (cd235a), cd45 (leukocytes), cd41a (platelets), cd144 (endothelial cells) and cd41 (monocytes) [35] were analysed. the study was composed of 37 p. vivax malaria patients between 15-66 years of age, 15 healthy donors and 12 ovarian cancer patients (an ev-inducing disease) [36]. the study concluded that most evs found in plasma during p. vivax infections are derived from platelets, rbcs and leukocytes. platelet-derived evs increased in a linear fashion, with the presence of fever at the time of blood collection [35]. the conclusion of these studies are somewhat varied and it is important to note that the number of patients was limited and the clinical profile, as well as the geographic location, may in part have contributed to the heterogeneity observed in the results. in addition, the method of isolation, storage and measurement of evs were not optimal. yet several laboratories have reported that the minimal size cut-off for measuring ev by flow cytometry is between 200500nm [37]. since evs range from 100 nm-1 um in size, it is likely that the smaller vesicles were not detectable by flow cytometry. additionally, antigen expression might have been too low in certain subsets of the samples, which would have prevented accurate measurements. technological advances are likely to offer better instruments for more accurate measurements in the future. in general, protocols used to prepare samples for ev analysis are relatively facile and based on differential centrifugations [38]. 3johan ankarklev, daisy hjelmqvist and pierre-yves mantel: uncovering the role of erythrocytederived extracellular vesicles in malaria: from immune regulation to cell communication the above mentioned studies have demonstrated a direct correlation between ev levels and disease severity; it was not, however, possible to assess the direct role of evs in malaria pathology based on the above described sets of data and further studies are mandatory to fully elucidate the role of evs in malaria. 2.1 the role of evs in malaria disease autopsy studies of brain tissue from patients who died of cerebral malaria revealed multifocal capillary obstruction by irbcs, platelets and leukocytes [39]. malaria is a complex disease in which the outcome is determined by a combination of several factors. despite intense research, there is still no clear understanding of the mechanisms leading to severe disease or death. however, obstruction of capillaries and a reduction in perfusion of essential organs, as well as leukocyte activation and inflammation, are thought to contribute to the disease [40]. an exaggerated immune response compromises the integrity of the bbb [41] and cm has a high mortality rate of about 20%, despite treatment [42]. infected rbcs sequester in capillaries and cause a considerable obstruction in blood flow, thereby compromising the perfusion of organs. the sequestration is mediated by the interactions of the plasmodial family of receptors, i.e., p. falciparum erythrocyte membrane protein 1 (pfemp1) with host receptors expressed on the host endothelial cells, such as icam1, cd36 and vcam1 [43]. during infection, the endothelium is further activated and as such, the expression of host receptors favouring sequestration of irbcs is increased. only mature parasites sequester in the capillaries, whereas the immature ring stages are found in circulation. adherence protects the parasite from destruction and clearance by the spleen as non-adherent mature stages are rapidly cleared [44]. parasites sequester in various organs including the heart, lung, brain, liver, kidney, subcutaneous tissues and placenta. in addition to the accumulation of irbcs, several histopathological studies have demonstrated excessive accumulation of leukocytes [45] and platelets [46] in the brain of deceased patients. all the vascular cells produce evs, including monocytes [47], rbcs [48], leukocytes [24], platelets and endothelial cells [29]. it is therefore highly likely that evs contribute to the pathology by triggering inflammation and promoting sequestration of irbcs to the endothelium. 2.2 the role of platelets as ev producers in vitro, platelets produce evs when stimulated by calcium ionophores, adp or thrombin/collagen. the platelet-evs can activate coagulation in vitro and participate in thrombus formation in vivo in a model of vessel injury through their surface expression of tissue factor and phosphatidylserine (ps) [49]. depending on the agonist used to stimulate vesiculation, the amount and composition of the evs will vary and therefore their effect on the recipient cells may vary as well, depending on the stimulus; for example, after thrombin stimulation platelets release evs enriched in mirnas [50]. platelets not only contribute to wound healing but also play a role in fighting infections, platelet-deficient or aspirin-treated mice are more susceptible to death when infected with p. chabaudi [51]. platelets have been found to bind to the irbcs and kill the parasites within post release of pf4 [52]. under some circumstances, platelets contribute to inflammation, for example, platelet-evs accumulate in the joint fluid of patients with rheumatoid arthritis. there, the evs are pro-inflammatory and can induce the secretion of il-1 from synovial fibroblasts [53]. furthermore, platelets play an important role in the induction of the clumping of irbcs [54] and in vitro induce the cytoadherence of irbcs to endothelial cells [55,56]. interestingly, platelet-evs bind to irbcs and transfer platelet antigens into the infected cells; binding occurs only to irbcs in a pfemp1 dependent-manner, whereas platelet-evs do not bind to uninfected rbcs. the platelet-evs dramatically increase the binding of irbcs to the endothelial cells [57]. therefore, platelet-evs contribute to sequestration and the avoidance of clearance of irbcs by the spleen. 3. activation of endothelial cells during malaria endothelial cells, when stimulated with tumour necrosis factor (tnf) or lps, are able to produce increased numbers of evs, thereby showing the role played by tnf in endothelial activation and the capability of cells to vesiculate in response to infectious stimuli. interestingly, ex vivo, the endothelial cells from patients with complicated malaria are more responsive to tnf-alpha stimulation and produce an elevated amount of evs [58], suggesting that some people may be genetically more prone to developing severe disease. 3.1 malaria in vivo models and the discovery of evs in severe disease the first direct evidence for a role of evs in the pathogenesis of severe malaria came from the mouse model of cm with p. berghei, using the atp-bindingcassette transporter 1 (abc1) knock-out mice. abc1 is a transporter involved in membrane-lipid turnover and promotes ca2+-induced exposure of ps at the membrane surface, an early step in microvesiculation. the abca1 gene is mutated in tangier disease, a disorder of free cholesterol efflux to high density lipoprotein [59]. hamon et al. discovered that the shedding of rbc evs is reduced by about 70% upon ionophores stimulation in the 4 j circ biomark, 2014, 3:3 | doi: 10.5772/58596 abc1-/mice [60]. the abc1 -/mice were completely resistant to cerebral malaria upon infection with the lethal strain p. berghei anka. all the abc1 deficient mice survived the neurological phase, whereas 90% of the wild-type mice died within the first seven days following infection. the immune response was impaired in the absence of abc1 and the mice had a lower plasma level of tnf-alpha. these mice also had a weaker upregulation of endothelial adhesion molecules in the brain micro-vessels, reduced leukocyte sequestration, as well as an ablated platelet accumulation. moreover, the number of evs was found to be dramatically reduced [61]. in addition to rbc, platelets and macrophages harbour a vesiculation defect upon stimulation by an agonist in vitro. interestingly, the wt and the ko showed comparable levels of evs prior to infection. however, the level of evs increased significantly in the wt at the time of the cerebral syndrome, whereas it remained low in the abca1 ko. after isolation, the evs from wild-type mice were more efficient at inducing clotting and tnf production from macrophages than the evs derived from the abca1 ko [61]. similarly, the administration of the low-molecular-weight thiol pantethine prevented the cerebral syndrome in p. berghei anka-infected mice. the protection was associated with an impairment on the part of the host's response to the infection, in particular, with a decrease of circulating evs and preservation of the bloodbrain barrier integrity without affecting parasite development [62]. 3.2 ev activation of innate immunity the interaction of malaria parasite-derived moieties with cells of the immune system is regarded as the initial step in the induction of the inflammatory response that determines the severity of the disease's condition. several parasite factors are believed to be released during egress and to induce a potent pro-inflammatory response. although the exact mechanisms and nature of these factors remain unknown, several “malaria toxins” have been identified. the plasmodium glycosylphosphatidylinositol (gpi) triggers a potent immune response by macrophages and the vascular endothelium [63,64]. in addition, the parasite digests haemoglobin and the resulting products, the hemozoin crystals, are coated with plasmodial dna that trigger tlr9 [65,66]. the at-rich dna can induce type i ifns in a tlr-independent fashion [67]. additional factors are likely to contribute to the immune modulation; in fact, another study demonstrated that evs, purified from the blood plasma of mice infected by malaria induced potent activation of macrophages in a toll-like receptor dependent manner. immunofluorescence staining revealed that evs contained significant amounts of parasite material, indicating that they were derived primarily from irbcs rather than platelets or endothelial cells [68]. furthermore, evs more potently induced macrophage activation than intact irbcs. macrophage activation by evs was mediated by a tlr-4and myd88dependent pathway [68]. a role for irbc-evs was further confirmed with the isolation of evs from in vitro cultured p. falciparum irbcs. these studies directly demonstrated that evs originate from irbcs and infection of the irbcs with p. falciparum increases the secretion of evs from irbcs by 15 fold when compared with uninfected rbcs [69,34]. proteomic analyses further demonstrated that evs contain malaria specific proteins; most of the plasmodial proteins are known to be exported and derived mainly from the maurer’s cleft, which are small organelles connected by actin filaments to the rbc membrane [70,69]. interestingly, small vesicles were found by electron tomography to be bound to the actin filaments. the maurer’s clefts serve as a platform for the export of parasite proteins to the rbc cell surface [71]. interestingly, evs interact with the innate immune system and are rapidly phagocytosed by macrophages, inducing their activation and secretion of tnf-alpha [69]. the tnf production due to macrophage activation following infection is a major source of tnf in vivo [72]. there is a positive correlation between tnf level and evs found in the plasma of patients with malaria [73]. in conclusion, these data indicate that irbc evs are highly likely to contribute to the inflammation observed in severe malaria and might therefore be a novel target for the development of therapies to prevent severe disease (figure 1). 4. rbc-derived evs in cell-to-cell communication in addition to the contribution of evs from different cell sources to malaria pathology, recent evidence indicates that evs derived from p. falciparum irbcs can regulate parasite density and mediate horizontal transfer of nucleic acids to regulate the rate of conversion into gametocytes, i.e., the transmission stage. to determine whether irbcs communicate and transfer information, regev-rudzki and colleagues used two strains expressing different drug resistance cassettes, which were linked to different fluorescent markers expressing red or green fluorescence. when the two transgenic strains (one containing a blasticidin (bsd) resistance marker and the other a wr resistance marker) were co-cultured in vitro, the parasites initially died. however, five days post initial drug treatment, recrudescence occurred and the surviving parasites contained both the green and red fluorescent markers, demonstrating the transfer of plasmids between the two transgenic strains. using a trans-well membrane system it was subsequently shown that the transfer of drug resistance is not dependent on cell to cell contact, but instead depends on soluble factors from irbcs smaller than 400 nm [74]. the transfer is inhibited by cytochalasin d, an inhibitor of actin polymerization and oryzalin [75]. finally, the transfer of 5johan ankarklev, daisy hjelmqvist and pierre-yves mantel: uncovering the role of erythrocytederived extracellular vesicles in malaria: from immune regulation to cell communication dna was demonstrated by using in vitro purified evs. vesicles could be observed budding at the surface of the irbcs by atomic force microscopy (afm) [74]. quantification of vesicles by nanosight in supernatants of synchronized parasite culture indicated that most evs were released during the late stages and the size of the vesicles varied from 50-250 nm [69]. the vesicular shape, size and lipid bilayer were further confirmed by electron microscopy and cryo-electron microscopy. fluorescent-labelled evs were internalized by irbcs and targeted towards the nucleus, and accordingly, the transferred plasmid dna was localized in the nuclear periphery, thereby suggesting that ev cargo might target gene regulation in the recipient cell. furthermore, in vitro analyses have shown that irbc evs induce sexual stage conversion in cultured parasites in a dose dependent manner. highly elevated levels of gametocytes were found in cultures where evs had been introduced [69,74]. the maurer’s clefts are important for the production of evs and the ptp2 knock-out parasites produce less evs as measured by afm. ptp2 is expressed on budding vesicles from the maurer’s clefts and is essential for pfemp1 export to the host cell membrane [76]. interestingly, the ptp2 ko parasites were also defective in the uptake of mvs, since transfer of the bsd gene was not possible using this strain. the localization of ptp2 on vesicles budding from the maurer’s clefts suggests that evs may rather be derived from the maurer’s clefts rather than from the host cell membrane, illuminating the possibility that different kinds of vesicles with diverse functions are released by irbcs (figure 2). figure 2. p falciparum infected rbcs (donor cells) secrete evs that are subsequently taken up by another irbc (recipient cell). evs act as a quorum sensing-like mechanism by inducing differentiation towards gametocytes. the stimuli that trigger ev release from the donor cell, as well as the mechanisms of uptake by the recipient cells are still unknown.. however, the maurer’s clefts seem to play a role particularly in the protein ptp2. the molecular mechanisms of differentiation are unknown; however, evs contain several factors such as dna, mrna, mirna, proteins and lipids that can potentially be involved. 5. quorum sensing-like mechanisms in plasmodium infections since irbc-derived evs are able to transfer nucleic acids from parasite to parasite and to induce differentiation to gametocytes, they have been proposed to exhibit quorum sensing-like mechanisms. previous investigations into bacteria have shown that prokaryotes use signalling molecules to control traits such as antibiotic production, nutrient storage, sporulation, biofilm formation and virulence factor secretion. this phenomenon, which was initially discovered in bacteria, has been termed 'quorum sensing' and in general refers to activities using signalling molecules to synchronize activities among large populations of cells. some basic principles can be distinguished in qs according to rutherford et al.; in the first stage, the bacteria produce the signalling quorum-sensing molecules. while at low cell density, the molecules diffuse without reaching the threshold required for detection. as the cell density increases, the cumulative production of molecules leads to a local high concentration sufficient for reaching the threshold necessary for detection and response. finally, after detection by specific receptors in the cells and activation of the expression of genes necessary for the cooperative behaviours, the qs molecules induce the release of more qs molecules. this positive feedback mechanism of induction further increases bacterial synchronicity [77]. the success of infection depends on the ability of the parasite to survive in its mammalian host and to ensure transmission to future hosts. overgrowth can damage the host and thereby limit the parasites transmission potential [78]. the quorum-sensing mechanism has been observed in vector-borne parasites such as trypanosoma brucei, which is the causative agent of african sleeping sickness. the parasites differentiate from replicating slender bloodstream forms to non-dividing stumpy forms, thereby limiting the parasite's population size and allowing survival of the mammalian host and establishment of a stable host-parasite relationship [79]. in the protozoan parasite t. brucei, a density-sensing mechanism activates the differentiation of proliferative slender cells to stumpy forms through the release of stumpy induction factor (sti) [80,81,82]. it would be a major advantage for parasites such as p. falciparum to communicate during blood-stage infection in order to enable populations to react to changing conditions in the host and to regulate cell density [83]. in conclusion, evs constitute a potential target for the control and management of severe p. falciparum infections; it also poses as a target for novel transmission blocking strategies of this important human pathogen. it is a major advantage for p. falciparum to communicate during the blood-stage infection in order to enable parasites at the population level to react to changing conditions in the host. 6 j circ biomark, 2014, 3:3 | doi: 10.5772/58596 6. the involvement of evs in gametocyte induction although evs can serve as vehicles for transferring plasmid dna from parasite to parasite, the real factor responsible for cell-cell communication and the induction of gametocytes is yet to be identified. several studies in other systems have pointed out a role for lipids, proteins, dna and rna in transfer of information mediated by evs. for example, evs may contain different forms of dna, genomic dna fragments [84] [85], mtdna [86] ssdna and cdna retrotransposon [87]. it has been shown that gdna can be transported by evs and increase the gdna-coding mrna and protein expression in the recipient cells [84]. for example, the bcr/abl hybrid gene was transferred from k562 evs to normal neutrophils and localized close to the nucleus, as demonstrated by dna-fish, thereby suggesting that tumours can transfer genetic mutations from cell to cell as a new avenue for expanding tumours [84]. evs can mediate the transfer of functional transmembrane proteins and proteases from the donor to the target cell. in the context of a tumour, the glioblastoma cells can share the oncogenic receptor egfrviii through evs, thereby favouring tumour growth [88] [89] by providing a functional receptor on the target cells. the ev-derived from a tumour can transfer the oncogenic receptor tyrosine kinase met to bone marrowderived cells to promote their education, mobilization and pro-invasive behaviour [90]. active wnt is secreted in exosomes during drosophila development and in human cells [91] also wnt11 secreted in fibroblast evs drive breast cancer cell invasive behaviour [92]. some pathogens use evs secretion to their own advantage to favour their growth and evade the immune system. gp63, a zn-metallo-protease secreted in evs by leishmania donovani, targets pre-mirna processor dicer1 to prevent mirnp formation in l. donovani interacting hepatic cells, thereby shutting down the lipid metabolism and promoting parasite growth [93]. the ccr5 chemokine receptors are transported on evs and transfer of ccr5+ evs by pbmcs to ccr5pbmcs render the ccr5cells susceptible to hiv infection [94]. additionally, evs contain mrna and mirna that can be transferred to recipient cells and regulate gene transcription in the recipient cell [10]. the rna profile of evs is different than the rna profile of the source cells and evs seem to lack ribosomal rna, while being enriched in small rna [95]. the role of mirna has been studied in a different context. the evs can transfer mirna between t cells and dendritic cells (dc) at the immunological synapse [96]. dcs release evs that contains mirna. upon maturation of the dcs, the mirna profile varies [97], regulating the transcription of genes in the target dcs; therefore, immature dc-evs have a different function than mature dc-evs. epstein barr viruses infect b cells and functional mature ebvencoded mirnas are secreted in evs by ebv-infected b cells and mediate repression of cxcl11/itac, an immunoregulatory gene down-regulated in primary ebv-associated lymphomas in recipient immature monocyte-derived dendritic cells [98]. platelets have been shown to contain an abundant and diverse array of micrornas and platelet-derived mvs are the most abundant evs in the blood circulation. upon activation, platelets release evs containing functional ago2:mir223 complexes [99], which can induce apoptosis after internalization by recipient cells (huvec) by down-regulating the expression of the insulin-like growth factor 1 receptor [50]. evs secreted by myotubes play a role in myogenesis by transferring mirna and down-regulating sirtuin1 in myoblasts [100]. interestingly the proteomics analysis of irbc-evs revealed the presence of human argonaute 2 as a component of irbc evs. furthermore, rna can be isolated from evs and is mainly composed of small rna, as shown by bioanalyzer analyses, thus raising the possibility that evs contain functional risc complexes that could be transferred to the recipient cell in order to regulate gene transcription. interestingly, it has recently been shown that rbc mirna can regulate that gametocytaemia. la monte and colleagues provided evidence for the presence of mirna in hbss and hbsa in patients with sickle cell anaemia, regulates gametocyte differentiation [101]. intriguingly, at a similar level as that seen by mantel et al., where evs from p falciparum irbcs were shown to have an important role in parasite density regulation [69]. it has been shown that evs from irbcs contain small rnas, suggesting a similar function as that seen in patients with sickle cell traits. the presence of functional mirnas in evs has also been found in other systems [98,99]. although plasmodium is not thought to possess the machinery for rnai pathway [102], it has been demonstrated that host mirnas regulate gametocytaemia. a strong link between the transcription factor apapi2 and sexual stage conversion has been indicated [103]. it would be highly interesting to try and identify the links between evs and epigenetic factors that influence gametocytogenesis. investigating the content of evs in terms of lipids, dna, rna and proteins will likely reveal the molecular pathways involved in the quorum sensing-like mechanisms in parasite communication and density regulation. 7. conclusion with escalating evidence that the role of evs are highly influential in malaria biology, it has become clear that investigations concerning synthesis, mechanisms of 7johan ankarklev, daisy hjelmqvist and pierre-yves mantel: uncovering the role of erythrocytederived extracellular vesicles in malaria: from immune regulation to cell communication uptake, content, as well as their direct role in transmission are highly important as part of an incentive for understanding malaria biology. the recently discovered link between evs and malaria sexual stage switching holds promising potential for solving the longstanding problem of hindering malaria transmission and ultimately eradicating malaria disease. 8. acknowledgements johan ankarklev is sponsored by a grant from the swedish research council (vr). 9. compliance with ethical research standards the authors declare no conflict of interest. 10. references [1] miller mb, bassler bl (2001) quorum sensing in bacteria. annu rev microbiol 55: 165-199. 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[103] campbell tl, de silva ek, olszewski kl, elemento o, llinas m (2010) identification and genome-wide prediction of dna binding specificities for the apiap2 family of regulators from the malaria parasite. plos pathog 6: e1001165. 11johan ankarklev, daisy hjelmqvist and pierre-yves mantel: uncovering the role of erythrocytederived extracellular vesicles in malaria: from immune regulation to cell communication exosomes and microvesicles ovarian cancer-derived exosomal fibronectin induces pro-inflammatory il-1 original research article safinur atay1, carolyn d. roberson2, cicek gercel-taylor3 and douglas d. taylor3,*   1 department of pathology & laboratory medicine, university of kansas medical center, kansas city, ks, usa 2 department of microbiology & immunology, university of louisville school of medicine, louisville, ky, usa 3 department of obstetrics, gynecology & women’s health, university of louisville school of medicine, louisville, ky, usa * corresponding author e-mail: ddtaylor@louisville.edu   accepted 19 feb 2013 © 2013 atay et al.; licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract  the  tumour  microenvironment  is  characterized by pro‐inflammatory profiles,  including  interleukin‐1  (il‐1β).  this  profile  is  generated  by  infiltrating  macrophages  following  interactions  with  tumours  or  their  components.  the  objectives  of  this  study  were  to  identify  whether  tumour  exosomes  could  induce  macrophage  il‐1β  production  and  the  mechanism  involved.  exosomes  were  isolated  from  ovarian  cancer  patients  and  ovarian  tumour  cells  by  chromatography  and  ultracentrifugation.  specific  exosomal proteins were defined by mass spectrometry  (ms)  and  confirmed  by  western  immunoblotting.  using macrophage‐like thp‐1 cells, induction of il‐1β  release was investigated by elisa. rgd peptides were  used  to  block  fibronectin  binding  by  thp‐1  51  integrin.  exosomes  isolated  from  ovarian  cancer  patients  and  from  ovarian  cancer  cells  were  demonstrated, by ms and immunoblotting, to express  fibronectin.  incubation  of  thp‐1  cells  with  these  exosomes  induced  pro‐inflammatory  cytokines,  in  particular  il‐1β.  blocking  of  thp‐1  binding  of  exosomal  fibronectin  with  rgd  peptides  abrogated  exosome‐mediated il‐1β production and down‐stream  phosphorylation  of  akt  and  c‐jun.  although  cancer  patients generally exhibit increased levels of il‐1β, the  underlying  mechanism  is  unclear.  here,  tumour‐ derived  exosomes  are  demonstrated  to  induce  pro‐ inflammatory  cytokine  in  macrophages  including  il‐ 1, whose induction is mediated by fibronectin.    keywords exosomes, fibronectin, il‐1, macrophages                                            1. introduction    macrophages have been  implicated  in phagocytosis and  tissue  remodelling  and  production  of  cytokines  and  chemokines  [1,  2].  unlike  many  cells  of  the  innate  immune  system,  macrophages  exhibit  a  high  degree  of  plasticity and can differentiate depending on the signals  in  their  environment  [3].  based  on  their  receptors  and  cytokine production, macrophages can be defined as m1  or m2 macrophages. pro‐inflammatory m1, or classically  activated,  macrophages  exhibit  up‐regulation  of  pro‐ inflammatory cytokines and chemokines, including tnf‐ α, il‐12, il‐6, ccl2 and il‐1β,  in addition to  increased  production  of  reactive  oxygen  species  and  nitrogen  intermediates  [4].  in  contrast,  anti‐inflammatory  m2,  or  alternatively  activated,  macrophages  are  polarized  by  stimuli  such  as  il‐4,  il‐13,  il‐10  or  glucocorticoid  1safinur atay, carolyn d. roberson, cicek gercel-taylor and douglas d. taylor: ovarian cancer-derived exosomal fibronectin induces pro-inflammatory il-1ß www.intechopen.com article www.intechopen.com exosomes microvesicles, vol. 1, 2:2013 hormones  [4]  and  up‐regulate  scavenger,  mannose  and  galactose  receptors.  they  are  il‐1  receptor  antagonists  and  downregulate  il‐1β  and  other  pro‐inflammatory  cytokines [5].     the malignant behaviours of tumour cells are defined by  the microenvironment, where macrophages represent the  major  population  of  immunologic  cells  inltrating  the  tumours. these  infiltrating macrophages are referred  to  as  tumour‐associated  macrophages  (tams).  while  initially it was believed that these macrophages exhibited  anti‐tumour  activity,  the  inltration  of  tam  into  tumours,  such  as  breast,  ovarian,  prostate  and  cervical  cancers,  has  been  correlated  with  poor  prognosis  and  short survival [6]. this adverse function of macrophages  has  been  attributed  to  their  immunosuppressive  nature  [7].  recent  studies  have  demonstrated  that  tams  promote  tumour  cell  proliferation,  invasion  and  metastasis, as well as angiogenesis, which is essential for  tumour growth [8]. tams secrete a variety of cytokines,  growth  factors  and  enzymes,  which  promote  tumour  metastasis and angiogenesis, such as vascular endothelial  growth  factor,  il‐8,  epidermal  growth  factor,  platelet‐ derived  growth  factor,  tumour  necrosis  factor  (tnf)‐α  and matrix metalloproteinases [9, 10].    cytokines  coordinate  the  immune  response  and  are  released  in  response  to  infection,  injury,  stress  or  trauma.  cytokines  regulate  differentiation  and  activation  of  various  immune  cells  and  coordinate  inflammatory reactions, such as the induction of acute‐ phase  proteins.  the  hyper‐activation  of  pro‐ inflammatory cytokines and/or the impairment of anti‐ inflammatory  cytokines  can  lead  to  pathological  conditions,  such  as  allergic  reactions,  sepsis  and  dysplasia.  il‐1,  tnf‐α  and  il‐17  are  prominent  pro‐ inflammatory  cytokines,  whereas  il‐10  and  tgf‐β  are  characterized by anti‐inflammatory properties. the pro‐ inflammatory phase is characterized by the production  of pro‐inflammatory cytokines, including interleukin 1  (il‐1)  [11].  il‐1  is  a  potent  multifunctional  pro‐ inflammatory  cytokine  produced  by  monocytes  and  tissue  macrophages.  il‐1  promotes  multiple  events,  such  as  attraction  and  activation  of  immune  cells,  stimulation  of  endothelial  cell  adhesion  molecule  expression,  and  enhanced  expression  of  extracellular  matrices and matrix‐degrading enzymes.     our  previous  work  has  demonstrated  that  exosomes  derived  from  a  first  trimester  extravillous  trophoblast  were  capable  of  inducing  the  migration  of  monocytes  and  “educating”  these  cells  to  produce  a  pro‐ inflammatory cytokine profile, including il‐1 [12]. il‐ 1  induction  by  exosomes  was  mediated  by  a  ligand‐ receptor  interaction  and  was  shown  not  to  require  exosome  uptake.  other  studies  have  shown  that  interactions  between  components  of  the  extracellular  matrix and macrophages lead to the production in pro‐ inflammatory cytokines [13]. proteins shown to  induce  pro‐inflammatory  cytokines  by  macrophages  include  thy‐1,  vitronectin,  integrins,  thrombospondin,  myosin  and  fibronectin.  fibronectin  is  present  in  the  extracellular matrix of  most  tissues,  where  it  supports  basic  cellular  processes  including  cell  adhesion,  migration,  survival  and  growth.  fibronectin  is  a  modular  protein  composed  of  independently  folded  domains (types i, ii and iii), which represent regions of  amino  acid  sequence  homology  [14].  within  the  extracellular matrix, fibronectin is polymerized and can  be  subjected  to  tensional  forces  generated  by  the  cells  that  expose  cryptic  activities  within  the  molecule,  including binding sites for integrins and growth factors  [15].  in  general,  fibronectins  are  a  class  of  large  glycoproteins  with  known  adhesive  activity  for  collagen,  fibrin,  heparin  and  cell  surfaces  and  are  associated with wound healing, platelet aggregation and  clotting, and cancer metastasis [16]. a specific function  in cancer biology has not been defined for fibronectin,  due  primarily  to  its  ubiquitous  expression  in  adult  human tissues and plasma. matsuura et al. identified a  group  of  fibronectin  isoforms,  containing an  o‐linked,  n‐acetyl‐galactosaminylated  hexapeptide  within  the  alternatively  spliced  type  iii  connecting  segment  [17]. these  unique  fibronectin  iosforms  have  been  identified  within  several  human  tumours  and  developmental tissues. these isoforms are designated as  oncofetal fibronectin and they appear to be distinct from  plasma and cellular fibronectins based on molecular size  and glycosylation patterns [18].     in the present study, we demonstrate the induction of il‐ 1  by  cancer‐derived  exosomes  and  investigate  the  mechanism  underlying  this  induction.  the  study  addressed the expression of exosomal components linked  with  the  induction  of  pro‐inflammatory  cytokines  by  macrophages. based  on ms  identification of  fibronectin  on tumour‐derived exosomes, the study investigated the  blockage  of  the  exosome‐mediated  events  by  rgd  analogues and the molecular mechanism linked with the  exosome‐associated  component(s)  responsible  for  the  induction of il‐1.    2. materials and methods     2.1 cell culture conditions    the ul‐o cell line was established from the ascites of a  patient with stage iii papillary serous adenocarcinoma of  the  ovary.  ul‐o  cells  were  maintained  in  75cm2  tissue  culture  flasks  in  20ml  hyclone  roswell  park  memorial  institute  (rpmi)  1640  culture  media  (thermoscientific)  supplemented  with  2mm  l‐glutamine,  10%  exosome‐ 2 exosomes microvesicles, vol. 1, 2:2013 www.intechopen.com depleted  foetal  bovine  serum  (fbs),  1mm  sodium  pyruvate,  0.1mm  nonessential  amino  acids  and  100units/ml  penicillin‐streptomycin  in  a  humidified  incubator at 37c with 5% co2. fbs‐associated exosomes  were  removed  by  ultracentrifugation  for  15  hours  at  100,000xg. the resulting supernatant was sterile  filtered  using a 0.22μm filter. thp‐1 acute monocytic leukaemia  cells  were  obtained  from  atcc  (rockville,  md,  usa)  and were maintained  in rpmi 1640 supplemented with  2mm  l‐glutamine,  10%  fbs,  1mm  sodium  pyruvate,  0.1mm  non‐essential  amino  acids,  0.05mm  2‐ mercaptoethanol and 100 units/ml penicillin‐streptomycin  in a humidified incubator at 37c with 5% co2.      2.2 exosome isolation from culture media and patient ascites    to  isolate  tumour  exosomes  from  culture  media,  ul‐o  cells were utilized. ul‐o cells were cultured for three to  four days (approximately 85% confluent and greater than  95%  viability  based  on  trypan  blue  exclusion).  the  conditioned  medium  was  centrifuged  at  400xg  for  ten  minutes to remove whole cells and this supernatant was  then  centrifuged  at  15,000xg  for  20  minutes  to  remove  large microvesicles and apoptotic vesicles. the resulting  cell  free  medium  was  concentrated  by  ultrafiltration  using an amicon stirred cell model 8200 with a molecular  weight cut‐off membrane of 500,000 daltons  (millipore,  billerica,  ma).  this  concentrated  material  was  then  chromatographically  separated  using  a  sepharose  2b  column  (2.5x30cm).  the  void  volume  fractions  were  pooled and centrifuged using a ti90 rotor (beckman) at  100,000  x  g  for  one  hour  at  4°c.  the  pellet  was  resuspended  in  pbs  and  the  vesicular  protein  concentration  determined  using  the  dc  protein  assay  (bio‐rad  laboratories,  hercules,  ca)  according  to  manufacturer’s  instructions.  this  vesicle  preparation  from ul‐o cells was termed oex.    to isolate exosomes from patient ascites, the ascites fluids  were centrifuged at 400xg for ten minutes to remove cells  and  the supernatant was centrifuged at 15,000xg  for 20  minutes to remove cell debris and apoptotic vesicles. the  resulting supernatant was concentrated by ultrafiltration  using an amicon stirred cell with a molecular weight cut‐ off  membrane  of  500,000  daltons  (millipore).  this  concentrated  material  was  then  chromatographically  separated using a sepharose 2b column (2.5x30cm). the  void  volume  fractions  were  pooled  and  centrifuged  at  100,000  x  g  for  one  hour  at  4°c.  the  pellet  was  resuspended  in  pbs  and  the  protein  concentration  determined using  the dc protein assay. to exclude  the  possibility  of  endotoxin  contamination  in  the  exosome  preparations  for  both  the  culture  and  ascites‐derived  vesicles,  a  lal  assay  (genscript,  piscataway,  nj)  was  performed  to  quantify  any  endotoxin  in  the  vesicle  preparations. for the exosome preparations used in these  studies,  endotoxin  concentrations  were  <0.2eu/ml,  including insufficient levels of endotoxin for the observed  activation effects. additionally, oexs were demonstrated  to  be  negative  for  il‐1  mrna  using  specific  primers  (sabiosciences)  and  protein  using  a  commercially  available il1‐β elisa kit (e‐biosciences, san diego, ca,  usa).    2.3 sds‐page and western immunoblot analysis of cell  culture lysate and culture and ascites‐derived exosomes    vesicle‐derived  proteins  or  proteins  from  total  cellular  lysates  (30μg  each)  were  resuspended  in  a  laemmli  sample  buffer.  this  suspension  was  boiled  and  loaded  onto  10%  sds‐page  gels.  after  electrophoretic  separation,  the  gels  were  stained  with  imperial  purple  protein stain (pierce chemical co, rockland, il) at room  temperature  for  two  hours  and  destained  overnight  in  double  distilled  water.  the protein  bands were  imaged  using  a  pharosfx  molecular  imager  and  quantity  one  software (biorad laboratories, hercules, ca).     for  western  immunoblot  analysis  [19],  10%  sds‐page  gels were electrophoretically transferred to nitrocellulose  membranes (biorad laboratories) for one hour, followed  by  blocking  with  5%  non‐fat  milk  in  tbs  with  0.05%  tween  20  (tbst)  for  one  hour.  membranes  were  incubated  overnight  at  4c  with  the  following  primary  antibodies:  anti‐fibronectin  (fn‐clone  tv‐1),  anti‐cd63  (clone  y‐18)  and  anti‐‐actin  (clone  c‐4)  (santa  cruz  biotechnology,  santa  cruz,  ca).  after  the  overnight  incubation,  membranes  were  washed  three  times  with  1xtbst and incubated for one hour with hrp‐conjugated  anti‐mouse  or  anti‐rabbit  igg  (biorad  laboratories)  at  room  temperature.  blots  were  developed  using  an  immun‐star  hrp  substrate  developing  kit  (biorad  laboratories)  and  exposing  x‐ray  film.  two  separate  western  blots  were  performed  for  each  target.  densitometric quantification of the resulting films for il‐ 1, caspase 1 and ‐actin was performed using un‐scan‐it  software (silk scientific, orem, ut).     2.4 dynamic light scattering analysis    exosome preparations were diluted to ~50μg/ml to yield  an  optimum  scattering  intensity  for  dynamic  light  scattering  measurements,  which  were  performed  on  a  malvern 4700 autosizer  (malvern  instruments ltd., uk)  employing  a  20mw  helium/neon  laser  (633nm),  at  a  constant  temperature of 25c. light scattering  from  the  sample was detected by a photomultiplier tube place at  90 to the incident laser beam. the translational diffusion  coefficient of the solutions was calculated from the time  autocorrelation  of  the  scattered  light  intensity  and  extracted  from  the  correlogram  using  the  method  of  cumulants applied  in the proprietary malvern software.  3safinur atay, carolyn d. roberson, cicek gercel-taylor and douglas d. taylor: ovarian cancer-derived exosomal fibronectin induces pro-inflammatory il-1ß www.intechopen.com the  diameter  of  the  exosomes  were  obtained  from  the  application of the stokes‐einstein equations [20].     2.5 mass spectrometry and proteomic analysis of   ascites‐derived exosomes    specific  protein  bands  from  sds‐page  gels  of  ascites‐ derived exosomal proteins were reduced, alkylated and  trypsinized  at  37c  overnight  with  shaking.  briefly,  tryptic  peptides  were  loaded  onto  a  1d  capillary  chromatography column comprised of a needle tip (100 x  365μm  fused  silica  capillary  with  an  integrated,  laser  pulled  emitter  tip)  packed  with  10cm  of  jupiter  5μm  rp300a  (phenomenex,  torrence,  ca).  peptides  were  eluted,  ionized and sprayed  into  the mass spectrometer  using a 120 minute gradient from 7% acetonitrile to 80%  acetronitrile  (plus  0.1%  formic  acid)  at  a  flow  rate  of  200nl/min. spectra were acquired with a ltq orbitrap  xl  linear  ion  trap  mass  spectrometer  (thermo  fisher  scientific,  waltham,  ma).  during  lc‐ms/ms  analysis,  the  mass  spectrometer  performed  data‐dependent  acquisition with a full ms scan between 300 and 2000m/z  followed by ms/ms scans (35% collision energy) on the  six most intense ions from the preceding ms scan. data  acquisition was performed using dynamic exclusion with  a  repeat  count  of  a  one  and  three  minute  exclusion  duration window.     the  acquired  mass  spectrometry  data  were  searched  against  a  translated  human  genome  database  (human‐ refseqxps) using the sequest algorithm assuming fixed  modification  of  cysteine  (+57  for  carabmidomethylation)  and variable oxidation of methionine (+16 to methionine).  database  analysis  was  performed  with  sequestsorcerer  (sage‐n research, san jose, ca). high‐probability peptide  and  protein  identifications  were  assigned  from  the  sequest  results  using  the  proteinprophet  (http://tools.proteomecenter.org/software.php)  and  sagen  sorcerer  statistical  platforms.  these  data  were  loaded  onto  scaffold  3  proteomic  analysis  software  to  qualitatively  and  quantitatively  compare  individual  lcms  analyses  and  provide  approaches  to  functional  annotation of data.    2.6 cytokine antibody array    to  define  the  effects  of  tumour‐derived  exosomes  on  macrophage  chemokine  and  cytokine  profiles,  their  production by thp‐1 cells was quantified with duplicate  arrays,  each  having  duplicate  spots  for  each  cytokine  using  proteome  profiler™  human  cytokine  antibody  array panel a arrays (r&d systems, minneapolis, mn)  according  to  the  manufacturerʹs  instructions.  thp‐1  monocytic cells were  incubated with 100μg/ml  tumour‐ derived  exosomes  or  were  untreated  for  20  hours.  the  cytokine  array  membranes  were  incubated  with  1ml  of  conditioned  media  from  each  sample,  diluted  1:3  and  15μl of cytokine array panel a detection antibody at 4°c  overnight. the membranes were then washed three times  with  20ml  of  1×  wash  buffer  and  incubated  with  horseradish  peroxidase‐conjugated  streptavidin  (1:2000‐ dilution). after 30 minutes, the membranes were washed  thoroughly  and  exposed  to  a  chemiluminescent  peroxidase substrate for five minutes in the dark before  imaging.  membranes  were  exposed  to  x‐ray  film  (research products international, mt prospect, il). as per  the  manufacturer’s  package  insert,  the  cytokine  array  data  on  developed  x‐ray  film  was  quantitated  by  scanning  the  film  on  a  transmission‐mode  scanner  and  analysing  the  array  image  file  using  image  analysis  software,  un‐scan‐it  gel  digitizing  software  version  6.1  (silk scientific corporation, orem, ut). positive controls  at three spots were used to identify membrane orientation  and  to normalize  the results from different membranes.  for each spot, the specific pixel level was determined by  subtracting  the  background  pixels  from  the  total  raw  pixel  levels.  to  quantify  relative  change  in  cytokine  levels  between  samples,  the  average  background‐ subtracted  mean  spot  pixel  densities  of  the  pair  of  duplicate  spots  representing  each  cytokine  was  determined  for  each  condition.  to  facilitate  further  analyses,  all  spots  in  the  arrays  were  quantified  and  their  specific  intensity  values  were  obtained  by  subtracting  the  background  intensity.  only  differences  in  cytokine  levels  that  were    two‐fold  compared  to  controls were considered significant.    2.7 elisa determination of il‐1 release    for  elisa  determination  of  il‐1  human  thp‐1,  cells  (1x106  cells/well)  were  incubated  with  exosomes  at  100μg/ml for six hours. for the elisa analysis, 1mm atp  was added for 30 minutes to induce mature il‐1 release.  culture supernatants for the treated human thp‐1 cells  were harvested and the il‐1 levels were measured using  il‐1 elisa kit (ebiosciences, san diego, ca) according  to the manufacturer’s instructions.    2.8 co‐culture of thp‐1 monocytes with integrin   function inhibitory peptides    to assess il‐1 production, thp‐1 cells (5x106 cells/well)  were  cultured  in  rpmi  1640  supplemented  media  (as  previously described) alone, co‐cultured with 100μg/ml  ul‐o  culture‐derived  exosomes    (oex),  or  co‐cultured  with  100μg/ml  ul‐o  culture‐derived  exosomes  (oex)  plus  peptide  mimics  of  fibronectin  binding  (sigma‐ aldrich,  st.  louis,  mo)  at  0.5mg/ml  for  30  minutes.  peptide mimics include: a negative control peptide (gly‐ arg‐ala‐asp‐ser‐pro‐lys)  that  does  not  block  integrin  binding (designated as cp), an antagonist peptide (gly‐ arg‐gly‐asp‐ser)  of  integrin  function  (designated  as  4 exosomes microvesicles, vol. 1, 2:2013 www.intechopen.com ant) and an  inhibitory peptide  (gly‐arg‐gly‐asp‐thr‐ pro)  of  fibronectin,  type  1  collagen  and  vitronectin  (designated  bfcv)  [15].  cultures  were  incubated  with  oex for six hours with or without the addition of 1mm  atp for 30 minutes at 37c. post incubation, supernatants  of  cultures  were  harvested  and  subjected  to  elisa  analysis  using  a  commercially  available  il‐1  kit  (ebiosciences, san diego, ca) to determine the amount of  il‐1 released.    2.9 kinetics of thp‐1, oex and integrin function   inhibitory peptide co‐cultures     thp‐1 cells were unstimulated, stimulated with oex, or  stimulated  with  oex  +  peptide  mimics  of  fibronectin  binding  (as previously described) for 0, 30, 60, 120, or 240  minutes.  total  intracellular  protein  extracts  were  prepared by adding 100μl of ripa buffer plus protease  and  phosphatase  inhibitors/106  cells  (thermoscientific).  the  extracts  were  incubated  on  ice  for  30  minutes  and  centrifuged  at  12,000rpm  for  ten  minutes.  protein  concentration  was  determined  using  the  dc  protein  assay (biorad laboratories). cellular protein (30μg) was  added  to  laemmli  reducing  buffer  and  boiled  for  five  minutes.  samples  were  electrophoresed  at  100v  for  1.5  hours  and  the  resulting  gel  was  transferred  to  nitrocellulose  membranes  (biorad  laboratories)  at  98v  for one hour on  ice. membranes were blocked with 5%  non‐fat dry milk in 1xtbs + 0.05% tween 20 (1xtbst) for  one hour and incubated on a rocker overnight at 4c with  anti  p‐akt2  (clone  b5),  anti  pser73  c‐jun  and  anti‐‐actin  (clone  c‐4).  all  antibodies  are  from  santa  cruz  biotechnology. after an overnight incubation, membranes  were  washed  three  times  with  1xtbst  and  incubated  at  room temperature for one hour with hrp‐conjugated anti‐ mouse  or  anti‐rabbit  igg  (biorad  laboratories).  subsequently, membranes were developed using immun‐ star hrp chemiluminescent kit (biorad laboratories) and  visualized  on  biomax  mr  film  (kodak).  western  blot  images  for  p‐akt2,  pser73  cjun  and  ‐actin  were  digitized  and relative change in expression of protein in comparison  to  the  control  was  quantified.  statistical  analyses  were  performed using the t‐test and calculated using graph pad  prism‐5.01 graphing and statistical software. a p‐value of  <0.05 was considered statistically significant.    2.10 statistical analysis    comparisons  between  exosome‐treated  and  untreated  cells  were  performed  using  a  t‐test  or  one‐way  anova  with  bonferroni’s  multiple  comparison  test,  calculated  using  graphpad  prism‐5  graphing  and  statistical  software. a p value of <0.05 was considered statistically  significant.      3. results     3.1 characterization of vesicles derived from   ascites and cell culture    assessment of vesicles derived from both in vivo and in  vitro  by  sds‐page  (figure  1a)  revealed  similar  composition  of  the  major  protein  components.  these  protein  bands  ranged  from  250kd  to  20kd.  the  electrophoretic protein pattern for vesicles isolated from  patient ul‐l and from culture media of ul‐o cells were  virtually identical. vesicles derived from patient ul‐tb  were  similar  to  vesicles  from  ul‐l  with  additional  bands  at  250kd  and  150kd.  the  size  of  these  vesicles  was  then  analysed  using  dynamic  light  scattering  (figure 1b)     vesicles  isolated  from  the  conditioned  media  of  ul‐o  cells  demonstrated  a  mean  diameter  of  112.6  ±  9.0nm.  this mean diameter size is within the general size range  described for exosomes and is consistent with our recent  findings with nanoparticle tracking analysis [21].    3.2 presence of fibronectin associated with patient   ascites‐derived vesicles     the protein composition of the vesicles isolated from the  ascites  of  ovarian  cancer  patients  was  defined  by  ms  sequencing. proteins from      figure 1. protein profiles obtained  from sds‐page of vesicles  isolated from patients ul‐l and ul‐tb ascites and from culture  media from ul‐o (oex). panel a: imperial purple stained gel of  ul‐l and ul‐tb ascites derived vesicles and oex vesicles. panel  b:  dynamic  light  scattering  size  determination  of  vesicles  isolated from cell cultures of ul‐o.   these vesicles were separated by sds‐page. to identify  vesicular  proteins,  bands  were  cut  from  the  gel  and  subjected  to  ms  sequencing.  three  specific  bands  produced clear sequences with high ion scores (figure 2).  of these 3 bands, band one was identified as fibronectin.     5safinur atay, carolyn d. roberson, cicek gercel-taylor and douglas d. taylor: ovarian cancer-derived exosomal fibronectin induces pro-inflammatory il-1ß www.intechopen.com   figure 2. mass spectrometric determination of specific proteins  associated with patient‐ascites derived vesicles. panel a presents  the imperial purple stained 10% sds‐page of vesicular proteins  from  ul‐l  and  ul‐tb.  panel b  shows  the  mass  spectrometry  defined  proteins  from  bands  one,  two  and  three  with  the  corresponding peptide count and ion score.     since  previous  studies  have  demonstrated  the  role  of  tumour cell‐associated fibronectin in the induction of il‐ 1β, the presence of fibronectin was confirmed on vesicles  isolated  in  vitro  from  conditioned  media  of  ul‐o  cells  (oex, figure 3a) and in vivo from ovarian cancer patients  (ul‐l,  ul‐o  and  ul‐tb,  as  well  as  normal  control,  figure 3b). these results confirm the enhanced presence  of  fibronectin  on  these  tumour‐derived  vesicles.  in  addition, specific markers were evaluated between ul‐o  cells and  their corresponding vesicles  (oex, figure 3a)  using  western  immunoblotting.  these  results  also  demonstrated  the  enriched  presence  of  cd63  on  the  vesicles, establishing their identity as exosomes.      figure 3. western immunoblot identification of specific protein  markers on cells and corresponding extracellular vesicles. panel  a presents markers associated with ul‐o cells and extracellular  vesicles released by ul‐o cells (oex). panel b presents presence  of  fibronectin  with  extracellular  vesicles  isolated  from  normal  human ab serm (normal) and three ovarian cancer patients.    3.3 exosome induction of il‐1 release by human macrophages     since the presence of tumours has been  linked with the  induction of a pro‐inflammatory microenvironment. the  initial question was whether exosomes derived from an  ovarian tumour would induce the thp‐1 cells to produce  a pro‐inflammatory profile. using the proteome profiler  human  cytokine  antibody  array  panel  a  array,  the  induction of multiple cytokines/chemokines by  tumour‐    derived  oex  was  evaluated  (figure  4).  when  thp‐1  monocytic cells were  incubated with 100μg/ml  tumour‐ derived  exosomes,  14  of  the  36  cytokines/chemokines  were induced by greater than two‐fold. of these, gro,  il‐1, il‐1, il‐8, il‐23, mcp‐1 (ccl2), mip1 and tnf  were  induced  by  more  than  20‐fold  (versus  the  control  treated). of particular interest was that il‐1 was induced  by  350‐fold  following  exposure  to  tumour‐derived  exosomes.     based  on  this  major  induction  of  il‐1,  the  exosome  component  that  might  mediate  this  pathway  was  evaluated.  based  on  our  previous  mass  spectrometric  evaluation of exosomal components, the potential role of  fibronectin  in  this  exosome‐induction  was  analysed.  fibronectin  has  been  reported  to  utilize  an  rgd  (arginine‐glycine‐aspartic  acid)  binding  motif  for  its  binding  to  integrins  (specifically  51)  localized  at  the  plasma  membrane  of  macrophages  to  induce  il‐1  production.  to  assess  the  role  of  exosome‐associated  fibronectin  on  il‐1  induction,  commercially  available  rgd  sequence  mimics  were  used.  incubation  of  oex  with thp‐1 cells resulted in the production and release of  il‐1.  the  negative  control  mimic  peptide  (cp),  which  does  not  block  integrin  binding  of  fibronectin,  was  included  as  a  negative  control.  in  figure  5,  cp  was  observed not to exhibit any effect on il‐1β production or  release.  in  contrast,  the  two  mimic  peptides  (ant  and  bfcv), known to inhibit fibronectin binding to integrins,  significantly  suppressed  il‐1  release  by  thp‐1  cells  (p<0.001).  in  contrast  to  previous  studies  using  trophoblast‐derived  exosomes,  il‐1  production  and  release  is  induced by  tumour‐derived exosomes  in both  the absence and presence of atp.      figure 4. exosome induction of cytokines/chemokines in thp‐1  cells, incubated with 100μg/ml oex (derived from ul‐o ovarian  tumour  cells)  for  20  hours.  the  plot  presents  the  average  background‐subtracted mean spot pixel densities of the pair of  duplicate spots representing each cytokine, divided by the value  for control thp‐1 cells. means and standard deviations from two  independent studies are shown  only values greater  than  two‐ fold were considered significant and shown on the graph.     6 exosomes microvesicles, vol. 1, 2:2013 www.intechopen.com   figure 5. exosome‐associated fibronectin mediates the increased  release of il‐1β. human thp‐1 were cultured in culture media  only  (thp)  or  pre‐cultured  with  the  various  rgd  mimic  peptides at 0.5mg/ml for 30 minutes. oex (exosomes from ul‐o  cells)  were  added  at  100μg/ml  for  6  hours,  followed  by  30  minutes with or without atp  (1mm) at 37°c. supernatants of  cultures were removed to determine the quality of il1β protein  released by elisa. means and standard deviations  from  three  independent studies are shown with p‐values calculated using  studentʹs t‐test. *** indicates level of significance (p < 0.001).        figure 6. phosphorylation of akt2 and cjun, following exposure  to  oex.  human  thp‐1  were  cultured  in  culture  media  only  (thp) or pre‐cultured with the various rgd mimic peptides at  0.5mg/ml for 30 minutes. oex were added at 100μg/ml for 0, 30  60, 120 and 240 minutes and thp‐1 cells were lyzed and levels of  the  phosphorylated  akt  and  c‐jun  were  defined  by  western  immunoblotting.  means  and  standard  deviations  from  three  independent  studies  are  shown  are  shown  with  p‐values  calculated using studentʹs t‐test (p value significant p < 0.05). the  bar at the 240 minute time point following oex treatment was  compared to untreated and  the peptide mimic treated samples  were  compared  with  the  oex  treated.  *  indicates  p<0.001,  **  indicated p<0.05, ns= not statistically significant.     the  il‐1  promoter  region  contains  ap1  and  c‐jun  transcription  factor  binding  sites.  thus,  to  define  the  kinetics and mechanisms of exosome induction of il‐1,  the time course of phosphorylation of akt2 and c‐jun in  the  presence  and  absence  of  the  rgd  mimics  was  investigated  (figure  6).  in  untreated  thp‐1  cells,  no  phosphorylation  was  observed  in  akt2  and  c‐jun  phosphorylation  showed  a  decreasing  trend  in  phosphorylation.  addition  of  oex  resulted  in  a  time‐ dependent phosphorylation of both akt2 and c‐jun. pre‐ incubation with the negative control peptide (cp) did not  significantly  alter  the  time‐dependent  phosphorylation.  in  contrast,  both  inhibitory  mimics  (ant  and  bvcf)  suppressed the phosphorylation of both akt2 and c‐jun  to background levels.     4. discussion    while previous studies have demonstrated that tumour‐ derived  exosomes  can  directly  suppress  activation  and  proliferation  of  cd4  and  cd8  lymphocytes  [22]  and  induce  t  regulatory  cells  [23]  and  myeloid  derived  suppressor  cells  [24],  this  study  focuses  on  their  consequences for macrophages. macrophages are critical  to both the innate and adaptive immune response, as they  rapidly  respond  to  changes  within  their  microenvironment.  in  general,  inflammation  from  pathogens  or  tissue  damage  can  activate  resident  macrophages to initiate or amplify the production of pro‐ inflammatory  cytokines  and  other  inflammatory  mediators  [25].  this  current  study  addresses  the  mechanism  underlying  exosome‐mediated  induction  of  il‐1  by  macrophages,  observed  in  cancer.  in  vitro,  macrophages can be phenotypically polarized to the m1  state  by  treatment  with  ifn‐γ  and  inducers  of  tnf‐α,  such as  lipopolysaccharide  (lps)  [26]. m1 macrophages  produce  pro‐inflammatory  cytokines  and  chemokines,  including  tnf‐α,  il‐12,  il‐6,  ccl2  and  il‐1β  [4,  5].  elevated  levels  of  il‐1β  are  present  in  m1  polarized  macrophages due to activation of the nf‐κb and mapk  pathways  [27],  while  no  il‐1β  protein  is  found  in  m2  polarized macrophages [28].    il‐1 exhibits profound effects on  immune cell function  during inflammation, resulting in investigations focused  on factors that control il‐1 expression. stimuli known to  regulate  il‐1  production  in  vitro  include  bacterial  lipopolysaccharide,  adherence  to  plastic,  certain  viral  infections  such  as  cytomegalovirus,  phorbol  myristate  acetate,  macrophage  ingestion  of  asbestos  fibres  and  certain  cytokines,  including  tumour  necrosis  factor‐,  granulocyte‐macrophage  colony  stimulating  factor  and  il‐1  itself  [29]. while  these  factors are  important  in  the  regulation of il‐1 expression, the mechanisms by which  they  modulate  il‐1  expression  in vivo  remain  unclear.  extracellular  matrices  (ecm)  are  also  capable  of  stimulating  the  expression  of  il‐1  [30].  fibronectin  is  highly  expressed  in  injured  tissues  and  appears  to  be  positioned  to  modulate  the  expression  of  il‐1  in  diseased  tissues  [30].  since  multiple  components  have  been implicated in the induction of the pro‐inflammatory  microenvironment  associated  the  cancer,  the  protein  composition  of  the  vesicles  isolated  from  the  ascites  of  ovarian  cancer  patients  was  analysed.  the  presence  of  fibronectin of vesicles  isolated from cancer patients and  7safinur atay, carolyn d. roberson, cicek gercel-taylor and douglas d. taylor: ovarian cancer-derived exosomal fibronectin induces pro-inflammatory il-1ß www.intechopen.com from vesicles obtained from ovarian cancer patients was  demonstrated  by  mass  spectrometric  sequencing  and  confirmed by western immunoblotting (figures 2 and 3).  the  ability  of  this  exosome‐associated  fibronectin  to  induce the release of il‐1β was further investigated. our  studies  demonstrate  the  exposure  of  thp‐1  cells  to  tumour‐derived  exosomes  induced  the  significant  production  and  release  of  pro‐inflammatory  cytokines/chemokines, including of particular note il‐1β  (figure 4).     in  vitro,  fibronectin  stimulates  the  expression  of  il‐1  mrna  and  its  translation  into  the  31kda  intracellular  precursor  protein,  along  with  secretion  of  the  17kda  active form in human mononuclear cells [31]. this effect  of fibronectin is mediated by the specific cell surface 51  integrin  receptor,  which  activates  poorly  understood  intracellular  signals  to  induce  il‐1  expression  [32].  fibronectin  contains  a  sequence,  termed  arg‐gly‐asp  (rgd),  which  promotes  its  attachment  to  integrin  receptors. monocytes and macrophages have been shown  to possess  fibronectin receptors  that recognize  the rgd  motif  and  mediate  pro‐inflammatory  cytokine  production. the effect of fibronectin has been shown to  be  dependent  on  binding  of  the  rgd  sequence  of  fibronectin  to  integrin  receptors,  as  this  effect  could  be  inhibited  by  integrin  receptor  blocking  peptides  (anti‐ rgd sequence mimics) [33].     il‐1β post‐translational processing and release is a highly  regulated  pathway  utilizing  a  multiprotein  complex,  termed the capase‐1 inflammasome [34]. il‐1β is initially  synthesized as a 34kd precursor protein that is cleaved to  the  biologically  active  17kd  form  by  active  caspase‐1.  inactive  pro‐caspase‐1  is  constitutively  present  in  both  m1  and  m2  macrophages  and  is  activated  within  m1  macrophages  by  self‐cleavage,  occurring  in  the  inflammasome complex. the nucleotide‐binding domain  and leucine‐rich repeat receptor containing pyrin domain  3  (nlrp3)  inflammasome  can  be  activated  by  specific  microbial motifs, by microbial toxins, by uric acid crystals  and by extracellular atp acting through the atp‐gated  plasma  membrane  ion  channel,  the  p2x7  receptor.  addition  of  extracellular  atp  to  macrophages  can  generally increase the release of mature il‐1. in contrast  to  our  previous  study  using  trophoblast‐derived  exosomes,  tumour‐derived  exosomes  do  not  require  addition of atp to induce release of il‐1 (figure 5). it is  unclear  whether  these  exosomes  carry  atp  or  utilize  a  distinct pathway to induce the production and release of  il‐1.  however,  these  possibilities  are  currently  under  investigation.     the  il‐1  promoter  region  contains  ap1  and  c‐jun  transcription  factor  binding  sites  that  are  critical  to  mediate  certain  pathologic  conditions.  our  results  demonstrate  that  tumour  exosomes  induce  il‐1  and  produce  phosphorylation  of  akt2  and  c‐jun  (figure  6).  the  addition  of  the  rgd  mimics  resulted  in  the  suppression  of  akt2  and  c‐jun  phosphorylation,  following  addition  of  ul‐o  exosomes.  these  findings  confirm  the  essential  role  of  exosome‐associated  fibronectin  in  the  regulatory  events  leading  to  the  promotion of il‐1β production and release.     while  other  studies  have  investigated  the  role  of  fibronectin and other components of  the ecms on  the  induction of il‐1, in the current study, we demonstrate  il‐1 induction by fibronectin associated with tumour‐ derived  exosomes.  this  study  with  ovarian  cancer‐ derived  exosomes  demonstrates  that  these  tumour  exosomes also express  fibronectin, which may account  for the pro‐inflammatory microenvironment observed in  cancer.  exposure  of  macrophages  to  ovarian  tumour‐ derived  exosomes  appears  to  “educate”  these  cells  to  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properly cited. abstract a successful strategy in regenerative medicine over the last decade has been the translation of stem cell therapy to repair diseased or damaged tissue in a wide range of indications, despite limited evidence attributing any therapeutic benefit to cell survival or differentiation. recent findings, however, have demonstrated that the conditioned media from stem cell cultures can produce similar efficacious effects compared to those observed for cells. this has led to the stem cell paracrine hypothesis, proposing that secreted factors released from the stem cells contribute significantly to their beneficial effects. it has been well documented that stem cells have the ability to release a range of growth factors, cytokines and chemokines relevant to their function; however, these factors are released at levels too low to account for the reported therapeutic effects. further purification of the conditioned media has since identified that not only are small molecules released by the stem cells, but so too are a large quantity of membrane-bound vesicles, including exosomes, in a functionally relevant manner. in this review, we present our current understanding and explore the evidence supporting the development of stem cell-derived exosomes as a cell-free regenerative medicine. keywords exosomes, characterization, isolation, therapeutic potential 1. introduction one major approach to regenerative medicine has been to replace lost tissue with new cellular material or facilitate the regeneration of damaged or diseased tissues by means of stem cell transplantation. over the last two decades, therapeutic options have progressed from the grafting of whole tissue (such as bone marrow) to the injection of autologous or allogeneic stem cell products derived from adipose, blood, bone and brain tissue. more recently, pluripotent cells derived from embryonic stem (es) cells or induced pluripotent stem (ips) cells, using cellular reprogramming technologies, have been the source of new potential cell therapies. the translation of stem cell therapies from bench to clinic has been 1indira vishnubhatla, randolph corteling, lara stevanato, caroline hicks and john sinden: the development of stem cell-derived exosomes as a cell-free regenerative medicine article j circ biomark, 2014, 3:2 | doi: 10.5772/58597 journal of circulating biomarkers remarkable, with thousands of trials currently registered in the national institutes of health clinical trials database (www.clinicaltrials.gov). the investigations span a broad range of indications, from microbial diseases, cancers and neoplasms, congenital, heart and blood disorders, nervous system diseases, immune disease (graft versus host, multiple sclerosis, arthritis and colitis), wounds and injuries. results from the early phase trials have demonstrated cell therapy as safe, feasible and in a number of indications potentially efficacious, for example, in cardiology [1], ischaemic stroke [2], peripheral ischaemia [3] and cancer [4]. however, for many of these therapies, evidence of a therapeutic benefit cannot be attributed to stem cell survival and/or differentiation [5-7]. the beneficial effects of cell implants without long-term cell survival together with observed trophic effects of the cell-conditioned culture medium suggest that secreted paracrine factors may be involved [8, 9]. small molecules (i.e., growth factors, chemokines and cytokines) are secreted at too low levels to account for the therapeutic efficacy reported [10]. however, the paracrine hypothesis has been strengthened by the recent discovery that stem cells release not only soluble factors but also extracellular vesicles which elicit similar biological activity to the stem cells themselves [11-14]. the most prominent of the extracellular vesicles are exosomes. exosomes have been shown to play a key role in cell-to-cell communication [15-20] and modulating cellular immunity [21-24]. recent data suggest that exosomes may physically link beneficial outputs from stem cells to neighbouring diseased or injured cells through the delivery and transfer of protein, bio-active lipid and nucleic acid cargo, inducing phenotypic and functional changes in the recipient cells that promote the activation of regenerative programs [25-28]. the paracrine hypothesis has inspired an alternative approach in regenerative medicine: namely, translating the potential clinical applications based on exosomes secreted by the stem cells rather than the stem cells themselves, an approach underpinned by the discovery of the control system for the transport and delivery of cellular cargo for which e. rothman, r.w. schekman and t. c. südhof received the 2013 nobel prize for medicine (www.nobelprize.org). 2. formation, release and function of exosomes stem cell-derived exosomes have now been identified and isolated from cell culture supernatants of several cell types with known therapeutic potential, including mesenchymal stromal (msc) [29-31], (bone marrow stem cells) mononuclear (mnc) [32], immune cells (dendritic and cd34+) [33, 34] and human neural stem cells (hnscs) [35, 36]. exosomes (30-100nm) can be distinguished from microvesicles (mvs) (100 nm-1μm) and apoptotic bodies (15 μm) by size and morphology [37-39]. exosomes are lipid bilayer-bound vesicles, released from cells after stimulation [40], and they express specific biomarkers independently of the cell source that will be detailed below. unlike mvs, which are derived from plasma membrane shedding, exosomes are secreted by multivesicular bodies (mvbs) [38, 41-44]. exosomes produced from a single cell can comprise a heterogeneous population of vesicles that may feature different markers, compositions and, potentially, different functions [40]. the heterogeneity of their composition, along with their stable physicochemical features, has led to the idea of using exosomes as natural nano-devices for the development of new therapeutic applications [45-48]. once released into the extracellular environment, exosomes may interact with recipient cells via adhesion to the cell surface mediated by lipid-ligand receptor interactions, internalization via endocytic uptake, or by direct fusion of the vesicles and cell membrane, which leads to the release of exosome content into the target cell [15, 49]. the net result of exosome-cell interactions is the modulation of the physiology of the target cell, induced through any of several different mechanisms, ultimately influencing the biology of tissue/the organism as a whole. the interactions involve many physiological processes, such as antigen presentation [50, 51], the transfer of rna [28] and tissue repair [30]. exosomes have been found to carry antigenic materials and express functional major histocompatibility (mhc) complexes, giving them the potential to mediate antigen-specific immune responses [52]. for example, tumour-derived exosomes captured by dendritic cells can induce the activation of antigenspecific t-cells not previously exposed to the tumour antigen [21, 23], and the function of follicular dendritic cells has been attributed to membrane-bound exosomes containing mhc class ii [53]. exosomes can also influence the biology of recipient cells via the delivery of bioactive content into target cells through the internalization of vesicles. the bilayer membrane encapsulation of exosomes provides a protected and controlled internal microenvironment, allowing cargo to travel long distances within tissues without degradation [27, 28, 54-56]. most recent developments implicate a major role for the horizontal transfer of proteins and genetic materials (such as mrna, microrna (mirna), pre-mirnas, and other non-coding rnas) in the reprogramming of recipient cells. es cellderived exosomes have been demonstrated to shuttle/transfer mrna and proteins to hematopoietic progenitors [26]. other studies have shown that adult stem cell-derived exosomes also shuttle selected patterns of mrna, mirna and pre-mirna associated with several cellular functions involved in the control of 2 j circ biomark, 2014, 3:2 | doi: 10.5772/58597 transcription, proliferation and cell immune regulation [57-60]. 3. exosomal features 3.1 membrane composition besides their characteristic morphology, exosomes are unique in their bilayer membrane protein and lipid composition compared to other extracellular vesicles. exosomal membrane components have been proposed as playing pivotal roles, such as signalling molecules in the uptake/fusion of exosomes with target cells and propagation of signalling in immune responses. for example, rab gtpase membrane proteins have been implicated in membrane trafficking and secretion [61,62]. 3.2 lipid rafts lipid rafts are structurally and functionally unique microdomains in the cell membrane surface which function as sites for key membrane activities, such as endocytosis, cell adhesion and membrane trafficking [63]. lipid rafts, tightly packed areas of sphingolipids and cholesterol compared to the surrounding areas, have been reported in both cellular and exosomal (including stem cell-derived) membranes [64]. as a consequence of the lipids’ composition and limited size, exosomes have a more rigid membrane than their parent cell [65]. high levels of ceramide (associated with biogenesis and release) have also been reported in exosomes [66]. lipid rafts reflect the endosomal origin of exosomes and represent a distinctive feature, which may be useful for their differentiation and isolation from other secreted microparticles [65]. 3.3 proteins exosome membranes comprise both ubiquitous and cell type-specific proteins that cover numerous biological functions. a complete database of exosomal proteins can be found at exocarta (www.exocarta.org) [67]. due to their endosomal origin, all exosomes contain membrane transport and fusion proteins (annexins, gtpases, flotillin), tetraspanins (cd9, cd63, cd81, cd82), heat shock proteins (hsp70, hsp90), proteins involved in multivesicle body biogenesis (alix and tsg101), lipidrelated proteins and phospholipases [29,68]. these proteins have been associated with lipid rafts. due to their size, lipid rafts can contain only a few proteins at one domain. to engage in a membrane function, lipid rafts cluster together and sequester specific sets of signalling and other proteins which serve as platforms to execute functions such as membrane trafficking and signalling [61]. proteins presented on the outer exoplasmic membrane layer are routinely used as identifying markers; however, there is wide variation in terms of protein profiles across exosomes from different cell sources. the most widelyused markers are tetraspanins, alix, flotillin, tsg101 and rab5b, which are detected using antibody-based techniques (such as western blot or elisa) for the rapid confirmation of the presence of exosomes. 3.4 cargo contents exosomal cargo can contain a variety of cytoplasmic nucleic acids, lipids and proteins, reflecting both the condition and origin of the parent or producer cell. as a result, exosomes exhibit some commonly shared contents and express distinctive molecules identifying their originating producer cell, but they also possess unique mixtures of proteins and rna. the complexity of cargo contents has been postulated as a significant factor of exosomal efficacy in treating complex tissue injuries such as those that affect multiple tissues and targets [69]. individual components in the cargo may not be equally or sufficiently efficacious, and the therapeutic efficacy of exosomes in relation to a specific injury may result from the synergy of a select permutation of individual exosome components. one aim of translational research is to assess the biochemical activities of these contents and identify the optimal permutations of these activities for therapeutic targets. there is a growing need for quick and easy methods for the analysis of exosomal cargo. the thorough molecular characterization of exosomes is hindered by the complexity of their composition, their high heterogeneity and their low abundance. to date, only a few studies have been conducted that have provided a systematic characterization of the protein and the genetic composition of stem cell-derived exosomes [29, 35, 57, 58, 69-71] (table 1). a number of proteins have been identified in the functional cargo of stem cell-derived exosomes which affect a variety of cellular functions [14, 18, 26, 49, 72, 73]. recent developments in protein separation techniques, spectrometry, immunological methods and bioinformatics have enabled the systematic characterization of mscderived exosomes [29, 68, 69] (table 1). these analyses have identified over 700 proteins that reflect: (1) the evolutionary conserved set of exosomal proteins (cd81, cd63, cd9 [tetraspanins], alix, tsg101) and those tissue or cell type-specific proteins; (2) integration of parental cellular processes and genes of self-renewal and differentiation; and (3) a list of proteins that can be associated with therapeutic potential [29]. 3indira vishnubhatla, randolph corteling, lara stevanato, caroline hicks and john sinden: the development of stem cell-derived exosomes as a cell-free regenerative medicine functional group / component constituent type (description) exosome source / reference surface receptors platelet-derived growth factor receptor beta (pdgf-rβ) epidermal growth factor receptor (egfr) plasminogen activator, urokinase receptor (plaur) hbm-msc [29] signalling molecules r-ras / n-ras mitogen-activated protein kinase 1(mapk1) guanine nucleotide binding protein 13 (gna13) guanine nucleotide binding protein (g protein), gamma 12 (gng12) cell division cycle 42 (cdc42) guanine nucleotide exchange factor (vav2) hbm-msc [29] ras-related rab proteins laminin-r 14-3-3 proteins hbm-msc [29] cell adhesion fibronectin 1 (fn1) ras gtpase-activating-like protein 1 (iqgap1) cd47 ezr integrins lectin, galactose binding, soluble 1 (lgals1) lectin, galactoside-binding, soluble, 3 (lgals3) hbm-msc [29] cd34 hcd34+ and mnc[34] msc-associated antigens cd63 hbm-msc [29]; hcd34+ and mnc [34] cd109, cd151 hbm-msc [29] cd248, and cd276 hbm-msc [29] cd13, cd73, cd105 hbm-msc [29] cd29, cd44, cd73, cd90, and cd105 positivity (msc markers) hbm-msc[29] cd9, cd81 hbm-msc[29], hcd34+ and mnc [34]; hesc-msc [64] raft-associated proteins transluminal protein (tsg101) hcd34+ and mnc [34]; hesc-msc [64] annexin v (bound to phosphatidylserine) hcd34+ and mnc [34] alix / actin interacting protein 1 (aip1) hesc-msc [30, 64, 104] heat shock protein 70kda 1a variant transferrin receptor hla-dr4 ezrin hnsc [35] raft-associated lipids gm1 gangliosides sphingomyelin ceramide hesc-msc [64] phosphatidylserine hcd34+ and mnc[36] novel protein cargo prostaglandin g/h synthase (cell proliferation) cathepsin f precursor (intracellular degradation and turnover of proteins) sarcoma antigen 1 (tumour marker) polymyositis/scleroderma (autoantigen to nuclear proteins) hnsc [35] 20s proteasome (degradation of misfolded proteins related to tissue damage in mi) hesc-msc [69] functional genetic cargo mirna126 and mirna 130a (angiogenesis) hcd34+ and mnc [34] mirna133b (stroke recovery) mouse msc [104, 111] mirna 96, mirna124 and mirna199a (differentiation) hbmmsc[115] mirna 451 (maintenance, differentiation) mirna 223 (regulator of cell cycle) hbm-msc and hlsc [58] mirna 335 (markers of cell of origin for bm-derived mscs) mirna 564 (selective expression in msc exosomes compared to mscs) hbm-msc[58] mirna 142-3p, mirna 501 (selective expression in hlsc exosomes compared to hlscs) hlsc[58] abbreviations: bm, bone marrow; hesc, human embryonic stem cells; hnsc, human neural stem cells; hlsc, human liver resident stem cells; hmsc, human mesenchymal stem cell; mi, myocardial infarction; mirna, micro-rna; mnc, mononuclear cells table 1. examples of functional components isolated from stem cell-derived exosomes 4 j circ biomark, 2014, 3:2 | doi: 10.5772/58597 4. proteomic characterization techniques such as shotgun proteomic analysis of the peptide mixtures from exosomal fractions have been used to identify proteins from hnscs [35]. a total of 103 proteins were detected in a sample of hnsc-derived exosomes, including previously reported heat shock 70 kda, transferrin receptor, hla dr4 and ezrin, and several as yet unreported such as anhydrolase-domaincontaining-8, g protein-coupled receptor associated sorting protein 1, the 70 kda heat shock protein 1a variant, and prostaglandin g/h synthase. rarer proteins such as cathepsin f precursor and sarcoma antigen 1 which were also detected were uniquely present in only one fraction (table 1). 5. genetic information in stem cell–derived exosomes mitochondrial dna, mrna and non-coding rnas such as mirna have been reported in stem cell-derived exosomes and may deliver horizontal gene transfer to target cells [11, 74, 75]. exosomes derived from human stem cells have been shown in vivo to deliver human mrna to mouse cells, resulting in protein translation [76, 77]. exosomes derived from es cells contain abundant mirna and have been demonstrated to transfer a subset of mirnas to mouse embryonic fibroblasts in vitro [60]. mirna is a class of small, non-coding rna molecules consisting of 19 to 24 nucleotides that have a variety of cellular properties associated with both physiological and pathological processes. they are involved in homeostasis, the post-translational modification of gene expression, oncogenesis and tumour suppression, rna and protein complex shuttling, and protection from rnase enzymatic degradation [20, 78-83]. exosomes provide a protected and controlled internal microenvironment, allowing mirna molecules to travel long distances within tissues without degradation [54]. evidence suggests that mirna packaging occurs non-randomly and that specific mirna populations are preferentially sorted and shuttled into mvs [84], and secreted contents could vary in response to different stimuli [58]. important questions, regarding how mirnas are sorted into exosomes (shedding vesicles and high density lipoprotein (hdl) particles), whether there is regulated specificity in this process, how secreted vesicles are taken up by recipient cells and whether specific cells are targeted, need to be addressed. the transfer of human mrna via exosomes and its translation into protein have been observed both in vitro and in vivo in diverse experimental models [28]. rna inactivation resulted in the significant loss of biological activities, suggesting that the transfer of nucleic acids has a relevant role [85]. however, exosomes also transfer regulators of transcription as they induce changes in the gene expression of the recipient cells, such as the upregulation of the bcl family genes and the downregulation of casp1 (caspase 1), casp8 (caspase 8) and lta (lymphotoxin α) genes involved in the “execution” phase of cell apoptosis [80]. moreover, the mirnas present in exosomes are candidates for the post-transcriptional regulation and induction of epigenetic changes in the recipient cells. mirnas shuttled by epc-derived mvs are the main effectors of the pro-angiogenic and renoprotective activities of mvs [79]. in fact, the non-specific as well as the specific depletion of mirnas in mvs obtained from diceror mirna126and mirna-296-knockdown epcs modify their biological effects [79, 86]. the number of secreted mirnas identified in stem cellderived exosomes has been expanding (table 1) and their role and biological/physiological significance is of considerable interest. stem cell types and their derived exosomes have distinct mirna profiles [34, 86, 87], and are listed in http://exocarta.org/mirna and http://www.mirbase.org. 5.1 producer cells to ensure the large scale supply required for clinical research and development, producer cell immortalization (either by natural selection or by genetic modification) and clonal isolation is a practical strategy to produce functionally bioactive exosomes for use as therapeutic agents or delivery vehicles [47, 88, 89]. immortalization ensures production stability and reduces the equivalence testing of new production lots. bioreactors and other specialized culture systems are also used for culture expansion and to increase the yields of exosomes and mvs [88, 90]. in respect of genetically modified producer cells, the immortalizing oncogene (e.g., c-myc) and its protein product are not trafficked into the exosomes [88]. while secreted exosomes from a range of cell types exhibit some common shared contents, they also express molecules that reflect their cells of origin [44, 58, 71] and their microenvironment (e.g., culture conditions) [37, 9193]. exosomes are released from producer cells with stable phenotypes. the possibility of changing the phenotype of the exosomes represents a novel therapeutic strategy for cell-free regenerative medicine. pre-treating the producer cells with different cytokine cocktails, etc., to drive a specific response [94], or stimulating/exposing producer cells in effect mimicking cells’ responses to a local diseased/damaged environment after implantation can result in exosomes therapeutically suited to specific indication, as in cancer vaccines [95]. 5indira vishnubhatla, randolph corteling, lara stevanato, caroline hicks and john sinden: the development of stem cell-derived exosomes as a cell-free regenerative medicine 5.2 preparation, isolation and manufacture of stem cell-derived exosomes efforts to harvest purified exosomes in clinically significant quantities are limited by the lack of tools and methods to differentiate them from microparticles in general and isolate different subsets. most cell types produce exosomes; those produced by cultured cells are isolated from the conditioned media (figure 1). additionally, the yield from different cell types varies significantly. within mscs, for example, the production of exosomes from es cell-derived mscs is up to seventimes that from cord bloodor bone marrow-derived mscs, suggesting a direct correlation between the donor cell ‘immaturity’ and exosome yield [96]. figure 1. overview of the isolation and manufacture of stem cellderived exosomes as yet, there is no agreed or standardized method for the preparation and purification of exosomes from cellconditioned culture media (table 2). mvs, exosomes, debris and culture media contaminants are all contained in the conditioned medium. serum-free defined media may be used to reduce contamination with a serum or its components. also, it is difficult to analytically distinguish exosomes from other membrane vesicles due to the limitations of commonly-used separation procedures, namely ultracentrifugation and flow cytometry [97]. the overall size of exosomes (<100 nm) has been an important criterion for distinguishing them from the other larger membrane-bound microvesicle secretions. among current methods (differential centrifugation, discontinuous density gradients, immunoaffinity, ultrafiltration and high performance liquid chromatography (hplc), differential centrifugation is the most commonly used for exosome isolation, which utilizes an increasing centrifugal force from 2000xg to 10,000xg to separate the mediumand larger-sized particles and cell debris from the exosome pellet at 100,000xg [33, 98-100]. although crude and non-specific, this method has been reported to allow for the significant separation/collection of exosomes from a conditioned medium. however, protein aggregates, genetic materials, particulates from media and cell debris are common contaminants. to enhance the specificity of exosome purification, sequential centrifugation has been combined with ultrafiltration [98], or equilibrium density gradient centrifugation in a sucrose density gradient or optiprep®, to provide for the greater purity of the exosome preparation (flotation density 1.1-1.2g/ml) [99] or sugar cushion [29, 33, 101]. the isolation of exosomes based on size is another option. ultrafiltration can be used to purify exosomes without compromising biological activity. membranes with different pore sizes such as 100 kda molecular weight cut-off (mwco) and gel filtration to eliminate smaller particles have been used to avoid the use of a nonneutral ph or non-physiological salt concentration [33, 100]. currently available tangential flow filtration (tff) systems are scalable (to >10,000l), purify and concentrate the exosome fractions [29] (www.pall.com), and are less time consuming than differential centrifugation. hplc can also be used to purify exosomes to homogeneouslysized particles and preserve their biological activity as the preparation is maintained at a physiological ph and salt concentration [68, 88]. although the purity of the exosome preparation is high, this method has poor scalability. 6 j circ biomark, 2014, 3:2 | doi: 10.5772/58597 isolation method indicator of exosome sample purity for clinical use advantages disadvantages differential centrifugation [22, 33, 98] ratio of total mhc class ii molecules per mg total protein (12% of total protein being mhc ii class); absence of non-exosomal proteins, haptoglobin and human serum albumin (<1-2% of overall) using sds-page and elisa; low levels of dna (<50ng/ml) [33] potential for sterile preparations [33, 98] time consuming; low recovery, poor reproducibility due to operator variability [33] pellet re-suspension difficult [99] contamination with other microparticles of overlapping size distribution, protein aggregates, particulates from media and cell debris [33, 100, 101] ultrafiltration (500 kda mwco hollow fibre) + centrifugation using sucrose –d20) density cushion (1.21g/cm3) [33] ratio of total mhc class ii molecules per mg total protein (12% of total protein being mhc ii class); absence of non-exosomal proteins, haptoglobin and human serum albumin (<1-2% of overall) using sds-page and elisa; low levels of dna (<50 ng/ml) [33] potentially high enrichment of exosomes; useful for clinical applications [33] hollow fibre cartridge removed dead cells and debris by using membranes with different pore sizes (i.e., 100kda mwco) the protein content of samples remained consistent [33] sterile preparations less time consuming than ultracentrifugation [100] filtration reduced scalability of the subsequent centrifugation method [99] ultrafiltration 100 kda mwco membrane (minimate tff capsule system, pall corporation, ann arbor, mi) and sequential ultracentrifugation onto a sucrose gradient [29] protein concentration (refractometry and bradford dye assay) of final exosomal preparation [29] used to isolate exosomes for protein characterization studies hplc + centrifugation [30, 88] presence of cd9 (western blot) and homogenous in size (dls), flotation density 1.11-1.16 g/ml, proteins mw range from 20 to 250 kda [30] 1.5 mg of exosomes from 1l of conditioned medium; doses of 0.3 or 0.4 μg per mouse [88] high throughput high purity (homogeneous sized particles) biological activity preserved as preparation is maintained at physiological ph and salt concentration [99] low yields expensive equipment and poor scalability [30, 99] differential ultracentrifugation (first using sugar cushion (30% sucrose/d2o cushion for i hour) [101] expressed as ratio of particle to protein high purity at 3.3 x1010 p/μg; impure if <1.5 x 109 p/μg [101] sugar cushion resulted in high purity and greater elimination of non-vesicular proteins [101] useful for high through put and clinical preparations[101] applicable for purification of large amounts of exosomes affinity beads [98, 102] not determined easy to use with fewer steps than centrifugation methods high specificity, dependent on choice of marker for immobilization [98] selection of exosome population subset; not intended for purification of large amounts of exosomes [98] difficulty in completely removing antibodies from sample and eluting fully functioning exosomes [98] polymer-based precipitation (exoquick) (www.systembio.com) number of particles within size range detected nanosight tracking system [100] potentially high yields fewer steps than centrifugation methods no method for removing polymers from exosome sample pellet difficult to re-suspend [100] flow field-flow fractionation (flfff)[35] not determined analytical technique for fractionation of exosomes, useful for morphological and protein characterization not for large-scale preparation of exosomes abbreviations: dynamic light scattering (dls); flow field-flow fractionation (flfff); high pressure liquid chromatography (hplc); kilodalton (kda); molecular weight cut off (nmwco); tangential flow filtration (tff) table 2. exosome isolation methods from cell culture media 7indira vishnubhatla, randolph corteling, lara stevanato, caroline hicks and john sinden: the development of stem cell-derived exosomes as a cell-free regenerative medicine immunoaffinity is a highly scalable technique using antibodies recognizing exosome-associated antigens conjugated to magnetic beads, chromatography matrices, plates or microfluidic devices [67, 88, 98, 102]. the antibodies can be used by themselves or in combination. for example, exotest® kits feature anti-cd63, -cd81 or cd9 antibodies immobilized on 96 well plates for exosome capture and characterization (www.hansabiomed.eu). other affinity-capture methods use lectins which bind to specific saccharide residues on the exosome surface (www.aethlonmedical.com). the targets or markers for affinity capture need to be identified to optimize the high specificity offered by this technique. exosomes can also be purified by using differential solubility in volume-excluding polymers, such as polyethylene glycols (pegs) and low-speed centrifugation or filtration. based on this concept, system biosciences has released a proprietary reagent called exoquick® that can be added to the conditioned cell media to precipitate the exosomes (www.systembio.com). the procedure has been reported as being fast and straightforward. however, it is not specific for exosomes and pellet re-suspension is difficult [100]. flow field-flow fractionation (flfff) is an elution-based technique that is used to separate and characterize macromolecules (e.g., proteins) and nanoto micro-sized particles (e.g., organelles and cells) and which has been successfully applied to fractionate exosomes from hnscs culture media [35]. defining the purity of an exosomal preparation is critical to defining the product prior to any translational development [101]. the production of clinical-grade exosome products would require current good manufacturing practice (cgmp). exosomal products are more stable and easier to store and transport to the clinical site than stem cells [47]. the use of clinical-grade immortalized stem cell lines can reduce the time required for cell production, allowing cell banking and scalability, and thereby reducing production costs, and can also be of value in a strategy for gmp manufacturing [68, 88]. also essential for the development of exosomes products are release assays, required for establishing the storage, shelf life and stability of the exosome products prior to clinical trials [46, 103]. 6. developing a stem cell-derived exosome product 6.1 therapeutics for clinical translation to be realized, it is necessary to characterize the physiochemical properties/phenotype of the purified exosome population along with the content and/or cargo of these vesicles in terms of proteins and small rnas (table 1). by addressing in an unbiased manner the genomic and proteomic content of the purified exosomes, it may be possible to predict the cellular targets and pathways that may be influenced. therapeutic approaches based on the delivery of mirnas are currently being explored for cerebrovascular, myocardial and peripheral ischaemia. msc-derived exosomes have been found to increase the mirna-133b level when exposed to middle cerebral artery occlusion (mcao) brain extracts [104]. mirna-133b is substantially down-regulated in the rat brain after mcao. the therapeutic approach is to increase mirna-133b in the brain tissue via exosomes to stimulate neurite outgrowth and improve functional recovery after stroke. similarly, msc-derived exosomes were also able to provide protection against myocardial ischaemia and treat myocardial infarction through the expression of higher levels of precursor forms of hsa-let-7b and hsa-let-7g mirnas. cd34+ stem cell-derived exosomes are described as inducing angiogenic activity in isolated endothelial cells and in murine models of vessel growth. the benefit of cd34+ cell therapy for functional recovery after ischaemic injury could be induced by the exosomemediated transfer of angiogenic factors to surrounding cells. preliminary data on the mechanism that may mediate the enhanced potency of cd34+-derived exosomes have shown that the pro-angiogenic mirnas 126 and 130a were highly expressed in cd34+ exosomes compared with other unselected mncs [34]. in another study, protein profiling led to the identification of 20s proteasome using the exocarta database as a candidate exosome protein, with the therapeutic potential to ameliorate tissue damage after myocardial infarction [69]. exosomes as active components of conditioned medium from human es cellderived mscs injected into swine and murine models of ischaemia /reperfusion injury reduced the infarct size [30]; from bone marrow-derived mscs, they accelerated repair in mouse models of acute kidney injury [77]; and from liver-derived stem cells, they induced proliferation and apoptosis resistance in cultured human hepatocytes [76]. many more detailed functional studies are required to discover other candidate proteins and their therapeutic potential. 6.2 clinical experience only one stem cell-derived exosome product has been used clinically to date [105]. in this first attempt, the administration of escalating doses of exosomes derived from donor bone marrow mscs into a patient suffering from severe therapy-refractory cutaneous and intestinal graft-versus-host disease grade iv was found to be well 8 j circ biomark, 2014, 3:2 | doi: 10.5772/58597 tolerated and led to a significant and sustainable decrease of symptoms. a small number of clinical trials are currently investigating dendritic cell-derived exosomes for immunotherapy or vaccination in severe cancers, such as non-small lung (phase ii, nct01159288) [106], glioma (phase i nct01550523) [107] and gastric (observational, nct01779583), and with phase i trials in non-small lung small lung ([108], colorectal [109] and melanoma [110] completed. interestingly, the use of plant-derived exosomes as vehicles to deliver curcumin to treat colon cancer is also under scrutiny (phase i, nct01294070). 6.3 advantages/limitations of exosome therapeutics the research to date indicates that stem cell-derived exosomes potentially have significant clinical utility. exosome-based, cell-free therapies in contrast to cellbased therapies in regenerative medicine can be generally easier to manufacture and prima facie safer, as they are non-viable and will not form tumours. by replacing the administration of live cells with their secreted exosomes, many of the safety concerns and limitations associated with the transplantation of viable replicating cells could be mitigated. stem cell-derived exosomes have generally been found to be less immunogenic than parental cells, as a result of a lower content of membrane-bound proteins, such as mhc complex molecules [10]. thus, ‘off-the-shelf’ products may be readily developed using exosomes with negligible alloimmunogenicity. cell-free products offer a number of advantages compared to cell transplantation. exosomes – in a manner similar to conventional biopharmacological products can be standardized and tested in terms of dose and biological activity. exosomes can be stored without potentially toxic cryopreservatives at -20°c for six months with no loss to their biochemical activity [100]. an important feature of exosomes is the encapsulation and protection of their contents from degradation in vivo, thereby potentially preventing some of the problems associated with small soluble molecules such as cytokines, growth factors, transcription factors and rnas, which are rapidly degraded [28]. the durability of exosomes in culture allows for the acquisition of large quantities of exosomes through their collection from a culture medium in which the exosomes are secreted over periods of time. scalable systems for exosome production to support large-scale commercially viable manufacturing processes need to be developed. while exosomes exhibit several attractive features as a therapeutic agent, there are also potential limitations to be considered as well. exosomes contain a mixture of biologically active molecules, some of which seem to have beneficial effects, whereas others might have detrimental (e.g., proinflammatory) effects under certain conditions. whether exosomes will turn out to be superior to angiogenic drugs or purified recombinant growth factors and other peptides within the context of cell-free approaches to tissue regeneration remains to be seen. in addition, these naturally-occurring secreted membrane vesicles are better tolerated in the body, as evidenced by their ubiquitous presence in biological fluids, and have an intrinsic homing ability. in contrast to cell-based therapy, stem cell-derived exosomes provide an ‘off-the-shelf’ therapeutic product and will incur less manufacturing and storage costs. similarly, most stem cells can be readily genetically modified, either by integrating viruses or episomal transfection. it is known that the overexpression or knock-down of mirnas can result in similar overor under-expression in exosomal cargo [79, 104, 111-113]. this has been exploited in preclinical studies where genetically modified exosomes have shown relevant therapeutic outcomes [78, 104, 111-114]. 6.4 clinical and regulatory considerations while the application of stem cell-derived exosomes remains largely untested by regulatory authorities in advance of clinical trials, it is likely that these products will be seen in a similar way to their parent cells. safety testing will focus on systemic bio-distribution, toxicity/tolerability and half-life studies. compared to stem cell therapy, exosomal products can be delivered more readily by less sophisticated and more conventional delivery devices as the formulation and stability will be similar to biologics. repeat administration and even selfadministration may also be possible in the future due to more straightforward product presentation. in addition, the much lower cost of such goods will be attractive to healthcare funders. 7. future scope the number of peer-reviewed publications focusing on exosomes has increased exponentially over the past decade. the newly founded international society for extracellular vesicles (isev) is seeing a rapid rise in the number of presentations on the therapeutic use of exosomes. much excitement and promise has been generated around the use of exosomes in cell-free regenerative medicine, which offers the potential for 9indira vishnubhatla, randolph corteling, lara stevanato, caroline hicks and john sinden: the development of stem cell-derived exosomes as a cell-free regenerative medicine safer, low risk products to cover a wider range of applications. databases such as exocarta have facilitated the sharing of data regarding the genomics and proteomics of exosomes. as this data continues to accrue, it will become important to determine which factors are most relevant to exosome functions. also key to exosome product translation will be advances in gmp supply and the production of clinical grade products, product characterization and potency 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[115] laine sk, alm jj, virtanen sp, aro ht and laitalaleinonen tk (2012) micrornas mir-96, mir-124, and mir-199a regulate gene expression in human bone marrow-derived mesenchymal stem cells. journal of cellular biochemistry 113: 2687-2695. 14 j circ biomark, 2014, 3:2 | doi: 10.5772/58597 article journal of circulating biomarkers analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization invited feature article shannon l. werner1, ryon p. graf1, mark landers1, david t. valenta1, matthew schroeder1, stephanie b. greene1, natalee bales1, ryan dittamore1 and dena marrinucci1* 1 epic sciences, inc., san diego, ca, usa *corresponding author(s) e-mail: dena@epicsciences.com received 30 january 2015; accepted 20 april 2015 doi: 10.5772/60725 © 2015 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the epic platform was developed for the unbiased detec‐ tion and molecular characterization of circulating tumour cells (ctcs). here, we report assay performance data, including accuracy, linearity, specificity and intra/interassay precision of ctc enumeration in healthy donor (hd) blood samples spiked with varying concentrations of cancer cell line controls (clcs). additionally, we demon‐ strate clinical feasibility for ctc detection in a small cohort of metastatic castrate-resistant prostate cancer (mcrpc) patients. the epic platform demonstrated accuracy, linearity and sensitivity for the enumeration of all clc concentrations tested. furthermore, we established the precision between multiple operators and slide staining batches and assay specificity showing zero ctcs detected in 18 healthy donor samples. in a clinical feasibility study, at least one traditional ctc/ml (ck+, cd45-, and intact nuclei) was detected in 89 % of 44 mcrpc samples, whereas 100 % of samples had ctcs enumerated if additional ctc subpopulations (ck-/cd45and ck+ apoptotic ctcs) were included in the analysis. in addition to presenting epic platform’s performance with respect to ctc enumeration, we provide examples of its integrated downstream capabilities, including protein biomarker expression and downstream genomic analyses at single cell resolution. keywords analytical validation, biomarker, circulating tumour cells, ctc, ctm, clinical feasibility, epic ctc platform, fluid biopsy, liquid biopsy, metastasis 1. introduction over 90 % of cancer-related deaths from solid tumours are caused by complications of tumour metastasis [1]: the translocation of primary tumour cells to a distant tissue, followed by adaptation to and colonization of the micro‐ environment of a secondary site to facilitate tumour cell proliferation and the macroscopic formation of metastatic lesions [2, 3]. circulating tumour cells (ctcs) are thought to represent the haematologic phase of tumour metastasis, 1j circ biomark, 2015, 4:3 | doi: 10.5772/60725 as ctc detection and enumeration are greater in metastatic patients than those with high-risk or benign disease [4, 5]. ctcs were first discovered in the late 1800s [5, 6] and exist in frequencies in the range of one in one billion blood cells [2, 5]. despite their rare nature, monitoring disease by the detection of ctcs has several key advantages over solid tissue biopsies [7]. ctcs are accessible via peripheral venous phlebotomy, which is less invasive to patients than solid tissue biopsies. in addition, some solid tissue biopsies require expensive radiographic imaging to guide biopsy needles, and can be potentially hazardous to patients who may already be weakened by current or previous history of cancer treatment. importantly, there can also be signifi‐ cant intra-patient tumour evolution over time [8-11], for which blood collection can represent a real-time fluid biopsy, and is more amenable for repeat sampling than tissue biopsies. the enumeration of ctcs has clinically validated prognos‐ tic value to predict progression free survival (pfs) and overall survival (os) in metastatic breast cancer [12], prostate cancer [13] and colorectal carcinoma [14] patients using the cellsearch platform. beyond enumeration, the molecular characterization of ctcs has potential to predict response to therapy [4, 15]. the integration of ctc enu‐ meration and biomarker expression analysis has been proposed for use in early clinical development of thera‐ peutics, as intermediate endpoints in clinical trials, and in stratification of patients for targeted therapy [16, 17]. ctc heterogeneity has been observed both within individual patients and across cohorts of patients, displaying a range of gene or protein expression signa‐ tures [4], cell size [18, 19], and cell density [9]. this fundamental inter and intra-patient heterogeneity has made it challenging to define a standard ctc definition and reference range for the development of ctc detection platforms. due to the rare nature of ctcs, several ctc detection and characterization methods utilize enrichment strategies to isolate ctcs from peripheral blood cells. such enrichment techniques rely on ctc expression of epithelial markers (epcam, cytokeratin), depletion of cells expressing a common leukocyte marker (cd45), selection of cells with specific physical properties (cell size, density, deformity), or a combination of epitope and physical property selection [16, 17]. positive selection of ctcs is the most common mechanism of ctc enrichment and is utilized by many technologies in development, including cellsearch, the only platform currently fda-cleared for prognostic applications [5, 16]. however, emerging literature suggests that ctcs display degrees of epithelial epitope plasticity, and have been reported to have more than 10 times less epcam expres‐ sion per cell than solid primary and metastatic tissue samples [20]. additionally, common epithelial cell surface markers (epcam, e-cadherin, cytokeratins) are often downregulated or absent in pluripotent cancer stem cells or cells undergoing epithelial-to-mesenchymal transition (emt) [21, 22]. in preclinical models, cells undergoing dedifferentiation or emt have been associated with increased motility, invasiveness and tumour aggressiveness [23-25]. thus, detection strategies that rely on epithelial marker enrichment might miss biologically relevant ctc subpo‐ pulations and hinder comprehensive analysis of ctc heterogeneity. to address this issue, some ctc detection platforms integrate negative selection, the depletion of cd45(+) cells from whole blood, as a method to enrich ctcs in an effort to detect epithelial marker-negative cells, and studies characterizing this strategy are ongoing [26-28]. alternatively, size exclusion methods select for cells that are larger than white blood cells and are not biased by cell surface marker expression [16, 29-31]. however, studies of prostate cancer, breast cancer, and melanoma ctcs have found considerable overlap between the lower limit of ctc diameter and median white blood cell diameter [18, 26, 32], which impacts the ability of size exclusion methods to detect small ctcs. size exclusion and micro-filtration systems can also reduce high ctc recovery due to cell membrane stress, thus reducing dynamic range and cell viability during the cell capture process [33], and some membrane filtration systems have been reported to show ctc capture variability, as well as frequent sample clogging on filters [34]. many emerging ctc detection modalities make use of microfluidics to select via size and deformity [35], aid enrichment by increasing epitope availability [32, 36, 37], or assist in immunomagnetic positive or negative epitope selection [26]. analogous to size exclusion and epitope selection, microfluidic techniques must utilize assump‐ tions about the physical nature of ctcs they are engineered to detect. without an established reference range or universal definition of ctc, any chosen parameters of ctc enrichment might bias sampling and miss biologically relevant ctcs. while microfluidic devices have displayed improved sensitivity of ctc detection and higher separa‐ tion efficiency compared to first-generation approaches [33, 38], one drawback is the potential for clogging [38, 39], which has important implications for accurate ctc enumeration for patients with high ctc burden, or for the detection of ctc clusters. additional drawbacks can include low sample throughput due to the complex integration of external electrical/magnetic fields, and prolonged processing time due to the device’s high fluidic resistance [34, 39]. additionally, ctc isolation using microfluidic chips typically requires a fresh blood sample to be processed within hours of patient blood draw at the clinical site, rather than allowing for shipment and blood sample processing at a centralized clia laboratory [26]. importantly, processing an entire fresh blood sample through a microfluidic chip may preclude the ability to store morphologically intact ctcs in a biorepository for retrospective biomarker analyses. 2 j circ biomark, 2015, 4:3 | doi: 10.5772/60725 to overcome the challenges outlined above, we developed an unbiased method to detect and characterize ctcs without cell enrichment, depletion or microfluidic manip‐ ulation, and with the added feature of being able to store samples in a central biorepository. while detection of ctcs has prognostic value for patient survival [5, 12-14], it is a comprehensive portrait of biomarker expression, hetero‐ geneity and clonal evolution that has been proposed as having great promise to derive clinically actionable ctc signatures for drug development, patient stratification and evaluation of drug resistance mechanisms [16, 17, 40]. to this end, the epic ctc platform was designed with integrated downstream capabilities for the evaluation of protein (immunofluorescence) and genetic (fish, ngs) biomarkers with single cell resolution. the following report describes the analytical performance of the epic ctc platform, clinical feasibility for the detec‐ tion of both traditional and non-traditional ctcs in mcrpc patients, and a description of the platform’s downstream biomarker analytic capabilities. 2. methods 2.1 sample receipt, processing and ctc enumeration samples were processed and analysed using the epic ctc platform (figure 1) as previously described [41-43]. briefly, blood samples were collected in 10 ml cell-free preservative blood tubes (streck, omaha, nebraska) and shipped to epic sciences. red blood cell (rbc) lysis was performed using ammonium chloride solution. follow‐ ing centrifugation, all nucleated cells were deposited on up to 12 glass slides per sample at a concentration of 3 x 106 cells/slide and frozen at -80 ºc until analysis. after thawing, two slides per sample were immunofluorescent‐ ly (if) stained with a cocktail of antibodies targeting cytokeratins (ck), cd45, and 4’,6-diamidino-2-phenylin‐ dole, dihydrochloride (dapi). slides were scanned by epic’s rapid fluorescent scanning method [43], which analyses each nucleated cell per slide using a propriet‐ ary algorithm developed within the context of haemato‐ pathology standards. in short, the algorithm utilizes 90 cellular parameters, including marker expression and cell morphology, to differentiate candidate ctcs from surrounding white blood cells (wbcs) [43]. candidate ctcs were identified and displayed in a web-based report, and trained technicians confirmed ctc candi‐ dates as being classified into one of the following categories (figure 2): traditional ctcs (figure 2a): defined as cells ck(+), cd45(-), intact dapi, and are generally larger and mor‐ phologically distinct from surrounding wbcs. small ctcs (figure 2b): defined as ck(+), cd45(-), intact dapi cells that are the same size or smaller than the size of neighbouring wbcs. figure 1. epic platform workflow for sample preparation, ctc enumeration and biomarker analysis. upon patient blood sample receipt at epic sciences, 1) whole blood is lysed and nucleated cells (3 x 106 per slide) are deposited onto 10-12 microscope slides and are frozen at -80 ºc until analysis. 2) two slides per patient sample are thawed and stained with a cocktail of antibodies including cytokeratin, cd45, dapi to perform ctc enumeration, and a fourth fluorescent channel is available for the evaluation of protein biomarker expression. 3) stained slides are scanned and 4) the resulting images are analysed using a multi-parametric digital pathology algorithm to detect ctc candidates and quantitate biomarker expression levels. ctc classifications are displayed in a web-based report and are confirmed by trained technicians. 5) ctc enumeration and biomarker expression results are compiled and reported. 3shannon l. werner, ryon p. graf, mark landers, david t. valenta, matthew schroeder, stephanie b. greene, natalee bales, ryan dittamore and dena marrinucci: analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization ctc clusters (figure 2c): defined as two or more adjacent ctcs, containing at least one traditional ctc, with shared cytoplasmic boundaries. ck(-) ctcs (figure 2d): defined as ck(-), cd45(-), with dapi intact. apoptotic ctcs (figure 2e): defined as ck(+), cd45(-) with a dapi pattern of chromosomal condensation and/or nuclear fragmentation/blebbing that is consistent with the classic definition of apoptosis [44]. figure 2. representative ctc subtypes detected by the epic platform. ctcs from prostate cancer patient samples were enumerated using the epic platform. representative 10x immunofluorescence images for the dapi (blue), cytokeratin (red), cd45 (green) channels are shown for ctc subtypes and the surrounding white blood cells (wbcs), with the three-channel merge to the far left of each image. classified ctc subtypes include a) traditional ctcs (ck+, cd45-, dapi+/intact), b) small ctcs (ck+, cd45-, dapi+/intact, with similar nuclear size to that of the surrounding wbcs), c) ctc clusters (two or more adjacent traditional ctcs that share cytoplasmic boundaries), d) ckctcs (ck-, cd45-, dapi+/intact), and e) apoptotic ctcs (ck+, cd45-, with dapi staining pattern consistent with chromosomal condensation and/or nuclear fragmentation). 2.2 analytical validation of ctc detection cultured cancer cell line cells (clcs; colo-205) were spiked into whole blood specimens from healthy donors (hd) at varying concentrations ranging from six-300 clcs/ slide (six slides each of six, 12, 25, 50, 100, 300 clcs/slide, and 24 additional slides were created for the 25 and 300 clcs/slide dilution). additionally, five slides of unspiked hd samples were prepared. all blood samples (spiked or unspiked) were processed as described above, and 3 x 106 nucleated cells were deposited per slide. slides were stained using the epic standard three-colour assay (ck/ cd45/dapi), and scanned using the epic ctc platform. one assay (staining/scanning) run consisted of three replicate tests of two slides per test (six slides total). following scanning and ctc classification, the number of clcs enumerated on two replicate slides was converted to clcs per millilitre of blood, and the resulting values were used to assess four critical assay performance characteris‐ tics (figure 3a): i. cell deposition repeatability and accuracy ii. linearity iii. specificity iv. precision/repeatability cell deposition repeatability and assay accuracy were evaluated by measuring the dapi counts per slide and calculating percent nucleated cell recovery for three replicate tests (two slides per test; six slides total) for each of six serial clc dilutions (six, 12, 25, 50, 100 and 300 clcs/ slide), and for five slides of unspiked healthy donor (hd) blood (zero clcs/slide). percent coefficient of variation (%cv) of dapi counts was calculated for each clc dilution tested. assay linearity was evaluated by plotting the actual clcs/ slide recovered versus the theoretical number of clcs/ slide for each of the clc concentrations tested, where each concentration was tested in triplicate tests (two slides per test). the linear regression was calculated. assay specificity was determined by measuring the number of clcs detected on the five unspiked healthy donor slides (zero clcs/ml), as well as in 18 healthy donor samples (two slides tested per sample) in a clinical feasibility analysis (figure 4). assay precision/repeatability was measured by calculating the percent coefficient of variation (%cv) of clc counts from the 25 clcs/slide and 300 clcs/slide dilutions. intraassay variability was measured for one operator who performed three tests on one day (two replicate slides/test; six slides total), whereas inter-assay variability was measured across three operators who performed five assay runs total (one run per day), with each run consisting of three tests of two slides per test (30 slides total). intraoperator repeatability was measured for one operator who performed three assay runs (one run per day), with each run consisting of three replicate tests of two slides/test (18 slides total). inter-operator repeatability was measured for three operators who performed one assay run each (18 slides total). 2.3 clinical feasibility of ctc detection forty-four (44) blood samples from all-comer metastatic castrate-resistant prostate cancer (mcrpc) patients were sent to epic sciences, processed onto slides, and two slides per sample were tested for ctc enumeration as described above. ctc enumeration (ctc/ml) was compared to that found for 18 healthy donor blood samples (two slides tested per sample), which also further addressed assay specificity. the prevalence of ctc clusters, ckctcs and apoptotic 4 j circ biomark, 2015, 4:3 | doi: 10.5772/60725 ctcs are also reported for the mcrpc cohort as percent of samples containing each subtype. 3. results 3.1 analytical validation to assess assay accuracy, linearity, specificity and preci‐ sion/repeatability, the ck(+), cd45(-), dapi(+) colo-205 cancer cell line control (clc) was spiked into healthy donor blood and processed onto slides as mock clinical samples. six (6) slides each of six clc dilutions (six, 12, 25, 50, 100, 300 clcs/3 x 106 wbcs per slide and 24 additional slides for both the 25 and 300 clcs/slide dilution) were prepared, as well as five slides of unspiked hd samples. a descrip‐ tion of the assay performance characteristics evaluated is described in figure 3a. 3.1.1 cell deposition repeatability, assay accuracy and linearity to evaluate the repeatability of the cell deposition onto slides, one operator performed three replicate ctc enu‐ meration tests (two slides/test; six slides total) for each of the six clc dilutions, as well as for five replicate unspiked hd slides, for a total of 41 slides. the numbers of nucleated cells per slide (dapi counts) were counted, and the percent recovery of nucleated cells per clc dilution was calculated (figure 3b). across 41 slides, the mean percent recovery was 88 % (2.64x106 nucleated cells/slide), with an observed coefficient of variation (%cv) of 9.7 %. the %cvs for each cell dilution were 6.7 % (zero clcs/slide), 6.3 % (six clcs/ slide), 3.0 % (12 clcs/slide), 7.0 % (25 clcs/slide), 2.7 % (50 clcs/slide), 3.8 % (100 clcs/slide), and 1.2 % (300 clcs/slide). assay linearity was evaluated by plotting the number of clcs recovered per slide for each of the spikein concentrations from three replicate tests (two slides/test; six slides total) against the calculated clc spike-in con‐ centration (figure 3c), and the linear regression was calculated. the assay was shown to be linear across all sample dilutions tested (r2=0.999). importantly, zero ctcs were enumerated in the unspiked healthy donor slides, thereby showing assay specificity. assay specificity is further established in the clinical feasibility testing of 18 healthy donor samples (two slides per sample tested), as shown in figure 4. figure 3. analytical validation of the epic platform. a) the analytical characteristics assessed to benchmark the performance of the epic ctc platform. varying concentrations of colo-205 cell line cells (clcs) were spiked into healthy donor blood, red blood cells lysed, and 3 x 106 nucleated cells were deposited onto slides, ranging from 0-300 clcs/slide. slides were stained with a cocktail of ck, cd45 and dapi antibodies, and assay accuracy, linearity, specificity and precision were determined as described in the methods. for each analysis, a “run” is comprised of three tests, with each test consisting of two replicate slides. b) the accuracy and repeatability of cell deposition was assessed calculating percent nucleated cell recovery (y-axis; mean ± sem) for one run each of six serial clc dilutions (six, 12, 25, 50, 100 and 300 clcs/slide), and for five slides of unspiked healthy donor (hd) blood (zero clcs/slide). c) assay linearity was characterized by plotting the actual clcs/slide recovered (y-axis) versus the theoretical number of clcs/slide (x-axis) for seven clc concen‐ trations (six slides tested/concentration), and the linear regression was calculated. assay specificity was determined by measuring the number of clcs detected on the unspiked healthy donor slides (zero clcs/ml). d) assay precision/repeatability was measured by calculating the percent coefficient of variation (%cv) for clc counts from the 25 clcs/slide and 300 clcs/slide dilutions. intra-assay variability was measured for one operator who performed one assay run, whereas inter-assay variability was measured across three operators who performed five assay runs total (one assay run per day). intra-operator repeatability was measured for one operator who performed three assay runs on separate days, whereas inter-operator repeatability was measured for thee operators who performed one assay run each. 5shannon l. werner, ryon p. graf, mark landers, david t. valenta, matthew schroeder, stephanie b. greene, natalee bales, ryan dittamore and dena marrinucci: analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization figure 4. clinical feasibility of ctc enumeration in metastatic castrateresistant prostate cancer patient samples. forty-four (44) mcrpc patient blood samples were tested for ctc enumeration using the epic platform, and the results were compared to those from 18 healthy donor (hd) blood samples. a) ctc incidence was calculated as ctc per millilitre (ctc/ml) of patient blood for traditional ctcs and ctc clusters (left, ctcs + clusters) and all ctc candidates (right; ctcs, ctc clusters, ckctcs, and apoptotic ctcs). each dot on the graph is representative of the ctc/ml value for that patient sample. b) summary of the range, median and mean ctc/ml values for 18 healthy donor and 44 mcrpc samples for traditional ctcs and ctc clusters (left) and all ctc candidates (right). 3.1.2 assay precision and repeatability precision of the epic platform was assessed intraand interassay as well as intraand inter-operator by calculating the observed variation in the enumeration of clcs in the 25 clcs/slide and 300 clcs/slide dilutions (figure 3d). one assay run consisted of three replicate tests, with two slides stained per test (six slides total) per clc concentration. intra-assay repeatability was assessed by calculating the %cv of clcs detected per millilitre (clcs/ml) of blood from slides prepared by a single operator who performed one assay run (six slides total) for both the 25 clcs/slide and 300 clcs/slide dilutions. the observed %cvs were calculated to be 20.7 % and 1.3 %, respectively. inter-assay repeatability was determined from calculating the %cv of clcs/ml blood detected based upon five assay runs performed by three operators on separate days, for a total of 30 slides evaluated per clc dilution. the resulting %cvs calculated were 4.7 % for 300 clcs/slide, and 17.3 % for 25 clcs/slide. intra-operator precision was evaluated for one operator who performed one assay run on three separate days (18 slides total), which resulted in %cvs of 3.1 % for 300 clcs/slide and 20.9 % for 25 clcs/slide. finally, inter-operator precision was assessed for three operators who performed one assay run each (18 slides total) on separate days. for this parameter, the observed %cv for the 300 clcs/slide dilution was found to be 5.3 % and 16.5 % for the 25 clcs/slide dilution. coefficient of variation for the detection of 300 clcs/slide increased from 1.3 % for samples analysed by a single operator in a single run to only 3.1 % when the single operator performed a single run on three separate days, whereas it increased to only 4.7 % when three operators performed five runs in total over five days. for the 25 clcs/ slide dilution, the %cv in clc detection was the same if an operator performed a single run on one day, or if he performed one run on three separate days (20.7 % and 20.9 %, respectively). similarly, the %cv in detection of 25 clcs/slide was found to be similar between a single operator who performed one run and multiple operators who performed runs on separate days. the variability for the detection of 25 clcs/slide was found overall to be higher than that for 300 clcs/slide, which is not unexpect‐ ed due to the inherent variability of performing serial cell dilutions, and the %cv was found to be at or under 20 % for all parameters tested. 3.2 clinical feasibility to establish clinical feasibility, we tested 44 all-comer metastatic castrate-resistant prostate cancer (mcrpc) samples, and compared ctc enumeration to that from 18 healthy donor samples (figure 4). traditional ctcs (ck+/ cd45-/intact nuclei) and clusters of traditional ctcs were detected in 89 % of the mcrpc samples analysed with a range of 0-20 ctc/ml (median=2.0 ctc/ml), whereas 23 % of samples were found to have ctc clusters. considering all possible ctc candidates (including non-traditional ck(-) ctcs and apoptotic ctcs), 100 % of the mcrpc samples had detectable ctcs (figure 4b) with a range of 1-28 ctc/ml (median=6.0 ctc/ml). in this cohort, we observed that 70 % of samples had ck(-) ctcs, whereas 77 % had apoptotic ctcs. in contrast, zero ctcs were enumerated in the 18 healthy donor samples tested, which further exhibits the specificity of the epic platform. so far, ctc and biomarker stability on patient slides have been demonstrated for up to one year, with studies ongoing to determine longer term storage stability (data not shown). 3.3 downstream capabilities of the epic platform in addition to ctc enumeration, the epic ctc platform was designed with integrated downstream capabilities for the evaluation of cell morphology characteristics, protein biomarker expression and genomic analyses (fish and ngs). the platform has an open fourth fluorescent channel for the evaluation of protein biomarker expression in patient ctcs (figure 1, step 2 of the ctc assay workflow), with a fifth channel currently in development. currently, a wide variety of fourth channel markers have been devel‐ oped targeting multiple disease indications, emt cell markers and drug sensitivities. using the epic platform, it is possible to simultaneously evaluate the expression of targetable protein biomarkers (if), the presence of specific driver genomic alterations (fish) and genome-wide copy 6 j circ biomark, 2015, 4:3 | doi: 10.5772/60725 number alterations (ngs) from a single tube of blood, and importantly, within individual patient ctcs. both fish and ngs analysis can be performed on single ctcs detected using the immunofluorescence assay workflow (figure 1). the potential to integrate the analysis of multiple biomarkers from a single patient blood sample, including genomic, protein and morphological endpoints, holds great promise for better understanding of disease progres‐ sion, heterogeneity and sensitivity/resistance to targeted therapies [16, 40, 45]. as proof of concept (figure 5), slides were created from healthy donor blood samples spiked with prostate cancer cell lines (vcap, lncap and pc3), and were analysed to confirm the presence of known protein and genetic markers associated with prostate cancer disease progression and/or resistance to targeted therapies. common molecular alterations in mcrpc include changes in androgen receptor (ar) signalling through alterations in ar protein expression levels and gene copy number variations, the presence of ar splice variants and mutations, and altered pi3k-axis signalling through pten gene deletions 46-49]. relative ar protein expression was analysed in vcap, lncap and pc3 clcs detected using the fourth channel capability of the epic platform (figure 5a), and confirmed with the relative ar expression known to be found in the high-, mediumand low-ar expressing cell lines, respec‐ tively. pten tumour suppressor gene loss was evaluated in these prostate cancer clcs by fish (figure 5b), where the signals for pten (green) and cep10 (red) can be compared between the clc (yellow circle) and the sur‐ rounding white blood cells (white carrots). in all three clc examples, the cep10 signals were found to be greater than two, which is indicative of polyploidy and is consistent with the tumour origin of the clcs. the vcap clc was shown to have pten non-deleted status (pten=cep10; three signals each), whereas lncap showed heterozygous pten loss (pten=2; cep10=4) and pc3 showed homozy‐ gous pten loss (pten=0, cep10=4). the presence of surrounding wbcs provide ample controls for evaluating the false-positive rate of detection of genetic alterations. additionally, ar gene copy number variation (cnv) was evaluated in single clcs using next-generation sequencing (ngs; figure 5c). as reported previously, ar gene amplification was found in the vcap clc [50, 51], but not the lncap or pc3 clcs. using the platform’s integrated downstream analysis, multiplexed evaluation of genomic figure 5. epic platform capabilities for the evaluation of protein and genetic biomarkers. human prostate cancer cell line control cells (clcs; vcap, lncap or pc3) were spiked into healthy donor blood, processed onto slides and stained with ck, cd45, dapi and n-terminal androgen receptor (ar) antibodies. additional slides were processed for pten loss by fish. subsequently, individual clcs were recovered and analysed for whole genome copy number variation by ngs. a) representative images (10x) of individual clcs detected, each with varying levels of ar expression (ar signal denoted in white). b) representative images of pten gene deletion status in clcs (yellow circles) and surrounding wbcs (white carrots), as determined by pten fish analysis. blue: dapi, red: cep10 signals, green: pten signals. the number of pten and cep10 signals found in each clc example are reported to the right of the image. c) comparison of log2 cnv (y-axis) found within isolated vcap (red), pc3 (grey), and lncap (blue) clcs across the x chromosome (x-axis). each data point represents the relative copy number within a 100,000 bp window normalized to healthy donor control wbc cnv. the highlighted window (yellow dotted line) contains the ar gene. 7shannon l. werner, ryon p. graf, mark landers, david t. valenta, matthew schroeder, stephanie b. greene, natalee bales, ryan dittamore and dena marrinucci: analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization and protein biomarkers within a patient sample and individual patient ctcs offers a unique opportunity to better understand mechanisms of resistance to therapy and to inform the optimization of targeted therapy regimens. as recently summarized by macaulay and voet, substantial advances in single cell genomic analyses for the detection of point mutations, copy number variation (cnv), loss of heterozygosity (loh) and structural variants have been made [52]. however, amplifying dna material via whole genome amplification (wga) has the inherent risk of creating bias and false-positive/negative results. an alternative strategy to increase dna quantity while preventing the complications associated with single cell analysis, would be to pool individual ctcs isolated from a patient sample by phenotypic subtype. however, this would negatively impact the ability to evaluate intrapatient heterogeneity. development of quality control (qc) criteria for evaluating the quality of both the ngs library and post-sequencing data has been described and imple‐ mented to avoid such false-positive and false-negative results [53]. additionally, secondary validation studies utilizing immunofluorescence to identify ctcs, followed by dna fish may confirm the incidence of specific genomic aberrations in patient ctcs, for which the epic platform is suitable. 4. discussion this study encompasses the analytical validation of the epic ctc detection platform where we assessed critical assay performance characteristics including assay accura‐ cy, linearity, specificity and precision/repeatability (intraassay, inter-assay, intra-operator and inter-operator). notably, the platform demonstrated a high percentage of nucleated cell recovery for all clc concentrations tested (figure 3b), and showed excellent assay linearity (r2 = 0.999) (figure 3c). furthermore, the assay is highly repeatable for detecting clcs at multiple dilutions within and across assay runs and multiple operators (figure 3d). the assay is also highly specific, in that zero ctcs were detected in unspiked healthy donor samples (figure 3c, figure 4). while in-depth clinical feasibility studies are underway, data from a small cohort of clinically confirmed all-comer mcrpc patient samples tested with the epic ctc platform showed that ≥1 traditional ctc/ml was detected in 89 % of patient samples, whereas 100 % of samples had ≥1 ctc/ ml when additionally considering the ck(-) and apoptotic ctc subpopulations. in this patient cohort, we observed the presence of multiple ctc subtypes, including ck(+) ctcs, ctc clusters, ck(-) ctcs and apoptotic ctcs. this is in contrast to the healthy donor samples tested, in which zero ctcs were enumerated in all 18 samples. in figure 5, we discuss the downstream capabilities of the epic plat‐ form, including methods to evaluate protein (immuno‐ fluorescence) and genetic (fish, ngs) biomarkers. in the last 10 years, great strides have been made with the technological development of ctc detection strategies, as well as advancements in the understanding of ctc biology, resulting in over 16,000 publications. however, further investigation of ctc utility in directing personal‐ ized medicine is warranted, and many studies and trials are now underway to address this. low ctc abundance poses a challenge with respect to having a statistically significant number of cells for biomarker analyses, and thus to the evaluation of tumour heterogeneity. low ctc incidence has been reported for non-metastatic or locally advanced prostate [32, 54] or locally advanced pancreatic adenocar‐ cinoma [55], and non-metastatic colorectal cancer [56], which may limit the utility of ctc assays for diagnosis of early stage disease. increasing the sensitivity of ctc detection may address this issue; however, increased sensitivity may result in the detection of false-positive results in healthy controls or in patients with benign disease [57]. additionally, some cancer indications such as ovarian [58] and nsclc [59] have reportedly low ctc numbers; however, the low abundance of ctcs identified may be a result of the ctc detection platform selected and reliance on using ctc enrichment strategies. emerging data show that ctcs are detected in larger numbers in these indica‐ tions when using epithelial marker-independent ap‐ proaches [60-62]. detection of ctcs with an epithelial-to-mesenchymal transition (emt) phenotype remains a challenge, and their functional and clinical relevance are still under investiga‐ tion. ctc platforms that rely on epithelial marker enrich‐ ment are ill-suited to detect these cells [20]; however, there is no universal biomarker that ensures the detection of mesenchymal ctcs. for example, some emt markers (i.e., vimentin) are expressed on the surrounding leukocytes, and ctcs show a broad range of phenotypes during emt and may express neither cytokeratins nor emt markers [4]. in addition to identifying additional mesenchymal mark‐ ers, it is unclear as to whether mesenchymal ctcs are even capable of seeding distal metastases, as these cells may be unable to undergo the reverse “mesenchymal-to-epithe‐ lial” transition [63]. ctcs may have an intermediate phenotype where they partially downregulate epithelial markers while partially upregulating mesenchymal markers, or express neither. these highly plastic cells suggest their stemness [64] but additional studies are warranted to evaluate their clinical significance. the epic ctc platform uses an unbiased ctc detection approach in which additional ctc subpopulations includ‐ ing ctc clusters, ck(-) ctcs, small ctcs and apoptotic ctcs are enumerated. while the biological significance of each ctc subtype is still under investigation, emerging literature suggests that each subtype may play an impor‐ tant role in tumour metastasis, epithelial-to-mesenchymal transition (emt), and as potential markers for cancer therapeutic responses. although not extensively validated as a biomarker, the presence of ctc clusters is reported to be a negative prognostic factor for survival in cohorts of small-cell lung cancer [65], as well as breast cancer and 8 j circ biomark, 2015, 4:3 | doi: 10.5772/60725 prostate cancer patients [66]. preclinical models suggest that ctc clusters might have survival advantages in blood circulation including resistance to programmed cell death and physical sheer forces [65-68]. there is limited publish‐ ed data on the biological relevance of ck(-) ctcs; however, it has been shown that ctcs of epithelial origin can display a range of both epithelial and non-epithelial gene biomark‐ er signatures [4, 32, 69, 70]. similarly, in-depth literature describing the biological significance of apoptotic ctcs is limited, although the enumeration of apoptotic ctcs has been explored as a potential drug response biomarker [71]. characterization of the prevalence and functional signifi‐ cance of these ctc subtypes as clinical markers for predicting sensitivity to targeted therapies and under‐ standing disease progression is of great interest, and is currently the subject of multiple ongoing clinical studies at epic sciences. an important feature of the epic platform is the capacity for the creation of a biorepository of patient slides, which may be frozen and stored for retrospective biomarker analyses. while one ctc test uses two slides, approximate‐ ly 10-12 slides are created per patient sample and the remaining slides are banked at -80 °c. in the wake of new biomarker discoveries and/or generation of biomarker hypotheses for a particular indication, banked patient slides may be evaluated retrospectively and correlated with clinical outcome data. so far, we have established patient slide stability for storage at -80 °c for up to one year with respect to their ability to retain ctcs and biomarker expression. longer-term stability studies of frozen patient slides are currently underway. while advancements in genomics and proteomics have contributed to our molecular understanding of cancer, emerging research also highlights inter-patient [72] as well as spatial [73-77] and temporal [8-11] intra-patient hetero‐ geneity, both of which limit the efficacy of targeted therapies and thus compromise patient outcomes. charac‐ terizing intra-patient heterogeneity has been posited as a means to intelligently guide precision-targeted therapies based on the current state of disease [7, 78, 79] and the development of resistance mechanisms to therapy. a comprehensive approach based on the detection of a panel of protein and genetic ctc biomarkers can give rise to a patient tumour’s molecular signature, which will not only inform the development of targeted therapeutic strategies, but will also allow for patient surveillance based upon molecular alterations over time. evaluation of biomarker panels in a liquid biopsy may provide the molecular landscape of primary and metastatic lesions, while offering the opportunity to evaluate molecular tumour evolution as a real-time film instead of a single frame snapshot. 5. conflict of interest authors slw, rpg, ml, dtv, ms, 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marrinucci: analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization cbx821819 1..5 research article suppression of cathepsin k biomarker in synovial fluid as a free-drug– driven process bennett ma 1 , gregg wesolowski 2 , bin luo 3 , traci lifsted 3 , keith wessner 3 , gary adamson 1 , helmut glantschnig 2 , and laura s lubbers 3 abstract cathepsin k (catk) inhibitors exhibited chondroprotective and pain-reducing effects in animal models, however, improvements were relatively modest at dose levels achieving maximal suppression of catk biomarkers in urine. in this report, a previously characterized catk inhibitor (mk-1256) is utilized to explore the potential of reduced target engagement and/or suboptimal exposure (free drug) as limiting factors to the pharmacological potential of catk inhibitors in the knee joint. following oral administration of mk-1256 at a dose level achieving maximal inhibition of urinary biomarker (helical peptide) in dogs, full suppression of the biomarker in synovial fluid was observed. subsequent tissue distribution studies conducted in dogs and rabbits revealed that mk-1256 levels in synovial fluid and cartilage were consistent with the free-drug hypothesis. reasonable projection (within twofold) of drug levels in these tissues can be made based on plasma drug concentration with adjustments for binding factors. these results indicate that the previously observed efficacies in the animal models were not limited by compound distribution or target engagement in the knee tissues. keywords cathepsin k, mk-1256, osteoarthritis, synovial fluid, cartilage date received: 19 april 2018; accepted: 1 december 2018 introduction osteoarthritis (oa) is a debilitating disease that is commonly characterized by joint pain and stiffness stemming from progressive cartilage loss, subchondral bone changes, osteophyte formation, and/or inflamed synovium. 1,2 excessive degradation of tissue matrices is thought to play an important role in the disruption of the joint homeostasis, as evidenced by elevated proteolytic cleavage of collagen observed in osteoarthritic cartilage along with accelerated turnover of subchondral bone and vertical trabeculae. 3 various hydrolytic enzymes catalyzing the breakdown of joint matrices have been implicated for their roles in oa progression, making them appealing targets for the development of disease-modifying anti-oa drugs (dmoads). 1,2 cathepsin k (catk), a cysteine protease displaying triple helical collagen hydrolase activity, is capable of disintegrating collagen type i (in bone) and type ii (in cartilage) and aggrecan. 3,4 catk inhibitors are effective bone antiresorptive agents for the treatment of osteoporosis in clinical trials, demonstrating improvements in bone mineral density and bone strength with robust reductions in collagen 1 department of pharmacokinetics, pharmacodynamics and drug metabolism, merck & co., west point, pa, usa 2 department of bone biology, merck & co., west point, pa, usa 3 department of pharmacology, merck & co., west point, pa, usa corresponding author: bennett ma, department of pharmacokinetics, pharmacodynamics and drug metabolism, wp75b-200, merck & co., west point, pa 19486, usa. email: bennett_ma@merck.com journal of circulating biomarkers volume 8: 1–5 ª the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454418821819 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://orcid.org/0000-0002-8494-9875 https://orcid.org/0000-0002-8494-9875 mailto:bennett_ma@merck.com https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454418821819 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage degradation biomarkers. 5,6 early assessments of catk inhibitors as dmoads in animal models revealed promising chondroprotective and pain-reducing effects. 7–9 however, the improvements were relatively modest (approximately 30–50%) when compared to maximal suppression (approximately 60–80%) observed for urinary biomarkers of catk-mediated collagen degradation. in an attempt to explore the potential of reduced target engagement and/or exposure as limiting factors for the observed efficacy, experiments were conducted to determine biomarker suppression and drug concentration at the knee joint using a previously characterized catk inhibitor mk-1256. 10 materials and methods in vivo dog and rabbit studies in vivo dog and rabbit studies were conducted in a similar manner as reported previously. 10 briefly, adult male beagle dogs received a daily oral dose of mk-1256 (at 7, 70, or 700 mg/kg; n ¼ 7 or 8 per dose group) prepared in 0.5% methylcellulose (mc) with 0.25% sodium dodecyl sulfate (sds) or control vehicle. on day 6, serial plasma samples were collected at 0, 1, 2, 4, 7 and 24 h post-dose with corresponding urine samples also collected in the same 24-h period. after the collection of the last plasma and urine samples on day 7, synovial fluid and articular cartilage samples from both knees were immediately harvested following euthanasia of dogs (n ¼ 4) dosed with 700 mg/kg mk-1256 and with control vehicle. distribution of mk-1256 to synovial fluid and articular cartilage was also studied in 9-monthold ovariectomized female new zealand white rabbits (approximately 3.5 kg body weight; n ¼ 5). an oral dose of 30 mg/kg mk-1256 was administered to the rabbits daily for 4 days, with serial plasma samples collected at 0, 1, 2, 4, 7, and 24 h post-dose on day 4. immediately following the collection of 24-h plasma sample, rabbits were euthanized and samples of synovial fluid and articular cartilage from both knees were harvested. all animal studies were conducted in compliance with iacuc policies. measurement of biomarker in urine and synovial fluid the bone resorption biomarker helical peptide (hp) in dog urine was measured using an enzyme-linked immunosorbent assay followed by normalization to creatinine level determined from the same sample. 10 for synovial fluid, samples were pretreated with bovine hyaluronidase (sigma, st louis, missouri, usa; 1 unit per 0.2 ml synovial fluid) at 37�c overnight was performed prior to quantification. quantitative analysis of mk-1256 in plasma, synovial fluid, and cartilage the concentration of mk-1256 in dog and rabbit plasma was determined by liquid chromatography–tandem mass spectrometry (lc-ms/ms) analysis. 10 this method was modified for synovial fluid, with samples diluted 1:10 (w/w) with control plasma prior to quantification. for cartilage harvested from knee joint of dog and rabbit, each milligram of tissue was enzymatically digested with 4 ml of phosphate-buffered saline containing 2 mg/ml each of collagenase (wako chemicals, richmond, virginia, usa) and hyaluronidase at 37�c for 24 h and then processed with a protein precipitation method followed by lc-ms/ms analysis. binding of mk-1256 to plasma proteins and to cartilage were performed as described by ma et al 10 and wang et al, 11 respectively. calculations prediction of drug concentration in synovial fluid and in cartilage was calculated with the following equations ½mk-1256�synovial fluid ¼ ½mk-1256�unbound; plasma fraction unbound of mk-1256 in synovial fluid ð1þ ½mk-1256�cartilage ¼½mk-1256�unbound; plasma � cartilage binding factor ð2þ in which the fraction unbound of mk-1256 in synovial fluid was calculated based on a 2.8-fold difference in protein content between synovial fluid (approximately 25 mg/ml) and plasma (approximately 69 mg/ml), 11–13 and the cartilage binding factor was determined by incubation of mk-1256 with bovine knee cartilage based on the method previously reported. 11 statistical analyses, including student’s t-test, were performed using the microsoft excel software package (redmond, washington, usa). results a dose-dependent decrease in urinary helical peptide (hp) level was observed following oral administration of mk1256 in dogs (figure 1(a)). this biomarker for collagen degradation was maximally suppressed on day 6 following a daily dose of mk-1256 at 700 mg/kg. full suppression of hp in synovial fluid was observed at this dose level (figure 1(b)), suggesting that mk-1256 exposure to the knee joint was sufficient to achieve full target engagement. direct measurement of (total) mk-1256 levels in synovial fluid and cartilage revealed lower drug concentrations in these tissues as compared to that in plasma (table 1), consistent with the anticipation that knee tissues exhibit less drug binding as a result of reduced protein content. with the apparent applicability of the free-drug hypothesis with regard to mk-1256 distribution in the knee, prediction of drug concentration in synovial fluid was performed with an 2 journal of circulating biomarkers estimated unbound fraction based on the protein content. projection of mk-1256 concentration in cartilage was obtained by applying a binding factor determined in vitro. predicted (total) drug concentrations in synovial fluid and cartilage were in reasonable agreement (within twofold) with the measured values (table 1). in a distribution study conducted in rabbits receiving a daily dose of 30 mg/kg mk-1256, the prediction drug concentrations in synovial fluid and cartilage were also within twofold of the observed values (table 1), indicating that the presented approach to predict drug concentrations in the knee tissues is applicable across species. discussion catk inhibitors have been suggested to display certain characteristics of dmoads, exhibiting chondroprotective and pain-reducing effects in various animal models. 7–9 in those cases, modest improvements were observed at dose levels achieving maximal suppression of urinary biomarkers for collagen degradation. to investigate the potential of reduced target engagement and/or drug level in the knee joint as contributing factors for limited efficacy, experiments were conducted in the current study to measure both the catk activity biomarker (hp) level and the distribution of a highly selective catk inhibitor (mk-1256) in the knee. maximal suppression of urinary biomarker was observed in dogs receiving a daily dose of mk-1256 at 700 mg/kg, consistent with the measured plasma drug concentration (cavg: 0.52 mm; table 1) being 43-fold higher than the reported half maximal effective concentration (12 nm). 10 at this dose level, full suppression of hp in synovial fluid was also achieved, suggesting that the unbound drug concentrations in synovial fluid and plasma closely resembled each other – a phenomenon that had been observed for several nonsteroidal antiinflammatory drugs (nsaids). 14,15 synovium is a highly vascularized membrane that encapsulates synovial fluid present in the articular cavity. 14,15 the porous nature of the membrane allows small molecules to diffuse in and out of the joint cavity efficiently. pharmacokinetic studies conducted in human with intra-articular administration of nsaids showed that these compounds efficiently diffused out of the synovial fluid, with half-life values observed in synovial fluid similar to those detected in plasma. 14,15 application of the non-compartmental analysis revealed that the area under the concentration–time curve (auc) measured in synovial fluid obtained after oral administration of nsaids was in good agreement with the plasma auc. consistent with this observation, the average (total) plasma drug concentrations determined in the current study were in reasonable agreement (within twofold) with the drug concentrations in synovial fluid after adjusting for protein binding (table 1). as the unbound drug concentration in synovial fluid approached equilibrium with the unbound fraction in plasma at steady state, full suppression of the biomarker in synovial fluid was observed at the dose level that achieved maximal inhibition of urinary biomarker (figure 1). while biomarker and the pharmacodynamics effect of mk-1256 was not directly assessed in articular cartilage, predicted and observed mk-1256 exposure data are congruent with inhibition of catk in this compartment in dogs and rabbits. taken together, the presented results in the animal models indicate that catk biomarker suppression in synovial fluid is a free-drug–driven process. early human pharmacokinetics studies conducted with nsaids showed evidence of applicability of free-drug hypothesis regarding compound distribution to the knee joint. 14 for compounds exhibiting extensive binding to plasma proteins (e.g. protein binding of >98%), drug concentration in synovial fluid in reference to that in plasma u ri n e h p /c r, μ g /m m o l c r 0 50 100 150 200 vehicle control 700 μg/kg 70 μg/kg 7 μg/kg p < 0.001 p < 0.01 p = 0.32 s y y n o v ia l fl u id h p , μ g /l 0 2 4 6 8 10 12 14 vehicle control 700 μg/kg p < 0.001 (a) (b) figure 1. biomarker suppression in dog urine (a) and synovial fluid (b). male beagle dogs received a daily oral dose of mk-1256 at 7, 70, or 700 mg/kg (n ¼ 7 or 8). urine samples were collected on day 6 while synovial fluid samples from both knees were immediately harvested 24 h after the last dose from the dogs in the high-dose group (700 mg/kg) and control group (n ¼ 4 per group). student’s t-tests were performed and the corresponding p values were reported. ma et al. 3 should be proportional to the (albumin) protein concentration ratio between these two matrices—an observation that was reported for inhibitors of matrix metalloprotease following oral administration of compound to rabbits. 11 with the consideration of a 2.8-fold difference in protein content between synovial fluid and plasma, drug binding to synovial fluid can be estimated from the plasma protein binding value determined in vitro. 13 this approach allows a projection of drug concentration in synovial fluid based on plasma drug level when equilibrium of unbound drug is reached between these matrices. reasonable prediction (within twofold) was observed in the current study as well as data reported by wang et al. 11 and day et al., 14 confirming the feasibility of the current approach to estimate drug level in synovial fluid based on plasma drug concentration and the unbound fraction of drug in plasma. in conclusion, the current study confirmed that the distribution of mk-1256 to the knee joint is consistent with the free-drug hypothesis, leading to full suppression of local catk biomarker in the synovial fluid at the dose level achieving maximal inhibition of urinary biomarker. the drug levels in synovial fluid and cartilage can be reasonably estimated based on plasma drug concentration with adjustments for binding factors. these results indicated that the observed efficacies in the animal models of oa were likely not limited by compound distribution or target engagement (i.e. catk inhibition) in the knee tissues. declaration of conflicting interests the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: all authors are current or former employees of merck & co., who were employed at the time the experiment was conducted. the authors alone are responsible for the writing and content of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was funded by merck & co. orcid id bennett ma 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article lilite sadovska1,2, cristina bajo santos1,2, zane kalniņa1 and aija linē1* 1 latvian biomedical research and study centre, riga, latvia 2 faculty of biology, university of latvia, riga, latvia lilite sadovska and cristina bajo santos contributed equally to this work. *corresponding author(s) e-mail: aija@biomed.lu.lv received 09 january 2015; accepted 12 march 2015 doi: 10.5772/60522 © 2015 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract extracellular vesicles (evs) have recently emerged as important mediators of intercellular communication. they are released in the extracellular space by a variety of normal and cancerous cell types and have been found in all human body fluids. cancer-derived evs have been shown to carry lipids, proteins, mrnas, non-coding and structural rnas and even extra-chromosomal dna, which can be taken up by recipient cells and trigger diverse physiological and pathological responses. an increasing body of evidence suggests that cancer-derived evs mediate paracrine signalling between cancer cells. this leads to the increased invasiveness, proliferation rate and chemoresistance, as well as the acquisition of the cancer stem cell phenotype. this stimulates angiogenesis and the reprogramming of normal stromal cells into cancer-promoting cell types. furthermore, cancer-derived evs contribute to the forma‐ tion of the pre-metastatic niche and modulation of antitumour immune response. however, as most of these data are obtained by in vitro studies, it is not entirely clear which of these effects are recapitulated in vivo. in the current review, we summarize studies that assess the tissue distribution, trafficking, clearance and uptake of cancerderived evs in vivo and discuss the impact they have, both locally and systemically. keywords extracellular vesicles, biodistribution, traffick‐ ing, tumour microenvironment, immunosuppression, metastatic niche 1. introduction extracellular vesicles (evs) are a heterogeneous population of nanosized membrane vesicles that are released in the extracellular space by almost all normal and cancer cell types. currently, three broad categories of evs have been defined. these are based on the mode of biogenesis: (i) exosomes, (ii) microvesicles and (iii) apoptotic bodies [1]. exosomes are evs of endocytic origin, which range from 50-150 nm in diameter. these are released into the extrac‐ ellular environment by a fusion of the multivesicular bodies (mvbs) with the plasma membrane. microvesicles (sometimes also referred to as ectosomes or microparticles) are large evs, ranging between 100-1000 nm in diameter, which are secreted by shedding or budding from the plasma membrane [2, 3]. recently, some cancer cells have been found to secrete very large evs (1-10 µm) called large oncosomes. these are due to the shedding of non-apoptotic plasma membrane blebs, which are characteristic of fastmigrating “aboeboid” tumour cells [4]. currently, there is 1j circ biomark, 2015, 4:2 | doi: 10.5772/60522 no consensus about whether these evs represent a subclass of microvesicles or an entirely new class of evs. apoptotic bodies are heterogeneous evs, which contain cytoplasm with condensed organelles and/or nuclear fragments. they are released into the surrounding extracellular space by apoptotic/dying cells [1]. in vivo, apoptotic bodies are quickly cleared by macrophages and other phagocytes [5]. although they have been found to carry mirnas, which can be functionally active in the recipient cells [6], they are structurally and functionally very different from live cellderived evs and will not be discussed in this review. although exosomes, microvesicles and oncosomes have distinct physical and biochemical properties, so far, no markers that can unambiguously distinguish these types of evs have been identified. furthermore, the current methods used for the fractionation of evs cannot reliably separate various types of evs [3]. furthermore, recent studies have uncovered substantial differences in the ev biogenesis of various cell types. this suggests that discrim‐ inating these types of evs could be more complex than initially thought [3, 7]. therefore, in the current review, we will use the term ev to designate all types of live cellsecreted vesicles. cancer-derived evs have been shown to carry a variety of lipids, proteins, mrnas, non-coding and structural rnas and even extra-chromosomal dna [8-10]. the molecular content of evs partially reflects that of the parent cells. however, studies have shown that they are enriched in certain molecules, indicating the existence of specific mechanisms that sort cargo into evs [11, 12]. overall, these mechanisms are poorly understood and are likely to be related to the mode of ev biogenesis. the sorting of specific proteins into evs can be mediated by the endosomal sorting complex for transport (escrt) machinery [13] or escrt-independent mechanisms such as tetraspanin [14] or ceramide-dependent pathways [15]. the sorting of rnas into evs can be mediated by the interaction of specific rna-binding proteins, such as hnrnpa2b1, with cis-acting elements in the rna sequence [12, 16]. evs can be taken up by recipient cells and trigger diverse biological effects. therefore, they have emerged as impor‐ tant mediators of intercellular communication, both in normal physiological processes and in the development of various diseases [8, 9, 17]. in cancer, evs have been shown to mediate paracrine signalling between cancer cells, crosstalk between tumour and microenvironment, contribute to the formation of the pre-metastatic niche and interfere with the anti-tumour immune response. however, most of the data regarding their role in cancer come from in vitro studies and it is not entirely clear which of the uptake mechanisms are recapitulated in vivo. most data are concerned with the fate of evs when they are internalized by various cell types, as well as what mechanisms govern the trafficking of evs in the body. in this review, we summarize studies that aim to assess the tissue distribu‐ tion, trafficking, clearance and uptake of cancer-derived evs in vivo. we also discuss the impact that they have been shown to have, both locally and systemically. 2. tissue distribution of cancer-derived evs evs have been found in various biological fluids, including blood, milk, urine, saliva, etc. [18]. here, they represent a heterogeneous mixture of evs derived from various cell types. several lines of evidence suggest that cancer-derived evs can be released into the circulation or other biofluids of cancer patients. at first, cancer patients have been found to have higher levels of circulating evs, compared to healthy controls [19-21]. secondly, evs isolated from the biofluids of cancer patients or tumour-bearing animals were shown to contain cancer-associated markers, such as melan-a [19], tyrp2 [22] and ca19-9 [23], and amplified or mutated oncogenes [9, 24]. however, until recently, very little was known about the half-life, clearance, trafficking and tissue distribution of cancer-derived evs in the body. data from in vivo studies that address these issues have only started to accumulate over the last few years. the main findings of these studies are summarized in table 1. in these studies, two different approaches for studying the biodistribution of evs have been exploited. one is based on administering exogenous evs into the circula‐ tion of experimental animals, while the other is based on tumour models that produce labelled evs endogenous‐ ly. most of the studies that used exogenously produced evs isolated them from a cell culture medium by differential ultracentrifugation and sucrose gradient, as described by thery et al. 2006 [25]. after the intrave‐ nous administration has been conducted, the evs are tracked in vivo. this is carried out either by labelling the evs with fluorescent membrane dyes, such as dii, pkh67 and pkh26 [22, 26-32], and loading them with superpar‐ amagnetic iron nanoparticles (spion5), allowing magnet‐ ic resonance tracking [33]; or by using evs engineered to display a membrane reporter [27, 30, 34]. ev reporter systems have been created by the genetic engineering of cell lines that produce evs with membraneanchored gaussia luciferase (gluc), biotin acceptor peptide (bap) [27] or green fluorescent protein (gfp) [34]. studies that are based on endogenously produced evs have used either genetically engineered cancer cell lines, which produced gfp-tagged cd63 [34, 35], or human cancer xenografts in mice, where cancer-derived evs were located by detecting human cd63 [32]. the ev detection methods vary depending on the method of ev labelling and the specific aim of each study. in vivo imaging system (ivis) is the method of choice for the analysis of ev tissue distribu‐ tion. however, studies that focus on the specific effects caused by cancer-derived evs in specific organs also used flow cytometry, microscopy, immunohistochemistry, gluc activity measurements, magnetic resonance, etc. 2 j circ biomark, 2015, 4:2 | doi: 10.5772/60522 cell line animal line ev labelling injection site detection method results ref. exogenously administered evs into cancer-free animals ts/a murine mammary tumour balb/c pkh67 i.v. flow cytometry evs are taken up by bone marrow cd11b +gr-1+ cells; suppress myeloid cell differentiation into dcs. [26] el-4 mouse lymphoma c57bl/6 irdye800 i.p. li-cor imager evs detected in the liver, lung, kidney and spleen, taken up by cd11b+gr-1+ cells. [28] b16-bl6 mouse melanoma c57bl/6 gluc-lactadherin i.v. via tail vein gluc activity measurement half-life of evs in the blood is ~2 minutes at 10 to 60 minutes after injection, evs are distributed mainly to the liver and lungs; at 4 hours – lungs and spleen. [30] balb/c gluclactadherin i.v. via tail vein las3000 ivis c57bl/6 pkh26 i.v. via tail vein fluorescent microscopy hek293t human embryonic kidney athymic nude mice gluc i.v. via retro-orbital vein bioluminescence imaging 30 minutes after injection, evs are distributed to the spleen, liver, lungs and kidneys; actively taken up by liver and lung cells but not spleen cells. evs are eliminated via hepatic and renal routes. [27] biotin-alexa680streptavidin i.v. via tail vein fmt imaging b16-f10 mouse melanoma c57bl/6 spion5 footpad in vivo mri evs home to the subcapsular sinus of lymph nodes. [33] mda-mb-231 human breast cancer nude mice dii i.v. via tail vein flow cytometry, if evs are internalized by macrophages in the lungs and brain, resulting in the activation of nf-κb pathway. [32] k562 human chronic myelogenous leukaemia sd rats n.a. i.v. via tail vein n.a. evs deliver hybrid bcr/abl dna to normal neutrophils; administration of evs induce cml phenotype in mice and rats. [36] nod/scid mice i.v. via tail vein n.a. exogenously administered evs into cancer bearing animals bsp73asml bdx rat pancreatic adenocarcinoma bdx rats n.a. footpad – first evs, then cells n.a. evs from metastatic cells support the metastatic spread of non-metastatic cells to lymph nodes and lungs. [37] b16-f10 mouse melanoma c57bl/6 albino; i.v. injection of melanoma cells dir footpad ivis evs home to sentinel lymph nodes and enhance migration of melanoma cells to ev-rich sites in lymph nodes. [29] b16-f10 and b16-f1 mouse melanoma c57bl/6 mice with orthotopic b16-f10 tumours pkh67 i.v. confocal microscopy evs home to the lungs, bone marrow, liver and spleen. evs enhance metastasis by bone marrow education via the transfer of met to the bone marrow progenitors. [22] hek293t human embryonic kidney rag2-/mice with breast cancer (hcc70) xenografts dir i.v. via tail vein ivis egfr-targeted evs home to tumour microenvironment and can deliver mirnas to egfr-expressing breast cancer cells. [31] hek293t human embryonic kidney athymic nude mice with glioma xenograft gluc i.v. via tail vein bioluminescence imaging similar amounts of evs are found in tumours, spleen and liver. [27] 4t1 mouse mammary tumour balb/c with 4t1 orthotopic tumour dir i.v., i.t. ivis200 evs are taken up in the liver and spleen, very little amounts travel to the tumour. slower uptake and clearance in mice with impaired innate immunity. intratumourally administered evs stay associated with tumour. [38] nude mice with 4t1 orthotopic tumour nod.cb17prkdcscid/j with 4t1 orthotopic tumour endogenously produced evs from genetically engineered cancer cell lines mmt-060562 mouse breast cancer; mdamb-231 human breast cancer nude mice with orthotopic mmt tumours or mdamb-231 xenografts cd63-gfp n.a. clsm imaging breast cancer cells secrete evs in the primary and metastatic tumour microenvironment and blood circulation; evs are taken up by cancer cells and cafs. [34] 3lilite sadovska, cristina bajo santos, zane kalniņa and aija linē: biodistribution, uptake and effects caused by cancer-derived extracellular vesicles cell line animal line ev labelling injection site detection method results ref. mda-mb-231 human breast cancer nude mice with mda-mb-231 xenograft human cd63 n.a. ihc evs are taken up by macrophages in the lung, brain and lymph nodes; induce inflammatory processes in tumour microenvironment and axillary lymph nodes.* [32] h460 human lung cancer nude mice with h460 xenograft hcd63-gfp n.a. immunomagnetic separation, rt-pcr human cancer-derived evs carry mrnas and are detectable in the blood and saliva. [35] gluc – gaussia luciferase; fmt – fluorescence mediated tomography; i.v. – intravenous; i.p. – intraperitoneal; ev – extracellular vesicle; dc – dendritic cell; bm – bone marrow; mdmc – monocyte derived myeloid cell; ivis – in vivo imaging system; spion5 – super paramagnetic iron oxide nanoparticles; mri – magnetic resonance imaging; pln – poplietal lymph node; if – immunofluorescence; clsm – confocal laser scanning microscopy; ihc – immunohistochemistry. table 1. studies investigating ev biodistribution and functions in vivo in cancer-free animals, exogenously administered cancerderived evs were distributed mainly to the liver, lungs, kidneys and spleen [28, 30] and were also detected in the lymph nodes [29, 33] and bone marrow [22, 26]. evs derived from human embryonic kidney cells (hek293t) were predominantly localized in the spleen, followed by the liver, lungs and kidneys. additionally, lower amounts of evs were also detected in the brain, heart and muscle [27]. however, when the ev-injected animals were trans‐ cardially perfused with pbs before collecting the organs, the highest amount of evs was detected in the kidneys and not the spleen, followed by the liver and lungs. this suggests that evs are actively taken up by the kidney, liver and lung cells but not the spleen cells. it also indicates that the accumulation of evs in the nonperfused spleen may be due to the uptake of evs by circulating lymphocytes and macrophages [27]. in the liver and lungs, they are likely to be taken up and degraded by phagocytic cells such as kuppfer cells and alveolar macrophages [27]. however, a portion of the evs may also be internalized by the kidney cells and released into the urine [27]. a recent study by cai et al. (2014) demonstrated that, when injected in rats eliciting some characteristics of cml, evs derived from the cml cell line k562 transferred bcr-abl hybrid gene to normal neutrophils. this suggests that ev-mediated transfer of oncogenes may represent a novel mechanism of tumourigenesis [36]. considering that cancer patients have substantially higher levels of evs in the blood than healthy individuals [19-21] and that cell-free rnas, part of which are likely to be packaged into evs, are remarkably stable in the patients’ blood [39], it seemed plausible that evs should be very stable in the biofluids. unexpectedly, when injected in the blood circulation of immunocompetent mice, murine melanoma-derived evs had a half-life of only approxi‐ mately two minutes. furthermore, they were cleared from the circulation within four hours [30]. similarly, when injected into athymic nude mice [27], hek293t-derived evs had a half-life of less than 30 minutes in vivo in most tissues. moreover, lymphoma-derived evs were taken up by cd11b+gr-1+ cells within one hour after the injection into immunocompetent mice [28]. such a short half-life of exogenously administrated evs was very surprising and has to be taken into account when designing studies for the identification of ev-associated cancer biomarkers. in tumour-bearing animals, both the exogenously admin‐ istered evs and endogenously produced evs have also been found to accommodate the tumour microenviron‐ ment, lymph nodes and bone marrow. however, in such studies, the largest part of intravenously administered evs was rapidly cleared from the circulation. exogenously administered hek293t-derived evs accumulated in xenograft tumours at similar levels than the liver and spleen at 60 minutes post-injection [27]. nevertheless, it remained unclear which cell types bind or internalize the evs. several other studies suggest that, in the tumour microenvironment, cancer-derived evs can be internalized by other cancer cells, as well as by surrounding cells like cancer-associated fibroblasts (cafs), endothelial cells and other stroma cells [31, 34, 40]. a number of studies have demonstrated that cancer-derived evs are released into the blood circulation and are trafficked to the lymph nodes, bone marrow and lungs. here, they promote metastatic niche formation and enhance the metastatic spread of cancer cells to these organs [22, 29, 34, 37]. furthermore, when taken up by macrophages, cancer-derived evs were shown to induce an inflammatory response [32]. taken together, these studies provide a solid basis for the concept that cancer-derived evs promote cancer development and progression in vivo by inducing various biological effects, both locally and systemically. 3. uptake of evs in tumour microenvironment it has become clear that, within the tumour microenviron‐ ment, the cellular composition is complex. furthermore, relationships between different cell types are no less sophisticated than those in any healthy organs [41]. therefore, the role of intercellular communication in the acquisition of various cancer phenotypes, invasive growth and metastasis and drug resistance is increasing‐ ly recognized. it can be mediated by soluble signalling molecules, cell-cell or cell-matrix adhesion, gap junc‐ 4 j circ biomark, 2015, 4:2 | doi: 10.5772/60522 tions and evs [42]. in the tumour microenvironment, cancer-derived evs have been shown to be taken up by other cancer cells and stromal cells, such as fibroblasts, as well as endothelial cells and tumour-infiltrated immune cells. evs can exert their effects in the recipient cells either by binding to the cell surface receptors or delivering their content inside the recipient cell. contra‐ ry to the single-molecule signals, evs have the potential to affect multiple signalling pathways inside the recipi‐ ent cell. hence, they provide more efficient means for phenotypic reprogramming or synchronizing the physiological state of the surrounding cells [43, 44]. internalization can occur either through a fusion with the cell membrane [45], endocytosis and micropinocytosis [46, 47] or phagocytosis [48]. however, it is not yet entirely clear which of these mechanisms lead to the degradation of ev components and which, eventually, result in the release of ev content into the cytoplasm. here, it can alter the physiological functions of the recipient cell. the recipient cell specificity is likely to be determined by the composition of adhesion molecules and lipid content on the surface of evs and the respec‐ tive ligands on the cellular surface [49-53]. for example, the internalization of glioblastoma-derived evs was found to depend on the expression of heparan sulfate proteoglycans on the recipient cells [50]. meanwhile, the uptake of rat tumour-derived evs by specific cell types depended on the expression of tetraspanin tspan8 and integrin α4 on the evs [54]. interestingly, low extracellu‐ lar ph (ph 6.0) has also been shown to increase the release and uptake of melanoma-derived evs [45]. this finding is particularly significant for understanding the ev function in the tumour microenvironment, as hypoxia and extracellular acidosis are common features of the vast majority of solid cancers. extracellular acidosis arises by switching metabolism to glycolysis, resulting in the increased production and excretion of acidic metabo‐ lites, such as lactic and carbonic acids, and can be as low as 5.9 [55]. recently, low ph-dependent ev-mediated elimination of cisplatin was also shown to serve as a mechanism of chemoresistance to cisplatin [56]. 4. paracrine effects caused by cancer-derived evs in the tumour microenvironment 4.1 cancer cell cross-talk the uptake of cancer-derived evs by other cancer cells can lead to increased invasiveness and metastatic potential [57-60], anchorage-independent growth [24, 61], prolifera‐ tion and chemoresistance [60, 62, 63], as well as inducing the epithelial-mesenchymal transition (emt) [59, 64] (figure 1). moreover, cancer-derived evs are shown to drive the oncogenic conversion of non-tumourigenic cells [65, 66]. these effects can be mediated by the transfer of functionally active proteins, such as hif1α [59], egfrviii [67], mirnas [61, 63] and possibly, other non-coding rnas, mrnas and fragments of genomic dna carrying various cancer genes and transposable elements [9, 68, 69]. in 1980, poste and nicolson provided the first experimental evidence that evs, shed from highly metastatic melanoma cells, could increase the metastatic potential of poorly metastatic melanoma cells [57]. later on, several independ‐ ent studies confirmed these findings in vitro and in vivo. a study by hao et al. (2006) demonstrated that, when i.v. was injected into c57bl/6 mice, the evs released from highly metastatic melanoma cell line were taken up by poorly metastatic cells, which acquired the capacity to form metastatic colonies in the lungs. similarly, evs derived from highly invasive (but not from non-invasive) triplenegative breast cancer cells significantly increased the proliferation, migration and invasion capacity of other breast cancer cell lines [60]. in line with this, evs from prostate cancer (pc) patients’ sera have been found to enhance the proliferation and invasion of pc cell lines [62]. a study by aga et al. (2014) demonstrated that evs derived from ebv-infected nasopharyngeal carcinoma contained the functionally active transcription factor, hif1α. further‐ more, the uptake of hif1α-containing evs (but not evs containing mutant hif1α) resulted in the downregulation of e-cadherin and upregulation of n-cadherin in recipient cells [59]. changes in eand n-cadherin expression are markers for emt that confer mesenchymal properties to epithelial cells. this is associated with invasion and metastasis, as well as the acquisition of cancer stem cell phenotype [70]. furthermore, evs from drug-resistant cell line variants have been shown to confer resistance to nonresistant pc or breast cancer cells [62, 63]. al-nedawi et al. (2008) demonstrated that glioma cells with a mutated form of epidermal growth factor receptor (egfrviii) release it via evs, which were taken up by indolent glioma cells [24]. this resulted in the activation of mapk and akt signalling pathways. it also led to changes in the expression of egfrviii-regulated genes, resulting in the morphological transformation and increase in anchor‐ age-independent growth capacity [24]. furthermore, breast and colorectal cancer (crc) cells have been shown to release amphiregulin (areg), an egfr ligand, via evs [71]. when taken up by breast cancer cells, ev-packaged areg displayed greater membrane stability and increased invasiveness. interestingly, the areg level in evs corre‐ lated with the mutant kras status of the donor cells [71]. later on, the same group demonstrated that mutant kras status affects the composition of the ev proteome. when taken up by crc cells with wt kras, evs derived from crc cells with mutated kras contained many tumourpromoting proteins, including kras and egfr, and enhanced colony formation [72]. another study demon‐ strated that evs derived from a hepatocellular carcinoma (hcc) were enriched in specific mirnas that could be taken up by other hcc cells. this resulted in the modula‐ tion of tak1 signalling pathway and enhanced anchorageindependent growth [61]. 5lilite sadovska, cristina bajo santos, zane kalniņa and aija linē: biodistribution, uptake and effects caused by cancer-derived extracellular vesicles a recent study by melo et al. (2014) highlights a novel mechanism that could potentially impact our understand‐ ing of the physiological role of cancer-derived evs. this study demonstrated that, in contrast to those produced by normal cells, breast cancer evs bear a risc-loading complex. this is associated with pre-mirnas that are capable of cell-independent mirna biogenesis. these evs were able to mediate the rapid and efficient silencing of target mrnas of recipient cells and, importantly, were shown to have a protumourigenic effect on non-tumouri‐ genic epithelial cells. it was demonstrated that the impact was dependent on the presence of dicer within cancer evs [65]. the authors proposed that cancer evs are capable of inducing an oncogenic “field effect” by subjugating neighbouring normal cells to cooperate in cancer progres‐ sion. in addition, another excellent study by abd elmageed et al. (2014) showed, for the first time, that evs produced by prostate cancer cells are capable of inducing a neoplastic transformation of tumour-trophic mesenchymal stem cells. the ev-primed adipose-derived tissue stem cells under‐ went a mesenchymal-to-epithelial transition, adopted genetic instability and oncogenic transformation, and were able to form tumours in vivo. this was achieved through the oncogenic factor transfer by prostate cancer cellderived evs, which included oncogenic mirnas, k-ras and h-ras transcripts and oncoproteins [66]. figure 1. local and systemic effects that are triggered by cancer-derived evs. locally cancer-derived evs have been reported to promote proliferation, invasiveness and chemoresistance, and to induce emt in cancer cells in a paracrine manner, and to stimulate angiogenesis and reprogramming of stromal cells into cafs. systemically, cancer-derived evs have been shown to contribute to the generation of metastatic microenvironment by reprogramming bmdcs, regulating gene expression in the lungs and lymph nodes and modulating anti-tumour immune response. bmdcs, bone marrow-derived cells; ecm, extracellular matrix; emt, epithelial-mesenchymal transition; caf, cancer-associated fibroblast; dc, dendritic cells; mdsc, myeloid-derived suppressor cell; nk, natural killer cell; tcr, t cell receptor. however, several recent findings have challenged the paradigm of the pro-tumourigenic role of cancer-derived evs, suggesting that cancer-derived evs can also have opposite or, so far, unknown roles. for example, a recent 6 j circ biomark, 2015, 4:2 | doi: 10.5772/60522 study by gabriel et al. (2013) demonstrated that evs derived from various cancer cell lines and pc patients’ plasma contained a functionally active tumour-suppressor protein pten that suppressed the proliferation of ptendeficient recipient cells [107]. as the authors did not detect it in the evs derived from normal cells and plasma from healthy individuals, they suggested that this might represent a mechanism used by cancer cells to downregu‐ late pten level [107]. meanwhile, another study found a functional pten protein in evs, produced by mouse embryonic fibroblasts and human embryonic kidney cells [108]. this suggests that this could be a more wide spread phenomenon, provoking questions regarding its biological significance in normal physiological processes and cancer. moreover, it remains to be determined which cell types actively take up pten-containing evs in vivo and whether they exert the same physiological effects in cancerous and normal cells. taken together, several lines of evidence strongly support the concept that cancer-derived evs act paracrinally to synchronize the physiological state in subpopulations of cells. they do this by delivering signalling molecules that are not endogenously expressed in the recipient cells and thus, driving the cancer progression. furthermore, they may even induce the acquisition of cancer cell phenotype in non-malignant cells. however, various ev subpopula‐ tions differ in their molecular content and may cause opposite effects in the recipient cells. thus, further studies that dissect the heterogeneity of evs and the recipient cell selectivity are urgently needed. 4.2 promotion of angiogenesis cancer-derived evs have also been shown to promote angiogenesis. a number of independent studies have demonstrated that cancer-derived evs can be taken up by the endothelial cells in vitro and in vivo. this results in morphological changes, migration and proliferation of endothelial cells, tube formation and neovascularization [40, 67, 73-78]. apparently, these effects can be mediated by the transfer of angiogenic proteins [73] and oncogenic proteins [67], as well as various mrnas and mirnas [40, 78, 79]. while the angiogenic proteins, such as angiogenin, il-6, il-8, timp-1 and vegf, are likely to impact in a paracrine manner, the delivery of oncogenic egfr to the endothelial cells was shown to trigger the endogenous expression of vegf, followed by the autocrine activation of vegf receptor-2 signalling [67]. several studies empha‐ size the role of ev-shuttled mirnas in the endothelial cell migration and neovascularization. a study by umezu et al. (2012) demonstrated that the uptake of leukaemia cellderived evs carrying mir-92a enhanced endothelial cell migration and tube formation [79]. meanwhile, zhuang et al. (2012) showed that tumour-secreted mir-9 triggered the activation of jak-stat pathway in the endothelial cells, resulting in enhanced migration and tumour angiogenesis [78]. furthermore, the administration of anti-mir-9 or jak inhibitors suppressed these effects in vitro and in vivo [78]. likewise, the treatment of mice carrying human carcinoma xenografts with diannexin, a drug that binds phosphati‐ dilserine and blocks ev exchange, resulted in the reduction of tumour growth rate and microvascular density [67]. collectively, these studies demonstrated, both in vitro and in vivo, that cancer-derived evs have a pro-angiogenic capacity and therefore, might represent an attractive target for therapeutic intervention. however, to date, it is not entirely clear at what stages of maturation endothe‐ lial cells are targeted by cancer-derived evs in vivo. 4.3 acquisition of caf phenotype among the different components of the tumour stroma, cancer-associated fibroblasts (cafs) are one of the main elements. cafs are defined as all the fibroblastic, nonneoplastic, non-vascular, non-epithelial and non-inflam‐ matory cells with a stable karyotype found in a tumour [80]. cafs promote tumour progression by secreting soluble growth factors, cytokines and chemokines that stimulate proliferation and migration of cancer cells, induce angio‐ genesis, modify tumour metabolism, stimulate acquisition of cancer stem cell phenotype and modulate the immune response [80, 81]. cafs can originate from resident tissue fibroblasts, mesenchymal stem cells, myofibroblasts and even epithelial and endothelial cells via emt or endmt, respectively [80]. this process is accompanied by persistent changes in their gene methylation pattern [82, 83]. once the caf phenotype is acquired, two autocrine signalling loops, mediated by tgf-β and sdf-1 cytokines, maintain them in this differentiation state in an autocrine manner [84]. a growing body of evidence suggests that cancer-derived evs play a crucial role in the reprogramming of these cells into cafs via the transfer of tgfβ. thus, for instance, tgfβ1 containing pc-derived evs was found to be taken up by primary lung fibroblasts, resulting in their differen‐ tiation into tumour-promoting cafs [85, 86]. these cells supported angiogenesis in vitro and tumour growth in vivo. interestingly, this effect could not be achieved by using soluble tgfβ1 and appeared to depend on heparan sulphate chains on the ev surface. moreover, ev-deficient (rab27a knock-down) cancer cells failed to achieve activa‐ tion of the tumour stroma [86]. likewise, gastric cancerderived evs were shown to trigger the differentiation of umbilical cord-derived mesenchymal stem cells into cafs by the transfer of tgfβ and the subsequent activation of tgfβ/smad pathway [87]. furthermore, breast cancerderived evs were found to convert adipose tissue-derived mesenchymal stem cells into cafs expressing various tumour-promoting factors [88]. hence, cancer-derived evs seem to play a crucial role in the induction of caf phenotype via the transfer of tgfβ. 7lilite sadovska, cristina bajo santos, zane kalniņa and aija linē: biodistribution, uptake and effects caused by cancer-derived extracellular vesicles 5. systemic effects caused by cancer-derived evs 5.1 formation of pre-metastatic niche the first evidence that cancer-derived evs can contribute to the generation of metastatic microenvironment was provided by jung et al. (2009) [37]. in this study, rats received injections of a conditioned medium containing evs and a soluble matrix obtained from highly metastatic pancreatic cancer cells, followed by the injection of the respective cancer cells. this showed that the conditioned medium promoted the settlement of a non-invasive variant of these cells in the lymph nodes and lungs. this suggested that highly metastatic cells deliver messages that elicit alterations in pre-metastatic organs. this allows the homing, settling and growing of poorly metastatic cells [37]. another study demonstrated that, when injected in the mouse footpad, mouse melanoma-derived evs, home to sentinel lymph nodes, enhance the migration of melanoma cells to the ev-rich sites in the lymph nodes. in the lymph nodes, evs were found to regulate the expression of a variety of genes involved in migration, extracellular matrix deposition and vascular proliferation [29]. moreover, evs derived from putative renal cancer stem cells were found to increase the number of lung metastases, when intrave‐ nously injected in scid mice [74]. an elegant study by peinado et al. (2012) demonstrated that, when injected in mice, evs derived from highly malignant mouse melano‐ ma, home to the lungs and bone marrow, enhance endo‐ thelial permeability at pre-metastatic sites and promote the development of distant metastasis. these evs were found to “educate” bone marrow-derived cells (bmdcs) by transferring the met oncoprotein, resulting in the activa‐ tion of the met pathway in bmdcs. they become condi‐ tioned to support tumour vasculogenesis, invasion and metastasis. furthermore, higher amounts of met were found in circulating evs isolated from patients with stage three and four melanoma than in healthy controls. this shows that this finding may also have relevance in a clinical setting [22]. a number of preclinical studies suggest that cancerderived evs have a systemic effect on the conditioning of a pre-metastatic niche and hence, set the basis for a new therapeutic strategy. however, it remains to be determined whether or not this type of signalling represents a common mechanism of metastasis in various human cancers. 5.2 modulation of anti-tumour immune response cancer-derived evs have been reported to both stimulate and suppress anti-tumour immune responses [11]. they are shown to contain tumour-associated antigens, such as cea and mart1, and are efficiently taken up by dendritic cells. in turn, these cross-present the antigens to cd8+ t cells, resulting in potent anti-tumour effects [89, 90]. moreover, as they bear mhc class i molecules, it has been suggested that they could directly stimulate cd8+ t cells [11]. in fact, several pre-clinical and clinical studies based on the immunization with cancer-derived evs, in combi‐ nation with various cytokines, have shown the induction of beneficial tumour-specific cd8+ t cell responses. such studies suggest that they may represent an attractive approach for cancer immunotherapy [91-94]. on the other hand, increasing evidence suggests that cancer-derived evs can suppress the anti-tumour immune response in a variety of ways. for instance, melanomaderived evs have been shown to be enriched for fasl and induced fas-mediated apoptosis in t cells [95]. subsequent studies have described similar immune evasion mecha‐ nisms, mediated by fasl or trail expression, on evs in prostate and colorectal cancer and glioma [96-99]. more‐ over, fasl expression can also lead to the tcr impairment due to the downregulation of cd3-ζ chain, which has been reported in ovarian [100, 101] and head and neck squamous carcinoma patients [98]. contrary to the effector t cells, cd4+cd25hightregs are resistant to fasl-induced apoptosis and cancer-derived evs have been reported to stimulate the expansion and suppressive functions of tregs [98, 102, 103]. ovarian cancer-derived evs have been shown to promote the proliferation of cd4+cd25+foxp3+t cells, convert cd4+cd25neg t cells into cd4+cd25+tregs and upregulate treg suppressor functions (e.g., production of perforin, granzyme b, il-10, etc.), when added to the culture of peripheral blood t cells obtained from healthy donors [102]. at least partially, this effect seems to be mediated by ev-transferred tgfβ1. this is because the pretreatment of malignant-effusion derived evs, with neutral‐ izing antibodies against tgfβ1, reduced the expansion and suppressive functions of tregs [104]. however, another recent study demonstrated that cd4+cd25hightreg expan‐ sion and il-10 secretion is promoted by cancer cell-secreted mir-214 that targets pten in cd4+ t cells [105]. other ev-mediated immunosuppressive mechanisms include t cell inhibition via the production of extracellular adenosine by ev-expressed cd39 and cd73 [106], and the downregulation of the activating receptor nkg2d on nk and cd8+ t cells by ev-transferred tgfβ [103, 107]. cancer-derived evs have also been shown to suppress the cytotoxic activity of nk cells by expressing mica, which triggers the downregulation of nkg2d from the cell surface and reduces nk cytotoxicity [108]. in addition, cancer-derived evs have multiple effects on myeloid precursors, dendritic cells and macrophages. cancer-derived evs have been shown to block the differ‐ entiation of myeloid precursors into dendritic cells and promote the generation of myeloid-derived suppressor cells via stat3 activation [26, 109, 110]. in turn, this results in the suppression of effector t cell proliferation, activation and cytolytic functions and the induction of treg cells [109, 111]. in macrophages, melanoma and breast cancerderived evs (but not those from non-cancerous cells) have been reported to activate nf-κb signalling and to alter the cytokine and chemokine profile, favouring the production 8 j circ biomark, 2015, 4:2 | doi: 10.5772/60522 of pro-inflammatory cytokines. however these changes were complex and not consistent with m1 or m2 polariza‐ tion [32, 112]. hence, the role of cancer-derived evs in the macrophage-mediated tumour-promoting or anti-tumour effects is not entirely clear and, presumably, may vary depending on their content and physiological state of their cell-of-origin. 6. concluding remarks and future directions collectively, these studies strongly support the para‐ digm of the cancer-promoting role of cancer-derived evs. they suggest that inhibition of the formation or uptake of cancer-derived evs or their components could be a novel therapeutic avenue. in fact, several studies have demonstrated that blocking the production or uptake of evs or specific mirnas carried by evs reduced tu‐ mour growth and angiogenesis. this clearly shows a therapeutic benefit [67, 78, 113]. in addition, natural or genetically engineered evs can be exploited as tools for delivery of virus-like particles or other gene therapy products, allowing to evade pre-existing neutralizing antibodies against the viral vectors and to increase transduction efficiency [114, 115]. moreover, cancer-derived evs seem to have very diverse effects on immune cells, which may lead to the stimulation or suppression of anti-tumour immune responses. hence, a deeper understanding of mechanisms and how they impact the functions of various immune cell subsets could help to develop novel strategies for shifting the balance towards immunostimulatory tumour microenvironment. however, it remains unclear whether it is possible to entirely stop cancer progression by inhibiting the forma‐ tion or uptake of cancer-derived evs. it seems likely that cancers differ in their ability to produce evs and in the degree to which they depend on the ev-mediated signal‐ ling. however, to the best of our knowledge, the levels of evs and their effects have not been systematically studied during the course of disease progression and compared among different cancer types. another layer of complexity is added by the heterogeneity of ev biogenesis and the composition of their molecular cargo. currently, there is great controversy regarding what types of evs each cell type produces and which of them carry molecular cargo that are capable of eliciting biologically significant effects. for instance, many studies have reported that exosomes are enriched in mirnas, suggesting that they function as vehicles for the intercellular transfer of mirnas [8, 116-120]. nonetheless, a recent study by chevillet et al. (2014) has challenged this view by demonstrating that, on average, most exosomes harbour less than one molecule of a given mirna. however, it remains unclear whether rare exosomes in the population carry many copies of a given mirna or whether a larger fraction of exosomes carries a low concentration of mirnas [121]. in this regard, a recent study by thakur et al. (2014) demonstrated that exosomes carry dsdna representing the whole genomic dna. however, only a subset (~10%) of exosomes contained dna [122]. hence, the characterization of ev subpopula‐ tions carrying cancer-derived molecular cargo seems to be of paramount importance for designing studies aimed at the discovery of ev-associated biomarkers and therapeutic targeting of evs. 7. conflict of interest the authors declare no conflicts of interest. 8. acknowledgements this study was supported by the latvian council of science (grant no. 625/2014). 9. references [1] kalra h, simpson rj, ji h, aikawa e, altevogt p, askenase p, bond vc, borras fe, breakefield x, budnik v, buzas e, camussi g, clayton a, cocucci e, falcon-perez jm, gabrielsson s, gho ys, gupta d, harsha hc, hendrix a, hill af, inal jm, jenster g, kramer-albers em, lim sk, llorente a, lotvall j, marcilla a, mincheva-nilsson l, nazarenko i, nieuwland r, nolte-'t hoen en, pandey a, patel t, piper mg, pluchino s, prasad ts, rajendran l, raposo g, record m, reid ge, sanchez-madrid f, schiffelers rm, siljander p, stensballe a, stoorvogel w, taylor d, thery c, valadi h, van balkom bw, vazquez j, vidal m, wauben mh, yanez-mo m, zoeller m, mathivanan s (2012) vesiclepedia: a compendium for extracellular vesicles with continuous community annotation. plos.biol. 10(12):e1001450. 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accepted: 15 june 2018 introduction cirrhosis of the liver is the histological development of regenerative nodules surrounded by fibrous bands in response to chronic liver injury, leading to portal hypertension and end-stage liver disease. causes of cirrhosis of the liver are multifactorial. despite varied etiology, the pathological characteristics which result in liver dysfunction are common. 1 in recent years, alcohol consumption has correlated with deaths from asymptomatic and self-limited fatty liver to cirrhosis of the liver. alcoholic liver disease (ald) progresses from steatosis to alcoholic hepatitis to fibrosis 1 department of biochemistry, sri devaraj urs medical college, sduaher, kolar, karnataka, india 2 department of medicine, sri devaraj urs medical college, sduaher, kolar, karnataka, india 3 department of community medicine, sri devaraj urs medical college, sduaher, kolar, karnataka, india 4 centre for cellular and molecular platforms, gkvk campus, bengaluru, karnataka, india corresponding author: kn shashidhar, department of biochemistry, sri devaraj urs medical college, sduaher, tamaka, kolar, karnataka, india. email: drshashikn1971@yahoo.co.in journal of circulating biomarkers volume 7: 1–9 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418788417 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:drshashikn1971@yahoo.co.in https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418788417 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage and frank cirrhosis (micronodular, occasionally mixed micronodular, and macronodular) and often occurs acutely against background chronic liver disease. 2 multifactorial pathogenesis plays a role in progression of the disease. accumulation of triacylglycerol in liver is an early and reversible effect of alcohol which increases peripheral lipolysis and alters liver redox potential leading to fatty acid synthesis. in ald, generated prooxidants enhance antioxidant system results in lipid peroxidation. 3 acetaldehyde generated from alcohol is highly reactive and toxic; it binds to phospholipids and amino acids, resulting in the formation of abnormal folding of proteins in endoplasmic reticulum (er) leading to er stress. 3 proliferation and activation of hepatic stellate cells (hscs) in ald are induced by kupffer cells and hepatocytes. chief mitogen for the activation of hscs is platelet-derived growth factor, which is produced by kupffer cells. kupffer cells induce collagen synthesis through the production of transforming growth factor b, tumor necrotic factor a (tnf-a), and reactive oxygen species (ros). activated hscs migrate and accumulate at tissue repair sites and secrete large amounts of extracellular matrix (ecm). hscs collagen synthesis is regulated at transcription and posttranscriptional levels. dysregulation of cytokine metabolism and activity is vital for alcohol-induced liver damage. tnf-a, a pro-inflammatory cytokine, is one of the key factors for pathophysiology of ald. hepatocytes induce fibrosis through the production of ros or apoptosis followed by regenerative nodular formation. 3,4 diagnosing the degree of disease is a crucial step for successful management of ald. despite the development of potential diagnostic tests for the past 50 years, liver biopsy is considered as the gold standard method for diagnosis of liver cirrhosis and provides useful information about damaging process, namely, steatosis, lobular inflammation, periportal fibrosis, mallory bodies, nuclear vacuolation, bile ductal proliferation, and fibrosis/cirrhosis. 2,5 limitation of liver biopsy is highly invasive; poor sample quality and tissue size make biopsy nonreproducible, and it depends on the experience of the pathologist leading to interobserver variations. risk allied for liver biopsy range from pain (84%) and hypertension, bleeding (0.5%), and damage to biliary system with approximately 0.01% mortality rate. 6 no single biomarker can establish alcohol to be the etiology of liver disease; existing biomarkers in clinical practice have narrow applicability due to lack of specificity and lack of sensitivity (distinguish intermediate stages) which prevent reliance on any single biomarker. 2,7 an ideal biomarker should be organ specific, a sensitive indicator for active damage, easily accessible in peripheral tissue, and cost-effective and should give insights for diagnosis, monitor the activity of disease, and assess therapeutic response. the determination of biomarkers could be an easy, noninvasive, and inexpensive method to monitor the progression of liver disease. this leads to urgency in the progression of biomarker discovery for cirrhosis of the liver with the help of technological advancement in “-omics” approach. discovery of biomarker candidates should be a simplified, unbiased, semiquantitative binary comparison between diseased and normal. 8 although individual sample analysis is recommended, pooling strategy with definite selection criteria from multiple individuals reduces sample number and cost. 9 alterations in protein domain due to ald which enters into circulation hold good for discovery of biomarker candidates. in the present study, we tried to discover protein biomarker candidates for alcoholic liver cirrhosis whose concentration may be altered due to changes in translation, posttranslational modifications, and/or degradation using two-dimensional electrophoresis (2de) after albumin depletion, followed by liquid chromatography–mass spectrometry (lc-ms). materials and methods samples blood samples were collected from 20 subjects: 10 clinically and diagnostically proven alcoholic liver cirrhotic subjects with varying degree and ageand gendermatched 10 healthy subjects (table 1) from r. l. jalappa hospital and research centre, attached to sri devaraj urs medical college, a constituent of sri devaraj urs academy of higher education, kolar, karnataka, india. individuals diagnosed with cirrhosis of the liver caused by ald based on clinical history and symptoms, namely, ascites, encephalopathy, jaundice, and altered biochemical parameters, were included in the study. individuals with diabetes and/or its complications, myocardial infarction, acute and chronic renal failure, pneumonia, and cancer were excluded from the study. collection of blood samples from cirrhotic liver subjects and healthy subjects was carried out after obtaining informed consent, and the study is approved by institutional ethical committee (dmc/klr/iec/61/2016-17). table 1. details of 20 blood samples (10 alcoholic liver cirrhotic subjects and ageand gender-matched 10 healthy subjects) used for discovery of biomarker candidates by proteomic approach. sample id gender age etiology sample id gender age etiology c1 m 36 na d1 m 36 ald c2 m 28 na d2 m 28 ald c3 m 62 na d3 m 62 ald c4 m 36 na d4 m 36 ald c5 m 35 na d5 m 35 ald c6 m 40 na d6 m 40 ald c7 m 70 na d7 m 70 ald c8 m 30 na d8 m 30 ald c9 m 62 na d9 m 62 ald c10 m 30 na d10 m 30 ald c: control; d: diseased (alcoholic liver cirrhosis); m: male; na: not applicable; ald: alcoholic liver disease. 2 journal of circulating biomarkers serum separation serum was collected from clotted blood using serum separator tubes centrifuged at 4000 r/min for 10 min. serum was stored at �20�c till further analysis. all samples were used for discovery of protein biomarker candidates after depletion of abundant albumin. desalting was carried out by acetone precipitation. 2de was carried out to find differentially expressed proteins between cirrhotic and healthy subjects. identified spots were characterized by lc-ms after in-gel trypsin digestion. reagents dye-based (cibacron blue) prefractionation albumin depletion kit was procured from thermo fisher scientific (waltham, massachusetts, usa). precast gels and other chemicals of analytical grade for sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), 2de, ingel trypsin digestion, and lc-ms were procured from biorad (hercules, california, usa) and sigma aldrich (st louis, missouri, usa). depletion of albumin resuspended resin (200 ml aliquot of resin) was transferred into spin column (column volume: 900 ml; 10-mm poresize polyethylene filter). bottom of the column was twisted off and placed in a 1.5-ml collecting tube. then, the sample is centrifuged at 12,000 � g for 1 min, the flow-through is discarded, and finally the spin column back is placed into the same collection tube. around 200 ml of binding/wash buffer was added to the spin column. then, the sample is centrifuged at 12,000 � g for 1 min, the flow-through is discarded, and, finally, the spin column back is placed into the new collection tube. about 50 ml of pooled serum sample (cirrhotic and healthy in separate columns) was added into resin and incubated for 2 min at room temperature. then, the sample is centrifuged at 12,000 � g for 1 min, flow-through is reapplied to spin column, and incubated for 2 min at room temperature. again, the sample is centrifuged at 12,000 � g for 1 min and flow-through is retained. spin column was placed in a new collection tube. resin was washed to release unbound proteins by adding 50 ml of binding/wash buffer for each 200 ml of the resin used. retained fractions of cirrhotic and healthy samples were run in sdspage for confirmation of albumin depletion. 10 sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) sds gel was prepared according to the standard protocol. samples from cirrhotic liver subjects and healthy subjects after depletion of albumin along with un-depleted samples were loaded in gel, and sds-page was carried out at 25 ma in 1� sds running buffer for confirmation of depletion along with molecular weight marker. after electrophoresis, gel was incubated in a fixing solution (40% methanol, 10% acetic acid) at room temperature for 20 min. gel was subjected for staining with sliver stain (0.1% silver nitrate, 36% formaldehyde) at room temperature for 20 min. excess staining solution was removed, and the gel was washed with 5% acetic acid.11–13 acetone precipitation acetone precipitation was carried out to remove excess salts which interferes electrophoretic run. protein samples after depletion of albumin (cirrhotic and healthy separately) were placed in acetone-compatible tubes. four times the sample volume of cold acetone (�20�c) was added into both tubes. the tubes were vortexed and incubated for 60 min at �20�c, followed by centrifugation at 13,000 � g for 10 min. then the supernatant was disposed carefully for the retention of protein pellet. 14 two-dimensional polyacrylamide gel electrophoresis albumin depleted and desalted protein pellet (200 mg) from cirrhotic and healthy subjects were diluted with rehydration buffer (8 m urea, 2% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate, 50 mm dithiothreitol (dtt), 0.2% w/v bio-lyte 3/10 ampholyte, bromophenol blue) and used separately for 2de with 7 cm (ph 4–7) nonlinear immobilized ph gradient dry strips (bio-rad). samples were left overnight for rehydration on 7 cm (ph 4–7) dry strips. isoelectric focusing was carried out at 250 v for 20 min, followed by 4000 v for 5 h at 20�c. proteins were separated by 8–16% precast polyacrylamide gels at 200 v for 40 min. after electrophoresis, gels were subjected to staining with silver stain (0.1% silver nitrate, 36% formaldehyde) at room temperature for 20 min. excess staining solution was removed, and the gel was washed with 5% acetic acid. 15 image analysis for image analysis, scanned gels were processed using pdquest 2-d analysis software (bio-rad). for differential analysis, the cirrhotic gel was compared with that of the healthy gel. differential expression of proteins present in both cirrhotic and healthy gels was considered significant when the fold change was least 2 and p � 0.05 with 95% confidence interval with the application of rank-sum test. in-gel digestion and peptide extraction excised spots were cut into cubes and transferred into a microcentrifuge tube, and 100 ml of destaining solution (100 mm ammonium bicarbonate/acetonitrile (1:1 vol/ vol)) was added and incubated for 30 min; 500 ml of neat acetonitrile was added and the tubes were incubated for 10 min until gel pieces shrink; 50 ml of dtt solution (10 mm dtt in 100 mm ammonium bicarbonate buffer) nallagangula et al. 3 was added to cover the gel pieces and incubated in 56�c thermostat for 30 min; and 500 ml of acetonitrile was added to the tubes and further incubated for 10 min. all the liquid was removed from the tube. following dtt treatment, to get reduction and alkylation of cystines and cysteines in the protein, 50 ml of iodoacetamide solution (55 mm iodoacetamide in 100 mm ammonium bicarbonate solution) was added to the tubes and incubated for 20 min at room temperature in dark. the gel pieces were again treated with acetonitrile for 10 min, and the entire liquid was removed from the tube. the gel pieces were saturated with trypsin buffer (13 ng/ml of trypsin in 10 mm ammonium bicarbonate in 10% acetonitrile) for 30 min. cold trypsin (20 mg of trypsin in 1.5 ml of ice-cold 1 mm hydrochloric acid) was added to the tubes and incubated overnight at 37�c. tubes were cooled to room temperature, gel pieces were centrifuged at 10,000 r/min for 1 min, and peptides were extracted in 100 ml of extraction buffer (1:2, 5% formic acid/acetonitrile) by incubating for 15 min at 37�c shaker, and the supernatant was withdrawn directly for lc-ms analysis. 16 mass spectrometric analysis mass spectrometric analysis of the extracted peptides was performed using nano lcms-ltq-orbitrap discovery (thermo scientific) coupled to nano lc (agilent 1200). the samples were reconstituted in 0.1% formic acid prior to injection; 70-min gradient run was set up using acetonitrile and water with formic acid as the mobile phase. ltq orbitrap discovery is a hybrid-type ms system with the ability to determine accurate m/z of intact precursors. the raw files post-ms run was analyzed using proteome discover software and mascot as search engine against human database. 17 results sds-page analysis for depletion of albumin immobilized resin form of cibacron blue was effective in binding abundant albumin from plasma/serum samples for depletion of significant amount. human serum albumin (hsa) from pooled serum samples of cirrhotic and healthy subjects was depleted using cibacron blue dye-loaded resin columns. proteins in the flow-through were analyzed by sds-page (figure 1) along with prestained molecular weight marker to investigate efficient depletion of hsa. sensitive staining, silver stain, was helpful for the detection of low nanogram proteins when compared with coomassie brilliant blue. silver-stained gel demonstrated significant amount of abundant albumin depletion from serum samples of both cirrhotic and healthy subjects. identification of biomarker candidates synthetic gel image representative of all features in the differential analysis comparing samples from cirrhotic and healthy is shown in figure 2. the image analysis software and statistical analysis found 46 spots in cirrhotic gel and 69 spots in control gel, of which 14 spots were identified with significant altered expression levels between cirrhotic and healthy based on quantitative ratio. these spots were figure 1. sds-page analysis for confirmation of albumin depletion (silver-stained gel). 1: normal pooled albumin depleted serum. 2: normal pooled serum. 3: cirrhotic liver pooled albumin depleted serum. 4: cirrhotic liver pooled serum. m: pre-stained molecular weight marker. sds-page: sodium dodecyl sulfate polyacrylamide gel electrophoresis. figure 2. comparison of 2de gel images representative of all features in differential analysis from alcoholic cirrhotic and healthy subjects. (a) 2de gel image of alcoholic liver cirrhotic subjects. (b) 2de gel image of healthy subjects. 2de: two-dimensional electrophoresis. 4 journal of circulating biomarkers excised, digested with trypsin, analyzed by lc-ms, and identified by mascot database. these spots contain more than one protein; among 14 spots, a total of 68 proteins were identified. many of the proteins were identified as the same protein in different locations on the gels and so among 68 proteins, we identified 46 candidate biomarkers for alcoholic liver cirrhosis. among 46 candidate biomarkers, 28 were identified based on protein score and clinical significance (table 2). among 28 protein biomarker candidates, 13 with increased expression and 15 with decreased expression were identified in alcoholic liver cirrhotic when compared to healthy subjects. serum concentrations of keratin isoforms were found to increase in alcoholic cirrhosis. immunoglobulins (igs), namely, polymeric immunoglobulin receptor isoform x1 and iggfc-binding protein precursor, were increased in cirrhotic liver when compared to healthy subjects but with low protein scores. along with 13 features whose expression is increased in cirrhosis of the liver, angiotensinogen preproprotein, a2-macroglobulin isoform x1, were found to increase compared to healthy subjects. serum albumin preproprotein, a-1-antitrypsin precursor and a-1-antichymotrypsin precursor showed decreased expression in alcoholic liver cirrhosis. concentrations of glycerol kinase isoform x1 and kininogen-1 isoform 1 precursor were decreased in cirrhotic liver compared to healthy subjects. discussion invasive liver biopsy is the gold standard diagnostic tool for liver fibrosis/cirrhosis with varied etiology and to distinguish between intermediate stages. reliable noninvasive biomarker with sensitivity and specificity is needed for diagnosis/prognosis and effective management of the disease. 8 in the present study, we used 2de followed by lcms for identification of biomarker candidates for alcoholic liver cirrhosis. for maximal detection of meaningful protein expression difference, cases and controls should differ absolutely in terms of disease of interest. simplified, unbiased binary comparison between diseased and healthy avoids contamination by other diseases and confounding table 2. proteomic biomarker candidates identified by 2de followed by lc-ms for alcoholic liver cirrhosis. biomarker candidate expression in ald mol. wt cal. pi physiological role keratin isoforms protects epithelial cells from damage or stress keratin type ii cuticular hb6 isoform x1 increases 62 6.37 keratin type i cuticular ha1 increases 47.2 4.88 keratin type ii cuticular hb5 isoform 1 increases 55.8 6.55 keratin type ii cytoskeletal 6c increases 60 8 keratin type ii cytoskeletal 2 epidermal increases 65.4 8 keratin type i cytoskeletal 9 increases 62 5.24 lumican precursor increases 38.4 6.61 collagen binding proteoglycan pigr isoform x1 increases 83.2 5.74 mediates transcellular transport of igs serotransferrin precursor increases 77 7.12 ferric ion binding protein ig lambda like polypeptide 5 isoform 1 increases 23 8.84 not known vitamin d binding protein isoform 3 precursor increases 55 5.63 vitamin d transport and storage haptoglobin isoform 1 preproprotein increases 45.2 6.58 binds free plasma hemoglobin transmembrane protein 201 isoform 1 increases 72.2 9.22 involved in nuclear movement during fibroblast polarization and migration a-1-antitrypsin precursor decreases 46.7 5.59 protease inhibitor hemopexin precursor decreases 51.6 7.02 scavenging heme apolipoprotein a-iv precursor decreases 45.3 5.38 chylomicrons and vldl secretion and catabolism cd5 antigen like isoform x1 decreases 38.7 5.66 key regulator of lipid synthesis zinc-a2-glycoprotein precursor decreases 34.2 6.05 lipid mobilization and fertilization dermcidin isoform 1 preproprotein decreases 11.3 6.54 antimicrobial activity a1-b-glycoprotein precursor decreases 54.2 5.86 not known glycerol kinase isoform x1 decreases 63.6 6.54 transfer of phosphate from atp to glycerol a2-hs-glycoprotein preproprotein decreases 39.3 5.72 role in endocytosis kininogen-1 isoform 1 precursor decreases 71.9 6.81 role in blood coagulation sex hormone binding globulin isoform1 precursor decreases 43.8 6.71 androgen transport protein a1-acid glycoprotein 1 precursor decreases 23.5 5.11 acute phase protein leucine-rich a2-glycoprotein precursor decreases 38.2 6.95 protein-protein interactions, signal transduction and cell adhesion a2-antiplasmin isoform x1 decreases 56.6 6.89 inactivating plasmin antithrombin-iii precursor decreases 52.6 6.71 serine protease inhibitor pigr: polymeric ig receptor; 2de: two-dimensional electrophoresis; lc-ms: liquid chromatography–mass spectrometry; ald: alcoholic liver disease; mol. wt: molecular weight; calc. pi: calculated isoelectric ph; igs: immunoglobulins. nallagangula et al. 5 factors which may alter the expression of protein results in false discovery of biomarker candidates. 18 discovery of biomarker candidates by proteomic approach is difficult, especially when the ph range is between 3 and 7, as abundant albumin interferes in identification and characterization of low abundant proteins by mass spectral and electrophoretic analysis. accurate protein biomarker candidate discovery was achieved after depletion of albumin using dye-based affinity columns. antibody-based immunoprecipitation is more robust for depletion of abundant proteins from plasma/serum and is suitable for identification of novel biomarker candidates. 19–21 depletion dilemma can be rectified using narrow ph (3–5.6) range and avoids interference of abundant proteins (albumin, transferrin, and igs) but chance to miss proteins whose isoelectric ph is in the alkaline range. 22 technological advancement in biomarker candidate discovery resulted in identification of protein biomarker candidates for chronic liver diseases (clds) with varied etiology (table 3). biomarker candidates identified require verification which demonstrates that the differential expression should remain detectable by assay to be used for validation. 18 despite numerous biomarker candidates identified, verification may be done only for few qualified candidates in terms of marker performance and reagent availability. 27 biomarker candidates that show significant expressional differences between diseased and healthy in discovery phase are prioritized. proteins that are secreted and/or present on cell surface and that act in cellular pathways and deregulated in ald should be considered for further validation. 9 in the present study, keratin isoforms showed upregulation in alcoholic liver cirrhosis. keratin is a fibrous structural protein that protects epithelial cells from damage and stress and regulates key cellular activities, namely, cell growth and protein synthesis. 28 lumican, leucine-rich repeat proteoglycan, constitute an important fraction of noncollagenous ecm proteins. it plays a major role in tissue homeostasis and modulates cellular functions, namely, cell proliferation, migration, and differentiation. 29 polymeric ig receptor (pigr) isoform x1 is a type-i transmembrane protein expressed from glandular epithelial cells of liver and breast. it mediates transcellular transport of polymeric igs. pro-inflammatory cytokines, namely, table 3. protein biomarker candidates identified by proteomic analysis for liver fibrosis. authors etiology of liver fibrosis type of sample proteomic techniques protein biomarker candidates identified white et al.23 hcv serum 2de, lc-ms a2 macroglobulin haptoglobin complement c4 serum retinol binding protein apolipoprotein a1 apolipoprotein a-iv bevin et al.24 hcv serum 2de, lc-ms a2 macroglobulin inter-a-trypsin inhibitor heavy chain h4 a-1-antichymotrypsin apolipoprotein l1 paraoxonase/aryleserase 1 zinc-a2-glycoprotein cd5 antigen-like protein b2 glycoprotein i bevin et al.25 hcv serum 2de, lc-ms, in-solution isoelectric focusing beta chains of c3 and c4 bevin et al.22 hcv serum 2de, lc-ms adiponectin, sex hormone binding protein 14-3-3 protein zeta/delta, complement c3dg immunoglobulin j chain apolipoprotein ciii corticosteroid binding globulin a2-hs-glycoprotein lipid transfer inhibitor protein haptoglobin-related protein katrinli et al.26 hbv liver tissue 2de, lc-ms apolipoprotein a1 pyruvate kinase glyceraldehyde 3-phosphate dehydrogenase glutamate dehydrogenase alcohol dehydrogenase transferrin, peroxiredoxin 3 keratin 5, annexin hcv: hepatitis c virus; hbv: hepatitis b virus; 2de: two-dimensional electrophoresis; lc-ms: liquid chromatography–mass spectrometry. 6 journal of circulating biomarkers interferon-g, tnf, and interleukin 1, which are the key regulators of pigr expression, upregulate in ald. 8,30,31 vitamin d-binding protein, a multifunctional protein that belongs to the albumin gene family can bind various forms of vitamin d (ergocalciferol, cholecalciferol, and calcifediol) for the transport. it is synthesized by hepatic parenchymal cells. 32 haptoglobin which is included in the existing noninvasive marker panel has showed increased expression in the present study as it is an acute phase protein. liver is the major site for the synthesis of haptoglobin; hepatic expression will be stimulated by upregulated il-6 in ald. 23,33,34 transmembrane protein 201 is involved in nuclear movement during fibroblast polarization and migration; actin-dependent nuclear movement is through association with transmembrane actin-associated nuclear lines. 35 serine protease inhibitors, a-1-antitrypsin (serpina1) and a-1-antichymotrypsin (serpina3), produced primarily in liver hepatocytes and released directly into the blood stream showed downregulation in alcoholic liver cirrhosis compared to healthy subjects. 36–38 hemopexin which showed decreased expression in ald is a single polypeptide chain of 439 amino acid residues with a molecular weight of 63 kda is expressed from liver, and it acts as a heme-scavenging protein. 39 apolipoprotein a-iv, even though not evident from liver, its expression, was also decreased in ald. downregulation of apolipoprotein aiv was reported in hepatic fibrosis in rat models. 23,40 cd5 antigen-like isoform x1, a key regulator of lipid synthesis, was downregulated in alcoholic liver cirrhosis, whereas upregulation was noted in liver cirrhotic patients in hepatitis c virus infection. 24 zinc-a2-glycoprotein, adipokine, which plays an important role in fat catabolism and which reduces insulin resistance, was downregulated in ald. 24,41 glycerol kinase, a phosphotransferase and a key enzyme in the regulation of glycerol uptake and metabolism, is involved in triglyceride and glycerophospholipid synthesis. glycerol kinase converts glycerol, a product of lipolysis to glucose in the liver, and shows downregulation in alcoholic liver cirrhosis. in the present study, a2-hs-glycoprotein has shown decreased expression in ald. it is a secretory protein expressed from liver and key regulator in inhibition of vascular calcification, bone metabolism regulation, control of protease activity, insulin resistance, and breast tumor cell proliferative signaling. 42 a-1-acid glycoprotein, an acute phase synthesized primarily in hepatocytes, which acts as a carrier of lipophilic compounds was down regulated in ald. 43 an serpinf2, a2-antiplasmin, synthesized in the liver as a single-chain glycoprotein with a molecular weight of 51 kda inhibits plasmin and was decreased in liver cirrhotic patients. 44 antithrombin iii, a member of the serpin family and an inhibitor of proteinases, namely, thrombin and factor xa, is primarily synthesized by hepatocytes and downregulated in ald. 45 our studies are also corroborated with the downregulation of a2-antiplasmin and antithrombin iii in ald. newly identified proteomic biomarker candidates for ald need validation and clinical assay optimization which require measurement of thousands of patient samples with narrow measurement coefficient of variation values. 18 concentration of proteins in serum/plasma ranges from picograms to nanograms per milliliter. enzyme-linked immunosorbent assay is the best alternative for quantification of these protein candidates for ald with high specificity and sensitivity capture and detection antibodies. newly developed assay requires analytical validation before evaluating clinical utility in terms of performance characteristics, namely, outcome studies, clinical requirement, proficiency testing, and goals set by regulatory agencies. 46 indicators of accuracy, precision, analytical measurement range, and reference intervals should be defined for newly discovered biomarker candidates. 47 after analytical validation of new methodology for protein of interest, biomarker candidate should confirm the performance characteristics in terms of consistency and accuracy in clinical evaluation to diagnose or predict the clinical outcome of ald. the newly identified biomarker candidate should satisfy specificity and sensitivity. evidencebased biomarker should fulfill regulatory requirements before introduced into clinical practice for ald. 18,48 conclusion with the help of technological advancement in “-omics” approach, we identified 28 protein biomarker candidates (13 with increased expression and 15 with decreased expression) for alcoholic liver cirrhosis. despite numerous biomarker candidates identified, verification may be done only for few qualified candidates that act in cellular pathways and deregulated in ald. these differentially expressed proteins between alcoholic cirrhosis and healthy subjects need to be validated to get the same differential expression detectable by assay to be used for validation. acknowledgements the authors thank dr kiranmayee p, sduaher for her timely suggestions to carry out this research work. declaration of conflicting interests the author(s) disclosed receipt of the following financial support for the research, authorship, and/or 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guillermina miranda-dı́az 1 , sonia sifuentesfranco 1 , ernesto germán cardona-muñoz 1 , adolfo daniel rodrı́guez-carrizalez 1 , geannyne villegas-rivera 2 , and luis miguel román-pintos 2 abstract oxidative stress induces nerve damage in type 2 diabetes mellitus and leads to diabetic polyneuropathy (dpn) and can affect the dna and antioxidant status. statins have pleiotropic, protective effects on the peripheral nerves of patients with diabetes. the aim of this study was to determine the effects of ezetimibe/simvastatin and rosuvastatin on dna damage in patients with dpn. this randomized, double-blind, placebo-controlled, clinical trial comprised outpatients from guadalajara, mexico. the inclusion criteria were either gender, age 35–80 years, type 2 diabetes, glycated hemoglobin �10%, diabetic polyneuropathy stage 1/2, and signed informed consent. patients who were taking antioxidant therapy or statins, had hypersensitivity to drugs, experienced organ failure, were pregnant or breastfeeding, or had other types of neuropathy were excluded. we assigned patients to placebo, ezetimibe/simvastatin 10/20 mg, or rosuvastatin 20 mg, and the primary outcomes were 8-hydroxy-20-deoxyguanosine (8-ohdg) for dna damage, 8-oxoguanine-dna-n-glycosilase (hogg1) for dna repair, and superoxide dismutase (sod). seventy-four patients were recruited. nine patients were included as negative controls. there were no differences in 8-ohdg between the healthy subjects (4.68 [3.53–6.38] ng/ ml) and the dpn patients (4.51 [1.22–9.84] ng/ml), whereas the hogg1 level was 0.39 (0.37–0.42) ng/ml in the healthy subjects and 0.41 (0.38–0.54) ng/ml in patients with dpn at baseline (p ¼ 0.01). sod decreased significantly in patients with dpn (5.35 [0.01–17.90] u/ml) compared with the healthy subjects (9.81 [8.66–12.61] u/ml) at baseline (p < 0.001). no significant changes in dna biomarkers were observed in any group between baseline and final levels. we noted a rise in hogg1 in patients with dpn, without modifications after treatment. there was a slight, albeit insignificant, increase in sod in patients who were on statins. keywords diabetic polyneuropathy, oxidative stress, statins, 8-hydroxy-20-deoxyguanosine, 8-oxoguanine-dna-n-glycosylase, superoxide dismutase date received: 30 march 2018; accepted: 10 september 2018 introduction diabetes mellitus (dm) is a heterogeneous, chronic, and progressive disease that is caused by insulin resistance and pancreatic b-cell dysfunction leading to extracellular hyperglycemia. 1 hyperglycemia promotes the characteristic macroand microvascular complications of dm by increasing oxidative stress through reactive oxygen species (ros) formation, 2,3 which in turn induces dna damage, 4,5 1 departament of physiology, institute of clinical and experimental therapeutics, university health sciences centre, university of guadalajara, guadalajara, jalisco, méxico 2 department of health-disease sciences as an individual process, tonala university centre, university of guadalajara, jalisco, méxico corresponding author: luis miguel román-pintos, department of health-disease sciences as an individual process, tonala university centre, university of guadalajara, lacandones 318, fracc. monraz, cp 44670, zapopan, jalisco, méxico. email: lmroman@yahoo.com journal of circulating biomarkers volume 7: 1–7 ª the author(s) 2018 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454418804099 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). http://orcid.org/0000-0002-5180-3810 http://orcid.org/0000-0002-5180-3810 mailto:lmroman@yahoo.com https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454418804099 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage including damage to the oxidized base, nonbase locations, and rupture of cross-links and dna chains. 5 further, fatty acid signaling pathways and oxidative stress defense against proinflammatory cytokines become dysregulated, as do dna repair mechanisms. 6 position 8 of guanine in dna is susceptible to rosinduced injury, causing oxidative dna damage, reflected by the increase in 8-ohdg, a sensitive and specific mutagenic biomarker that effects dna damage. 7 dna repair through base excision is achieved through hogg1 to reduce the noxious effects of 8-ohdg. 8 ros can also impair cellular defense and compromise the antioxidant status, thus promoting oxidative stress-induced injury. 9 diabetic polyneuropathy (dpn) is the most frequent microvessel complication in type 2 dm patients, developing in nearly 110 million people worldwide. 10 its prevalence increases approximately 10% one year after dm is diagnosed and can be found in 50%, 25 years after the diagnosis of type 2 dm. 11 several groups have described the mechanisms of peripheral nerve dysfunction, secondary to oxidative stress-induced damage in dm. 12 statins (3-hydroxy-3-methylglutaryl coenzyme a inhibitors) are potent serum cholesterol reducers that have pleiotropic, protective effects on peripheral nerves in patients with dm. 13,14 epidemiological studies have reported that dyslipidemia is a risk factor for dpn. 15 simvastatin has antioxidant activity against hydroxyl radicals. 16 by adding ezetimibe to simvastatin, greater cardiovascular protection is achieved, secondary to a reduction in low-density lipoprotein cholesterol. 17 rosuvastatin upregulates antioxidant defense in vitro and reduces the production of ros-dependent nicotinamide adenine dinucleotide phosphate; 18 thus, it diminishes oxidative stress in patients with dyslipidemia. 19 we aimed to determine the effects of ezetimibeþ simvastatin and rosuvastatin on oxidative stress-induced dna damage and superoxide dismutase (sod) in patients with dpn. materials and methods in this randomized, double-blinded, placebo-controlled, clinical trial, patients took one daily dose of placebo (calcined magnesia), ezetimibe þ simvastatin 10/20 mg, or rosuvastatin 20 mg at night for 16 weeks. we also included nine clinically healthy individuals to establish normal values of the biomarkers. randomization was performed through computer-based assignment of codes. a pharmacist packed the medication in vials, containing 30 units each, similar in appearance, size, and form. we oversaw delivery of the vials every 4 weeks, which had been tagged with the assigned codes. each patient received a diary to record the time and date that the pills were administered, to ensure adherence. every month, the diary was revised, and a pill count was performed to guarantee adherence and medication safety. laboratory safety profiles (creatine phosphokinase and liver and kidney function) were analyzed each month, and those who had significant adverse reactions were eliminated from the study. the subjects were outpatients who had been recruited from the primary care clinics of guadalajara and its surrounding areas. the inclusion criteria were either gender, age 35–80 years, type 2 dm that had been diagnosed per the american diabetes association criteria, 20 glycated hemoglobin (hba1c) �10%, dpn as confirmed by dyck,21 and signed informed consent. the exclusion criteria were patients who had been taking any antioxidant therapy or statins for the past 3 months, hypersensitivity to the test substances, liver/kidney failure, pregnancy/breastfeeding, and other types of neuropathy. we rejected patients if they were nonadherent to the pharmacological treatment (less than 80% of pill intake) or when a statin was necessary, acute complications of dm, or evidence of severe impairment that prevented continuation with the treatment. the sample size was calculated, based on the comparative effects of simvastatin and atorvastatin on oxidative stress markers and antioxidant status. 22 the type i error was 0.05, and the type ii error was 0.20; the mean sod in the control group was 106.87 versus 75.63 in the experimental group. the standard deviation (sd) was 14 mmol/l, with two treatment groups, an allocation rate of 1:1, and two tailed. as a result, there were 19 patients in each group. after blood samples had been collected in two tubes (one with 0.1% ethylenediaminetetraacetic acid and the other dry), we separated the plasma and serum by centrifugation at 2000 r/min for 10 min at room temperature; then, the samples were stored at �80�c until use. the synergy ht (biotek, winooski, vermont, usa) microplate reader was used to perform all optical density readings. 8-hydroxy-20-deoxyguanosine we used the enzyme-linked immunosorbent assay kit (8-hydroxy-2-deoxyguanosine; no. ab10124 abcam ® , cambridge, uk) according to the manufacturer’ instructions. first, the plasma sample, enzyme immunoassay (eia) buffer, standards, and 8-ohdg-ache tracer were added to all the wells except for the blank. then, we added the 8ohdg monoclonal antibody and incubated the plate for 18 h at 4�c. the plate was washed with buffer for the recommended times, and 200 ml ellman’s reactive was added to each well. the optical density was read at 405 nm, and levels of 8-ohdg were reported in nanograms per milliliter. 8-oxoguanine-dna-n-glycosylase-1 the repair of oxidative damage to dna was measured using a commercial kit (human-8-oxoguanine-dnaglycosylase; mbs702793 mybiosource ® , san diego, california, usa) according to the manufacturer’s instructions. the reagents and samples were prepared for the indicated dilutions. we added 100 ml of plasma and standards to the 2 journal of circulating biomarkers wells and incubated the plate at 37�c. the biotinylated antibody was aggregated and then incubated. we performed the corresponding washes and added the horseradish peroxidase–avidin, substrate, and stop solution at the appropriate times. the optical density was read at 450 nm, and the levels were reported in nanograms per milliliter. superoxide dismutase we measured o�2 that was generated by xanthine oxidase and hypoxanthine through the reaction of tetrazolium salts using a commercial kit according to the manufacturer’s instructions (sod no. 706002, cayman chemical company ® , ann arbor, michigan, usa). we diluted the serum samples 1:5 in sample buffer and added 10 ml of the sample to 200 ml of detector (1:400 dilution). after slow agitation, 20 ml of xanthine oxidase was added to the wells. then, the microplate was incubated for 20 min at room temperature, and the absorbance was read at 440 nm. levels were reported in units per milliliter. statistical analysis we reported categorical variables as frequencies and percentages and analyzed them by �2 or fisher’s exact tests as needed. we performed kolmogorov–smirnov and shapiro–wilk tests to determine the distribution of the main variables and concluded that they did not follow a normal distribution; thus, we expressed continuous variables as median (min � max). we used wilcoxon test for intragroup comparisons and kruskal–wallis test for between-group comparisons, followed by mann–whitney u test as the post hoc analysis. we performed an intention-to-treat analysis. results were significant at a two-sided p-value of <0.05, with the 95% confidence interval. ethical considerations the helsinki statements in the 64th general assembly in fortaleza, brazil, in october 2013 were followed, in agreement with national and international laws. the protocol was revised and approved by the local research and ethics committee of the university of guadalajara, under registry cei/044/2014 and international clinical trial number nct02129231 (clinicaltrials.gov). results demographics a total of 107 patients were evaluated—18 were taking statins, 5 did not sign the informed consent form, 8 had another type of neuropathy, and 2 had end-stage renal disease. the remaining 74 subjects were randomly allocated to one of the three groups (figure 1). the mean age was 54.55 + 10.65 years, and none of the patients was aged under 30 years. the female:male ratio was 1.5:1. gender, age, weight, height, body mass index, and duration of diabetes were similar between all treatment groups (table 1). metabolic parameters the baseline plasma glucose level in the placebo group was 186.40 + 70.94 mg/dl versus 146.49 + 49.75 mg/dl in the ezetimibe/simvastatin group and 188.59 + 64.80 mg/ dl in the rosuvastatin group (p ¼ 0.07). hyperglycemia was persistent, despite medical treatment, and did not differ between groups (p ¼ 0.44). the hba1c level in the placebo group was 8.83 + 1.76%, compared with 7.92 + 1.51% in the ezetimibe/ simvastatin group and 8.86 + 1.99% in the rosuvastatin group (p ¼ 0.16), and did not differ significantly at the end of the trial (p ¼ 0.12). baseline total cholesterol levels were 211.43 + 56.24 mg/dl for the placebo group, 210.71 + 50.77 mg/dl for the ezetimibe/simvastatin group, and 216.81 + 39.45 mg/ dl for the rosuvastatin group (p ¼ 0.80). the levels after the intervention were 202.35 + 38.53 mg/dl, 130.35 + 48.11 mg/dl, and 141.12 + 41.89 mg/dl, respectively (p < 0.001). the baseline triglyceride levels for the placebo, ezetimibe/simvastatin, and rosuvastatin groups were 240.13 + 131.48 mg/dl, 239.22 + 249.19 mg/dl, and 217.08 + 121.32 mg/dl, respectively (p ¼ 0.55); the final levels were 242.39 + 142.56 mg/dl, 162.26 + 105.97 mg/dl, and 167.75 + 114.40 mg/dl (p ¼ 0.03). laboratory safety parameters underwent minimal changes and did not affect creatine phosphokinase or liver or kidney function during the treatment period (data not shown). the median and range for plasma 8-ohdg in healthy subjects was 4.68 (3.53–6.38) ng/ml versus 4.51 (1.22– 9.84) ng/ml for patients with dpn (p ¼ 0.79). the baseline levels for the placebo, ezetimibe/simvastatin, and rosuvastatin groups were 5.00 ng/ml (1.99–9.84), 3.99 ng/ml (1.22–9.84), and 4.06 (3.41–7.57) ng/ml, respectively (p ¼ 0.57). after treatment, these levels were 4.50 ng/ml (1.83–19.63), 4.17 ng/ml (2.34–7.23), and 4.22 ng/ml (2.20–9.84) (p ¼ 0.89). the normal value of hogg1 in the healthy subjects was 0.39 ng/ml (0.37–0.42) and 0.41 ng/ml (0.38–0.54) for patients with dpn (p ¼ 0.01). the baseline levels were 0.41 ng/ml (0.38–0.54), 0.41 ng/ml (0.38–0.47), and 0.40 ng/ml (0.39–0.53) for the placebo, ezetimibe/simvastatin, and rosuvastatin groups (p ¼ 0.65), compared with 0.41 ng/ml (0.38–0.45), 0.41 ng/ml (0.38–0.49), and 0.41 ng/ml (0.38–0.51) (p ¼ 0.92), respectively, after the intervention. sod levels in healthy subjects were 9.81 u/ml (8.66– 12.61) versus 5.35 u/ml (0.01–17.90) in dpn patients (p < 0.001). the baseline sod levels were 5.73 u/ml (2.56– 17.90) for the placebo group, 4.61 u/ml (1.99–7.97) for carrillo-ibarra et al. 3 http://clinicaltrials.gov the ezetimibe/simvastatin group, and 5.72 u/ml (0.01– 9.41) for the rosuvastatin group (p ¼ 0.56). the final levels were 7.64 u/ml (0.12–17.69), 4.34 u/ml (1.51–18.09), and 5.23 u/ml (0.01–25.81) (p ¼ 0.19) (table 2). discussion the baseline clinical and metabolic characteristics of patients with type 2 dm and dpn were similar between all groups. with regard to dyslipidemia, as expected, there was a significant reduction in the lipid profiles of patients who were treated with either statin, but rosuvastatin was more efficient in improving the triglyceride levels. although our aim was not to examine metabolic parameters, glycemia status and lipid profile are variables that can alter antioxidant/oxidative homeostasis. studies have shown a reduction in the urinary levels of 8-oh2dg in patients with insulin-dependent and non– insulin-dependent dm; 23,24 further, higher levels of this biomarker have been implicated in the severity of microvascular complications in dm, suggesting a direct effect of 8-oh2dg in dpn. 25 tonolo et al. reported a significant figure 1. flow chart diagram of patients. 4 journal of circulating biomarkers decrease in urinary 8-ohdg in patients with type 2 dm, microalbuminuria, and hypertension after 4-year treatment with simvastatin. 26 abe et al. observed a decline in urinary 8-ohdg after 6 months of treatment with rosuvastatin in patients with diabetic nephropathy. 23 however, we failed to note any difference in the plasma levels of 8-ohdg in patients with dpn versus the healthy subjects or any change after 16 weeks of treatment with statins. these findings suggest that the baseline plasma 8-ohdg is similar in those with and without diabetes, at table 1. baseline demographic and clinical characteristics of healthy subjects and patients in each treatment group. a variable healthy subjects (n ¼ 9) placebo (n ¼ 24) ezetimibe/simvastatin (n ¼ 25) rosuvastatin (n ¼ 25) p value female gender, n (%) 4 (44.4) 17 (70.8) 16 (60.0) 13 (52.0) 0.40 age (years) 55.56 + 5.55 54.71 + 9.63 55.00 + 12.06 53.96 + 10.48 0.78 age-group, n (%) 0.55 31–40 0 (0) 1 (4.2) 4 (16.0) 4 (16.0) 41–50 1 (11.1) 6 (25.0) 4 (16.0) 2 (8.0) 51–60 6 (66.7) 8 (33.3) 6 (24.0) 9 (36.0) 61–70 2 (22.2) 8 (33.3) 9 (36.0) 10 (40.0) >70 0 (0.0) 1 (4.2) 2 (8.0) 0 (0.0) weight (kg) 72.32 + 10.22 73.71 + 11.43 75.40 + 13.85 76.93 + 18.73 0.83 height (m) 1.62 + 0.12 1.58 + 0.09 1.59 + 0.10 1.62 + 0.12 0.74 bmi (kg/m2) 28.08 + 4.08 29.29 + 4.31 29.37 + 4.11 29.04 + 4.65 0.94 nutritional status, n (%) 0.38 normal 2 (22.2) 4 (16.7) 4 (16.0) 4 (16.0) overweight 5 (55.6) 10 (41.7) 8 (32.0) 11 (44.0) obesity class i 2 (22.2) 7 (29.2) 12 (48.0) 6 (24.0) obesity class ii 0 (0.0) 3 (12.5) 0 (0.0) 4 (16.0) obesity class iii 0 (0.0) 0 (0.0) 1 (4.0) 0 (0.0) duration of type 2 dm (years) — 10.08 + 8.34 10.24 + 6.64 12.08 + 8.33 0.67 duration of dpn symptoms (years) — 0.96 + 1.39 2.40 + 3.3 1.68 + 2.51 0.41 hypertension, n (%) — 16 (66.7) 15 (60.0) 8 (32.0)* 0.04 sbp (mmhg) 125 + 15 142 + 25 143 + 26 135 + 17 0.68 dbp (mmhg) 76 + 8 84 + 11 81 + 10 81 + 7 0.43 active smoking, n (%) 1 (11.1) 9 (37.5) 8 (32.0) 12 (48.0) 0.50 dm treatment, n (%) 0.40 metformin — 5 (20.8) 5 (20.0) 4 (16.0) glyburide — 0 (0.0) 1 (4.0) 3 (12.0) metformin/glyburide — 15 (62.5) 13 (52.0) 11 (44.0) insulin — 1 (4.2) 2 (8.0) 0 (0.0) insulin and oad — 1 (4.2) 2 (8.0) 5 (20.0) other oad — 2 (8.3) 2 (8.0) 2 (8.0) bmi: body mass index; dm: diabetes mellitus; dpn: diabetic polyneuropathy; sbp: systolic blood pressure; dbp: diastolic blood pressure; oad: oral antidiabetic. avalues are represented as mean + sd, unless otherwise specified; p for treatment groups (healthy subjects not included). *p < 0.05 versus placebo and ezetimibe/simvastatin. significant values are shown in bold. table 2. baseline and final levels of 8-ohdg, hogg1, and sod in patients with dpn.a 8-ohdg (ng/ml) hogg1 (ng/ml) superoxide dismutase (u/ml) baseline final p baseline final p baseline final p healthy subjects 4.68 (3.53–6.38) 0.39 (0.37–0.42) 9.81 (8.66–12.61) placebo 5.00 (1.99–9.84) 4.50 (1.83–19.63) 0.81 0.41 (0.38–0.54)* 0.41 (0.38–0.45)* 0.33 5.73 (2.56–17.90)* 7.64 (0.12–17.69)* 0.20 ezetimibe/ simvastatin 3.99 (1.22–9.84) 4.17 (2.34–7.23) 0.93 0.41 (0.38–0.47) 0.41 (0.38–0.49)* 0.86 4.61 (1.99–7.97)** 4.34 (1.51–18.09)* 0.23 rosuvastatin 4.06 (3.41–19.63) 4.22 (2.20–9.84) 0.18 0.40 (0.39–0.53)* 0.41 (0.38–0.51)* 0.69 5.72 (0.01–9.41)** 5.23 (0.01–25.81)* 0.23 sod: superoxide dismutase; dpn: diabetic polyneuropathy; 8-ohdg: 8-hydroxy-20-deoxyguanosine; hogg1: 8-oxoguanine-dna-n-glycosilase. a values are represented as median (min–max). *p < 0.05 versus healthy subjects. **p < 0.01 versus healthy subjects. significant values are shown in bold. carrillo-ibarra et al. 5 least in our study groups. moon et al. reported similar results in patients with atherosclerotic cerebrovascular disease, wherein 99 patients with acute or chronic stroke received rosuvastatin 20 mg/day for 1 month. they evaluated the antioxidant effects of rosuvastatin on malondialdehyde (mda), oxidized ldl (oxldl), protein carbonyl content (pco), and 8-ohdg (oxidative damage in lipids, proteins, and dna) but did not observe any significant reduction in 8-ohdg or pco, whereas mda and oxldl declined significantly. 24 few studies have examined the response to the concentration of hogg1 following the use of antioxidants. an observational study in patients with acute myocardial infarction reported that there were no differences in the concentrations of hogg1 between patients who were receiving statin therapy or not. 26 in our study, the concentrations of hoog1 were significantly elevated in the three groups of dpn versus the healthy controls. this finding suggests this repair enzyme is upregulated in response to oxidative damage and that concentrations within the normal ranges of 8-ohdg can be offset by the rise in hoog1. sod is the first-line of defense against ros. 27 in experimental and clinical research, a decrease in the activity of antioxidant enzymes, sod, catalase, and glutathione peroxidase favors the development of microvascular complications in dm. 28,29 the baseline activity of sod was reduced in patients with dpn compared with the healthy subjects before treatment initiation, consistent with other clinical investigations. 30 however, statins did not improve sod activity significantly in our study. in streptozotocininduced diabetic rats, a significant increase in sod and catalase was reported with simvastatin treatment, 31 but we could not reproduce these results in patients with type 2 dm and dpn. the baseline level of hoog1 was increased in our study, likely due to counteracting mechanisms of dna oxidative stress-induced damage; however, we would need more data on signaling pathways to make a definitive conclusion. ezetimibe/simvastatin and rosuvastatin might provide antioxidant benefits by reducing dyslipidemia; however, statins did not reduce 8-ohdg or hoog1 plasma levels. there was a slight increase in sod in patients who were on statins, but this change was not significant, perhaps because the study was too short to induce relevant changes in uncontrolled patients with a long duration of diabetes. acknowledgments we thank the laboratory staff of hospital civil of guadalajara “dr juan i. menchaca” for their aid in performing the metabolic parameters. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) received no financial support for the research, authorship, and/or publication of this article. orcid id luis miguel román-pintos http://orcid.org/0000-0002-5180-3810 references 1. bloomgarden zt. american diabetes association annual meeting, 1997. type 2 diabetes. diabetes care 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a new target for statin therapy. atherosclerosis 2011; 219(2): 753–760. 26. tonolo g, velussi m, brocco e, et al. simvastatin maintains steady patterns of gfr and improves aer and expression of slit diaphragm proteins in type ii diabetes. kidney int 2006; 70(1): 177–186. 27. aruoma oi, grootveld m, and bahorun t. free radicals in biology and medicine: from inflammation to biotechnology. biofactors 2006; 27(1–4): 1–3. 28. spanidis y, mpesios a, stagos d, et al. assessment of the redox status in patients with metabolic syndrome and type 2 diabetes reveals great variations. exp ther med 2016; 11(3): 895–903. 29. coppey lj, davidson ep, dunlap ja, et al. slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve. int j exp diabetes res 2000; 1(2): 131–143. 30. kasznicki j, kosmalski m, sliwinska a, et al. evaluation of oxidative stress markers in pathogenesis of diabetic neuropathy. mol biol rep 2012; 39: 8669–8678. 31. el-azab mf, hazem rm, and moustafa ym. role of simvastatin and/or antioxidant vitamins in therapeutic angiogenesis in experimental diabetic hindlimb ischemia: effects on capillary density, angiogenesis markers, and oxidative stress. eur j pharmacol 2012; 690(1–3): 31–41. carrillo-ibarra et al. 7 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true 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, melissa m chu 1 , nigel d toussaint 1,2 , michael mx cai 1,2 , tim d hewitson 1,2 , and stephen g holt 1,2 abstract the recently discovered klotho proteins have roles in a diverse range of metabolic processes with the oldest protein, aklotho, implicated in various cellular pathways in energy, glucose, and phosphate metabolism. circulating soluble klotho (skl), derived from membrane a-klotho cleavage, not only has effects on ion channels and insulin signaling pathways, but is inversely associated with mortality. effects of physical exercise on skl have not been well studied. the effect of a single high-intensity standardized exercise on skl and serum phosphate (spi) levels in healthy adults was investigated. a standard bruce protocol treadmill exercise was undertaken by 10 fasting healthy volunteers. skl, spi, and blood glucose levels were measured in samples collected 1-week prior, immediately pre (tpre), 0 (tpost), 30 (t30), 240 (t240) min, and 1-week after exercise. median (interquartile range) age of participants was 47.5 (44–51) years; five (50%) were male. all study participants achieved at least 90% predicted maximum heart rate (mhr). skl increased acutely after exercise (tpre median 448 pg/ml vs. tpost median 576 pg/ml; p < 0.01). there was a nonsignificant spi decline at t30 (tpre 0.94 + 0.12 mmol/l vs. t30 0.83 + 0.22 mmol/l). exercise led to a reduction in blood glucose by t240 with median glucose levels at tpre, tpost, t30, and t240 of 6.0, 6.5, 6.3, and 5.7 mmol/l, respectively. in conclusion, a single high-intensity exercise session is associated with a transient increase in skl, a delayed reduction in blood glucose, and a nonsignificant decrease in spi levels in healthy adults. the evaluation of long-term effects of cardiovascular fitness programs on skl and spi in healthy individuals and disease cohorts are required to identify potential lifestyle modifications to help improve chronic disease management and long-term outcomes. keywords exercise, soluble klotho, phosphate date received: 18 february 2018; accepted: 2 july 2018 introduction klotho was originally identified as an antiaging protein almost 20 years ago. genetic studies identified the klotho (kl) gene highlighting that over-expression extended life span and deletion produced complex phenotypes in animals similar to human aging. 1 the kl gene encodes a 1012 amino acid long protein, which is expressed in different tissues, although mostly in the kidney especially within the distal convoluted tubule. 1,2 the gene product is commonly referred to as a-klotho, to differentiate it from two other subsequently discovered members of the klotho family: b-klotho and g-klotho. while all three are single-pass transmembrane proteins of different lengths and share a 1 department of nephrology, the royal melbourne hospital, melbourne, vic, australia 2 department of medicine, the university of melbourne, parkville, vic, australia corresponding author: sven-jean tan, department of nephrology, the royal melbourne hospital, grattan street, parkville, vic 3052, australia. email: jean.tan@mh.org.au journal of circulating biomarkers volume 7: 1–7 ª the author(s) 2018 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454418794582 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:jean.tan@mh.org.au https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454418794582 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage substantial degree of homology, 3 they appear to have different physiological actions and play a role in a diverse range of metabolic processes. 1,4–8 a-klotho exists in two forms—membrane-bound klotho (mkl) and soluble klotho (skl). the actions of mkl and skl differ, with mkl within the kidney predominantly involved in phosphate regulation and renal tubular phosphate reabsorption. 6 circulating skl is the cleaved extracellular domain of mkl (approximately 130 kda) and can act as a soluble paracrine or endocrine mediator. 9–12 skl has also been directly implicated in phosphate regulation. 13 it displays enzymatic activity that may be important in regulating other ion channels (predominantly involving potassium and calcium transport). 14–16 skl has been implicated in peroxisome proliferator-activated receptor-g (pparg) regulation and insulin antagonism and has been shown to be anti-fibrotic, possess antioxidant, and tumor-suppressive properties. 17–20 distinguishing between the various autocrine, paracrine, and endocrine actions of a-klotho has been challenging due to its cellor organ-specific actions as well as its effects on a wide range of metabolic pathways. 20 b-klotho augments fgf-19 and fgf-21 signaling; is found in the liver, gall bladder, pancreas, colon, and adipose tissue; and participates in bile acid metabolic pathways. 7,8 g-klotho is coupled to fgf19 and is found in the eye, adipose, and kidney, although its function is not clearly understood. 3 the cleavage of b-klotho, relative contributions of the three proteins to circulating skl, the degree of overlap, and mutual influence between the three klotho proteins are unclear. 20 nonetheless, higher skl levels have been associated with improved survival 21 and lower levels with increased cardiovascular and all-cause mortality. 22 physical exercise is known to delay the effects of aging and to protect from cardiovascular disease, diabetes, and cancer. 23,24 however, the mechanisms through which exercise exerts these beneficial effects are not fully elucidated. it is, therefore, plausible that skl may mediate some of the benefits of exercise, given the many parallels between physiological effects of exercise and the pleiotropy exhibited by skl described above. there have been two previous studies demonstrating increased skl levels in women only, one in response to long-term exercise 25 and the other in response to acute, high-intensity exercise. 26 we aimed to investigate the effect of an acute high-intensity exercise on serum klotho, glucose, and phosphate in both healthy male and female adults. methods study population this single-center study recruited healthy adult volunteers aged >18 years old to participate in a single-intervention study. exclusion criteria included history of cardiovascular disease, chronic kidney disease, diabetes mellitus, psychological or medical illness precluding informed consent, and physical handicap impairing ability to undertake a treadmill intervention. male and females were age-matched with equal numbers of males and females recruited. informed consent was obtained from all individual participants included in the study. this study was approved by the local human research ethics committee (hrec 2015.097) and conducted in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. exercise intervention all participants were nonsmokers and given routine instructions on how to prepare for a standard exercise test (e.g. instructed to dress with appropriate footwear for the exercise intervention). participants fasted from midnight, the night prior to the exercise intervention. none of the study participants were on treatment with medications that could affect exercise capacity or exercise responses. a treadmill-based exercise stress test (est) was performed following a standard bruce protocol. 27 in brief, 12-lead electrocardiogram (ecg) electrodes were placed on the torso with limb leads placed on shoulders and below the level of the umbilicus on respective sides, ensuring good skin contact to reduce skin resistance. all participants started at low workload treadmill speed. a gradual and continuous increase in workload was instituted every 3 min corresponding to the bruce protocol. an approximately 0.8 mph increase in treadmill speed and 2% ramp incline occurred from stage i (1.7 mph and 10% incline) to stage v (5.0 mph and 18% incline) and a smaller increase in speed occurred for the final stage (vi—5.5 mph and 20% incline). mhr was estimated for each individual participant using the following formula: mhr ¼ [220–age(years)]. a target of 85% of mhr was applied for the est. participants were instructed to advise investigators and est technician if premature termination of the est was required. all participants were monitored in the resting phase after the completion of the est for a minimum of 6 min until blood pressure, heart rate, and ecg tracing approximate baseline values. all participants were provided with a standard meal at 60 min post est. participants were also instructed to refrain from strenuous exercise or activity 1-week pre and post the intervention. data and sample collection baseline measurements including blood pressure, heart rate, and 12-lead ecg were performed on all participants in supine and standing resting positions prior to the est. ecg recording was continued throughout the est in accordance with the standard protocol, with standing blood pressure measurements recording at 3-min intervals. 2 journal of circulating biomarkers baseline tests for hemoglobin, blood glucose level, serum biochemistry, and klotho were measured immediately prior to exercise intervention (tpre). blood samples were also collected 1 week prior to est (td-7), 0 (tpost), 30 (t30), 60 (t60), 120 (t120), 240 (t240) min; 1 day (tdþ1); and 1 week post est (tdþ7) for repeat evaluation of serum phosphate (spi) and blood glucose levels. skl was measured at td-7, tpre, tpost, t30, t240, and tdþ7. biochemical analysis routine biochemistry was performed at a central laboratory using standard techniques. spi was measured via the olympus au7000 platform (beckman coulter, inc., brea, california, usa) using molybdate or the chromogen arsenazo iii, respectively. the analytical range for spi was 0.32–5.00 mmol/l with analytical coefficient of variation (cv) <2.5%. blood collected in a serum separator tube (sst™; bd biosciences, franklin lakes, new jersey, usa) was allowed to stand at room temperature for 30 min prior to centrifugation (10 min, 4�c, 3000 � g) and aliquoted for storage at �80�c until batched analysis. serum skl concentrations were measured using the ibl skl elisa kit (immuno-biological laboratories co., ltd, gunma, japan) according to the manufacturer’s protocol. based on duplicate measurements, the intra-assay and inter-assay cvs for this study were 3.1 and 7.2%, respectively. blood glucose levels were tested at the above time points using a calibrated freestyle optium h ™ (abbott diabetes care, doncaster, vic, australia) blood glucometer and accompanying blood glucose test strips. statistical analysis continuous variables have been reported using mean and standard deviation or median and interquartile range (iqr) as appropriate. student’s t-test or mann–whitney tests were used first to evaluate differences between male and females as appropriate. friedman’s test with dunn’s multiple comparison was used to assess changes in skl and blood glucose levels across all time points measured. wilcoxon-signed rank test was used to evaluate individual time points against either tpre or tpost. repeated measures anova was used to assess changes in spi. all statistical analyses were performed with spss statistics version 24.0 (ibm corp., armonk, new york, usa). all graphics have been created with graphpad prism 7 for macintosh (la jolla, california, usa). a p-value of <0.05 was considered significant unless otherwise stated. results a total of 10 participants were included in the analysis. all participants were nonsmokers and none were on treatment with medications that could affect exercise capacity or exercise responses (such as beta-blockers or calcium channel blockers). five participants (50%) were male. median (iqr) age of participants was 47.5 (44–51) years. baseline (resting/fasting) characteristics of study participants are presented in table 1. all participants had baseline blood pressure, heart rate, hemoglobin, glucose, and serum creatinine values that were within the expected range for age and sex. males exhibited higher resting systolic blood pressures (137 + 9.1 vs. 125 + 5 mmhg (females); p ¼ 0.032). males demonstrated a trend toward lower baseline skl levels compared with females (423 (415–614) pg/ml in males vs. 669 (448–1095) pg/ml in females; p ¼ 0.117). all participants achieved at least 90% estimated mhr, and 9/10 reached 100% mhr, with a mean duration of 12.3 min of exercise. all exercise was terminated by the participants with fatigue, and no ecg changes suggesting ischemia were noted at any time during or at least 6 min into recovery. no adverse events were recorded during or following the est. mean heart rate and blood pressure readings throughout the est are shown in figure 1. table 1. baseline characteristics of study participants (n ¼ 10).a clinical parameter total (n ¼ 10) males (n ¼ 5) females (n ¼ 5) age (years) 49 (44.75–50.75) 47 (44–50) 50 (48–51) gender (male; n, %) 5/10 (50%) 5 5 resting heart rate (bpm) 84.3 + 10.9 86.2 + 10.1 82.4 + 12.6 resting systolic blood pressure (mmhg) 131 + 9.4 137 + 9.1 125 + 5* resting diastolic blood pressure (mmhg) 83 + 7.1 83 + 4.5 83 + 9.7 hemoglobin (g/l) 141.1 + 16.7 154.6 + 4.8 124.3 + 5.3 serum calcium (mmol/l) 2.38 + 0.05 2.38 + 0.04 2.38 + 0.06 spi (mmol/l) 0.98 + 0.12 0.92 + 0.12 1.03 + 0.11 serum creatinine (mmol/l) 71.5 + 11.1 79.0 + 8.6 64.0 + 8.0 egfr (ml/min/1.73 m2) 98.9 +12.1 99.4 + 11.0 98.3 + 14.4 serum skl (pg/ml) 483 (423–767) 423 (415–614) 669 (448–1095) blood glucose reading (optima glucometer; mmol/l) 5.07 + 0.42 5.36 + 0.15 4.78 + 0.41 egfr: estimated glomerular filtration rate; spi: serum phosphate; skl: soluble klotho. a t-test or mann–whitney u-test performed for continuous variables between gender groups. *p < 0.05 compared to males. tan et al. 3 changes in skl for study participants throughout the study period are presented in figure 2 and table 2. a significant increase after the est in skl was seen (tpre median 483 (423–766) pg/ml vs. tpost 602 (514–831) pg/ ml; p < 0.01) but levels returned to baseline by t30 (497 (445–746) pg/ml). changes in spi and blood glucose are presented in figure 3. a nonsignificant trend toward reduction in spi post est was observed, with spi at tpre compared to t30 (tpre 0.94 + 0.12 mmol/l vs. t30 0.83 + 0.22 mmol/l; p > 0.05). this reduction was sustained until t120, 0.77 + 0.17 mmol/l (figure 3). median glucose levels at tpre, tpost, t30, and t240 were 6.0, 6.5, 6.3, and 5.7 mmol/l, respectively. an initial nonsignificant rise in blood glucose was seen at tpost following est (p ¼ 0.26 vs. tpre). this was followed by a reduction in blood glucose observed at the earliest at t120, median glucose level 5.8 mmol/l (p ¼ 0.65 vs. tpost). this reduction was sustained at t240 (p ¼ 0.013 vs. tpost; figure 3). discussion a standardized high-intensity physical exercise in healthy adults causes a transient but immediate increase in skl levels and a delayed reduction in blood glucose levels. two other studies have reported an increase in skl following exercise, and neither of these showed the time course of this rise and were performed in postmenopausal women undergoing a 12-week aerobic exercise program 25 or in men and women after a 20-min run. 26 in contrast to this study, the latter failed to demonstrate an increase in skl level in men who were recruited to the same study. however, all participants in this latter study 26 were involved in regular physical activity with a minimum of 1-year resistance and aerobic training prior to the study. there are limited studies in this area and the effect of gender has not been widely investigated. it has been suggested though that preexisting fitness level and type of exercise, aerobic versus anaerobic, could impact on the intensity of skl inflection following the prescribed exercise. 28,29 the study presented here included a standardized est in order to individualize exercise according to age of participants by targeting mhr and included healthy participants who were not allowed to undertake physical activity in the 1 week prior to the intervention. skl measurements were performed at more than one time point allowing temporal evaluation in skl change following the intervention. this provides novel additional information compared to the prior two reports, 25,26 demonstrating that a single episode of high-intensity exercise is not only associated with an increase in skl in both men and women but is not sustained. multiple mechanistic pathways have been considered in the exercise-induced elevation of circulating skl levels. physical exercise has been reported to upregulate pparg,30 downregulate angiotensin-ii type-1 receptor (at1 r), 31 and oxidative stress 32 in animal models. in turn, these pathways have been linked to increased renal klotho mrna expression and skl levels, 33–35 thereby potentially increasing circulating skl levels. notably, the time lag in animal models between physical exercise/activity and biochemical evaluation ranged from 6 to 16 weeks, 30–32 while the follow-on in vitro pparg and oxidative stress effects on klotho mrna have been reported ranging between 12 h and 48 h 33,34 and in vivo effects of at1 r blockade on klotho mrna between 4 days and 14 days. correspondingly, the clinical study by matsubara et al. demonstrated figure 1. changes in blood pressure and heart rate with est intervention. est: exercise stress test. figure 2. change in skl levels in males (black lines) and females (dotted lines) following est (percentage change from tpre). **p < 0.005 versus tpre. #p < 0.05 and ##p < 0.005 versus tpost. skl: soluble klotho; est: exercise stress test. 4 journal of circulating biomarkers an increase in skl levels following a 12-week exercise intervention, 25 where the aforementioned mechanisms are likely to be implicated. we have shown that acute and transient increases in circulating skl occur suggesting more acute physiological processes may be involved, such as a rapid activation of cleavage enzymes or a release of preformed skl within seconds to minutes of strenuous exercise. a trend toward lower spi levels post exercise was observed in this study, and while lower spi levels may be in response to the increased skl levels mediating phosphate excretion, 13 the apparent reduction in spi levels occurs promptly in response to physical activity. both increase and decrease in spi levels have been reported previously following physical activity, despite consistently documented increases in parathyroid hormone level. 36–38 these discrepancies may reflect the different physical exercise programs that the respective study participants were subjected to including a prolonged (5-h) low-intensity bicycle ergometer exercise, a 7-day high-intensity field exercise, and maximal exercise capacity testing prior to and after a 6-week endurance program. 36–38 however, the reduction in spi may simply reflect extracellular pi depletion related to uptake into muscle which outstrips resupply. the wide variation in these prescribed interventions limits the ability to compare these results and also alludes to the wide variation and day-to-day fluctuations that occur normally when measuring extracellular spi. furthermore, no “phosphate sensor” has ever been reported in humans so to invoke changes in skl levels due to changes in spi remains highly speculative and this study provides little further insight into such a mechanism. lower glucose levels were also detected at t240 post est and this is consistent with well-established evidence showing improvements in insulin sensitivity after a single bout of physical exercise, 39,40 although the intensity and duration of such activity to achieve maximal beneficial outcome for the healthy population as well as those with insulin resistance continues to be debated. 41 physical exercise, however, may be one of the key stimulators of skeletal muscle glut4 expression. 42 taken together, the data presented here might suggest a number of pathways implicated in beneficial changes of physical activity with a central role for glucose and energy metabolism, pparg and skl. there are several limitations to this study. the cohort is small in size and, therefore, the findings from this study require validation within a larger cohort. although there is a lack of representation of patients with chronic disease in this study such as chronic kidney disease, diabetes, and coronary heart disease, the study findings in healthy adults are encouraging. the standardized meal provided to the study participants following the est in a fasting state was intended to standardize the glucose load, though may have affected the statistical significance of the glucose and phosphate results following t60. further studies are required to quantify the long-term effects of standardized cardiovascular fitness and high-intensity exercise programs on skl, spi, and blood glucose in healthy individuals. subsequently, studies are needed to evaluate the same programs in disease cohorts in order to identify beneficial lifestyle modifications that could improve chronic disease management and long-term outcomes. conclusion in summary, this study demonstrated that a single shortduration episode of high-intensity physical exercise is table 2. median skl levels following est in males (m) and females (f). a time point td-7 tpre tpost t30 t240 tdþ7 skl (n ¼10; pg/ml) 484 (442–748) 483 (423–766) 602** (514–831) 497## (445–746) 485# (449–738) 517 (419–781) skl (n ¼ 5 m; pg/ml) 455 (414–509) 423 (415–614) 596* (508–704) 464# (423–626) 468 (416– 603) 507 (416–603) skl (n ¼ 5 f; pg/ml) 718 (468–971) 669 (448–1095) 765* (527–126) 722# (440–1006) 649# (446–1041) 671 (436–1148) skl: soluble klotho; est: exercise stress test. a friedman’s test with dunn’s multiple comparison was used to assess changes in skl across all time points measured. wilcoxon-signed rank test was used to evaluate individual time points against either tpre or tpost. *p < 0.05 versus tpre. **p < 0.005 versus tpre. # p < 0.05 versus tpost. ## p < 0.005 versus tpost. figure 3. changes in median blood glucose level (black line) and mean serum phosphate level (dotted line) following est (percentage change from tpre). #p < 0.05 compared to tpost for blood glucose level. est: exercise stress test. tan et al. 5 associated with an acute and transient increase in skl, a delayed glucose reduction, and with spi levels showing a trend to acute reduction also. future studies, both larger standardized interventional clinical studies and basic science research, are critical to improve the understanding of the mechanisms involved in benefits of physical exercise and also to enable translation of these concepts to guide clinical practice especially in cohorts with lower skl or hyperphosphatemia, both of which are associated with impaired glucose tolerance and poorer survival. the relative benefit of exercise in the low baseline fitness group should also be investigated as this may be the group that benefits most from the flow-on effects of skl rise. authors’ note the contents of this article are solely the views of the individual authors and do not reflect the views of nhmrc or the jacquot foundation. acknowledgment the authors would like to thank ms sarra byrns for her technical assistance with conducting est. author contributions sjt designed and conducted the clinical study, undertook the assays, performed the data analysis, and prepared the manuscript. mmc conducted the clinical study and contributed to manuscript. ndt, mmxc, and sgh aided with the study design and critically appraised the manuscript. tdh provided assistance with the assays and contributed to manuscript. declaration of conflicting interests the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: sjt has received speaking honoraria from shire. sgh has received research funding or honoraria from amgen, baxter, gilead, novartis, and shire. ndt has received consultancy fees, honoraria and research funding from amgen, sanofi, and shire pharmaceuticals. mmc, mmxc, and tdh have no conflict of interests to declare. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: sjt is a current recipient of a national health and medical research council (nhmrc) postgraduate research scholarship. mmxc is a current recipient of a combined nhmrc and jacquot foundation postgraduate research scholarship. ndt is supported by a jacquot foundation research establishment award. ethical approval all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. references 1. kuro-o m, matsumura y, aizawa h, et al. mutation of the mouse klotho gene leads to a syndrome resembling ageing. nature 1997; 390(6655): 45–51. 2. li sa, watanabe m, yamada h, et al. immunohistochemical localization of klotho protein in brain, kidney, and reproductive organs of mice. cell struct funct 2004; 29(4): 91–99. 3. hu mc, shiizaki k, kuro-o m, et al. fibroblast growth factor 23 and 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increase in serum parathyroid hormone levels after prolonged physical exercise. med sci sports exerc 1988; 20(2): 122–125. 39. devlin jt and horton es. effects of prior high-intensity exercise on glucose metabolism in normal and insulinresistant men. diabetes 1985; 34(10): 973–979. 40. mikines kj, sonne b, farrell pa, et al. effect of physical exercise on sensitivity and responsiveness to insulin in humans. am j physiol 1988; 254(3 pt 1): e248–e259. 41. adams op. the impact of brief high-intensity exercise on blood glucose levels. diabetes metab syndr obes 2013; 6: 113–122. 42. richter ea and hargreaves m. exercise, glut4, and skeletal muscle glucose uptake. physiol rev 2013; 93(3): 993–1017. tan et al. 7 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice jcb j circ biomark 2021; 10: 26-29issn 1849-4544 | doi: 10.33393/jcb.2021.2329original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu controls in the serum of children with high-risk/high-stage neuroblastoma (1). we developed and validated a sensitive and specific high-performance liquid chromatography (hplc)/tandem mass spectrometry assay to quantify the circulating lipoforms of g d2 , and we are evaluating g d2 as a circulating tumor biomarker for neuroblastoma. the c 18 lipoform (18-carbon fatty acid chain length in ceramide) is the predominant form of g d2 in the plasma of patients with neuroblastoma. interpreting the results of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. the steady-state circulating concentration of a tumor biomarker is determined by its rate of production by the tumor and by its clearance. slow clearance results in accumulation of the biomarker in the plasma and enhances the sensitivity for detecting low tumor burden. however, a slowly cleared biomarker is not responsive to rapid changes in tumor burden (e.g., surgical resection). for example, an α-fetoprotein (afp) concentration exceeding 100,000 ng/ml can take more than 2 months to fall into the reference range after complete resection of an afp-producing tumor. conversely, a rapidly eliminated tumor biomarker will not accumulate in the circulation, but its concentration may better reflect changes in tumor burden in real time. pharmacokinetics of the disialoganglioside, g d2 , a circulating tumor biomarker for neuroblastoma, in nonhuman primates frank m. balis1, cynthia lester mccully2, christine m. busch1, elizabeth fox3, katherine e. warren2 1children’s hospital of philadelphia, philadelphia, pa usa 2pediatric oncology branch, national cancer institute, nih, bethesda, md usa 3st. jude children’s research hospital, memphis, tn usa abstract background: the ganglioside g d2 is a potential circulating tumor biomarker for the childhood cancer neuroblastoma. interpreting the levels of a circulating tumor biomarker depends in part on a knowledge of the biomarker’s clinical pharmacology. methods: we studied the plasma and cerebrospinal fluid (csf) pharmacokinetics of the c 18 lipoform of g d2 in two nonhuman primates with indwelling subcutaneous csf lateral ventricular reservoir systems. g d2 was quantified with a validated high-performance liquid chromatography (hplc)/tandem mass spectrometry assay. g d2 was administered as a short intravenous infusion and frequent plasma and csf samples were drawn over 72 hours. results: g d2 plasma concentration declined monoexponentially with a half-life of 16 hours. clearance was 0.0136 and 0.0131 l/h and volume of distribution (v d ) was 0.035 and 0.038 l/kg in the two animals. v d was equivalent to plasma volume. greater than 98% of g d2 in plasma is in a bound form consistent with its known association with lipoproteins and accounting for its limited volume of distribution. g d2 did not cross over from plasma into the csf. conclusions: the pharmacokinetic profile of g d2 is favorable for a circulating tumor biomarker. this study demonstrates the value of characterizing the clinical pharmacology of circulating biomarkers to better understand their clinical behavior. keywords: biomarker, ganglioside, neuroblastoma, pharmacokinetics introduction the ganglioside g d2 is a constituent of the plasma membrane of neuronal cells and is also expressed on the surface of the childhood cancer, neuroblastoma, and other cancers of neuroectodermal origin, such as melanoma. g d2 has a lipid domain (ceramide) that inserts into the plasma membrane and a 5-membered glycan domain that contributes to the glycocalyx on the cell surface. the glycan domain contains 2 sialic acid groups that are fully ionized in the physiological ph range. g d2 circulates in low nanomolar concentrations in children, but its concentration is 30-fold elevated compared to received: august 6, 2021 accepted: november 22, 2021 published online: december 3, 2021 corresponding author: frank m. balis, md children’s hospital of philadelphia 3501 civic center blvd., ctrb4024 philadelphia, pa 19104 usa balisf@chop.edu https://doi.org/10.33393/jcb.2021.2329 https://creativecommons.org/licenses/by-nc/4.0/legalcode balis et al j circ biomark 2021; 10: 27 © 2021 the authors. published by aboutscience www.aboutscience.eu we studied the pharmacokinetics and cerebrospinal fluid (csf) penetration of g d2 after intravenous administration of the ganglioside to two nonhuman primates (nhps) with indwelling subcutaneous csf lateral ventricular reservoirs that allow for rapid, serial csf sampling (2). methods chemicals purified human brain-derived g d2 was purchased from emd millipore corp. (billerica, ma) and contains two dominant lipoforms of g d2 —d18: 1-18:0 (c 18 , molecular weight 1674.9 g/mol) and d18: 1-20:0 (c 20 ). g d2 was dissolved in a small volume of dimethyl sulfoxide (dmso), diluted in sterile normal saline, and filter sterilized through a 22-micron filter. a sample of the filtered drug solution was assayed for content of the c 18 lipoform of g d2 to quantify the administered dose. animals this study was approved by the national cancer institute animal care and use committee. two adult male rhesus monkeys (macaca mulatta), weighing 8.0 and 8.6 kg, respectively, were humanely utilized for this study and were cared for in accordance with the national research council guide for the care and use of laboratory animals, 8th edition (3). animals were socially housed when possible. both subjects had previously undergone implantation of an indwelling lateral ventricular catheter, which was attached to a subcutaneously implanted csf reservoir (2). the subjects also had subcutaneously implanted femoral venous access ports for sampling blood. each subject had a veterinary physical and neurological examination and blood chemistries and complete blood counts performed prior to g d2 administration to ensure they were physiologically and neurologically within normal limits. after g d2 administration the subjects were observed for adverse events daily for 2 weeks and had biweekly clinical chemistries and complete blood counts. g d2 was administered as a 10-minute intravenous infusion through a temporary catheter in the cephalic or saphenous vein. blood was serially sampled from the femoral venous access port and csf was sampled from the subcutaneous csf reservoir. sample times and sample processing blood (3 ml) and csf (0.3 ml) were collected prior to the g d2 infusion, at the end of the 10-minute infusion, and 0.25, 1, 2, 4, 6, 8, 24, 48, and 72 hours postinfusion. blood was collected in heparinized tubes and placed on ice. plasma was separated by centrifugation at 4°c. plasma and csf were stored frozen at −80°c until assayed. sample analysis the concentration of the c 18 lipoform of g d2 in plasma and csf samples was quantified using a previously reported, validated hplc/tandem mass spectrometry method with a lower limit of quantification of 3 nm (4). human brain-derived g d2 , which is made up of approximately 60% c 20 and 40% c 18 lipoforms, was used to construct the standard curves for the assay. pharmacokinetic analysis a one-compartment pharmacokinetic model with first-order elimination was individually fit to the plasma concentration-time data from the 2 subjects using phoenix nlme v.8.3 (certara, princeton, nj). model parameters are clearance (cl) and volume of distribution (v d ). the elimination rate constant (k el ) was derived from cl/v d , the half-life from 0.693/k el , the area under the concentration-time curve (auc) from dose/cl, and the mean residence time (mrt) from 1/k el . protein binding human plasma was spiked with human brain-derived g d2 to achieve a 200 nm concentration of the c 18 lipoform. spiked plasma was centrifuged through a vivaspin 500 concentrator with a 300,000 molecular weight cutoff (mwco) polyethersulfone (pes) membrane (sartorius, goettingen, germany). g d2 concentration was measured in the concentrate that remained above the pes membrane and the effluent that passed through the pes membrane. results the predose concentrations of c 18 g d2 in the 2 subjects were 5.0 and 3.7 nm in plasma and 4.0 and 8.9 nm in csf. the predose plasma concentrations in the subjects are similar to g d2 concentrations in control human plasma (1). end-ofinfusion g d2 plasma concentrations were 1,390 and 1,090 nm, and concentrations declined in the plasma in a mono exponential fashion (fig. 1). pharmacokinetic parameters for fig. 1 plasma concentration-time curve for the c 18 lipoform of g d2 in 2 nonhuman primates after a short intravenous infusion. pharmacokinetics of the circulating biomarker, g d2 28 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb c 18 g d2 for the 2 subjects are listed in table i. the administered dose of g d2 was estimated from the volume of drug solution administered and the concentration of c 18 g d2 in the postfiltration drug solution. intersubject variability in the pharmacokinetic parameters was minimal. the volume of distribution of g d2 approximated plasma volume (blood volume in adult rhesus macaques is 0.062 l/kg (5)). clearance of g d2 from the circulation was slow with a half-life of 16 hours. c 18 g d2 concentrations remained at or near baseline levels in csf throughout the 72-hour sampling period. the peak csf concentration in animal 1 was 9.0 nm at the end of the infusion (baseline concentration was 4.0 nm), and csf concentrations in animal 2 did not exceed the baseline concentration of 8.9 nm. more than 98% of c 18 g d2 in human plasma (200 nm) was retained in the plasma concentrate (above the membrane) after centrifugation through the vivaspin 500 concentrator with a 300,000 mwco pes membrane. the effluent g d2 concentration was 4.5 nm after 15 minutes of centrifugation. conclusions the disialoganglioside g d2 is expressed in the cell membrane of neuroblastoma tumor cells and is shed into the circulation in patients with high-risk/high-stage disease (1,6). prospective studies are ongoing to assess its potential utility as a circulating tumor biomarker for high-risk neuroblastoma. we characterized the pharmacokinetics of g d2 in a nhp csf access model that has proven to be predictive of human plasma and csf disposition for a wide variety of drugs (7) in order to better understand the clinical behavior of circulating g d2 . the pharmacokinetic parameters from this study should prove useful for interpreting serial g d2 concentrations monitored over the course of a patient’s disease. the pharmacokinetic characteristics of g d2 are favorable for a circulating tumor biomarker. the 16 hours half-life in plasma indicates that g d2 should be rapidly responsive to changes in tumor burden. after treatment, a new steadystate concentration should be reached within 3 to 4 days (5 half-lives), suggesting that plasma g d2 concentration could be useful for assessing treatment effect in near real time. the limited volume of distribution, which is equivalent to plasma volume in nhps, enhances sensitivity because the g d2 is concentrated in (limited to) the compartment from which it is being measured. if g d2 were more widely distributed throughout the body, the concentration in plasma would be proportionally lower. ultrafiltration of plasma spiked with g d2 showed that it circulates in a bound form with a large molecular weight. this is consistent with the previously reported association of g d2 and other gangliosides with lipoproteins, which have molecular weights in excess of 3,000 kda (8). g d2 is not detectable in lipoprotein-depleted plasma and is primarily associated with low-density lipoproteins (ldls) (8). binding of g d2 to ldl and, to a lesser extent, other lipoproteins accounts for the volume of distribution being limited to plasma volume, and likely also accounts for the lack of g d2 penetration into the csf in the nhp csf access model. detecting g d2 in the csf of patients with neuroblastoma could be an indicator of brain or meningeal tumor spread even in the presence of high plasma g d2 concentrations. the anti-g d2 monoclonal antibody, dinutuximab, is a component of the frontline treatment of neuroblastoma. circulating g d2 could theoretically bind to dinutuximab and block the binding of the antibody to g d2 on the surface of tumor cells. dinutuximab binds to the glycan portion of g d2 that resides on the cell surface. the configuration of g d2 in ldl is likely to be similar to that in the cell membrane with the polar glycan portion on the surface of the lipoprotein complex and the ceramide portion extending into the nonpolar core. therefore, even though g d2 is essentially all bound to lipoproteins in the circulation, the antigenic glycan portion may still be exposed on the surface for binding to dinutuximab. dinutuximab immunotherapy is currently administered in the final phase of neuroblastoma therapy, when plasma g d2 concentrations are likely to be low, but pilot studies are ongoing to investigate the use of dinutuximab in the initial phase of therapy when circulating g d2 concentrations are likely to be higher in some patients. binding of the antibody to lipoprotein-associated g d2 could lower the efficacy of dinutuximab by limiting the amount of antibody available to bind to tumor cells. the pharmacokinetic profile of g d2 is favorable for a circulating tumor biomarker. with its relatively short half-life, plasma g d2 concentrations should reflect changes in tumor burden with a minimal lag time, and the limited volume of distribution translates into higher concentrations in plasma, improving its sensitivity. this study demonstrates the value of characterizing the clinical pharmacology of circulating biomarkers to better understand their clinical behavior. the use of the nhp model that is predictive of human pharmacokinetics provided the opportunity to study the pharmacokinetics of g d2 after administration of a known dose and without interference from endogenous production. we plan to confirm the results using a more limited sampling approach in children with neuroblastoma after definitive treatment. table i pharmacokinetic parameters for the c 18 lipoform of g d2 in nonhuman primates after a short intravenous infusion animal weight (kg) dose (nmol) volume of distribution (l/kg) clearance (l/h) k el (h−1) auc (nm × h) half-life (h) mrt (h) 1 (zb39) 8.6 402 0.0354 0.0136 0.0448 29,500 15.5 22.3 2 (zj57) 8.0 313 0.0384 0.0131 0.0427 23,800 16.2 23.4 k el = first-order elimination rate constant; auc = area under the concentration-time curve; mrt = mean residence time. balis et al j circ biomark 2021; 10: 29 © 2021 the authors. published by aboutscience www.aboutscience.eu disclosures conflict of interest: the authors declare no conflict of interest. financial support: this project was funded by alex’s lemonade stand center of excellence in drug development and clinical pharmacology award and a grant from cure childhood cancer. references 1. balis fm, busch cm, desai av, et al. the ganglioside g d2 as a circulating tumor biomarker for neuroblastoma. pediatr blood cancer. 2020;67(1):e28031. crossref pubmed 2. lester mccully cm, bacher j, macallister rp, et al. development of a cerebrospinal fluid lateral reservoir model in rhesus monkeys (macaca mulatta). comp med. 2015;65(1):77-82. pubmed 3. guide for care and use of laboratory animals. 8th ed. washington, dc: the national academies press 2011. online 4. busch cm, desai av, moorthy gs, fox e, balis fm. a validated hplc-ms/ms method for estimating the concentration of the ganglioside, g d2 , in human plasma or serum. j chromatogr b analyt technol biomed life sci. 2018;1102-1103:60-65. crossref pubmed 5. hobbs tr, blue sw, park bs, greisel jj, conn pm, pau fk. measurement of blood volume in adult rhesus macaques (macaca mulatta). j am assoc lab anim sci. 2015;54(6):687693. pubmed 6. ladisch s, wu zl, feig s, et al. shedding of gd2 ganglioside by human neuroblastoma. int j cancer. 1987;39(1):73-76. crossref pubmed 7. baratz e, mccully c, shih j, warren k. comparison of pharmacokinetic parameters between non-human primates and human patients. neuro-oncol. 2018;20(suppl 2):i157. crossref 8. valentino la, ladisch s. localization of shed human tumor gangliosides: association with serum lipoproteins. cancer res. 1992;52(4):810-814. pubmed https://doi.org/10.1002/pbc.28031 https://www.ncbi.nlm.nih.gov/pubmed/31612589 https://www.ncbi.nlm.nih.gov/pubmed/25730761 http://grants.nih.gov/grants/olaw/guide-for-the-care-and-use-of-laboratory-animals.pdf https://doi.org/10.1016/j.jchromb.2018.10.010 https://www.ncbi.nlm.nih.gov/pubmed/30368044 https://www.ncbi.nlm.nih.gov/pubmed/26632777 https://doi.org/10.1002/ijc.2910390113 https://www.ncbi.nlm.nih.gov/pubmed/3539825 https://doi.org/10.1093/neuonc/noy059.582 https://www.ncbi.nlm.nih.gov/pubmed/1737341 jcb j circ biomark 2022; 11: 1-4issn 1849-4544 | doi: 10.33393/jcb.2022.2328original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu axis, has a key role in the pathophysiology of ckd-mbd, as it is involved in calcium, phosphorus, and calcitriol homeostasis, as well as in mechanisms of cellular aging (2). fgf-23 is a glycoprotein of 252 amino acids (32 kda), encoded by the homonymous gene located on chromosome 12. fgf-23 exerts its biological activity through interaction with one of four specific receptors (fgfr 1-4) by a paracrine mechanism (3). fgf-23 is metabolized to its inactive c-terminal and n-terminal fragments and intact fgf-23 (ifgf23) represents its biologically active form (4). at the cellular level, fgf-23 acts by binding to an fgf-23-fgfr complex with klotho. although the receptors are ubiquitously expressed, klotho expression is restricted primarily at the level of renal tubules, parathyroid glands, and choroid plexus, determining the tissue specificity of fgf-23 (2,5). fgf-23 acts at the level of renal tubules by reducing phosphorus reabsorption and inhibiting the expression of the sodium/phosphate cotransporter (npt2a and npt2c) on the cell membrane in the proximal tubule. it also reduces the conversion of 25-oh-vitamin d (25-oh-vitd) to its active form 1,25(oh) by inhibiting the expression of 1α-hydroxylase and increases its degradation by increasing the activity of 24-hydroxylase. in ckd there is a progressive increase in fgf-23 levels associated with reduced renal function. several factors may contribute to this phenomenon, such as hyperphosphatemia, hypercalcemia, secondary hyperparathyroidism, and klotho deficiency (6). serum levels of fgf-23 increase in the early stages of ckd as a compensatory mechanism to prevent the onset of hyperphosphatemia and secondary diagnostic utility of fgf-23 in mineral bone disorder during chronic kidney disease luisa albanese1, gemma caliendo1, giovanna d’elia1, luana passariello1, anna maria molinari1,2, claudio napoli3,4, maria teresa vietri1,2 1unity of clinical and molecular pathology, aou, university of campania “luigi vanvitelli”, naples italy 2department of precision medicine, university of campania “luigi vanvitelli”, naples italy 3department of advanced medical and surgical sciences (damss), university of campania “luigi vanvitelli”, naples italy 4clinical department of internal medicine and specialistic units, aou, university of campania “luigi vanvitelli”, naples italy abstract our data confirm that intact fibroblast growth factor 23 (ifgf-23) concentration is increased in patients with chronic kidney disease (ckd) and that it increases with disease progression (stages i-v). therefore, ifgf-23 could be considered an early biomarker in the course of chronic kidney disease-mineral bone disorder (ckd-mbd), which has several aspects that make it potentially useful in clinical practice. the availability of an automated method for ifgf-23 assay may represent an added value in the management of the patient with ckd-mbd already from the early stages of the disease, before the increase of the routinely used laboratory parameters, 1-84 parathyroid hormone (pth) and 25-oh-vitamin d (25-oh-vitd), which occur in more advanced stages of the disease. keywords: bone density, ckd, fgf-23 introduction the term “chronic kidney disease-mineral bone disorder” (ckd-mbd) is defined as a systemic disorder of bone and mineral metabolism due to ckd, which occurs in the presence of one or a combination of the following conditions: alterations in laboratory parameters (calcemia, phosphoremia, parathormone, vitamin d); abnormalities in turnover, mineralization, volume, linear growth, or bone strength; vascular or soft tissue calcifications secondary to ckd (1). the three alterations present in ckd-mbd have different prevalence in patients; that is, metabolic alterations are the first to appear followed by bone alterations and vascular calcifications. recently, molecules that are produced by the bone and kidney have been identified. these paths play a pivotal role in the mechanisms of bone and cardiovascular alterations during ckd-mbd. fibroblast growth factor 23 (fgf-23)/klotho axis, in addition to the parathyroid hormone (pth)/vitamin d received: august 4, 2021 accepted: december 4, 2021 published online: january 8, 2022 corresponding author: maria teresa vietri department of precision medicine university of campania “luigi vanvitelli” via l. de crecchio, 7 80138 naples italy mariateresa.vietri@unicampania.it https://doi.org/10.33393/jcb.2022.2328 https://creativecommons.org/licenses/by-nc/4.0/legalcode fgf-23 in mbd during ckd2 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb hyperparathyroidism (7). even transient increase in phosphoremia, in the early stages of ckd, stimulates fgf-23 production, which then tends to normalize phosphoremia but at the same time causes a reduction in 1,25(oh)vitd resulting both in increased parathormone synthesis and secretion (8,9). extrarenal manifestations of fgf-23 in ckd can be klothodependent and/or klotho-independent, receptor action of fgf-23 that does not require the presence of the membrane coreceptor. klotho-independent effects of fgf-23 in ckd are expressed at the cardiac level, with hypertrophy of left ventricular myocytes, and at the hepatic level, with increased synthesis of proinflammatory cytokines (10). during renal failure, effects of both klotho-dependent and -independent fgf-23 are instead expressed at the level of the central nervous system, the immune system, and the vascular system. enzyme immunoassays, used until now to assay fgf-23, recognized both the c-terminal portion and the intact form of fgf-23. the current availability of a new automated chemiluminescence immunoassay (clia) for the determination of the intact molecule only allows overcoming these methodological limitations, favoring a routine use of the fgf-23 assay in clinical practice, in association with parameters already used routinely such as parathormone and vitamin d (11,12). the aim of the present descriptive study was to assess the circulating levels of ifgf-23 in patients with ckd at various stages in specialist follow-up with the liaison ifgf-23 assay. methods sixty-three patients (35 males and 28 females, aged 20-80 years) diagnosed with ckd-mbd were selected; all underwent renal transplantation. the patients were hospitalized at the teaching hospital of the university of campania “luigi vanvitelli” of naples (italy) and signed an informed consent at the admission time. patients aged <18 years, with inflammatory bowel disease, acute infectious or inflammatory disease, or advanced neoplasia were excluded from the study. circulating levels of ifgf-23, 1-84 pth, 25-oh-vitd, creatinine, and albumin were assayed for each patient. blood samples were collected at 8 am from fasting patients, centrifuged, and stored at −20°c until assayed. 1-84 pth, 25-oh-vitd, and ifgf-23 were assayed using chemoluminescence (clia) methods on the liaison xl platform (diasorin spa, italy). albumin and creatinine were assayed by the colorimetric method (architect abbott diagnostics). reference values for 1-84 pth ranged from 4.6 to 58.1 pg/ ml, whereas for 25-oh-vitd it was <10 ng/ml (deficiency), 10-30 ng/ml (insufficiency), 30-100 ng/ml (sufficiency). the reference range for ifgf-23 was 25.1-95.5 pg/ml; for creatinine it was 0.72-1.25 mg/dl for males and 0.571.11 mg/dl for females; and for albumin it was 3.5-5.2 g/dl. glomerular filtrate was estimated using clearance of creatinine according to the egfr-epi equation for the classification of patients at a given stage of ckd. thus, of the 63 patients with egfr between 16 and 104 ml/min/1.73 m2, 10 had stage i, 19 stage ii, 22 stage iii, 7 stage iv, and 5 stage v ckd. statistical analysis data are reported as means ± standard deviation (sd), unless otherwise stated. comparison between the groups was performed using χ2 test. significance was assumed for p-values less than 0.05. results about 39.7% (25/63) of patients with ckd had ifgf-23 levels above normal (>95.5 pg/ml), 14.3% (9/63) of patients had 1-84 pth values increased beyond the normal range (>58.1 pg/ml), and 7.9% (5/63) had insufficient 25-oh-vitd values (<10 ng/ml). table i shows the number of patients distributed in the different stages of ckd with ifgf-23 and 1-84 pth values above the cutoff and with 25-oh-vitd deficiency. table i percentage of patients with increased values of ifgf-23 and 1-84 pth and 25-oh-vitd deficiency divided into the five stages of ckd patients with ckd (total 63) ifgf-23 (>95.5 pg/ml) 1-84 pth (>58.1 pg/ml) 25-oh-vitd (<10 ng/ml) stage i (n = 10) 2/10 (20%) – – stage ii (n = 19) 5/19 (26.3%) 2/19 (10.5%) – stage iii (n = 22) 9/22 (40.9%) 4/22 (18.2%) 2/22 (9%) stage iv (n = 7) 6/7 (85.7%) 1/7 (14.3%) 2/7 (28.6%) stage v (n = 5) 3/5 (60%) 2/5 (40%) 1/5 (20%) ckd = chronic kidney disease; ifgf-23 = intact fibroblast growth factor 23; pth = parathyroid hormone; 25-oh-vitd = 25-oh-vitamin d. specifically, 20% of stage i, 26.3% of stage ii, 40.9% of stage iii, 85.7% of stage iv, and 60% of stage v ckd patients had ifgf-23 levels above the cutoff. no statistically significant differences are found among the ckd stages (p<0.05). values of 1-84 pth above the normal range were observed in 10.5% of stage ii, 18.2% of stage iii, and 14.3% and 40% of stage iv and stage v ckd patients, respectively. for stage i ckd patients, increased values of 1-84 pth were not found. in addition, insufficient 25-oh-vitd values were found in 9%, 28.6%, and 20% of patients at ckd stages iii, iv, and v, respectively. table ii shows the percentages of patients with increased serum values of ifgf-23,1-84 pth, and 25-oh-vitd deficiency divided according to the number of years since transplantation (<10 years, 11-20 years, and >20 years). thirty-two of the 63 patients had received transplantation for <10 years, 20 for 11-20 years, and 11 for >21 years. values of ifgf-23 beyond the normal range were found in 43.7% of organ transplant patients received less than 10 years, 35% received between 11 and 20 years, and 36% received >20 years. albanese et al j circ biomark 2022; 11: 3 © 2022 the authors. published by aboutscience www.aboutscience.eu increased values of 1-84 pth have been detected in 18.7% of transplant patients received less than 10 years, 10% of transplant patients received 11-20 years, and 9.1% of transplant patients received more than 20 years. a 25-oh-vitd deficiency existed in 9.4% of patients who had received transplantation for less than 10 years and 10% of patients who had received transplantation for more than 11 years. no deficient 25-oh-vitd values were found for patients who had been transplanted for more than 20 years. discussion increased serum fgf-23 levels from the earliest stages of ckd represent the first indicator of alterations in mineral metabolism. increased levels of fgf-23, 1-84 pth, and phosphorus with a concomitant reduction in serum levels of active vitamin d are closely related to reduced glomerular filtration rate (gfr). the increase in fgf-23 concentration may be caused by the abnormal stimulation of its secretion exerted by substances released from the damaged renal parenchyma or by the effects exerted by the uremic state on bone mineralization processes. other shreds of evidence, instead, suggest the crucial role that phosphorus plays in the uncontrolled stimulation of fgf-23 secretion (3). limitation of our study is the distribution of samples by disease stage. out of 63 enrolled patients, 41 had ckd stages ii-iii. despite this limitation, our results show increased fgf-23 levels in a high percentage of patients with ckd, which is in line with what has been recently reported (12). moreover, our data show that the percentage of patients with ifgf-23 values above reference limits increases with disease progression (stages i-v). in the early stages of ckd, increasing ifgf-23 could represent a compensatory mechanism to prevent the establishment of hyperphosphatemia and secondary hyperparathyroidism. starting from stage iv, with a further reduction of residual renal function, klotho levels are also reduced, resulting in peripheral resistance due to the fgf-23 action and further increase in the ifgf-23 levels. renal transplantation represents the therapy of choice in ckd, allowing, compared to dialysis, a better quality of life and increased survival (13-15). indeed, it is reported that after renal transplantation the concentration of fgf-23 decreases rapidly. here, we noticed a correlation between plasma ifgf-23 concentration and years since transplantation. indeed, ifgf-23 values above the normal limit were found in 43.7% of patients transplanted less than 10 years, in 35% of patients transplanted 10-20 years, and in 36% of patients who had received transplantation for more than 20 years. in addition, increased levels of 1-84 pth have been observed in 18.7% of patients after renal transplantation less than 10 years while 10% of patients with transplantation for 10-20 years, and 9% of patients with transplantation for more than 20 years. these data indicate, as has been pointed out, that after renal transplantation, persistent hyperparathyroidism is associated with elevated ifgf-23 levels, which may occur with inhibition of the synthesis of the active form of vitamin d, resulting in the persistence of elevated levels of 1-84 pth. the persistence of high levels of ifgf-23 after renal transplantation can be interpreted as a mechanism of compensation and adaptation to the new metabolic balance created in the early posttransplant phases. factors that may favor the persistence of high levels of fgf-23 after transplantation may include immunosuppressive therapy: corticosteroids, calcineurin inhibitors, and mammalian target of rapamycin (mtor) inhibitors, which stimulate the production of ifgf-23 (16). conclusions the availability of an automated method for ifgf-23 assay may represent an added value in the management of the patient with ckd-mbd already from the early stages of the disease, before the increase of the routinely used laboratory parameters, 1-84 pth and 25-oh-vitd, which occur in more advanced stages of the disease. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. authors contribution: all authors contributed equally to this manuscript. references 1. moe s, drüeke t, cunningham j, et al; kidney disease: improving global outcomes (kdigo). definition, evaluation, and classification of renal osteodystrophy: a position statement from kidney disease: improving global outcomes (kdigo). kidney int. 2006;69(11):1945-1953. crossref pubmed 2. hu mc, shiizaki k, kuro-o m, moe ow. fibroblast growth factor 23 and klotho: physiology and pathophysiology of an endocrine network of mineral metabolism. annu rev physiol. 2013;75(1):503-533. crossref pubmed 3. ho bb, bergwitz c. fgf23 signalling and physiology. j mol endocrinol. 2021;66(2):r23-r32. crossref pubmed 4. shimada t, urakawa i, isakova t, et al. circulating fibroblast growth factor 23 in patients with end-stage renal disease treated by 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salivary cortisol levels are influenced by daily glycemic oscillations in type 2 diabetes. endocrine. 2016;53(1):220-6. crossref. pubmed 16. economidou d, dovas s, papagianni a, pateinakis p, memmos d. fgf-23 levels before and after renal transplantation. j transplant. 2009;2009:379082. crossref pubmed https://doi.org/10.1038/ki.2012.176 https://www.ncbi.nlm.nih.gov/pubmed/22622492 https://doi.org/10.1146/annurev.med.051308.111339 https://www.ncbi.nlm.nih.gov/pubmed/20059333 https://doi.org/10.1038/ki.2011.47 https://www.ncbi.nlm.nih.gov/pubmed/21389978 https://doi.org/10.1001/jama.2011.826 https://www.ncbi.nlm.nih.gov/pubmed/21673295 https://doi.org/10.1016/j.tem.2018.08.006 https://www.ncbi.nlm.nih.gov/pubmed/30217676 https://doi.org/10.1007/s00774-011-0306-4 https://www.ncbi.nlm.nih.gov/pubmed/21898178 https://doi.org/10.1007/s00223-017-0307-y https://www.ncbi.nlm.nih.gov/pubmed/28761972 https://doi.org/10.2217/fca.11.24 https://www.ncbi.nlm.nih.gov/pubmed/21797745 https://doi.org/10.1016/j.trre.2021.100609 https://www.ncbi.nlm.nih.gov/pubmed/33706201 http://https//doi.org/10.1007/s12020-015-0777-5 https://pubmed.ncbi.nlm.nih.gov/26511948/ https://doi.org/10.1155/2009/379082 https://www.ncbi.nlm.nih.gov/pubmed/20107581 jcb j circ biomark 2021; 10: 9-13issn 1849-4544 | doi: 10.33393/jcb.2021.2212original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu are among the main strategies to improve survival rates (3). the first step in the diagnosis is to distinguish between non– small cell lung cancer (nsclc) and sclc, which has a poorer prognosis (4,5). despite lung cancer diagnosis being only based on histology, different serum biomarkers have been proven to be useful tools for the diagnosis, prognosis and follow-up, but there are currently no guidelines or standards on their application in clinical practice (6,7). the national academy of clinical biochemistry (nacb) laboratory medicine practice guidelines (8) recommend carcinoembryonic antigen (cea), squamous cell carcinoma–related antigen (sccag), neuron-specific enolase (nse), cytokeratin 19 fragment 21-1 (cyfra 21.1), carbohydrate antigen 125 (ca 125) and progastrin-releasing peptide (progrp) as routine markers of lung cancer. nse is considered the tumor marker of choice in sclc diagnosis, but it is not pathognomonic because its effectiveness is related to the stage of the tumor (9). circulating progastrin-releasing peptide in the diagnosis of small cell lung cancer (sclc) and in therapeutic monitoring vittoria barchiesi1, vittorio simeon2, claudia sandomenico3, monica cantile4, dionigio cerasuolo5, paolo chiodini2, alessandro morabito3, ernesta cavalcanti5 1department “campania centro”, a.o.r.n. “antonio cardarelli”, napoli italy 2medical statistics unit, university of campania “luigi vanvitelli”, napoli italy 3thoracic medical oncology unit, istituto nazionale tumori irccs, “fondazione g.pascale”, napoli italy 4pathology unit, istituto nazionale tumori irccs, “fondazione g.pascale”, napoli italy 5laboratory medicine unit, istituto nazionale tumori irccs, “fondazione g.pascale”, napoli italy abstract introduction: progastrin-releasing peptide (progrp), a precursor of grp, has been recently reported as a putative circulating biomarker for differential diagnosis between non–small cell lung cancer (nsclc) and sclc. we evaluated the diagnostic effectiveness of progrp to differentiate patients with nsclc and sclc and the usefulness of combined measurement of progrp and neuron-specific enolase (nse) for diagnosing sclc. methods: serum progrp, nse, cytokeratin 19 fragment 21-1 (cyfra 21.1), squamous cell carcinoma antigen (scc ag) and carcinoembryonic antigen (cea) were prospectively collected and measured in patients with a new diagnosis of lung cancer. serum progrp was also measured in healthy subjects. the serum progrp, nse, cyfra 21.1 and cea concentrations were determined by an electrochemiluminescence immunoassay and the serum scc ag concentration was determined by an automated immunofluorescence assay. differences between progrp and nse in patients with sclc and nsclc were evaluated and compared using mann-whitney test. results: a total of 77 patients affected by sclc (n = 17) and nsclc (n = 60) were enrolled in the present study. moreover, 50 cases of healthy subjects were analyzed for progrp. sclc patients showed a significantly higher progrp (1,484 pg/ml; range 168-3,777) levels compared to nsclc patients (45 pg/ml; range 31.7-60.6), p<0.0001. in healthy subjects the median progrp level was 36.1 (28.8-43.5) pg/ml, significantly lower than sclc patients. progrp showed a higher specificity when compared to nse, with a difference in proportion of 47.5% (95% confidence interval 32.5% to 62.5%, p<0.001). serial measurements of progrp in sclc patients showed a decrease in responsive chemotherapy patients. conclusions: progrp is an accurate biomarker for diagnosis of sclc and for discrimination of sclc from nsclc. keywords: lung cancer, progrp, serum biomarker introduction lung cancer is one of the most common and malignant tumors, with high morbidity and mortality worldwide, and its incidence has been increasing, especially among women, in the last decades (1,2). the early diagnosis and accurate staging of lung cancer for immediate and appropriate treatment received: november 23, 2020 accepted: june 15, 2021 published online: july 7, 2021 corresponding author: ernesta cavalcanti laboratory medicine unit istituto nazionale tumori irccs, “fondazione g.pascale” 80100 napoli italy e.cavalcanti@istitutotumori.na.it https://doi.org/10.33393/jcb.2021.2212 https://creativecommons.org/licenses/by-nc/4.0/legalcode progrp in sclc diagnosis10 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb progrp is a precursor of gastrin-releasing peptide (grp), an active hormone involved in the physiological digestive process. grp is a 27-amino-acid peptide homologous to the c-terminal of bombesin isolated from porcine stomach, in fetal as well as neonatal lung tissue and in primary lung cancer, particularly in sclc (10-13). grp cannot be used as a biomarker due to its instability (half-life of grp is 2 min), while progrp is a stable protein with a half-life of 19-28 days. recently, circulating progrp has been reported as a putative biomarker for differential diagnosis between nsclc and sclc (14-18). the aim of the present study was to evaluate the diagnostic effectiveness of progrp to differentiate patients with nsclc and sclc, the usefulness of combined measurement of progrp and nse for the diagnosis of sclc, the comparison of diagnostic efficacy of progrp vs. a combined panel of tumor markers and to establish the reference values of progrp in healthy patients. methods patients serum progrp, nse, cyfra 21.1, scc ag and cea were prospectively collected and measured in patients with a new diagnosis of lung cancer admitted to the thoracic medical oncology of the national cancer institute “g. pascale” of naples. moreover, serum progrp was measured in healthy subjects collected at the unit of transfusional medicine of the national cancer institute “g. pascale” of naples. the present study was approved through the ethics committee and all patients completed an informed consent. assay serum samples were taken in vacutainer tube sst ii advance and analyzed in less than 1 hour. serum progrp, nse, cyfra 21.1 and cea concentrations were determined by an electrochemiluminescence immunoassay on a cobas c6000 automated analyzer (roche diagnostics). serum scc ag concentration was determined by an automated immunofluorescence assay on kryptor compact plus (thermo scientific). for all analytes the reference range was set according to the data sheet manufacturer. statistical analysis data were described as reported: continuous variables as mean and standard deviation, or median and interquartile range if distribution was not symmetric; categorical variables as number and percentage. differences of diagnostic markers progrp and nse in patients with sclc and nsclc were evaluated and compared using mann-whitney test. successively, progrp and nse were categorized and defined positive using these criteria: cutoff >100 pg/ml for progrp; >17 ng/ml for nse. furthermore, the algorithm proposed by molina et al. (19), taking into consideration scc, progrp, nse, cyfra and cea, was used to classify patients. for each biomarker (progrp, nse and algorithm), the diagnostic measures, such as sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv), were calculated to evaluate the accuracy in discriminating sclc from nsclc. pairwise comparison of diagnostic measures was performed using mcnemar’s test (20). receiver operating characteristic (roc) curve analysis was calculated to define new threshold values for progrp and nse in our population. the area under the roc curve (auc) was used to quantify accuracy and define sensitivity and specificity. for descriptive purpose, progrp values (in log scale) in sclc patients were collected and reported for the whole treatment period. all tests were two-tailed and a p-value <0.05 was considered statistically significant. all data were analyzed using r software 3.3.1 (r foundation for statistical computing, vienna, austria). results progrp serum level in lung cancer patients a total of 77 patients affected by lung cancer, 17 sclc and 60 nsclc, were enrolled in the present study between july 2015 and july 2017; moreover, 50 cases of healthy subjects were collected from the unit of transfusional medicine, at the national cancer institute “g. pascale” of naples, italy. the study design is shown in figure 1. the mean age of the 17 sclc patients was 63.4 ± 8.6 years and there were 10 (58.8%) male and 7 (41.2%) female patients. the 60 nsclc patients were older (68.4 ± 9.1 years) and with higher proportion of male patients (65%) (tab. i). the median (interquartile range [iqr]) progrp level in the total population was 47.3 (34.3-87.3) pg/ml. sclc patients showed (tab. i and fig. 2) a significantly higher progrp (1,484 [168-3,777] pg/ml] levels compared to nsclc patients (45 [31.7-60.6] pg/ml) (mann-whitney test, p<0.0001). in addition, nse was significantly higher in sclc than nsclc patients fig. 1 flow chart of the study design. barchiesi et al j circ biomark 2021; 10: 11 © 2021 the authors. published by aboutscience www.aboutscience.eu (55 [34-107.4] ng/ml versus 17.7 [13-24] ng/ml), whereas cyfra 21.1 was lower in sclc patients. there were no differences for cea and scc biomarkers. in healthy subjects the median progrp level was 36.1 (28.8-43.5) pg/ml, significantly lower than sclc patients (data not shown). progrp and nse in lung cancer histological types and therapeutic response diagnostic accuracy of each biomarker is reported in table ii. according to lung cancer histological type, progrp showed a sensitivity of 82.4% (95% confidence interval [ci], 56.6-96.2) with a specificity of 93.3% (95% ci, 83.8-98.2). ppv and npv were 77.8% and 94.9%, respectively. nse biomarker and algorithm (nse + progrp) showed the following value of accuracy: sensitivity (100% and 86.7%, respectively), specificity (45% and 96.6%, respectively), ppv (34% and 86.7%, respectively) and npv (100% and 96.6%, respectively). pairwise comparison of progrp with nse and algorithm highlighted no differences in terms of sensitivity. progrp showed a higher specificity when compared to nse, with a difference in proportion of 47.5% (95% ci 32.5% to 62.5%, exact mcnemar’s test p<0.001). progrp and algorithm measures revealed a similar performance for both of them. on roc curve analysis, auc for progrp and nse was comparable with 0.86 (95% ci, 0.71-1) and 0.92 (95% ci, 0.840.99), respectively (fig. 3). serial measurements of progrp in 12 sclc patients showed a decrease in responsive patients and they are depicted in figure 4. discussion in our study, median progrp levels were significantly higher in patients with sclc than in those with nsclc or healthy subjects. plasma progrp at cutoff level of 100 pg/ml showed a high sensitivity and specificity (82.4% and 93.3%, respectively) in identifying patients with sclc, with a specificity higher than nse. ppv and npv were 77.8% and 94.9%, respectively. moreover, responsive patients presented a decrease in progrp levels. these results confirm the accuracy of progrp in clinical practice in the diagnosis of sclc. in 2011, a meta-analysis of 5,146 patients enrolled in 11 clinical trials, including 1,095 with sclc, concluded that table i patients’ characteristics and values of different analytes total patients (n = 77) sclc (n = 17, 22%) nsclc (n = 60, 78%) age, years  mean (sd) 67.3 (9.16) 63.4 (8.6) 68.4 (9.11) sex, n (%)  female 28 (36.40) 7 (41.2) 21 (35.00)  male 49 (66.60) 10 (58.8) 39 (65.00) creatinine, mg/dl  median (iqr) 0.82 (0.67-0.93) 0.82 (0.68-0.9) 0.82 (0.66-0.96) ldh, u/l  median (iqr) 418 (336-560) 510 (376-633) 415 (330-543) ast, u/l  median (iqr) 17 (13-22) 17 (13-22.5) 17 (14-22) alt, u/l  median (iqr) 16 (12-25) 16 (11-20) 16 (12-26) bilirubin, mg/dl  median (iqr) 0.5 (0.4-0.7) 0.5 (0.4-0.6) 0.5 (0.4-0.7) cea, ng/ml  median (iqr) 7.9 (3.1-38.9) 4.6 (3-17.9) 8.7 (3-42.6) cyfra, ng/ml  median (iqr) 5.4 (3.2-12.7) 3.4 (2.3-6.4) 7 (3.3-13.7) nse, ng/ml  median (iqr) 19 (14.2-27.6) 55 (34-107.4) 17.7 (13-24) scc, ng/ml  median (iqr) 0.5 (0.1-0.8) 0.2 (0.1-0.6) 0.5 (0.2-1.1) progrp, pg/ml  median (iqr) 47.3 (34.3-87.3) 1484 (168-3777) 45 (31.7-60.6) progrp (log10)  mean (sd) 1.9 (0.8) 3 (1) 1.7 (0.3) alt = alanine aminotransferase; ast = aspartate transaminase; cea = carcinoembryonic antigen; cyfra 21.1 = cytokeratin 19 fragment 21-1; iqr = interquartile range; ldh = lactate dehydrogenase; nsclc = non–small cell lung cancer; nse = neuron-specific enolase; progrp = precursor of gastrin-releasing peptide; scc = squamous cell carcinoma; sclc = small cell lung cancer; sd = standard deviation. fig. 2 progrp and nse levels in sclc and nsclc. on the left: progrp level in sclc and nsclc patients; on the right: nse level in sclc and nsclc patients. nsclc = non-small-cell lung cancer; nse = neuron-specific enolase; progrp = progastrin releasing peptide; sclc = small cell lung cancer. values were reported in log10 scale. progrp in sclc diagnosis12 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb progrp appeared to be a promising marker for sclc, with a sensitivity of 71.6% and a specificity of 92.1% (21). however, the poor pooled estimates of sensitivity, the wide range of sensitivity and specificity estimates across studies and the high degree of inconsistency made the conclusions of the meta-analysis weak. the levels of six tumor markers, cyfra21-1, cea, nse, ca125, progrp and scc, were evaluated in 392 chinese patients affected by lung cancer (including 308 with nsclc and 84 with sclc), in 116 patients with benign lung diseases and in 144 healthy controls (22). the results showed that the levels of nse and progrp were significantly higher in the sclc group than in the nsclc group and that the sensitivity (at 95% specificity) of nse, progrp and the combination of the two markers for differential diagnosis of nsclc and sclc was 71.9%, 90.6% and 90.8%, respectively. in a chinese study, plasma progrp levels were prospectively measured in 75 sclc patients, and they were significantly higher than those of 234 nsclc patients (1058.0 vs. 37.46 pg/ ml, p<0.001) (23). in this study, progrp showed 87.8% sensitivity and 91.5% specificity, at a cutoff level of 65.7 pg/ml. moreover, change of progrp levels before and after chemotherapy was analyzed. in patients with sclc who were followed through the treatment, the median progrp levels of the responders decreased after chemotherapy (p<0.001). a retrospective italian study evaluated serum progrp levels in 37 patients with sclc and 28 patients with advanced nsclc (24). median progrp level was 919 pg/ml in sclc and 32 pg/ml in nsclc (p<0.0001). in this study, progrp showed 86.4% sensitivity and 96.4% specificity, at a cutoff level of 77.8 pg/ml. moreover, in patients with extended disease, median progrp was 46-fold higher than in patients with limited disease (p = 0.004), notwithstanding all the limitations of a statistical analysis conducted over a small-size population. the results of our study support the use of progrp at diagnosis to discriminate sclc from nsclc or nonmalignant disease. strengths of our analysis are the prospective design of the study and the inclusion also of health controls to determine the reference values of progrp in healthy subjects, not evaluated to date in other studies. furthermore, our study highlights that the diagnostic efficiency of the test is equivalent to that demonstrated by the association of tests included in the diagnostic algorithms, suggesting that progrp can be considered a valid test to reduce time and costs. limitations of our study are the small number of patients enrolled in the study and, in particular, of those with sclc evaluated with serial measurements of progrp during treatment. a larger study to confirm the predictive role of progrp reduction in early identification of responsive sclc patients to first-line treatment with chemotherapy combined with immunotherapy is planned. in conclusion, progrp is an accurate biomarker for the diagnosis of sclc and for discriminating sclc from nsclc. table ii diagnostic accuracy of progrp, nse and their combination in discriminating sclc from nsclc sn sp ppv npv progrp 82.4 (56.6-96.2) 93.3 (83.8-98.2) 77.8 (52.4-93.6) 94.9 (85.9-98.9) nse 100 (80.5-99.9) 45 (32.1-58.4)* 34 (21.2-48.8) 100 (87.2-100) algorithm 86.7 (59.5-98.3) 96.6 (88.3-99.6) 86.7 (59.5-98.3) 96.6 (88.3-99.6) npv = negative predictive value; nse = neuron-specific enolase; ppv = positive predictive value; progrp = progastrin-releasing peptide; sn = sensitivity; sp = specificity. *exact mcnemar’s test p<0.001. fig. 3 receiver operating characteristics (roc) curves of progrp and nse. nse = neuron-specific enolase; progrp = progastrin-releasing peptide. area under the curve (auc) were reported. fig. 4 serial measurement of progrp in sclc patients and correlation with therapeutic response. black point with solid line describes patients with complete response to therapy. gray point with dashed line describes patients with partial response to therapy. progrp = progastrin-releasing peptide; sclc = small cell lung cancer. barchiesi et al j circ biomark 2021; 10: 13 © 2021 the authors. published by aboutscience www.aboutscience.eu further studies should confirm its utility also for treatment and monitoring of sclc patients. abbreviations auc = area under the curve; cea = carcinoembryonic antigen; cyfra 21.1 = cytokeratin 19 fragment 21-1; grp = gastrin-releasing peptide; iqr = interquartile range; nacb = national academy of clinical biochemistry; npv = negative predictive value; nsclc = non–small cell lung cancer; nse = neuron-specific enolase; ppv = positive predictive value; progrp = precursor of gastrin-releasing peptide; roc = receiver operating characteristics; scc ag = squamous cell carcinoma antigen; sclc = small cell lung cancer. acknowledgments the authors are 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and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu syndrome in patients with severe sars-cov-2. this may cause harmful tissue damage, multiple organ failure and hypercoagulability, and is associated with poor clinical outcomes (1). conversely it is known that people with immune deficiency have an increase in mortality when admitted to hospital with covid-19 (2). a range of serum autoantibodies, such as nucleolar antinuclear antibodies (anas), antineutrophil cytoplasmic antibody (anca), anti-cyclic citrullinated peptide, and antiphospholipid autoantibodies, have already been detected in severe sars-cov-2 patients and linked to disease severity, reflecting immune system dysregulation in patients with severe sars-cov-2 lung disease (3-5). it is not yet clear, however, whether patients who exhibit such robust immune response to sars-cov-2 have higher background levels of antibody and autoantibody responsiveness when compared to patients who develop mild disease (6), and for how long the level of autoantibodies persist. one form of antibody response to the development of abnormal cell surface characteristics is tumour-associated autoantibodies. these proteins are produced early in tumorigenesis, being measurable up to 5 years before the development of clinical symptoms (7). they represent biologically amplified markers, assessment of background levels of autoantibodies as a prognostic marker for severe sars-cov-2 infection frank m. sullivan1, agnes tello1,2, petra rauchhaus3, virginia hernandez santiago1, fergus daly1 1division of population and behavioural sciences, st andrews university medical school, st andrews united kingdom 2edinburgh clinical trials unit, usher institute, university of edinburgh, edinburgh united kingdom 3tayside clinical trials unit, university of dundee, dundee united kingdom abstract background: patients with more severe forms of sars-cov-2 exhibit activation of immunological cascades. participants (current or ex-smokers with at least 20 years pack history) in a trial (early diagnosis of lung cancer, scotland [ecls]) of autoantibody detection to predict lung cancer risk had seven autoantibodies measured 5 years before the pandemic. this study compared the response to covid infection in study participants who tested positive and negative to antibodies to tumour-associated antigens: p53, ny-eso-1, cage, gbu4-5, hud, mage a4 and sox2. methods: autoantibody data from the ecls study was deterministically linked to the eave ii database, a national, real-time prospective cohort using scotland’s health data infrastructure, to describe the epidemiology of sarscov-2 infection, patterns of healthcare use and outcomes. the strength of associations was explored using a network algorithm for exact contingency table significance testing by permutation. results: there were no significant differences discerned between sars-cov-2 test results and earlycdt-lung test results (p = 0.734). an additional analysis of intensive care unit (icu) admissions detected no significant differences between those who tested positive and negative. subgroup analyses showed no difference in covid-19 positivity or death rates amongst those diagnosed with chronic obstructive pulmonary disease (copd) with positive and negative earlycdt results. conclusions: this hypothesis-generating study demonstrated no clinically valuable or statistically significant associations between earlycdt positivity in 2013-15 and the likelihood of sars-cov-2 positivity in 2020, icu admission or death in all participants (current or ex-smokers with at least 20 years pack history) or in those with copd or lung cancer. keywords: covid-19, current or ex-smokers, lung cancer, mortality prediction, serum biomarkers received: september 7, 2021 accepted: april 20, 2022 published online: may 3, 2022 corresponding author: frank m. sullivan division of population and behavioural sciences st andrews university medical school st andrews united kingdom fms20@st-andrews.ac.uk introduction patients infected with covid-19 show a range of immune responses, from weaker immune responses in asymptomatic individuals, to symptomatic patients showing a varying degree of immune dysregulation. these may be manifested by increased levels of interleukins, c-reactive protein and d-dimer, along with lymphopenia, monocytosis and neutrophilia. extremely high levels of proinflammatory cytokines can lead to a cytokine storm and macrophage activation https://doi.org/10.33393/jcb.2022.2337 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:fms20@st-andrews.ac.uk sullivan et al. j circ biomark 2022; 11: 25 © 2022 the authors. published by aboutscience www.aboutscience.eu increasing the detectable signal for the corresponding level of antigen (8). they persist in the circulation with half-lives of typically up to 30 days (9). the earlycdt-lung test is an enzyme-linked immunosorbent assay (elisa) that measures seven autoantibodies, each with individual specificity for the following tumourassociated antigens (taas): p53, ny-eso-1, cage, gbu4-5, hud, mage a4 and sox2 (10). a sample is positive if at least one autoantibody is elevated above a predetermined cutoff (11). the test has been developed throughout the preclinical, clinical assay validation and retrospective biomarker development pathway stages. in cohort studies, it has demonstrated a specificity of 91% and sensitivity of 41%. the early diagnosis of lung cancer scotland (ecls) study was a phase iv biomarker trial using earlycdt-lung followed by imaging in 12,208 smokers and ex-smokers aged 50-75 at risk of developing lung cancer recruited from general practices in scotland (12,13). a total of 6,088 participants in the intervention arm received the earlycdt-lung test at the baseline visit and 598 (9.8%) had a positive autoantibody result. in the 2-year analysis of the ecls trial, earlycdt-lung was shown to reduce late stage presentations of lung cancer. we have investigated whether the production of autoantibodies in response to cell surface abnormalities in cancer, as measured by the baseline earlycdt-lung test in the ecls trial, was associated with more severe disease in at-risk participants (current and former smokers) who then developed a sars-cov-2 infection 5-6 years later. methods participants aged 50-75 who were current or ex-smokers with at least 20 years pack history were recruited to ecls between december 2013 and april 2015, and all baseline assessments of plasma antibody levels occurred during this time (14). sars-cov-2 status and outcome data for ecls participants during 2020 were obtained from the eave ii database, which is a national, real-time prospective cohort using scotland’s health data infrastructure, to describe the epidemiology of sars-cov-2 infection, patterns of healthcare use and outcomes (15,16). data from both sources was linked using scotland’s community health index (chi) number at the university of dundee’s health informatics centre (hic) (17,18). the strength of associations was explored using a network algorithm for exact contingency table significance testing by permutation. this approach is appropriate for the sparseness of the data here, where an approximate chi-squared analysis would provide severely discrepant outputs. (for 2 × 2 contingency tables, the network algorithm reduces identically to fisher’s exact test.) results there were no significant differences discerned between sars-cov-2 test results and earlycdt-lung test results (positive/negative) (p = 0.734); or likewise between sars-cov-2 test results and earlycdt-lung test results (positive/negative/ control) (p = 0.779); or finally between sars-cov-2 test results and treatment (tested/not tested) (p = 0.587). an additional analysis of intensive care unit (icu) admissions detected no significant differences between those who tested positive and negative. there was no difference in covid-19 positivity or death rates amongst those diagnosed with lung cancer with positive and negative earlycdt-lung test results (tab. i). table i sars-cov-2 test results by earlycdt-lung test result result of sarscov-2 test positive negative control n % n % n % positive 9 6.7 86 7.8 84 7.0 negative 126 93.3 1021 92.2 1110 93.0 total 135 100 1107 100 1194 100 patient deceased no 131 97.0 1072 96.8 1155 96.7 yes 4 3.0 35 3.2 39 3.3 total 135 100 1107 100 1194 100 in table ii, nil significance was found. table ii outcomes in at-risk participants (current and former smokers) with lung cancer earlycdt-lung test result stage testpositive testnegative not tested total n % n % n % n % stage 3 0 (0.0) 1 (14.3) 4 (44.4) 5 (27.8) stage 4 0 (0.0) 1 (14.3) 0 (0.0) 1 (5.6) other 2 (0.0) 5 (71.4) 5 (55.6) 12 (66.7) total 2 (100) 7 (100) 9 (100) 18 (100) covid result n % n % n % n % test-positive 0 (0.0) 1 (14.3) 1 (11.1) 2 (11.1) test-negative 2 (100.0) 6 (85.7) 8 (88.9) 16 (88.9) total 2 (100) 7 (100) 9 (100) 18 (100) or** = 0.00 (0.00, 66.5) p = 1.0 hospitalized* n % n % n % n % no 0 (0.0) 4 (57.1) 3 (33.3) 7 (38.9) yes 2 (100.0) 3 (42.9) 6 (66.7) 11 (61.1) total 2 (100) 7 (100) 9 (100) 18 (100) or** = 0.00 (0.00, 4.20) p = 0.44 death* n % n % n % n % no 2 (100.0) 6 (85.7) 9 (100.0) 17 (94.4) yes 0 (0.0) 1 (14.3) 0 (0.0) 1 (5.6) total 2 (100) 7 (100) 9 (100) 18 (100) or** = 9999 (0.015, 9999) p = 1.0 *event within 28 days of a covid test. **odds ratio (test-positive vs test-negative). no association between previous autoantibody expression and development of sars-cov-226 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table iii shows no difference in covid-19 positivity or death rates amongst those diagnosed with chronic obstructive pulmonary disease (copd) with positive and negative earlycdt results. table iii outcomes in at-risk participants (current and former smokers) with copd earlycdt-lung test result covid result test positive testnegative not tested total n % n % n % n % positive 1 (6.6) 3 (2.6) 9 (8.3) 13 (5.4) negative 15 (93.8) 113 (97.4) 100 (91.7) 228 (94.6) total 16 (100) 116 (100) 109 (100) 241 (100) or** = 2.51 (0.0913, 24.13) p = 0.407 hospitalized* n % n % n % n % no 9 (56.3) 68 (58.6) 58 (53.2) 135 (56.0) yes 7 (43.8) 48 (41.4) 51 (46.8) 106 (44.0) total 16 (100) 116 (100) 109 (100) 241 (100) or** = 0.908 (0.312, 2.858) p = 1.0 death* n % n % n % n % no 16 (100.0) 110 (94.8) 105 (96.3) 231 (95.9) yes 0 (0.0) 6 (5.2) 4 (3.7) 10 (4.1) total 16 (100) 116 (100) 109 (100) 241 (100) or** = 9999 (0.176, 9999) p = 1.0 *event within 28 days of a covid test. **odds ratio (test-positive vs test-negative). discussion and conclusions no clinically valuable or statistically significant associations between earlycdt-lung positivity in 2013-15 and the likelihood of sars-cov-2 positivity in 2020, icu admission or death were found. this was true for the entire study cohort and in subgroup analyses of at-risk participants (current and former smokers) with lung cancer and copd. this is in contradistinction to those exhibiting the nucleolar immunofluorescence pattern where a significant association with interstitial lung sars-cov-2 disease has been demonstrated (19). strengths of the study include the community-based sampling of the ecls cohort, large numbers of the cohort who had a covid test validated by laboratory and outcome assessment. weaknesses include the time which had elapsed between the initial trial and the onset of the pandemic, as well as small numbers of study subjects who were in the subgroup analyses. some studies have shown that some routine clinical laboratory tests, such as lymphocyte count, lactate dehydrogenase and d-dimer are known to be affected in patients with covid-19 (20), with lymphopenia, raised lactate dehydrogenase and elevated d-dimer being associated with worse disease severity and outcomes (21-23). other studies have shown significant differences in inflammatory markers amongst patients who required icu admission compared to patients who have not, and markers of infection and inflammation such as c-reactive protein, procalcitonin, and ferritin which are, as expected, correlated with severe disease (24-27). this hypothesis-generating study did not find a clear association between the expression of 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https://www.ncbi.nlm.nih.gov/pubmed/32492406 https://doi.org/10.1177/20503121211050755 https://www.ncbi.nlm.nih.gov/pubmed/34659766 https://doi.org/10.1186/s12879-022-07144-5 https://www.ncbi.nlm.nih.gov/pubmed/35189826 https://doi.org/10.1016/j.jcrc.2021.09.023 https://pubmed.ncbi.nlm.nih.gov/34808527/ © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu jcb issn 1849-4544 doi: 10.33393/jcb.2020.2164 j circ biomark 2020; 9(1): 8-12 original research article complementary tools such as lung ultrasound (lus) and cardiac biomarkers might aid in the diagnostic approach. lus is highly sensitive for pulmonary congestion assessment in hf (1), since the number and distribution of b-lines denote the amount of extravascular fluid in the lung. current guidelines included natriuretic peptides to minimize hf diagnosis complexity, especially in the nonacute setting when echocardiography is not immediately available. in recent years, several cardiac biomarkers have been described, reflecting different active pathogenic pathways in hf (2). the association of b-lines and n-terminal pro-brain natriuretic peptide (nt-probnp) has been characterized in decompensated acute hf patients. nevertheless, there are few data on outpatients, and no data with other cardiac biomarkers. accordingly, we investigated the correlation between b-lines and different biomarkers in outpatients with suspicion of hf lung ultrasound and biomarkers in primary care: partners for a better management of patients with heart failure? mar domingo1-3, laura conangla1-3, josep lupón1,4,5, asunción wilke2,4, gladys juncà1, elena revuelta-lópez1,5,6, xavier tejedor7, antoni bayes-genis1,4,5 1heart failure unit and cardiology department, hospital universitari germans trias i pujol, badalona spain 2primary care service barcelonès nord i maresme, catalan health institute, badalona spain 3institut universitari d’investigació en atenció primària jordi gol (idiap jordi gol), barcelona spain 4department of medicine, universitat autònoma de barcelona, barcelona spain 5cibercv, instituto de salud carlos iii, madrid spain 6icrec research program, germans trias i pujol health science research institute, can ruti campus, badalona spain 7biochemistry service, hospital universitari germans trias i pujol, badalona spain abstract introduction: the association of pulmonary congestion assessed by lung ultrasound (lus) and biomarkers—other than n-terminal pro-brain natriuretic peptide (nt-probnp)—is uncertain. methods: we investigated the relationship between total b-line count by lus and several biomarkers in outpatients with suspicion of heart failure (hf). primary care patients with suspected new-onset nonacute hf were evaluated both with a 12-scan lus protocol (8 anterolateral areas plus 4 lower posterior thoracic areas) and 11 inflammatory and cardiovascular biomarkers. a cardiologist blinded to lus and biomarkers except nt-probnp confirmed hf diagnosis. after log-transformation of biomarkers’ concentrations, unadjusted and adjusted correlations were performed. results: a total of 170 patients were included (age 76 ± 10 years, 67.6% women). hf diagnosis was confirmed in 38 (22.4%) patients. after adjustment by age, sex, body mass index, and renal function, total b-line sum significantly correlated with nt-probnp (r = 0.29, p < 0.001), growth/differentiation factor-15 (gdf-15; r = 0.23, p = 0.003), high-sensitive troponin t (hstnt; r = 0.36, p < 0.001), soluble interleukin-1 receptor-like 1 (sst2; r = 0.29, p < 0.001), cancer antigen 125 (ca-125; r = 0.17, p = 0.03), high-sensitivity c-reactive protein (hscrp; r = 0.20, p = 0.009), and interleukin (il)-6 (r = 0.23, p = 0.003). in contrast, il-33 (r = −0.01, p = 0.93), il-1β (r = −0.10, p = 0.20), soluble neprilysin (snep; r = 0.09, p = 0.24), tumor necrosis factor-alpha (tnf-α; r = 0.07, p = 0.39), and tnf-α receptor superfamily member 1a (tnfrsf1a; r = 0.14, p = 0.07) did not. conclusions: total b-line sum correlated significantly, although moderately, with congestion and several inflammation biomarkers. unexpectedly, the highest correlation found was with hstnt. keywords: biomarkers, congestion, diagnosis, heart failure, lung ultrasound, primary care introduction heart failure (hf) diagnosis is challenging in ambulatory patients, since signs and symptoms are mild and can be related with other diseases, and even to natural aging. received: june 11, 2020 accepted: september 4, 2020 published online: october 16, 2020 corresponding author: antoni bayes-genis heart institute, hospital universitari germans trias i pujol department of medicine, universitat autònoma de barcelona carretera del canyet s/n 08916, badalona spain abayesgenis@gmail.com domingo et al 9 © 2020 the authors. published by aboutscience in the primary care setting. we hypothesized that biomarkers with multiple bio-profiling other than nt-probnp might be associated with pulmonary congestion by lus. methods study design and patients the present study is a biomarker subanalysis of a prospective cohort of ambulatory patients >50 years old, referred by their primary care physician to nt-probnp test for suspected new-onset non-acute hf (july 2015 to january 2018) (3). we excluded patients with established hf diagnosis, pulmonary fibrosis, or radiological pachypleuritis. the study was performed in accordance with the declaration of helsinki; the local ethics committee approved the research protocol and informed consent was obtained from all subjects. procedures all inclusion visits were scheduled in a centralized setting, where the primary care physician investigator (lc, md, aw) evaluated the patients, focusing on framingham criteria, and performed lus. blood samples were collected for nt-probnp measurement (xt), and serum aliquots were stored at −80°c prior to assay (er-l). at a subsequent visit, a cardiologist investigator (gj) assessed all participants and performed a transthoracic doppler echocardiogram. this physician confirmed hf diagnosis, following the european society of cardiology guidelines. the cardiologist had access to the patients’ electronic records, including the primary care investigator visit and nt-probnp, but was blinded to lus and other biomarkers. assays biomarker panel nt-probnp, high-sensitive troponin t (hstnt) and growth/ differentiation factor-15 (gdf-15) were measured by cobas elecsys® kits (roche diagnostics). cancer antigen 125 (ca125) was tested by architect ca 125 ii assay (abbott diagnostic). high-sensitivity c-reactive protein (hscrp) was measured by hscrp reagent (beckman coulter). human soluble neprilysin (nep) and soluble interleukin-1 receptor-like 1 (sst2) were measured by human soluble neprilysin/cd10 elisa kit (aviscera bioscience) and presage® st2 (critical diagnostics) assays, respectively. interleukin (il)-1β, il-33, il-6, tumor necrosis factor-alpha (tnf-α), and tnf-α receptor superfamily member 1a (tnfrsf1a) were tested by quantikine® immunoassay kits (r&d systems). nt-probnp was analyzed after collection. the rest of the biomarkers were analyzed in the first or second freeze-thaw cycle. lung ultrasound lus was performed with a pocket device (v-scan simple model with a sectorial phased array transducer; general electric®) and interpreted bench side. lus was performed with patient in a seated position; 8 anterolateral thoracic areas plus 4 posterior lower areas were examined. each of the 12 areas was classified according to the number of b-lines in the sagittal scan. a thoracic area was considered positive if ≥3 b-lines were observed. pleural effusion was considered as 10 b-lines. lus congestion was defined as 2 out of 6 positive scans in each hemithorax. transthoracic doppler echocardiography echocardiographic study was performed using an ie33 ultrasound system (philips medical systems; andover, massachusetts) with a s5-1 sector transducer (5.1 mhz bandwidth), and analyses were performed with an echopac. statistical analysis categorical values are described as absolute numbers (percentages) and continuous variables as means (standard deviations) or medians [interquartile ranges], depending on whether data distribution was normal as assessed by normal q-q plots. to assess the relationship of total b-line sum acquired by lus with biomarkers’ concentrations, pearson correlation was used after logarithmic transformation of biomarker levels; afterward, partial correlations adjusted by age and sex, and finally by age, sex, body mass index, and estimated glomerular filtration rate (egfr) were performed. analyses were performed using statistical package for the social sciences (spss) 24. a two-sided p < 0.05 was considered significant. results table i shows baseline characteristic and biomarker values of the 170 patients included. they were elderly, predominantly women, obese or overweight, and mainly in new york heart association (nyha) class ii. hf diagnosis was confirmed in 38 (22.4%) patients, and only one had left ventricular ejection fraction (lvef) < 40%. patients with hf diagnosis had higher levels of all biomarkers except il-33, il-1β, and soluble neprilysin (snep). they also had a higher number of total b-line count (p < 0.001). although 85% of patients had exertional dyspnea, only 17.1% had crackles, 9.4% orthopnea, and 3.5% paroxysmal nocturnal dyspnea. correlations between total b-line sum and studied biomarkers are shown in table ii. unadjusted analyses showed that total b-line sum was significantly associated with nt-probnp, gdf-15, hstnt, sst2, ca-125, hscrp, il-6, and tnfrsf1a (r range 0.18-0.35), while il-33, il-1β, snep, and tnf-α levels were not associated with total b-line sum. after the adjustments for the four covariates, r values tended to slightly decrease except for hstnt and tnfrsf1a that lost statistical significance. discussion bedside lus has appeared as a step forward for hf diagnosis, and biomarkers other than nt-probnp are currently lung ultrasound and biomarkers for heart failure diagnosis in primary care10 © 2020 the authors. published by aboutscience table i demographic, clinical characteristics and biomarker levels of patients total n = 170 hf diagnosis n = 38 no hf diagnosis n = 132 p-value age, years 76 ± 10.4 81.2 ± 8.3 74.4 ± 10 <0.001 female sex, n (%) 115 (67.6) 23 (60.5) 92 (69.7) 0.29 lvef, % 63 ± 5.8 59.9 ± 7.2 63.8 ± 5 <0.001 comorbidities, n (%)  hypertension  diabetes mellitus  copd  valvular heart disease  myocardial infarction  atrial fibrillation  obesity (bmi >30 kg/m2)  egfr < 60 ml/min/1.72 m2 132 (77.6) 43 (25.3) 19 (11.2) 6 (3.5) 15 (8.8) 19 (11.2) 84 (49.4) 48 (28.2) 36 (94.7) 12 (31.6) 7 (18.4) 3 (7.9) 7 (18.4) 16 (42.1) 20 (52.6) 18 (47.4) 96 (72.7) 31 (23.5) 12 (9.1) 3 (2.3) 8 (6.1) 3 (2.3) 64 (48.5) 30 (22.7) 0.004 0.31 0.11 0.10 0.02 <0.001 0.68 0.003 functional class, n (%)  i  ii  iii 20 (11.8) 116 (68.2) 34 (20.0) 2 (6.1) 18 (54.5) 18 (39.4) 18 (13.6) 98 (74.2) 16 (12.1) <0.001 exertion dyspnea 145 (85.3) 36 (94.7) 109 (82.6) 0.06 orthopnea 16 (9.4) 8 (21.1) 8 (6.1) 0.005 paroxysmal nocturnal dyspnea 6 (3.5) 2 (5.3) 4 (3.0) 0.51 lung crackles 29 (17.1) 11 (28.9) 18 (13.6) 0.03 total b-line sum 5.6 ± 10.1 14.1 ± 15.0 3.2 ± 6.4 <0.001 biomarkers  nt-probnp, ng/l  gdf-15, ng/l  hstnt, ng/l  sst2, ng/ml  ca-125, u/ml  hscrp, mg/l  il-33, pg/ml  il-1β, ng/ml  il-6, pg/ml  snep, ng/ml  tnf-α, pg/ml  tnfrsf1a, ng/ml 202 (104-640) 1708 (1175-2511) 11.9 (6.7-21.7) 27.5 (21.8-37.6) 13.7 (9.7-22.7) 3.4 (1.9-7.3) 93.7 (93.7-601.2) 0.34 (0.27-0.44) 4.4 (2.9-7.2) 0.209 (0.062-0.605) 56.3 (49.9-67.5) 1.88 (1.45-2.39) 1350 (666-3551) 3133 (2040-4075) 24.8 (13.8-39.9) 39.6 (32-56.6) 21.4 (11.4-55.4) 5.3 (2.3-19.2) 93.7 (93.7-548) 0.33 (0.25-0.45) 6.3 (4.3-14.3) 0.206 (0.062-0.465) 60.8 (52.7-71.1) 2.39 (1.72-3.56) 148 (88-289) 1470 (1113-2117) 9.6 (5.9-15.9) 25.2 (20.7-33.8) 13 (8.8-20) 3.1 (1.7-5.8) 93.7 (93.7-632.3) 0.35 (0.28-0.43) 3.9 (2.8-5.9) 0.208 (0.062-0.630) 55.1 (49.1-66.1) 1.75 (1.42-2.21) <0.001 <0.001 <0.001 <0.001 <0.001 0.005 0.78 0.60 <0.001 0.62 0.02 <0.001 data are expressed as mean (standard deviation), median (percentiles 25th-75th), or absolute numbers (percentages). bmi = body mass index; ca-125 = cancer antigen 125; copd = chronic obstructive pulmonary disease; egfr = estimated glomerular filtration rate; gdf-15 = growth differentiation factor 15; hf = heart failure; hscrp = high-sensitivity c-reactive protein; hstnt = high-sensitivity troponin t; il = interleukin; lvef = left ventricular ejection fraction; nt-probnp = n-terminal pro-brain natriuretic peptide; nyha = new york heart association; snep = soluble neprilysin; sst2 = soluble interleukin-1 receptor-like 1; tnf-α = tumor necrosis factor α; tnfrsf1a = tnf receptor superfamily member 1a. under investigation. although there is a growing interest on both, added value for better patient diagnosis and management has scarcely been studied. in our study, we assessed the correlation between b-lines and a biomarker panel in primary care outpatients with new-onset nonacute hf suspicion. our results showed that (i) total b-line sum observed by lus was significantly—although moderately— associated with several biomarkers of active pathogenic pathways in hf, especially with those related to congestion and inflammation; and (ii) hstnt, a biomarker related to domingo et al 11 © 2020 the authors. published by aboutscience myocardial injury, was mainly and unexpectedly associated with total b-line sum. hf diagnosis can be difficult at early stages, especially in women and old patients with comorbidities. our patients were elderly, mainly women, and not very symptomatic. less than 10% of patients had lung congestion symptoms and only 17% crackles. in this context, hf was only confirmed in 22.4% of patients since framingham criteria, despite being highly specific, have a poor sensitivity for hf diagnosis in ambulatory patients. hand-held devices could be easily incorporated in primary care and have shown a good correlation with standard ultrasound equipment for b-line detection. in our study, we used the 8-zone technique adding 4 posterior zones since these areas are the first that show signs of congestion and could add accuracy in outpatients. as expected and according to previous studies in acute hf, total b-line sum was significantly associated with ntprobnp levels. however, we projected a highest correlation, since increased intracardiac filling pressures often precede lung congestion. nevertheless, in mildly symptomatic primary care patients, pulmonary congestion is not always present unlike hemodynamic dysfunction. sst2 levels also correlated with b-line count in a similar level that nt-probnp. st2 is a member of the il-1 receptor family linked to myocardial fibrosis and adverse remodeling, both related to diastolic dysfunction and increased end-diastolic pressures that can contribute to pulmonary congestion. sst2 has been described as a 3-in-1 biomarker and provides insight into the hemodynamic, inflammatory, and pro-fibrotic/remodeling burden of the myocardium (4). total b-line sum also correlated with gdf-15, a marker of cell injury inflammation, oxidative stress, and hypoxia. these results are consistent with previous studies where gdf-15 may indicate a greater systemic inflammatory response in old patients and those with hf and preserved ef (5), as was the population of our study. it is remarkable that these two biomarkers, being in part inflammatory biomarkers but also associated with other several pathogenic pathways in hf, correlated with total b-line sum in a greater degree than the more “pure” inflammatory ones (hscrp, il-1β, il-33, il-6, tnf-α, and tnfrsf1a). maybe the more remarkable finding was the high correlation of total b-line sum with hstnt, a biomarker of myocardial injury frequently elevated in patients with hf without coronary ischemia. unexpectedly, hstnt correlation was even higher than that observed with nt-probnp and sst2. recently, myhre et al (6) showed that high-sensitive cardiac troponin t (hs-ctnt) concentrations were associated with worse diastolic function, suggesting that high levels of hs-ctnt may serve as an early marker of subclinical alterations in diastolic function that may lead to a predisposition to hf. finally, although there was a statistically significantly correlation between total b-line sum and ca-125, we anticipated a higher correlation, since both are surrogates of pulmonary and systemic congestion (7), respectively. congestion plays a major role in acute hf syndromes; however, it is known that severity and organ distribution are largely heterogeneous. in fact, our primary care patients showed low percentages of congestion signs or symptoms. limitations of our study include the limited sample size and the low incidence of hf since our target was primary care patients with mild symptoms and suspicion of hf. these facts might have an impact on the external validity of the study. also hf diagnosis was performed by a single cardiologist. although larger studies in diverse populations are needed, our data are hypotheses generating correlations between b-lines, a surrogate of pulmonary congestion, and of biomarkers in hf patients. conclusion in primary care outpatients with new-onset nonacute hf suspicion, total b-line sum is significantly—although moderately—associated with several biomarkers of congestion and inflammation, and remarkably with hstnt. table ii correlations between total b-line sum and studied biomarkers nt-probnp gdf-15 hstnt sst2 ca-125 hscrp il-6 il-33 il-1β snep tnf-α tnfrsf1a unadjusted r 0.32 0.27 0.35 0.32 0.21 0.22 0.27 –0.02 –0.08 0.10 0.09 0.18 p-value <0.001 <0.001 <0.001 <0.001 0.007 0.004 <0.001 0.81 0.28 0.21 0.26 0.02 adjusted by age and sex r 0.29 0.23 0.34 0.30 0.19 0.21 0.24 –0.01 –0.11 0.09 0.08 0.15 p-value <0.001 0.003 <0.001 <0.001 0.02 0.007 0.002 0.93 0.18 0.24 0.32 0.06 adjusted by age, sex, bmi, and egfr* r 0.29 0.23 0.36 0.29 0.17 0.20 0.23 –0.01 –0.10 0.09 0.07 0.14 p-value <0.001 0.003 <0.001 <0.001 0.03 0.009 0.003 0.93 0.20 0.24 0.39 0.07 *estimated by ckd-epi (chronic kidney disease epidemiology collaboration). bmi = body mass index; ca-125 = cancer antigen 125; egfr = estimated glomerular filtration rate; gdf-15 = growth differentiation factor 15; hscrp = high sensitivity c-reactive protein; hstnt = high-sensitivity troponin t; il = interleukin; nt-probnp = n-terminal pro-brain natriuretic peptide; snep = soluble neprilysin; sst2 = soluble interleukin-1 receptor-like 1; tnf-α = tumor necrosis factor α; tnfrsf1a = tnf receptor superfamily member 1a. lung ultrasound and biomarkers for heart failure diagnosis in primary care12 © 2020 the authors. published by aboutscience acknowledgments the authors thank jaume barallat and adriana cserkóová for their valuable technical support in sample processing. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this work was supported by la marató de tv3 [pi 201510.10], the primary healthcare university research institute idiap-jordi gol, and the catalan society of family physicians (camfic). references 1. platz e, jhund ps, campbell rt, mcmurray jj. assessment and prevalence of pulmonary oedema in contemporary acute heart failure trials: a systematic review. eur j heart fail. 2015;17:906-916. 2. berezin ae. prognostication in different heart failure pheno types: the role of circulating biomarkers. j circ biomark. 2016;5:6. 3. conangla l, domingo m, lupón j, et al. lung ultrasound for heart failure diagnosis in primary care. j card fail. 2020 jun 6:s10719164(19)31822-6. doi: 10.1016/j.cardfail.2020.04.019. online ahead of print. 4. pascual-figal da, bayes-genis a, asensio-lopez mc, et al. the interleukin-1 axis and risk of death in patients with acutely decompensated heart failure. j am coll cardiol. 2019;73:1016-1025. 5. wollert kc, kempf t, wallentin l. growth differentiation factor 15 as a biomarker in cardiovascular disease. clin chem. 2017;63:140-151. 6. myhre pl, claggett b, ballantyne cm, et al. association between circulating troponin concentrations, left ventricular systolic and diastolic functions, and incident heart failure in older adults. jama cardiol. 2019;4:997-1006. 7. núñez j, bayés-genís a, revuelta-lópez e, et al. clinical role of ca125 in worsening heart failure: a biostat-chf study subanalysis. jacc heart fail. 2020;8:386-397. jcb j circ biomark 2021; 10: 14-19issn 1849-4544 | doi: 10.33393/jcb.2021.2220short communication journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu mainly bone marrow-derived cells and suggests a greater influence of bone marrow health status on individual crp profiles and, most likely, vice versa. conversely, high diagnostic potential of the crp, in particular for the assessment of bone marrow damage (bmd), can be deduced yet still, which entails a steep gain in knowledge about character and functionality of individual rare cells (7). one of the most promising markers to be associated with bmd is the circulating erythroblast (ceb). this cell type is a known and common part of the crp, yet has been rarely investigated for clinical applications in cblb (6). we have earlier reported on the occurrence of immature ceb-like cells in healthy donors and suggested the association of respective findings above baseline with underlying pathologies affecting the bone marrow (8). our data presented support of the hypothesis that increased levels of ceb predict bmd caused by chronic and systemic diseases. we herein report a new finding of erythroid cell abnormality with respect to poly-nucleation, cell pairing, and aggregation in association with systemic pathologies. the implications of the findings of, in particular, morphologically aberrant ceb would represent cytological evidence of bmd and be far-reaching for care in systemic diseases, for example, for the evidence-based prediction of distant invasion in cancer. as a first step into this research, we intend to describe circulating erythroblast abnormality associated with systemic pathologies may indicate bone marrow damage stefan schreier1-3, prapaphan budchart3, suparerk borwornpinyo3,4, wichit arpornwirat5, wannapong triampo2,6 1school of bioinnovation and bio-based product intelligence, faculty of science, mahidol university, rama vi rd, bangkok thailand 2thailand center of excellence in physics, ministry of higher education, science, research and innovation, bangkok thailand 3premise biosystems co., ltd. bangkok thailand 4excellent center for drug discovery, faculty of science, mahidol university, rama vi, rd, bangkok thailand 5department of oncology, bangkok hospital, bangkok thailand 6department of physics, faculty of science, mahidol university, bangkok thailand abstract background: the circulating rare cell population is diverse and rich in diagnostic information. its characterization and clinical exploitation by cell-based liquid biopsy is an ongoing research task. bone marrow is one of the major contributors to the peripheral blood rare cell population and, consequently, determines individual rare cell profiles thus depending on bone marrow health status. bone marrow damage has been associated with aggressive or late-stage systemic diseases and egress of various bone marrow cells into the blood circulation. the association of quantity and heterogeneity of circulating erythroblast with bone marrow damage is of particular interest. methods: circulating cd71high/cd45-/hoechsthigh blast cells from healthy, noncancerand cancer-afflicted donors were enriched by cd45 depletion and analyzed by immunofluorescence microscopy. results: a new finding of aberrant and mitotic circulating erythroid-like cells that appear similar across blood donors afflicted with various systemic pathologies is reported. further presented is a classification of said erythroblast-like cells in nine subcategories according to morphological differences between phenotypically similar cells. conclusion: aberrant and mitotic bone marrow-derived rare circulating erythroid-like cells can be detected in the blood of afflicted individuals but not in healthy donors, suggesting the cause of bone marrow damage. keywords: bone cancer, bone marrow damage, circulating rare cells, erythroblast, liquid biopsy introduction advancement in cell-based liquid biopsy (cblb) comprises aspects of technology and biology (1,2). the latter often focused on the clinical translation of biomarkers mostly comprising known phenotypically specified cell types, such as epithelial, endothelial, or mesenchymal cells (3-5). a new chapter may have been opened with the awareness of the so-called circulating rare cell population (crp) that holds the potential of cell discovery as well as of comprehensive cell analysis similar to a cytological investigation (6). crp includes all kinds of non-hematopoietic but also rare hematopoietic received: december 7, 2020 accepted: july 14, 2021 published online: august 31, 2021 corresponding author: stefan schreier school of bioinnovation and bio-based product intelligence faculty of science mahidol university rama vi rd, bangkok 10400 thailand stefan.scr@mahidol.edu https://doi.org/10.33393/jcb.2021.2220 https://creativecommons.org/licenses/by-nc/4.0/legalcode schreier et al j circ biomark 2021; 10: 15 © 2021 the authors. published by aboutscience www.aboutscience.eu and classify the newly found ceb abnormalities as part of crp associated with systemic disorders and provide first-hand evidence of the association with bmd. theory of liquid biopsy-based bmd detection in cancer, bmd is an expected consequence of invasion by aggressive tumor cells referred to as disseminated tumor cells (dtc) and may entail a systemic response by way of egress of bone marrow cellular components that forms abnormal crp profiles (6). consequently, bmd detection in the setting of a cancer diagnosis is predictive of aggressive distant invasive disease. relevant circulating cell types being indicative of and highly sensitive to slightest bmd are erythroid precursors that vary in quantity and maturation state upon bone marrow imbalances (8). ceb concentrations may vary in the range of <1 to 2 × 105 cells/ml depending on age, lifestyle in the lower concentration regimen, and disease type and severity in the higher concentration regimen, respectively. the pathological mechanism behind erythroblast egress as well as abnormalities is not well understood in particular for nonerythroid pathologies. we suggest at least two pathologies at work. one is the dysregulation of erythropoiesis, another is a dysfunctional blood barrier. we theorize that a change in the micro-environment of the so-called erythroid islands (9) at the vascular niche is caused by stress, for example, by invasion and nesting of aggressive dtc. stress may be caused by inducing hypoxic conditions or by interaction with the erythroblast committed central macrophage or the erythroblasts themselves (10,11). hypoxia develops in growing tumors, suggesting that active/aggressive dtc may induce hypoxia in the bone marrow micro-environment as well and may outstrip their ability to take up oxygen and nutrients from their environment by diffusion. tumor-induced hypoxia is associated with poor prognosis (12). furthermore, dtc are theorized to be in competition with erythroblasts for physical interaction with the central macrophage, and then changing the activation state of the central macrophage in the erythroid island most likely causing inflammatory cell recruitment, dysregulation of erythroblast proliferation and maturation, and destabilization of the vascular integrity. furthermore, erythroblast cell cycle defects may be caused by altered mitochondrial function (13). methods patient cases nonspecific systemic pathologies were chosen to investigate the presence of ceb abnormality comprising a lateand early-stage breast cancer sample taking liquid biopsy before palliative treatment and after surgery and during chemoadjuvant therapy treatment, respectively. the collection further comprised an extensive stage small cell lung cancer (sclc) patient taking a liquid biopsy during 3rd line chemotherapy therapy and a stage 4 non-small cell lung adenocarcinoma before palliative treatment. another liquid biopsy was taken from a stage 3 head and neck cancer patient during chemotherapy. furthermore, subjectively healthy persons with known underlying pathologies included three individuals afflicted with diabetes type ii, osteoporosis, and thrombocytosis, respectively. informed consent was sought from the patient at the time of the blood draw in accordance with institutional review board (irb) protocol. detection platform the cblb procedure has been described in our previous publication with modifications (8). in brief, 5 ml blood taken from 17 healthy donors and 4 cancer patients was subjected to pre-enrichment by red blood cell lysis yielding highly purified nucleated cells in the range of 1.8e7 to 5.5e7 cells. the cell suspension was further enriched by cd45 depletion assay employing an automated magnetic cell enrichment platform (walderbach i, sanolibio gmbh, muenchen). the resulting cell suspension comprised carry-over leukocytes counting 3000 cells on average and all sorts of cd45-negative rare cells, among them ceb. in order to visualize their presence, the enriched cell suspension was subjected to cell membrane staining by conjugate antibodies reactive against cd71 and cd45 in both cases and nucleus staining by hoechst blue dna stain. for analysis, the sample suspensions were loaded into a 386-well plate suitable for high-resolution image recording at 40× magnification using the operetta system (perkinelmar) and recording a bright field channel, channels for uv, green, and yellow fluorescence emission. columbus analysis software served as screening and image analysis tool. cells were identified as ceb in case of high expression of cd71 along the membrane, high nucleus density as well as complete absence of cd45 marker signal. results we have identified known but also thus far unreported ceb morphologies exclusively in diseased individuals that follow in principle typical descriptions of erythroblast morphology and erythroid-specific cd71high/cd45-/hoechsthigh phenotype. detected cell morphologies could be recognized across diseases and therefore grouped into four main morphological distinct types which could be further divided into a total of nine subtypes (fig. 1). type 1 ceb represents normal erythroblasts and were found commonly in healthy donor blood at low concentrations, which can be further grouped into small (type 1a) and large (type 1b) erythroblasts owing to the size differences to the erythroblast maturation process (8). type 1a is the most abundant cell type in all samples and represents the matured circulating counterpart of otherwise bone marrow dwelling orthochromatic erythroblasts with diameters in the range of 6.5 µm to about 12.4 µm. type 1a cells corresponded to the commonly described appearance with dense nuclei and a high nucleus to cytoplasm ratio (n/c ratio). class 1b denotes less frequent large ceb with diameters exceeding 12.4 µm as previously described (8). apart from the size, immaturity is represented by a low-density chromatin nuclei in the diameter of about 6 µm to 10 µm and exhibiting a low n/c. type 1 ceb may reflect abnormal erythropoiesis by cell elevation above normal concentrations only (tab. i). type 1 ceb could be further divided into subtypes 1c and 1d comprising new implications on cancer invasiveness assessment16 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table i patient findings patient type patient description type 1a type 1b type 1c type 1d type 2a type 2b type 3a type 3b type 4 1. breast cancer, late stage treatment-naive recurrent breast cancer and bone, liver metastasis 3219 13 46 10 257 1 515 2 1260 2. breast cancer, early stage postsurgery baseline, stage 2, lymph node negative 2 none none none 2 none none none none mid-term act 62 3 6 none 1 none 2 none none 3. small cell lung cancer, extensive stage 3 months 3rd regimen, brain metastasis 385 10 4 none 36 none 6 2 4 4. lung adenocarcinoma, late stage treatment naive, stage 4 81 9 none none 4 none 4 none none 5. head and neck, advanced stage under treatment 5 none none none none none 1 none none 6. diabetes healthy 6 none none none 1 none 1 none none 7. thrombocytosis stable thrombocytosis, healthy 2705 2 none none none none 1 none none 8. osteoporosis osteoporosis healthy 9 none 1 none 1 none none none none 9. healthy data from schreier et al. 2018 (n = 15) 7.5* <1 none none none none none none none act = adjuvant chemotherapy. numbers denote cell concentrations per 5ml whole blood. fig. 1 gallery of cd71high/cd45-/hoechsthigh circulating erythroblast (ceb) types. from left to right and upper to lower. the first two images represent normal erythroblasts measuring 10.5 μm and 7 μm in diameter, respectively. next is shown a class 1b large ceb with low chromatin density measuring 14 μm in major diameter. next is shown a megaloblast presenting irregular chromatin texture and is followed by a macronormoblast. the following two images represent symmetric type 2a binucleated ceb. the next image represents an asymmetric aberrant type 2a binucleated ceb. next is shown a type 2b ceb and is characteristic of a binucleated and multilobulated nucleus. the next two images represent symmetric mitotic ceb of class 3a. next is shown a type 3b mitotic ceb with apparent chromatin bridging and a type 3b ceb showing symmetric and asymmetric mitosis. the last two images represent type 4 ceb aggregates revealing nucleus variations in size and maturation. schreier et al j circ biomark 2021; 10: 17 © 2021 the authors. published by aboutscience www.aboutscience.eu so-called megaloblasts and macro-normoblasts, respectively. the finding of such abnormal morphologies in the circulation represents evidence of dyserythropoiesis and predicts a pathological state of the bone marrow (14,15). megaloblasts show nucleocytoplasmic asynchrony and moderate to high and intracellular chromatin heterogeneity and high n/c ratio. macro-normoblasts show no nucleocytoplasmic asynchrony, total condensation nuclei in a diameter of about 4 µm to 6 µm, and a very low n/c ratio. type 2 ceb denotes an apparently relatively frequent morphological abnormality in both nonmalignant and malignant diseases and is characterized by symmetric multi-nucleation in a single cell most likely ascribed to asynchronous mitosis. type 2 ceb may also vary in size and shape and includes aberrant cell types containing nuclei that differ in size and maturation (fig. 1). we further divided type 2 ceb into binucleated (type 2a) and multinucleated or multinuclear lobulation single cells (type 2b) following world health organization (who) descriptions of bone marrow erythroblast abnormality (16). different underlying pathologies and/or stages may be causative of the number of nuclei in a single cell, for example, in the classification of congenital dyserythropoietic anemia in which types i–iv directly correlate with the number of erythroblast nuclei (17). myelodysplastic syndromes may produce three or more nucleated, yet mostly binucleated erythroblasts (15). type 3 ceb denotes cells in synchronous mitosis at various stages from metaphase till cytokinesis and seems to occur across disease types as frequent as type 2a ceb. this type could be further divided into symmetric (type 3a) and aberrant (type 3b) cell morphologies. the latter includes ceb pairs showing internuclear bridging or containing nuclei that differ in size, number, and maturation (fig. 1). type 4 comprises cells appearing in groups of at least three round or oval, normal or binucleated cells and represents the morphological aspect of a cell cluster. these cells may be seen to be in division or constitute a breakaway of aggregated erythroblasts from an erythroid island within the bone marrow. type 4 ceb is only found in late-stage malignancies. apart from types 1a and b, all other herein reported types were not found in the healthy cohort (8) (tab. i). therefore ceb types 1c, d, 2, 3, and 4 are herein referred to as abnormal and would denote bone marrow defects to some extent. according to table i, the ceb profile with regard to heterogeneity and cell number may be positively correlated with disease severity, suggesting a relatively high sensitivity toward slightest physiological imbalances of the bone marrow. discussion crp is expected to convey a wealth of diagnostic information ascribed to lesion-associated cell egress into the blood stream or to tissue repair or maintenance. it is good to say that the rarest population of cells is most decisive for, and thus informative of, our health. rare cells associated with tissue repair may comprise bone marrow-derived progenitor and stem cells. those rare cells associated with lesions may comprise respective tissue-derived mature somatic cells, such as epithelial or endothelial cells. the diagnostic exploitation of a single, a few, or all detectable circulating rare cells in a sample is referred to as cblb. our investigation in that field, in particular on the circulating cd71high phenotype, has revealed a staggering variety of morphologies across different diseases that are seemingly not associated with pathological erythropoiesis and seem to correspond to findings of erythroid abnormality as could be found in the bone marrow (15). erythrobast abnormality has been rarely reported to circulate, and to the best of our knowledge only in case of diseases with underlying pathological erythropoiesis (18). apart from systemic diseases, treatment by chemotherapy is known to elevate ceb concentration (11), suggesting that ceb abnormality in patient 3 may be caused or influenced by chemotherapy. the two samples of patient 2 before and during chemoadjuvant therapy, respectively, would also support chemotherapy-induced ceb elevation. we would like to take the opportunity to suggest possible clinical usefulness of our findings. the assumption is that ceb abnormality would denote sufficient cytological evidence to diagnose bone marrow defects and would allow us to grade bmd based on cell quantity and type. cell aberration, in particular, and/or the finding of type 4 ceb could provide evidence for the association with more severe bmd as has been found exclusively in metastatic cancer (patients 1 and 3). our thoughts about ceb abnormality and the association with bmd may be supported by investigations of the bone marrow pathology reporting similar erythroblast abnormality in case of severe bone marrow diseases, such as leukemia or hereditary anemia (18-20). when applied to cancer, in particular early stage cancer patients may benefit from correct assessment of individualized distant invasion profiles (21) however, conventional functional imaging methods are not capable of detecting potentially hazardous distant micro-metastasis. also, the detection of circulating tumor cells (ctcs) only predicts metastases based on statistics (22,23). moreover, bone marrow biopsy may provide pathological evidence of distant disease development at the individual level, yet is not routinely performed given the patient risk by the procedure and low discrimination power between dormant and active invasion. positive findings of tumor cell dissemination at earliest stages were commonly reported from bone marrow biopsies (24-26). despite the evidence to support prognostic significance in early-stage breast cancer patients (27-30), bone marrow micro-metastases assessment (bmma) as prognostic marker for relapse was found invariant to existing treatment decision making and, thus, was not adopted into clinical routine ever since (31). henceforth, the diagnostic question of bmma and single-cell invasion must be shifted from the status of distant invasion to distant aggressiveness, that is, bmd. the detection of ceb via cblb could be a wellsuited and better alternative to bmma given the improved stratification power between dormant or inactive and active tumor cell invasion. we can expect from our data that dormant metastases would not produce marked ceb profiles and, vice versa, active disease would generate detectable ceb profiles. finally, our bmd detection platform may tap into the exciting notion of ctc/dtc-guided treatment decision based on tumor evolution (32). new implications on cancer invasiveness assessment18 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb conclusion the study investigated circulating erythroid nucleated cells in the blood of healthy and afflicted individuals. bone marrow-derived erythroblasts were identified in each blood donor by positive cd71 staining and typical erythroblast morphology confirming commonness as part of the blood rare cell population. furthermore, abnormal erythroblast-like cells as known to the pathologist from bone marrow biopsies were found only in diseased individuals in this study. this report is mainly dedicated to the new finding of aberrant and mitotic erythroblast-like cells that are so far unknown to circulate in the blood of diseased individuals foremost in cancer patients. the cell abnormality could be coarsely classified into synchronous and asynchronous mitosis, respectively, and is likewise suspected to be bone marrow-derived, consequently indicating various degrees of bmd. a new cancer biomarker panel could be established in cblb when used in combination with circulating epithelial cells as a potential detection and grading platform of cancer-associated active distant invasion. acknowledgments we would like to give special thanks to dr. kanakorn runglodvatana from vajira hospital, bangkok, for providing head and neck cancer patient blood donation. ethics approval and consent to participate the study subject titled “advancing cell-based liquid biopsy” was approved by the mahidol university central irb, mahidol university, with protocol number 2019/197.3007. all healthy donors were appointed, informed, and consented in written form. in case of the three cancer patient cases, study information was provided by the treating doctor and consent was given by the patient verbally, respectively during out patient consultation, due to very limited visitation time. abbreviations act = adjuvant chemotherapy; bmd = bone marrow damage; bmma = bone marrow micro-metastases assessment; cblb = cell-based liquid biopsy; ceb = circulating erythroblast or circulating erythroblast-like cell; ctc = circulating tumor cell; dtc = disseminated tumor cells; h&n = head and neck cancer; n/c ratio: nucleus to cytoplasm ratio; rcp = rare cell population; sclc = small cell lung cancer. disclosures conflict of interest: ss and sb are shareholders of the companies involved in the development and manufacturing of cell separation technology and biomarkers as was employed in this work. financial support: the study was partly funded by mahidol uni versity. authors’ contribution: ss conceived and designed the study, carried out imaging analysis, and drafted the manuscript. prb carried out experimentation and took part in image 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https://doi.org/10.1055/a-0753-7331 https://www.ncbi.nlm.nih.gov/pubmed/30792547 cbx807827 1..10 research article adipose mesenchymal stem cells– derived exosomes attenuate retina degeneration of streptozotocin-induced diabetes in rabbits a safwat1, d sabry2, a ragiae3, e amer4, rh mahmoud5, and rm shamardan6 abstract this study aimed to evaluate the effect of mesenchymal stem cells (mscs)–derived exosomes in retina regeneration of experimentally induced diabetes mellitus (dm) in a rabbit model. exosomes are extracellular vesicles that contain many micrornas (micrnas), mrnas, and proteins from their cells of origin. dm was induced by intravenous (iv) injection of streptozotocin in rabbits. mscs were isolated from adipose tissue of rabbits. exosomes were extracted from mscs by ultracentrifugation. exosomes were injected by different routes (iv, subconjunctival (sc), and intraocular (io)). evaluation of the treatment was carried out by histopathological examination of retinal tissues and assessment of micrna-222 expression level in retinal tissue by real-time polymerase chain reaction. histologically, by 12 weeks following sc exosomal treatment, the cellular components of the retina were organized in well-defined layers, while io exosomal injection showed well-defined retinal layers which were obviously similar to layers of the normal retina. however, the retina appeared after iv exosomal injection as irregular ganglionic layer with increased thickness. micrna-222 expression level was significantly reduced in diabetic controls when compared to each of healthy controls and other diabetic groups with iv, sc, and io routes of injected exosomes (0.06 + 0.02 vs. 0.51 + 0.07, 0.28 + 0.08, 0.48 + 0.06, and 0.42 + 0.11, respectively). we detected a significant negative correlation between serum glucose and retinal tissue micrna-222 expression level (r ¼ �0.749, p ¼ 0.001). we can associate the increased expression of micrna-222 with regenerative changes of retina following administration of mscs-derived exosomes. the study demonstrates the potency of rabbit adipose tissue–derived mscs exosomes in retinal repair. so, exosomes are considered as novel therapeutic vectors in mscs-based therapy through its role in shuttling of many factors including micrna-222. keywords exosomes, mesenchymal stem cells, micrna, diabetes mellitus date received: 22 december 2017; accepted: 11 september 2018 1 department of ophthalmology, faculty of medicine, al-azhar university, cairo, egypt 2 department of medical biochemistry and molecular biology, faculty of medicine, cairo university, cairo, egypt 3 department of supplementary science histology and cell biology, faculty of oral and dental medicine, future university, new cairo, egypt 4 department of medical biochemistry, faculty of pharmacy, al ahram canadian university, egypt 5 department of medical biochemistry and molecular biology, faculty of medicine, fayoum university, faiyum, egypt 6 department of anatomy, faculty of medicine, fayoum university, faiyum, egypt corresponding author: d sabry, department of medical biochemistry and molecular biology, faculty of medicine, cairo university, cairo 11562, egypt. email: dinasabry@kasralainy.edu.eg journal of circulating biomarkers volume 7: 1–10 ª the author(s) 2018 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454418807827 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:dinasabry@kasralainy.edu.eg https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454418807827 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage introduction diabetic retinopathy (dr) is one of the serious complications of diabetes mellitus (dm), 1 which leads to severe visual impairment especially in late stages of the disease. 2 hyperglycemia and defects in insulin signaling pathways result in many pathological processes which end by the development of dr. 3 dr involves the presence of microvascular defects, neuroretinal dysfunction, and degeneration of the retina. 4 mesenchymal stem cells (mscs) were isolated and purified from many sources, such as bone marrow, adipose tissue, fetal membranes, embryo, and cord blood, and could be further utilized in tissue repair including the retina. 5–8 mscs are multipotent cells which have the possibility to transform into many types of cells involving cardiovascular, neurogenic, endothelial cells, and adipocytes. 9 mscs have been utilized as therapeutic agents because of their direct role in tissue regeneration and their strong anti-inflammatory properties which were discovered more recently. 10 moreover, mscs have been applied in many clinical attempts for management of retinal degeneration. 11 exosomes are extracellular vesicles (evs) of endosomal origin and are characterized from other vesicles derived from the cell by their origin, size, composition, and shape. 12 accordingly, exosomes are present in supernatants of cell culture. cd81 and cd63 proteins have been utilized as specific markers of exosomes. 13–16 it has been proved that exosomes may play a crucial role in transfer of mrna, micrornas (micrnas), and proteins. 13,14 it was found that presence of specific micrnas, such as micrna-175p, micrna-126, micrnas-221/222, and micrna-296, involved in the regulation of angiogenesis. 17,18 angiogenesis, abnormal growth of retinal blood vessels, was associated with the severity of dr. mir-221/mir-222 family sounds to negatively regulate angiogenesis by binding to the c-kit receptor. 19 the role of micrna-222 in regulating the neovascularization observed in dr was detected by targeting signal transducer and activator of transcription 5a (stat5a). additionally, it is indicated that stat5a is mostly regulated by micrna-222 during inflammationassociated neoangiogenesis. 20 thus we focused on detecting the expression of micrna-222. recently, more research have investigated the functions of exosomes . 21 our present study investigated the therapeutic effect of micrna-222-containing exosomes in treatment of retinal degeneration after their injection in experimental rabbit model with dm induced by streptozotocin (stz). materials and method mscs isolation and expansion the rabbit fat tissue was dissected and transported to 50 ml falcon tubes and weighted. for enzymatic dissociation, an equal volume of 250 u/ml of collagenase type ii was added to the fat tissue and incubated at 37�c for 90 min under agitation in shaking water bath. 22 by using a sterile strainer, cell suspensions were filtered. the filtered cells were centrifuged at 300 � g for 5 min and resuspended in expansion medium (a-modified eagles medium [mem] supplemented with 100 u/ml penicillin/streptomycin and 10% fetal calf serum) (r&d systems, abingdon, uk). cells quantification was assessed by cell counter and cells were plated at 4000 cells/cm 2 for 7 days. on day 7, cells were trypsinized, counted, and replated in expansion medium at the density of 2000 cells/cm 2 for another period of 7 days (end of passage 1). the expansion was performed till reaching third passage. isolation of exosomes exosomes were isolated from supernatant of first, second, and third passages of mscs cultured in a-mem deprived of fetal bovine serum (fbs). after centrifugation at 2000 � g for 20 min to remove debris, cell-free supernatant was centrifuged at 100,000 � g (ultracentrifuge of beckman coulter optima l 90 k) for 1 h at 4�c, washed in serumfree medium 199 containing hepes 25 mm (sigma, st louis, missouri, usa), and subjected in the same conditions to a second ultracentrifugation. the protein content of exosomes pellet was quantified by the bradford method (bio-rad, hercules, california, usa). 23 exosomes labeling with pkh26 exosomes were labeled with pkh26 dye for their in vitro tracing by fluorescence microscopy according to the manufacturer’s recommendations (sigma). following ultracentrifugation, the exosomes pellet was diluted with pkh26 kit solution to 1 ml and then 2 ml of fluorochrome was added to this suspension and incubated at 38.5�c for 15 min. after that, 7 ml of serum-free high glucose-modified eagles medium (hg-dmem) was added to the suspension and then it was ultracentrifuged for second time at 100,000 � g for 1 h at 4�c. the final pellet was resuspended rapidly in hg-dmem and stored at �80�c for future injection in experimentally induced rabbit. 24 western blot for characterization of exosomes the antibody used was antigen affinity-purified polyclonal sheep igg anti-rabbit cd81 (catalog no. 0349509; biolegend, san diego, california, usa). protein was isolated from isolated exosomes using radioimmunoprecipitation assay buffer. twenty nanograms of protein were loaded and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4–20% polyacrylamide gradient gels. following incubation in 5% nonfat dry milk, tris hydrochloride, 0.1% tween 20 for 1 h, cd81 polyclonal– monoclonal antibody was added to one of the membranes including specimen samples and incubated at 4�c overnight. appropriate secondary antibody was incubated for 2 h at room temperature. after being washed twice n 1 � 2 journal of circulating biomarkers tbs-t, densitometric analysis of the immunoblots was performed to quantify the amounts of cd81 against housekeeping protein b-actin by image analysis software on the chemidoc mp imaging system (version 3) produced by bio-rad. experimental induction of diabetes animals were handled and cared for in accordance with the guide of laboratory animals published by the us national institute of health (nih publications no. 8023, revised 1978) and approved by the ethics committee for animal experimentation at faculty of medicine, cairo university. male rabbits (1000–1200 gm) were injected by stz at a dose of 60 mg/kg of the body weight intravenously. stz induced diabetes within 3 days by damaging the beta cells. the experiment was carried out on rabbits and was divided as follows: group 1: rabbits received the standard diet, that is, control group (n ¼ 3); group 2: rabbits were injected with stz, that is, diabetic control (n ¼ 3); group 3: diabetic rabbits injected systemically with exosomes (100 mg protein/ml) labeled with pkh26 (intravenous (iv)) (n ¼ 3); and group 4: diabetic rabbits injected locally with exosomes labeled with pkh26 (subconjunctival (sc) and intraocular (io)) (n ¼ 3/each route of injection). at 4, 8, and 12 weeks from stz injection and administration of exosomes, venous blood was collected from the retroorbital vein to assess serum glucose and insulin. all rabbits were euthanized with carbon dioxide narcosis, and the retinal tissues were collected for histopathological examination and real-time polymerase chain reaction (rtpcr) analysis. histopathological examination tissue specimens were collected from euthanized rabbits of all groups. tissue sections (4–6 mm) were prepared. tissues were divided into two groups. the first group was examined for tracing of injected exosomes labeled with pkh26. the other group was washed with phosphate-buffered saline and fixed overnight in 10% buffered neutral formalin at 4�c. serial micrometer sections of the dissected tissues were stained with hematoxylin and eosin. rna extraction rna was extracted from retinal tissues using mirneasy extraction kit (qiagen, valencia, california, usa); manual instruction was available online at http://asmlab.org/public/ files/mirneasy-mini-kit-en.pdf. real-time polymerase chain reaction conversion of individual micrnas into the corresponding cdna was done with a taqman microrna real-time reverse transcriptase (rt) kit (assay id 000397, catalog no. 4427975; applied biosystems, foster city, california, usa) according to the manual manufacturer’s protocol; was accessible at https://tools.thermofisher.com/content/sfs/man uals/cms_042167.pdf. for micrna detection reaction, a forward primer and a 30 universal reverse primer were provided by the rt kit. rabbit u6 snrna was amplified as an internal control. qpcr using sybr green master mix (applied biosystems) was performed employing the applied biosystems instrument with software version 2.1 (stepone™). the amplification conditions were as follows: 1 cycle of 50�c for 2 min and 1 cycle of 95�c for 10 min, followed by 40 cycles of 95�c for 30 s and finally 60�c for 1 min. specificity was verified by melt curve analysis. the data were expressed in cycle threshold (ct). the pcr data sheet involves ct values of assessed micrna-222 and internal control (u6 snrna). the relative quantitation of micrna was normalized by ddct method. statistical analysis of data the obtained data were organized and statistically analyzed using spss software statistical computer package version 18 (spss inc., chicago, usa). for quantitative data, the mean and standard deviation were measured. one-way analysis of variance was used to test the difference about mean values of measured parameters among groups, multiple comparison between pairs of groups were performed using tukey honest significant difference (post hoc range test). pearson correlation was run to identify the relation of relative expression levels of micrna-222 with serum glucose and insulin levels. significance was adopted at p � 0.05. results serum glucose and insulin levels we first investigated serum glucose concentrations (table 1) and insulin levels (table 2) in different groups of study. the diabetic controls had significantly higher concentrations of serum glucose than other groups involved in the study, healthy controls, diabetic with iv injected exosomes, diabetic with sc and io administrated exosomes (322.9 + 41.4 vs. 85.6 + 10.7, 164.8 + 30.4, 177.2 + 12.3, and 185.4 + 29.0, respectively). serum insulin levels were significantly reduced in diabetic control group than each of healthy controls, diabetic with sc and diabetic with io injected exosomes groups (7.0 + 1.9 vs. 40.2 + 10.5, 30.8 + 2.5, and 29.3 + 2.6, respectively). mscs and exosomes characterization adipose tissue–derived mscs were cultured through first, second, and third passages where they were characterized using inverted microscope by their specific spindle fusiform shape in different passages. exosomes were analyzed by electron microscopy which appeared as micrographs for spheroidal exosomes with a formal size of less than 100 nm and contained homogenously sized particles (figure 1). safwat et al. 3 http://asmlab.org/public/files/mirneasy-mini-kit-en.pdf http://asmlab.org/public/files/mirneasy-mini-kit-en.pdf https://tools.thermofisher.com/content/sfs/manuals/cms_042167.pdf https://tools.thermofisher.com/content/sfs/manuals/cms_042167.pdf the isolated exosomes had specific membrane markers such as cd81 which was scanned versus b-actin in different passages by western blot analysis (figure 2(a)). the detected levels of cd81 showed a gradual increase in isolated exosomes while moving from one passage to another. table 1. comparison of serum glucose levels between different studied groups. glucose (mg/dl) healthy control (n ¼ 3) diabetic control (n ¼ 3) diabetic þ exosomes (iv) (n ¼ 3) diabetic þ exosomes (sc) (n ¼ 3) diabetic þ exosomes (io) (n ¼ 3) mean + sd 85.6 + 10.7 322.9 + 41.4 164.8 + 30.4 177.2 + 12.3 185.4 + 29.0 p1 — 0.00 a 0.00 a 0.00 a 0.00 a p2 0.00a — 0.00a 0.00a 0.00a p3 0.00 a 0.00 a — 0.29 0.41 p4 0.00a 0.00a 0.29 — 0.34 p5 0.00 a 0.00 a 0.41 0.34 — sd: standard deviation; iv: intravenous; sc: subconjunctival; io: intraocular; p1: comparison with control group; p2: comparison with diabetic group; p3: comparison with diabetic þ exosomes (iv) group; p4: comparison with diabetic þ exosomes (sc) group; p5: comparison with diabetic þ exosomes (io) group. a p � 0.05 is considered significant. table 2. comparison of serum insulin levels between different studied groups. insulin (miu/ml) healthy control (n ¼ 3) diabetic control (n ¼ 3) diabetic þ exosomes (iv) (n ¼ 3) diabetic þ exosomes (sc) (n ¼ 3) diabetic þ exosomes (io) (n ¼ 3) mean + sd 40.2 + 10.5 7.0 + 1.9 34.5 + 10.9 30.8 + 2.5 29.3 + 2.6 p1 — 0.00a 0.16 0.013a 0.00a p2 0.00 a — 0.00 a 0.00 a 0.00 a p3 0.16 0.00a — 0.23 0.25 p4 0.013 a 0.00 a 0.23 — 0.47 p5 0.00a 0.00a 0.25 0.47 — sd: standard deviation; iv: intravenous; sc: subconjunctival; io: intraocular; p1: comparison with control group; p2: comparison with diabetic group; p3: comparison with diabetic þ exosomes (iv) group; p4: comparison with diabetic þ exosomes (sc) group; p5: comparison with diabetic þ exosomes (io) group. ap � 0.05 is considered significant. figure 1. ultrastructure of exosomes. exosomes isolated from mscs. they are heterogeneous in size, ranging from 30 nm to 100 nm. they are directly frozen and observed by cryo-electron microscopy without chemical fixation or contrasting. exosomes appear round and are visualized with improved resolution (arrows). scale bar 100 nm. mscs: mesenchymal stem cells. figure 2. (a) western blot scanning densitometry of cd81 versus b-actin protein levels in mscs-derived exosomes in different passages. lane 1: passage 1, lane 2: passage 2, lane 3: passage 3. (b) mean of ratio of cd81 versus b-actin protein levels in mscs-derived exosomes in different passages. cd81 showed a significant increased level in passage 2 when compared to passage 1 and significant elevated levels in passage 3 when compared to passage 2. p1: passage 1; p2: passage 2, and p3: passage 3. mscs: mesenchymal stem cells. 4 journal of circulating biomarkers quantitative expression of cd81 against b-actin showed a significant increase in passage 2 when compared to passage 1 (2.43 + 0.41 vs. 1.44 + 0.19, p < 0.0001) and a significant increase in passage 3 when compared to passage 2 (3.37 + 0.40 vs. 2.43 + 0.41, p < 0.0001) (figure 2(b)). evaluation of exosomal uptake by retinal tissue exosomes labeled with pkh26 dye, red fluorescence chromophore inserting into the lipid bilayers of exosomes, were incorporated in degenerated retinal tissue which became clearly visible under fluorescent microscopy as shown in figure 3. histopathological results histological examination of retina tissue of induced diabetic rabbit (positive control) demonstrated the presence of abnormal thickness of all retina layers (ganglionic layer (gl), inner plexiform layer (ipl), outer plexiform layer (opl), outer nuclear layer (onl)) with well-observed degenerative areas in both gl and ipl (figure 4(a)). furthermore, other retina tissue showed well-observed hemorrhage in ipl and onl (figure 4(b)). toward the end of experimental duration, retina tissue of that group showed complete damage of gl and ipl with wellobserved hemorrhage and abnormal thickness of all other retinal layers (figure 4(c)). following sc microvesicles (mvs) injection (sc group), retina tissue showed wellorganized retinal layers with well-observed vacuoles in onl, gl, and ipl (figure 5(a)). moreover, at the end of experimental duration, retinal tissue showed normal architecture (figure 5(b)). following io mvs injection (io group), retina tissue showed degeneration and vacuoles in gl and ipl (figure 6(a)). these findings were markedly improved after the third dosage posttreatment (figure 6(b)). however, retina after being iv injected with exosomes (iv group) demonstrated irregular gl with increased thickness of all other layers (figure 7). gene expression analysis of micrna-222 level to analyze the therapeutic effect of mscs exosomes in regeneration of induced diabetic retinal tissue damage, relative gene expression levels of micrna-222 in retina were investigated by rt-pcr in different groups of our study (table 3 and figure 8). it was found that micrna222 was significantly decreased in diabetic control group compared to each of healthy controls, diabetic with iv injected exosomes, diabetic with sc and diabetic with io administrated exosomes groups (0.06 + 0.02 vs. 0.51 + 0.07, 0.28 + 0.08, 0.48 + 0.06, and 0.42 + 0.11, respectively). p values were <0.001 in relation of diabetic control group to each of the previous other groups, respectively. it was found that the level of micrna-222 was significantly figure 3. mscs-derived exosomes labeled with pkh26 under fluorescent microscopy. mscs: mesenchymal stem cells. figure 4. (a) photomicrograph of retina tissue of positive control (diabetic eye) showing abnormal thickness of all retina layers (gl, ipl, opl, onl) with well-observed degenerative areas in both gl and ipl, h&e �100. (b) photomicrograph of retina tissue of positive control (diabetic eye) showing hemorrhage in ipl and onl, h&e �100. (c) photomicrograph of retina tissue of positive control (diabetic eye) showing complete damage of gl and ipl layer with well-observed hemorrhage and abnormal thickness of all other retinal layers, h&e �100. gl: ganglionic layer; ipl: inner plexiform layer; opl: outer plexiform layer; onl: outer nuclear layer; oll: outer limited layer rod and cone; h&e: hematoxylin and eosin. safwat et al. 5 elevated in diabetic group with sc injected exosomes than diabetic iv injected exosomes group (0.48 + 0.06 vs. 0.28 + 0.08, p < 0.001). also, we detected that the level of micrna-222 in diabetic group associated with io exosomes was significantly increased than diabetes with iv exosomes (0.42 + 0.11 vs. 0.28 + 0.08, p < 0.001). however, there was no increase in relative micrna-222 level in diabetic group with io exosomes than in diabetic group with sc route of injected exosomes (0.42 + 0.11 vs. 0.48 + 0.06). the increased expression level of retinal micrna-222 in healthy controls in relation to other diabetic groups involved in the study (diabetic controls, diabetic with iv, sc, and io routes of administrated exosomes) indicated the role of high glucose conditions in reducing micrna-222 expression level. there is a significant negative correlation between serum glucose concentrations and gene expression levels of micrna-222 (r ¼ �0.749, p ¼ 0.001) (figure 9) and a significant positive correlation between serum insulin and micrna-222 levels (r ¼ 0.736, p ¼ 0.002) (figure 10) in different groups of study. discussion the reestablishment of damaged, lost, and aging tissues in the human body is the main target of regenerative medicine. various types of stem cells have been tested in both animal models and human clinical studies for its utilization in tissue regeneration. 25 the current study demonstrates that systemic, sc and io administration of rabbit adipose mscs exosomes can induce repair of diabetic retinal degeneration in a model of rabbits. dr is a common complication of diabetes which if left untreated can lead to loss of vision. 26,27 hyperglycemia impairs retinal microvasculature, which leads to increased permeability, blood leakage to the extravascular space, figure 6. (a) photomicrograph of retinal tissue after io mvs injection showing degeneration and vacuoles in gl and ipl, h&e �100. (b) photomicrograph of retinal tissue after io mvs injection showing well-defined components of retinal layer, h&e �100. gl: ganglionic layer; ipl: inner plexiform layer; h&e: hematoxylin and eosin; mvs: microvesicles; io: intraocular. figure 5. (a) photomicrograph of retina tissue of sc mvs–injected groups showing well-organized retinal layers with well-observed vacuolation in onl, gl, and ipl, h&e �100. (b) photomicrograph of retina tissue of sc mvs–injected groups at the end of experimental duration with retinal tissue showing normal architecture, h&e �100. gl: ganglionic layer; ipl: inner plexiform layer; onl: outer nuclear layer; oll: outer limited layer rod and cone; inl: inner nuclear layer; h&e: hematoxylin and eosin; sc: subconjunctival; mvs: microvesicles. 6 journal of circulating biomarkers continuous decrease in retinal blood flow, and closure of these microvessels. 2 many studies are supporting the role of mscs in promoting tissue regeneration involving retinal degenerative diseases 11 without the need of cellular differentiation. the effect of mscs-dependent therapy is mostly due to the paracrine secretion of trophic factors, exosomes. 28–30 exosomes have therapeutic effects in mediating tissue repair, through its role in the transport of specific mrna from one cell to other cells. this leads to the production of multiple proteins from each mrna that is transported to these cells. 31 furthermore, it was detected that mscs-derived exosomes contain other regulatory elements, micrnas. 32 many studies demonstrated that the released micrnas from exosomes between cells can directly inhibit or stimulate the transcription of target genes and lead to differential expression of these protein-coding genes which may be involved in different biological processes. 20,33–37 therefore, exosomes act as a very powerful tool by which the cells can transport messages to others. 31 in our study, we proved the existence of exosomes through measurement of cd81 by western blot assay. cd81 is one of tetraspan proteins family, cd9, cd63, cd81, and cd82. the membranes of exosomes are very rich in cd81 due to their endosomal origin. 38 as the expansion of mscs was done until reaching third passage, we detected that cd81 showed a significant increased level in the second passage than in the first passage. also, the relative quantitation of cd81 to b-actin protein was significantly elevated in the third passage compared to second and first passages, and these results confirm that the isolated molecules from mscs-conditioned media are exosomes. the current study showed the presence of well-observed edema, complete damage of retina with detached sclera from underlying muscles and extensive hemorrhage in retinal layers in diabetic controls by histological examination. by iv administration of rabbit adipose mscs exosomes at 4, 8, and 12 weeks following stz injection, induced irregular gls with increased thickness of retina appeared. however, sc stem cell–derived exosomes injection restored the cellular components of the retina which were organized in well-defined layers. also, our results showed that io exosomes infusion encouraged the regeneration of retina which appeared as well-defined retinal layers similar to layers of normal retina, and that occurred over a period of 12 weeks. these results indicate the vital therapeutic role of mscs-derived exosomes in regenerative changes in retinal tissue. however, the duration of up to 12 weeks was chosen in many studies where this period was found to be sufficient to detect a complete regenerative response in a model of rats. 32,39 it is worth to be mentioned that human mscs-derived exosomes do not have major histocompatibility complex proteins of classes i and ii as it was detected by previous studies, so we can utilize exosomes derived from human mscs in the future without the need of induction of immunosuppression. 32–34 in the current study, we revealed that micrna-222 has a highly significant reduced expression level in retinal tissue of diabetic control group compared with other groups administrated with mscs exosomes as well as the healthy controls. we detected a significant negative association between the expression level of micrna-222 and serum glucose levels in the groups of our study. so we can suspect that hyperglycemia leads to decreased micrna-222 expression. previous studies showed that hyperglycemic conditions cause reduction of micrna-222 expression in microparticles derived from endothelial cells in coronary artery disease. 40,41 it has been detected that micrna-222 acts as a critical modulator of vascular smooth muscle cells proliferation by targeting p27kip1 and p57kip2 genes. 42 moreover, micrna-222 can inhibit neovascularization which may occur in advanced stages of dr by regulating stat5a protein expression. 20 these effects of micrna222 appear to be specific to endothelial cells and vascular smooth muscle cells. 43 it was found that micrna-222 is highly expressed in endothelial cells present in normal vessels, while decreased expression levels were detected in endothelial cells of atherosclerotic blood vessels exposed to inflammatory stimuli. the increased numbers of new vessels with low micrna-222 expressing atherosclerotic plaques were determined. 20 so this study could demonstrate for the first time, to our knowledge, the important role of exosomal shuttled micrna-222 in retinal tissue repair. reduced micrna222 expression levels in retinal tissue of stz-induced diabetes were associated with the development of severe retinal damage and extensive hemorrhage in different layers of retina of this group by histopathological figure 7. photomicrograph of retinal tissue after iv mvs injection showing well-defined layers with increase in all layer thickness (ipl, inl, onl, and oll) and losing their regular form, h&e �100. ipl: inner plexiform layer; inl: inner nuclear layer; onl: outer nuclear layer; oll: outer limited layer rod and cone; h&e: hematoxylin and eosin; mvs: microvesicles; iv: intravenous. safwat et al. 7 examination. furthermore, exosomal-mediated transfer of micrna-222 by iv, sc, and io routes caused increased expression level of micrna-222 in retinal tissue of these groups and these results were associated with regenerative changes of retina we detected. thus, we can provide evidence that micrna-222 has a crucial role in retinal tissue regeneration which may occur via its effect in regulation of vascular cell biology. 40 in conclusion, adipose mscs-derived exosomes act as biological mediators in retinal tissue repair through its role in delivering micrna-222 to retinal cells. so exosomes containing micrnas may provide a novel therapeutic procedure to prevent or delay the dr which is a major complication of dm and may lead to severe impairment of vision. however, further studies are needed to know the mechanism by which hyperglycemia caused decrease in expression level of micrna-222 and how administration figure 9. correlation of plasma micrna-222 expression level with serum glucose in all studied groups. table 3. comparison of quantitative micrna-222 gene expression between different groups. micrna222 healthy control (n ¼ 3) diabetic control (n ¼ 3) diabetic þ exosomes (iv) (n ¼ 3) diabetic þ exosomes (sc) (n ¼ 3) diabetic þ exosomes (io) (n ¼ 3) mean + sd 0.51 + 0.07 0.06 + 0.02 0.28 + 0.08 0.48 + 0.06 0.42 + 0.11 p1 — 0.00 a 0.00 a 0.33 0.015 a p2 0.00a — 0.00a 0.00a 0.00a p3 0.00 a 0.00 a — 0.00 a 0.00 a p4 0.33 0.00a 0.00a — 0.12 p5 0.015 a 0.00 a 0.00 a 0.12 — sd: standard deviation; iv: intravenous; sc: subconjunctival; io: intraocular; p1: comparison with control group; p2: comparison with diabetic group; p3: comparison with diabetic þ exosomes (iv) group; p4: comparison with diabetic þ exosomes (sc) group; p5: comparison with diabetic þ exosomes (io) group. a p � 0.05 is considered significant. figure 8. relative quantitation of micrna-222 expression levels in all studied groups. micrna-222 showed marked decrease in diabetic group with retinal degenerative changes in comparison to other groups involved in the study. 8 journal of circulating biomarkers of exosomes was associated with reduced serum glucose levels. also, other components of evs are needed to be explored in each of diabetic and healthy subjects to detect their mechanisms of action and to study the 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476–487. 43. liu x, cheng y, yang j, et al. cell-specific effects of mir221/222 in vessels: molecular mechanism and therapeutic application. j mol cell cardiol 2012; 52(1): 245–255. 10 journal of circulating biomarkers << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice jcb j circ biomark 2023; 12: 12-16issn 1849-4544 | doi: 10.33393/jcb.2023.2453original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu infection, or lesions identified on a surveillance ultrasound in patients with cirrhosis, chronic hbv infection, or prior hcc (2). the main two markers for hcc are alpha-fetoprotein (afp) and new biomarker: protein induced by vitamin k absenceii or antagonist (pivka-ii). these markers play an important role in diagnosis, differential diagnosis, and prognosis. a nomogram of both afp and pivka-ii can be used to differentiate hcc and intrahepatic cholangiocarcinoma with an area under the receiver operating characteristic (roc) curve of 95.1% (3). both biomarkers can be used as a prognostic factor after several treatments such as radiofrequency, surgical resection, embolization, or radiotherapy (4-12). pivka-ii level lower than 25 mau/ml or equal after radiotherapy had long progression-free survival (p = 0.004) (4). additionally, both biomarkers can indicate tumor size, tumor differentiation, vascular invasion, and those treated with hepatitis b/c antiviral agents (13-16). as hcc is a lethal cancer with 5-year survival rate of under 15%, abdominal ultrasonography is used for hcc surveillance (17,18). however, its sensitivity was low at 63% (19). biomarkers were studied and used as an alternative pivka-ii or afp has better diagnostic properties for hepatocellular carcinoma diagnosis in high-risk patients tanita suttichaimongkol1, manoon mitpracha2, kawin tangvoraphonkchai1, phuangphaka sadee1, kittisak sawanyawisuth1, wattana sukeepaisarnjaroen1 1department of medicine, faculty of medicine, khon kaen university, khon kaen thailand 2division of gastroenterology, department of medicine, khon kaen regional hospital, khon kaen thailand abstract background: hepatocellular carcinoma (hcc) is a lethal cancer. two biomarkers were used for hcc diagnosis including alpha-fetoprotein (afp) and protein induced by vitamin k absence-ii or antagonist (pivka-ii). however, data on biomarkers and hcc diagnosis are not consistent. this study aimed to evaluate if pivka-ii, afp, or a combination of both biomarkers had the best diagnostic properties for hcc. methods: this was a prospective study and enrolled patients 18 years or over with a high risk for hcc. afp and pivka-ii levels were calculated for hcc diagnosis. diagnostic properties of both biomarkers were reported with sensitivity, specificity, and a receiver operating characteristic (roc) curve. results: there were 260 patients with high risk for hcc in this cohort. of those, 219 patients were diagnosed with hcc: confirmed by biopsy in 7 patients (2.69%) and by imaging in the others. median values of afp and pivka-ii were 56 ng/ml and 348 mau/ml, respectively. pivka-ii level of 40 mau/ml had sensitivity of 80.80%, while afp of 10 ng/ml had sensitivity of 75.80%. a combination of pivka-ii at 100 mau/ml or over and afp of 11 ng/ml gave sensitivity of 60.30%. the roc curve of pivka-ii plus afp was significantly higher than the afp alone (0.855 vs. 0.796; p = 0.027), but not significantly different from the pivka-ii alone (0.855 vs. 0.832; p = 0.130). conclusion: pivka-ii may have more diagnostic yield for hcc compared with afp. it can be used alone without a combination with afp. keywords: hepatitis b virus, hepatitis c virus, sensitivity, specificity received: july 8, 2022 accepted: february 1, 2023 published online: february 17, 2023 corresponding author: kittisak sawanyawisuth department of medicine, faculty of medicine khon kaen university khon kaen, 40002 thailand kittisak@kku.ac.th introduction hepatocellular carcinoma (hcc) is a common cancer. it is the sixth most common cancer and the second highest in cancer-related death globally. in 2015, there were 854,000 new cases and 810,000 deaths in the world (1). there were three common causes of hcc: hepatitis b virus (hbv), alcohol, and hepatitis c virus (hcv). these causes related to hcc deaths in 33%, 30%, and 21%, respectively (1). high-risk patients for hcc were patients with cirrhosis, chronic hbv https://doi.org/10.33393/jcb.2023.2453 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:kittisak@kku.ac.th suttichaimongkol et al j circ biomark 2023; 12: 13 © 2023 the authors. published by aboutscience www.aboutscience.eu diagnostic method particularly for hcc including afp and pivka-ii (20-22). the advantage of afp is widely available, while pivka-ii is highly specific to hcc. data on biomarkers and hcc diagnosis are not consistent. a report from the usa found that afp was more sensitive than pivka-ii (70% vs. 66%) in patients with hcc compared with patients with cirrhosis (23). on the other hand, a study from nigeria found that pivka-ii was more sensitive than afp (96.8% vs. 62.9%) in patients with hcc vs. control with benign liver disease (24). a study from korea recommends to use pivka-ii combined with afp to diagnose hcc in patients with hbv infection (25). these results showed that data on biomarkers and hcc diagnosis are not consistent and varied among countries, in which more data on this issue are required. this study aimed to evaluate if pivka-ii, afp, or a combination of both biomarkers had the best diagnostic properties for hcc. materials and methods this was a prospective study conducted at university hospital, khon kaen university, thailand. data were collected from the hcc project. the inclusion criteria were patients 18 years or over with a high risk for hcc. the high risk for hcc was defined by the american association for the study of liver diseases (aasld) guidelines for hcc management (17): cirrhosis or presence of liver nodule(s) of 1 cm or over in size. those with pregnancy, obstructive jaundice, vitamin k, or warfarin administration and presence of extrahepatic malignancy were excluded. the study protocol was approved by the ethics committee in human research, khon kaen university, thailand (he621134). this study was a part of hcc project of khon kaen university, thailand. eligible patients provided a written informed consent prior to study participation. data were collected as follows: baseline characteristics, laboratory results, and radiographic findings. baseline characteristics included age, sex, etiology of cirrhosis, comorbid diseases, and the child-pugh score for cirrhosis. laboratory tests in the study were platelet count, serum creatinine, prothrombin time, liver function test, afp, and pivka-ii. all samples were tested for afp and pivka-ii by using a test kit (µtaswako i30; fujifilm wako pure chemical corporation). radiographic findings were numbers of liver mass, largest mass size (cm), and portal vein invasion. hcc was diagnosed by either confirmation by pathological findings or radiographic findings of arterial hypervascularity followed by venous and/or delayed phase or washout of contrast (17). statistical analyses descriptive statistics were used to calculate mean (1st3rd interquartile ranges) or number (percentage) of the study population. afp and pivka-ii levels were calculated for hcc diagnosis by logistic regression analysis. results were shown as various cutoff points with their diagnostic properties including sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, a roc curve, and an area under the roc curve with 95% confidence interval (ci). all statistical analyses were performed using stata software version 10.1. results there were 260 patients with a high risk for hcc in this cohort. of those, 219 patients were diagnosed with hcc: confirmed by biopsy in 7 patients (2.69%) and by imaging in the others. the other 41 patients had a diagnosis of dysplastic nodule (25 patients; 60.98%), regenerative nodule (8 patients; 19.51%), hemangioma (5 patients; 12.20%), liver cyst (1 patient; 2.44%), fibronodular hyperplasia (1 patient; 2.44%), and hepatic adenoma (1 patient; 2.44%). baseline characteristics and laboratory results are shown in tables i and ii. the median age was 58 years with male predominance (81.15%). the most common cause of hcc was hcv (43.85%) with a proportion of cirrhosis of 98.46%: mostly child-pugh score class a (81.15%). the median largest size of liver mass was 3.5 cm. the median values of afp and pivka-ii were 56 ng/ml and 348 mau/ml, respectively. the pivka-ii level of 40 mau/ml had sensitivity of 80.80% with 75.60% specificity, while afp of 10 ng/ml had sensitivity of 75.80% with 65.90% specificity (tabs. iii and iv). a combination of pivka-ii at 100 mau/ml or over and afp of 11 ng/ml gave sensitivity of 60.30% with 92.70% specificity. the roc curve of pivka-ii plus afp was significantly higher than the afp alone (0.855 vs. 0.796; p value = 0.027), but not significantly different from pivka-ii alone (0.855 vs. 0.832; p value = 0.130), as shown in figure 1. table i baseline characters of patients with a high risk for hepatocellular carcinoma (n = 260) factors median (1st-3rd quartile) or number (percentage) age, years 58 (54-63) male sex, n (%) 211 (81.15) etiology hbv 98 (37.69) hcv 114 (43.85) hbv plus hcv 6 (2.31) nafld 14 (5.38) ald 27 (10.38) aih 1 (0.38) comorbid diseases none 193 (74.23) diabetes 38 (14.62) hypertension 12 (4.62) cirrhosis 256 (98.46) child-pugh score a 211 (81.15) child-pugh score b 36 (13.85) child-pugh score c 9 (3.46) aih = autoimmune hepatitis; ald = alcoholic liver disease; hbv = hepatitis b virus; hcv = hepatitis c virus; nafld = nonalcoholic fatty liver disease. pivka-ii and afp in hcc14 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table ii laboratory results of patients with a high risk for hepatocellular carcinoma (n = 260) factors median (1st-3rd quartile) or number (percentage) platelet ´103/mm3 145 (102-220) creatinine (mg/dl) 0.94 (0.80-1.15) prothrombin time (sec) 12.3 (11.5-13.4) albumin (g/dl) 3.9 (3.4-4.4) bilirubin (mg/dl) 1.0 (0.6-1.6) alanine aminotransferase (u/l) 51 (29-86) aspartate transaminase (u/l) 74 (40-133) alkaline phosphatase (u/l) 135 (88-196) alpha-fetoprotein (ng/ml) 59 (6-1,598) pivka-ii (ma/ml) 348 (31-10,442) radiographic findings number of liver nodules, n (%) 1 156 (90.00) 2 46 (17.69) 3 12 (4.62) ≥4 46 (17.69) largest size (cm) 3.5 (1.8-7.5) portal vein invasion, n (%) no invasion 181 (69.62) main portal vein invasion 56 (21.54) non-main portal vein invasion 23 (8.85) pivka-ii = protein induced by vitamin k absence-ii or antagonist. table iii performance characteristics of protein induced by pivka-ii (mau/ml) for diagnosing hcc in patients with a high risk for hcc cutoff sensitivity specificity lr+ lr≥40 80.80 (75.00-85.80) 75.60 (59.70-87.60) 3.31 (1.93-5.70) 0.25 (0.18-0.35) ≥60 78.50 (72.50-83.80) 80.50 (65.10-91.20) 4.03 (2.15-7.52) 0.27 (0.20-0.36) ≥80 75.30 (69.10-80.90) 82.90 (67.90-92.80) 4.41 (2.24-8.70) 0.30 (0.23-0.39) ≥100 72.10 (65.70-78.00) 87.80 (73.80-95.90) 5.92 (2.59-13.50) 0.32 (0.25-0.40) ≥120 69.40 (62.80-75.40) 87.80 (73.80-95.90) 5.69 (2.49-13.0) 0.35 (0.28-0.44) hcc = hepatocellular carcinoma; lr+ = positive likelihood ratio; lr= negative likelihood ratio; pivka-ii = vitamin k absence-ii or antagonist. table iv performance characteristics of afp (ng/ml) for diagnosing hcc in patients with a high risk for hcc cutoff sensitivity specificity lr+ lr≥10 75.80 (69.60-81.30) 65.90 (49.40-79.90) 2.22 (1.44-3.42) 0.37 (0.27-0.51) ≥12 73.10 (66.70-78.80) 70.70 (54.50-83.90) 2.50 (1.54-4.04) 0.38 (0.28-0.51) ≥14 69.40 (62.80-75.40) 73.20 (57.10-85.80) 2.59 (1.55-4.32) 0.42 (0.32-0.55) ≥20 65.30 (58.60-71.60) 78.00 (62.40-89.40) 2.97 (1.66-5.34) 0.45 (0.35-0.57) ≥200 44.70 (38.00-51.60) 90.20 (76.90-97.30) 4.59 (1.79-11.80) 0.61 (0.52-0.72) afp = alpha-fetoprotein; hcc = hepatocellular carcinoma; lr+ = positive likelihood ratio; lr= negative likelihood ratio. fig. 1 receiver operating characteristic (roc) curves and their area under the roc curves (95% confidence interval) of protein induced by vitamin k absence-ii or antagonist (pivka-ii), alpha-fetoprotein (afp), and the combination of both biomarkers to predict hepatocellular carcinoma (hcc) in patients with a high risk for hcc. discussion this cohort found that pivka-ii was more sensitive for hcc diagnosis than afp and can be used alone without a combination with afp. the results of this study were comparable with two previous studies to detect hcc in cirrhosis patients with liver nodules of 1 cm or over (26-28). note that this study had larger study population than the other two studies (260 vs. 128 vs. 90). with larger sample size in this study, pivka-ii was more sensitive to detect hcc than afp (80.80% vs. 75.80%) by the cutoff point of 40 mau/l and 10 ng/ml, respectively. compared with the study from france, the sensitivity of pivka-ii was comparable (80.80% vs. 77%) as well as the cutoff point (40 vs. 42 mau/l). however, this study had different results compared with the us study (23), in which afp had suttichaimongkol et al j circ biomark 2023; 12: 15 © 2023 the authors. published by aboutscience www.aboutscience.eu better sensitivity than pivka-ii in hcc with a sample size of 208 patients. afp at 10.9 ng/ml had higher sensitivity at 66% compared with 56% sensitivity of pivka-ii at 221.5 mau/ml. these differences may be due to different control. in the previous study, patients with cirrhosis without liver mass served as controls, while non-hcc patients were cirrhotic patients with liver mass in this study. another possible explanation is the property of pivka-ii, which is an indicator of microvascular invasion (26). high pivka-ii of over 90 mau/ml had higher risk of microvascular invasion by 3.5 times (95% ci 1.08, 11.8; p value 0.043). in this study, 30.38% of patients had portal vein invasion, which may be an indicator of microvascular invasion (tab. ii). in this study, we found that pivka-ii can be used for hcc diagnosis without a need to combine with afp. these results were different from the study from korea (25). the areas under the roc curve of these combinations were significantly different from pivka-ii alone (0.912 vs. 0.870) or afp alone (0.902 vs. 0.812) for those with liver cirrhosis in the previous study. once again, these differences may be due to different study population. the previous study enrolled patients with hbv infection and categorized into three groups: non-cirrhotic hbv infection, cirrhosis without hcc, and hcc group (no data whether cirrhosis or not). additionally, the cirrhosis group in the previous study may or may not have liver nodules like in this study. note that hbv infection was accounted in only 37.69% in this study. another explanation is different cutoff points for pivka-ii and afp. the cutoff points for these two markers in the previous study were 40 mau/ml and 25 ng/ ml, while the cutoff points in this study were 100 mau/ml and 11 ng/ml. note that a combination of these two biomarkers had lower sensitivity but higher specificity. even though both afp and pivka-ii are useful diagnostic markers for hcc, a previous report found that they may not be a good marker for small hcc nodules less than 2 cm as they have sensitivity of approximately 50% (29). however, they may be used for hcc detection particularly in hepatitis virus-related hcc (22,30). a study from china found similar findings as this study but different cutoff for both afp and pivka-ii (31). a combination of afp and pivka-ii model is better than afp alone but comparable with pivka-ii alone. therefore, pivka-ii may be used alone without a combination with afp to diagnose hcc. compared with benign liver disease, this study had pivka-ii and afp cutoff points at 40 mau/ml and 10 ng/ml while the chinese study had cutoff points of 43.47 mau/ml and 21.47 ng/ml for pivka-ii and afp. the different cutoff points may be from different study population and sample size. this study had larger sample size and most patients (80%) had hbv or hcv as a cause, while the chinese study did not show causes of hcc. another study conducted with liver cirrhosis as a control group also found that similar findings of pivka-ii alone were comparable with a combination of pivka-ii and afp for hcc diagnosis (32). there are some limitations in this study. first, etiologies of cirrhosis in this study are varied; hcv was the most common cause (43.85%) followed by hbv infection (37.69%). the results of this study may not be applicable for other countries with different causes of cirrhosis or hcc. second, we used different cutoff points for a combination of pivka-ii and afp as discussed earlier. third, some associated factors with hepatitis virus or fatty liver such as sleep apnea were not studied (33-38). no predictor for hcc was studied as well as systematic review (39-42). finally, note that control group in this study were those with high risk for hcc: presence of liver mass of 1 cm or more in size. as this study enrolled highrisk patients for hcc with various causes, various child-pugh score classification, various liver nodule sizes, these may lead to possible selection biases. further studies are necessary before considering these biomarkers even for a general evaluation of the hcc diagnosis. in conclusion, pivka-ii may have more diagnostic yield for hcc compared with afp. it can be used alone without a combination with afp. acknowledgments the authors would like to thank the division of research and graduate studies, and the fundamental fund of khon kaen university, khon kaen, thailand. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. authors contribution: all authors 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www.aboutscience.eu/jcb © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu and magnetic resonance imaging (mri). these techniques are costly, and their efficiency and accuracy in treatment evaluation may strongly affect patient outcomes (3). measuring differences in serum tumor markers has been established as a tool for evaluation of therapy effectiveness of different cancers (4,5). cancer antigen 15-3 (ca 15-3) and carcinoembryonic antigen (cea) are the most frequent serum tumor markers used for breast cancer, even though their functionality remains controversial (4,6). breast cancer markers are applied for therapy response speculations, after initial therapy observation, and as prognostic indicators. cea is expressed in the majority of human lung, pancreatic, and gastric cancers, in addition to breast carcinoma (7). measurements of cea in breast cancer are suggestive of lymph node involvement and tumor size. consequently, cea concentrations above 7.5 μg/l are linked to a higher possibility of subclinical metastases (8). the normal range of cea levels was connected to a significantly better prognosis of patients at the time of diagnosis compared to those with elevated levels (9). studies propose cea as a useful marker for monitoring treatment response including chemotherapeutic ones (10-12). diagnostic impact of cea and ca 15-3 on chemotherapy monitoring of breast cancer patients diya hasan department of allied medical sciences, zarqa college, al-balqa applied university, zarqa jordan abstract introduction: serum tumor markers have emerged as an effective tool to determine prognosis and treatment efficiency in different cancer types. this study aimed to explore the chemotherapy monitoring efficiency and prognostic sensitivity of tumor-associated cancer antigen 15-3 (ca 15-3) and carcinoembryonic antigen (cea) in early (ii) and late (iv) clinical stage breast cancer. methods: ca 15-3 and cea serum levels were assessed in 56 breast cancer patients at early (n = 26) and late (n = 30) clinical stages with these primary inclusion criteria: those who received adjuvant chemotherapy ac (adriamycin and cyclophosphamide) or ac-t (adriamycin and cyclophosphamide followed by taxane) regimens and possessed tumors negative for human epidermal growth factor receptor 2 (her2) based on a particleenhanced turbidimetric assay. results: ca 15-3 had a higher elevation than cea in the pretreatment group of breast cancer patients when compared to healthy controls. late-stage patients showed higher positive serum levels than early-stage patients for both markers, with a preference for ca 15-3 over cea. ac-t chemotherapy regimen treatment in both clinical stages revealed a significantly higher level of both markers as compared to the ac regime, with a preference for ca 15-3 over cea in late stage. both markers were significantly higher in the late-stage group as compared to early-stage groups for both chemotherapy regimens. conclusions: ca 15-3 is more efficient as a prognostic monitoring marker than cea and reveals a positive connection between chemotherapy regimen system and staging, with increased observability in late-stage patients. keywords: breast cancer, ca 15-3, cea, chemotherapy, prognosis received: june 29, 2022 accepted: october 3, 2022 published online: november 7, 2022 corresponding author: dr. diya hasan amman, gardenz street 0795071521 amman jordan diyahasan@bau.edu.jo introduction breast cancer is the most common cancer among jordanian females, accounting for 22.4% of cancer cases (1). after being diagnosed with breast cancer, the patient treatment plan includes a combination of surgery, radiation, hormone therapy, and chemotherapy. disease progress is evaluated according to consistent measures (2) based on alterations in the size of the quantifiable lesions. during chemotherapy treatment, metastatic breast cancer is generally examined using imaging techniques such as positron emission tomography (pet), computed tomography (ct), https://doi.org/10.33393/jcb.2022.2446 https://creativecommons.org/licenses/by-nc/4.0/legalcode chemotherapy monitoring by serum markers58 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb ca 15-3 (muc1) is a cell surface glycoprotein derived from the muc1 gene. it’s expressed on the surface of various epithelial cell types and overexpressed in 90% of breast cancer cases (13). the elevated level of ca 15-3 is used to determine the relapse potential of breast cancer patients, and as a tool for therapeutic response evaluation at late stages (14). ca 15-3 preoperative concentrations of early breast cancer patients have a notable relation to predict their outcomes (15). using serum tumor markers (ca 15-3 and cea) permits the early identification of up to 60-80% of breast cancer patient metastasis (16). the serum levels of ca 15-3 and cea were shown to be beneficial in the management of breast cancer patients and could aid as prognostic indicators and for observing disease development (17). this study investigated the clinical importance of serum tumor markers ca 15-3 and cea for monitoring jordanian breast cancer patient responses to different chemotherapy regimens and their correlation with different clinical stages in addition to their prognostic value sensitivity. materials and methods patient cohort fifty-six female breast cancer patients were involved in this study using these main inclusion criteria: (1) human epidermal growth factor receptor 2 (her2)-negative and (2) received adjuvant chemotherapy regimen ac (adriamycin and cyclophosphamide) or ac-t (adriamycin, cyclophosphamide, taxane). table i shows the patient characteristics. patients were categorized as follows: 26 patients (46.4%) were graded as stage ii and 30 patients (53.5%) were graded as stage iv, patients who did not meet the criteria in table 1 were excluded. stage ii or less was considered as early stage and stage ii and above was considered as late stage as stated by the american joint committee on cancer (ajcc) staging system (18). the median age between the two groups did not show any significant difference (p = 0.232). table i patient features and treatment method patient parameter n (%) age (years) median 49 range 43-55 gender female clinical stage  stage ii 26 (46.4%)  stage iv 30 (53.5%) chemotherapy regime  ac × 4 29 (51.7%)  ac × 4 followed by t × 4 27 (48.2%) histological type idc her2 receptor negative a = adriamycin; c = cyclophosphamide; her = human epidermal growth factor receptor; t = taxane; idc = invasive ductal carcinoma. primary chemotherapy the first group of ac regimen consisting of 14 stage ii and 15 stage iv patients was treated with 4 cycles of adriamycin 50 mg/m2 and cyclophosphamide 1000 mg/m2 on day 1, which was repeated every 21 days. the second group of ac-t regimen consisting of 12 stage ii and 15 stage iv patients received the previous chemotherapy regimen ac, followed by 4 cycles of taxane 80 mg/m2 every 21 days. the flowchart of patients is presented in figure 1. fig. 1 flowchart of patients’ selection. ac (adriamycin & cyclophosphamide) or ac-t (adriamycin & cyclophosphamide followed by taxane) regimes treatments. (based on the oncology decision according to treatment guidelines). sample collection ethical approval for this study was acquired from al bashir hospital, amman, jordan (#3345), and written informed consent was collected from all patients. cea and ca 15-3 blood samples were gathered from patients for diagnosis after the third month of treatment protocol (fourth cycle). cea and ca 15-3 measurements the serum was isolated by centrifugation (2,500 rpm for 10 minutes) of patients’ blood samples. serum cea and ca 15-3 levels were determined using an electrochemiluminescence immunoassay system (modular analytics e170, cobas e601; roche, germany): a particle-enhanced turbidimetric assay for cea and immunoturbidimetric assay for ca 15-3. marker assays were done using the commercial kits for cea (elecsys cea, cobas, roche, germany) and ca 15-3 (elecsys ca 15-3, cobas, roche, germany). a cut-off point of <5.0 μg/l (cea) and <25.0 u/ml (ca 15-3) was used as indicated by the roche diagnostic kit brochure. the cea and ca 15-3 readings of 20 healthy females (con −) with inclusion criteria–does not have any type of cancer or chronic diseases, age ≥18 years, not on any type of medication, and 16 prechemotherapy breast cancer female patients (con +)—were obtained from bio-lab laboratories. statistical analysis statistical analysis was performed using spss software. a t-test and fishers test were performed to find out possible hasan j circ biomark 2022; 11: 59 © 2022 the authors. published by aboutscience www.aboutscience.eu marker-level variations between target groups. to see if the differences in proportions were statistically significant, the chi-square test was utilized. when possible, the odds ratio was utilized to assess the relationship. results this study was planned to determine the correlation between cea and ca 15-3 levels’ elevation significance on monitoring response to jordanian breast cancer female patients’ treatment with different chemotherapy regimens at early and late clinical stages. cea and ca 15-3 levels in breast cancer the cea and ca 15-3 serum levels were measured in all samples using elisa. the serum levels of cea (1.7 μg/l) and ca 15-3 (18.7 u/ml) were significantly increased (fig. 2; p = 0.0005 and p = 0.0001), respectively, in the pre chemotherapy group (con +) compared to the healthy group (con −): cea (1.09 μg/l) and ca 15-3 (8.7 u/ml). the presented data revealed differentiation between cea and ca 15-3 serum-level elevation of studied groups (fig. 2), as we observed a stronger increase of ca 15-3 level (fig. 2b) compared to cea level (fig. 2a). these results imply that cea and ca 15-3 levels can be used efficiently to anticipate breast cancer tendency and provide a convenient detection method for breast cancer with a preference for ca 15-3 over cea in sensitivity. positive serum levels of cea and ca 15-3 in all-inclusive patient samples, elevated positive serum levels found in early and late stages were identified as follows: for cea (ii ac 4/14 [29%]), (ii ac-t 4/11 [36%]), (iv ac 7/14 [50%]), and (iv ac-t 9/14 [64%]) of the breast cancer cases used the cut-off point <5.0 μg/l. as for ca 15-3 (ii ac 5/14 [35.7%]), (ii ac-t 7/11 [63.6%]), (iv ac 10/14 [71%]), and (iv ac-t 9/14 [64%]) of the breast cancer cases used the cut-off point <25 u/ml (fig. 3). in total 8/25 (32%) of stage ii and 16/28 (57%) of stage iv patients had higher levels of cea than the cut-off point, while for ca 15-3 12/25 (48%) of stage ii and 19/28 (68%) of stage iv patients had higher levels of ca 15-3 than the cut-off point (fig. 3). in our study, a combined chemotherapy regimen demonstrated higher positive serumlevel percentages for both markers as compared to a single chemotherapy regimen in the early-stage patient group; this elevation was notably stronger for ca 15-3 in comparison to cea. the same result was obtained for cea in the latestage patient group; however, ca 15-3 behaved differently as positive serum levels were higher in a single chemotherapy regimen compared to a combined regimen. overall, the late-stage patient group showed higher positive serum-level percentages compared to the early-stage group for both markers with a preference for ca 15-3 over cea. these findings suggest that the serum levels of ca 15-3 might be more beneficial for observing chemotherapy response in advanced tumors than early diagnosis as compared with cea. fig. 2 difference in serum level between healthy non-cancer group (control –) and breast cancer patients groups before chemotherapy (control +) of (a) cea (b) ca 15-3 markers. (*significant increase differences for the serum levels of cea and ca 15-3 (****p = 0.0005 and ****p = 0.0001) respectively in pre-chemotherapy group (con +) compared to healthy ones (con –)). fig. 3 positive serum levels of carcinoembryonic antigen (cea) and cancer antigen (ca) 15-3 markers for both chemotherapy regimens (ac and ac-t) in early (ii) and late (iv) clinical stages. ac = adriamycin and cyclophosphamide; ac-t = adriamycin and cyclophosphamide followed by taxane. cea and ca 15-3 levels based on chemotherapy type furthermore, the marker serum-level response and its association with the type of chemotherapeutic treatments in both early and late stages were investigated. comparing the chemotherapy monitoring by serum markers60 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb cea levels between both treatment regimens ac (mean rank: 2.41) and ac-t (mean rank: 2.65) in early stage, the ac (mean rank: 8.16) and ac-t (mean rank: 17.45) in late stage showed no significant changes (p = 0.71 and p = 0.41), respectively (fig. 4a). conversely, the ca 15-3 levels behaved differently where it had shown a significant change (p = 0.056) comparing the ac-t (mean rank: 144.39) to ac (mean rank: 25.55) at late stage but not at early stage (p = 0.089) ac (mean rank: 25.00) and ac-t (mean rank: 43.27) (fig. 4b). this elevation in response to a combined chemotherapy regimen was observed to be stronger in the case of ca 15-3 in comparison to cea and more specifically in the late-stage patient group. significant change (p = 0.022) in stage iv ac but not in stage iv ac-t (p = 0.081) (fig. 5a). the ca 15-3 level comparison ([ii (mean rank: 25.00) and iv (mean rank: 49.35) stages of ac treatment], [ii (mean rank: 34.27) and iv (mean rank: 130.96) stages of ac-t treatment]) showed a significant change in stage iv compared to stage ii in both ac and ac-t treatments (p = 0.005 and p = 0.044), respectively (fig. 5b). fig. 4 effect of chemotherapy regime type ac or ac-t in both early (ii) and late (iv) clinical stages on serum level response of (a) cea and (b) ca 15-3 markers. (*significant increase for the ca 15-3 serum level (*p = 0.05) comparing the ac-t to ac at late-stage iv). cea and ca 15-3 levels based on clinical stage analysis was performed to check if the cea and ca 15-3 elevation response was associated with breast cancer progression in terms of different clinical stages (early and late) in each of the studied chemotherapeutic regimens. comparing cea levels between ii (mean rank: 2.41) and iv (mean rank: 5.26) stages of ac treatment, ii (mean rank: 2.65) and iv (mean rank: 5.83) stages of ac-t treatment, there was a fig. 5 effect of both clinical stages early (ii) and late (iv) in association with chemotherapy regime type ac or ac-t on serum level response of (a) cea and (b) ca 15-3 markers. (*significant increase for the cea serum level (*p = 0.02) for ac treatment at late-stage iv), (*significant increase for the ca 15-3 serum level in late stage iv in both ac and ac-t treatments (*p = 0.005 and* p = 0.044) respectively. the results revealed a significant connection between a change in marker level and clinical staging, as both cea and ca 15-3 were significantly elevated in the late-stage patient group; more so than in the early-stage group in both chemotherapeutic regimens (fig. 5). collectively, these data suggest that cea and ca 15-3 are predictive of chemotherapy response among different regimens throughout treatment and show differences between both early and late stages. discussion breast cancer is the most frequent cancer among jordanian women. this study evaluated the significance of hasan j circ biomark 2022; 11: 61 © 2022 the authors. published by aboutscience www.aboutscience.eu using ca 15-3 and cea for monitoring different chemotherapy regimens since assessing prognosis using diagnostic markers is believed to help in patients’ therapeutic response anticipation, which is vital for evaluating the course of therapy and to avoid the side effects of worthless and inefficient treatments. in the present study, ca 15-3 had shown a higher elevation as compared to cea as both markers were significantly elevated in breast cancer patients at the time of diagnosis in comparison to healthy controls. these results imply that both ca 15-3 and cea markers can efficiently predict breast cancer susceptibility and deliver benefit for breast cancer prognosis detection. the combination of both tumor markers (ca 15-3 and cea) is important in breast cancer (19). ca 15-3 has better prognostic significance in relation to cea (20). however, some studies have indicated that the prognostic value of ca 15-3 is lower than that of cea (21), which demonstrates marker contradictory association in breast carcinogenesis (22). some studies have described the changes of ca-15-3 and cea levels to be independent regardless of the breast cancer stage (23); however, our results showed that elevated serum levels of cea and ca 15-3 above the cut-off point were identified in 8 (32%) and 12 (48%) of early-stage patients, and 16 (57%) and 19 (68%) of late-stage patients. more notable serum levels were elevated in the late stage than in the early stage with a preference for ca 15-3 over cea. ca 15-3 and cea elevation levels have been described as connected with clinic pathological parameters including advanced tumor, node, metastasis (tnm) stage (24). the author analyzed the clinical impact of ca 15-3 and cea to breast cancer patients with different chemotherapy regimens and in terms of different tumor clinical stages. this study found that ca 15-3 and cea levels were shown to be higher in late stage and combined regimen as compared to early stage and single regimen, mainly with a preference for ca 15-3 at late stage over cea. these results suggest that ca 15-3 and cea serum levels can be an indicator for stage and chemotherapy regimen systems. additionally, they suggest the clinical importance of ca 15-3 for follow-up as a prognostic variable during chemotherapy treatment of breast cancer patients. ca 15-3 serum levels showed variations among breast cancer stages (9). ca 15-3 levels increase in all types of tumors; however, in breast cancer, it continues to increase as the tumor develops (25). studies have reported that alterations of tumor marker levels are associated with a patient’s therapeutic response, in addition to imaging method evaluation (26). ca 15-3 flaring (125% over the baseline) has been noticed in breast cancer patients after chemotherapy and has been linked with higher chances of disease development (27). increased ca 15-3 levels were found for locally progressed breast cancer patients who received primary chemotherapy (ac or ac-t regimen), which is an indicator of a poor response to treatment (28). the analysis of ca 15-3 levels upregulating during the first 4-6 weeks of a new therapy should be considered because false initial rises can happen (29-33). the chemotherapy influence on the temporary elevation of ca 15-3 followed by its decline could be a consequence of unsuitable early cancelation or change of chemotherapy regimen (30,34,35). previous studies indicated no connection between a breast cancer patient’s prior treatment and cea levels (36). based on our data, the cea levels changed during the course of treatment and stages. cea elevation for colorectal cancer patients has been noticed in the first 4-6 weeks after beginning chemotherapy (37,38). in addition, a chemotherapy regimen based on both irinotecan and oxaliplatin was found to induce a cea flare and was correlated with good prognosis for colorectal cancer patients (39). the mechanisms by which chemotherapy induces cea during cancer treatment remain to be elucidated. several factors were described to have an influence and connection including hypothyroidism and inflammatory diseases (40,41). in some protocols cea combined with ca 15-3 is used to observe the chemotherapy response for breast cancer patients, as it flares in the first 4-8 weeks of therapy as previously noted (42). the current study promotes the option of monitoring ca 15-3 and cea during adjuvant chemotherapy for breast cancer patients in jordan as its results could contribute to treatment evaluation and be beneficial for customizing chemotherapeutic regimens in the future. conclusions in conclusion, monitoring serum ca 15-3 and cea levels for jordanian breast cancer patients undergoing chemotherapy treatment provides prognostic indication and clinical information for evaluating tumor response as both markers had elevated levels with a preference for ca 15-3 over cea in sensitivity. acknowledgments the author acknowledges the biomedical scientists at biolab diagnostic laboratories, represented by dr. amid abdelnour, for their support of this work. 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https://doi.org/10.1097/maj.0b013e318065b023 https://www.ncbi.nlm.nih.gov/pubmed/17570994 https://www.ncbi.nlm.nih.gov/pubmed/12820311 https://doi.org/10.1200/jco.2007.14.2364 https://www.ncbi.nlm.nih.gov/pubmed/17954709 © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu jcb issn 1849-4544 doi: 10.33393/jcb.2020.2121 j circ biomark 2020; 9(1): 1-7 original research article et al (2) reported that from 2011 to 2014 there were 60,000 reported deaths due to pri. pri management is also expensive: management of hospital-acquired pri (hapi) alone has been reported to cost us healthcare over $26 billion annually (3). management costs for community-acquired pri have yet to be determined but may be presumed to be at least $8 billion (30% of hapi costs). spinal cord injury (sci) is defined as damage to any part of the spinal cord or nerves at the end of the spinal canal. traumatic sci occurs suddenly due to a blow or cut to the spine, such as can occur in a motor vehicle accident or fall. sci often leads to permanent loss of strength, sensation, and function below the site of the injury. a complete sci will result in total loss of all motor and sensory function below the level of injury, while with an incomplete sci there can be some sensory and/or motor function retained. during the acute and subacute stages following injury, there can be some neurorecovery. once neurorecovery has plateaued, the condition is considered to be chronic sci (4). this is most commonly considered to be 1 year following traumatic injury. exploring adipogenic and myogenic circulatory biomarkers of recurrent pressure injury risk for persons with spinal cord injury kath m. bogie1,2, katelyn schwartz2, youjin li3, shengxuan wang3, wei dai3, jiayang sun4 1case western reserve university, departments of orthopaedics and biomedical engineering, cleveland, ohio usa 2louis stokes cleveland veterans affairs medical center (lscvamc), research service, cleveland, ohio usa 3case western reserve university, department of population & quantitative health sciences, cleveland, ohio usa 4department of statistics, george mason university, fairfax, virginia usa abstract purpose: to investigate linkages between circulatory adipogenic and myogenic biomarkers, gluteal intramuscular adipose tissue (imat), and pressure injury (pri) history following spinal cord injury (sci). methods: this is an observational repeated-measures study of 30 individuals with sci. whole blood was collected regularly over 2-3 years. circulatory adipogenic and myogenic gene expression was determined. imat was defined as above/below 15% (imatd) or percentage (imat%). pri history was defined as recurrent pri (rpri) or pri number (npri). model development used r packages (version 3.5.1). univariate analysis screened for discriminating genes for downstream multivariate and combined models of averaged and longitudinal data for binary (rpri/ imatd) and finer scales (npri/imat%). results: for adipogenesis, krüppel-like factor 4 was the top rpri predictor together with resistin and cyclin d1, and sirtuin 2 was the top imat predictor. for myogenesis, the top rpri predictor was dysferlin 2b, and pyruvate dehydrogenase kinase-4 was the top imat predictor together with dystrophin. conclusion: circulatory adipogenic and myogenic biomarkers have statistically significant relationships with pri history and imat for persons with sci. biomarkers of interest may act synergistically or additively. variable importance rankings can reveal nonlinear correlations among the predictors. biomarkers of interest may act synergistically or additively, thus multiple genes may need to be included for prediction with finer distinction. keywords: adipogenesis genes, circulatory biomarkers, myogenesis genes, recurrent pressure injury, spinal cord injury introduction pressure injuries (pris) are defined as localized damage to skin and underlying soft tissue that develops due to intense and/or prolonged pressure or pressure combined with shearing (1). pri can present as intact skin or as open ulcers and can take months or years to heal once developed. development and/or recurrence of a pri limits activities of daily living, often leading to hospitalization and even death. padula received: april 9, 2020 accepted: august 18, 2020 published online: september 21, 2020 corresponding author: kath m. bogie louis stokes cleveland veterans affairs medical center 10701 east blvd cleveland, oh 44106 usa kmb3@case.edu adipogenic and myogenic circulatory biomarkers in spinal cord injury patients2 © 2020 the authors. published by aboutscience persons with sci remain at risk for pri development throughout their lifetime and often develop community-acquired pri. pri remain one of the most devastating secondary complications for persons living with sci. it remains unclear why some persons with sci suffer from a continuous cycle of recurring pri, while others remain pri free. the national pressure injury advisory panel has recognized that some pri might be unavoidable due to complex and systemic intrinsic factors (5). in response to these intrinsic factors, biomarkers that signal tissue health status are released into circulation. circulatory messenger ribonucleic acid (mrna) biomarkers related to fatty metabolism, which are valuable risk indicators for pri risk, have previously been reported (6). factors that regulate and control downstream biomarker production related to fatty metabolism, specifically adipogenesis and myogenesis, are thus of interest for determining the risk of developing recurrent pri (rpri). the study objective was to evaluate relationships between circulatory adipogenic and myogenic biomarkers, pri history, and muscle composition, in persons with chronic sci, by secondary analysis of preexisting blood samples. methods a repeated-measures study design was employed. individuals with complete or incomplete sci were recruited: exclusion criteria included having an open pelvic region pri and presence of a systemic disease. a comprehensive demographic profile was obtained at enrolment. whole blood samples were obtained every 6 to 12 months over 2-3 years. the study was carried out at a tertiary care facility. all clinical study activities were reviewed and approved by the local institutional review board and by the us army human research protection office. blood sample collection whole blood samples were collected for quantitative reverse transcription polymerase chain reaction (rt-qpcr) analysis. samples were frozen immediately in a −80°c freezer prior to further processing. samples from participants who met the eligibility criteria of either having no pri history or having a history of rpri were selected for secondary analysis focused on circulatory adipogenic and myogenic biomarkers (fig. 1). rt-qpcr analysis rna was extracted from whole blood samples using qiazol lysis reagent and the qiagen mini kit (qiagen, valencia, ca) following the rna extraction protocol provided in each kit. immediately following completion of rna isolation, sample concentration was measured using the nanodrop nd-2000. sample yields between 25 and 200 ng/μl of rna and a260:a280 ratios between 1.8 and 2.0 were used for further processing. rna integrity number (rin) was determined using fluorometric quantification (qubit, fisher scientific singapore) and confirmed using the agilent bioanalyzer. verification of 18s and 28s ribosomal rna integrity was analyzed by agarose gel electrophoresis and agilent bioanalyzer electropherograms. the thermocycler was used for complementary deoxyribonucleic acid (cdna) synthesis; 200 ng rna from each sample was mixed with buffer ge and nuclease-free water from participants with blood samples available n = 39 eligible participants n = 30 ineligible participants n = 9 blood sample passed quality control n = 12 participants with no pri history n = 12 rt-qpcr analysis of blood sample n = 12 participants with history of recurrent pri n = 18 blood sample passed quality control n = 18 rt-qpcr analysis of blood sample n = 18 fig. 1 observational study: flow chart for secondary analysis of blood samples for model development bogie et al 3 © 2020 the authors. published by aboutscience the qiagen rt2 first strand kit (qiagen, valencia, ca). at stage 2, the rt mix (qiagen, rt2 first strand kit, valencia, ca) was added to the rna solution and the sample was left to run for the remaining stages. nuclease-free water (qiagen, rt2 first strand kit, valencia, ca) was added after cdna synthesis was completed. the cdna was mixed with nuclease-free water (qiagen, valencia, ca) and sybr green rox qpcr master mix (qiagen, valencia, ca) based on the qiagen rt2 profiler pcr array handbook. commercially available custom rt2 profiler pcr array plates (pahs-049za and pahs-099za) were employed to determine expression of over 100 genes involved in adipogenesis and myogenesis. rt-qpcr analysis was run using a 96-fast plate sybr green qpcr protocol on an abi viia7 platform (applied biosystems, foster city, ca, usa) by the standard curve method. each well contained 25 μl total reaction volume. the protocol included 1 cycle for 10 minutes at 95°c to activate the hotstart dna taq polymerase followed by 40 cycles of 2-step rt-qpcr, including 15 seconds at 95°c and 1 minute at 60°c where fluorescence data collection was performed. ramp rates were adjusted to 1°c/s. lastly, a 1-cycle melting curve verified rt-qpcr specificity according to the rt2 profiler pcr array handbook. raw ct (cycle threshold) values were loaded into qbase+ software (biogazelle, zwijnaarde, belgium) to compute calibrated normalized relative quantities (cnrq) for further data analysis. inhibition testing used rt control and positive pcr control wells on the custom array plates. ct values across all pcr array runs were analyzed for sufficient quality by the value of the c t ppc wells of 20±2. values outside this threshold were eliminated from analysis. general dna contamination was observed by analyzing the raw ct values of the human genomic dna contamination (hgdc) well on each plate and eliminating sample runs with values less than 35 ct in the hgdc well. a control ct value less than 35 indicated the presence of detectable genomic dna contamination as recommended by the qiagen rt2 profiler pcr array handbook. references genes (beta-2-microglobulin, actin, hypoxanthine phosphoribosyltransferase-1, and large ribosomal protein p0) were included in custom plate arrays from qiagen. normalization methods were automated through qbase+ software. gluteal intramuscular adipose tissue (imat) content was determined following a previously developed standardized protocol (7). demographics repeated evaluations were completed for 30 participants with complete or incomplete sci. study participants had either a history of multiple pri (group i) or no history of pri (group ii). study groups were comparable with respect to standard demographic measures as shown in table i. statistical methods as with most studies using multivariate pcr arrays, not all genes were present for all samples. merely deleting any gene (the column variable) or any observation (the row variable) with any missing values would both eliminate too many observations and lead to a biased sample, since there is a low detection limit (ldl) for each gene. in the current study, genes with more than 70% missing were excluded from all analysis. to overcome the ldl issue, missing values for the remaining genes were imputed before a downstream analysis and the missing information directly incorporated in the next stage analysis. the imputed values were determined to be an estimated conditional mean of gene expression, given that the expression is known to be smaller than the detection limit defined by qbase+, or a scaled minimum observed value, as suggested by nie et al (8). the minimum observed values of normalized expressions were negative if gene activity was less than the normalization factors. we examined the effect of imputation by the conditional mean, and 1, √2, and 2 times the minimum observed. it was found that each approach gave similar results. hence, the results using √2 as the scaling factor are presented. comprehensive analyses were carried out on data with longitudinal repeated measures and on data averaged over time to find the factors that influence prl outcomes. meaningful outcomes were defined as the number of prl (nprl) and the percent of imat (imat%) since imat has been found to be a major risk factor for prl in earlier analysis (7,9). the dichotomized versions of these two outcomes were “with or without pressure injury” (rprl) and “below or above 15% imat” (imatd). data analyses were performed using statistical software program r (version 3.5.1) with packages rpart (for tree-based modeling), lme4 (for linear mixed effects modeling), geepack (for generalized estimating equation [gee] modeling), glmnet (for least absolute shrinkage and selection operator [lasso] on generalized linear models), and glmmlasso (for table i study participant demographics group i pri group ii no pri n 29 18 12 age at injury range 22-75 22-71 mean 46.3 50.2 age at study range 33-81 30-74 mean 57.5 57.6 sex male 16 89% 11 92% female 2 11% 1 8% duration of injury range 9 mo-46 y 1 mo–34 y mean 13.8 y 7.5 y neurological level above t6 12 67% 9 75% below t6 6 33% 3 25% asia level complete (ais = a) 7 39% 2 17% incomplete (ais = b,c,d) 11 61% 10 83% ais = american spinal injury association impairment scale; pri = pressure injury. adipogenic and myogenic circulatory biomarkers in spinal cord injury patients4 © 2020 the authors. published by aboutscience lasso on generalized linear mixed models). due to the large number of genes relative to the number of subjects (large p problem), our comprehensive analyses included univariate, multivariate, and combined analyses. univariate analyses each gene was examined to see if it was discriminated between the dichotomized outcomes of interest, specifically rpri and imatd. the statistical methods for group comparisons are given below: group comparison via confidence interval on data averaged over time for each gene, the tand bootstrap confidence intervals (cis) of each group mean were used to check the ci overlap for contrasting groups. the absence of any overlap approximated a significant group mean difference at 0.05, based on the ci levels recommended by payton et al (10). group comparison via survival models gene expression data can be considered as left-censored data, that is, values below the detection limit are censored. thus, survival models were applied treating data without missing values as an event and data with missing values as censored. in this case, “longer survival time” is equivalent to higher gene expression values. both a parametric procedure based on the log-normal distribution and the kaplan–meier, a nonparametric survival analysis procedure, were used to check group differences based on rpri and imatd for each gene. variable importance rankings from the parametric model are presented for important adipogenesis (tab. ii) and myogenesis genes (tab. iii). genes selected by this prescreening procedure were then included for downstream analyses using multivariate and combined approaches. multivariate analyses tree-based models and penalized regression procedures using lasso are suitable for finding important factors for large-p data. for averaged data over time, tree-based models on dichotomized rpri and imatd including all eligible genes were first performed. tree-based models handle missing values by using surrogates. the variable importance ranking from these models is indicative of the candidate genes for the final modeling. lasso preforms variable selection and prediction from a complex dataset simultaneously. two lasso r packages were used: glmnet for averaged data and glmmlasso for longitudinal data with a random intercept. variables of interest were also analyzed on the finer scale. poisson regression was applied to npri. gaussian regression was applied to the logit transformed imat%. for averaged data, the penalization level was set by both fivefold cross-validation (cv) and minimizing the bayesian information criterion (bic). for longitudinal data the table ii binary outcomes: variable importance ranking for adipogenesis genes gene name standard abbreviation ranking (a) rpri as outcome imat% 6.01 kruppel-like factor 4 klf4 3.21 retinoblastoma 1 rb1 2.25 cyclin-dependent kinase inhibitor 1a cdkn1a 1.50 fatty acid synthase fasn 1.50 insulin receptor substrate 2 irs2 1.50 lamin a/c lmna 1.50 (b) imatd as outcome sirtuin 2 sirt2 4.80 complement factor d (adipsin) cfd 2.40 cyclin-dependent kinase inhibitor 1b cdkn1b 1.92 insulin receptor substrate 2 irs2 1.92 sterol regulatory element binding transcription factor 1 srebf1 1.92 tafazzin taz 1.92 imatd = below or above 15% intramuscular adipose tissue; rpri = recurrent pressure injury. table iii binary outcomes: variable importance ranking for myogenic genes gene name standard abbreviation ranking (a) rpri as outcome imat% 6.01 dysferlin 2b dysf2b 2.76 mitogen-activated protein kinase 1 mapk1 2.76 lamin a/c lmna 2.25 calpain 3 capn3 1.88 akt serine/threonine kinase 1 akt1 1.50 mitogen-activated protein kinase 14 mapk 14 1.50 caspase 3, apoptosis related casp3ar 1.26 adrenoceptor beta 2 adrb2 1.26 myocyte enhancer factor 2c mef2c 1.26 (b) imatd as outcome pyruvate dehydrogenase kinase 4 pdk4 5.09 lamin a/c lmna 2.78 matrix metallopeptidase 9 mmp9 2.78 transforming growth factor beta 1 tgfb1 2.78 ribosomal protein s6 kinase, polypeptide 1 rps6ka1 2.31 utrophin utrn 2.31 imatd = below or above 15% intramuscular adipose tissue; rpri = recurrent pressure injury. bogie et al 5 © 2020 the authors. published by aboutscience penalization level was set only by minimizing bic due to high computational cost in using cv. gees using the selected genes from longitudinal lasso were also fit to serve as a complementary “sanity check” for the effects of the selected genes. as above, a possibly significant effect was defined as having a p< 0.05. a marginal effect was defined as having a p>0.05 but <0.10. regression combining the results from univariate and multivariate analyses genes found to be important from either the univariate analyses or one of the multivariate analyses were selected into a preliminary candidate set. a further combined analysis was then conducted using generalized linear mixed models to evaluate the feasibility of a more parsimonious model. three candidate analysis procedures/models were applied (tree, glmm lasso, and combined glmm) using both binary (rpri/imatd) and finer scales (npri/imat%) on averaged data and longitudinal data with a random intercept. all the reported poisson glmm models passed the “over-dispersion” test. results following sci, there is a loss of muscle mass and changes in muscle fiber type (11), which has some similarities to sarcopenia. acutely following injury, there is a rapid and dramatic loss of muscle mass over a period of weeks or months. it has also been shown that composition of the paralyzed muscle also changes (9): dystrophic-type changes occur in muscles with the lean muscle tissue replaced by adipose tissue, that is, fatty infiltration, or imat, increases. these changes in composition continue, even several years following injury. imat has been reported to increase with aging in healthy adults (12), with an increase of around 3% annually for persons over 50 years (13). however, following sci, the rapid imat accumulation seen in some individuals is occurring both more quickly and at a younger age than would be expected for sarcopenic muscle changes (9). higher imat compromises tissue resilience by reducing the overall microvascularity of the affected composite muscle tissue. the remaining blood vessels are also more prone to occlude under load. these changes thus impair the response to applied loads and increase the risk of tissue breakdown and pri development. changes in adiposity do not appear to be associated or correlated with clinical or demographic factors such as level or extent of injury (9,14). it is also important to clarify that in the same way as cholesterol levels vary independent of body mass index (bmi), so higher levels of imat deposition are not correlated with sci-adjusted bmi. there is growing appreciation that there is increased subclinical inflammatory activity following sci (15-17), which has recently been confirmed by functional genomics (18). when inflammation is prolonged by dysregulation, it can have harmful effects on tissue. however, prior work has shown that while inflammatory biomarkers can be detected in the circulation of persons with sci, they are not discriminatory for rpri risk (6). the role of fatty infiltration, or intramuscular adipose tissue, has only recently been reported (7,9). relationships between adipogenic and myogenic circulatory biomarkers and rpri in persons living with sci have not previously been reported. in the current study, results from different models were relatively consistent for identifying most important genes with a statistically significant effect on pri history or imat. all the genes with a significant p value (<0.05) for the regression modeling combining univariate and multivariate analyses were considered together with those that have a marginal p value (<0.10) from most tests. adipogenic circulatory biomarkers using rpri as the outcome measure, imat% is the top variable and krüppel-like factor (klf4) the top adipogenesis gene that complements imat in predicting prl (tab. iia). klf4 supports energy demand in skeletal muscle (19) and has been suggested as a potentially significant inhibitor of adipose biology (20). when imatd was defined as the primary model outcome, the adipogenesis tree model indicated sirtuin 2 (sirt2) was the critical determinant (tab. iib). the importance of sirt2 as an adipogenesis gene for imat is also shown by both glmm with a lasso penalty and the combined glmm analysis (tab. v). sirt2 inhibits lipid synthesis, playing a role in glyceroneogenesis or fat deposition into adipose tissue (21). thus, increased sirt2 expression may play a role in imat deposition over time following sci. in the combined glmm analysis for npri, a finer scale than rprl, in addition to imat, important adipogenesis genes included gata binding protein 2 (gata2), resistin (retn), and cyclin d1 (ccnd1) (tab. iv). we also found retinoblastoma 1 (rb1) to be a significant factor in models fit for rpri (tab. iia) and in the multivariate adipogenesis model using imat% as outcome (tab. v). rb1 expression is an important factor in adipocyte differentiation (22). it is negatively associated with bmi in able-bodied individuals, but positively associated with adipogenesis regulators such as peroxisome proliferator-activated receptor gamma (pparγ). current results suggest rb1 levels are reduced in environments that limit adipogenesis. complement factor d (cfd), also known as adipsin, is an important factor in the binary adipogenesis model with imatd as outcome (tab. iib). table iv poisson generalized linear mixed model using npri as outcome for adipogenesis (conditional r2 = 0.73) gene name standard abbreviation estimate std. z value pr(>|z|) (intercept) 0.359 0.346 1.04 0.2990 imat% 2.479 0.852 2.91 0.0036 gata binding protein 2 gata2 0.355 0.890 0.4 0.6900 resistin retn 1.116 0.694 1.61 0.1080 cyclin d1 ccnd1 0.582 0.246 2.36 0.0180 months −0.001 0.016 −0.05 0.9599 imat = intramuscular adipose tissue; npri = recurrent pressure injury number. adipogenic and myogenic circulatory biomarkers in spinal cord injury patients6 © 2020 the authors. published by aboutscience cfd is exclusively expressed in adipose tissue and is a ratelimiting component of the alternative complement pathway that regulates innate immune response (23). myogenic circulatory biomarkers for myogenesis, in addition to pri, the top gene that works with imat is dysferlin 2b (dysf2b) to predict rpri (tab. iiia) and pyruvate dehydrogenase kinase 4 (pdk4) for predicting imatd (tab. iiib). although included on a myogenesis panel, pdk4 also contributes to glucose metabolism regulation, facilitating glyceroneogenesis and triacylglycerol storage (24). the current models also showed pdk4 to be a useful but not significant factor in multivariate myogenesis models with imat as the outcome (tab. vii). the binary myogenesis tree model (tab. iiia) indicates that dysf2b expression is a myogenic determinant for rpri. lack of dysf2b causes changes in myofiber repair, alters calcium homeostasis, and causes chronic muscle inflammation (25). muscle loss due to myofiber replacement with fibrotic or adipogenic tissue is well known in chronic muscle injuries, but has not previously been studied in persons with chronic sci. muscle damage and regeneration are exacerbated by increased levels of fibro/adipogenic precursors (faps), which cause adipogenic replacement of muscle fibers in tissues with mutated dysferlin (25). glmm with a lasso penalty indicated dysf2b as an important gene in the multivariate myogenesis models for nprl (tab. vi). for the combined glmm analysis, dystrophin (dmd) also appeared to be more significant in the combined model when logit(imat) is the outcome measure (tab. vii). pdk4 was also included in the final models but with p values larger than 0.10 (tab. vii). limitations this exploratory study involved a moderately sized cohort followed for up to 3 years. variations between univariate and multivariate models for adipogenesis indicate some difference in pathways for pri development and imat. for myogenesis, the results across all analyses are more consistent. further work is needed to clarify the impact on pri risk over time of genes of interest involved in adipogenesis and myogenesis. conclusions persons with sci have a near-normal life expectancy if they do not develop severe secondary complications, in particular pri. while this is a lifetime risk, it also appears that susceptibility for this devastating secondary complication is unique for each individual. these differences are not clearly associated with either level of injury or american spinal injury association impairment scale (ais) grade. this exploratory analysis confirms that imat is a major indicator for rpri risk. circulatory adipogenic and myogenic biomarkers have statistically significant relationships with pri history and imat. the models indicated the importance of each gene based on the sum of the improvements in all nodes. variable importance rankings can reveal nonlinear correlations among the predictors. biomarkers of interest may act synergistically or additively, thus multiple genes may need to be included for prediction with finer distinction. table v linear mixed model using logit(imat) as outcome for adipogenesis (conditional r2 = 0.98) gene name standard abbreviation estimate std. t value pr(>|t|) (intercept) −2.252 0.408 −5.53 0.00001 nuclear receptor coactivator 2 ncoa1 2.453 1.054 2.33 0.0518 resistin retn −0.964 0.420 −2.29 0.0631 sirtuin 2 sirt2 1.486 0.599 2.48 0.0448 sirtuin 3 sirt3 −2.621 0.584 −4.49 0.0034 retinoblastoma 1 rb1 −3.328 0.648 −5.14 0.0015 retinoid x receptor, alpha rxra 2.658 0.648 4.10 0.0074 months −0.051 0.010 −5.08 0.0017 imat = intramuscular adipose tissue. table vi poisson generalized linear mixed model using npri as outcome for myogenesis (conditional r2 = 0.75) gene name standard abbreviation estimate std. z value pr(>|z|) (intercept) −0.220 0.297 −0.74 0.4595 imat% 2.674 0.859 3.11 0.0019 dysferlin 2b dysf2b 1.061 0.692 1.53 0.1252 hexokinase 2 hk2 −1.132 1.002 −1.13 0.2587 interleukin 1, beta il-1b 1.138 0.806 1.41 0.1578 months −0.003 0.016 −0.18 0.8608 imat = intramuscular adipose tissue; npri = recurrent pressure injury number. table vii linear mixed model using logit(imat) as outcome for myogenesis (conditional r2 = 0.72) variable name standard abbreviation estimate std. t value pr(>|t|) (intercept) −2.067 0.376 −5.49 0.000003 matrix metallopeptidase 9 mmp9 0.641 0.621 1.03 0.3100 pyruvate dehydrogenase kinase 4 pdk4 0.173 0.644 0.27 0.7910 dystrophin dmd 0.401 0.194 2.07 0.0490 months 0.002 0.018 0.12 0.9040 imat = intramuscular adipose tissue. bogie et al 7 © 2020 the authors. published by aboutscience data availability statement data cannot be shared publicly due to the policies of our institution. data may be made available to researchers who meet the criteria for access to confidential data following review by the institutional data access committee (contact via dr. km bogie). disclosures conflict of interest: the authors report grants from cdmrp spinal cord injury research program and grants from craig h. neilsen foundation during the conduct of the study. financial support: the work described in this article was supported by the cdmrp spinal cord injury research program (grant no. w81xwh-14-1-0618) and the craig h. neilsen foundation (grant no. 315537). references 1. henzel mk, bogie km. medical management of pressure ulcers in patients with spinal cord disorders. in: kirshblum s, lin vw, eds. spinal cord medicine, 3rd ed. springer 2018;516-543. 2. padula wv, pronovost pj, makic mbf, et al. value of hospital resources for effective pressure injury prevention: a cost effectiveness analysis. bmj qual saf. 2019 feb;28(2):132-141. 3. padula wv, delarmente ba. the national cost of hospital acquired pressure injuries in the united states. int wound j. 2019 jun;16(3):634-640. 4. burns as, marino rj, flanders ae, flett h. clinical diagnosis and prognosis following 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limb girdle muscular dystrophy 2b. nat commun. 2019 jun 3;10(1):2430. jcb j circ biomark 2021; 10: 20-25issn 1849-4544 | doi: 10.33393/jcb.2021.2327review journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu published a study on cytokine profiling in patients with ancaassociated vasculitis (aav) (6). serum from healthy subjects and from patients with aav or other autoimmune-mediated diseases was analyzed for concentrations of certain proand anti-inflammatory mediators. it became apparent that serum il-33 was significantly higher in aav subjects than in controls. also, aav patients with necrotizing glomerulonephritis (ngn) differed from aav subjects without ngn. these findings led to the hypothesis of il-33 as a potential biomarker in acute renal diseases, namely in aki. the respective study has been initiated and is ongoing. the current article will summarize the most important references on il-33 as a diagnostic tool and risk predictor in genetic and acquired diseases. interleukin-33—origin and physiology interleukin-33 (il-33) belongs to the il-1 family of cytokines (7). common feature of the latter is a beta-trefoil structure, which has been identified in proteins such as il-1alpha and -beta, il-1ra, and il-18. il-33 was initially found within endothelial cell nuclei of so-called human high endothelial venules (hev). baekkevold et al (8) described the protein as nf (nuclear factor)-hev. later, pichery and colleagues (9) detected il-33 within the nuclei of murine cells in various tissues, such as epithelial cells, lymphoid organs, brain, and embryonic tissue. herein, the cytokine was expressed in a constitutive manner. in contrast, blood vessels did not show constitutive il-33 expression. within nuclei, il-33 binds to chromatin (10); in the extracellular space however, it interacts with st2. the latter exists as membrane-bound and soluble isoform (sst2), respectively (11). interleukin-33 mediates so-called alarmin function. alarmins, which are represented by extracellularly secreted proteins of various serum il-33 as a biomarker in different diseases: useful parameter or much need for clarification? stefan erfurt1, meike hoffmeister2, stefanie oess2, katharina asmus1, oliver ritter1, susann patschan1, daniel patschan1 1 department of medicine 1, university hospital brandenburg, medical school (mhb) theodor fontane, brandenburg germany 2 institute of biochemistry, brandenburg medical school (mhb) theodor fontane, brandenburg germany abstract interleukin-33 (il-33), a member of the il-1 family, is critically involved in the modulation of the activity of a diverse range of immunocompetent cells. essential roles have been implicated in cardioprotection, in both innate and adaptive immune responses in mucosal organs, and in the maintenance of adipose tissue cells. over the past 10 years, several studies evaluated the usability of il-33 as a biomarker in diseases of inflammatory and noninflammatory origin. our group is currently evaluating the predictive role of serum il-33 in acute kidney injury (aki). the aim of the article is to discuss selected studies on il-33 in different diseases and its potential role as a biomarker molecule. keywords: biomarker, il-33, inflammatory diseases, noninflammatory diseases introduction the identification of biomarkers for either early diagnosis or risk prediction in various diseases remains a fundamental goal in the whole field of medicine. a sophisticated discussion of all or of at least the most important findings in recent years is impossible in one single article that is intended to focus on one potential marker molecule. the literature on biomarkers in even one particular disease, such as acute kidney injury (aki), is vast. for illustrative purposes, it needs to be mentioned that more than 100 candidate molecules have been investigated in aki subjects over the past 20 years (1). only few parameters (e.g., ngal, kim-1 (2,3)) or combinations of two proteins such as the product of urinary insulin-like growth factor-binding protein (igfbp-7) and tissue inhibitor of metalloproteinase (timp-2) (4,5) have been proven as reliable tools for diagnostic/prognostic purposes under certain aki-associated conditions. however, no parameter can truly replace serum creatinine for measuring excretory kidney function in daily clinical practice, despite creatinine far from being an “optimal” biomarker. in 2019, our group received: august 3, 2021 accepted: november 10, 2021 published online: november 30, 2021 corresponding author: daniel patschan zentrum innere medizin 1 universitätsklinikum brandenburg medizinische hochschule brandenburg hochstraße 29 14770 brandenburg germany daniel.patschan@mhb-fontane.de https://doi.org/10.33393/jcb.2021.2327 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:daniel.patschan@mhb-fontane.de erfurt et al j circ biomark 2021; 10: 21 © 2021 the authors. published by aboutscience www.aboutscience.eu origin and phenotype, indicate cell/tissue damage. subsequently, immune cells are activated. in this context, il-33 has been shown to modulate the activity of mast cells, group 2 innate lymphoid cells (ilc2s), t helper 2 cells, eosinophils, basophils, dendritic cells, macrophages, and others (12). essential roles of the cytokine have been implicated in cardioprotection (11), in both innate and adaptive immune responses in mucosal organs (13) and in the maintenance of adipose tissue cells (via immune cell modulation) (14). quantification of serum il-33 the data on il-33 quantification by either elisa (enzymelinked immunosorbent assay) or multiplex technologies are heterogeneous. by far not all groups initially succeeded in measuring serum levels of the cytokine. firstly, krychtiuk and colleagues (15), who identified low serum il-33 to be predictive in intensive care-treated patients, did not detect the protein in 57 out of 223 subjects. in 2016, ketelaar et al (16) reported the results of serum il-33 quantification by using four different elisa kits (quantikine and duoset, r&d systems, respectively; adi-900-201, enzo life sciences; skr038, genway biotech inc, san diego, usa). serum samples were drawn from asthma patients. the authors included different numbers of samples per individual kit: quantikine— n = 45; duoset—n = 17; adi-900-201—n = 17; skr038—n = 22). surprisingly, the percentages of samples above the lower detection limit (lld) were 0 (zero) in two kits (adi-900-201 and skr038). the quantikine kit showed only 2% of all samples above the lld, the duoset kit was successful in at least 76%. comparable findings were reported by asaka et al (17), who also employed the quantikine kit. difficulties in il-33 quantification were also reported by rivière and colleagues (18). in this context, it needs to be noted that erythrocytes contain il-33 in relevant concentrations. thus, hemolysis may increase serum il-33 concentrations and potentially adulterate the findings in biomarker-related studies (19). aberrant il-33 in diseases: elevation genetic disorders behairy and colleagues (20) evaluated 60 infants with cholestasis, assigned to one of two subgroups with versus without biliary atresia (ba and non-ba group). subjects belonging to the first group showed significantly higher serum il-33. also, il-33 was higher in both groups as compared to healthy controls. finally, il-33 correlated with aspartate and alanine aminotransferase (alt), and with serum bilirubin in a positive manner, respectively. since cytokine levels additionally correlated with the fibrosis stage, the authors concluded a potential (pathogenic) role of il-33 in ba disease progression. cardiovascular diseases a study in subjects with chronic kidney disease (ckd) was published in 2017 (21). two-hundred and thirty-eight ckd patients were followed up for 24 months. both serum il-33 and st2, the circulating isoform of the il-33 receptor, were quantified and associated with cardiovascular events and parameters of endothelial dysfunction (flow-mediated vasodilation, fmd). the two analytes increased with decreasing estimated glomerular filtration rate and elevated levels were associated with impaired fmd and cardiovascular risk. nevertheless, bao et al (22) failed to show increased il-33 in ckd subjects. in experimental studies tgf-β ko (knock out) enhanced il-33, and while its inflammatory regulation varies with the disease, in the heart it protects against pathological remodeling after infarction, myocyte hypoxia, pressure overload, and increased ca2+ release from the sr (sarcoplasmic reticulum) which is tunneled to mitochondria via mitochondrial ryr, leading to stimulation of mitochondrial adenosine triphosphate production (23). autoimmune-mediated diseases in 2010, mu et al published data on rheumatoid arthritis (ra) (24). three groups were evaluated: healthy controls, ra subjects, and patients with osteoarthritis. the cytokine was not detectable in controls and osteoarthritis but in almost 100 ra subjects (42.2%). cytokine concentrations correlated with the rf and anti-ccp titers, respectively, and anti-tnfalpha therapy reduced il-33 serum levels. il-33 elevation in ra was also shown by hong and colleagues (25). borsky et al (26) performed a study in psoriasis vulgaris subjects, intended to quantify serum levels of alarmins. the latter act as danger signals. the following factors were analyzed: hmgb1, il-33, s100a7, and s100a12. in total, 63 psoriasis patients and 95 controls were included. all four proteins were elevated in a significant manner. correlations between serum concentrations and disease activity were however not identified. our group studied subjects with aav and with other autoimmune-mediated diseases (6). serum concentrations of the following parameters were measured: il-1β, il-6, il-17 a, il-17 f, il-21, il-22, il-23, tnf-α, scd40l, il-4, il-10, il-25, il-31, il-33, and inf-γ. a total number of 62 aav subjects were included in the study (39 females; 23 males). forty-five subjects were pr3+, 17 subjects showed anca specificity for mpo (myeloperoxidase). serum il-33 was elevated in aav and ssc (systemic sclerosis). in aav, higher levels were found in non-ngn. minaga et al (27) investigated a rarer type of disease, type 1 autoimmune pancreatitis (type 1 aip), belonging to the heterogeneous group of igg4-related disease (igg4-rd). the study revealed higher levels of interferonalpha and of il-33 in type 1 aip subjects in comparison to individuals with chronic pancreatitis of other origin and to healthy controls. bakr et al (28) analyzed serum il-33 in patients with pemphigus vulgaris. affected subjects showed higher cytokine levels than healthy controls. also, serum levels significantly correlated with the activity of the disease (measured by the pemphigus disease area index, pdai). neoplasia a study in patients with newly diagnosed prostate cancer was published in 2019 (29). one-hundred and fifty individuals were included. serum il-33 was higher than in controls, serum il-33 as biomarker22 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb particularly in subjects with higher disease stages. nevertheless, cytokine concentrations did not differ between certain predefined genotypes. as a matter of fact, elevated il-33 has also been documented in other malignant diseases such as gastric cancer (30), endometrial cancer (31), non-small cell lung cancer (32), and breast cancer (33). sepsis sepsis may be regarded as an archetype of a widespread inflammatory disease. in 2016, çekmez and colleagues (34) reported data from 128 septic children (20 of these were controls). finally, 68 individuals were excluded since blood cultures were negative. serum il-33 differed significantly between the two groups of controls vs sepsis (1.1 ± 0.28 ng/ ml vs 5.23 ± 1.80 ng/ml; p = 0.01). two years later, another study performed in septic children (neonates) (35) showed comparable findings with a rapid il-33 increase at the first day after diagnosis, followed by a decrease over time (days 3 and 7). others although immune-mediated mechanisms are substantially involved in the pathogenesis of asthma bronchiale, the disorder has been placed in this section. bahrami and colleagues (36) included 61 affected children and 63 healthy controls in their analyses and found serum il-33 to be significantly elevated. blood cytokine levels increased with increasing disease severity. the predictive value of serum il-33 in polytraumatized subjects was analyzed by halát and colleagues (37). the specific aim was to evaluate whether il-33 is suitable for the prediction of pulmonary complications. the study included 130 patients, serum samples were obtained at the time of hospital admission and 2 days later. subjects that developed both parenchymal lung injury and subsequent acute respiratory distress syndrome (ards) showed particularly high serum il-33 at the time of admission. it was hypothesized that the cytokine is involved in promoting sustained lung injury in traumatized patients. sundnes et al (38) also published a trial in traumatized individuals (n = 136). plasma samples were collected at the time of hospital admission and several hours later (2, 4, 6, and 8 hours). the analyses showed a rapid and transient il-33 increase in a subset of patients. the latter presented with more intense tissue injuries and a higher degree of coagulopathy. another study related to serum patterns of alarmins (danger-associated molecular patterns—damps) focused on schizophrenia. the following molecules were quantified: il-33, sst2, hmgb1, and s100b (39). the study included 68 patients suffering from chronic schizophrenia and 29 healthy controls. all four analytes were higher in the disease group. it was concluded that alarmins may be involved in latent neuroinflammation underlying/perpetuating schizophrenia. yuan and colleagues focused on chronic hepatitis b (chb) (40). a total number of 130 chb patients were included, 48 cases were defined as stable, 50 patients showed progression to acute-on-chronic liver failure (aclf), 30 individuals also progressed but with overall milder dynamics (pre-aclf). the highest il-33 levels were detected in aclf patients, associations were found with serum alt, aspartate transaminase, and the model endstage liver disease (meld) score, respectively. the combination of serum il-33 and the meld score was predictive with regard to the 90-days mortality. thus, il-33 was proposed as prognostic marker in chb-associated liver disease. in 2021, venkataraman et al (41) published multiplex immune assay data from children with “paediatric inflammatory multisystem syndrome temporally associated with sarscov-2 infection (pims-ts).” numerous cytokines were elevated, including il-33. duan and colleagues analyzed gout patients and found increased il-33 in respective subjects with even higher levels in those individuals without impaired kidney function (42). also, il-33 was negatively correlated with markers of kidney dysfunction. aberrant il-33 in diseases: suppression cardiovascular diseases experimental data showed neuroprotective effects of il-33 (43,44). therefore, chen et al (45) evaluated the relationship between serum il-33 and the risk for hemorrhage transformation (ht) of acute ischemic stroke (ais) in humans. more than 150 individuals were included, none of these received thrombolytic therapy. patients were assigned to the ht group if any radiographic signs of ht became apparent during the first 2 weeks after ais diagnosis. multivariate regression analysis identified il-33 as an independent ht predictor (lower serum il-33 levels were associated with higher ht risk). the authors expanded their analyses in the context of poststroke depression (46) and found a comparable (inverse) association between serum il-33 and the risk for depression after ais. together, the data confirmed the concept of il-33-mediated neuroprotection, even in humans. in this context, an important study by li et al (47) evaluated long-term outcome variables of stroke patients in relation to initial serum il-33. higher cytokine levels were associated with a better 2-year outcome of affected individuals. low serum il-33 was finally also shown to be predictive in patients with intracerebral hemorrhage (48). a study in patients with heart failure with reduced ejection fraction (hf-ref) was published by segiet et al (49). one hundred and fifty-five hf-ref individuals (males: 106, females: 49) were included, the mean left ventricular ejection fraction was 32.13 ±12.8%. patients displayed lower mean serum il-33 as compared to controls. also, cytokine levels were lower in hf-ref of ischemic origin as opposed to hf-ref of other etiology. autoimmune-mediated diseases a rarer autoimmune-mediated disorder (at least in central europe), behçet’s disease was evaluated by koca et al (50) who included 117 affected individuals. while patients’ serum il-33 did not differ from control levels, active disease states were associated with lower il-33. nevertheless, certain allelic variations did not differ in serum il-33. erfurt et al j circ biomark 2021; 10: 23 © 2021 the authors. published by aboutscience www.aboutscience.eu others in contrast to the study by halát et al (37), who found il-33 to be elevated in polytraumatized patients, another investigation performed in critically ill subjects showed opposite findings. krychtiuk and colleagues (15) evaluated the predictive value of both il-33 and sst2 with regard to the 30 days mortality in 223 intensive care patients. il-33 was not detectable at all in 57 subjects. serum il-33 concentrations below the median strongly predicted death in an independent manner. lower-than-normal il-33 concentrations were also documented in osteoporosis (51) and in amyotrophic lateral sclerosis (52). finally, hasan et al (53) showed a negative correlation between il-33 and the body mass index. they also found il-33 to negatively correlate with hba1c in nondiabetic individuals. no aberrant il-33 in diseases cardiovascular diseases firouzabadi and colleagues (54) measured serum il-33 and the soluble isoform of its receptor (sst2) in 44 heart failure patients (n = 25 under carvedilol treatment vs n = 19 without beta-blocker therapy) and in 22 healthy controls. the analyses did not reveal any differences between any of the three groups, which led to the conclusion that cardioprotective effects of carvedilol are most likely not mediated via the il-33/ssts axis. two other studies related to acute cardiovascular diseases did also not show any differences in serum il-33 between controls and patients. demyanets et al (55) investigated subjects with stable angina, nstemi (nonst-segment elevation myocardial infarction), or stemi in comparison to healthy controls. dhillon and colleagues (56) analyzed nstemi subjects. table i summarizes studies on serum il-33 in diseases, assigned to one out of three groups: elevation/suppression or constant il-33. conclusions the data on serum il-33 in human diseases are heterogeneous. on the one hand, methodological difficulties in measuring serum il-33 may occur and must always be put into consideration if low levels or no protein at all is detectable. the majority of studies in which il-33 quantification table i studies in serum il-33 in inflammatory and noninflammatory disorders il-33 elevation il-33 suppression constant il-33 biliary atresia (20) ht of ais (45) heart failure (54) ckd (21) poststroke depression (46) ckd (22) rheumatoid arthritis (24) long-term outcome after stroke (47) stable angina pectoris, nstemi, stemi (55) rheumatoid arthritis (25) intracerebral hemorrhage (48) nstemi (56) psoriasis vulgaris (26) hf-ref (49) prostate cancer (29) behçet’s disease (50) gastric cancer (30) critically ill subjects (15) endometrial cancer (31) osteoporosis (51) non–small cell lung cancer (32) amyotrophic lateral sclerosis (52) breast cancer (33) increased body weight (53) sepsis in infants (34) sepsis in infants (35) asthma bronchiale in children (36) polytrauma (37) trauma (38) anca-associated vasculitis (6) pemphigus vulgaris (28) type 1 autoimmune pancreatitis (27) schizophrenia (39) hepatitis b-associated acute-on-chronic liver failure (40) pims-ts (41) gout (42) references appear according to the order in the text. ais = acute ischemic stroke; ckd = chronic kidney disease; hf-ref = heart failure with reduced ejection fraction; ht = hemorrhage transformation; il = interleukin; nstemi = non-st-segment elevation myocardial infarction; pims-ts = pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 infection. serum il-33 as biomarker24 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb succeeded showed higher blood concentrations of the cytokine. however, to identify a pattern that potentially allowed conclusions on the exact role of serum il-33 in inflammatory and noninflammatory diseases is quite difficult. we cited a total number of 21 studies that reported il-33 elevation under pathological circumstances. thirteen out of these 21 investigations evaluated diseases with distinct inflammatory pathogenesis (exceptions: ckd, ba, schizophrenia, five malignancies). two out of 10 studies reporting lower il-33 were nevertheless also performed in inflammatory disease states (behçet’s disease, critically ill subjects). thus, it may be concluded that elevation of serum il-33 should be expected with higher probability in inflammatory than in noninflammatory disorders. the references on malignancies with elevated il-33 were all referring to carcinomas. activation of the immune system is known to significantly occur in malignant disorders. it is therefore not surprising that diseases such as heart failure with preserved or reduced ejection fraction, ischemic stroke, or even most cases of aki are not associated with increased serum il-33. regarding the heterogeneity of diseases with either il-33 elevation or suppression, it is hardly possible to propose the molecule as specific biomarker at the moment. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. authors’ contributions: se searched for literature and corrected the article. he also performed experiments related to il-33. mh corrected the article and is involved in a project related to il-33. so provided additional references and helped in structuring the article. ka 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https://doi.org/10.1371/journal.pone.0095055 https://www.ncbi.nlm.nih.gov/pubmed/24751794 https://doi.org/10.1016/j.ahj.2011.03.025 https://www.ncbi.nlm.nih.gov/pubmed/21641364 article journal of circulating biomarkers onward to 2016 editorial shidong jia1, antonio chiesi2 and winston patrick kuo1,3* 1 predicine holdings ltd, hayward, ca, usa 2 exosomics siena, siena, italy 3 biopharma research council, tinton falls, nj, usa *corresponding author(s) e-mail: wpkuo1@gmail.com doi: 10.5772/62278 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. as we embrace for another exciting year (2016), we are thrilled to announce jcb has been indexed in the doaj (directory of open access journals) (https://doaj.org/toc/ 1849-4544)! this was attributable to the number of quality published original research articles in 2015 with the help of our associate editors and editorial board. we published several papers covering each of the main topics within the scope of the journal from cell-free dna, circulating tumour cells and extracellular vesicles to circulating proteins. in addition, we opened a new technology forum presenting the latest technology and published its first article entitled, “analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization” by shannon werner and her colleagues. over the past year, we were proud to announce several ongoing developments such as the expansion of our associate and editorial board with key opinion leaders in the circulating biomarker field including: lawrence rajendran (university of zurich, switzerland) as part of our associate editors team, and quentin qiang liu (dalian medical university) and shlomit kenigsberg (create fertility centre, canada) as members of our editorial board. jcb is now media partners with both selectbio and the cambridge healthtech institute (chi). jcb has arranged a discount for our readers when registering for selectbio and chi events, which by the way, have many presenters that are members of our associate editors and editorial board. additional information about such opportunities can be found on jcb’s news page (bit.ly/1p9exvd). as an ongoing partnership/collaboration with the biophar‐ ma research council (http://www.biopharmaresearch‐ council.org/), the editor-in-chief, winston patrick kuo organized three events around the “microbiome”, two as a webinar series and the other, a conference hosted by the national cancer institute. in the first webinar, we were able to utilize our editorial team to suggest the theme and present in the first session “introduction to the micro‐ biome” by alexander “sasha” vlassov, phd, and as well as other talks from camilia martin, md, ms of the harvard medical school, and floyd dewhirst, phd, of the forsyth institute. the second webinar was held as a four-session webinar series in april 2015 entitled “short course in the microbiome”. the goal of the short course was to provide insights at an introductory level, yet also a broad overview of the field in the form of an exchange between researchers from academia and industry in the clinical applications of the oral and gut microbiome. we had world renowned speakers including rob knight from ucsd, camilia martin from the harvard medical school, and romina goldszmid and k. leigh greathouse both from the nih. they covered topics related to the “system biology ap‐ proach”, “spatially explicit maps”, “microbiome and cancer therapy” and a “round-up of the latest news”. a meeting dispatch was published online and is available on the journal of circulation biomarker’s site (http://www.inte‐ 1j circ biomark, 2016, 5:2 | doi: 10.5772/62278 https://doaj.org/toc/1849-4544 https://doaj.org/toc/1849-4544 bit.ly/1p9exvd http://www.biopharmaresearchcouncil.org/ http://www.biopharmaresearchcouncil.org/ http://www.intechopen.com/journals/journal-of-circulating-biomarkers/short-course-in-the-microbiome chopen.com/journals/journal-of-circulating-biomarkers/ short-course-in-the-microbiome). the event at the nci was themed “altering the microbiome: can it impact health?”. the purpose of this was to discuss how the host micro‐ biome may be altered and whether such approaches can result in a positive impact on the host. we learned that the preparation process required about six months of coordi‐ nation. we plan to continue several webinar series on circulating tumour cells and cell-free circulating dna later this year. as we strive to publish the best science in all fields of circulating biomarkers, the editorial team plans to focus on additional topics such as cell-free dna and disruptive technologies, among others. jcb accepts original and review articles as well as editori‐ als, perspectives, short research reports, protocols and methods, notes to the editor, letters to the editor and meeting dispatch reports. we would like to mention that intech have decided that jcb will not apply any article processing charges for authors whose research papers have been accepted for publication in volume 5/2016. manu‐ script processing and peer-reviewing for jcb is conducted entirely online. the editorial manager facilitates the manuscript processing time, reducing costs and providing a better experience for our authors and reviewers. we have received numerous positive comments on this evolving electronic system, and we are indebted to intech open access publisher for managing this process for us. for more information, please visit our new manuscript submission system at http://www.editorialmanager.com/exo/ default.asp. in addition, we are looking to expedite the acceptance to publishing time for our authors and will request informal opinions from our editorial board for borderline cases. as an ongoing effort, jcb will continue to work on part‐ nering with scientific societies, and academic and industry leaders to advance the field and to allow free access to knowledge in the circulating biomarker space. it’s our great pleasure to thank the editorial team and publishers who make this possible. 2 j circ biomark, 2016, 5:2 | doi: 10.5772/62278 http://www.intechopen.com/journals/journal-of-circulating-biomarkers/short-course-in-the-microbiome http://www.intechopen.com/journals/journal-of-circulating-biomarkers/short-course-in-the-microbiome http://www.editorialmanager.com/exo/default.asp http://www.editorialmanager.com/exo/default.asp © 2020 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). any commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu jcb issn 1849-4544 doi: 10.33393/jcb.2020.2163 j circ biomark 2020; 9(1): 13-19 original research article cancers remain dependent on androgen receptor signaling for growth and that these orally administered agents can improve progression-free survival and overall survival (1-7). aatts have quickly become the most common treatment modalities in crpc. abiraterone and enzalutamide received approvals in 2011 and 2012, apalutamide in 2019, and darolutamide the same year (1-7). since then, a recent analysis of medical records of 2,559 mcrpc patients in the united states from january 2013 to september 2017 found that 1,980 (77%) received life-prolonging therapies, of whom 1,294 (65%) received an aatt as the first-line therapy. of the 969 patients who received a second-line treatment, 523 (54%) received an aatt, with sequential use of aatts commonly practiced (8). the second most common class of drugs utilized are taxanes, such as docetaxel or cabazitaxel (8). while these two drug classes are the most commonly utilized in the management of mcrpc in the united states, the choice between an aatt and a taxane is often empiric without a predictive biomarker guidance (8). in this setting, the most clinically validated treatment selection biomarker development of an immunofluorescent ar-v7 circulating tumor cell assay – a blood-based test for men with metastatic prostate cancer david lu1,2, rachel krupa1, melissa harvey1, ryon p. graf1, nicole schreiber3,4, ethan barnett3, emily carbone3, adam jendrisak1, audrey gill1, sarah orr1, howard i. scher5,6, joseph d. schonhoft1 1epic sciences, san diego, california usa 2exact sciences, madison, wisconsin usa 3department of medicine, memorial sloan kettering cancer center, new york, new york usa 4prostate cancer clinical trials consortium, new york, new york usa 5genitourinary oncology service, department of medicine, memorial sloan kettering cancer center, new york, new york usa 6department of medicine, weill cornell medical college, new york, new york usa abstract introduction: here we describe the development of a protein immunofluorescent assay for the detection of nuclear-localized androgen receptor variant 7 (ar-v7) protein within circulating tumor cells (ctcs) identified in patient blood samples. used in the clinic, the test result serves as a validated biomarker of futility for patients with progressing metastatic castration-resistant prostate cancer (mcrpc) who are treated with androgen receptor targeted therapies (aatt) in whom nuclear-localized ar-v7 ctcs are identified and have received level 2a evidence in the 2019 national cancer center network (nccn) guidelines (v1.0). methods: assay development was completed on the epic sciences rare cell detection platform using control cell lines of known ar-v7 status and clinical testing of mcrpc patient samples obtained at the decision point in management. results and conclusions: using these samples, all assay parameters, scoring criteria, and clinical cutoffs for positivity were prospectively selected and locked. after assay lock, blinded clinical validation testing was initiated on multiple, independent, clinical cohorts as reported by scher et al (jama oncol. 2016;2:1441-1449; jama oncol. 2018;4:1179-1186) and armstrong et al (j clin oncol. 2019;37:1120-1129). keywords: ar-v7, castration-resistant prostate cancer, circulating tumor cells (ctcs), predictive biomarkers introduction the last decade has seen a dramatic expansion of the therapeutic options for men with recurrent metastatic castration-resistant prostate cancers (mcrpcs). in particular, next-generation androgen receptor targeted therapies (aatt) including abiraterone, enzalutamide, apalutamide, and darolutamide have unequivocally demonstrated that many crpc received: june 9, 2020 accepted: september 4, 2020 published online: october 23, 2020 corresponding author: joseph d. schonhoft, phd epic sciences 9381 judicial dr. san diego, california 92121, usa joseph.schonhoft@epicsciences.com nuclear-localized ar-v7 in ctcs14 © 2020 the authors. published by aboutscience to date is the presence of nuclear-localized androgen receptor splice variant 7 (ar-v7) protein detected in the circulating tumor cells (ctcs) of metastatic crpc patients using the epic sciences platform, receiving level 2a evidence in the 2019 national cancer center network (nccn) guidelines. the assay is performed at epic sciences as a laboratory developed test (ldt), reimbursed by the centers for medicare and medicaid services (cms) and top private payers, has received accreditation by the college of american pathologists (cap), and approval from the new york state department of health (nysdoh). the first study to demonstrate clinical utility of the epic sciences nuclear-localized ar-v7 test was a blinded cross sectional study consisting of mcrpc patients treated with aatt and taxanes from memorial sloan kettering cancer center (mskcc) (9). both treatment-naïve (first line) and pretreated (second line or greater) patients were considered, and the results showed superior survival times for patients in whom nuclear-localized ar-v7 ctcs were identified who received a taxane compared with aatt and that favorable prostatespecific antigen (psa) responses were observed only in the patients treated with taxanes. this study was followed by a blinded prospective validation that included mcrpc patient samples from three centers (10). here, only second-line mcrpc-treated patients, those who had failed one prior therapy, were considered and ar-v7 biomarker-positive patients were observed to have favorable survival on taxanes compared with aatt in a risk-adjusted model, confirming our initial report. the third study, prophecy (nct02269982) (11), was a prospectively designed multicenter blinded validation that included firstand second-line mcrpc patients with a poor prognosis based on a validated nomogram that included pretreatment levels of lactate dehydrogenase (ldh), alkaline phosphatase (alk), or presence of visceral metastases among other features. all patients were treated with aatt. here again, patients with nuclear-localized ar-v7 ctcs had a shorter time to radiographic progression, shorter overall survival, and an unfavorable psa response (11). here we present the development data for the epic sciences nuclear-localized ar-v7 assay. analytical and clinical feasibility testing, including control cell line screening, antibody testing, and initial patient feasibility testing, were executed to achieve assay lock prior to the commencement of the three clinical utility studies. materials and methods ar-v7-positive cell line engineering a lentiviral vector containing both ef1a-ar-v7 and rsvpuromycin inserts was transduced into a pc3 (ar-negative) parental cell line to create a stable ar-v7 expressing cell line. target sequence integration into the cell line genome was confirmed using genomic template polymerase chain reaction (pcr). ar-v7 transfected cells were maintained under puromycin selection during culture to ensure the stable expression of ar-v7 protein. transfected subclones were assessed via quantitative pcr (qpcr), western blotting and immunofluorescent staining to confirm ar-v7 protein expression. cell culture all cell lines were cultured using sterile techniques and complete cell culture media. to manufacture control slides for assay development and subsequent assay validation, cells were trypsinized into single-cell suspension, counted, and spiked into normal donor (nd) blood collected into streck bct at known quantities. qpcr qpcr primer pairs specific for the ar n-terminal domain (arntd), ar-v7 cryptic exon (arv7ce), and the ar c-terminal domain (arctd) were used to assess appropriate gene expression and messenger ribonucleic acid (mrna) splicing profiles in each cell line. cells were harvested and assayed for gene expression using a single cell-to-ct kit (ambion) as per manufacturer protocol. real-time quantitative (qrt)-pcr was performed with taqman probes for arntd, arctd (thermofisher), or a probe set specific to arv7ce (custom design) with a quantstudio 7 (thermofisher). all samples were analyzed for 18s (thermofisher) as an internal rna quality control. western blotting whole cell lysates were prepared using nupage lds sample buffer supplemented with 0.5% beta-mercaptoethanol and protease inhibitor. cells were harvested via scraping from tissue culture dishes at approximately 80% confluency and sonicated via probe sonicator followed by incubation at 95°c for 5 minutes. protein lysates (40 µg/well) were run on nupage 4%-12% tris-glycine precast gels and transferred using the iblot dry blotting system. overnight incubation with ar-v7 primary antibody followed by detection via westerndot 625 goatantirabbit kit (cat# w10142, thermofisher) was used for visualization of ar-v7 protein. all western blotting reagents and equipment was sourced from thermofisher and ar-v7 antibody was sourced from abcam (clone ep343). laboratory-derived sample and patient blood sample processing laboratory-derived (ld) control samples were created using nd blood collected into streck cell-free blood collection tubes and spiked with either positive (gs3 or 22rv1) or negative (du145 or pc3) cell line cells (clcs). contrived ld samples were used to create unequivocal positive and negative samples to mimic patient samples of known ar-v7 status. these samples were accessioned and processed identically to patient samples. for patient samples, mcrpc patient blood (7.5 ml) was collected in streck tubes at mskcc and processed at epic sciences within 48 hours. all patients gave informed consent to an institutional review board (irb)-approved protocol before blood draw. for both ld and patient samples, red blood cells were lysed, and approximately 3 million nucleated cells were dispensed onto glass microscope slides, lu et al 15 © 2020 the authors. published by aboutscience fixed, and stored at −80°c as previously described (9-13). all testing presented in this study was performed prior to the start of the three clinical validation studies. nd blood sample processing nd blood samples were acquired from a local blood collection site (the scripps research institute normal blood donor service, la jolla, ca). blood was drawn into streck tubes from male donors and shipped to epic sciences for processing. immunofluorescent staining briefly, the immunofluorescence (if) staining components of the assay are as follows and as previously reported (9-13). analytical validation of the epic sciences platform for rare cell detection has been previously reported (12). all nucleated cells deposited onto a slide are detected with dapi, a dye that fluoresces upon binding to deoxyribo nucleic acid (dna) and stains the cell nucleus. white blood cells (wbcs) are detected using an anti-cd45 antibody that is directly conjugated to alexa fluor™ 647. ck proteins in ctcs are detected using a panel of mouse immunoglobulin g (igg)1 anti-ck antibodies visualized with a secondary goat antimouse igg1 antibody conjugated to alexa fluor™ 555. the rabbit monoclonal anti-ar-v7 antibody is recognized by a secondary goat antirabbit antibody conjugated to horseradish peroxidase (garhrp), which catalyzes covalent binding of tyramide conjugated to alexa fluor™ 488. immunofluorescent scanning stained slides are scanned using the epic sciences automated scanning platform. the platform consists of a highthroughput microscope slide scanning system that collects images at 10× magnification for each of the four fluorescent channels used in the ar-v7 assay (dapi, ck, cd45, ar-v7). all nucleated cells found on each glass slide are imaged and mean fluorescent intensities (mfis) are quantified. final outputs from the fluorescent scanner include cell-level images enabling visualization of subcellular biomarker localization, mfis for each biomarker, and unique coordinates to allow for the relocation of cells of interest for genomics or reimaging purposes. imaging algorithms using the images captured by the epic sciences platform, proprietary image analysis algorithms classify ctcs based on cellular morphology and biomarker mfis detected from each of the four channels corresponding to dapi, ck, cd45, and ar-v7. exposure times are automatically varied to maximize signal in each field of view. for this reason, the signal quantified in a clc or ctc subpopulation is normalized to the average signal observed in a representative population of wbcs on the slide. the nuclear localization of ar-v7 signal (nl-arv7) is determined based on its colocalization with dapi (13). on a ctc level, results are binary for the purposes of this assay. a ctc with ar-v7 fluorescent signal above background that colocalizes with dapi is considered ar-v7 positive. trained technical reviewers confirm the algorithm classification to identify the presence or absence of ar-v7-positive (nuclearlocalized) ctcs. results control cell line characterization of ar-v7 mrna and protein expression to enable ar-v7 if assay development, appropriate control cell lines (pc3, du145, and 22rv1) were screened to confirm ar-v7 mrna and protein expression levels. consistent with previous reports, pc3 and du145 cells lacked detectable ar gene expression and were negative for both ar mrna and protein (fig. 1a, b). 22rv1 cells are known to express several ar splice variants (14), and because of this heterogeneity, mrna transcripts corresponding to the arntd-, arv7ce-, and arctd-specific sequences were detected, reconfirming the expression of both full-length ar and ar-v7 splice variants (fig. 1a). to create an unequivocal control without the limitations of the inherent heterogeneity and lot-to-lot variability of ar variant expression in 22rv1s, we engineered a cell line to constitutively express only the ar-v7 variant. lentiviral transfection of a pc3 parental line was used to generate several candidate subclones for constitutive ar-v7-expressing cell lines in which three were selected (gs1, gs3, gs8). while all three subclones expressed the ar-v7 transcript, only the gs3 line was found to be positive for ar-v7 protein via if staining (fig. 2a) and western blot (fig. 1b). notably, full-length ar is not expressed in gs3 cells as evidenced by lack of detectable arctd transcript (fig. 1a). for subsequent ar-v7 if assay development, both pc3 and du145 cells were utilized as negative controls to confirm test specificity, while 22rv1 and gs3 cells were used as positive controls. ar-v7-specific immunofluorescent assay development ar-v7 if assay development utilized the epic sciences existing rare cell detection platform as previously described (12). using ld samples containing either positive (gs3 or 22rv1) or negative (pc3, du145) cells spiked into nd blood, or nd blood alone, if staining conditions were developed and subsequently optimized (fig. 2). primary antibody titration results observing relative fluorescent signal in each clc as a function of primary antibody concentration are shown in figure 2b, c. upon completion of preliminary primary and secondary antibody titration curves, extensive guard banding studies were performed to confirm optimal assay parameters including primary and secondary antibody concentrations, tyramide concentration, fixation reagent concentration, fixation reagent incubation time, and wash buffer incubation times (data not shown). final assay conditions were selected based on optimal signal-to-background ratios, yielding median nuclear-localized ar-v7 in ctcs16 © 2020 the authors. published by aboutscience ar-v7 fluorescent signals of 8.4-, 17,and 127-fold above background in du145, 22rv1, and gs3 cells, respectively (fig. 2c). ar-v7 signal in positive control cells was observed to be consistently and predominantly localized in the cell nucleus, whereas signal in du145 and pc3 was largely undetectable and if low levels were present it was non-nuclear (example images, fig. 2a). therefore, hereafter, we require nuclear-specific localization of ar-v7 to call a cell positive. by comparison, similar criteria for nuclear localization have been applied to ar-v7 expression in tissue (15-17). finally, we tested 21 nd blood donors and 0/21 (0%) were found to have ar-v7-positive ctcs. 0.0 2.0 4.0 6.0 8.0 ar n-terminal domain mrna expression fo ld c h an g e to 2 2r v1 n.d. 0.0 0.5 1.0 1.5 ar c-terminal domain mrna expression 0 20 40 60 80 100 ar-v7 mrna expression pc3 du145 22rv1 gs3 pc3 du145 22rv1 gs3 pc3 du145 22rv1 gs3 n.d. n.d. fo ld c h an g e to 2 2r v1 fo ld c h an g e to 2 2r v1 n.d. n.d. n.d. n.d. fig. 1 control cell line characterization of androgen receptor variant 7 (ar-v7) messenger ribonucleic acid (mrna) and protein expression. (a) quantitative polymerase chain reaction analysis using ar n-terminal (arntd), ar c-terminal (arctd), and ar-v7 (arv7ce)specific primer sets. pc3 and du145 cell lines are negative for all ar-specific mrna sequences (nd: not detected). while 22rv1s expresses relatively low levels of ar-v7 transcript, ~90-fold relative abundancy was observed in ar-v7 stably transfected gs3 cells. furthermore, no arctd-specific transcript was observed in gs3s, confirming no full-length ar mrna expression. mrna abundance is quantified as fold expression compared to 22rv1. 18s is used as an internal control. (b) western blot confirms mrna expression profiles. du145 and pc3 cells are ar-v7-negative, while abundant protein was observed in gs3 and 22rv1 cell lysates. gs1 and gs8 cells, alternative ar-v7 stably transfected subclones, did not produce ar-v7 protein and were discarded. lower weight nonspecific bands (~72 kda, 50 kda) were observed in some cell lines. histone h3 is used as a loading control. ba lu et al 17 © 2020 the authors. published by aboutscience 1 2 4 8 16 32 64 128 256 512 017836e 017836f 017836g 017836h 017836j 017836k 017836l 017836m 017387a 017387b 017387c 017387d 017387e 017387f 017387g 017387h slide id cr at io [anti-arv7 1°] 1 μg/ml 3 μg/ml 5 μg/ml pc3 (ar-v7 negative)gs3 (ar-v7 expressing) 1 2 4 8 16 32 64 128 256 512 018465b 018465c 018465d 019344r 019344s 019344t 019349a 019349b 019349c 018465e 018465f 018465g 019344u 019344v 019345a 019349d 019349e 019349f 018465h 018465j 018465k 019345b 019345c 019345d 019349g 019349h 019349j slide id cr at io clc 22rv1 du145 gs3 2 μg/ml 3 μg/ml 4 μg/ml fig. 2 androgen receptor variant 7 (ar-v7)-specific immunofluorescent assay development. optimization of an immunofluorescent circulating tumor cell (ctc) assay detecting the presence of nuclear-localized ar-v7 protein was developed using control cell lines. nuclearlocalized staining was observed in 22rv1 and gs3 cells, whereas pc3 and du145 cells are consistently negative for nuclear ar-v7 signal as seen in representative images (a). representative microscopy images of 22rv1, gs3, pc3, du145 cell lines. (b) antibody titration curves comparing gs3 (ar-v7 expressing) and pc3 (ar-v7 negative). each data point represents the mean fluorescence signal relative to the local background of a single cell detected (cratio). (c) titration curves comparing gs3, 22rv1, and du145 at 2, 3, and 4 µg/ml of ar-v7 primary antibody. at the selected primary antibody concentration of 3 µg/ml, typical fluorescent signals observed in du145, 22rv1, and gs3 were 8.4-, 17-, and 130-fold above background, respectively. immunofluorescent signals in negative controls (i.e., pc3, du145) are nonzero due to a combination of cellular autofluorescence, instrument noise, and nominal nonspecific (non-nuclear) antibody binding. a b c scoring criteria for ar-v7-positive cells before initiation of patient feasibility testing, scoring criteria were defined. for a ctc to be scored as ar-v7 positive, it must: (1) be negative for cd45 staining (blood lineage marker); (2) have ck positivity or cellular morphology indicative of epithelial (or nonhematopoietic) lineage; (3) have an intact nucleus without signs of apoptosis; and (4) have ar-v7 staining with clearly defined nuclear-localized signal. existing clinical data indicated that ar-v7 would be more likely to be expressed in patients who had previously failed treatments in the metastatic setting (18). to assess the general clinical feasibility of the ar-v7 assay to potentially detect physiologically relevant levels of ar-v7 protein in ctcs, 27 mcrpc patient samples were tested. these samples were obtained from patients who had previously failed at least one line of treatment in the metastatic setting, were progressing, and in need of a therapy change. samples were not selected based on known ar-v7 status. furthermore, this cohort was used strictly for development purposes and was not included in any subsequent clinical validation testing. twenty-two of 27 (82%) patients harbored ctcs, 14/27 (51.8%) of patients harbored ctcs with nuclearlocalized expression, and patients with ar-v7-positivity qualitatively demonstrated substantial heterogeneity, in terms of ar-v7 protein expression localization and intensity within their observed ctc populations (fig. 3). based on these data, a cutoff of at least 1 ar-v7-positive ctc detected in a patient sample was prospectively chosen as the criterion for ar-v7 test positivity. for each patient sample here, 6 million total nucleated cells were analyzed per patient sample. the detection of at least 1 positive cell was used for all subsequent clinical validation studies (9-11). furthermore, a nuclear ar-v7 fluorescence intensity of at least 3.2-fold above background was also prospectively selected as a criterion for ar-v7 positivity based on a qualitative assessment of the mcrpc patient ctcs, that is, it was not possible to determine ar-v7 localization below this threshold. therefore, patient samples containing ctcs with ar-v7 if intensity below 3.2, those with non-nuclear-localized signal above 3.2, and those samples in which ctcs are not detected are classified as negative. discussion in this report we present assay development results for the epic sciences nuclear-localized ar-v7 test. importantly, all studies presented and all assay parameters were locked prior to initiation of any of the three clinical utility studies (9-11). the assay specifically detects the ar-v7 splice variant protein lacking the ligand binding domain in the nucleus of ctcs. control cell line experiments demonstrate the assay’s ability to distinguish between the ar-v7 truncated protein from the full-length protein and other splice variants within ctcs. analysis of blood samples from patients with progressing mcrpc in need of a change in therapy for progressing disease were used to develop a patient scoring criteria and to demonstrate the ability of nuclear-localized ar-v7 in ctcs18 © 2020 the authors. published by aboutscience 2 16 128 001467 011444 010236 002787 009516 008507 010012 002532 008241 004419 009494 002009 010044 004711 004321 003277 006474 002182 004491 005071 009922 002401 003368 003863 004826 006085 010898 a rv 7 si g n al t o n o is e ra ti o single ctc ctc cluster inidividual blood tube id from a mcrpc patient composite composite composite dapi dapi dapi ck ck ck cd45 cd45 cd45 ar-v7 ar-v7 ar-v7 nuclear localized ar-v7 positive composite composite composite dapi dapi dapi ck ck ck cd45 cd45 cd45 ar-v7 ar-v7 ar-v7 low signal relative to local background and/or signal is non-overlapping with dapi channel nuclear localized ar-v7 negative a b c fig. 3 scoring criteria for androgen receptor variant 7 (ar-v7) positive cells. samples from 27 progressing mcrpc patients in need of a therapy change tested with the locked clinical trial assay prior to conduct of clinical utility studies. (a) dot plot where every dot is one circulating tumor cell (ctc, blue) or ctc cluster (green); the y-axis indicates the immunofluorescence intensity and the x-axis indicates the unique sample identifier. dashed line indicates the analytical cutoff for signal intensity, which combined with the presence of nuclearlocalized signal constitutes a positive. representative ar-v7-positive (b) and ar-v7-negative (c) ctc images with the following panels left to right: 4-color composite, dapi, ck, cd45, ar-v7. exposure times are varied to maximize signal and fluorescence intensities are reported relative to the local background. the locked assay to specifically detect the ar-v7 protein in ctcs from patients. the three independent clinical studies performed after assay-lock showed the clinical utility of the epic sciences nuclear-localized ar-v7 assay in providing information for guiding treatment selection for men with progressing mcrpcs (9-11). specifically samples were obtained at the pertinent decision point in management and collectively show that patients in whom nuclear-localized ar-v7 protein is identified in ctcs harbor tumors that are resistant to aatts, such as abiraterone acetate or enzalutamide, and have a better chance at response and have longer survival times when treated with taxanes, such as docetaxel or cabazitaxel. other assays are currently available for ar-v7 detection of ctcs in the blood; however, only the nuclear-localized ar-v7 assay described here has met the ballman criteria (19) for a predictive biomarker to date, in which two treatment groups, aatts and taxanes, have been directly compared and a quantitative statistical interaction between biomarker, treatment group, and overall survival has been observed such as in the two studies reported by scher et al (9,10). the most recent study, prophecy (11), a multicenter prospective trial, enrolled patients treated in the first and second lines with aatts and directly compared ar-v7 protein expression by the nuclear-localized ar-v7 assay with the detection of ar-v7 mrna using qpcr from isolated ctcs’ affinity enriched from blood: the johns hopkins ar-v7 assay (18). the nuclear-localized protein expression ar-v7 assay identified fewer ar-v7-positive patients (11% of patients having nuclear-localized ar-v7 protein vs. 24% having detectable ar-v7 mrna in isolated ctc), although had a higher specificity of identifying nonresponse (0% vs. 11%; 50% psa decline). while it is possible that some cells express ar-v7 mrna, but not the functional proteins, both methods of ar-v7 detection showed a strong association with poor survival and measures of response, and lu et al 19 © 2020 the authors. published by aboutscience increased in frequency at progression, providing further evidence that the ar-v7 splice variant is a mechanism of aatt resistance (11). the ar-v7 protein is a transcription factor and is required to bind to dna in order to activate the ar signaling axis, and with this in mind, prior tissue-based assessments of ar-v7 have generally required the criteria for nuclear localization (15-17). similarly, for ctcs it is necessary that the ar-v7 protein is nuclear localized to make a positive biomarker call and in prior clinical outcome analysis it was found that the nuclear localization is required for making treatment decisions between aatts and taxanes (13). aatts, such as abiraterone, enzalutamide, apalutamide, and darolutamide, have been shown to extend life in crpc (1-7); however, these treatments are not curative and nearly all patients eventually progress (20). it is hypothesized that because the ar-v7 splice variant lacks the ligand binding domain it can track into the nucleus and activate the ar signaling axis in the absence of androgens. however, other mechanisms of resistance exist, including other forms of ar reactivation, ar bypass signaling, and ar independent disease (20), and it is likely that ar-v7 expression may cooccur with many, highlighting the need for more comprehensive biomarkers that can be assessed in real time. needed are additional blood-based ctc-based if assays or genomic biomarkers assayed in ctcs or in circulating tumor dna (ctdna) to provide a more comprehensive biomarker panel to further inform therapy choice, where there are too few predictive biomarkers; the ctc nuclear-localized ar-v7 assay and tissue-based assessment of brca alterations for parpi treatment are of only a few biomarkers to be reimbursed for physician choice in therapy selection to date. disclosures conflict of interest: d. lu, r. krupa, m. harvey, r. graf, j. schonhoft, a. jendrisak, a. gill, s. orr are or were employees of epic sciences during the writing, data collection, and analysis. h.i. scher is a consultant/advisory board member for ambry genetics corporation, amgen, essa pharma, janssen biotech, janssen research & development, onclive insights, menarini silicon biosystems, physicians education resource, sanofi aventis, and wcg oncology; and he has received institutional research funding from epic sciences, illumina, janssen diagnostics, menarini silicon biosystems, and thermofisher. no other disclosures are reported. financial support: epic sciences provided facilities, materials, and services to perform isolation and sequencing of circulating tumor cells. work at memorial sloan kettering cancer center was supported by the sidney kimmel center for prostate and urologic cancers, and funded in part by the nih/nci cancer center support grant to msk (p30 ca008748), the nih/nci spore in prostate cancer grant to msk (p50 ca092629), the prostate cancer foundation, and the prostate cancer clinical trials consortium. references 1. beer tm, armstrong aj, rathkopf de, et al. enzalutamide in metastatic prostate cancer before chemotherapy. n engl j med. 2014;371:424-433. 2. scher hi, fizazi k, saad f, et al. increased survival with enzalutamide in prostate cancer after chemotherapy. n engl j med. 2012;367:1187-1197. 3. ryan cj, smith mr, fizazi k, et al. abiraterone acetate plus prednisone versus placebo plus prednisone in chemotherapy naive men with metastatic castration-resistant prostate cancer (cou-aa-302): final overall survival analysis of a randomised, double-blind, placebo-controlled phase 3 study. lancet oncol. 2015;16:152-160. 4. de bono js, logothetis cj, molina a, et al. abiraterone and increased survival in metastatic prostate cancer. n engl j med. 2011;364:1995-2005. 5. ryan cj, smith mr, de bono js, et al. abiraterone in metastatic prostate cancer without previous chemotherapy. n engl j med. 2013;368:138-148. 6. smith mr, saad f, chowdhury s, et al. apalutamide treatment and metastasis-free survival in prostate cancer. n engl j med. 2018;378:1408-1418. 7. fizazi k, shore n, tammela tl, et al. darolutamide in nonmetastatic, castration-resistant prostate cancer. n engl j med. 2019;380:1235-1246. 8. george dj, sartor o, miller k, et al. treatment patterns and outcomes in patients with metastatic castration-resistant prostate cancer in a real-world clinical practice setting in the united states. clin genitourin cancer. 2020;18:284-94. 9. scher hi, lu d, schreiber na, et al. association of ar-v7 on circulating tumor cells as a treatment-specific biomarker with outcomes and survival in castration-resistant prostate cancer. jama oncol. 2016;2:1441-1449. 10. scher hi, graf rp, schreiber na, et al. assessment of the validity of nuclear-localized androgen receptor splice variant 7 in circulating tumor cells as a predictive biomarker for castration resistant prostate cancer. jama oncol. 2018;4:1179-1186. 11. armstrong aj, halabi s, luo j, et al. prospective multicenter validation of androgen receptor splice variant 7 and hormone therapy resistance in high-risk castration-resistant prostate cancer: the prophecy study. j clin oncol. 2019;37: 1120-1129. 12. werner sl, graf rp, landers m, et al. analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization. j circ biomark. 2015;4:3. 13. scher hi, graf rp, schreiber na, et al. nuclear-specific ar-v7 protein localization is necessary to guide treatment selection in metastatic castration-resistant prostate cancer. eur urol. 2017;71:874-882. 14. hu r, dunn ta, wei s, et al. ligand-independent androgen receptor variants derived from splicing of cryptic exons signify hormone-refractory prostate cancer. cancer res. 2009;69:16-22. 15. efstathiou e, titus m, wen s, et al. molecular characterization of enzalutamide-treated bone metastatic castration-resistant prostate cancer. eur urol. 2015;67:53-60. 16. qu y, dai b, ye d, et al. constitutively active ar-v7 plays an essential role in the development and progression of castration resistant prostate cancer. sci rep. 2015;5:7654. 17. welti j, rodrigues dn, sharp a, et al. analytical validation and clinical qualification of a new immunohistochemical assay for androgen receptor splice variant-7 protein expression in metastatic castration-resistant prostate cancer. eur urol. 2016; 70:599-608. 18. antonarakis es, nakazawa m, luo j. resistance to androgen-pathway drugs in prostate cancer. n engl j med. 2014;371: 2234. 19. ballman kv. biomarker: predictive or prognostic? j clin oncol. 2015;33:3968-3971. 20. watson pa, arora vk, sawyers cl. emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer. nat rev cancer. 2015;15:701-711. jcb j circ biomark 2022; 11: 28-35issn 1849-4544 | doi: 10.33393/jcb.2022.2386original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu interleukin-33 (il-33) belongs to the interleukin-1 family of cytokines (5). the cytokine was initially found within endothelial cell nuclei of so-called human high endothelial venules (hev). pichery and colleagues (6) detected the protein within the nuclei of murine cells in various tissues, such as epithelial cells, lymphoid organs, brain, and embryonic tissue. within nuclei, il-33 binds to chromatin (7); in the extracellular space however, it interacts with st2. the latter exists as membranebound and soluble isoform (sst2), respectively (8). il-33 has been shown to modulate the activity of several immunocompetent cells such as mast cells, group 2 innate lymphoid cells (ilc2s), t helper 2 cells, eosinophils, basophils, dendritic cells, macrophages, and others (9). early aki recognition remains difficult, although new biomarkers have been identified in recent years (10). future diagnostic criteria will most likely include markers of structural kidney damage (11). until then, aki is being diagnosed according to the 2012 published “kdigo clinical practice guidelines for acute kidney injury” (12). experimental data suggest a critical role for il-33 in the pathogenesis of aki (13,14). also, several studies evaluated il-33 and sst2 as biomarkers in inflammatory and soluble il-33 receptor predicts survival in acute kidney injury stefan erfurt1, meike hoffmeister2,3, stefanie oess2,3, katharina asmus1, susann patschan1,3, oliver ritter1,3, daniel patschan1,3 1 department of internal medicine i – cardiology, nephrology and internal intensive medicine, brandenburg university hospital, brandenburg medical school (theodor fontane), brandenburg an der havel germany 2institute of biochemistry, brandenburg medical school (theodor fontane), brandenburg an der havel germany 3 faculty of health sciences (fgw), joint faculty of the university of potsdam, the brandenburg medical school theodor fontane and the brandenburg technical university cottbus-senftenberg, cottbus germany abstract introduction: the prediction of acute kidney injury (aki)-related outcomes remains challenging. herein we prospectively quantified soluble st2 (sst2), the circulating isoform of the il-33 receptor, in hospitalized patients with aki. methods: in-hospital subjects with aki of various etiology were identified through the in-hospital aki alert system of the brandenburg university hospital. sst2 was measured within a maximum of 48 hours from the time of diagnosis of aki. the following endpoints were defined: in-hospital death, dialysis, recovery of kidney function until demission. results: in total, 151 individuals were included in the study. the in-hospital mortality was 16.6%, dialysis therapy became mandatory in 39.7%, no recovery of kidney function occurred in 27.8%. sst2 was significantly higher in nonsurvivors (p = 0.024) but did not differ in the two other endpoints. the level of sst2 increased significantly with the severity of aki. further differences were detected in subjects with heart insufficiency (lower sst2), and in patients that required icu treatment, or ventilatory therapy, or vasopressors (all higher). conclusions: the current study suggests sst2 as biomarker of “acute distress”: it predicts post-aki survival and substantially increases in subjects with a higher degree of cumulative morbidity under acute circumstances (e.g., icu therapy, vasopressor administration). keywords: acute kidney injury, il-33, soluble st2, mortality, biomarker received: february 17, 2022 accepted: may 16, 2022 published online: june 6, 2022 corresponding author: daniel patschan zentrum für innere medizin 1, kardiologie, angiologie, nephrologie klinikum brandenburg, medizinische hochschule brandenburg hochstraße 29 14770 brandenburg germany d.patschan@gmail.com introduction acute kidney injury (aki) occurs with increasing frequencies at hospitals in central europe and the united states. it is being estimated that up to 18% of all hospitalized subjects develop aki during the treatment course (1). the in-hospital mortality of hospital-acquired aki has been reported to vary from 10% to 20% (1-3), with exceptionally low survival rates under intensive care conditions (4). https://doi.org/10.33393/jcb.2022.2386 https://creativecommons.org/licenses/by-nc/4.0/legalcode erfurt et al j circ biomark 2022; 11: 29 © 2022 the authors. published by aboutscience www.aboutscience.eu noninflammatory diseases. the literature on il-33 reveals heterogenous findings, including protein elevation or suppression, or constant serum il-33, depending on etiology and course of the disease (15-17). also, il-33 quantification has been associated with substantial difficulties. lately we summarized the literature on the topic (18). for instance, ketelaar et al. (19) used 4 different elisa kits (quantikine and duoset r&d systems, respectively; adi-900-201 enzo life sciences; skr038 genway biotech inc san diego usa) for analyzing serum samples from asthma patients. the percentages of samples above the lower detection limit (lld) were 0 (zero) in two kits (adi-900-201 and skr038). also, the quantikine kit showed only 2% of all samples above the lld, the duoset kit in contrast was successful in at least 76%. similar observations were made by asaka et al. (20), who also employed the quantikine kit. finally, riviere and colleagues (18) reported difficulties in il-33 quantification as well. regarding il-33 and sst2 in conjunction, two studies were performed in aki subjects so far. the first study revealed sst2 as an early predictor of acute kidney injury in patients with myocardial infarction (21). the second investigation showed sst2 to be aki predictive in subjects undergoing cardiac surgery (22). herein, we prospectively analyzed serum sst2 levels in patients with newly onset aki of various etiology. three endpoints were defined: in-hospital death, the need for dialysis, and recovery of kidney function until demission. methods setting this prospective observational study was conducted at the brandenburg university hospital in brandenburg an der havel, germany. the hospital is part of the brandenburg medical school. study population and design the study was approved by the local ethics committee of the brandenburg medical school theodor fontane in october 2019 (file no. e-01-20190820). all recruited participants were hospitalized patients of the university hospital brandenburg. patients of multiple medical departments with newly onset aki were included from may 2020 to june 2021. aki was defined according to criteria 1 or 2 of 2012 revised kdigo classification (23). the third criterion (urine output of below 0.5 ml/kg/h for at least 6 hours) was not applied since information on urine production was not available in all subjects. serum il-33 and sst2 levels were determined once at the time of initial diagnosis of aki. all patients were over 18 years of age, were not previously receiving renal replacement therapy at the time of blood collection, and signed written informed consent. preexisting chronic renal failure requiring dialysis, terminal disease with a strictly palliative treatment regimen, suspected or active covid-19 disease, and age less than 18 years resulted in exclusion from the study. the aki etiology was identified according to respective criteria for sepsis (24), cardiorenal syndrome types 1 or 3 (25), and hepatorenal syndrome (26). the diagnosis of obstruction was made by ultrasound analysis, the diagnoses of drug-induced, contrast-associated, and postsurgery aki were made according to the history. volume depletion or prerenal aki was diagnosed if other causes were unlikely and if the patient presented clinical symptoms of volume depletion (e.g., dry skin in conjunction with low blood pressure and tachycardia). blood sampling and preanalytics an automated aki alert system has been implemented at the brandenburg university hospital in 2018. elevated serum creatinine levels (according to the kdigo criteria 1 or 2) that are measured during daily laboratory checks are registered by an electronic algorithm and transmitted to the nephrologist in charge. the messages exclusively contain a patient-related number and do not allow to identify individuals without the in-hospital database. after written informed consent was obtained, a standardized venous blood sample was collected in two 3.5 ml serum tubes (bd vacutainer® sst™ ii advance). blood was collected in the supine position with as little venous congestion as possible to avoid hemolysis. in patients with central venous line, blood was collected from that catheter. the filled blood tubes were stored upright for 30 minutes to maintain the clotting time specified by the manufacturer. this was followed by centrifugation at 1,400 g for 10 minutes at room temperature. samples were stored in plastic tubes at constant −22°c until analysis. quantification of serum sst2 the quantification of sst2 was performed by using a commercially available kit: human st2/il-33r quantikine elisa kit (dst 200, r&d). the assay detects free and il-33-complexed st2. analyses were performed in duplicates according to the manufacturer’s instructions. sample predilution was adjusted individually to the concentrations. the range of assay sensitivity was 2.45-13.5 pg/ml. reference blood samples from healthy adults were used for comparison. endpoints three primary endpoints were defined: in-hospital death, the need for dialysis, and recovery of kidney function until demission. the second criterion (need for dialysis) was fulfilled if one or more dialysis treatment sessions became mandatory. dialysis was performed as hemodialysis, or hemodiafiltration, or slow extended daily dialysis (sledd), or as continuous veno-venous hemodiafiltration (cvvhd(f)). the respective procedure was chosen by the nephrologist in charge. renal recovery was defined according to the criteria published by fiorentino et al (27). it was diagnosed, if the last serum creatinine concentration did not differ from the initial value by more than 50%. statistics initially, results of sst2 quantification were tested for normality with the kolmogorov-smirnov test. since data were not distributed normally, the mann-whitney test was applied sst2 predicts aki survival30 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb for comparisons between two groups. comparisons between three or more groups were performed with the kruskal-wallis test. the results are given as median + the interquartile range (iqr). correlations were analyzed by calculating the pearson correlation coefficient. a p-value of below 0.05 was considered as statistically significant. the youden index (specificity + sensitivity −1) was employed for the identification of cut-off values, sensitivities and specificities were extracted from roc (receiver operating characteristic) curves. statistical analyses were performed with the following applications: wizard for macos (version: 2.0.9, developer: evan miller, 2021) or graphpad prism (version 9.3.1). results baseline characteristics and outcomes in total, 151 subjects were included in the study (females 62, males 89). the mean age of all individuals was 74.9 ± 13.4 years. the mean in-hospital treatment time was 16.2 ± 10.9 days. in-hospital mortality was 16.6%. dialysis therapy became mandatory in 39.7%. renal recovery occurred in 72.2%. all patient characteristics are summarized in table i. etiology and severity of aki the most frequent aki etiology was sepsis with 23.6%. other etiologies were volume depletion; cardiorenal; contrast induced (or associated); hepatorenal; drug-induced; obstruction; combined. more than 60% were diagnosed with akin (acute kidney injury network (28)) stage iii (tab. i). soluble st2 sst2 was quantified once, at the time of aki diagnosis plus a maximum of 48 hours in some individuals. serum st2 levels correlated negatively with age (p = 0.004; r = −0.233) but not with the duration of in-hospital treatment (p = 0.228) (fig. 1). females did not significantly differ from males (p = 0.407). only five patients were younger than 40 years. out of these, only one subject showed sst2 levels of higher than 2 × 105 pg/ml as opposed to 25% of the individuals with age 60 or higher. endpoints aki patients with in-hospital death showed significantly higher serum sst2 at the time of diagnosis as compared to surviving subjects (146,100 [iqr 97,420-233,700] vs. 74,325 [iqr 40,030-192,900] pg/ml; p = 0.024) (fig. 2). additional analysis revealed a sst2 concentration of >86,110 pg/ml as cut-off (sensitivity 84%; specificity 55.56%). the risk of in-hospital death was 15.4% in subjects that reached the cut-off (prediction intervals 10.8%-21.4%). patients requiring dialysis did not differ from those without the need for renal replacement therapy (123,550 [iqr 57,050-287,000] vs. 75,890 [iqr 38,560-189,600] pg/ml; p = 0.083) (fig. 2). subjects with renal recovery did not differ from patients without recovery (78,410 [iqr 43,520-192,900] vs. 132,900 [iqr 42,650-258,200] pg/ml; p = 0.48) (fig. 2). etiology and aki stage sst2 did not differ between all aki types of a certain etiology (p = 0.2). the three most frequent entities (septic aki; aki due to volume depletion; cardiorenal aki) were compared with all other entities combined. however, sst2 did not differ in any of the three analyses (septic aki, p = 0.4; aki due to volume depletion, p = 0.6; cardiorenal aki, p = 0.39). sst2 significantly differed between the akin stages (28), the marker gradually increased from stage i to iii (i: 51,830 [iqr 32,310146,100] vs. ii: 73,620 [iqr 35,650-191,200] vs. iii: 128,500 [iqr 53,060-267,000] pg/ml; p = 0.014). the significance levels between akin stages i, ii, and iii were: i vs. ii p = 0.99; ii vs. iii p = 0.35; i vs. iii p = 0.01 (fig. 3). table i patients’ characteristics variable result age (years ± sd) 74.9 ± 13.4 gender (females/males) 62/89 in-hospital treatment (days ±sd) 16.2 ± 10.9 aki etiology (%) sepsis 23.6 volume depletion 23.6 cardiorenal 20.1 contrast-induced 12.5 hepatorenal 2.8 drug-induced 1.4 postsurgery 1.4 obstruction 0.7 combined 18.5 morbidities preexisting ckd (%) 73.5 arterial hypertension (%) 88.4 diabetes mellitus (%) 48 coronary artery disease (%) 42.6 preexisting heart insufficiency (%) 55.6 pulmonary disease (%) 24.7 obesity (%) 49 history of neoplasia (%) 27.8 dialysis initiated (%) 39.7 in-hospital death (%) 16.6 recovery of kidney function (no/yes in %) 27.8/72.2 icu treatment (%) 32.5 ventilatory therapy (%) 17.2 vasopressor therapy (%) 16.6 erfurt et al j circ biomark 2022; 11: 31 © 2022 the authors. published by aboutscience www.aboutscience.eu morbidities sst2 did not differ between patients with hypertension or diabetes mellitus as compared to subjects without the respective morbidity (p = 0.117 and p = 0.4). the same applied for pulmonary disease (one or more of the following diagnoses: chronic obstructive pulmonary disease, asthma, other), obesity, history of neoplasia, and coronary artery disease (p = 0.934, p = 0.247, p = 0.738, and p = 0.249). patients with preexisting heart insufficiency, however, displayed lower serum sst2 than those without heart insufficiency (68,365 [iqr 39,280-134,000] vs. 144,850 [iqr 52,510-309,400] pg/ml; p = 0.005) (fig. 4). treatment course patients that required treatment at the intensive care unit showed higher sst2 than subjects without icu therapy (170,200 [iqr 63,970-364,800] vs. 65,745 [iqr 35,620157,700] pg/ml; p < 0.001) (fig. 3). subjects that required ventilatory or vasopressor therapy displayed higher sst2 also (181,950 [iqr 107,700-369,800] vs. 73,620 [iqr 40,030187,200] pg/ml; p < 0.001 and 175,500 [iqr 107,700306,500] vs. 74,755 [iqr 40,030-188,400] pg/ml; p = 0.004) (fig. 5). discussion in the current study, we identified sst2 as novel predictor of survival in subjects with hospital-acquired aki. to establish new biomarkers in aki remains a fundamental goal in clinical nephrology. the majority of biomarker studies aimed (and still aim) to find parameters that allow aki recognition as early as possible. among the most widely studied molecules are neutrophil gelatinase-associated lipocalin (ngal), kidney injury molecule-1 (kim-1), liver-fatty acid binding protein (l-fabp), and the product of urinary tissue inhibitor of metalloprotease-2 (timp-2) and insulin-like growth factor-binding protein 7 (igfbp7) (schrezenmeier and colleagues provided an excellent summary (10).). p=0.004 ss t2 (p g/ m l) 0 2×105 4×105 6×105 8×105 10×105 12×105 age (years) 10 20 30 40 50 60 70 80 90 100 fig. 1 soluble st2 in relation to the age of all included subjects. serum levels of the protein correlated negatively with age. fig. 2 primary endpoints. a) survival; b) dialysis; c) recovery of kidney function. respective p-values are displayed (the bold p-value in “a” indicates a statistically significant difference). a b c survival death 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.024 no dialysis dialysis 0 500000 1000000 1500000 ss t 2 (p g/ m l) p=0.083 co m ple te re co ve ry inc om ple te re co ve ry no re co ve ry 0 500000 1000000 1500000 ss t 2 (p g/ m l) p=0.059 fig. 3 soluble st2 in relation to the acute kidney injury stage according to akin. serum levels of the protein gradually increased from stages i to iii. akin i akin ii akin iii 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.014 sst2 predicts aki survival32 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb fig. 4 morbidities. in total seven morbidities were analyzed in detail: arterial hypertension (a); diabetes mellitus (b); coronary artery disease (cad – c); heart insufficiency (hi – d); pulmonary disease (pd – e); obesity (f); history of neoplasia (g). comparisons were always made between subjects with versus without the respective diagnosis. the only difference in soluble st2 that reached the level of statistical significance was detected between subjects with versus without preexisting heart insufficiency (higher in subjects without the disease – d). a no hypertension hypertension 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.117 b no diabetes diabetes 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.4 c no cad cad 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.249 d no hi hi 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.005 e no pd pd 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.934 f no obesity obesity 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.247 g no neoplasia neoplasia 0 500000 1000000 1500000 ss t2 (p g/ m l) p=0.738 several studies additionally evaluated the prognostic value of certain marker molecules, particularly regarding the prediction of in-hospital survival. hall and colleagues (29) found the urinary concentrations of ngal, kim-1, and il-18 as predictive for the composite endpoint of aki progression and in-hospital death. all markers were measured instantly if the aki criteria were fulfilled. a 2011 published study evaluated both the diagnostic and prognostic potency of urinary ngal (30). subjects that reached the primary (composite) endpoint (aki progression, dialysis, and death) showed higher ngal levels at the time of inclusion. survival prediction through both urinary ngal and kim-1 was also shown by nickolas et al (31). our findings did not only reveal higher sst2 in nonsurvivors but also gradually increased serum levels from aki stages i to iii according to kdigo (12). serum st2 was also higher in subjects that either required vasopressors erfurt et al j circ biomark 2022; 11: 33 © 2022 the authors. published by aboutscience www.aboutscience.eu or ventilatory therapy or icu treatment in general. thus, elevated protein levels are apparently associated with a higher degree of cumulative morbidity under acute circumstances. regarding permanent or preexisting diseases, the only difference occurred between subjects with versus without chronic heart insufficiency. firouzabadi et al (16) failed to show higher or lower sst2 levels in heart insufficiency as opposed to healthy subjects. the individuals in this particular study did however not suffer from aki. regarding cardiac disease, soluble il-33 receptor (sst2) has been shown to correlate with myocardial inflammation and fibrosis in rats with acute myocardial infarction (32). in our study, the predictive value of sst2 was limited to the category survival. tung and colleagues in contrast identified sst2 to be aki predictive in patients with st-segment elevation myocardial infarction (33). in our study cohort, subjects with versus without recovery of kidney function or dialysis did not differ in sst2. the recovery process was only assessed through serum creatinine, a marker that exclusively reflects the amount of glomerular filtration and by no means any adaptive or maladaptive responses within the renal tissue. several studies included outcome analyses of post-aki kidney function. koyner and colleagues (34) identified il-18, urinary albumin to creatinine ratio, and plasma ngal to be associated with a higher risk of aki progression. comparable to our study, measurements were performed in close timely relation to aki onset (at the day of aki diagnosis, at least akin stage i). however, subjects exclusively received cardiac surgery. caironi and colleagues (35) measured plasma proenkephalin a 119-159 (penkid) in >900 septic subjects in order to identify associations with aki onset and recovery of kidney (albumin italian outcome sepsis – albios – trial). plasma penkid was shown as useful not only in aki but also in postaki recovery prediction. the most intriguing difference to our study was the inclusion of subjects with sepsis only. the same applies for the 2018 published kid-sss study (kidney in sepsis and septic shock study), which evaluated the same marker, measured within the first 24 hours after icu admission (36). more than 580 were included. penkid levels were associated with major adverse kidney events (makes); low levels were suggestive for rapid recovery of kidney function. as opposed to sst2, penkid reflects the amount of glomerular filtration with high sensitivity. in an observational cohort study, schunk et al (37) measured the urinary dickkopf-3 (dkk-3)/creatinine ratio in patients that received cardiac surgery. some patients participated in the so-called “renalrip multicenter trial”. in this particular cohort, a urinary dickkopf-3 (dkk-3)/ creatinine ratio of >471 pg/mg was associated with higher risks for aki and persistent renal dysfunction. dkk-3 is particularly secreted by stressed tubular epithelial cells (38). in the 2020 published ruby study finally (39), urinary elevation of the c-c motif chemokine ligand 14 (ccl14) was shown to be predictive for persistent stage iii aki. in the same year, members of the “acute disease quality initiative consensus conference” published “recommendations on acute kidney injury biomarkers” (40). consensus statement number 9 suggests, “… novel biomarkers can be used for prediction of duration and recovery of aki.” the recommendation received grade c (weak grade). subsequently, the authors particularly discussed the penkid and dkk-3 data. whether sst2 will presumably serve as marker of recovery prediction in aki or not still needs to be elucidated more in detail. herein, a heterogeneous group of aki subjects was included, suffering from acute kidney dysfunction of various etiology. the data presented in the current study anyhow suggest a role of sst2 as biomarker of “acute distress”: it predicts post-aki survival and substantially increases in subjects with a higher degree of cumulative morbidity under acute circumstances (e.g., icu therapy, vasopressor administration). in this respect, two studies a b c no icu icu 0 500000 1000000 1500000 ss t 2 (p g/ m l) p<0.001 no vt vt 0 500000 1000000 1500000 ss t 2 (p g/ m l) p<0.001 no vasopressors vasopressors 0 500000 1000000 1500000 ss t 2 (p g/ m l) p=0.004 fig. 5 treatment course. sst2 differed in all tested categories: icu therapy (a), ventilatory (b), and vasopressor therapy (c). the interleukin-33 receptor was detected in significantly higher concentrations if patients received respective measures. icu = intensive care unit; vt = ventilatory therapy. sst2 predicts aki survival34 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb already evaluated the prognostic role of sst2 in sepsis (41,42). the limitations of the current study shall be mentioned. prehospital creatinine values were missing in many subjects. the aki definition according to kdigo (12) did not consider urine volumes since respective information was missing in too many individuals. also, follow-up data after hospital demission were not available. finally, it needs to be evaluated whether or not impaired kidney excretory function potentially modulates circulating sst2 per se. a recently initiated study in sepsis/septic shock will hopefully clarify this particular aspect. in summary, sst2 may become clinically useful for risk stratification in aki patients in the future. a respective study should therefore exclusively focus on aki subjects treated under intensive care conditions. in any case, sst2 has for sure been identified as new candidate for risk prediction in aki. acknowledgment the authors thank jana friedrich for technical assistance. disclosures conflict of interest: the authors declare that they have no conflict(s) of interest. financial support: the study was supported by the jackstädt-stiftung. ethics statement: the study was formally approved by the ethics committee of the medical school of brandenburg (no.: e-0120190820). author contributions: se collected all samples and all patient-related clinical data. he also performed all measurements of sst2. he also assisted in writing. mh provided substantial knowledge and experimental expertise regarding quantification of sst2. so provided substantial knowledge regarding quantification of sst2. ka helped to identify patients and collected patient-related clinical data. sp prepared figures and collected references. or assisted in data analysis and manuscript writing. dp designed the study, 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https://doi.org/10.1007/s00134-010-1773-0 https://www.ncbi.nlm.nih.gov/pubmed/20151106 cbx668791 1..6 research article circulating cell-free dna is a predictor of short-term neurological outcome in stroke patients treated with intravenous thrombolysis alejandro bustamante1, fernando mancha2, hada c macher3, teresa garcı́a-berrocoso1, dolors giralt1, marc ribó4, juan m guerrero3, and joan montaner1,2 abstract circulating cell-free dna (cfdna) has been described as a prognostic marker for several diseases. its prognostic value for short-term outcome in stroke patients treated with intravenous thrombolysis remains unexplored. cfdna was measured on admission in 54 tissue plasminogen activator (tpa)-treated patients and 15 healthy controls using a real-time quantitative polymerase chain reaction assay. neurological outcome was assessed at 48 h. predictors of neurological improvement were evaluated by logistic regression analysis, and the additional predictive value of cfdna over clinical variables was determined by integrated discrimination improvement (idi). stroke patients presented higher baseline cfdna than healthy controls (408.5 (179–700.5) vs. 153.5 (66.9–700.5) kilogenome-equivalents/l, p ¼ 0.123). a trend towards lower cfdna levels was found in patients who neurologically improved at 48 h (269.5 (143.3–680) vs. 504 (345.9–792.3) kilogenome-equivalents/l, p ¼ 0.130). in logistic regression analysis, recanalization at 1 h and cfdna < 302.75 kilogenome-equivalents/l was independently associated with neurological improvement after adjustment by age, gender and baseline national institutes of health stroke scale score. the addition of cfdna to the clinical predictive model improved its discrimination (idi ¼ 21.2% (9.2–33.3%), p ¼ 0.009). these data suggest that cfdna could be a surrogate marker for monitoring tpa efficacy by the prediction of short-term neurological outcome. keywords stroke, biomarkers, circulating dna, thrombolysis, outcome, neurological improvement date received: 14 june 2016; accepted: 11 august 2016 introduction stroke represents the fifth cause of death and one of the leading causes of disability worldwide. 1 nowadays, intravenous thrombolysis with tissue plasminogen activator (tpa) remains the only approved treatment for acute stroke. 2 objective tests predicting tpa response in acute stroke patients, and therefore, short-term neurological outcome, might be used to guide physicians in the indication of endovascular therapies in tpa non-responders or to optimize resources improving patient’s allocation. circulating cell-free dna (cfdna) is found in plasma under several conditions, 3,4 cell death being the major factor 1 neurovascular research laboratory, institut de recerca, hospital universitari vall d’hebron. universitat autonoma de barcelona, barcelona, spain 2 stroke program, institute of biomedicine of seville, hospital universitario virgen del rocio, seville, spain 3 department of clinical biochemistry, hospital universitario virgen del rocio (ibis/csic/sas/university of seville), seville, spain 4 stroke unit, department of neurology, hospital universitary vall d’hebron, barcelona, spain corresponding author: joan montaner, hospital vall d’hebron, passeig de la vall d’hebron, 119-129, 08035 barcelona, spain. email: joan.montaner@vhir.org journal of circulating biomarkers volume 5: 1–6 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416668791 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 3.0 license (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:joan.montaner@vhir.org https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454416668791 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage accounting for cfdna release. 5 several studies have assessed the predictive value of cfdna as a prognostic marker for long-term outcome in stroke patients 6,7 as well as its role in differentiating between ischaemic and haemorrhagic stroke. 8 however, these studies enrolled few tpa-treated patients. therefore, hypothesizing that high cfdna level table 1. baseline characteristics and univariate predictors of neurological improvement at 48 h. neurological improvement all (n ¼ 54) yes (n ¼ 33) no (n ¼ 21) p value age (years) 77.0 (70–82) 77.5 (71.5–82) 77 (56.5–83) 0.804 gender (male) 44.4% 45.5% 42.9% 0.851 hypertension 68.5% 66.7% 71.4% 0.713 diabetes 20.4% 24.2% 14.3% 0.497 dyslipidaemia 35.8% 42.4% 25.0% 0.200 atrial fibrillation 48.1% 48.5% 47.6% 0.951 tobacco use 13.2% 15.2% 10.0% 0.697 coronary disease 25.9% 33.3% 14.3% 0.119 previous stroke 13.0% 6.1% 23.8% 0.096 previous disability 3.8% 3.2% 4.8% 0.999 baseline nihss 14.2 + 7.1 13.5 + 5 15.3 + 9 0.429 sbp 154 + 27 156 + 27 152 + 29 0.591 dbp 81 + 14 82.9 + 14 81.5 + 15 0.0.928 glycaemia 117.5 (97–164) 120 (100–169) 112 (92—129) 0.188 proximal occlusion 44.4% 45.5% 42.9% 0.851 ocsp taci 63.0% 72.7% 47.6% 0.041* paci 25.9% 24.2% 28.6% poci 11.1% 3.0% 23.8% laci 0% – – toast atherothrombotic 18.5% 12.1% 28.6% 0.115 cardioembolic 57.4% 57.6% 57.1% lacunar 0% – – undetermined 22.2% 30.3% 9.5% uncommon 1.9% 0% 4.8% time to tpa (min) 183 + 74 176 + 65 195 + 87 0.383 1-h recanalization 39.2% 54.8% 15.0% 0.004* nihss: national institutes of health stroke scale; sbp: systolic blood pressure; dbp: diastolic blood pressure; ocsp: oxfordshire stroke project classification; taci: total anterior circulation infarct; paci: partial anterior circulation infarct; poci: posterior arterial circulation infarct; laci: lacunar infarct; toast: trial of org 10172 in acute stroke treatment classification; tpa: tissue plasminogen activator. *p < 0.05. figure 1. differences in baseline cfdna levels between different groups. box plots represent median (interquartile range) of cfdna levels between the following comparisons: (a) stroke patients (n ¼ 54) versus healthy controls (n ¼ 15). (b) patients with neurological improvement at 48 h (n ¼ 33) versus patients without neurological improvement at 48 h (n ¼ 21). cfdna: circulating cell-free dna. 2 journal of circulating biomarkers might reflect higher cell death, we aimed to explore the prognostic value of cfdna in acute stroke patients treated with tpa regarding short-term neurological outcome. methods patients and clinical evaluation our target group consisted of acute ischemic stroke patients admitted to the stroke unit of our centre within the first 4.5 h after symptoms onset. fifty-four patients with nonlacunar stroke involving the middle cerebral artery or the basilar artery territories and 15 ageand gender-matched healthy controls were evaluated. all patients received recombinant tpa in a standard dose, and recanalization was assessed at the end of tpa infusion by transcranial doppler with the thrombolysis in brain ischaemia score. 9 all patients underwent a complete workup on neurological assessment and ancillary tests as previously published. 10 neurological outcome was assessed at 48 h, and neurological improvement was defined as a decrease of four or more points in the national institutes of health stroke scale (nihss). 11 written informed consent was obtained from all patients or relatives, and the study protocol was approved by the local ethics committee. blood sampling and cfdna measurement blood samples were taken on admission, before tpa administration (<4.5 h), centrifuged (8 min–2000 � g) and dna from 400 ml of serum was extracted using magna pure and nucleic acid isolation kit i (roche diagnostics, basel, switzerland). cfdna was measured using quantitative polymerase chain reaction (pcr) analysis (lightcycler 480 real-time pcr; roche diagnostics) using lc480 probes master kit (roche diagnostics). b-globin hydrolysis probes system comprises primers beta-globin-354f and beta-globin-455 r and a dual-labelled fluorescent probe betaglobin-402 t. pcr conditions were 95�c for 5 min and 61�c for 15 min for 48 cycles. the final size of the amplicon was 102 bp. the experiment was repeated twice to evaluate the reproducibility of the results. statistical analysis spss v15.0 [chicago, spss inc.] software was used for statistical analysis. normality was assessed by kolmogorov–smirnov table 2. factors associated with cfdna levels. yes no p value gender (male) 383 (173–1435) 346 (159–628) 0.121 hypertension 383 (158–665) 405 (173–999) 0.999 diabetes 185 (127–688) 383 (179–913) 0.512 dyslipidaemia 383 (167–913) 346 (164–657) 0.978 atrial fibrillation 384 (172–672) 309 (151–801) 0.972 tobacco use 597 (237–1447) 346 (158–673) 0.373 coronary disease 321 (147–808) 383 (172–650) 0.961 previous stroke 639 (102–2045) 383 (166–657) 0.632 previous disability 617 (135–1099) 383.5 (167–688) 0.943 proximal occlusion 408 (206–665) 309 (115–801) 0.470 ocsp taci 252 (143–578) 0.041 paci 657 (180–999) poci 1099 (432–1135) laci – toast atherothrombotic 179 (100–371) 0.433 cardioembolic 433 (173–808) lacunar – undetermined 297 (172–575) uncommon – 48-h improvement 227 (135–688) 457 (309–927) 0.123 correlation coefficient p value age r ¼�0.075 0.589 baseline nihss r ¼�0.096 0.492 sbp r ¼ 0.072 0.612 dbp r ¼ 0.095 0.503 glycaemia r ¼�0.167 0.242 time to tpa r ¼�0.065 0.646 nihss: national institutes of health stroke scale; sbp: systolic blood pressure; dbp: diastolic blood pressure; ocsp: oxfordshire stroke project classification; taci: total anterior circulation infarct; paci: partial anterior circulation infarct; poci: posterior arterial circulation infarct; laci: lacunar infarct; toast: trial of org 10172 in acute stroke treatment classification; tpa: tissue plasminogen activator. *p < 0.05. bustamante et al. 3 test. normally distributed variables were analysed by student t test, and non-normally distributed variables were analysed by mann–whitney u or kruskal–wallis tests. in the univariate analysis, intergroup differences were assessed by pearson w2 test for categorical variables. cut-off points were obtained from receiver operating characteristic curves. to build predictive models, all clinical variables associated with neurological improvement at p value <0.15 in univariate analysis (oxfordshire stroke project classification, trial of org 10172 in acute stroke treatment classification, 1-h recanalization, coronary artery disease and previous stroke) were included in a forward stepwise logistic regression analysis. afterwards, cfdna was added by enter method to build a second predictive model. for both models, odds ratios (ors) were adjusted by age, gender and baseline nihss score. comparisons between models were made with integrated discrimination improvement (idi) index. 12 results demographic data are summarized in table 1. cfdna levels trended to be higher in stroke patients compared to controls (408.5 (179–700.5) vs. 153.5 (66.9–700.5) kilogenome-equivalents/l, p ¼ 0.123; figure 1(a)). there were no significant associations between cfdna levels and baseline characteristics of stroke patients (table 2), neither baseline stroke severity or aetiology, although patients with previous stroke and those with previous disability had non-significant higher cfdna levels. regarding neurological outcome, 33 patients (61.1%) improved at 48 h. no patient developed tpa-related complications such as symptomatic haemorrhagic transformation or malignant oedema. patients who improved at 48 h trended to have lower cfdna levels (286.5 (152–688) vs. 526 (382.8–927.5) kilogenome-equivalents/l, p ¼ 0.123; figure 1(b)). a cut-off point of cfdna <302.75 kilogenomeequivalents/l was found to have 81% sensitivity and 55% specificity for the prediction of 48 h neurological improvement. patients with cfdna levels below this cut-off point were significantly more prone to improve at 48 h than those with higher levels (81.8% vs. 46.7%, p ¼ 0.010). in logistic regression analysis, after adjusting by age, gender and baseline nihss score, the presence of arterial recanalization 1 h after tpa (adjusted or (adjor) ¼ 43.8(3.1–620.9), p ¼ 0.005) and cfdna levels <302.75 kilogenome-equivalents/l (adjor ¼ 27.1(2.63–279.2), p ¼ 0.006) were the only independent predictors of 48 h improvement in our cohort. when models including or not the biomarker were compared, assessing cfdna levels resulted in improved discrimination (idi ¼ 21.2% (9.2–33.3%), p ¼ 0.0005; figure 2, table 3). discussion our study showed that baseline cfdna was an independent predictor of neurological improvement at 48 h in stroke patients treated with tpa. moreover, cfdna <302.75 kilogenome-equivalents/l improved the prediction of neurological improvement regarding clinical variables and after adjustment by age, gender and baseline nihss score. although higher cfdna levels have been previously associated with long-term disability and mortality after stroke, 6,7 a diagnostic test, which is able to identify early those patients with a higher chance of short-term neurological improvement, could be easily used by clinicians, to indicate early the rescue procedures (activation of interventional teams, inclusion in clinical trials) for patients with a low chance to improve after intravenous tpa treatment or, conversely, patients with a high chance to improve could be safely discharged from scarce resources such as stroke unit’s beds. high levels of cfdna in acute stroke patients have been associated also with infarct volume or baseline neurological impairment, which seems logical while cfdna is an indicator of cell death. 13,14 in accordance with these data, we found that patients with previous stroke and those with previous disability had higher cfdna levels, which may indicate a sustained inflammatory response or a greater figure 2. comparison between predictive models for 48 h neurological improvement. light bars represent the predictive ability of the clinical model, composed by 1-h recanalization adjusted by age, gender and baseline national institutes of health stroke scale score. dark bars represent the predictive ability of the second model, constructed by adding cfdna levels <302.75 kilogenome-equivalents/l over the only clinical model. the integrated discrimination improvement index was 21.2% (9.2–33.3%), p ¼ 0.0005. cfdna: circulating cell-free dna. 4 journal of circulating biomarkers level of cell death in these patients, although these results did not reach statistical significance. the main finding of our study, however, was an association with short-term outcome. since this association has been noted in tpatreated patients, it might not indicate just a higher infarct size or neurological deficit, but a greater bbb damage as has been described, 8 and therefore a higher chance of complications related to this breakdown in tpa-treated patients such as haemorrhagic transformation or oedema. however, high cfdna levels have also been associated with other conditions related to cell death. 3,4 although in our study cfdna levels were not related to baseline comorbidities or vascular risk factors, we cannot exclude that these differences were related to some conditions not assessed in the present study. our study has some limitations. first, our sample size was relatively small and this fact could be responsible for missing some associations, such as a less number of patients developing neurological worsening or none with tpa-related complications. second, our data from shortterm neurological outcome has been derived from a cohort of tpa-treated patients and extrapolation of the results to all stroke patients should be carefully interpreted. third, final infarct volume was not measured, and so this parameter could not be used for further adjustments, although some other parameter reflecting similar information such as baseline nihss score was used to adjust the model. besides these limitations, cfdna could represent a surrogate marker for monitoring tpa efficacy with the prediction of short-term neurological outcome, improving the prediction that may be done with clinical information. however, our results are still of preliminary nature, and future studies are needed to replicate these results, as well as the possible underlying association with symptomatic haemorrhagic transformation. compliance with ethical research standards all research on human subjects presented in this article was conducted in accordance with the ethical research standards prescribed by the responsible national/institutional committee on human experimentation and with the wma declaration of helsinki as of its seventh revision in 2013. informed consent was obtained from all human subjects participating in the study. acknowledgement neurovascular research laboratory takes part in the spanish stroke research network invictus (rd12/0014/0005). declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work has been funded by instituto de salud carlos iii, grant fis pi15/354, co-financed by the european regional development fund (feder). ab is supported by a rio hortega contract cm13/00265 from the instituto de salud carlos iii. references 1. mozaffarian d, benjamin ej, go as, et al. american heart association statistics committee and stroke statistics subcommittee. heart disease and stroke statistics–2016 update: a report from the american heart association. circulation 2016; 133: e38–e360. table 3. logistic regression analysis and additional predictive value of the model including cfdna for 48-h improvement. a 48-h improvement clinical model clinical þ cfdna model logistic regression 1 h recanalization 9.8 (2.05–46.78); p ¼ 0.004* 43.84 (3.10–620.91); p ¼ 0.005* nihss admission 0.92 (0.83–1.02); p ¼ 0.095 0.93 (0.82–1.05); p ¼ 0.221 age 1.02 (0.97–1.08); p ¼ 0.471 1.01 (0.94–1.09); p ¼ 0.735 gender (male) 2.23 (0.56–8.83); p ¼ 0.254 7.22 (0.75–69.60); p ¼ 0.087 cfdna – 27.1 (2.63–279.2); p ¼ 0.006* idi statistics idi events – 8.9% idi non-events – 12.3% idi – 21.2% (9.2–33.3) p value reference 0.0005* cfdna: circulating cell-free dna; ocsp: oxfordshire stroke project classification; toast: trial of org 10172 in acute stroke treatment classification; adjor: adjusted odds ratio; nihss: national institutes of health stroke scale; idi: integrated discrimination improvement. athe table represents the comparison between either predictive models, including or not the biomarker cfdna. for the logistic regression analysis, variables entered on step 1 (p < 0.05) were ocsp, toast, 1 h recanalization, coronary disease and previous stroke. the table shows just the variables entered in the final step. adjors and 95% confidence intervals are given for each of the variables. *p < 0.005. bustamante et al. 5 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice jcb j circ biomark 2021; 10: 1-8issn 1849-4544 | doi: 10.33393/jcb.2021.2194original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2021 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu infections and deaths exceed 17.8 million and 680,000, respectively. the first studies that analyzed the clinical complications associated with this disease were from china. in this area, many of the patients had mild to moderate symptoms (80%), about 14% had a severe disease course (dyspnea, o 2 saturation ≤93%, pulmonary infiltrates), and about 6% presented with critical progression characterized by respiratory failure, septic shock, and/or multiorgan failure (3). the data accumulated so far from more than 10,000 patients in the european union and in the new york city area show that among the confirmed cases, 30% required admission and 4% required care in intensive care units (icus) due to their critical condition (4,5). in turn, it was observed that mortality is particularly high in the subgroup of patients with advanced age and preexisting comorbidities, among which obesity, hypertension, and diabetes are frequently found (6). it is noteworthy that patients without these associated comorbidities can also present with a critical or severe course of the disease. therefore, the search for early biomarkers to assess the severity of the pathology and its clinical progression is currently necessary to rationalize the use of hospital resources in icus and reduce the mortality associated with covid-19. value of clinical laboratory test for early prediction of mortality in patients with covid-19: the bgm score laura macias-muñoz1*, robin wijngaard1*, bernardino gonzález-de la presa1, josé luis bedini1,3, manuel morales-ruiz1-3, wladimiro jiménez1-3 1department of biochemistry and molecular genetics, biomedical diagnostic center, hospital clinic, barcelona spain 2 institut d’investigacions biomèdiques august pi i sunyer (idibaps), centro de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd), barcelona spain 3department of biomedicine, university of barcelona, barcelona spain *the first two authors are co-authors. both contributed equally to this study. abstract background: covid-19 causes high mortality and long hospitalization periods. the aim of this study was to search for new early prognostic strategies accessible to most health care centers. methods: laboratory results, demographic and clinical data from 500 patients with positive sars-cov-2 infection were included in our study. the data set was split into training and test set prior to generating different multivariate models considering the occurrence of death as the response variable. a final computational method called the bgm score was obtained by combining the previous models and is available as an interactive web application. results: the logistic regression model comprising age, creatinine (crea), d-dimer (dd), c-reactive protein (crp), platelet count (plt), and troponin i (tni) showed a sensitivity of 47.3%, a specificity of 98.7%, a kappa of 0.56, and a balanced accuracy of 0.73. the cart classification tree yielded tni, age, dd, and crp as the most potent early predictors of mortality (sensitivity = 68.4%, specificity = 92.5%, kappa = 0.61, and balanced accuracy = 0.80). the artificial neural network including age, crea, dd, crp, plt, and tni yielded a sensitivity of 66.7%, a specificity of 92.3%, a kappa of 0.54, and a balanced accuracy of 0.79. finally, the bgm score surpassed the prediction accuracy performance of the independent multivariate models, yielding a sensitivity of 73.7%, a specificity of 96.5%, a kappa of 0.74, and a balanced accuracy of 0.85. conclusions: the bgm score may support clinicians in managing covid-19 patients and providing focused interventions to those with an increased risk of mortality. keywords: bgm score, clinical biochemistry, covid-19, mortality prediction, risk score, serum biomarkers introduction the sars-cov-2 virus emerged in the last quarter of 2019 in wuhan, the capital of hubei province of china. the disease caused by sars-cov-2 virus, named covid-19 by the world health organization, has spread rapidly and globally causing a pandemic with unprecedented clinical, humanitarian, and economic repercussions (1,2). in the absence of reliable data on worldwide seroprevalence, the number of confirmed received: october 26, 2020 accepted: december 15, 2020 published online: february 8, 2021 this article includes supplementary material corresponding author: manuel morales-ruiz department of biochemistry and molecular genetics hospital clinic villarroel 170 barcelona 08036 spain morales@clinic.cat https://doi.org/10.33393/jcb.2021.2194 https://creativecommons.org/licenses/by-nc/4.0/legalcode bgm score for covid-19 mortality prediction2 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb several clinical laboratory markers, such as lymphocyte (lymph) count, lactate dehydrogenase (ldh), and d-dimer (dd), are altered in patients with covid-19 (7). other studies have shown significant differences in the concentration of cytokines in blood (interleukin [il]-6, tumor necrosis factor [tnf]-γ, il-8, il-2r) among patients who have required icu admission and patients who do not (8). in turn, infection biomarkers such as c-reactive protein (crp), procalcitonin (pct), and ferritin (fer) increased significantly with the severity of the disease (8). however, despite the research efforts made in the field of laboratory tests, reliable algorithms with early prognostic value have not yet been generated to establish the risk of future complications in patients infected with sars-cov-2. the lack of accurate early prognostic algorithms based on central laboratory testing for covid-19 has spurred researchers to direct their efforts toward the use of omics tools in the search for potential biomarkers. in a first study published by shen et al (9), the combination of proteomics and metabolomics allowed the identification of a panel of 22 proteins and 7 metabolites with predictive power to differentiate mild vs. severe covid-19 with 94% accuracy. a second study published in cell systems (10) showed that european researchers identified 27 differentially expressed proteomics biomarkers associated with different grades of covid-19 severity in hospitalized patients. despite these positive advances, the technical complexity of these omics tools and their high cost limit their applicability in the clinical arena. in the context of the vast scope of the sars-cov-2 crisis and until we achieve sufficient immunization coverage of the population, we believe that the search for new early prognostic strategies must prioritize their applicability and accessibility to most health care centers. in this line, the objective of our study was to generate predictive algorithms for early stratification of patients with covid-19 who may be at the risk of developing severe complications. to this aim, we designed a retrospective cross-sectional single-center study in which we evaluated different predictive algorithms for mortality considering demographic factors, clinical factors, and standard laboratory tests usually present in most central clinical laboratories. materials and methods patient population five hundred patients with covid-19 confirmed by real-time reverse transcription polymerase chain reaction (rt-pcr) in nasopharyngeal exudates were included in this retrospective study. these patients required hospitalization in an icu, internal medicine, or pneumology ward in our hospital between march and june 2020. the clinical and laboratory data that we collected in our database were the first information available within 48 hours after admission of the patient. demographic and clinical data were obtained from our hospital information system (sap patient management). the variables included were: age, sex, smoking and drinking habits, asthma, chronic obstructive pulmonary disease (copd), diabetes mellitus, dyslipidemia, obesity, hypertension, heart failure, ischemic heart disease, hospitalization days, icu stay, and in-hospital death. this study was approved by the ethical committee of the hospital clinic of barcelona and was conducted following the ethical principles of the 1975 declaration of helsinki. the data set is available at the online repository figshare with doi:10.6084/ m9.figshare.13252277. laboratory measurements blood samples were collected in lithium heparin-, ethylenediaminetetraacetic acid-, and citrate-coated blood collection tubes for biochemical, hematological, and coagulation testing, respectively. after centrifugation at 3,000 rpm for 15 minutes, plasma samples were immediately processed. alkaline phosphatase (alp), alanine aminotransferase (alt), aspartate aminotransferase (ast), total bilirubin (tbil), creatinine (crea), fer, gamma-glutamyl transferase (ggt), glucose (glu), ldh, crp, pct, and troponin i (tni) were measured using an atellica solution automated analyzer (siemens healthineers, tarrytown, ny, usa). the intra-assay and inter-assay coefficient of variation was lower than 6% and 8%, respectively, in all cases. hematological parameters (including hemoglobin [hb] and counts of white blood cells [wbc], neutrophils [neu], lymph and platelets [plt]) were analyzed without centrifugation using an advia 2120 (siemens healthineers, tarrytown, ny, usa). finally, the sysmex 5100 (sysmex, kobe, japan) was used for dd, prothrombin time (pt), and partial thromboplastin time (ptt) analysis. all the parameters were measured in the core laboratory of the hospital clinic of barcelona according to the manufacturer’s instructions. statistical analysis categorical variables were expressed as numbers and percentages and compared using the chi-square test. continuous variables were expressed as median and interquartile range (iqr) and were compared by the mann-whitney wilcoxon test. the strength of the relationship between the laboratory parameters was assessed using the pearson or spearman correlation coefficients. the multivariate statistical analyses conducted were logistic regression (lr) (11), classification tree (ct) through the cart algorithm (12), and artificial neural network (nnet) (13). missing data were imputed via bagged tree models (11), and the data set was then split into a training and test set. the optimal parameter for each model was determined in the training set, calculating the best averaged predictive performance after 10-fold cross-validation. additionally, to the previous multivariate models, we generated a computational method, called the bgm score, which provides the survival probability of a patient with covid-19 considering the variables age, crea, dd, crp, plt, and tni. we modulated the survival probability of the bgm score as a probabilistic event depending on the survival probability given by the lr (p s(lr) ), the ct (p s(ct) ), and the nnet (p s(nnet) ) models generated from our data set. further, the p s(lr) , p s(ct) , macias-muñoz et al j circ biomark 2021; 10: 3 © 2021 the authors. published by aboutscience www.aboutscience.eu and p s(nnet) were multiplied by their corresponding model accuracies (ac (lr) , ac (ct) , and ac (nnet) ; respectively), giving the following equation for the bgm score survival probability: p s(bgm) = (ac (lr) × p s(lr) ) ∩ (ac (ct) × p s(ct) ) ∩ (ac (nnet) × p s(nnet) ). additionally, the following constraints were applied to the p s(bgm) to incorporate the best predictive features of the lr, ct, and nnet models: p ac p ac p ac ps s s s( ) ( ) ( ) ( ) ( ) ( ) ( ) . . bgm lr lr lr lr ct ct= × < × ≥ × <0 0 5 0 5 0.. ; . . ( ) ( ) ( ) ( ) ( ) ( 5 1 0 5 0 5 = × ≥ × < × ac p ac p ac p s s s ct ct nnet nnet nnet nneet) .≥      0 5 p ac p ac p ac p ac s s s s( ) ( ) ( ) ( ) ( ) ( ) ( ) ( . .bgm lr lr ct ct ct ct= × < × × ≥ 0 5 0 5 nnnet nnet lr lr nnet nne ) ( ) ( ) ( ) ( ) ( . ; . × <      = × < × p ac p ac p s s s 0 5 0 5 tt ct ct nnet nnet ) ( ) ( ) ( ) ( ) . . ac p ac p s s × < × ≥ 0 5 0 5 sensitivity, specificity, positive predictive value, negative predictive value, kappa, total accuracy, and balanced accuracy ([sensitivity+specificity]/2) were calculated for each model considering only the test set (99 cases). all the statistical analyses were performed using public libraries from the comprehensive r archive network (cran; http://cran.rproject.org) rooted in the open-source statistical computing environment r, version 3.6 (http://www.r-project.org/). a p-value <0.05 was considered statistically significant. we wrote an interactive web application using the r shiny package (14) that implements the four multivariate models that we generated in our study. this web application can be used to calculate the survival probability for a patient with covid-19 and is freely available at the link “https://bgm-hoc. shinyapps.io/shiny_covid_clinic/.” results five hundred subjects with a confirmed diagnosis for covid-19 formed the study population. overall, the median age of the patients was 64 years, 42.6% were female, and patients were discharged within a median of 10 days. the most common comorbid conditions were hypertension (44.2%), dyslipidemia (31.2%), and diabetes mellitus (18.8%). among the patients recruited, 23.4% required icu, and 19.4% died during follow-up. the demographic, clinical, and laboratory results of the patients corresponding to the first 48 hours after admission are summarized in tables i and ii. we evaluated a panel of 12 biochemical, 5 hematological, and 3 coagulation biomarkers for each patient. as shown in figure 1, we detected the presence of high significant correlations in ast-alt (r = 0.9, p < 0.001) and wbc-neu (r = 0.8, p < 0.001). to avoid the presence of multicollinearity bias in multivariate analysis, we excluded the variables ast and neu for future calculations. we included the rest of the biochemical, demographic, and clinical variables in the multivariate lr model that we designed considering the occurrence of death as a response variable and that we generated performing 10-fold cross-validation. among all the explanatory table i demographic and clinical characteristics of the patients at the first 48 hours after admission total n = 500 nonsurvivors n = 97 (19.4%) survivors n = 403 (80.6%) p-value female, n (%) 213 (42.6%) 42 (43.3%) 171 (42.4%) 0.96752 age, median (iqr) 64 (54-76) 80 (72-86) 61 (50-72) 6.50e-25 active smoker, n (%) 25 (5.0%) 6 (6.2%) 19 (4.7%) 0.73589 active alcohol consumer, n (%) 15 (3.0%) 4 (4.1%) 11 (2.7%) 0.69568 asthma, n (%) 25 (5.0%) 5 (5.2%) 20 (5.0%) 1.00000 copd, n (%) 26 (5.2%) 8 (8.2%) 18 (4.5%) 0.21092 diabetes, n (%) 94 (18.8%) 30 (30.9%) 64 (15.9%) 0.00111 dyslipidemia, n (%) 156 (31.2%) 41 (42.3%) 115 (28.5%) 0.01247 obesity, n (%) 33 (6.6%) 4 (4.1%) 29 (7.2%) 0.38628 hypertension, n (%) 221 (44.2%) 64 (66.0%) 157 (39.0%) 2.63e-06 atrial fibrillation, n (%) 37 (7.4%) 10 (10.3%) 27 (6.7%) 0.31576 heart failure, n (%) 23 (4.6%) 11 (11.3%) 12 (3.0%) 0.00112 ischemic heart disease, n (%) 31 (6.2%) 14 (14.4%) 17 (4.2%) 0.00045 icu admission, n (%) 117 (23.4%) 30 (30.9%) 87 (21.6%) 0.06921 hospitalization days, median (iqr) 10 (6-18) 6 (3-11) 12 (7-20) 9.86e-09 copd = chronic obstructive pulmonary disease; icu = intensive care unit; iqr = interquartile range. bgm score for covid-19 mortality prediction4 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb variables included initially in the model (variables shown in tabs. i and ii) only age, crea, dd, crp, plt, and tni were early independent predictors of mortality after admission (tab. iii), according to the model selection rule based on the akaike information criterion (aic). this multivariate model yielded a sensitivity of 47.3%, a specificity of 98.7%, a kappa of 0.56, and a balanced accuracy of 0.73 for identifying patients with a high risk of mortality. we obtained these performance characteristics using a validation set of 99 cases that were not used for training the model. despite the high negative predictive value of the lr model (0.9), we observed a low sensitivity that suggests that the model was sensitive to class imbalance. to improve our classification performance without downsampling, we next generated two additional multivariate models based on a different paradigm of categorization, cts with the cart algorithm (the implementation in r is called rpart) and nnets. considering all the variables from tables i and ii, the cart ct yielded tni (cutoff value of 44 ng/l), age (cutoff value of 79 years), dd (cutoff value of 700 ng/ml), and crp (cutoff value of 15 mg/dl) as the most potent early predictors for stratifying patients with a high vs. low risk of mortality (fig. 2). these cutoff values were similar to their reference intervals implemented for clinical diagnosis (tni < 45.2 ng/l, dd < 500 ng/ml, and crp < 1 mg/dl). this model outperformed the lr model with a sensitivity of 68.4%, a specificity of 92.5%, a kappa of 0.61, and a balanced accuracy of 0.80 for identifying patients with a high risk of mortality in our validation set. despite this performance comparison, one common characteristic in these models was that both yielded clinical laboratory measurements as the most powerful predictors of mortality in patients with covid-19. in general, the outputs of lr and ct are intuitive and easy to implement as predictive algorithms in the clinical setting. however, nnets are black boxes regarding the contribution of the explanatory variables to the output of the response table ii biochemical, hematological, and coagulation parameters determined within the first 48 hours after admission total (n = 500) nonsurvivors (n = 97) survivors (n = 403) p-value normal range n median (iqr) n median (iqr) n median (iqr) alp, u/l 491 (98.2%) 68 (55-90) 94 (96.9%) 76 (58-108) 397 (98.5%) 68 (45-86) 0.00342 46-116 alt, u/l 492 (98.4%) 29 (19-50) 95 (97.9%) 25 (18-48) 397 (98.5%) 29 (19-51) 0.30541 5-40 ast, u/l 487 (97.4%) 38 (27-60) 93 (95.9%) 49 (31-73) 394 (97.8%) 37 (26-56) 0.00974 5-40 tbil, mg/dl 490 (98.0%) 0.5 (0.4-0.7) 93 (95.9%) 0.6 (0.4-0.9) 397 (98.5%) 0.5 (0.4-0.7) 0.01930 0.2-1.2 crea, mg/dl 500 (100%) 0.89 (0.71-1.1) 97 (100%) 1.11 (0.87-1.83) 403 (100%) 0.86 (0.69-1.04) 4.60e-10 0.3-1.3 fer, ng/ml 383 (76.6%) 602 (266-1278) 71 (73.2%) 914 (376-1533) 312 (77.4%) 559 (240-1190) 0.00221 15-200 ggt, u/l 491 (98.2%) 39 (25-78) 94 (96.9%) 39 (27-92) 397 (98.5%) 40 (24-76) 0.43835 5-40 glu, mg/dl 500 (100%) 107 (96-130) 97 (100%) 125 (104-160) 403 (100%) 105 (95-123) 9.95e-07 65-110 ldh, u/l 477 (95.4%) 316 (244-418) 87 (89.7%) 432 (276-583) 390 (96.8%) 301 (240-395) 6.99e-07 <234 crp, mg/dl 499 (99.8%) 7.3 (3.4-15.1) 97 (100%) 14.3 (7.9-22.8) 402 (99.8%) 6.3 (2.8-11.9) 1.51e-12 <1 pct, ng/ml 416 (83.2%) 0.11 (0.05-0.25) 77 (79.4%) 0.37 (0.17-1.05) 339 (84.1%) 0.09 (0.04-0.18) 2.40e-19 <0.5 tni, ng/l 410 (82.0%) 8.5 (3.9-22.8) 77 (79.4%) 45.0 (20.1-112.1) 333 (82.6%) 6.8 (3.2-14.9) 1.05e-21 <45.2 hb, g/l 500 (100%) 137 (126-147) 97 (100%) 130 (114-143) 403 (100%) 139 (128-148) 0.00015 120-170 plt, ×109/l 500 (100%) 180 (137-227) 97 (100%) 166 (112-220) 403 (100%) 182 (146-231) 0.00130 130-400 wbc, ×109/l 500 (100%) 6.0 (4.5-7.7) 97 (100%) 7.2 (5.4-9.8) 403 (100%) 5.8 (4.4-7.3) 1.89e-05 4-11 lymph, ×109/l 500 (100%) 0.8 (0.6-1.1) 97 (100%) 0.6 (0.4-0.9) 403 (100%) 0.9 (0.6-1.2) 5.10e-11 0.9-4.5 neu, ×109/l 500 (100%) 4.6 (3.2-6.3) 97 (100%) 5.7 (4.5-8.3) 403 (100%) 4.2 (3.1-5.7) 5.17e-08 2-7 dd, ng/ml 450 (90.0%) 700 (400-1300) 79 (81.4%) 1500 (800-4350) 371 (92.1%) 600 (400-1000) 1.96e-11 <500 pt, sec 273 (54.6%) 12.8 (12.1-13.6) 57 (58.8%) 13.1 (12.3-14.3) 216 (53.6%) 12.8 (12.1-13.5) 0.02284 9.9-13.7 ptt, sec 225 (45.0%) 29.7 (27.4-31.8) 55 (56.7%) 29.1 (26.7-31.3) 170 (42.2%) 30.0 (27.9-32.0) 0.11795 23.5-32.5 alp = alkaline phosphatase; alt = alanine aminotransferase; ast = aspartate aminotransferase; crea = creatinine; crp = c-reactive protein; dd = d-dimer; fer = ferritin; glu = glucose; ggt = gamma-glutamyl transferase; hb = hemoglobin; ldh = lactate dehydrogenase; lymph = lymphocyte count; neu = neutrophil count; pct = procalcitonin; plt = platelet count; pt = prothrombin time; ptt = partial thromboplastin time; tbil = total bilirubin; tni = troponin i; wbc = white blood cell count. macias-muñoz et al j circ biomark 2021; 10: 5 © 2021 the authors. published by aboutscience www.aboutscience.eu variable. therefore, we have to limit the selection of variables to generate manageable nnet models applicable to most clinical settings regardless of their limitations in their laboratory tests portfolio. in this context, and with the intention intending of improving our lr model, we generated an artificial nnet including only the variables that remained as early independent predictors of mortality in the lr model: age, crea, dd, crp, plt, and tni. supplemental figure 1 shows the optimal architecture of the neural model that was obtained after 10-fold cross-validation. the nnet model yielded a sensitivity of 66.7%, a specificity of 92.3%, a kappa of 0.54, and a balanced accuracy of 0.79 for identifying patients with a high risk of mortality in our validation set. this performance was comparable to that achieved by our previous ct algorithm. the three algorithms we generated can be divided into two groups considering their sensitivity and specificity. the model with the highest specificity was lr, while the ct and nnet models presented lower specificity but a higher sensitivity. these differences in predictive accuracy led to our developing a new hybrid model in combination with the lr, ct, and nnet models to incorporate the best predictive features of each. as described in the material and methods, our model, called the bgm score, calculates a survival probability for patients with covid-19 by multiplying the survival probabilities of the three previous models corrected by their accuracies. we assessed the bgm score performance in terms of prediction accuracy over the validation set, yielding a sensitivity of 73.7%, a specificity of 96.5%, a kappa of 0.74, and a balanced accuracy of 0.85 for the prediction of covid19 patients who died. figure 3 shows the statistical comparison of the accuracies of the four models, where it can be fig. 1 correlation plot. the plot shows the correlation between all the clinical laboratory results obtained within the first 48 hours after patient admission. the bar on the right depicts the equivalence between the color code, and the value of the correlation coefficients shown for each pair of laboratory parameters. alp = alkaline phosphatase; alt = alanine aminotransferase; ast = aspartate aminotransferase; crea = creatinine; crp = c-reactive protein; dd = d-dimer; fer = ferritin; glu = glucose; ggt = gamma-glutamyl transferase; hb = hemoglobin; ldh = lactate dehydrogenase; lymph = lymphocyte count; neu = neutrophil count; pct = procalcitonin; plt = platelet count; tbil = total bilirubin; tni = troponin i; wbc = white blood cell count. table iii logistic regression coefficients for predicting the response variable “survival vs. death” for patients with covid-19 odd ratio std. error z-statistic p-value age 2.012 1.281 5.665 1.47e-08 crea 2.573 1.012 2.465 0.01370 dd 2.086 1.000 2.493 0.01266 crp 2.012 1.828 4.942 7.71e-07 plt 0.697 1.079 –3.025 0.00249 tni 1.210 1.000 2.051 0.04031 crea = creatinine; crp = c-reactive protein; dd = d-dimer; plt = platelet count; tni = troponin i. bgm score for covid-19 mortality prediction6 © 2021 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb seen that the bgm score model significantly outperformed all the other models. our hybrid model corroborates the prognostic value of the clinical laboratory tests for patients with covid-19. a web application with the implementation of the bgm score model is available at “https://bgm-hoc.shinyapps.io/ shiny_covid_clinic/”. discussion this retrospective study identified risk factors for death in hospitalized patients with covid-19. older age, lower lymph and plt in addition to higher crea, dd, crp, and tni were independent risk factors for death among patients. taking this into account, we developed a predictive algorithm for mortality (bgm score) considering standard laboratory tests usually present in most central clinical laboratories. concerning biochemical, hematological, and coagulation parameters, our findings are in consonance with those previously described. for instance, a recent published work by sisó-almirall et al (15) revealed that ldh, dd, and crp were the most important laboratory parameters significantly associated with adverse outcomes, evaluated as death or icu admission. alterations in coagulation parameters, in particular high dd level and low plt, have been linked with severe covid19 patients (16,17). these disorders reflect the hypercoagulable state present in poor prognosis, which could promote microthrombosis in the lungs, as well as in other organs (18). elevated tni levels are frequent in patients with covid-19 and have been significantly associated with fatal outcomes. several mechanisms may explain this phenomenon: viral myocarditis, cytokine-driven myocardial damage, microangiopathy, and unmasked coronary artery disease. sars-cov-2 uses angiotensin-converting enzyme 2 (ace2) as its entry receptor and subsequently downregulates ace2 expression. this mechanism may complicate the clinical course mediated through inflammatory response, endothelial dysfunction, and microvascular damage (19). early monitoring of immunological biomarkers is an important basis to guide treatment strategies in covid-19. in this study, crp was the only immunological biomarker assessed significantly related to mortality. recently, a meta-analysis including 16 independent studies highlighted the importance of crp as a possible biomarker for mortality due to covid-19 infection (20). furthermore, the study by wang (21) showed that crp levels were positively correlated with lung lesion and disease severity in the early stage of covid-19. lymphopenia is a common feature in patients with covid-19. significant decreases in t-cell counts have been observed in patients with severe disease (22). up to now, the underlying mechanisms leading to the observed lymphopenia are little known and better understanding will provide insight into better management of such patients (23). a high serum crea level is a frequently observed complication in nonsurvivor inpatients (24). it has been described that around 20% of patients admitted to an icu require renal replacement therapy 15 days after illness onset (25). previous studies reported comorbidities to be one of the most important risk factors associated with increased disease severity (6,7,26). likewise, our study reported a significant association between mortality and some of the collected comorbidities, including diabetes, dyslipidemia, hypertension, heart failure, and ischemic heart disease. despite these differences found between survivors and nonsurvivors, none of our prediction models did include any comorbidity since the clinical laboratory measurements were stronger predictors of mortality in patients with covid-19. fig. 2 multivariate classification tree analysis. troponin i (tni), age, d-dimer (dd), and c-reactive protein (crp) were the most powerful predictors. the number 0 represents survival and 1 represents death. for each square (leaves), survival and death probabilities are represented with decimal numbers at the left and the right side, respectively, and the percentage represents the number of cases that is split between the leaves of tree partitions. units: tni, ng/ml; age, years; dd, ng/ml; crp, mg/dl. fig. 3 comparison of the accuracy values of the different multivariate models. logistic regression (lr), decision tree (dtree), neural network (nnet), and the bgm models. the plot shows the accuracy value for each model (horizontal line) and their corresponding 95% confidence interval (vertical lines). *p < 0.05 vs. all the models, and #p < 0.05 vs. the logistic regression model. macias-muñoz et al j circ biomark 2021; 10: 7 © 2021 the authors. published by aboutscience www.aboutscience.eu age was the only nonlaboratory-related variable associated with symptom aggravation in our study. this is in accordance with previous publications reporting age to be the most important predictor of death in patients with covid-19 (27). immunosenescence is defined as the declined ability of elderly patients to react properly upon infection, to initiate and maintain an adequate protective immune response, and to develop immunological memory (28). thus, the severity of viral infections (e.g., influenza, respiratory syncytial virus) is notably increased among older adults compared to younger individuals, and more acute and long-term sequelae often develop as a result (29,30). other studies have been published using machine learning models to predict the mortality in covid-19 patients, and some of them were included in systematic reviews and metaanalysis (31). our study presents some common points with these publications since the predictors used in the bgm score were also identified to be relevant predictors of mortality in other models, supporting their significant association with adverse patient outcomes. one of our study’s strengths is that we have used a relatively large sample size compared with the research items cited in the meta-analysis, including a substantial number of nonsurvivors. also, our final algorithm, the bgm score, only includes a small number of simple laboratory measurements, which makes our model easy to implement in the routine clinical practice. it’s noteworthy that all the variables of our study were collected in the first 48 hours of admission. hence, our model can provide an early detection of patients at high risk of death, favoring early interventions. our study has several limitations. first, it was a retrospective single-center study, which may lead to biased results. second, we recruited only patients with moderate or severestage disease and not asymptomatic or mild-stage disease. therefore, prospective multicenter studies including patients with various stages of disease are warranted to confirm the reliability of the bgm score model. the effects the pandemic is causing on medical resources worldwide highlight the need to develop early predictor models capable of detecting which patients can be managed safely at district hospital or can benefit from domiciliary hospitalization programs and which ones will need intensive care. therefore, identifying risk factors at presentation that predict the likelihood of disease progression will be useful to: (1) increase the therapeutic effect in patients with a risk of higher disease progression and (2) reduce the mean hospitalization time in patients not at risk. to conclude, we have developed an easy-to-use model comprising biochemical, hematological, and coagulation parameters presented in most clinical laboratories able to predict the survival probability of a patient with covid-19 with high accuracy. this web tool may support clinicians in managing this infectious disease and providing focused interventions to patients with covid-19 at a higher risk of death. disclosures financial support: this work was supported by grants to mm-r (pid2019-105502rb-100) and to wj (rti2018-094734-b-c21) from dirección general de investigación científica y técnica and agència de gestió d’ajuts universitaris i de recerca (sgr 2017/2019). the work was cofinanced by feder of european union. the centro de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd) is funded by instituto de salud carlos iii. conflict of interest: the authors have no financial relationships to disclose relevant to this study. references 1. the lancet. emerging understandings of 2019-ncov. lancet. 2020;395(10221):311. crossref pubmed 2. zhu n, zhang d, wang w, et al; china novel coronavirus investigating and research team. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med. 2020;382(8): 727-733. crossref pubmed 3. world health organization. report of the who-china joint mission on coronavirus disease 2019 (covid-19). online (accessed september 2020). 4. european centre for disease prevention and control. novel coronavirus disease 2019 (covid-19) pandemic: increased transmission in the eu/eea and the uk – sixth update. online 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abstract myasthenia gravis (mg) is an autoimmune disease leading to varying degrees of skeletal muscle weakness. it is caused by specific antibodies directed against definite components in the postsynaptic membrane at the neuromuscular junction (nmj), such as the acetylcholine receptor (achr) and the muscle-specific kinase (musk) receptor. in clinical practice, mg patients may be classified into three main subgroups based on the occurrence of serum autoantibodies directed against achr or musk receptor or antibody-negative. as the mg subgroups differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies, they could benefit from targeted treatment as well as the detection of other possible disease biomarkers. we performed proteomics on plasma fractions enriched in low-abundance proteins to identify potential biomarkers according to different autoimmune responses. by this approach, we evidenced a significant reduction of vitronectin in mg patients compared to healthy controls, irrespective of the autoantibodies nmj target. the obtained results were validated by monoand two-dimensional western blotting analysis. vitronectin is a multifunctional glycoprotein involved in the regulation of several pathophysiological processes, including complement-dependent immune response, coagulation, fibrinolysis, pericellular proteolysis, cell attachment, and spreading. the pathophysiological significance of the reduction of plasma vitronectin in mg patients has yet to be fully elucidated. it could be related either to a possible deposition of vitronectin at nmj to counteract the complement-mediated muscle damage at this level or to a parallel variation of this glycoprotein in the muscle extracellular matrix with secondary induced alteration in clustering of achrs at nmj, as it occurs with variation in concentrations of agrin, another extracellular matrix component. the clinical value of measuring plasma vitronectin has yet to be defined. according to present findings, significantly lower plasma values of this glycoprotein might be indicative of an impaired complement-dependent immune response. keywords myasthenia gravis, autoimmunity, vitronectin, proteomics, biomarkers date received: 19 february 2019; accepted: 18 august 2019 1 department of biomedical sciences, university of sassari, viale san pietro, sassari, italy 2 department of medical, surgical and experimental sciences, university of sassari, viale san pietro, sassari, italy 3 diagnostic laboratory of neuroimmunolgy, u.o. neurologia iv, i.r.c.c.s. fondazione istituto neurologico “c. besta”, milano, italy corresponding author: marilena formato, dipartimento di scienze biomediche, university of sassari, viale san pietro 43, 07100 sassari, italy. email: formato@uniss.it journal of circulating biomarkers volume 8: 1–9 ª the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454419875912 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://orcid.org/0000-0002-8356-894x https://orcid.org/0000-0002-8356-894x mailto:formato@uniss.it https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454419875912 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage introduction myasthenia gravis (mg) is a chronic autoimmune neuromuscular disease characterized by varying degrees of weakness of the skeletal muscles caused by autoantibodies against some proteins of the neuromuscular junction (nmj) involved in nerve signal transduction. 1,2 according to the autoantibodies target, 3–5 patients can be subgrouped in anti-muscle acetylcholine receptor antibody positive (achrab þ ), with a prevalence of about 80% (70% of cases detectable with routine assays and 5–10% with more-sensitive, cell-based assays); anti-musclespecific kinase antibody positive (muskab þ ), with a prevalence up to 10% (more frequent in the mediterranean area than northern europe, possibly owing to a combination of genetic and environmental factors); and anti-lowdensity lipoprotein receptor-related protein 4 antibody positive (lrp4 þ ), detected in 1–5% of patients with mg of any type and in 7–33% of mg patients without achr and musk antibodies. there is also a minority of patients called double seronegative (dsn) patients, because they do not have detectable antibodies against achr or musk. other known autoantibodies targets are agrin, titin, the voltage-gated k þ channel kv1.4, ryanodine receptor, collagen q, and cortactin. finally, there is a further subtype of patients in which the target of the autoimmune response is not known yet. three main mechanisms underline the loss of functional achrs directly resulting in the destruction of muscle end plate: the binding of divalent antibodies causing crosslinking of achrs and accelerated internalization and degradation of the receptor; direct sterical blocking of acetylcholine binding sites; complement-mediated lysis of postsynaptic membrane resulting in functional impairment of achrs and reduction of voltage-gated na þ channels number, leading to increased action potential threshold. 6 musk protein is essential for nmj formation and agrininduced achr clustering. 7 musk antibodies have been demonstrated to inhibit achr clustering. 8 moreover, they are able to cause experimental autoimmune mg. 9,10 recently, lrp4 has been identified as a new autoantigen in dsn patients. 11,12 lrp4 acts as a receptor for agrin 13 and is required for agrin-induced activation of musk, achr clustering, and nmj formation. 14 actually, no commercial tests for lrp4 antibodies are available and standardization of clinical setting has not been achieved yet. 3 mg patients differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies 3 accounting for the significant variability among mg subgroups. however, targeted therapeutic and prognostic studies are rare. in this respect, identification of new disease biomarkers could help in tailoring specific treatments for clinical management of this pathology. the aim of this study was to identify potential plasma biomarkers of mg in patients with different autoimmune responses, by applying two-dimensional electrophoresis (2-de) analysis coupled with mass spectrometry (ms) to a plasma fraction enriched in low-abundance proteins. we applied a recently developed technology, based on hexapeptide combinatorial ligand libraries, to increase the low-abundance proteins concentration with simultaneous minimal capture of the high abundance species. 15 materials and methods sample collection forty-two consecutive patients diagnosed with generalized mg referred to the neuroimmunological laboratory of the neurological clinic (department of medical, surgical and experimental sciences, university of sassari) for autoantibody testing were recruited in the period from january 2016 to december 2016. clinical features or severity of disease were evaluated according to the myasthenia gravis foundation of america (mgfa) clinical classification. 16 in particular, the diagnosis of mg was clinically based on fluctuating muscle weakness, worsening with repetitive activities while improving with resting, clearly responding to acetylcholinesterase inhibitors in the absence of other alternative diagnoses with or without findings of impairment of neuromuscular transmission on single fiber emg and on the repetitive stimulation test of motor nerves. all patients were under symptomatic therapy with pyridostigmine, an inhibitor of acetylcholinesterase, at the recruitment. furthermore, some of them were under immunosuppressive therapy with corticosteroids and/or azathioprine, as reported in table 1. twenty-four healthy volunteers, matched for age and sex with patients, were enrolled as controls. informed consent was obtained before enrolment. exclusion criteria were the presence of renal, hematological, liver, or other associated chronic autoimmune diseases. peripheral venous blood samples were drawn into plastic syringes, under sterile conditions, and immediately transferred into tubes containing ethylenediamine tetraacetic acid�k3-salt (k3 edta). plasma was separated from whole blood by centrifugation at room temperature at 1000 � g for 10 min and stored at �80�c until assayed. anti-achr and anti-musk antibodies were measured either by radioimmunoassay kit (rsr limited, avenue park, pentwyn, cardiff, uk) or enzyme linked immunosorbent assay (elisa) kit (rsr limited). lrp4 autoantibodies were detected by a cellbased assay with hek293 cells transfected with human lrp4 fused to green fluorescent protein. human igg were detected with cy3-conjugated anti-human igg antibody. cells were examined on laser scanning confocal microscope system (nikon instruments europe b.v). of 42 patients, 18 were achrab þ , among which 2 patients were also found positive for anti-lrp4 antibodies, 8 patients were muskab þ , and 16 were negative for both anti achr and musk antibodies and referred to as dsn, 2 of which were positive for anti-lrp4 antibodies. all study participants gave informed written consent to the study. institutional review board approval was obtained 2 journal of circulating biomarkers (protocol no. 2195/2/ce, 09/12/2015). the study was conducted in accordance with the ethical principles of the current declaration of helsinki. plasma low-abundance proteins enrichment to reduce the very high dynamic range of protein concentration in plasma and to perform differential protein expression analysis of the lower expressed fraction, proteominer™ enrichment kit (bio-rad, hercules, california, usa), which exploits nonspecific interactions with a large, highly diverse bead-based library of combinatorial peptide ligands, was used according to the manufacturer’s instructions with slight modifications. 17 the hexapeptides baits are potentially able to interact with any protein present in a complex proteome. the system works in large overloading conditions in which abundant proteins saturate their ligand sites on beads, whereas trace proteins are table 1. characteristics of 42 mg patients and relative abundances of plasma vitronectin obtained by two-dimensional electrophoresis analysis. patientsa age gender mgfa clinical classification immunosuppressive therapy vitronectin (relative abundance) achrabþ 1 65 f iib corticosteroids 456.19 2 61 f iia — 26,970.9 3 64 f iia corticosteroids, azathioprine 4249 4 85 m iia dsn 343.65 5 80 f iiib — 7479.01 6 91 f iiib — 7812.01 7 79 f iia azathioprine 299.87 8 80 f iva — 907.01 9 50 f iiib — 8500.03 10 40 f ivb corticosteroids 3484.04 11 51 m iiib — 387.54 12 61 f iiib corticosteroids, azathioprine 6030.09 13 43 f iiia corticosteroids, azathioprine 16,526.1 14 83 m ii — 4273.02 15 53 f iiia corticosteroids, azathioprine 7659 16 84 f iib corticosteroids, azathioprine 7852.04 17* 89 m ii — 586.07 18* 67 f iiib — 1860 muskabþ 19 84 f iv corticosteroids 15,019.9 20 52 f iib corticosteroids 1488 21 50 f iiib corticosteroids, azathioprine 17,971.6 22 49 f iiib corticosteroids, azathioprine 1739.08 23 79 f iiib — 940.08 24 45 f iiib — 2023.05 25 82 f iiib — 2680.05 26 68 m iib corticosteroids 5560 dsn 27 50 m ii — 8363.08 28 63 f iiib — 1783.04 29 63 f iia corticosteroids 5334.09 30 69 f iia — 2627.02 31 89 m iib — 5180.03 32 69 m iib corticosteroids, azathioprine 1281.01 33 91 m iiia — 566.1 34 59 m iia — 313.06 35 47 m iia — 12,596.4 36 83 f iib azathioprine 1357.01 37 28 f iia 13,856.5 38 64 m iiib corticosteroids, azathioprine 2726.02 39 60 f iia — 13,573.2 40 91 m iib — 6443.07 41** 47 f iia corticosteroids, azathioprine 7620.02 42** 76 m iib corticosteroids, azathioprine 1661.04 mg: myasthenia gravis; achrab þ : acetylcholine receptor antibody positive; mgfa: myasthenia gravis foundation of america; lrp4ab þ : lipoprotein receptor-related protein 4 antibody positive; muskab þ : anti-muscle-specific kinase antibody positive. aall patients were under symptomatic therapy with pyridostigmine, an inhibitor of acetylcholinesterase, at the recruitment. *achrabþ/lrp4abþ patients. **dsn/lrp4abþ patients. lepedda et al. 3 enriched, thus reducing the dynamic protein concentration range. 15 it means that, by means of this process, the most abundant plasma proteins, including apo ai, are “equalized” and, therefore, they do not differ among enriched samples. briefly, 1 ml plasma sample was incubated with the functionalized beads for 2 h at room temperature followed by two consecutive elution steps with 150 ml of an elution buffer containing 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (chaps), 8 m urea, and 5% acetic acid. then, eluate was concentrated and dialyzed against a buffer containing 8 m urea and 4% chaps using amicon ultra-0.5 ml centrifugal filter units (millipore, billerica, massachusetts, usa). protein concentration was determined using dc protein assay kit (bio-rad), according to the manufacturer’s instructions, using bovine serum albumin as the standard. proteins were stored at �20�c until analysis. differential protein expression analysis two-dimensional electrophoresis (2-de) was performed as previously described. 17–19 briefly, 300 mg of proteins was applied to 70 mm immobilized ph gradient (ipg) strips (ph 3–10, bio-rad) for 6 h by rehydration loading at 20�c and subsequently focused at 50 ma/ipg strip for 22 kvh at 20�c in a protean isoelectric focusing (ief) cell (biorad). following ief, proteins were in-gel reduced by incubating ipg strips with a 50 mm tris buffer containing 6 m urea, 30% glycerol (v/v), 3% sodium dodecyl sulfate (sds) (w/v), and 1% dithiothreitol (dtt) (w/v), followed by ingel alkylation, using the same solution containing 2.5% iodoacetamide (w/v) in place of dtt. each step was performed keeping strips under continuous shaking for 15 min. then ipg strips were sealed, with 0.5% low melting point agarose (w/v) in sds running buffer at the top of second dimension gels (8 � 7� 0.1 cm3). sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page) was carried out on 12%t, 3%c polyacrylamide gels for 15 min at 50 v followed by 90 min at 150 v in a mini-protean tetra cell (bio-rad). gels were then fixed in 30% ethanol (v/v) and 2% phosphoric acid (v/v) solution for 1 h, washed twice in 2% phosphoric acid (v/v) solution for 10 min, equilibrated in 18% ethanol (v/v), 2% phosphoric acid (v/ v), and 15% ammonium sulfate (w/v) solution for 30 min, and then stained in the same solution added with 0.02% coomassie brilliant blue g250 (w/v) for 48 h. gel images were acquired using gs800 calibrated densitometer (biorad) at 36.3 mm resolution. image analyses were performed using pdquest 2-d analysis software v8.0.1 (bio-rad). 2-de proteome maps were normalized by assigning to each detected spot a relative value corresponding to the ratio of single spot volume to the volume of all spots in the gel after background subtraction. in-gel digestion and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (maldi-tof ms) analysis protein identification was obtained by a ms analysis service (nurex s.r.l., sassari, italy). spots of interest were excised from gels with sterile pipette tips, destained with a solution containing 2.5 mm nh4hco3 and 50% acetonitrile (v/v), dehydrated with 100% acetonitrile, and dried at room temperature before proteolytic treatment. tryptic digestion was performed by incubating dried spots in 5 mm nh4hco3 buffer containing 10 ng/ml trypsin overnight at 37�c. the resulting peptides were mixed with an equal volume of a-cyano-4-hydroxycinnamic acid saturated solution (40% acetonitrile (v/v) and 0.1% trifluoroacetic acid (v/v)) and applied as a microcrystalline thin film onto a stainless steel 96-spot maldi target. mass analyses were performed using a maldi micromx-mass-spectrometer (waters, manchester, uk), according to the tuning procedures suggested by the manufacturer. peak lists were generated using protein lynx global server v.2.2.5 (waters) and searched against the swiss-prot human database (version 57.4) using mascot (http://www.matrixscience.com). research parameters included taxa (homo sapiens), trypsin digest, monoisotopic peptide masses, iodoacetamide modifications, one missed cleavage by trypsin, and a mass deviation of 50 ppm. only protein identifications with significant mascot scores (p < 0.05) were taken into consideration. western blotting analysis western blotting (wb) analysis was performed as previously described 17–19 ; 5 mg of low-expressed plasma proteins–enriched fraction and an equivalent of 0.25 ml of plasma from untreated samples were resolved by sdspage on 5%t, 3%c stacking gel, and 12%t, 3%c polyacrylamide separating gel. with regard to 2-de wb analysis, 50 mg of pooled enriched fractions and 1 ml of pooled plasma samples from each mg patients subgroup and from control group were resolved as described above. subsequently, resolved proteins were electroblotted onto hybond-p pvdf membranes (ge healthcare, uppsala, sweden) at 250 ma for 1.5 h. afterward, membranes were incubated with blocking solution (pbs, 0.1% tween-20, 3% nonfat dry milk) for 1 h at room temperature, followed by overnight incubation at 4�c, either with a rabbit monoclonal anti-vitronectin (clone ep873y, ab45139, millipore), diluted 1:20,000 with blocking solution, or with a goat polyclonal anti-apolipoprotein ai antibody (ab740, millipore), diluted 1:4,000, for data normalization. then, after 30-min washing (pbs, 0.1% tween-20), membranes were incubated with anti-rabbit (dilution 1:5,000, bio-rad) or anti-goat (dilution 1:5,000, bio-rad) horseradish peroxidase (hrp)-conjugated secondary antibody solution for 1 4 journal of circulating biomarkers h at room temperature. following further membrane washing, proteins were revealed by enhanced chemiluminescence (amersham ecl prime western blotting detection reagent; ge healthcare) using chemidoc xrs system (bio-rad) for data acquisition. densitometric analysis was performed using quantity one 4.6.3 software (bio-rad). statistical analysis student’s t-test analysis was performed to evaluate variations in protein expression levels among mg subgroups and healthy controls. statistical significance was set at p < 0.05. results patients with clinical diagnosis of mg were classified into three subgroups, achrab þ , muskab þ , and dsn patients, based on the presence of antibodies against different nmj targets (table 1). among them, four patients were also positive for anti-lrp4 antibodies. as the presence of high-abundance proteins in plasma samples (e.g. albumin and igg) makes the detection of mediumand lowabundance proteins extremely challenging, 15 we performed an enrichment in low-expressed proteins using hexapeptide ligand libraries. following sample elution, an additional dialysis step was necessary to remove acetic acid from the elution buffer and optimize isoelectric focusing/separation in the first dimension of 2-de. 17 this approach led to a reduction of the most abundant plasma proteins (85% of albumin was depleted) and to an increase of the less represented ones, with a total number of protein spots detected of about 2.4 times higher in treated samples, enabling the differential expression analysis of the less represented proteins (figure 1). comparative 2-de analysis of lowabundance proteins enriched plasma samples, using pdquest 2-d analysis software v8.0.1, coupled with maldi-tof ms, allowed us to identify vitronectin as differentially expressed between mg patients and healthy controls (uniprotkb/swiss-prot accession number: p04004.1). in particular, vitronectin was significantly reduced in all mg patients subgroups as compared to healthy controls (�54% in achrabþ, p ¼ 0.012; �58% figure 1. representative 2-de maps of plasma low-abundance proteins-enriched fractions from healthy controls (a), achrabþ mg patients (b), muskabþ mg patients (c), and dsn mg patients (d) after mass compatible coomassie brillant blue g-250 staining. molecular weight standards (all blue prestained protein standards, bio rad) were run alongside focused strips in the second dimension. circled spots train, corresponding to vitronectin, was differentially expressed between mg patients and controls. 2-de: twodimensional electrophoresis; achrabþ: acetylcholine receptor antibody positive; muskabþ: anti-muscle-specific kinase antibody positive; mg: myasthenia gravis. lepedda et al. 5 in muskab þ , p ¼ 0.010; �65% in dsn, p ¼ 0.002) (figure 2), but no significant difference was evidenced in vitronectin expression among mg subgroups. furthermore, no association between vitronectin expression and severity of disease, according to mgfa clinical classification, or immunosuppressive therapies was found (table 1). one-dimensional wb analysis, confirming 2-de findings, showed a significant down-expression of vitronectin in all myasthenic patients (figure 3) being �39% in achrabþ (p < 0.001), �40% in muskabþ (p < 0.001), �43% in dsn (p < 0.001) enriched samples, �57% in achrabþ (p < 0.001), �70% in muskabþ (p < 0.001), and �67% in dsn (p < 0.001) whole plasma samples. these reductions were observed in mg patients irrespective of their autoimmune response. apo ai expression was used for wb data normalization. further confirmation came from twodimensional wb analysis on pooled plasma samples both as a whole and after enrichment treatment (figure 4). discussion mg is an autoimmune disorder mainly mediated by autoantibodies against postsynaptic membrane proteins at the figure 2. bar chart reporting relative abundances of vitronectin obtained by 2-de maps image analysis of enriched plasma samples from healthy controls, achrabþ mg patients, muskabþ mg patients, and dsn mg patients. data are reported as mean + sd. *achrabþ versus controls, �54%, p value ¼ 0.012; **muskabþ versus controls, �58%, p value ¼ 0.020; ***dsn versus controls, �65%, p value ¼ 0.002. 2-de: two-dimensional electrophoresis; achrabþ: acetylcholine receptor antibody positive; mg: myasthenia gravis; muskabþ: anti-muscle-specific kinase antibody positive; sd: standard deviation. figure 3. wb analysis of vitronectin expression in both enriched ((a) and (c)) and whole ((b) and (d)) plasma samples from healthy controls, achrabþ mg patients, muskabþ mg patients and dsn mg patients. (a) bar chart reporting relative abundances of vitronectin in enriched fractions normalized for apolipoprotein ai levels, obtained by image analysis of wb profiles. (b) bar chart reporting relative abundances of vitronectin in whole plasma samples obtained by image analysis of wb profiles. (c) mono-dimensional wb profiles of both vitronectin and apolipoprotein ai in enriched plasma fractions. (d) mono-dimensional wb profiles of vitronectin in whole plasma samples. data are reported as mean + sd. *achrabþ vs controls, �39%, p value < 0.001; ** muskabþ vs controls, �40%, p value < 0.001; ***dsn vs controls, �39%, p value < 0.001; #achrabþ vs controls, �57%, p value < 0.001; ##muskabþ vs controls, �70%, p value < 0.001; ###dsn vs controls, �67%, p value < 0.001. 6 journal of circulating biomarkers nmj, such as achr and musk receptor. the diagnosis relies on both electrophysiology and the detection of specific autoantibodies in serum. there is a subset of clinically diagnosed myasthenic patients, referred to as dsn, who are negative for the presence of both antibodies. as mg patients subsets differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies, targeted therapies should be mandatory. 3 in this respect, the identification of new tissue or humoral biomarkers of mg might be of great value for both diagnosis and specific treatment. our aim was to identify potential plasma markers associated with the immune derangements of the different mg subtypes. to this end, we applied 2-de coupled with ms, which allows for comparing the expression/abundance of hundreds of proteins at the same time, to identify differentially expressed plasma proteins between mg patients and healthy controls, following plasma enrichment of lowabundance protein components. to date, only few studies have dealt with identification of molecular markers in mg disease by proteomic analysis, but none of them used plasma-enriched samples to detect mediumand lowabundance proteins. 20–22 by this approach, we documented a statistically significant reduction of plasma vitronectin expression in all mg patients subgroups as compared to healthy controls that did not correlate neither with the severity of the disease nor with the immunosuppressive therapy. although it could represent a limitation of our study, vitronectin reduction in plasma of mg patients, described for the first time in this study, might be interesting and deserves further studies addressed to the evaluation of its relevance/roles in the pathogenesis or progression of mg disease. vitronectin, also known as serum spreading factor, is a multifunctional glycoprotein present in blood, urine, and figure 4. 2-de wb analysis of enriched plasma sample pools from healthy controls (a), achrab þ mg patients (b), muskab þ mg patients (c), and dsn mg patients (d). circled trains of spots correspond to vitronectin. wb: western blotting; achrabþ: acetylcholine receptor antibody positive; mg: myasthenia gravis; 2-de: two-dimensional electrophoresis; muskab þ : anti-muscle-specific kinase antibody positive. lepedda et al. 7 amniotic fluid, 23 which plays a role in the regulation of both complement and coagulation systems. 24–26 it is also part of the extracellular matrix, substantially enriched at sites of injured, fibrosing, inflamed, and cancer tissues. 27 in addition, it contributes to tissue remodeling and healing by regulating proteolysis, cell adhesion, migration, and survival in the injured and stressed tissue. 28–31 interestingly, significantly reduced serum vitronectin concentrations were found associated with extreme physical stress in healthy people, 32 a condition comparable, in some way, to muscle fatigue in mg, as well as with liver disease, renal disease, and systemic lupus erythematosus (sle). 33 however, the pathophysiological significance of this reduction has not been elucidated up to now, although it has been suggested that the lower levels of plasma vitronectin in chronic liver disease might be in relation to its higher levels in injured tissues undergoing healing process. 34 in fact, the structure and function of vitronectin are locationdependent. 35 soluble monomers of this glycoprotein exist in plasma, and insoluble oligomers are associated with the extracellular matrix where they may interact with several cell surface receptors, including the urokinase-type plasminogen activator receptor and the av integrin receptor subclass. 35 conversion of vitronectin from a solutionphase to a matrix-phase protein seems to occur via interactions with plasminogen activator inhibitor-1, by which vitronectin is able to bind several partners, including complement proteins. 35 the pathophysiological significance of the reduction of vitronectin in plasma of mg patients that we have documented is unknown. on account of the aforementioned findings, it could be related either to a possible deposition of vitronectin at nmj to counteract the complement-mediated muscle damage at this level or to a corresponding variation of this glycoprotein in the muscle extracellular matrix with secondary induced alteration in clustering of achrs at nmj, as it occurs with variation in distribution of agrin, an extracellular heparan sulfate proteoglycan. 36 indeed, both vitronectin and agrin act on the same cells surface receptors, integrins. 28 elucidation of the underlying causes related to reduced plasma vitronectin expression could be provided by immunohistochemical analyses on patient muscle biopsies, and this surely deserve further studies. the clinical value of measuring plasma vitronectin remains to be defined. vitronectin is present in normal plasma at concentrations of 200–400 mg/ml.28 considered its involvement in immune defense via interaction with the terminal complex of complement, and the low expression of vitronectin in plasma of mg patients and other autoimmune diseases, such as sle, plasma values of this glycoprotein below 200 mg/ml might be indicative of an impaired complement-dependent immune response. conclusions by a proteomic approach on plasma samples enriched in low-abundance proteins, we evidenced a significant reduction of vitronectin expression in all mg patients subgroups compared to healthy controls, irrespective of the autoantibodies target and the severity of the disease. as it cannot allow for discriminating between the different mg clinical manifestations, it could represent a limitation of this study. nonetheless, due to vitronectin involvement in a number of biological activities, its plasma reduction in mg, described for the first time in our study, might be interesting and deserves further studies. in particular, the potential roles of this glycoprotein in the impaired complementdependent immune response and/or in clustering the achrs at nmj should be addressed. acknowledgment the authors wish to thank dr socrates j tzartos for support in lrp4 data, laboratory of molecular neurobiology and immunology, department of biochemistry, hellenic pasteur institute (athens, greece). declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: aj lepedda thanks regione autonoma della sardegna for its finantial support (por—fse 2014-2020—asse prioritario 3 “istruzione e formazione”—obiettivo tematico: 10, priorità d’investimento: 10ii, obiettivo specifico: 10.5, azione dell’accordo di partenariato 10.5.12—c.u.p. j86c18000270002). the authors thank the university of sassari for its financial support (fondo di ateneo per la ricerca 2019). orcid id antonio junior lepedda https://orcid.org/0000-0002-8356894x references 1. drachman db. myasthenia gravis. new engl j med 1994; 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/pagesize [612.000 792.000] >> setpagedevice cbx786159 1..16 review placental protein 13: an important biological protein in preeclampsia ranjeeta gadde1, dayanand cd1, and sr sheela2 abstract placental protein 13 (pp13), a glycan binding protein predominantly expressed in syncytiotrophoblast, dimeric in nature, lacks n-terminal signal peptide, bypasses the endoplasmic reticulum, and secretes into maternal circulation as exosomes or microvesicles. pp13 has jelly roll fold conformation with conserved carbohydrate recognition domain which specifically binds to b-galactosides of the glycan receptors during placentation. pp13 binds to glycosylated receptors on human erythrocytes and brings about hemagglutination by the property of lectin activity; other functions are immunoregulation and vasodilation during placentation and vascularization. the gene lgals13 located on 19q13.2 comprising four exons expresses a 32-kda protein with 139 amino acid residues, pp13. impaired expression due to mutation in the gene leads to a nonfunctional truncated pp13. the low serum levels predict high risk for the onset of preeclampsia or obstetric complications. hence, pp13 turned to be an early marker for risk assessment of preeclampsia. the recombinant pp13 and monoclonal antibodies availability help for replenishing pp13 in conditions with low serum levels and for detection and prevention of preeclampsia, respectively. keywords placental protein 13, preeclampsia, eclampsia, jelly roll fold, syncytiotrophoblast date received: 30 december 2017; accepted: 28 may 2018 introduction preeclampsia is a pregnancy-specific disorder characterized by hypertension and proteinuria after 20 weeks of gestation. 1 although the exact etiology of the disorder is still not known, the impairment in the early placentation is associated with onset of preeclampsia that complicates up to 2–8% of all the pregnancies.2 prediction, understanding of underpinning mechanism and prevention of preeclampsia, is still not clear. hence, preeclampsia and eclampsia are the leading causes of maternal, perinatal morbidity, and mortality. 3,4 placental protein 13 (pp13) is a carbohydrate binding protein synthesized in the syncytiotrophoblast, which is involved in early placentation process. 5,6 it is a member of galectin family with a conserved carbohydrate recognition domain (crd). 7–9 the specificity of this site for b-galactosides-containing glycoconjugates10–12 is established and plays a significant role in biological events such as implantation and embryogenesis. 13,14 the biological specificity of the pp13 present in the apical membrane of the syncytiotrophoblast to the glycans of the membrane and extracellular matrix proteins such as annexin ii is a primary requisite for the placental implantation to the endometrium. 15 pp13 also binds to band g-actin within trophoblasts, which facilitates the migration of trophoblasts toward the placental bed and also increases the release of prostacyclins for vascular remodeling of maternal spiral arteries in early placentation. 16 from the immunological point of view, for an effective placentation, pp13 induces the apoptosis of 1 department of biochemistry, sri devaraj urs medical college, kolar, india 2 department of obstetrics and gynecology, sri devaraj urs medical college, kolar, india corresponding author: dayanand cd, department of biochemistry, sri devaraj urs medical college, sduaher, tamaka, kolar, karnataka 563101, india. email: cd8905@yahoo.co.in journal of circulating biomarkers volume 7: 1–16 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418786159 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:cd8905@yahoo.co.in https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418786159 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage maternal t cells to progress the deepening of implantation. 17 the expression of pp13 is also important for differentiation and syncytialization of the villous trophoblast which is vital for the release of placental hormones and immune proteins for embryo development and immune tolerance. 14,18 during pregnancy, various factors are the reason for the release of pp13 into the maternal circulation. the available studies indicated the low levels of pp13 in serum/plasma generally in first trimester of pregnancy. however, the levels gradually increase as gestation progresses. 19–22 the exact reason for the decreased pp13 levels in first trimester and increased levels in the third trimester is not known which is in line with the etiology of the preeclampsia. preeclampsia is associated with impaired placentation, 23,24 evidence of low serum levels of pp13, and other heterogeneous causes, hence became a major cause for morbidity and mortality of fetus and mother. 25,26 therefore, an attempt was made to summarize the information on pp13 in this article. historical perspective hans bohn (1928–2014) gets the credit for successful isolation of factor xiii (fibrin stabilizing factor) of blood coagulation system from human placenta. after discovery, he developed a quantification method to demonstrate factor xiii deficiency that affects fibrin cross-linkage and fibrin stabilizing property and is associated with wound healing after injury or surgery. 27 during isolation and purification procedure of factor xiii, he noticed that a side fraction yielded human placental lactogen. throughout this period, there was a rise in the research interest related to physiological and pathological aspects of human placenta. 28 this trend motivated him to explore the content of human placenta from a biochemical perspective. the galectin generally is numbered sequentially and hence the names for galectin 1–12 were assigned. 29 the new member of the galectin family pp13 has been designated as galectin 13. 28 pp13 is one among the 56 proteins isolated from the placenta by hans bohn. later on, in 1983, a research contribution by his coworkers led to the purification and characterization of placental tissue proteins 13 and 17. 30 he characterized many pps for their physicochemical characteristics and also developed immunoassay to quantify these proteins of diagnostic significance. several pps such as pp4 (annexin-v), pp5 (tissue factor pathway inhibitor-2), pp10 (plasminogen activator inhibitor-2), and pp13 (galectin 13) were studied for their amino acid sequence and biological functions with respect to biological regulators of pregnancy process. 31–33 thereafter, he continued his research in collaboration with other scientists to investigate sequencing, structural, and molecular biological characterization of several pps including pp13. this research contribution significantly improved the understanding of biological role and diagnostic importance in pregnancy complication, malignancies, and other placental and pregnancy-related protein research. subsequently, pp research continued to add more information for further discoveries and improvements in clinical diagnostics and patient care. biosynthesis, secretion, and physical and chemical characteristics of pp13 biosynthesis of pp13 biosynthesis of pp13 occurs on free ribosomes in the cytoplasm of syncytiotrophoblast and is present in soluble form in the brush border apical plasma membrane as evident by staining techniques. 34,35 the placental gene lgals13 located on the long arm of chromosome 19 at loci q13.2 encodes pp13 protein exclusively. 36 lgals13 gene and its transcribing unit codes for pp13 at 50 end that has promoter region followed by comprising four exons designated as e1–e4 (figure 1). the full length of cdna encoding human placental pp13 from expression library (gen bank; acc. no: af117383.1) predicted the molecular mass and the amino acid composition of the cloned protein which corresponds to 578 bp insert with a 417-bp open reading frame encoding a 139-amino acid protein. 37,38 secretion of pp13 nascent pp13 lacks n-terminal signal sequence; hence, it bypasses the translocation route to endoplasmic reticulum and golgi bodies. instead, pp13 utilizes the “non-classical” secretory pathway or unconventional routes to reach the maternal circulation either through vesicular shedding or direct translocational system. studies showed that in the syncytiotrophoblast, pp13 is highly co-localized with cytoskeletal protein actin, annexin ii, placental alkaline phosphatase, a glycosylphosphatidylinositol-anchored lipid raft resident protein, and cd71, a non-raft plasma membrane protein. 28 the actin cytoskeleton polymerization and associated motor proteins are the driving force for variety of cellular processes for transportation of galectins. 39,40 the various secretory routes utilized by galectins in the syncytiotrophoblast are either direct translocational or extra cellular vesicular transport in the form of microvesicles (40–100 nm) and exosomes (0.1–1 mm).41–44 a lot has been already published on the release of pp13 by shedding syncytiotrophoblast microparticles. 28,35 a recent study has indicated that the amount of pp13 released via the exosomes and microvesicles is actually decreased in preeclampsia. the process regulating the release of these organelles has not been extensively investigated and is not well known. therefore, future studies must evaluate pp13 biomarker potential in association with syncytiotrophoblast extracellular vesicles and exosomes. 45 even though pp13 primarily originated from placenta, it has been demonstrated that its expression was also noticed in human healthy liver, spleen, kidney, and bladder tissues 2 journal of circulating biomarkers and also in few pathological conditions of liver adenocarcinoma, neurogenic tumor, and malignant melanoma. 46 physical and chemical characteristics of pp13 human pp13 is the member of the b-galactoside binding soluble-type galectin super family and is a relatively small protein with a molecular weight of 32 kda consisting of 139 amino acid residues. it is a homodimer stabilized by disulfide bonds. each polypeptide chain molecular weight is 16 kda. 47 the primary structure of pp13 has 69% homology to human eosinophil charcot–leyden crystal structure (galectin 10). 48 the secondary structure is similar to prototype galectins such as galectin 7 and galectin 10 or charcot–leyden crystal protein. 49,50 the c-terminal crd is of chimera-type galectin (galectin 3). 51 the typical structural alignment of these proteins showed five-stranded (f1–f5) and six-stranded (s1–s6 a/ s6b) b-sheets stabilized by one or two alpha-helices at their ends (figure 3). the structural variation in sequence of primary structure of pp13 and galectin 10 is evident but the secondary structure is found to be identical except for f1 beta-sheet which is longer by one residue in case of pp13. from repository, the model was available from brookhaven protein data bank source with account number if87. 46 the dimeric nature of pp13 consists of two structurally similar disulfide bonds between four cysteine residues (cys19, cys92, cys136, and cys138) present on the surface of pp13. the alignment of galectin crd motif showed highly conserved sequences of 13 residues, of which 8 residues (arginine 53 (arg53), asparagine 65 (asn65), tryptophan 72 (trp72), glu75, arginine 55 (arg55), his57, val63, and thr77) play an important role in sugar binding. from these eight residues, four residues are of identical type (val63, asn65, trp72, and glu75) and three are conservatively substituted (arg53, arg55, and his57) in pp13. 46 with respect to substrate automatic docking with crd region particularly on the concave face of s4–s6 b-sheets, several carbohydrates as ligand to protein conformations were analyzed. they are n-acetyllactosamine, lactose, mannose, n-acetylgalactosamine, galactose, and so on, from which n-acetyllactosamine has more binding energy (�26.9 kcal/mole) due to high van der waals forces and figure 1. location, arrangement, and structure of lgals13 gene.53 gadde et al. 3 strong stacking interaction. this interaction has affinity to highly conserved trp72 residue and strong hydrogen bond with conserved arg55, asn65, and glutamine 75 (gln75) residues in crd of pp13. this results in pp13 specificity for the sugar provides the cellular selection of binding molecules and its orientation toward binding. figure 2. expression, secretion, and functions of pp13. (1) bio-synthesis: pp13 transcripts are translated on the free ribosomes in the cytoplasm of syncytiotrophoblast and dimerized as prototype pp13 with formation of carbohydrate recognition domain. (2) intracellular protein–protein interactions: actin cytoskeleton and motor proteins in the cytoplasm interaction drive the translocation of pp13 to the apical membrane of the syncytiotrophoblast and extracellular space. (3) interaction with cell surface glycans and lattice formation: pp13 cross-links with glycoconjugates on cell surfaces and forms galectin–glycan lattice. (4) cross-linking and signaling. (5) cell–cell interactions: the cross-linked dimeric pp13 and lattice lead to intracellular signaling cascade to promote cell–cell interactions. (6) cell–matrix interactions: binding to b-galactoside residues of extracellular matrix proteins generates response to pp13 functions. (7) cytoplasm of syncytiotrophoblast. (8) apical membrane of syncytiotrophoblast. (9) extracellular matrix. (10) decidual membrane. pp13: placental protein 13.28 figure 3. (a) pp13 “jelly-roll” fold structure indicating fiveand six-stranded b-sheets linked by two alpha helices. (b) eight amino acid residues on the carbohydrate recognition domain of pp13. (c) stereoscopic view of pp13 molecule with bound n-acetyllactosamine to the residues in the carbohydrate recognition domain (a455, asn65, and gln75) and the tryptophan ring (trp72) playing a key role in stacking interactions46 pp13: placental protein 13; asn65: asparagine 65; gln75: glutamine 75; trp72: tryptophan 72. 4 journal of circulating biomarkers computational analysis indicated that the positions of serine and tyrosine phosphorylation sites are serine 48, tyrosine 41, and tyrosine 80 close to the highly conserved crd. 46 pp13 is a glycoprotein with a 0.6% carbohydrate content and has the electrophoretic mobility same as that of albumin; isoelectric point (pi) is in the range of 4.7–4.8 and sedimentation coefficient is 3.1 svedberg or s value. the primary structure of pp13 has two linear polypeptide chains. each chain has methionine at the c-terminal and asparagine at the amino terminal. 30 annexin ii and b/gactin were identified as ligands for pp13 by the mass spectrometry; hence, these proteins are considered to play a key role in placentation and maternal artery remodeling, respectively. 52 structure of lgals13 gene and its mutations the lgals13 gene encoding for pp13 is expressed by the placental syncytiotrophoblast on chromosome 19 (19q13.1). lgals13 gene and its transcribing unit codes for pp13 at 50 end that has promoter region followed by comprising four exons designated as e1–e4, which consist of base pairs in e1(60 bp), e2 (72 bp), e3 (211 bp), and e4 (251 bp) spaced by introns. intronic regions vary between 499 bp and 1834 bp in length. exon 4 and part of exon 3 of lgals13 gene exclusively code for the entire crd. 53 many studies reported that downregulated placental expression of lgals13 is due to mutations in the gene, characterized by single nucleotide polymorphism (snp) and single nucleotide deletion (delt221 mutation) in exon 3, mutations of the exon–intron boundaries (dex-2 mutation), and an snp in promoter region. the dna variants result in the expression of truncated protein which cannot bind carbohydrates. the implication of altered, misfolded, and nonfunctional protein may be associated with a variety of obstetrical syndromes such as preeclampsia where the normal implantation and placentation are generally disrupted. in support of this genetic evidence, decreased pp13 levels and its messenger rna (mrna) expression of lgals13 in the first trimester of pregnancy, 36,54–57 snp (221delt) with low pp13 level is seen in adverse pregnancy conditions 58–60 and intronic polymorphisms are associated with pp13 level and impaired of lysophospholipase activity. 52,59–61 the promoter variants with snps were found in women with increased risk to develop preeclampsia. 62–64 transcription factor activating enhancer binding protein 2 alpha (tfap2a) is a protein encoded by human tfap2a gene. it binds to specific dna sequence and recruits transcription machinery. it specifically binds to g regions at �98 position than to a in the promoter region and induces promoter expression as revealed by pp13 promoter reporter expression studies after transfecting bewo cells with pp13 having “a” or “c” in the �98 promoter position.65 decreased placental expression of glial cell missing-1 and estrogen-related receptor g genes, encoding transcription factors, regulates lgals13 expression in the syncytiotrophoblast which is associated with trophoblast fusion and syncytium formation. 18 according to the available literature, the following few studies clearly demonstrate that decreased pp13 level or impairment in expression and its possible genetic causes are seen in women with preterm preeclampsia. ongoing research to determine whether the presence of polymorphic pp13 variants may serve to improve our understanding of the underlying pathophysiology is of paramount importance. the mutation in the promoter region and the exons of lgals13 gene is responsible for lower expression of pp13 mrna and the low levels of plasma pp13 in the first trimester. therefore, genetic analysis of lgals13 and plasma pp13 level is one among the several biomarkers studied to understand preeclampsia and its complications at early stage to facilitate antenatal checkup. quantification of pp13 the quantity of pp13 from serum, plasma, tissue fluids, and placental tissue was carried out by employing immune-based technique—enzyme-linked immunosorbent assay (elisa) which is currently widely employed procedure for pp13 assay from biological samples. the results published from this assay method in several research studies generate varied information in different trimesters of normotensive pregnancies and subjects likely to develop preeclampsia and eclampsia. measurement of pp13 is not a routine investigation in clinical medicine but gained much attention in pregnancyrelated research. in many studies, quantification of pp13 in biological sample was done either by elisa 19–22,66–76 or by dissociation-enhanced lanthanide fluorescence immunoassay (delfia) methods. 77–86 the underlying principle of elisa for the estimation of pp13 is an enzyme-linked antibody sandwich method. the test principle is that the microtiter plate is pre-coated with an antibody specific to pp13. standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to pp13. avidin conjugated to horseradish peroxidase is added to each microplate well and incubated. after the addition of tetramethylbenzidine substrate solution, only those wells that contain pp13, biotin-conjugated antibody, and enzyme-conjugated avidin will exhibit a change in color. the enzyme–substrate reaction is terminated by the addition of sulfuric acid solution and the color change is measured at a wavelength of 450 nm. the concentration of pp13 in the samples is then determined by comparing the absorbance of the samples with the standard curve. delfia technique is also used to estimate pp13 in many research contexts. the unit for representing the quantification is picogram per milliliter or nanogram per milligram of tissue. it is reported that the normal term placenta yields gadde et al. 5 nearly 3.7 mg of pp13. 28 the advantage of elisa method over delfia is that the reducing agent dithiothreitol in the sample dilution buffer of the elisa keeps the pp13 in monomer form which prevents its affinity to the sugar residues of various blood proteins especially of the erythrocytes. functions of pp13 placental implantation placental implantation involves attachment of the blastocyst to the endometrial epithelium and subsequent invasion in order to form a functional placenta. it occurs through the trophoblast, an outer layer of blastocyst, that subsequently develops into syncytiotrophoblast, which is involved in implantation and placentation. 87 it is known that placentation also involves pp13, a member of group of pregnancy-related proteins. it is highly expressed in placenta at maternal–fetal interface and may play an important role in the maintenance of pregnancy (e.g. embryo implantation, maternal–fetal immune tolerance, placentation, and vascular remodeling). the fact that structural and functional characteristics of this protein and its role are important in placental development and regulation pathways is receiving increased interest in recent times. 88 the carbohydrate binding specificities were extensively studied by numerous extracellular methods such as solid phase assays, 89,90 surface plasmon resonance, 91 fluorescence polarization, 92 frontal affinity chromatography, 93 and isothermal calorimetry. 94 b-human chorionic gonadotropin induced trophinin is an intrinsic membrane protein expressed on the apical membrane of the trophectoderm cells which is involved in adhesion of blastocyst to the endometrial epithelium during implantation process. 95,96 in addition to this, binding of pp13 to the glycosylated receptor is also important for adhesion and signaling in placentation. based on sugar binding assay results, it has been found that pp13 has the strongest binding affinity to the n-acetyllactosamine, mannose, and n-acetylglucosamine residues of carbohydrate moiety in glycoproteins and glycolipids present on placenta. 97 an automatic docking study done using flexx module of sybyl determined that the carbohydrate molecules such as n-acetyllactosamine, lactose, mannose, n-acetylgalactosamine, and galactose are located at different ligand positions at the crd. 98–100 another study has also reported that all the carbohydrate conformations were characterized by their binding energies 101 and pp13 has the highest van der waals interaction with n-acetyllactosamine (�26.9 kcal/mol). a strong stacking interaction with the aromatic ring of trp72 and three strong hydrogen bond interactions (<2.5 å) with arg55, asn65, and gln75 residues in crd of pp13 were detected. the conserved arg53 and arg55 in the crd play a critical role in the interactions between pp13 and the lactose moiety. mannose has the lowest van der waal interaction with the pp13 but no hydrogen bond interactions were reported (table 1). 46 sugar binding assays to determine the affinity property found that pp13 exists as homodimer stabilized by disulfide, a bond linkage, which is favorable for hemagglutination in terms of lectin activity. reducing agents diminish dimerization and reduce sugar binding affinity and hemagglutination activity. this unique property of pp13 affects its biological activity in the placenta. 102 in vivo, placentally expressed purified pp13 was found to be phosphorylated. experiments done with placental pp13/galectin 13 showed that it contains phosphorylation sites. computational analysis indicated sites for serine/tyrosine kinase phosphorylation at positioning ser48 (44–57) and tyr41 (37–45) and tyr80 (76–84) close to crd domain. the carbohydrate binding affinity was observed to be the same in pp13-b (placentally expressed phosphorylated) and pp13-r (bacterially expressed non-phosphorylated). the only possible linkage of phosphorylation is to lysophospholipase activity. since limited information is available on phosphorylation and functional properties of pp13, it is necessary to establish the exact role of phosphorylated pp13. 103 regarding the hemagglutination process, several research reports (table 2) showed that pp13 has the tendency to bind to b-galactosides located on the terminal positions of abo blood group antigens. immunoassay staining intensity of placental tissue showed variable pp13 binding to rbc antigens of respective blood groups a, b, and o. 34 research reports pertaining to relationship between different blood groups and preeclampsia risk indicated mixed results regarding the effect of blood groups on preeclampsia severity. a review of a few studies looking at this subject suggests higher risk of preeclampsia with a blood group, 104,105 with o blood group, 106,107 and with ab blood group. 34,105,108–113 yet a few more studies suggested no relationship between them. 114–118 a limited number of studies explored the association of different blood groups with decreased bioavailability of pp13. 34,113,119 further research and large cohort studies are required to table 1. van der waals energies of pp13-ligand complexes. ligand van der waals energy (kcal/mol) number of hydrogen bonds amino acid residues involved in hydrogen bonding n-acetyl-dlactose amine �765.2 3 arg55, asn65, gln75 lactose �759.4 2 arg55(2) n-acetyl-dgalactosamine �751.9 2 arg55(2) galactose �751.4 3 arg53(2), asn65 mannose �741.5 0 — pp13: placental protein 13; arg55: arginine 55; asn65: asparagine 65; gln75: glutamine 75; arg53: arginine 53. 6 journal of circulating biomarkers investigate the effect of blood groups on the availability of pp13 and its association with preeclampsia. immunological function maternal immunological response is essential for the establishment, maintenance, and completion of a healthy successful pregnancy. the human semi-allogeneic fetus is not rejected by the maternal immune system, despite expressing paternal antigens due to many fetal, maternal, and placental mechanisms that have been implicated in aiding fetal tissues in escaping maternal immune attacks. the immunological barrier (placenta and maternal decidua) plays a crucial role in acceptance of the fetus and making the decidua more receptive for the invading fetus. of the numerous mechanisms involved, uterine natural killer cells (regulate the trophoblast invasion through secretion of angiogenic growth factors), 120 cytokines and chemokines (interleukin-8 and interferon inducible protein10), 121 dendritic cells, 122 macrophages, 123 the complement system, 124 toll-like receptors, 125 decidualized endometrium secretion, 126 the trophoblast human leucocyte antigen (hla) expression pattern, 127 fas ligand, 128 indoleamine 2,3-dioxygenase, 129 b7 family, 130 t helper cells, 131 and t regulatory cells 132 are all implicated in modulating immune responses for an effective implantation. the inadequate maternal–fetal immune tolerance is one of the proposed mechanisms leading to preeclampsia. breakdown of maternal–fetal tolerance also leads to other pregnancy-specific autoimmune diseases, bleeding complications during the first trimester, pregnancy-induced hypertension, and preterm or recurrent miscarriages. 133 so apart from the abovementioned mechanisms, human placenta-specific galectins expressed by the syncytiotrophoblast at the maternal–fetal interface are key regulator proteins of the immune responses. they confer immune tolerance to the semi-allogeneic fetus by bringing about the apoptosis of maternal t lymphocytes to sustain placentation. 134,135 pp13 drains through the decidual veins in the form of exosomes/microvesicles into the decidua where it attracts and activates maternal immune cells, diverting them away from maternal spiral arteries to facilitate uninterrupted spiral artery remodeling. pp13 plays a unique role in early pregnancy by forming decidual crystal-like aggregates within decidual zones of necrosis. these zones are associated with necrotic and apoptotic immune cells which are cd45ro memory t cells, cd68 þ macrophages, and cd57 þ large granular lymphocytes. the decidual zones of necrosis peak at 7–8 weeks of gestation when placental circulation is not yet established and diminish after the table 2. research studies showing association of blood groups a, b, and o with preeclampsia. s.no. author year study design results 1 may 1973 case control association of blood group a with preeclampsia than o blood group 2 scott and beer 1976 case control no association between abo blood group and preeclampsia risk 3 amin et al. 1989 case control blood group o as the risk factor for preeclampsia 4 spinillo et al. 1995 case control maternal ab blood group associated with increased risk of severe preeclampsia 5 witsenburg et al. 2005 case control abo blood group never observed as risk in pregnancy complications (preeclampsia, hellp syndrome, and pih). 6 clark and wu 2008 prospective cohort no effect of abo blood type on the risk of preeclampsia 7 hiltunen et al. 2009 nested case control preeclampsia risk associated with ab blood group 8 than et al. 2011 case control maternal blood group ab contributes to less pp13 bioavailability and increased risk of preeclampsia 9 lee et al. 2012 cohort ab blood group associated with high risk of developing preeclampsia than o blood group 10 phaloprakarn and tangjitgamol 2013 case control blood groups a and ab were associated with increased risk for preeclampsia 11 hentschke et al. 2014 case control no association between blood groups and preeclampsia 12 seyfizadeh et al. 2015 case control abo blood group associated with unfavorable outcomes of pregnancy 13 manjunatha and anita 2015 crosssectional ab blood group has highest preeclampsia risk 14 avci et al. 2016 case control ab blood group with decreased availability of pp13 associated with higher risk of preeclampsia 15 elmugabil et al. 2016 case control blood group o with higher risk for preeclampsia 16 reisig et al. 2016 case control blood group a with increased risk of preeclampsia 17 mital et al. 2016 case control ab blood group with higher risk of preeclampsia. 18 aghasadeghi and saadat 2017 case control no association between abo blood groups and preeclampsia risk 19 beyazit et al. 2017 case control no association between blood groups and preeclampsia pp13: placental protein 13. pih: pregnancy induced hypertension. gadde et al. 7 completion of spiral artery remodeling at about 10–14 weeks of gestation. the diversion of these immune cells facilitates uninterrupted trophoblast invasion and remodeling of the maternal spiral arterioles. 17 as the demands of the growing fetus for oxygen and nutrients increase as the pregnancy progresses, the transformation of the maternal vasculature becomes critical. impaired spiral artery remodeling may be linked to an immune maladaptation syndrome, preeclampsia followed by failure of the fetus to reach its optimal growth, and intrauterine growth retardation (iugr). 136–139 among the studied recombinant galectins, the apoptotic effects of pp13 on freshly isolated activated cd3 þ t cells were stronger when compared to galectin 1. the pp13/galectin 13 functional role with respect to t cell apoptosis is similar to that of the mechanism behind t cell apoptosis caused by galectin 1. 28 however, the similar effect was not found with truncated pp13 due to lack of crd function and t cell apoptotic activity. hence, structural integrity of pp13 is crucial in sugar binding and the regulation of cell–matrix interactions for successful human placentation. thus, inadequate placental expression and structural derangement with altered function of pp13 result in adverse conditions during pregnancy and poor obstetric outcome. 36 the involvement of pp13 as pro-inflammatory function demonstrated in in vitro study where pp13 triggered the secretion of cytokines and chemokines into the culture medium from mononuclear cells isolated from peripheral blood of pregnant women. thus, varied action of pp13 with respect to apoptotic and pro-inflammatory function studied in in vitro experiments. 28 regulation of blood pressure the role of pp13 in embryo implantation in maternal–fetal interface has been reported. but animal experiments and studies in humans regarding the role of pp13 in blood pressure regulation are lacking. nonetheless, there are studies which suggest that pp13 contributes to vasodilation and remodeling of uteroplacental vasculature and improves pregnancy outcome. 140–142 the exact molecular mechanism of vasodilation and the unidentified responsive element that influences endothelial nitric oxide synthase and cyclooxygenase enzymes in preeclampsia model are yet to be elucidated even though prostaglandins do play an important role in vascular remodeling during early placentation. 142 introduction of pp13 to cultured trophoblasts elicited depolarization of the trophoblast membrane by calcium ions leading to liberation of linoleic and arachidonic acids from the trophoblast membrane lipids and this leads to increased amount of prostacyclin and thromboxane synthesis. 50 the in vivo vasodilatory effects of pp13 in preeclamptic animal models and the levels of pp13 correlated with nitric oxide and prostaglandin levels. this needs to be further investigated in order to evaluate the potential therapeutic use of pp13 in humans for improving blood flow and pregnancy outcome in hypertensive disorders of pregnancy or preeclampsia. recombinant pp13 recombinant pp13 (pp13-r) was genetically engineered and produced by hy-laboratories (hylabs rehovot, israel). the basis for pp13 sequence construct has been validated by sanger sequencing information published by national centre for biotechnology information, usa. the same sequence constructs are expressed in transfected escherichia coli. the expressed protein was harvested and affinity purified, verified by sds page, hplc, and immunoblot using pp13-specific monoclonal antibodies. several in vivo and in vitro extensive studies explored the functional aspects of recombinant pp13 in biomedical research. in a protein conformation study, pp13-r was reported as a homodimer stabilized by disulfide bonds possessing binding strength to different sugars in nonreducing conditions. however, the reducing conditions favorable for the monomeric form lack binding ability to sugars. 58 in vitro study on hemagglutination of human erythrocytes described the binding affinities of common recombinant pp13 (pp13-r) and truncated recombinant pp13 (trpp13). pp13-r has the strongest binding affinity to the b-galactosides on the erythrocyte surface of the blood group ab and weakest affinity to erythrocytes of blood group b. however, this is not exhibited by trpp13 due to altered crd. this provides evidence that conserved crd is crucial to exhibit lectin activity. 28 the major in vitro study information is that the immune response of recombinant galectin was studied and noted that among the galectins, pp13-r had the strongest apoptosis-inducing effect on the freshly isolated maternal t cells. this observation laid the strong foundation to understand the central role of pp13 in maintaining immune tolerance at the maternal–fetal interface. 36 in an experimental animal model, the role of crd of pp13 was elucidated in pregnancy by comparing the wildtype recombinant pp13 and truncated variant on administering both the variants to pregnant rats. the significance of wild-type pp13 in regulating blood pressure and expanding uteroplacental vasculature was noted. this suggests that pp13 may have a potential therapeutic role during pregnancy risk. 65 phosphorylation of the protein side chains of pp13 expressed and purified from placenta indicated the specific biological function and necessitates further research in order to understand the role that the phosphorylated protein plays and its mechanism. the inherent biological property restored on wild-type pp13 and pp13-r studied and compared by phosphorus-31 nuclear magnetic resonance analysis and found that both variants exhibited weak endogenous lysophospholipase activity. the affinity purified wild-type pp13 and pp13-r when incubating with 8 journal of circulating biomarkers protein extracts from human placenta or wrl-68 fetal hepatic cell lines identified annexin-ii and b/g-actin as specifically bound proteins for pp13. 101 the role of pp13-r on the cardiovascular system in gravid and nongravid rodents showed, for the first time, the influence of pp13 on vasodilation, uterine artery remodeling, and in improving uteroplacental blood flow as well as adaptation of the maternal vasculature to pregnancy. the same author also studied the role of pp13-r on pregnant rats in lowering blood pressure, venous remodeling, and improving fetal growth. 140 administration of truncated pp13 variant resulted only in a hypotensive effect with loss of venous remodeling and increase in placenta and pup weights. 79 in a recent animal study, the role of pp13-r in endothelial signaling pathways of vasodilation of resistance arteries from pregnant and nonpregnant rats was studied and concluded that pp13 may become useful therapeutically in improving blood flow and pregnancy outcomes in hypertensive pregnancies. 142 screening performance preeclampsia is one of the most common and lifethreatening disorders of pregnancy affecting a total of 8.5 million women worldwide. preeclampsia is responsible for 18% of maternal deaths and up to 40% of fetal mortality. despite extensive research, preeclampsia still lacks a safe, cost-effective, reliable, early means of diagnosis, or prediction. pp13/galectin 13 produced by the syncytiotrophoblast during pregnancy is involved in normal placentation. in normal pregnancies, serum levels of pp13 slowly rise with gestational age. several studies have reported that decreased serum levels of pp13 in the first trimester increase the risk of subsequently developing preeclampsia. measurement of pp13 levels as a first trimester screening marker for preeclampsia may provide an opportunity for identification of women destined to develop early-onset preeclampsia. romero et al. 69 studied a cohort of 300 patients, of which 50 developed preeclampsia. serum pp13 concentration in the first trimester was significantly lower in patients who developed preterm and early-onset preeclampsia than in those with normal pregnancies; and at 80% specificity, a cutoff of 0.39 multiples of median (moms) had a sensitivity of 100% for early-onset preeclampsia and 85% for preterm preeclampsia. based on these results, it was concluded that the first trimester maternal serum pp13 concentrations can be used in the risk assessment for preeclampsia. gonen et al. 15 studied a cohort of 1366 pregnant women and subsequently 20 were diagnosed with preeclampsia. at 6–10 gestational weeks, pp13 levels were significantly lower among the preeclampsia group with a median of 0.28 mom (95% confidence interval (ci) 0.15–0.39, p < 0.004). using a cutoff of 0.40 mom, the sensitivity was 80%, a false positive rate (fpr) was 20%, and odd ratio was 16.0 (95% ci 18.2–169.2). this study reported that pp13 in the first trimester alone or in combination with the slope between the first and second trimesters may be a promising marker for assessing the risk of preeclampsia. nicolaides et al. 66 studied pp13 as a biochemical marker for the early onset of preeclampsia at 11 þ 0 to 13 þ 6 weeks of gestation. at an fpr of 10%, pp13 showed a prediction rate of 80% as a single biochemical marker. in combination with doppler ultrasound pi, the prediction rate increased to 90%. spencer et al. 68 conducted a nested case-control study to evaluate whether the measurement of maternal serum pp13 and pregnancy associated plasma protein-a (papp-a) at 11 þ 0 to 13 þ 6 weeks of gestation alone or in combination with the second trimester uterine artery pulsatility measured by doppler velocimetry is useful in predicting those women who will develop preeclampsia. there were 446 controls and 44 cases with early preeclampsia where delivery was induced prior to 35 weeks. in addition, there were 44 cases with preeclampsia in which delivery was not induced before term. median pp13 levels for controls, all cases, and early preeclampsia cases were 176.9, 121.9, and 111.7 pg/ml, with moms of 1.00, 0.69, and 0.63, respectively (p < 0.001). the first trimester pp13 levels may be useful in predicting preeclampsia and early preeclampsia, and the accuracy of the method increases when coupled with the second trimester doppler pi measurement. further studies are required to establish the real value of pp13 in the first trimester screening for preeclampsia. plasma pp13 has also been used in combination with urinary glycosaminoglycans/proteoglycans as early markers for preeclampsia by de muro et al. 20 a total of 62 women were enrolled in the study, of which 24 presented complications such as preeclampsia, proteinuria, and hypertension during pregnancy. plasma levels of pp13 were significantly reduced in the group of women who went on to develop complications compared with controls (p ¼ 0.022). the reduced plasma levels of pp13 and the alteration of the relative content of urinary glycosaminoglycans and proteoglycans observed in their study could be a promising tool for the prediction of preeclampsia in an early stage of pregnancy. moslemizadeh et al. 75 conducted a prospective nested case-control study that recruited 1500 pregnant women and 100 women developed preeclampsia and represented the case group. of 100 women with preeclampsia, 66 cases have mild preeclampsia, while 34 cases have severe preeclampsia. serum pp13 levels along with papp-a were measured in the first and second trimesters and were significantly lowered in women who developed preeclampsia (p < 0.001). the cumulative value of all the four variables with cut-off points of 238.5 has sensitivity and specificity of 91.0% and area under curve of 0.968. the study concluded that measuring pp13 with papp-a in both trimesters of pregnancy is advantageous for the prediction of the incidence of preeclampsia. gadde et al. 9 wortelboer et al. 81 conducted a nested case-control study to investigate the predictive value of pp13 along with other biochemical markers such as pregnancy associated plasma protein a (papp-a), b-human chorionic gonadotropin (b-hcg) and placental growth factor(plgf) and desintegrin adam 12 in the first trimester of pregnancy. pp13 and plgf were reduced in women with preeclampsia, with median of 0.68 and 0.73 mom, respectively (p < 0.0001 for both). this study demonstrates that pp13 and plgf in the first trimester might be promising markers in risk assessment for early preeclampsia/hellp syndrome. odibo et al. 73 conducted a prospective cohort study of pregnant women followed from the first trimester to delivery as part of a first trimester screening study for adverse pregnancy outcomes. this study was conducted on 477 women of whom 42 women were diagnosed with preeclampsia. for a fixed fpr of 20%, pp13, papp-a, and mean uterine artery pulsatility index identified 49, 58, and 62%, respectively, of women who developed any form of preeclampsia. pp13 was best in predicting early onset preeclampsia with a sensitivity of 79% at an fpr of 20% and can be considered as individual predictors of women at risk to develop preeclampsia. svirsky et al. 76 conducted a cohort study to determine the first trimester maternal serum pp13 in singleton versus twins with and without severe preeclampsia. in twins, the median was 1.74 mom (n ¼ 76) versus 1.74 in unaffected twins (p ¼ 0.10) and 2.26 (n ¼ 6) for mild preeclampsia (p ¼ 0.30). among singletons with severe preeclampsia, the median was 0.44 mom (n ¼ 26, p < 0.0001), and for mild preeclampsia 0.62 (n ¼ 17, p < 0.001). based on the results, they concluded that pp13 was higher in twins than singletons corresponding to larger placental mass. beljan et al. 22 conducted a prospective study to detect whether the first trimester serum pp13 and copeptin can predict preeclampsia in advanced age nulliparous women. this study confirmed a significant contribution for the combination of two biomarkers for the prediction of preeclampsia (p ¼ 0.007). decreased level of pp13 and an increased level of copeptin can predict preeclampsia with a sensitivity of 66.7% and a specificity of 97.3%, with an fpr of 2.7%. el sherbiny et al. 74 conducted a cohort study on 100 women to assess the value of pp13 as an early marker for screening of preeclampsia and to correlate with the pp13 mrna. the maternal serum pp13 level in the preeclamptic group was (157.9 + 45.5 pg/ml), which is significantly lower than that of the control group (225 + 67.3 pg/ml), with a highly statistically significant difference (p < 0.0001). the frequency of maternal pp13 was also lower in the preeclamptic group (28%) compared to that in the control group (76%), with a highly statistically significant difference (p < 0.0001). this study concluded that serum pp13 assay and pp13 mrna expression are reliable markers for early detection of preeclampsia and was recommended doing it as a routine investigation during the first trimester. khalil et al. 71 conducted a nested case-control study to evaluate whether the first trimester maternal serum pp13 can predict preeclampsia in women with a priori high risk. women who developed preeclampsia had significantly lower (p < 0.001) pp13 moms compared with controls. pp13 moms for controls and preeclampsia cases were 1.0 (range 0.0–10.0) and 0.4 (range 0.0–7.0), respectively (p < 0.001). at an mom cutoff of 0.53, for an fpr of 10%, sensitivity was 50% for preeclampsia at term (>37 weeks), 62% for preterm preeclampsia (<37 weeks), and 71% for early onset preeclampsia (< 34 weeks). based on these results, the first trimester pp13 can predict preeclampsia in women at increased priori risk and predicts early-onset better than late-onset disease. chafetz et al. 67 performed a prospective, nested casecontrol study to evaluate the first trimester serum pp13 as a screening test for preeclampsia and intrauterine growth restriction. the median first trimester pp13 level was 132.5 pg/ml in the control subjects (n ¼ 290). median pp13 levels were significantly lower among women who had preeclampsia (27.2 pg/ml; p < 0.001), iugr (86.6 pg/ml; p < 0.001), and preterm delivery (84.9 pg/ml; p ¼ 0.007). when pp13 was expressed as multiples of the gestational age-specific medians among the control subjects, the moms were 0.2 for preeclampsia, 0.6 for iugr, and 0.6 for preterm delivery (p < 0.001 for each disorder compared with subjects). receiver operating characteristic analysis yielded areas under curve of 0.91, 0.65, and 0.60 for preeclampsia, iugr, and preterm delivery, respectively. at a 90% specificity rate, the corresponding sensitivities were 79, 33, and 28%, respectively. this study concluded that maternal pp13 levels in the first trimester are a promising diagnostic tool for the prediction of preeclampsia with high sensitivity and specificity. huppertz et al. 70 conducted a prospective longitudinal study with 41 normal pregnant women, 18 cases with preterm delivery or cervix insufficiency, and 4 with developing late-onset preeclampsia. six hundred and sixty-six blood samples were obtained every 2–4 weeks starting from 5 weeks to 8 weeks of gestation (10–12 samples/ patient) and tested for serum pp13. in normal pregnant women delivering at term, median maternal serum pp13 levels were increasing from 166 pg/ml to 202 pg/ml and 382 pg/ml in the first, second, and third trimesters, respectively. preeclamptic women had significantly reduced pp13 levels in the first trimester (mom of 0.14 at 7–8 weeks; p ¼ 0.005 compared to normal). pp13 in the third trimester was significantly higher compared to normal at 35–36 weeks with pp13 mom of 1.79. this preliminary study indicates that low levels of pp13 in early pregnancy indentify at risk pregnancies, whereas high levels precede the syndrome in late pregnancy and suggest syncytiotrophoblast necrosis. 10 journal of circulating biomarkers a large number of cohort studies are required to determine the study replicability and to establish pp13 measurements as an early screening indicator in larger population. however, the exact reason for lower level of pp13 bioavailability in the first trimester of the susceptible subjects likely to develop preeclampsia is still unknown. despite considerable amount of research, development of sensitive, stable, reliable, and cost-effective biochemical analytes is of great importance. future perspectives reliable early screening for preeclampsia is one of the most important challenges in prenatal care, since preeclampsia is a serious complication and an important cause of maternal and perinatal morbidity and mortality with a prevalence of 2–8%2 resulting in about 50,000–60,000 deaths annually worldwide. 143 it is responsible for 18% of maternal deaths and up to 40% of fetal mortality.6 according to world health organization, the mortality rate of preeclampsia in developing countries is seven times higher than developed countries. 144 pp13 is produced by the syncytiotrophoblast throughout pregnancy. the significantly reduced levels in the first trimester make it a promising tool for identifying women at increased risk of preeclampsia and scope for research to find the exact cause for low levels of pp13 at the first trimester. however, whether it has a better predictive value when used alone or should be included in the highly sensitive panel of biomarkers needs to be determined by conducting larger prospective studies in different populations. data generated in animal studies suggest that pp13 has a beneficial effect in regulating blood pressure and the potential use of this galectin as a therapeutic drug. administering pp13 to patients at risk for preeclampsia may be helpful in reducing the incidence of preeclampsia and in improving obstetrical outcomes. an extensive research is necessary to study the effect of pp13 in arterial expansion and a thorough understanding of the mechanistic pathway in preeclamptic animal or human model. hence, there is a scope to evaluate the efficacy of recombinant pp13 supplementation in alleviating pregnancy complications at conception. thus, the outcome would gain momentum in advanced preeclampsia research through clinical trials. the pp13 structure has additional domains known to possess lysophospholipase activity and phosphorylation sites. the role of these functional domains in relation to normal pregnancy and in pregnancy complications still needs to be studied further. summary the overall aim of this review article is to summarize the properties and biological functions of pp13 in normal pregnancy and in preeclampsia. pp13 is a 32-kda protein localized in the brush border membrane of the syncytiotrophoblast lining at the common fetal–maternal blood spaces of the placenta. as a “prototype” galectin, it has a single sugar binding domain or crd which emerges into the extracellular space and has lectin-like activity. the dimerized phosphorylated protein functions as a regulator of blood flow and blood pressure and has special immune functions at the fetal–maternal interface. pp13 induces different levels of hemagglutination, which depend on the type of blood group antigen, and it is involved in multistep process in pregnancy that supports trophoblast invasion as well as generation of maternal systemic endothelial effect. further data are needed to establish whether dysregulation of pp13 levels in the maternal circulation in early pregnancy can serve as a reliable screening, predictive, and prognostic biomarker for preeclampsia. animal experimental results which found beneficial effects of pp13 in regulating blood pressure suggest the potential use of this galectin 13 as a therapeutic drug in preeclampsia. however, a large prospective and longitudinal studies are needed to confirm this observation. although advanced research is focusing on molecular and epigenetic changes in preeclampsia, future study designs should first identify a highly sensitive and specific serum marker. pp13 is known to be involved in different stages of pregnancy ranging from trophoblast invasion to vasodilation of the maternal vasculature needed for the increased blood flow to the fetus. administration of recombinant pp13 in women with low serum levels may prevent impaired placental development in preeclampsia. further research is required to completely understand the role of pp13 in the development of preeclampsia. the diagnosis of preeclampsia based on biomarkers might be useful in identifying patients at risk, monitoring disease progression to provide effective and timely interventions. conclusion pp13, a placental galectin protein, is a homodimeric, phosphorylated protein localized in syncytiotrophoblast. by its crd, it exerts erythrocytes agglutination or lectin activity through sugar binding capabilities. it is an immune regulatory, vasodilatory protein with weak lysophospholipase activity involved in placentation process. the low levels of blood pp13 acts as a new biological protein for individualized risk assessment and drug design target in management of preeclampsia. preparation of monoclonal antibodies and recombinant pp13 helps in research for detection and replenishing the pp13 level in preeclampsia has attracted much attention. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. gadde et al. 11 funding the author(s) received no financial support for the 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issn 1849-4544 www.aboutscience.eu/jcb © 2022 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu occupational factors (1). despite excellent control of local disease, prognosis remains poor due to metastatic progression affecting ~50% of patients (2-4). mortality rates for um are unchanged over the past decades (1). extracellular vesicles (evs) have emerged as biomarkers in various cancers and provide valuable clinical information (5,6). their use as a biomarker assay has gained interest as a new tool for monitoring of cancer patients; however, standardization and validation of evs as a biomarker are needed (4,7). evs are small lipid bilayer particles released from all types of cells and found in different body fluids, most commonly the blood, but have also been detected in aqueous humor (ah) (8,9). evs are classified mainly into exosomes (50-100 nm), microvesicles (100-1000 nm), and apoptotic bodies (50 nm-2 µm) based on their biogenesis, number, size, distinct biological functions and markers (10-16). exosomes are a constitutive and abundant component of the vitreous (17). evs are involved in the transfer of biological macromolecules to recipient cells, and modulating various physiological introduction uveal melanoma (um) is a primary intraocular tumor in adults that accounts for less than 5% of all melanoma cases (1,2). the incidence of um has remained stable at ~5.1 per million since the 1970s with subtle differences depending on geographic location, as well as environmental and characterization of extracellular vesicles isolated from different liquid biopsies of uveal melanoma patients carmen luz pessuti1, deise fialho costa1,2, kleber s. ribeiro1,3, mohamed abdouh2, thupten tsering2, heloisa nascimento1, alessandra g. commodaro1, allexya affonso antunes marcos1, ana claudia torrecilhas3, rubens n. belfort1, rubens belfort jr1, julia valdemarin burnier2,4,5 1department of ophthalmology, federal university of são paulo, vision institute, ipepo, são paulo brazil 2cancer research program, mcgill university health centre research institute, montreal, quebec canada 3department of pharmaceutical sciences, federal university of são paulo, diadema, são paulo brazil 4department of oncology, mcgill university, montreal, quebec canada 5experimental pathology unit, department of pathology, mcgill university, montreal, quebec canada abstract purpose: uveal melanoma (um) is the most common intraocular malignant tumor in adults. extracellular vesicles (evs) have been extensively studied as a biomarker to monitor disease in patients. the study of new biomarkers in melanoma patients could prevent metastasis by earlier diagnosis. in this study, we determined the proteomic profile of evs isolated from aqueous humor (ah), vitreous humor (vh), and plasma from um patients in comparison with cancer-free control patients. methods: ah, vh and plasma were collected from seven patients with um after enucleation; ah and plasma were collected from seven cancer-free patients with cataract (cat; control group). evs were isolated using the membrane-based affinity binding column method. nanoparticle tracking analysis (nta) was performed to determine the size and concentration of evs. ev markers, cd63 and tsg101, were assessed by immunoblotting, and the ev proteome was characterized by mass spectrometry. results: mean ev concentration was higher in all analytes of um patients compared to those in the cat group. in the um cohort, the mean concentration of evs was significantly lower in ah and plasma than in vh. in contrast, the mean size and size distribution of evs was invariably identical in all analyzed analytes and in both studied groups (um vs. cat). mass spectrometry analyses from the different analytes from um patients showed the presence of ev markers. conclusion: evs isolated from ah, vh, and plasma from patients with um showed consistent profiles and support the use of blood to monitor um patients as a noninvasive liquid biopsy. keywords: aqueous humor, extracellular vesicles, liquid biopsy, plasma, proteomic analysis, uveal melanoma, vitreous humor received: february 3, 2022 accepted: may 27, 2022 published online: june 27, 2022 this article includes supplementary material. corresponding author: carmen luz pessuti department of ophthalmology federal university of são paulo porto street, 69 são paulo, 09416-020  brazil luz.pessuti@unifesp.br https://doi.org/10.33393/jcb.2022.2370 mailto:luz.pessuti@unifesp.br https://creativecommons.org/licenses/by-nc/4.0/legalcode luz pessuti et al j circ biomark 2022; 11: 37 © 2022 the authors. published by aboutscience www.aboutscience.eu and pathological processes, such as pathogen dissemination and regulation of the host immune system (18-20). recent studies have shown that tumor cells release large amounts of evs that can be uptaken by malignant and stromal cells, inducing tumor progression (21,22). they have been shown to play a major role in mediating metastasis, ranging from oncogenic reprogramming of malignant cells to the formation of pre-metastatic niches (23-25). furthermore, our group and others have shown that cancer-derived exosomes can transfer bioactive molecules such as proteins, dna, mrnas, and mirnas to recipient cells, thereby changing their function (26-29). in ocular and cutaneous melanoma, the concentration of evs and proteins is increased in patients compared to healthy individuals and has been shown to correlate with disease progression (30,31). moreover, the profile of circulating ev-derived mirnas is often altered in human cancers, and evs from um patients have been shown to contain mir-146, a potential circulating marker in um (32). recently, we have reported that the number of evs produced and the profile of tumor-associated proteins vary between normal melanocytes and um cell lines, and also between primary and metastatic um cell lines (33). evs released by metastatic melanoma cells were enriched in proteins (9,10,23) involved in the pre-metastatic niche formation (25), suggesting their role in preparing the environment for colonization by circulating tumor cells (ctcs). there is a lack of detailed characterization of evs in this disease as well as in nonblood-based liquid biopsy. in this study, our aim was to determine the proteomic profile of evs isolated from ah, vitreous humor (vh), and plasma from patients with um and to compare with cancer-free control patients. materials and methods patients a total of 14 participants were enrolled for this study: 7 patients diagnosed with primary um, and 7 healthy controls undergoing cataract surgery at the department of ophthalmology, federal university of são paulo (unifesp/ epm), brazil. the patients were recruited from july 2019 to december 2019 at the department of ophthalmology of the unifesp/epm. the clinical characteristics of the study population are described in table i. this study was approved by the ethics committee investigational review board (cep number 2198149) and adhered to the principles of the declaration of helsinki and resolution 196/96 of the ministry of health, brazil. informed consent was obtained from all participants. sample collection ah and plasma samples were collected from um patients and controls. additionally, vh samples were collected from um patients. peripheral blood (10 ml) was collected in ethylenediaminetetraacetic acid (edta) tubes. the tubes were centrifuged for 10 minutes at 1,900 × g), and plasma were collected. vh and ah samples from um patients were collected from the enucleated eyes after the surgery with a syringe and fine needle. in the control group, ah samples were collected during cataract surgery. all routine surgical procedures were followed. all collected samples were kept at −80°c until the experimental procedure. table i clinical features of patients enrolled in this study patients sex age (years) cell types size tnm cat1 male 70 n/a n/a n/a cat2 female 77 n/a n/a n/a cat3 female 82 n/a n/a n/a cat4 female 77 n/a n/a n/a cat5 female 75 n/a n/a n/a cat6 male 76 n/a n/a n/a cat7 female 63 n/a n/a n/a um1 male 72 mixed um, predominance of spindle cells affecting the ciliary body and choroid 1.9 × 0.6 pt4e um2 female 86 epithelioid choroidal melanoma 1.2 × 1.1 pt3b um3 female 53 mixed choroidal melanoma, predominance of spindle cells 1.3 × 1.0 pt3a um5 male 63 mixed um, predominance of spindle cells infiltrating the choroid and ciliary body 1.2 × 1.5 pt3b um6 female 61 mixed um, predominance of epithelioid cells 1.0 × 0.8 pt2 um8 female 65 mixed um, predominance of spindle cells infiltrating the choroid and ciliary body 2.8 × 0.7 pt4b um9 female 39 mixed choroidal melanoma, predominance of epithelioid cells 1.5 × 1.2 pt3a size refers to tumor size (base diameter × thickness [cm × cm]). cat = cataract, control group; n/a = not applicable; tnm = tumor, node, metastasis; um = uveal melanoma. https://creativecommons.org/licenses/by-nc/4.0/legalcode characterization of extracellular vesicles isolated38 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb ev purification and characterization the protocol for ev isolation was performed according to the guidelines of the international society for extracellular vesicles (isev) (10). samples were centrifuged at 16,000 × g for 10 minutes at 4°c to eliminate cellular debris. then, ev isolation was performed using the exoeasy maxi kit (qiagen, valencia, ca, usa) according to the manufacturer’s instructions (10,11,34-36). isolated evs were diluted 100 × in phosphate buffered saline (pbs) and analyzed by nanoparticle tracking analysis (nta) using the nanosight ns300 instrument (malvern analytical, uk). pbs was used as a diluent. samples and diluent were read in triplicates for 30 seconds at 20 frames per second. the nta 3.2 software was used to estimate the concentration and size of the particles. immunoblotting evs isolated from patients and controls were lysed in ripa buffer containing complete mini protease inhibitors (sigma) at 4°c for 30 minutes. samples were sonicated for 2 seconds (three times), and spun at 13,000 × g for 30 minutes at 4°c. protein concentrations were quantified by the bca assay (thermo fisher scientific). protein samples were processed for immunoblotting and mass spectrometry (ms). ev-derived proteins (20 µg) were separated using 12% miniprotean® precast polyacrylamide gel (bio-rad). proteins were transferred onto polyvinylidene difluoride (pvdf) membranes (bio-rad). membranes were blocked for 1 hour at room temperature with 5% nonfat dry milk in 1x tris-buffer saline with 0.05% tween 20 (tbst). membranes were probed with anti-tsg101 (abcam; 1:1,000) and anti-cd63 (abcam; 1:1,000), anti-alix (thermofisher scientific 1:1,000), anti-β-actin (sigma 1:1,000), anti-tenascin c (abcam 1:1,000), anti-vimentin (abcam 1:500) primary antibodies, followed by horseradish peroxidase (hrp)-conjugated goat anti-rabbit (sigma 1:1,000) and goat anti-mouse (sigma 1:3,000) secondary antibodies. membranes were washed five times for 10 minutes each time after each incubation and developed using ecl prime western blot detection (ge healthcare). protein signals were visualized using the chemidoc xrs + system. ms analysis ms analysis was performed in nine samples [ah (n = 3), plasma (n = 3), and vh (n = 3)] from um-5, um-6, and um-8 patients; 20 µg of ev proteins from each sample was loaded onto a single stacking gel band to remove contaminants such as lipids, detergents, and salts. each sample was run in duplicate. the gel band was reduced with dtt (dithiothreitol), alkylated with iodoacetic acid, and digested with trypsin. extracted peptides were resolubilized in 0.1% aqueous formic acid and loaded onto a thermo scientific acclaim pepmap (75 μm inner diameter × 2 cm, c18 3 μm particle size) precolumn and then onto an acclaim pepmap easy-spray (75 μm inner diameter × 15 cm with 2 μm c18, 2 µm beads) analytical column separation using a dionex ultimate 3000 uhplc at 250 nl/min with a gradient of 2-35% organic (0.1% formic acid in acetonitrile) over 3 hours. peptides were analyzed using a thermo orbitrap fusion ms operating at 120,000 resolution (full width at half maximum in ms1) with higher energy collisional dissociation (hcd) sequencing (15,000 resolution) at top speed for all peptides with a charge of 2+ or greater. the ms raw data were converted into *.mgf format (mascot generic format) for searching using the mascot 2.6.2 search engine (matrix science) against human protein sequences (uniprot 2019). the database search results were loaded onto scaffold q+ scaffold_4.10.0 (proteome sciences) for spectral counting, statistical treatment, data visualization, and quantification. protein threshold >99%, peptide threshold >95%, and two of a minimum number of unique peptides were applied in scaffold q+ to increase the confidence level of identified proteins. additional filters such as p-value cut-off of 0.05 and a fold-value change of ≥2 were used to identify the differential expression of proteins. the identified protein list in scaffold was exported to microsoft excel and uploaded into the david bioinformatics database (version 6.8) for gene ontology (go) analyses (i.e., biological process, cellular component, and kegg pathway). in addition, bioinformatic analysis and vesiclepedia database (37) search were performed using the funrich software (version 3.1.3) (37-39). statistical analysis statistical analysis was performed using the graphpad software (prism, version 5.00 for windows; graphpad, san diego, ca). the mann-whitney test was used to determine the statistical difference between respective groups. the results are expressed as mean ± standard deviation (sd). a p-value < 0.05 was considered significant. results characterization and isolation of evs from plasma, ah, and vh evs were isolated from the plasma, ah, and vh of um patients, and ah and plasma of cataract patients. immunoblotting analysis showed the expression of ev markers cd63, tsg101, and alix with different expression levels depending on the analyzed samples (fig. 1a, b, and supplementary figure a). the expression of cd63 and alix was higher in um evs than in cat evs (fig. 1a, and supplementary figure a). moreover, the expression of both cd63 and tsg101 was higher in evs isolated from vh and plasma than in evs isolated from ah (fig. 1a, b). nta from all samples showed that evs ranged from 80 to 442 nm in size, with similar 10 percentile mean (d10) size (133 nm, 135 nm, and 139 nm) in plasma, ah, and vh, respectively (fig. 1c, d). when analyzing sizes of isolated um evs, no difference was observed in all samples: 219 ± 26 nm (range: 168-241) in plasma, 211 ± 37 nm (range: 173-265) in ah, and 216 ± 71 nm (range: 110-314) in vh (fig. 1d). also, no difference was observed in the average size of evs from ah and plasma between the um and cat groups (fig. 1d). in the um cohort, the concentration of evs ranged from 2.6 × 109 to 9 × 1010 particles/ml in ah, vh, and plasma samples (fig. 1e). the mean concentration of evs in vh (6.6 × 1010 particles/ml) was significantly higher when compared to ah luz pessuti et al j circ biomark 2022; 11: 39 © 2022 the authors. published by aboutscience www.aboutscience.eu fig. 1 characterization of evs derived from ah, vh, and plasma. a,b) proteins isolated from the different assay evs (seven um samples and two cat samples) were analyzed by western blot for the expression of specific ev markers (i.e., cd63 and tsg101). c) nanosight analyses of evs. representative size distribution histograms showing data of evs from ah, plasma, and vh. note that mean ev sizes are similar. histograms are displayed as averaged ev concentration (black line) and the variation between four repeated measurements indicating ±1 standard error of the mean (red outline). d) mean size of evs isolated from ah and plasma of um (n = 7) and cataractsuffering (cat, n = 7) patients, and from vh of um patients (n = 7). e) concentrations of evs isolated from different analytes of seven um patients. **p ˂ 0.01. f,g) concentrations of evs isolated from ah (f) and plasma (g) of um (n = 7) and cataract-suffering (cat, n = 7) patients. ***p ˂ 0.001. h and i) the concentrations of evs isolated from the plasma of um patients (n = 7) were plotted against ocular tumor size (base diameter (h) and thickness (i)). no correlation was found as shown by the correlation coefficient (r). legend close to graph d applies to graphs d, f, and g. ah = aqueous humor; cat = cataract; ev = extracellular vesicle; vh = vitreous humor; um = uveal melanoma. (1010 particles/ml, p < 0.01) and plasma (2.7 × 1010 particles/ ml, p < 0.01) (fig. 1e). no difference was observed in the concentration of ah-derived evs between the um and cat groups. in contrast, the concentration of plasma-derived evs was significantly higher in um patients than in the cat control group (p < 0.001) (figs. 1f, g). notably, we did not find any correlation between the concentrations of evs isolated from um patients and ocular tumor size (fig. 1h, i). ev protein cargo from plasma ah and vh to gain an in-depth understanding of the protein cargo in evs isolated from the different analytes, we performed whole proteomic analysis by ms. for this purpose, we focused our analysis on evs isolated from three um patients (um-5, um-6, and um-8). our goal from this analysis was to determine whether these evs carried common protein cargo and characterization of extracellular vesicles isolated40 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb fig. 2 plasma-derived ev protein cargo mirrored that of evs isolated from ah and vh of um patients. venn diagram analyses. a) the majority of proteins isolated from evs derived from the different analytes were shared with data published in vesiclepedia database. b) evs isolated from the three analytes shared 209 proteins (39%). c-e) analyses of ev protein cargo in the same analytes from different donors. note that these evs shared 106 proteins (33%, c) in the aqueous humor, 181 proteins (44%, d) in the vitreous humor and 247 proteins (73%, e), which is in the same range of those shared between evs from the three analytes (39%, see b). data were collected from three um patient analytes repeated twice each (um5-1, um5-2, um6-1, um6-2, um8-1, and um8-2). ah = aqueous humor; ev = extracellular vesicle; vh = vitreous humor; um = uveal melanoma. also the nature of those proteins. we identified 542 proteins of which 498 (92%) overlap with ev proteins previously reported in the vesiclepedia database (supplementary table a, list of ev-contained proteins identified by ms screening) (fig. 2a) (37). as a readout for the purity of isolated evs, we detected proteins that are specific to the tissue of origin (i.e., complement and coagulation factors in evs from the plasma, melanocyte protein pmel and htra1 in the vh, and betaand gamma-crystallin in the ah) (tab. ii). in addition, protein cargo detected in isolated evs included typical ev protein signatures such as escrt components cd81, cd63, cd9, hla, annexins and syntenin (supplementary table a, list of ev-contained proteins identified by ms screening). moreover, herein, we report the presence of 44 novel proteins not previously reported in the vesiclepedia database (37); 2 are present in all evs, 4 are present in evs from plasma and vh, 4 are present in evs from vh and ah, and the rest are unique to evs from a single analyte (tab. iii). interestingly, 209 (39%) of the identified proteins were shared between evs from the three assays (fig. 2b). in addition, when we analyzed each analyte separately, we observed that evs from the three samples shared 106 (33%) proteins in ah, 181 (44%) in vh, and 247 (73%) in plasma (fig. 2c-e). proteins by go analysis in specific biological processes of the proteins found in our proteomic analyses data from um patients, 344 proteins were detected from plasma evs, 334 in evs from ah, and 421 in evs from vh (fig. 2b). to identify the physiological processes to which these proteins were associated, clustering was conducted into go categories using the david bioinformatics platform (fig. 3). characterization by biological process highlighted categories related to retina homeostasis, regulation of apoptosis, cell growth, and the activation of pathways involved in cancer cell biology (i.e., mapk/erk cascades). in addition, of the highly expressed proteins, several clustered in the categories of cell-cell adhesion and movement of cell or subcellular component (fig. 3a). when clustering the proteins based on cellular component, we found they grouped into ev categories (i.e., vesicles) (fig. 3b). molecular functions clustering using kegg pathway analysis revealed that isolated evs were enriched for proteins related to immune escape from cancer, such as those involved in complement and coagulation cascades, and proteins involved in cell metabolic activities and interaction with extracellular matrix (ecm). particularly, a panel of proteins clustered in the pi3k-akt signaling pathway and the proteoglycan group were exclusively present in plasma-isolated evs (fig. 3c). luz pessuti et al j circ biomark 2022; 11: 41 © 2022 the authors. published by aboutscience www.aboutscience.eu table ii protein readout for the purity of isolated evs spectrum count identified proteins id ah vh p plasma coagulation factor v fa5 2 0 146 c4b-binding protein alpha chain c4bpa 1 7 127 coagulation factor ix fa9 1 16 79 von willebrand factor vwf 0 0 72 coagulation factor x fa10 2 11 70 multimerin-1 mmrn1 0 0 33 platelet glycoprotein ib alpha chain gp1ba 0 2 22 c4b-binding protein beta chain c4bpb 0 0 14 serum amyloid p-component samp 0 7 14 c-reactive protein crp 1 1 13 sushi, von willebrand factor type a, egf and pentraxin domain-containing protein 1 svep1 0 0 2 serum amyloid a-1 protein saa1 0 0 1 vh pigment epithelium-derived factor pedf 37 111 13 retinol-binding protein 3 ret3 3 111 0 melanocyte protein pmel pmel 0 27 1 serine protease htra1 htra1 0 8 0 retinaldehyde-binding protein 1 rlbp1 0 2 0 retinoschisin xlrs1 0 2 0 interphotoreceptor matrix proteoglycan 1 impg1 0 1 0 ah and vh opticin opt 12 12 0 ah beta-crystallin b1 crbb1 179 2 1 alpha-crystallin a2 chain crya2 146 0 0 alpha-crystallin b chain cryab 139 7 0 gamma-crystallin s crygs 97 1 0 beta-crystallin a3 crba1 76 0 0 beta-crystallin a4 crba4 52 0 0 gamma-crystallin c crgc 44 0 0 gamma-crystallin d crgd 42 0 0 retinal dehydrogenase 1 al1a1 41 0 0 filensin bfsp1 2 0 0 phakinin bfsp2 2 0 0 data are derived from three patients (um-5, um-6, and um-8) and samples were run in duplicates. ah = aqueous humor; ev = extracellular vesicle; id = alternative name; p = plasma; vh = vitreous humor. um arises from melanocytes of the uveal tract (25,34). evs isolated from the ah and vh may contain proteins reflective of um cells. we pooled our data from intraocular-derived evs by focusing on proteins that regulate tumor growth and oncogenesis (tab. iv). this identified a panel of proteins that are mainly involved in protecting cells against apoptosis, controlling cell growth, promoting angiogenesis, and inducing cell spreading (i.e., clusterin, alpha-enolase, fibulin-1, cathepsin, hsp, ecm1, met, and gas6). moreover, vimentin (an intermediate filament protein that is overexpressed in epithelial tumors such as ums) was detected in vh-derived evs (tab. iv) (36,38-40). plasma-isolated evs were also enriched in proteins involved in the regulation of cell proliferation (i.e., sparc, tenascin, plexin) and cell survival (i.e., clusterin), and the metastatic process such as metastatic niche organization (i.e., ecm1, ecm2, emilin, c-reactive protein [crp], oncoprotein-induced transcript 3 [oit3], and integrins) (tab. v, and characterization of extracellular vesicles isolated42 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table iii newly characterized protein from evs isolated from plasma, aqueous humor, or vitreous humor identified proteins id p vh ah complement c4-b co4b y y y beta-crystallin b1 crbb1 y y y vitamin k-dependent protein c proc y y n immunoglobulin j chain igj y y n l-selectin lyam1 y y n neuropilin-2 nrp2 y y n soluble scavenger receptor cysteine-rich domain-containing protein ssc5d srcrl y n n extracellular matrix protein 2 ecm2 y n n plexin domain-containing protein 1 pldx1 y n n retinol-binding protein 3 ret3 n y y opticin opt n y y beta-1,4-glucuronyltransferase 1 b4ga1 n y y wnt inhibitory factor 1 wif1 n y y beta-ala-his dipeptidase cndp1 n y n receptor-type tyrosine-protein phosphatase zeta ptprz n y n macrophage colony-stimulating factor 1 receptor csf1r n y n serpin e3 serp3 n y n cadherin-related family member 1 cdhr1 n y n clusterin-like protein 1 clul1 n y n retinaldehyde-binding protein 1 rlbp1 n y n retinoschisin xlrs1 n y n adipocyte plasma membrane-associated protein apmap n y n left-right determination factor 2 lfty2 n y n neuronal cell adhesion molecule nrcam n y n interphotoreceptor matrix proteoglycan 1 impg1 n y n triggering receptor expressed on myeloid cells 2 trem2 n y n cathepsin l1 catl1 n y n endothelial lipase lipe n y n bpi fold-containing family b member 4 bpib4 n y n semaphorin-3b sem3b n y n zinc transporter zip12 s39ac n y n tsukushin tsk n y n beta-crystallin a3 crba1 n n y beta-crystallin a4 crba4 n n y gamma-crystallin c crgc n n y gamma-crystallin d crgd n n y gamma-crystallin b crgb n n y beta-crystallin b3 crbb3 n n y filensin bfsp1 n n y protein s100-b s100b n n y secreted frizzled-related protein 3 sfrp3 n n y phakinin bfsp2 n n y dna polymerase theta dpolq n n y protein kinase c-binding protein nell2 nell2 n n y data are derived from three patients (um-5, um-6, and um-8) and samples were run in duplicates. ah = aqueous humor; id = alternative name; n = absent; p = plasma; vh = vitreous humor; y = present. luz pessuti et al j circ biomark 2022; 11: 43 © 2022 the authors. published by aboutscience www.aboutscience.eu fig. 3 gene ontology classification of ev protein cargo. the most enriched categories in biological process (a), cellular component (b), and molecular function (c) are shown. data were collected from three um patient analytes repeated twice (um5-1, um5-2, um6-1, um6-2, um8-1, and um8-2). ev = extracellular vesicle; um = uveal melanoma. characterization of extracellular vesicles isolated44 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb supplementary figure a) (41-44). these data suggest that, while ahand vh-isolated proteins govern in situ um growth and motility, those contained in the plasma-derived evs are more involved in um cell metastatic organotropism and the maintenance of the metastatic niche. discussion evs have been reported to regulate many aspects of physiological and pathological processes such as cancer. they carry substances that mirror the content of their cell of origin and have the capability to exhibit different biological functions on recipient cells via trafficking of different factors, that is, nucleic acids, proteins, lipids (10,21,44-52). evs released from tumor cells promote cell proliferation, migration, invasion, angiogenesis, and metastases (54,57-63). ev cargo could be used as circulating biomarkers in liquid biopsy, mainly in the context of cancer. in the present study, we determined the proteomic profile of evs isolated from ah, vh, and plasma from patients with um in comparison with cancer-free control patients. table iv protein cargo from aqueous humoror vitreous humorderived evs involved in cell proliferation, survival, and invasion spectrum count identified proteins alternative name ah vh clusterin clus 50 175 pigment epithelium-derived factor pedf 37 111 alpha-enolase enoa 30 7 vitronectin vtnc 29 100 gamma-enolase enog 7 2 cathepsin d catd 6 64 fibulin-1 fbln1 6 4 myocilin myoc 6 heat shock protein hsp 90-alpha hs90a 5 10 galectin-1 leg1 3 heat shock protein hsp 90-beta hs90b 3 4 extracellular matrix protein 1 ecm1 2 5 growth arrest-specific protein 6 gas6 2 cd44 antigen cd44 2 5 c-reactive protein crp 1 1 plexin domain-containing protein 2 pxdc2 3 ras-related protein rab-1a rab1a 1 vimentin vime 14 cathepsin b catb 8 hepatocyte growth factor receptor met 6 cadherin-related family member 1 cdhr1 6 fibronectin finc 38 periostin postn 4 legumain lgmn 3 cathepsin f catf 3 ah = aqueous humor; ev = extracellular vesicle; vh = vitreous humor. data are derived from three patients (um-5, um-6, and um-8) and samples were run in duplicates. table v protein cargo from um plasma-derived evs involved in cell proliferation and survival, and metastatic niche organization identified proteins alternative name spectrum count fibronectin finc 130 vitronectin vtnc 109 clusterin clus 57 integrin alpha-iib ita2b 30 endoplasmin enpl 28 integrin beta-3 itb3 22 sparc sprc 22 nidogen-1 nid1 16 vinculin vinc 14 tenascin tena 13 pigment epithelium-derived factor pedf 13 c-reactive protein crp 13 heat shock protein hsp 90-alpha hs90a 10 fibulin-1 fbln1 9 heat shock protein hsp 90-beta hs90b 7 endoplasmic reticulum chaperone bip bip 7 cd44 antigen os = homo sapiens cd44 6 heat shock cognate 71 kda protein hsp7c 4 extracellular matrix protein 1 ecm1 3 plexin domain-containing protein 2 pxdc2 2 extracellular matrix protein 2 ecm2 2 beta-parvin parvb 2 caveolae-associated protein 2 cavn2 2 ras-related protein rab-1a rab1a 1 hepatocyte growth factor activator hgfa 1 oncoprotein-induced transcript 3 protein oit3 1 emilin-1 emil1 1 vascular endothelial growth factor receptor 3 vgfr3 1 plexin-b1 plxb1 1 integrin beta-1 itb1 1 alpha-enolase enoa 1 protein s100-a8 s10a8 1 protein s100-a9 s10a9 1 data are derived from three patients (um-5, um-6, and um-8) and samples were run in duplicates. ev = extracellular vesicle; um = uveal melanoma. luz pessuti et al j circ biomark 2022; 11: 45 © 2022 the authors. published by aboutscience www.aboutscience.eu the size and distribution of evs detected in the three samples were consistent with exosomes (10). in the blood samples, a significantly higher concentration of evs was found in um patients compared to the control group. this is in agreement with our recent observations that um cell lines shed more evs than normal choroidal melanocytes (33). another study suggests that ev has potential roles in cancer progression and invasion (11). interestingly, the mean concentration of evs in vh from um patients was higher when compared to plasma and ah, which seems normal as um takes place in the posterior segment of the eye. we showed that evs derived from ah, vh, and plasma were positive for cd63 and tsg101 markers. besides, the expression of cd63 was higher in um evs in comparison with evs isolated from samples of control group. our data corroborate with a study that demonstrated high levels of cd63 in exosomes isolated from plasma of melanoma patients (53). also a study showed exosomal marker tsg101 was detected in plasma-derived exosome from ovarian cancer patients (21). we observed that the plasma ev proteomic cargo resembles that of evs obtained from ah and vh. although we found that only 209 proteins (39%) were shared between evs from the three samples (a value that reached 221 proteins [49%] and 279 proteins [57%] when taking into account only ah vs. plasma and vh vs. plasma, respectively), this is not surprising as the plasma is the common carrier of evs from different tissues. moreover, proteomic mining of isolated evs from um group identified a set of proteins involved in oncogenesis (i.e., regulation of cell proliferation and survival, promotion of angiogenesis, and cell invasion) and metastasis (i.e., cell spreading and metastatic niche organization) (36,38-43,54). for example, sparc abrogation has been reported to reduce cell proliferation in um (41). cathepsin, a lysosomal acid proteinase, was reported to be involved in different cancer types, especially in regulating um invasion potential (36,40). galectin has been shown to facilitate cell migration, to promote metastasis, and to be a hallmark for cancer aggressiveness (55,56). oit3 is involved in the development and function of the liver, which is the primary site for um metastasis (54). in addition, several integrins were detected in the isolated evs from the um group. these proteins are involved in adhesion to extracellular matrix components and specific organotropism of metastasizing cancer cells (43,64). the integrins present in the ev preparations demonstrate an upregulation of various signal transduction molecules such as s100-a. it has been shown that exosome-derived integrins are internalized by target cells and activate src phosphorylation and proinflammatory s100 gene expression (64). furthermore, evs from melanoma were found to upregulate s100 proteins in recipient target cells, resulting in vascular leakiness and promotion of metastasis (31,65). other proteins found in the datasets such as heat shock proteins and crp are indicators of worse prognosis in um (38,42). in addition, melanocyte-specific type i transmembrane glycoprotein (pmel) was enriched in evs from vh and less in evs from plasma. this protein is released by proteolytic ectodomain shedding and may be used as a melanomaspecific blood marker (5,6,67,68). interestingly, the recovered protein cargo contained factors involved in cell proliferation, cell survival, oncogenesis, cell invasion, and metastatic niche organization. together, these data suggest that plasma from um patients could be used as liquid biopsy platform for patient diagnosis and noninvasive monitoring. using clustering analysis based on go biological process, categories consistent with retinal homeostasis and activation of intracellular pathways involved in cancer cell biology were identified (i.e., mapk/erk cascades). almost all ums are characterized by mutations in one of gnaq, gna11, plcb4, or cysltr2 genes, and these are upstream activators of the mapk/erk cascade (66). one limitation of this study is the low number of analyzed samples for the proteomic characterization (three um samples). however, the consistency of the data between the analyzed samples makes the conclusions valuable. unfortunately, due to the lack of material, performing differential protein expression analysis is not possible at this stage. studies including more samples are in progress to address this weakness. liquid biopsy is already distinguishing cancer-free individuals from non-small cell lung cancer patients and pancreatic ductal adenocarcinoma by the quantitative analysis of exosomal mir-21 and mir-10b, respectively (67). intra-ev metabolites from prostate cancer patients before and after prostatectomy revealed novel biomarkers (68). one must remember that not only tumor cells release exosomal rna to affect biological functions but also many normal cells will secrete the same exosomal rna physiologically (69). as mentioned before, exosomal integrins could be used to predict organ-specific metastasis (64). therefore, therapy supported by liquid biopsy could be driven in a premature way in case of early metastasis diagnosis or even somehow by targeting and blocking cancer pre-metastatic ev development. certainly, this promising new tool has to be used with caution, and further studies are needed. in conclusion, it has been observed that vh is significantly enriched in evs when compared to ah and plasma in um patients. ev concentrations in plasma and ah from um patients was higher when compared to those in the cataract group. proteomic analysis demonstrated that evs from the different samples shared a panel of proteins, suggesting that circulating um evs mirrored the in situ shed of evs (i.e., ah and vh). evs isolated from ah, vh, and plasma from patients with um showed consistent profiles and support the use of blood to monitor um patients as a noninvasive liquid biopsy. acknowledgments the authors would like to acknowledge the technical expertise and scientific support of the proteomics facility of the muhc-ri, especially lorne taylor and amy wong. disclosures financial support: this work was supported by cnpq process no. 429571/2018-6, fapesp, and capes. rbj is an investigator for cnpq brazil. conflict of interest: the authors report no conflict of interest. data availability: all data generated and analyzed during this study are included in this manuscript. characterization of extracellular vesicles 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accepted 18 february 2016 doi: 10.5772/62579 © 2016 the author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract urine exosomes (extracellular vesicles; evs) contain (micro)rna (mirna) and protein biomarkers that are useful for the non-invasive diagnosis of various urological diseases. however, the urinary tamm-horsfall protein (thp) complex, which forms at reduced temperatures, may affect ev isolation and may also lead to contamination by other molecules including micrornas (mirnas). there‐ fore, we compared the levels of three mirnas within the purified ev fraction and thpprotein-network. urine was collected from healthy donors and evs were isolated by ultracentrifugation (uc), two commercial kits or sepharose size-exclusion chromatography (sec). sec enables the separation of evs from protein-complexes in urine. after uc, the isolation of ev-mirna was compared with two commercial kits. the ev isolation efficiency was evaluated by measuring the ev protein markers, alix and tsg101, cd63 by western blotting, or mir-375, mir-204 and mir-21 by rt-qpcr. by using commercial kits, ev isolation resulted in either low yields or dissimilar mirna levels. via sec, the evs were separated from the protein-complex fraction. importantly, a different ratio was observed between the three mirnas in the protein fraction com‐ pared to the ev fraction. thus, protein-complexes within urine may influence ev-biomarker studies. therefore, the characterization of the isolated ev fraction is important to obtain reproducible results. keywords urine extracellular vesicles, ultracentrifuga‐ tion, tamm-horsfall protein, protein-complex, microrna, size-exclusion chromatography 1. introduction extracellular vesicles (evs) are small spherical structures (30-200 nm) that are composed of a lipid bilayer. different classes of evs have been assigned, and definitions and proposed nomenclature, like exosomes, microvesicles and apoptotic bodies, have been discussed [1–3]. evs are actively released from cells and contain proteins and nucleic acids that partly reflect the cellular content [4]. evs are involved in intercellular signalling [5,6] and can be found in biofluids such as urine [7]. as biofluids are easily accessible, evs have gained wide interest as a source of biomarkers for diagnostic or prognostic measures [8]. urine evs are derived from epithelial cells lining within the urinary tract [9] and are very stable [10]. ev isolation methods are increasingly becoming available, providing 1j circ biomark, 2016, 5:4 | doi: 10.5772/62579 faster, easier and less labour intensive techniques for ev isolation. however, these commercially available kits all need to be evaluated and isolated fractions need to be characterized. for isolating urine evs, the presence of the tam horsfall protein (thp; uromodulin), which forms polymers at lower temperatures, may decrease the ev yield. thp has been described to trap evs. therefore, evs may pellet at a lower speed centrifugation [11]. however, it has not yet been studied to what extent the thp-protein complex binds free mirnas, and how this contributes to the levels of detected mirnas within evs. therefore, we investigat‐ ed to what extent thp polymers contribute to the expres‐ sion of mirna within evs. using size-exclusion chromatography (sec), we separated the urine protein complexes from the ev fraction. interestingly, the protein complexes contained mirnas and, of the three tested mirnas, we found a different relationship compared to that within the evs, indicative of a different biomarker profile. therefore, when using any type of urine ev isolation method, the contribution of protein-complexes when performing biomarker identification studies should be carefully considered. 2. methods 2.1 urine ev isolation urine was collected from healthy individuals and stored at -80˚c. the urine evs were isolated by differential (ul‐ tra)centrifugation (uc), as previously described [5]. to break down the thp-polymers, dithiothreitol (200 mg/ml; dtt) was added where indicated for 1 h at 37˚c to the thp pellet (15,000 x g pellet), and the supernatant was used for ev isolation by uc as, previously described [12]. for a comparative analysis, both norgen biotek urine ev rna isolation kit (#47200, norgen biotek, thorold, on, canada) and life technologies ev isolation kit (# 4484452 life technologies, mulgrave, vic, australia) were used, according to the manufacturer’s instructions. the ultra‐ structural evaluation of the urine evs was performed by a transmission electron microscopy (tem), as previously described [4]. 2.2 the mirna expression levels rna was isolated by trizol, as previously described [4]. the mirna expression was determined by rt-pcr, according to the manufacturer’s instructions and as previously described [4]. we selected mir-21, mir-204 and mir-375 (life technologies, grand island, my, usa; mir-21 #000397, mir-204 #000508 and mir-375 #000564), which were described to be present in the urine evs. 2.3 protein analyses western blot was performed, as previously described [5]. for the measurement of cd63 protein levels, non-reduced conditions were used. the membranes were incubated with mouse anti-alix (1:500; cell signaling), goat antitsg101 (1:1000; santa cruz biotechnology, usa), rabbit anti-thp (1:1000; santa cruz biotechnology) or mouse anti-cd63-antibody (1:1000; santa cruz biotechnology). the overall protein yield was determined by coomassie brilliant blue (r-250, merck, france) staining on the total gels, as previously described [5]. 2.4 size exclusion chromatography sec was performed according to the protocol described by böing et al. [13], with small modifications. in brief, sephar‐ ose (cl-4b/2b 30 ml, ge healthcare; uppsala, sweden) was washed with pbs containing 0.32% trisodiumcitrate (ph 7.4, 0.2 µm filtered). subsequently, the tip of a 10 ml plastic syringe (becton dickinson (bd), san jose, ca) was stuffed with a nylon stocking (20 denier, hema, amster‐ dam, the netherlands). the syringe was stacked with 10 ml washed sepharose to create a column with a diameter of 1.6 cm and height of 6.2 cm. the urine was centrifuged 500 x g 15 minutes and 1.5 ml of it was loaded on the column, followed by elution with pbs/0.32% citrate (ph 7.4, 0.02 µm filtered). the eluate was collected in 26 sequential fractions of 0.5 ml. each fraction was stored at -80˚c until further processing. 3. results 3.1 ultracentrifugation and commercial ev isolation kits as uc is very labour intensive, we determined whether commercial ev isolation kits are easier to use improved the ev yield. to determine the ev isolation yields using the commercial norgen kit and life technologies (lt)-kit, we compared the expression of mir-21 and mir-375. ev isolation using both kits was considered to be very user friendly, fast and easy. for both kits, we slightly modified the protocol to remove debris from the samples. we then added one additional 15,000 x g centrifugation step, as commonly used for uc protocols. however, after using the norgen kit (10 ml urine), both of the tested mirnas were below the detection limit (figure 1a). the output of this kit is a purified rna fraction, though hardly any small rnas were detected. this indicates that the isolation efficacy was very poor (figure 1b). evs isolation by the lt-kit resulted in higher mirna levels (figure 1c) by using only 5 ml of urine as a starting material. increasing the volume of urine, while not increasing the solution given by lt, did not result in an increase in the levels of the tested mirnas. this is probably related to the maximum binding capacity obtained with 5 ml urine. the mirna levels in the evs after uc (in which sevenfold more urine was used) were >20 times higher compared to the lt-kit (figure 1c). when the evs were isolated from the unbound fraction from the ltkit (supernatant) by uc, we measured the high mirna levels that seemed to be in relation with the total mirna levels that were present in the sample. 2 j circ biomark, 2016, 5:4 | doi: 10.5772/62579 3.2 thp traps evs to determine the contribution of protein complexes to the rna signal, we determined the level of ev-capture within the thp-network. storage at reduced temperatures activates the thp-complex formation. we determined the effect of thp on ev isolation and compared the equal input volumes before isolation as normalization (figure 1d). thp was found at comparable levels when the urine was kept at either 4˚c or -80˚c (figure 1d). the ev marker, cd63, was detected in both the ev (uc) and thp-pellet fraction (15,000 x g fraction), indicating that evs may, indeed, be present in both fractions (figure 1d). however, the ev marker, tsg101, was not detected in the thpfraction, which indicates that the ev number in this fraction was low (figure 1d). to determine the amount of evs that were captured in the thp network, we performed an electron microscopy. a small fraction of evs were trapped in the thp-network (figure 1e). the number of evs in the thp pellet was slightly increased after storage at -80˚c, as well as the presence of thp-complexes, compared to urine storage at 4˚c, indicating that thp reduced ev isolation to figure 1 wachalska et al. a ultracentrifugation 35 ml urine norgen kit 10 ml urine 36 34 30 28 26 24 22 20 m ir n a e x p re s s io n ( c t) 32 38 c 34 30 28 26 24 22 20 m ir n a e x p re s s io n ( c t) 32 1 2 3 4 1 2 3 4 1 2 3 4 ultra 35 ml lt-kit 5 ml lt-kit 5 ml supernatant 100 kda 45 kda 4˚c -80˚c 4˚c -80˚c exosomes thp thp tsg101 cd63 d b 40 20 0 40 20 0 [fu] [fu] norgen ultracentrifuge 4 20 60 100 150 [nt] 4 20 60 100 150 [nt] e 4˚c -80˚c 40 20 0 [fu] ultracentrifuge + dtt 4 20 60 100 150 [nt] f g m ir n a e x p re s s io n ( c t) 20 21 22 23 24 25 26 27 28 mir-21 mir-204 mir-375 ev-fraction without dtt ev-fraction with dtt ** * * 20 21 22 23 24 25 26 27 28 mir-21 mir-204 mir-375 ev-fraction without dtt ev-fraction with dtt ev-fraction without dtt ev-fraction with dtt figure 1. urine extracellular vesicle isolation methods. a. expression of mir-21 and mir-375 in urine extracellular vesicles, comparing ultracentrifugation with norgen kit. b. small rna profile of isolated extracellular vesicles after norgen and ultracentrifugation. c. expression of mir-21 in urine of four different donors, comparing ultracentrifugation (ultra) to that of the life technologies kit (lt) and the supernatant (sn). d. western blot showing the expression of thp, tsg101 and cd63 in extracellular vesicles and thp fraction. e. em pictures showing extracellular vesicles trapped within the thp-network, after storage of 4c or -80c. size bar = 200 nm. f. the effect of the addition of 200 mg/ml dtt to the thp-pellet on the smallrna profile before extracellular vesicles isolation by ultracentrifugation. g. mir-21, mir-204 and mir-375 expression levels after releasing extracellular vesicles from the thp network by the addition of dtt, showing an increase in the expression levels. each value represents the mean +/sd of three donors. ** p<0,01, *p<0,05. 3magda wachalska, danijela koppers-lalic, monique van eijndhoven, michiel pegtel, albert a. geldof, andrea d. lipinska, r. jeroen van moorselaar and irene v. bijnsdorp: protein complexes in urine interfere with extracellular vesicle biomarker studies a small extent (figure 1e). after breaking down the thp network by incubation with dtt, small rna levels were highly increased in the ev fraction obtained after uc (figure 1f), compared to normal ultracentrifugation only (figure 1b). the levels of mir-375, mir-204 and mir-21 in the evs isolated with uc, combined with dtt-breakdown fraction, was increased by ~10 times (figure 1g). 3.3 size exclusion chromatography besides evs, protein-complexes are released by a break‐ down of the thp-polymer network after adding dtt. by single-step sec, evs can be separated from the protein complexes, with urine as the starting material and without adding denaturating chemicals to the samples [13]. within serum, it has been described that evs are present in fractions nine and 10 and proteins in fractions 16-32 [13]. in the fractions 16-23, we observed proteins, which is in agreement with the observations of nieuwland et al. [13] (figure 2a). the exosomal marker, cd63, was observed in fractions nine and 10, and slightly in fractions 11-13 (figure 2b). by em, we confirmed the presence of 100 nm sized evs in fraction 9 and 10 (figure 2c), similar to observations in human serum [13] and plasma (pegtel et al., unpublished data). to determine the mirna expression levels and ratio, we performed rt-qpcr and detected mirnas within the evs, and the ratio between fractions nine, 10 and 11 were comparable (figure 2d). on the other hand, in the proteinfractions, the ratio between the measured mirnas was different compared to the ev fraction (figure 2d-e). this indicates that, in urine, mirna containing protein com‐ plexes may contain a distinctive set of biomarkers com‐ pared to those found within evs. 4. discussion the results from the present study demonstrate that polymeric-thp, which is formed at reduced temperatures in urine, traps evs and contains mirnas. these proteincomplexes have a different mirna ratio of mir-21, mir-375 and mir-204 compared to the ev fraction. there‐ fore, when using urine evs as a source for biomarker identification studies, both commercial ev isolation methods and adding reducing agents, such as dtt, should be carefully checked for contamination with protein-bound rna. a b fraction 9 fraction 10 c d figure 2 wachalska et al. evs proteins proteins proteins evs ev-fraction protein-fraction 29 32 35 38 9 1 0 1 1 1 9 2 0 2 1 mir-204 mir-21 mir-375 0.0 0.5 1.0 1.5 2.0 e v fr a c ti o n p ro te in fr a c ti o n mir-204 mir-21 mir-375 29 32 35 38 9 1 0 1 1 1 9 2 0 2 1 mir-204 mir-21 mir-375 29 32 35 38 9 1 0 1 1 1 9 2 0 2 1 mir-204 mir-21 mir-375 ev-fraction protein-fraction m ir n a e x p re s s io n ( c t) m ir n a r a ti o e figure 2. contribution of proteins to extracellular vesicle isolations. a. coomassie staining of the lysate of 26 fractions from size exclusion, compared to the supernatant, purified extracellular vesicle (evs isolated by ultracentrifugation) and thp-pellet. b. western blot showing cd63 levels in each of the 26 fractions from size exclusion, compared to the supernatant, purified extracellular vesicles (ultracentrifugation) and thp-pellet. c. electron microscopy pictures of extracellular vesicles isolated within fractions 9 and 10. d. expression of mir-204, mir-21 and mir-375 in extracellular vesicles fractions 9-11 and protein fractions 19-21. e. changed ratio between mir-204 and mir-375 related to mir-21, indicating that the protein fraction contains a different subset of mirnas than the extracellular fraction. 4 j circ biomark, 2016, 5:4 | doi: 10.5772/62579 evs are secreted vesicles that are considered potential sources for biomarkers. urinary evs isolation usually contains a two-step differential centrifugation process. during an initial 15,000xg run, high-density membranes and thp polymers are removed. after the second step at 100,000×g, urinary evs are normally found in the pellet [15]. previously, it was demonstrated that storage at -20˚c dramatically decreased the number of evs [10], while longterm storage at -80˚c or short-term storage at 4˚c resulted in comparable ev yield [10,12], which is in agreement with our observations. the two tested commercial kits provided a more easy and rapid method. however, repeated testing of the norgen kit resulted in extreme low rna yields. unfortunately, this kit does not allow the characterization of the isolated fraction, as the output is rna only. the kit of life technologies was extremely easy and fast, though isolation of evs was inefficient. furthermore, both of the isolation kits make use of a protocol that does not include a high-speed pre-centrifugation step (15,000 x g). there‐ fore, protein complexes are potentially also captured using the kits tested in this study. the presence of thp in urine may reduce ev isolation yields. thp is a major protein component of urine [16]. thp polymer had a rope-like structure, in agreement with the observation of others [12,17]. polymeric thp has been described to entrap large amounts of evs, which may pellet at high speed [14], reducing ev yields. dtt can release evs from the thp polymeric network [10,12]. however, monomeric thp will be present in the ev fraction [12,18,19], potentially interfering with proteomic and rnaassays. we observed evs in the thp-pellet, though, compared to the total ev fraction, the number was rela‐ tively limited. it is most likely that mirnas are trapped by thp network, which contribute to the higher biomarker signals observed in previous studies, compared to the content in evs. by destroying this network with a reducing agent, evs are released, as well as protein-complexes that contain (micro)rna. therefore, thp may interfere with measuring the expression of markers when changing ev isolation methods. in conclusion, urine evs are a potential source of the identification of biomarkers. storage of urine and further processing may affect the biomarker profile due to the contribution of protein-complexes that also contain rna. we show that the contribution of proteincomplexes in urine, such as thp, requires detailed charac‐ terization before proceeding with rna or protein-based marker profiles for disease detection. 5. conflict of interest the authors report no conflict of interest. 6. acknowledgements this study was financially supported by stichting pon, stichting vumc/cca, worldwide cancer research #15-1005 and grant umo-2011/03/d/nz6/00923 from the polish national science centre. 7. references [1] van der pol e, böing an, harrison p, sturk a, nieuwland r. classification, functions, and clinical relevance of extracellular vesicles. phar‐ macol. rev. 2012;64:676–705. 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[19] mitchell pj, welton j, staffurth j, court j, mason md, tabi z, clayton a. can urinary exosomes act as treatment response markers in prostate cancer? j. transl. med. 2009;7:4. 6 j circ biomark, 2016, 5:4 | doi: 10.5772/62579 jcb j circ biomark 2023; 12: 17-25issn 1849-4544 | doi: 10.33393/jcb.2023.2502review journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu causative aneurysm has been obliterated (1,2). the pathophysiology of dci is complex and multifactorial; however, it is not yet fully understood. as a result, an accurate diagnostic test and effective treatment are both lacking. biomarkers may not only help to further elucidate the pathophysiology of dci, but could also aid in optimizing early diagnosis and prompt treatment. even though the present literature is brimmed with biomarker studies of dci, accurate biomarkers of dci have not yet been established. in 2018, an extensive systematic review proposed a panel analysis of 6 selected genetic and 15 protein biomarker candidates for further prospective validation of dci prediction (3). however, this review used a very broad definition of dci, including also dci defined solely as angiographic vasospasm on digital subtraction angiography or increased flow velocities on transcranial doppler (tcd) ultrasonography. historically, angiographic vasospasm was considered the sole cause of dci; however, in the past decades research has laboratory biomarkers of delayed cerebral ischemia following subarachnoid hemorrhage: a systematic review maud a. tjerkstra1, homeyra labib1, bert a. coert1, rené spijker2,3, jonathan m. coutinho4, w. peter vandertop1, dagmar verbaan1 1amsterdam umc, university of amsterdam, department of neurosurgery, neuroscience amsterdam, amsterdam, netherlands 2medical library, amsterdam umc, university of amsterdam, amsterdam public health, amsterdam, netherlands 3 cochrane netherlands, julius center for health sciences and primary care, university medical center utrecht, utrecht university, utrecht, netherlands 4amsterdam umc, university of amsterdam, department of neurology, neuroscience amsterdam, amsterdam, netherlands maud a. tjerkstra and homeyra labib contributed equally. abstract delayed cerebral ischemia (dci) substantially contributes to disability and death in subarachnoid hemorrhage (sah) patients; however, its pathophysiology is incompletely understood and diagnostic and therapeutic strategies are lacking. biomarkers may help to elucidate the pathophysiology, optimize early diagnosis, or provide treatment targets. we systematically searched pubmed and embase on october 13, 2021, for studies that evaluated at least one laboratory biomarker in patients with dci, using the most up-to-date definition of dci as proposed by a panel of experts in 2010. quality of studies was assessed using the newcastle-ottawa scale or cochrane collaboration’s risk of bias assessment tool. biomarkers of clinical and radiological dci were analyzed separately. results were meta-analyzed if possible, otherwise narratively reviewed. biomarkers were classified as significant, inconclusive, or nonsignificant. we defined validated biomarkers as those with significant results in metaanalyses, or in at least two studies using similar methodologies within the same time interval after sah. the search yielded 209 articles with 724 different biomarkers; 166 studies evaluated 646 biomarkers of clinical dci, of which 141 were significant and 7 were validated biomarkers (haptoglobulin 2-1 and 2-2, adamts13, vwf, nlr, p-selectin, f2-isoprostane); 78 studies evaluated 165 biomarkers of radiological dci, of which 63 were significant and 1 was a validated biomarker (lpr). hence, this review provides a selection of seven biomarkers of clinical dci and one biomarker of radiological dci as most promising biomarkers of dci. future research should focus on determining the exact predictive, diagnostic, and therapeutic potentials of these biomarkers. keywords: biomarker, delayed cerebral ischemia, subarachnoid hemorrhage received: october 8, 2022 accepted: march 20, 2023 published online: april 5, 2023 this article includes supplementary material corresponding author: maud annabel tjerkstra department of neurosurgery amsterdam umc, university of amsterdam meibergdreef 9 po box 22660, 1100 dd, amsterdam the netherlands m.a.tjerkstra@amsterdamumc.nl introduction delayed cerebral ischemia (dci) is an important and potentially preventable contributor to disability and death in subarachnoid hemorrhage (sah) patients in whom the https://doi.org/10.33393/jcb.2023.2502 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:m.a.tjerkstra@amsterdamumc.nl biomarkers of dci after sah18 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb shown that dci and vasospasms are two different entities (4). therefore, the combined use of both vasospasm and dci in the 2018 review may have misguided the selected panel of biomarkers. in 2010, a panel of experts proposed a strict definition of dci, divided into a clinical and radiological definition, to be used as outcome measure and to enable more reliable metaanalyses of future clinical trials and observational studies (4). we used these definitions of dci to systematically review the current literature on laboratory biomarkers of dci and selected a new panel of the most promising biomarkers. the selected biomarkers may be useful for the prediction or diagnosis of dci, either individually or combined. methods the preferred reported items for systematic review and meta-analysis (prisma) 2020 guidelines were followed. this review was not registered and a review protocol was not prepared. search strategy and selection criteria pubmed and embase were searched for relevant studies from january 1, 1985, to october 13, 2021. the search strategy can be found in the supplemental data (supplementary material, page 2-3). two authors (mat and hl) independently selected eligible studies. titles and abstracts were screened for potential eligibility when covering primary studies on biomarkers of dci in sah patients either written in dutch or english. of the selected studies, full texts were screened and studies were considered eligible if they met the following criteria: (1) patients with sah of whom (2) at least 95% were diagnosed with a causative aneurysm, (3) measurement of at least one laboratory biomarker (4) during hospital admission, (5) using either the radiological or clinical definition of dci as proposed in 2010 (criteria listed in tab. i), and (6) the studies had to perform, or allow for, statistical analysis on the biomarker in question and dci. exclusion criteria were: (1) studies with mixed clinical and radiological dci cohorts, in which patients with either clinical or radiological dci could not be distinguished from the mixed cohort, (2) biomarker levels in patients with and without dci were inconsistent (e.g., different numbers in tables/figures and text), (3) no level of statistical significance was given, nor could be calculated, (4) studies in children only, (5) the same patient population (similar years and hospital of patient inclusion) was used by more than one study and, within those studies, the same biomarker was measured and compared similarly between dci and no dci patients. in this case, the study with the smallest sample size, and if sample sizes were equal the most outdated study, was excluded. lastly, we checked studies on nimodipine administration. in 1988 and 1989, two randomized controlled trials showed efficacy of nimodipine in preventing dci, resulting in a shift in sah management (5,6). therefore, we excluded studies with sah patients before 1985. sah cohorts between 1985 and 1990 were only included if nimodipine administration was explicitly mentioned. all papers after 1990 were included, unless nimodipine was described explicitly as being not part of the treatment protocol. table i criteria of the clinical and radiological definition of dci clinical dci defined as: • �a� decrease� in� level� of� consciousness� (described� by� a� decrease� of�the�glasgow�coma�scale�score�or�an�increase�of�the�national� institutes�of�health�stroke�scale�score)�or • �focal�neurological�impairment�(e.g.,�aphasia,�motor�impairment,� sensory�deficits,�neglect)� excluded if: • �vague�definitions�of�dci,�e.g.,�“clinical�deterioration,”�“neurological� deterioration,”� “worsening� of� neurological� condition,”� without� specifically�mentioning�either�decreased�level�of�consciousness� or focal neurological impairment • �the�dci�cohort�also�includes�patients�with�solely�signs�of�vasospasm� without�clinical�symptoms�of�dci.�for�example,�patients�with:�  • arterial�narrowing�on�ct-a�or�dsa  • increased�flow�velocities�on�tcd radiological dci defined as: • new�ischemia/infarction/stroke�or • secondary�ischemia/infarction/stroke�or� • �ischemia/infarction/stroke� due� to� dci� (or� associated� dci� terms,� including�delayed�ischemic/neurological�deficit,�[clinical]�vasospasm,� secondary�ischemia/infarction) excluded if: • �defined� as� “presence� of� ischemia/infarction/stroke”� without� defining�“new,”�“secondary,”�or�“due�to�dci/associated�terms�of� dci”� • �presence�of�ischemia/infarction/stroke�determined�by�an�imaging� modality other than noncontrast ct or mri, e.g., ct perfusion, ct-a) • �other�nonimaging�methods�used�to� assess� ischemia/infarction/ stroke,�e.g.,�brain�tissue�oxygenation�monitoring�and�micro�dialysate biomarkers �ct�=�computed�tomography;�ct-a�=�ct-angiography;�dci�=�delayed�cerebral�ischemia;�dsa�=�digital�subtraction�angiography;�mri�=�magnetic�resonance�imaging;� tcd = transcranial doppler. tjerkstra et al j circ biomark 2023; 12: 19 © 2023 the authors. published by aboutscience www.aboutscience.eu data extraction the same authors (mat and hl) independently extracted baseline sah characteristics (age, gender, world federation of neurological societies [wfns] grade and fisher grade), the study’s definition of dci, the number of patients with and without dci, and biomarker-related data. if studies addressed both clinical and purely radiological dci separately, biomarker-related data were extracted for both definitions. the biomarker-related data concerned laboratory biomarkers only (clinical and radiological biomarkers [heart rate, blood pressure, temperature, tcd velocities, computed tomography (ct) perfusion variables etc.] were left out of consideration). biomarker data had to concern levels of individual biomarkers or ratios of two biomarkers, that is, combinations of three or more biomarkers, such as decision tree analysis or a self-made calculated score based on multiple biomarkers, were left out of consideration. data included the biomarker medium (blood, cerebrospinal fluid [csf], microdialysate [md], urine, or other), timing of measurement(s), and all statistical analyses regarding the biomarker and the occurrence of dci. if the biomarker medium was not mentioned, it was assumed to be blood. a biomarker measured in multiple media, for example, in blood and csf, was considered as two separate biomarkers. nongenetic biomarkers were stratified into biomarker groups, which were based on the multifactorial pathophysiology of dci as described by a previously published systematic review (7). of biomarkers with multiple functions, we aimed to select the function which the authors of the study considered as dci-related and subsequently categorized the biomarker into that specific biomarker group. nongenetic biomarkers which could not be stratified to any biomarker group related to the pathophysiology of dci were categorized as “other.” within the “other” group, we aimed to cluster biomarkers with similar functions. genetic biomarkers were categorized as a separate group. the extracted data were compared and discrepancies were discussed with a third author (dv) and solved. quality assessment two authors (mat and dv) independently assessed the quality of included studies using the newcastle-ottawa scale (nos) for case control and cohort studies and the cochrane collaboration’s risk of bias assessment tool for randomized trials. studies could obtain a maximum of eight or nine points, depending on the use of a nonexposed cohort. the cochrane collaboration’s risk of bias assessment tool comprises seven criteria, which can be scored as high, low, or unclear risk of bias. discrepancies were discussed with a third opinion (wpv) and solved. data interpretation and analysis biomarkers of clinical dci (primary outcome) were analyzed separately from biomarkers of radiological dci (secondary outcome). we categorized biomarkers into significant and nonsignificant biomarkers. biomarkers, which were studied by one study only, were considered significant if at least one measurement was significantly associated with dci (p < 0.05) and nonsignificant if all measurements yielded nonsignificant results. if a biomarker was investigated by more than one study, the classification into significant and nonsignificant was either based on a biomarker-specific meta-analysis, if methodologically possible (similar type of analyses and timing of measurement), or by descriptive comparison. metaanalyses were performed only if all relevant studies, which investigated the biomarker in question on a specific point in time after sah, could be included, using review manager 5.4. for continuous data we used the inverse variance method with random effects model to calculate weighted mean differences with 95% confidence intervals. for dichotomous data we used the mantel-haenszel method with random effects model to calculate estimated odds ratio with 95% confidence intervals. a pooled effect size was estimated for each specific timing of biomarker measurement. descriptive comparison of the studies was done by summarizing the results of all relevant studies in a biomarker-specific figure, which was then used to classify the biomarkers into significant, inconclusive, and nonsignificant. the biomarker-specific figures display the author, year, number of patients with and without dci, the type of analyses, and the results divided into categories of sampling timing (day 0-3, 4-8, 9-13, and >14 after sah). the method used for classification of biomarkers into significant, inconclusive, and nonsignificant based on the biomarker-specific figures is explained in the supplemental data (supplementary material, page 4). in order to select the most promising biomarkers of dci, we evaluated whether significant biomarkers were validated by a second study. a validated biomarker was defined as a biomarker with significant results in at least two studies using similar methodologies within the same time interval after sah, without any other study showing contradictory results. biomarkers of which the meta-analysis of all relevant studies yielded a significant association were considered validated, too. additionally, biomarkers that had only been investigated by one study were differentiated from those biomarkers that were investigated by a high-quality study. we defined high quality as a maximum (eight or nine) or maximum minus one point (seven out of eight or eight out of nine) quality assessment score. data have been reported according to the prisma reporting guidelines for systematic reviews and meta-analyses. results included studies after removing duplicates, the search yielded a total of 5,820 studies. we excluded 3,900 studies during abstract screening and 1,711 studies during full-text screening, after which 209 articles (204 cohort and 4 case-control studies, 1 randomized controlled trial) remained (fig. 1). baseline characteristics of all included studies are listed in table s1 of the supplemental data (supplementary material, page 5-12). altogether, the 209 studies measured 724 different biomarkers in 25,933 sah patients (not corrected for overlapping biomarkers of dci after sah20 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb patient cohorts). a summary of the nos quality scores is listed in table ii, and the quality scores of individual studies are displayed in the baseline characteristics table (table s1, page 5-12). the four case-control studies scored three, four (n = 2) and seven of nine points. the randomized controlled trial scored low risk on three of seven items. clinical dci biomarkers of clinical dci were studied by 166 studies comprising 646 different biomarkers (fig. 2) in 20,983 sah patients of whom 5,810 (28%) developed dci. most biomarkers (588 of 646, 91%) were investigated by one study and 58 (9%) by more than one study (range 2 to 14, median 3). the distribution of the biomarkers among the biomarker groups is shown in table iii. in total, 141 biomarkers (n = 88 nongenetic, n = 53 genetic) were classified as significantly associated with clinical dci (table s2, page 13-34). a summary of studied and significant biomarkers and specific trends observed within each biomarker group are displayed in table iii. of the significant biomarkers, 121 were investigated by one study and 20 by more than one study. meta-analyses were possible for five biomarkers (haptoglobin 1-1, 2-1, and 2-2, adam metallopeptidase with thrombospondin type 1 motif 13 [adamts13] and fig. 1 prisma 2020 flow diagram for systematic reviews. table ii summary of the newcastle-ottawa scale quality assessment scores of 204 cohort studies quality score 134 studies without control group (maximum 8 points) 70 studies with control group (maximum 9 points) 1 point none none 2 points 7 studies 2 studies 3 points 24 studies 8 studies 4 points 20 studies 16 studies 5 points 35 studies 20 studies 6 points 34 studies 14 studies 7 points 10 studies 7 studies 8 points 4 studies 3 studies 9 points – none von willebrand factor [vwf]). the meta-analyses showed that sah patients with haptoglobin polymorphism 2-1 were less likely to develop dci and patients with haptoglobin polymorphism 2-2 were more likely to develop dci (8-10). dci patients had lower adamts13 levels on day 1 and higher levels of vwf on day 4 after sah, compared to patients without tjerkstra et al j circ biomark 2023; 12: 21 © 2023 the authors. published by aboutscience www.aboutscience.eu fig. 2 flowchart of all biomarkers, divided into clinical and radiological dci. adamts13 = adam metallopeptidase with thrombospondin type 1 motif 13; lpr = lactate/pyruvate ratio; nlr = neutrophil/lymphocyte ratio; vwf = von willebrand factor. table iii number of studied and significant biomarkers and specific trends observed within each biomarker group of clinical dci biomarker group biomarkers of clinical dci specific trends observed among significant biomarkers within biomarker groupstudied significant nongenetic biomarkers 225 88 brain injury 16 8 all�indicated�more�brain�injury�in�patients�with�dci.�no�specific�trend�in�time.� coagulation�cascade 33 15 all�indicated�increased�platelet�activity,�hypercoagulability,�or� hyperfibrinolysis.�no�specific�trend�in�time.� cortical�spreading�depressions 2 1 excitotoxicity�in�first�48�h�after�sah. endothelial injury 3 2 both indicated more endothelial injury in dci. inflammation�and�immune� system 83 29 25�of�29�biomarkers�indicated�enhanced�inflammation�or�immune� response.�most�frequently�observed�in�first�couple�of�days�following�sah. oxidative�stress 18 7 5�of�7�biomarkers�indicated�increased�oxidative�stress�in�patients�with� dci.�no�specific�trend�in�time.� vascular�tone 22 9 6�of�9�biomarkers�indicated�vasoconstriction,�2�indicated�vasodilation,�1� was�significant�with�unknown�point�estimate.� volemic�status 10 2 both increased and decreased diuresis and natriuresis were observed in dci. other 38 14 subcategories�were:�cerebral�metabolism,�gender-specific�growth� factors, lipid metabolism, oxygen-carrying capacity, and stress response. genetic biomarkers 421 53 genomic microrna methylation polymorphism rna single�nucleotide�polymorphism  5  51  44  13  15 293  1  0 36  3  6  7 function�often�unclear,�some�were�involved�in�processes�of�oxidative� stress,�vascular�tone,�volemic�status,�stress�response,�inflammation�and� coagulation,�among�others. dci�=�delayed�cerebral�ischemia;�rna�=�ribonucleic�acid;�sah�=�subarachnoid�hemorrhage. dci, in two studies (11,12). the results of the remaining 53 significant biomarkers that were investigated by multiple studies that could not be meta-analyzed were summarized in biomarker-specific figures. based on the biomarker-specific figures, three other biomarkers (neutrophil/lymphocyte ratio [nlr] and p-selectin in blood and f2-isoprostane in urine) had significant results in two or more studies using similar methodologies and timing of measurement, and were considered validated biomarkers. four studies on nlr showed a proinflammatory state on admission and preoperatively in patients with dci compared to patients without dci (13-16). p-selectin, a marker of endothelial activation, was found to have a significantly larger increase during dci onset, compared to paired samples in the non-dci group in two studies biomarkers of dci after sah22 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb (17,18). f2-isoprostane in urine on day 3 after sah was significantly associated with the occurrence of dci in two studies (19,20). the quality of studies from which we found the seven validated biomarkers is listed in table iv. both metaanalyses and biomarker-specific figures can be found in the supplemental data (supplementary material, figure s1-s58, page 35-45). among the 121 significant biomarkers investigated by one study only, 5 (n = 4 nongenetic, n = 1 genetic) biomarkers were investigated by high-quality studies: brain natriuretic peptide (21), osteopontin (22), tenascin c (23), and neutrophil/ albumin ratio (24) in blood and endothelial nitric oxide synthase (snp rs2070744 t-786c) using buccal swabs (25). radiological dci biomarkers of radiological dci were studied by 78 studies comprising 165 different biomarkers (fig. 2) in 10,282 sah patients of whom 2,254 (22%) developed dci. most biomarkers (146 of 165, 88%) were investigated by one study and 19 (12%) by more than one study (range 2 to 8, median 2). the distribution of the biomarkers among the biomarker groups is shown in table v. in total, 63 biomarkers (n = 39 nongenetic, n = 24 genetic) were classified as significantly associated with radiological dci table iv quality of relevant studies of validated biomarkers validated biomarker studies quality of studies clinical dci adamts13 li,�2017�(11) tang,�2015�(12) 3/8 4/9 f2-isoprostane wiśniewski,�2020�(19) wiśniewski,�2017�(20) 6/9 6/9 haptoglobulin�2-1 kim,�2018�(8) leclerc,�2015�(9) ohnishi,�2014�(10) 3/8 5/8 6/8 haptoglobulin�2-2 kim,�2018�(8) leclerc,�2015�(9) ohnishi,�2014�(10) 3/8 5/8 6/8 nlr liu,�2020�(13) al-mufti,�2019�(14) wu,�2019�(16) tao,�2017�(15)� 6/8 6/8 7/8 5/8 p-selectin wang,�2011�(17) frijns,�2006�(36) 7/9 5/8 vwf li,�2017�(11) tang,�2015�(12) 3/8 4/9 radiological dci lpr kofler,�2020�(26) torné,�2020�(27) 4/8 2/8 adamts13�=�adam�metallopeptidase�with�thrombospondin�type�1�motif�13;� dci = delayed�cerebral�ischemia;�lpr�=�lactate/pyruvate�ratio;�nlr�=�neutrophil/ lymphocyte�ratio;�vwf�=�von�willebrand�factor. (supplementary material, table s2, page 13-34). a summary of the studied and significant biomarkers and specific trends observed within each biomarker group is given in table v. of the significant biomarkers, 56 were investigated by one study and 7 by multiple studies. meta-analyses were not possible. based on the biomarker-specific figures, one biomarker, lactate/pyruvate ratio (lpr) in blood, was considered validated as two independent studies showed that an lpr higher than 40 was associated with dci, measured during the first weeks following sah (26,27). the quality of the two studies on lpr was 4/8 and 2/8 (table 4). the biomarker-specific figures can be found in the supplemental data (supplementary material, figure s59-s77, page 46-48). among the 56 significant biomarkers investigated by one study, 7 nongenetic blood biomarkers were investigated by a high-quality study: monocyte lfa-1, monocyte mac-1, monocyte psgl-1, neutrophil lfa-1, neutrophil mac-1, neutrophil psgl-1, and osteopontin (22,28). clinical vs. radiological dci of all biomarkers, 89 were studied for both clinical and radiological dci (supplementary material, table s2, page 13-34). eight blood biomarkers were significantly associated with both clinical and radiological dci (d-dimer, platelet activating factor, interleukin-17, lectin complement pathway activity, osteopontin, brain natriuretic peptide, urea/creatinine ratio and serine proteinase inhibitor family e member 1 snp rs2227631 + rs1799889 + rs6092 + rs6090 + rs2227684 + rs7242). forty-five biomarkers were classified as nonsignificant for both clinical and radiological dci. of the remaining 36 biomarkers, the classification into significant, inconclusive, or nonsignificant differed between clinical and radiological dci. discussion this review has selected seven biomarkers of clinical dci (haptoglobin polymorphisms 2-1 and 2-2, adamts13, nlr, p-selectin, and vwf in blood and f2-isoprostane in urine) and one biomarker of radiological dci (lpr in md) as most promising biomarkers of dci. of note, the majority of significant biomarkers (121/141 of clinical dci and 56/63 of radiological dci) were only evaluated by one study, and although being potential biomarkers, especially the 12 biomarkers from high-quality studies, are in need of validation. in 2018, a review on biomarkers of dci yielded a panel of 21 predictive biomarkers (3); however, this review had three substantial limitations, which may have misguided the selected panel of biomarkers: (1) patients with solely angiographic vasospasm and increased flow velocities on tcd ultrasound, as indication of dci, were also included, (2) the results of biomarkers measured in different media (csf and blood) were combined, and (3) no differentiation between predictive and diagnostic biomarkers was made. in our present review, the most up-to-date definition of dci was strictly applied during the selection of eligible articles and a biological substance measured in multiple media was considered a separate biomarker for each medium. as a result, our review yielded less studies (209 vs. 270). although we found more biomarkers (724 vs. 257), our review yielded tjerkstra et al j circ biomark 2023; 12: 23 © 2023 the authors. published by aboutscience www.aboutscience.eu only eight validated biomarkers, as opposed to 21 by jabbarli et al., of which two (adamts13 and nlr) were in agreement (3). the discrepancy between the panels of biomarkers selected by both reviews emphasizes the importance of a uniform definition of dci, especially in the absence of solid diagnostic criteria. the eight validated biomarkers of dci are all related to known pathways of the multifactorial pathophysiology of dci. in clinical dci, increased platelet aggregability, thereby potentially leading to the formation of microthrombi, was observed by decreased admission levels of adamts13 and increased levels of vwf on day 4 after sah. as dci usually occurs between days 4 and 10 after sah (4), the early onset of significantly differing levels makes these biomarkers potentially predictive of dci. notwithstanding, the quality of the studies was rather low (nos scores of 3/8 and 4/9), therefore a high-quality study is necessary to further elucidate matters. most inflammation and immune system biomarkers seem to be significantly elevated early in the course of sah. accordingly, elevated nlr (a validated biomarker) in the first couple of days following sah was found to be significantly associated with clinical dci in four independent studies. but, the exact predictive ability, including determination of an optimal cut-off value with accompanying accuracy, specificity, and sensitivity, remains to be determined. whether nlr could also serve as a preventive treatment target is debatable, as nlr is a highly nonspecific biomarker and nonspecific anti-inflammatory therapies have thus far proven disappointing for the prevention of dci (29). p-selectin was found to significantly increase during clinical dci onset in two studies, which suggests that p-selectin may be a potential candidate for diagnosis or therapeutic treatment, for example, p-selectin antagonism. thus far, therapies targeting p-selectin have only been performed in experimental settings of ischemic stroke, showing a reduction in infarct volume and better clinical outcome (30), and myocardial infarction, showing significantly reduced myocardial damage after percutaneous coronary intervention (31). p-selectin antagonism has not been studied in sah, yet. three validated biomarkers of oxidative stress were found for clinical dci in our review: f2-isoprostane in urine and genetic biomarkers haptoglobin polymorphism 2-1 and 2-2. genetic markers often require extensive processing time and table v number of studied and significant biomarkers and specific trends observed within each biomarker group of radiological dci. biomarker group biomarkers of radiological dci specific trends observed among significant biomarkers within biomarker groupstudied significant nongenetic biomarkers 100 39 brain injury  6  2 all�indicated�more�brain�injury�in�patients�with�dci.�no�specific�trend� in�time. coagulation�cascade  16  9 all four biomarkers of primary hemostasis indicated increased platelet�activation/aggregation,�three�of�four�secondary�hemostasis� biomarkers showed hypercoagulability, one biomarker showed hyperfibrinolysis�in�patients�with�dci.�no�specific�trend�in�time.� cortical�spreading�depressions – – – endothelial injury  4  2 both biomarkers indicated increased endothelial injury in dci. inflammation�and�immune� system  33 15 12�of�15�inflammation�and�immune�system�markers�indicated� increased�inflammation�and�elevated�immune�response�in�patients� with�dci.�no�specific�trend�in�time.� oxidative�stress  4  2 both�biomarkers�indicated�increased�oxidative�stress�in�patients�with� dci. vascular�tone  8  1 vasoconstriction�between�days�0�and�14�after�sah�in�dci.� volemic�status  2  2 more diuresis and natriuresis were observed in dci. more dci in hyponatremia vs. no hyponatremia. other  27  6 subcategories�were:�angiogenesis,�cerebral�metabolism,�lipid� metabolism, stress response, and protein metabolism. genetic biomarkers  63 24 genomic microrna methylation� polymorphism rna single�nucleotide� polymorphism  3  28 –  5 –  27  0 19 –  1 –  4 function�often�unclear;�however,�four�biomarkers�were�involved�in� processes�of�coagulation,�inflammation,�and�vascular�tone. dci�=�delayed�cerebral�ischemia;�rna�=�ribonucleic�acid;�sah�=�subarachnoid�hemorrhage. biomarkers of dci after sah24 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb are therefore unsuitable for acute diagnosis; however, early determination of genetic profiles, for example, on admission, may aid in stratification of patients at high risk of clinical dci. as such, the haptoglobin polymorphisms 2-1 and 2-2 may be useful for patient stratification, and could perhaps provide a potential preventive treatment target, for example, the administration of exogenous haptoglobulin (32). for f2-isoprostane, whether the observed increased urine levels have preceded the onset of clinical dci in all patients is unknown; therefore, we cannot distinguish whether these biomarkers are predictive or diagnostic. further research should be performed to explore the temporal relationship of these markers in regard to clinical dci development. the remaining validated biomarker, lpr in md, allows for continuous neuromonitoring providing direct metabolic information, which can be used to detect an ischemic state, that is, radiological dci. since dci currently is a clinical diagnosis per exclusion, lpr may be a helpful additive in comatose patients, as it may detect metabolic distress in an early phase already, before hypodensities are visible on noncontrast head ct and when ischemic brain injury may still be preventable. however, this invasive technique yields metabolic information only locally, resulting in a detection failure of at least 20% (27). the difficulty of dci is in the incomplete understanding of its pathophysiology and lack of hard diagnostic criteria. currently, two definitions are used to identify patients with dci: clinical and radiological dci (4). even though adherence to one definition yields a different selection of patients than the other, both are strongly related to poor clinical outcome at follow-up, therefore both seem to be relevant (33-35). differences in the two definitions are clearly observed in this review, too, as 36 of 89 biomarkers studied for both definitions showed dissimilar results. the clinical definition seems to be the most relevant, given that early detection and correction of altered biomarkers may prevent the onset of clinical deficits. however, not all patients are neurologically assessable, therefore in comatose patients we currently rely on the radiological definition for dci diagnosis. considering that radiological appearance of ischemia or infarction is not immediately visible, once radiological dci is diagnosed the treatment window for ischemic disease has often elapsed. diagnosing radiological dci based on biomarkers may lead to earlier detection of radiological dci, perhaps even within the treatment window. limitations of this review are the heterogeneity in definitions used for dci in literature, which complicates the inclusion of relevant studies, the methodological heterogeneity among included studies, which has resulted in a largely narrative review with a suboptimal method used for classification of biomarkers, and a high proportion of poor quality studies. the quality of studies was not implemented in our method used for classification. as a result, even though the results of studies of validated biomarkers have been reproduced by a second group, some of the selected validated markers rely on studies with relatively poor quality (e.g., adamts13, vwf, and lpr). strengths of this review are the strict use of the most up-to-date definition of dci and the inclusion of all types of laboratory markers in blood as well as csf. conclusion in this review, seven biomarkers of clinical dci (haptoglobin polymorphisms 2-1 and 2-2, adamts13, nlr, p-selectin, and vwf in blood and f2-isoprostane in urine) and one biomarker of radiological dci (lpr in md) were selected as the most promising biomarkers of dci. future research should focus on determining the exact predictive or diagnostic abilities of these biomarkers, and exploring whether these biomarkers could serve as preventive or therapeutic treatment targets for dci. disclosures credit author statement: maud a. tjerkstra conceptualization, methodology, formal analysis, investigation, writing—original draft homeyra labib conceptualization, methodology, formal analysis, investigation, writing—review and editing bert a. coert writing— review and editing rené spijker conceptualization, methodology, 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international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu introduction epigenetics is defined as hereditary changes in gene expression without altering the deoxyribonucleic acid (dna) sequence (1). methylation of cytosine in the dna sequence and the biochemical changes of histones are two critical mechanisms in the epigenetics that play an important role in gene regulation, differentiation, and carcinogenicity (2-5). another mechanism that affects epigenetics and the gene expression is microribonucleic acids (mirnas). mirnas are non-coding endogenous rnas with a length of 20 to 25 nucleotides. these molecules can bind to untranslated 3′ regions (utrs) and suppress the expression of messenger rnas (mrnas) at the posttranscriptional level by pairing a specific base sequence (6,7). mirnas bind to their target mrnas and regulate their stability and/or translation. if mirnas bind completely to their target sequence on the the role of micrornas in covid-19 with a focus on mir-200c hadi sodagar1, shahriar alipour1,2, sepideh hassani1, shiva gholizadeh-ghaleh aziz1, mohammad hasan khadem ansari1, rahim asghari3,4 1department of biochemistry, faculty of medicine, urmia university of medical sciences, urmia iran 2student research committee, urmia university of medical sciences, urmia iran 3department of internal medicine, school of medicine, urmia university of medical sciences, urmia iran 4 hematology, immune cell therapy and stem cells transplantation research center, clinical research institute, urmia university of medical sciences, urmia iran abstract objective: epigenetics is a quickly spreading scientific field, and the study of epigenetic regulation in various diseases such as infectious diseases is emerging. the microribonucleic acids (mirnas) as one of the types of epigenetic processes bind to their target messenger rnas (mrnas) and regulate their stability and/ or translation. this study aims to evaluate non-coding rnas (ncrnas) with a focus on mir-200c in covid-19. in this review, we first define the epigenetics and mirnas, and then the role of mirnas in diseases focusing on lung diseases is explained. finally, in this study, we will investigate the role and position of mirnas with a focus on mir-200c in viral and severe acute respiratory syndrome–related coronavirus (sars-cov2) infections. methods: systematic search of medline, pubmed, web of science, embase, and cochrane library was conducted for all relative papers from 2000 to 2021 with the limitations of the english language. finally, we selected 128 articles which fit the best to our objective of study, among which 5 articles focused on the impact of mir-200c. results: due to the therapeutic results of various drugs in different races and populations, epigenetic processes, especially mirnas, are important. the overall results showed that different types of mirnas can be effective on the process of various lung diseases through different target pathways and genes. it is likely that amplified levels of mir-200c may lead to decreased angiotensin-converting enzyme-2 (ace2) expression, which in turn may increase the potential of infection, inflammation, and the complications of coronavirus disease. conclusion: mir-200c and its correlation with ace2 can be used as early prognostic and diagnostic markers. keywords: covid-19, epigenetic, lung diseases, mir-200c, mirnas received: november 5, 2021 accepted: february 22, 2022 published online: march 21, 2022 corresponding author: shahriar alipour department of biochemistry, faculty of medicine urmia university of medical sciences 11km sero road 0098 urmia iran alipour.sh@umsu.ac.ir shiva gholizadeh-ghaleh aziz department of biochemistry, faculty of medicine urmia university of medical sciences 11km sero road 0098 urmia iran gholizadeh.sh@umsu.ac.ir https://doi.org/10.33393/jcb.2022.2356 https://creativecommons.org/licenses/by-nc/4.0/legalcode sodagar et al j circ biomark 2022; 11: 15 © 2022 the authors. published by aboutscience www.aboutscience.eu mrna, they can lead to degradation; but in case of binding incorrectly, translational suppression of their target genes occurs by a mechanism that has not yet been fully understood (8). each mirna is predicted to have multiple gene targets and each mrna may be regulated by more than one mirna (9,10). the mirnas play a vital role in many important biological processes, including cell proliferation (11), growth (12), differentiation (13), apoptosis (14), metabolism (15), aging (16), signal transduction (17), and viral infections (18). it is estimated that about one-third of genes and their pathways are regulated and controlled by mirnas. briefly, mirnas have a remarkable effect on the genomic and epigenetic mechanisms (19,20). the role of mirnas in diseases focusing on lung diseases the mirnas involve in the development, progression, prognosis, diagnosis, and evaluation of therapeutic response in human diseases (21). in recent years, altered expression of the mirnas has been identified in many human cancers (22), cardiac hypertrophy and failure (23), metabolic disorders (24), immune system–related diseases, and inflammation (9). also, the mirnas have been studied in lung homeostasis, functional development, and various pulmonary diseases including asthma, chronic obstructive pulmonary disease (copd), cystic fibrosis (cf), idiopathic pulmonary fibrosis (ipf), and lung cancer (25) (tab. i). in recent years, an increasing amount of research has shown the impact of mirnas in the progress of pulmonary diseases (43). our knowledge of the role of mirnas in lung diseases has developed step by step. the role of mirnas in the unique pulmonary cells is thought to be essential in understanding the mechanism of lung function and disease pathogenesis (25). more recently, many studies have begun to report the effects of mirna transfer via extracellular vesicles. in lung diseases, this transfer was indicated to be facilitated via the intercellular communication between many types of cells in the respiratory system including endothelial cells (44), bronchial epithelial cells (45), mesenchymal stem cells, and others (46). table i relationship between mirna types and their target genes in different lung diseases refmeasurement typesampleexpression in diseasegene targetmirnadisease (26,27)quantitative pcrpbuprunx3mir-145 asthma (49)qrt-pcrserumupil-12mir-21 (50)qrt-pcrhbsmcsdownrhoamir-133a (28)rt-pcrbecuptgfβr2mir-19a (48)rt-pcrmacrophages—monocytesupil-13ra1mir-155 (29)qrt-pcrlung upsmad7mir-15b copd (30)rt-pcr/northern blotplfdowncox-2mir-146a (31)rt-pcrlung downbimmir-24-3p (32)high-throughput microarraypbmcsupnfkbiamir-93-5 (33)rt-pcr lungdowntom1mir-126 cf (34)qrt-pcr cell lineupcftrmir-145 (61)quantitative pcr cell culturedownsin3amir-138 (35)rt-pcrbronchial tissuesupano1mir-9 (36)microarrayslung downhmga2let-7d ipf (37)mirna array/northern blottinglung upsmad, smad7mir-21 (38)mir arrayhltdowntgf-β1mir-200c (39)taqman mirna assayserumuptgf-βmir-199a-5p (40) bioinformatics analysis and luciferase reporter assay lungdownslc22a18mir-137 lung cancer (41)qrt-pcr and western blottissuesdowntgfβr2mirrna-34a (42)rt-pcrlung cancer tissuedowne2f3mir-449a (55)rt-pcrtissuedownzeb1mir-200 bec = human bronchial epithelial cells; cf = cystic fibrosis; cftr = cystic fibrosis transmembrane channel; copd = chronic obstructive pulmonary disease; hlt = human lung tissue; ipf = idiopathic pulmonary fibrosis; mirna = microribonucleic acid; pb = peripheral blood; plf = primary lung fibroblast; qrt-pcr = quantitative reverse transcription polymerase chain reaction; pbmc = peripheral blood mononuclear cell; hbsmc = human bronchial smooth muscle cells. covid-19: role of mrnas and mir-200c16 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb the mirnas in asthma asthma is a chronic inflammatory disease of the lungs that is often associated with clinical features such as airway hyperresponsiveness (ahr), airflow obstruction, excessive mucus secretion, and airway wall structural changes (remodeling) (47). interleukin (il)-13 and transcription factor signal transducer-and-activation-of-transcription-6 (stat6)-operated path ways have been shown to play a significant role in regulating the prominent asthma features, for example, ahr and remodeling. mir-155 has been shown to be upregulated in order to target directly the transcription of the il-13 receptor a1 (il13ra1) in human macrophages, reducing the levels of il13ra1 protein and decreasing the levels of activated stat6, which is vital in regulating the il-13 signaling pathway (48). inhibition of mir-21 leads to a decrease in th2 cytokine levels (il-4, il-5, and il-13), the number of inflammatory airway leukocytes and ahr (49). downregulation of mir-133a was followed by an increased expression of rhoa and subsequently increased bronchial hyperactivity in a murine model of asthma (50). elevated expression of the mir-155 has also been indicated in murine models of asthma. additionally, by using antagomir against mir-145, the mucus secretion, th2 cytokine production, and eosinophil infiltration in the airways decreased (51). the mirnas in lung cancer dysfunction of mirnas is often identified in malignancies, including lung tumor. lung cancer is the leading cause of cancer-related mortality worldwide and to date the roles of mirnas in lung cancer have been specified and reviewed widely along with the other diseases. histologically, lung cancer can be mostly divided into small cell (sclc) and non–small cell lung cancer (nsclc). the latter is more common and is subclassified into squamous, adenocarcinoma, and large-cell carcinoma (52). recent sequencing studies have exposed a very large number of targets for each single mirna. by regulating the posttranscriptional gene expression, mirnas strongly involved in wide-ranging pathways with the main effect are on the progressive and carcinogenesis routes (53,54). concisely, various mirnas that are recognized as either oncogenes or tumor suppressors in lung cancer are also involved in the immune system response, for instance, the mir-200 family. the low expression of the mir-200 family members in human early-stage lung adenocarcinomas has been correlated with upregulation of pd-l1 (55) and cd8+ t-cell immunosuppression and metastasis, which resulted in the reduction of tumor load. this finding greatly supported the role of mir-200 as a tumor suppressor. the mirnas in copd copd is an inflammatory progressive lung disease that is prompted by chronic inflammation exposure of the airways to stimuli including cigarette smoking and other noxious gases. an increasing number of studies have demonstrated that injured cells such as endothelial and epithelial cells participate seriously in the pathogenesis of copd (56). the exposure of the respiratory epithelial cells to the harmful agents like cigarette smoke leads to the release of proinflammatory and inflammatory cytokines such as il-1, il-6, il-8, and tumor necrosis factor (tnf)-α (57,58). the mirnas and cf in the caucasian community, cf is the most frequent deadly hereditary disease. it is caused by a recessive mutation in the cftr (cystic fibrosis transmembrane channel) gene, which codes for a chloride channel (59). mirnas can target cftr directly or indirectly for regulating cf. several mirnas can complementarily and directly regulate cftr expression such as mir-145 (via smad3 and tgf-β), mir-223 (via cftr mrna), mir-9 (via anoctamin 1), and mir-494 (via solute carrier family 12member 2 (slc12a2)), alone or together. however, mir-509-3p and mir-494 downregulate cftr expression (60). some mirnas like mir-138 can also repress the biosynthesis intermediary actors, such as the transcription factor sin3a (sin3 transcription regulator family member a) and cftr (61). the mirnas and infections recent advances in molecular mechanisms point to the importance of mirnas in the lung and respiratory infections. acute viral respiratory infections (avris) are the most common causes of acute respiratory symptoms (62). changes in the regulation of mirna expression in the epithelial cells of human rhinovirus (hrv), influenza (iv), human metapneumovirus, human coronavirus, and respiratory syncytial virus infections are associated with the pathogenesis of acute respiratory diseases (63). for example, the expression of host mirnas changes in response to iv stimulation. these mirnas directly or indirectly target viral and host genes to regulate virus replication, stimulate or suppress innate immune responses and cell apoptosis during the viral infection (64,65). iv increases the expression of mir-4276 by upregulating two proteins involved in the apoptotic pathway, cas9 and cocx6c (74), and eventually leads to increased virus replication and apoptosis. furthermore, a number of specific cellular mirnas in iv-infected cells including mir-323, mir491, and mir-654 target the protected region of viral pb1 gene to prevent the virus from replicating in mdck cells (76). another mechanism in iv infection is the altered expression of cellular mirnas and their effect on important signaling pathways associated with the immune system (66). in hrv infections, mirnas result in antiviral responses by modulating the immune response (mir-128 and mir-155) as well as controlling virus entry into the infected lung cells (mir-23b) (67). rsv causes viral respiratory disease in infants and young children (68), modulating the expression of host cell mirnas for antiviral responses and virus replication similar to the mirnas mentioned above (69,70). for instance, mir-125a regulates nuclear factor kappa b (nf-κb) signaling pathway by suppressing a20 inhibitor protein (ccl5) as an important cytokine in both innate and compatible immune systems (71). coronaviruses cause a wide range of respiratory infections, from mild upper respiratory tract infections to severe lower respiratory tract infections (72). table ii shows the four major sodagar et al j circ biomark 2022; 11: 17 © 2022 the authors. published by aboutscience www.aboutscience.eu table ii relationship between different types of mirnas and their target genes in well-known viral lung infections refpathwayseffects on gene regulation gene targetmirnaviral disease (74)–  inhibits cox6c and caspase-9 and promoting viral replication –  up–  cox6c–  mirna-4276 influenza virus (75)–  inhibits replication of virus –  up–  pb1–  mir-323, mir-491 (76)–  reduces virus replication by degrading m1 mrna –  up–  m1–  let-7c (77)–  negatively regulates innate immune and inflammatory responses –  up–  traf6–  mir-146a-5 (78)–  regulates virus entry –  down–  ap1g1–  mir-576-3p (79)–  suppresses iav replication –  down–  hdac8–  mir-21-3p (64)–  regulates antiviral response –  up–  mapk3, irak1 –  mir-132, mir-200c (80)–  regulates the immune response against rv-1b and inhibits virus replication –  up–  smad2, egfr –  mir-128 , mir-155 rhinoviruses (81)–  prevents viral infection by decreasing the vldlr –  up–  vldlr–  mir-23b (82)–  antiviral host response –  upccl7, socs3 let-7f rsv (83-85)–  induces mirnas to involve in the immune response pathways such as nf-kb and type i ifns –  up–  il-13, tlr4, runx2  mir-30, let-7i (86)–  promotes viral replication –  down–  ngf, trka mir-221 (87)–  inhibits nf-κb signaling pathway and results in reducing macrophage activation –  down–  tnfaip3 mir-125a (88)n protein of virus binds to mir-9 and modulates nf-kb expression up–  nf-kbmir-9oc43 coronavirus (89,90)–  suppresses viral replication that may aid evasion of immune surveillance until successful infection of other cells up–  virulent proteins, including n, s, m, and e mir-17, mir-574-5p, mir-214sars (91)–  mirna-mrna network significantly impacts mers-cov replication up–  map3k9, myo15b, spock1   mir-16-1-3p, mir-26a-13p, mir-425-5p, mir-1275, mir-2277-5p , mir-500b-5p, mir627-5p, mir-1257, mir-1275 mers (92)–  these mirnas may downregulate viral gene expression resulting in the inhibition of viral replication up–  viral mrnamir628-5p, mir-18a-3p, hsa-mir332-3p mers (93)–  acts as a negative regulator of nf-κb as the transcription factor of the il-6 gene downil-6 mir-146a-5psarscov2 –  overexpression of mir-200c induces downregulation of ace2 in human cells upace2 mir-200c (94)–  targets sars-cov2 genome upsars-cov2 orf1a/b  mir-1202 (95)–  expression of let-7d-5p negatively correlates with tmprss2 expression 91 up–  tmprss2let-7d-5p il = interleukin; mers = middle east respiratory syndrome; mirna = microribonucleic acid; nf-κb = nuclear factor kappa b; rsv = respiratory syncytial virus; sars-cov = severe acute respiratory syndrome–related coronavirus; iav = influenza a viruses. covid-19: role of mrnas and mir-200c18 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb categories of the pulmonary virus families and some of the most important mirnas that change the expression of the genes involved in infections with these viruses. severe acute respiratory syndrome coronaviruses (sars-cov) use host cell mirnas to escape removal by the immune system (89). in middle east respiratory syndrome coronavirus (merscov) infection, cellular mirnas act as an antiviral therapeutic agent (92). the functional mechanisms of mirnas in sars-cov2 as the causative agent of covid-19 are diverse. for example, increased mir-200c expression in the disease downregulates the expression of angiotensin-converting enzyme (ace2) protein that is the receptor essential for the virus entry into the cell (73). association of mir-200c with the genes involved in inflammation (ace2, il-6) mir-200c-3p is a member of the mir-200 family with two clusters mir-200a/b/429 and mir-200c/141. the mir200c-3p is one of the most important mirnas of the second cluster. studies on the mir-200 family have shown that it has a variety of roles in cancer progression, drug resistance, and oxidative stress (96,97). the results of various studies have revealed the crucial role of the mir-200c epithelial-mesenchymal transmission, proliferation, metastasis, apoptosis, autophagy, and therapeutic resistance in several types of cancer (98). the mir-200c is also measured as a biomarker to predict disease progression, diagnosis, and response to therapy in several cancers, both in tissues and in body fluids (blood, urine) (96). studies using mirnas can contribute not only to the understanding of virus-host interactions but also to the stratification of the different severities of covid-19. in this sense, mir-200c-3p, which has been associated with viral infections, including influenza a, offers itself as a candidate for the study of covid-19. the analysis of its expression in groups of patients presenting different levels of disease aggressiveness could contribute to a better screening of patients affected by sars-cov2. thus, in pimenta’s study, which aimed to analyze the expression of mir-200c-3p in saliva samples from patients with covid-19, the results showed that the expression pattern of mir-200c-3p increased with disease severity (99). furthermore, the significant impact of mir-200c-3p in acute respiratory distress syndrome (ards) was discovered, which proposes it as a potential factor in sars-cov-2 research and is considered as a potential diagnostic agent for sars-cov-2 studies (100). in a study of the h5n1 avian influenza virus (aiv) ace, serum levels of mirna-200c-3p were found to increase in the virus causing acute pulmonary injury and ards. this mirna binds to the 3′-utr locus of the ace2 gene, and inhibits the expression of this protein and thus exacerbates the disease (100-102). the ace2 gene was first identified from complementary dna in the left ventricle of the human heart (102). ace2 inactivates angiotensin ii (ang ii) by cleavage and produces ang 1-7 (103). ang ii binds to type 1 and type 2 ang ii receptors with high affinity and is involved in regulating blood pressure, body fluid balance, inflammation, cell proliferation, hypertrophy, and fibrosis (104-106). ace2 has been shown to neutralize the development of severe ards caused by aiv, coronavirus, and sepsis in mice (106). ace2 has also been reported as a receptor for the sars-cov2 virus to enter the pneumocytes (107). the role of mir-200c in lung inflammation and lung diseases mir-200c, alongside with mir-141, is placed in the intragenic zone of chromosome 12. mir-200c family has beneficial effects on preventing drug resistance, cancer development, and oxidative stress. it consists of two clusters: (1) mir-200c/141 cluster including mir-141-3p and mir-141-5p, mir-200c-3p, mir-200c-5p on chromosome 12p13.31; (2) mir-200a/ b/429 cluster including mir-200a-3p, mir-200a-5p, mir200b-3p, mir-200b-5p, and mir-429 on chromosome 1p36.33 (108). mir-200, like ace2, is greatly expressed in the epithelial cells of the pneumocytes, mainly in type ii alveolar epithelial cells. the expression of mir-200 has a crucial role in the differentiation of type ii alveolar epithelial cells in fetal lungs, which are important components of the renin-angiotensin system signaling pathway all over the body. mir-200 displays several important effects in the body such as anti-remodeling, anti-inflammatory, and anti-proliferative through reduction of angiotensin ii levels (fig. 1) (109). remarkable points in this issue are about controlling covid-19 patients’ mortality rates and disease severity, by upregulating ace2 levels with using angiotensin receptor blockers or ace2 blockers (110). mir-200 is the exact and direct target of ace2 at 3′-utr of ace2 mrna which by binding to its locus results in the depression of ace2 expression as a receptor responsible for ards incidence. normally, ace2 catalyzes the conversion of agii to ag1-7. later, ag1-7 binds to mitochondrial assembly (mas) receptors resulting in ag1-7 protective effects including anti-proliferation, anti-necrotic and anti-hypertrophic as well as vasodilation and declining of proinflammatory cytokine secretion. sars-cov2 inhibits this pathway and worsens agii adverse effects on lung tissue during the acute phase of the disease. it was reported that sars-cov2 induces the secretion of il-6, tnf-α, il-1β (102,111-113). activation of nf-κb pathway, an important factor in ards pathogenesis, is one of the noticeable pathways leading to the upregulation of mir-200c-3p. increased expression of mir-200c-3p occurred when the ace2 expression decreased (100) (fig. 1). these mechanisms include increased mir-200c expression, inhibition of ace2 expression, by affecting ace2 protein outside the cell, and by inhibition of other anti-inflammatory functions, all of which are shown in the figure. (1) increased mir-200c expression that sars-cov-2 inhibit ace2 indirectly by regulating mir-200c and directly inhibiting ace2 expression, (2) by affecting the ace2 gene, (3) ace2 protein outside the cell, and (4) by inhibiting other anti-inflammatory functions, all of which are shown in the figure. in addition, mir200c can also reduce ace2 expression, thereby reducing ace2 expression and reducing its function. according to research results, the reduction in disease severity in covid-19 patients associates with the correlation between low expression of ace2 and high levels of mir-200c-3p in the lungs and the upper respiratory tract (114,115). sodagar et al j circ biomark 2022; 11: 19 © 2022 the authors. published by aboutscience www.aboutscience.eu recent studies about the entrance of sars-cov-2 to the host cells imply that some mirnas can actually control the expression of ace2 and tmprss2, which are potentially of high effect in sars-cov-2 pathogenesis (116). several pathways have been studied about the effect of epigenetics on the regulation of ace2/tmprss2 expression levels in respiratory diseases. the epigenetic repression of mirna transcription can control their regulatory regions. for instance, lysine-specific demethylase 5b (jarid1b, encoded by the kdm5b gene) was displayed to suppress the transcription of mir-200 family including mir-141, mir-200a, mir-200b, mir-200c, and mir-429. hsa-mir-125a/hsa-let-7e mirnas inhibit the transcription of mir-200 family through stimulating h3k4me3 histone, which demethylases the mirnas of this family. therefore, hsa-mir-125a-5p via binding to mir-200 family pursues 3′-utr of ace2 mrna and results in the enhancement of ace2 gene expression while 3′-utr of the tmprss2 is targeted by hsa-let-7e-5p. concludingly, jarid1b epigenetic activity doesn’t directly regulate the expression of ace2 and tmprss2 (116). scientists have investigated if promoting h3k4me3 demethylation is caused by repression of the transcription of the let-7e and mir-125a via jarid1b gene (117); for example, the upregulation of jarid1b in lung cancer cell line a549 concluded threefold depression of mir-200a and mir-200c expression, while jarid1b knockdown enhanced 1.5-fold their conserved and stable levels (118). the experimental data show the presence of controlling network containing mir-125a/let-7e/mir-200 families, ace2/tmprss2 as well as histone demethylase jarid1b, and further point a new way for signaling pathway for ace2 expression. in one report, the single-cell rna sequencing data analysis sharply indicated that in the majority of human cells ace2 and tmprss2 are not expressed without jarid1b. so, for better understanding, the viral infection pathogenesis needs to be investigated in the regulatory network related to the expression of jarid1b, ace2, and tmprss2 in human respiratory epithelial cells (116). according to cellular ontologies research on 24 mirnas, for evaluating the mirnas targeting sars-cov-2 host cell receptor ace2, it was revealed that mir-429, mir-200a-3p, mir-210-3p, mir-200b-3p, and mir-200c-3p were highly expressed in the respiratory epithelial cells and mir-200c-3p exists abundantly in the cells including endo-epithelial cell, epithelial cells, respiratory epithelial cells, leukocytes, hematopoietic cells, and myeloid leukocytes. also, mir-200b fig. 1 mir-200c and ace2 mechanism of function in the pathogenesis of covid-19. sars-cov2 induces inflammation and severe ards through four mechanisms: (1) virus indirectly leads to ace2 downregulation by enhancing mir-200c expression. (2) virus directly inhibits ace2 gene expression. (3) sarscov2 inhibits binding of ace2 protein to its receptor on the lung cells. (4) sars-cov2 inhibits the anti-inflammatory effects of ace2. ace2 = angiotensin-converting enzyme-2; ards = acute respiratory distress syndrome; covid = coronavirus; sars-cov = severe acute respiratory syndrome– related coronavirus. covid-19: role of mrnas and mir-200c20 © 2022 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb and mir-200c were discovered to be extremely conserved (119). in clinical trials, mir-200 and its correlation with ace2 can be used as early prognostic and diagnostic markers. its location on the upstream of ards signaling pathways may reduce the morbidity and mortality rates of covid-19 via epigenetic procedures, which can be so beneficial for human survival. conclusion at present, there is no exact treatment for covid-19. due to the importance of mirnas in pulmonary diseases, mainly the infectious viral diseases as well as sars-cov-2, they can be potential candidates of targeted therapy in sars-cov-2 in order to reduce the morbidity and mortality rates of this disease as mir-200c and its correlation with ace2 can be used as early prognostic and diagnostic markers. however, further research must be carried out to reveal the exact effect of mir-200c in the pathogenesis of covid-19 in order to be used clinically. authors’ contributions hs was responsible for the largest share in writing the article. sa and sg-ga conceptualized and wrote the article and article design. however, sa share has been higher. sa contributed in review and editing of final submitted version. mhka and ra contributed in methodology, data validation and writing original draft of this article. acknowledgments the authors would like to thank the officials of urmia university of medical sciences and the student research committee for their support of this project. disclosures conflict of interest: the authors declare no conflict of interest. financial support: this review article was partly supported by urmia university of medical 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https://doi.org/10.1007/s11033-021-06378-x https://doi.org/10.1371/journal.pone.0235987 https://doi.org/10.1074/jbc.m111.304865 https://www.ncbi.nlm.nih.gov/pubmed/21969366 https://doi.org/10.4161/cc.25142 https://www.ncbi.nlm.nih.gov/pubmed/23759590 https://doi.org/10.1016/j.mgene.2020.100831 https://www.ncbi.nlm.nih.gov/pubmed/33224734 cbx899214 1..13 research article analytical validation of the cellmax platform for early detection of cancer by enumeration of rare circulating tumor cells pratyush gupta, zulfiqar gulzar, ben hsieh, austin lim, drew watson, and rui mei abstract the cellmax (cmx®) platform was developed to enrich for epithelial circulating tumor cells (ctcs) in the whole blood. this report provides assay performance data, including accuracy, linearity, limit of blank, limit of detection (lod), specificity, and precision of enumeration of cancer cell line cells (clcs) spiked in cell culture medium or healthy donor blood samples. additionally, assay specificity was demonstrated in 32 young healthy donors and clinical feasibility was demonstrated in a cohort of 47 subjects consisting of healthy donors and patients who were colonoscopy verified to have colorectal cancer, adenomas, or a negative result. the cmx platform demonstrated high accuracy, linearity, and sensitivity for the enumeration of all clc concentrations tested, including the extremely low range of 1 to 10 cells in 2 ml of blood, which is most relevant for early cancer detection. theoretically, the assay lod is 0.71 ctcs in 2 ml of blood. the analytical specificity was 100% demonstrated using 32 young healthy donor samples. we also demonstrated precision across multiple days and multiple operators, with good reproducibility of recovery efficiency. in a clinical feasibility study, the cmx platform identified 8 of 10 diseased subjects as positive (80% clinical sensitivity) and 4 of 5 controls as negative (80% clinical specificity). we also compared processing time and transportation effects for similar blood samples from two different sites and assessed an artificial intelligence-based counting method. finally, unlike other platforms for which captured ctcs are retained on ferromagnetic beads or tethered to the slide surface, the cmx platform’s unique airfoamenabled release of ctcs allows captured cells to be transferred from a microfluidic chip to an eppendorf tube, enabling a seamless transition to downstream applications such as genetic analyses and live cell manipulations. keywords circulating tumor cell (ctc), liquid biopsy, biomarker, colorectal cancer, metastasis, cellmax ctc platform, microfluidics, analytical validation date received: 3 july 2019; accepted: 10 december 2019 introduction in the united states, the risk of developing cancer in one’s lifetime is 40%,1 with most cancers detected too late for treatments to have any significant impact on survival. the world health organization defined the current dilemma the best, “ . . . we cannot treat our way out of the cancer problem.” there is a need for “more commitment to the prevention and early detection” of cancer. 2 colorectal cancer (crc) in particular is a disease, afflicting countries cellmax life, sunnyvale, ca, usa corresponding author: rui mei, cellmax life, 1271 oakmead parkway, sunnyvale, ca 94085, usa. email: rui@cellmaxlife.com journal of circulating biomarkers volume 8: 1–13 ª the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454419899214 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (https://creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://orcid.org/0000-0001-7903-2846 https://orcid.org/0000-0001-7903-2846 mailto:rui@cellmaxlife.com https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454419899214 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage with a high human development index, and crc incidence is positively correlated with increasing prosperity. early detection of crc disease and recurrence has been shown to significantly improve overall survival, inclusive of colorectal polyp or adenoma detection. 3,4 there exists an unmet medical need for a novel test that can detect precancerous lesions or early stage diseases. circulating epithelial cells, commonly referred to as circulating tumor cells (ctcs), provide rich and varied information in the context of detecting and staging cancer. this information can be mined for early detection, characterization of metastasis, and treatment monitoring. 5,6 ctcs were first discovered in the late 19th century. 7 they present an opportunity for diagnosis of cancer via liquid biopsy performed on less than 10 ml of blood from a single blood draw, superseding solid tissue biopsy and related invasive procedures. 8 this is of great benefit to patients weakened by the disease and possibly by chemotherapy. secondly, the cost of testing can be lowered since radiographic image guidance is not required and less time needs to be invested by skilled health-care personnel. thirdly, peripheral venous phlebotomy is a more suitable technique for the repeat sampling required to monitor disease progression or recurrence. monitoring cancer by the detection of ctcs has several key advantages over solid tissue biopsies. 9,10 ctcs in the peripheral blood are involved in tumor metastasis, with greater numbers detected in metastatic patients compared to patients with localized and benign disease. 11,12 ctcs increase in the hematologic phase of tumor metastasis. a tumor that grows larger than 2 mm 13 may undergo angiogenesis and shed tumor cells that enter the vascular system and migrate to distant locations. clinical validation has supported the prognostic value of ctc enumeration to predict progression-free survival and overall survival in metastatic breast cancer, 14,15 prostate cancer, 16,17 and colorectal carcinoma. 12 beyond enumeration, the molecular characterization of ctcs has the potential to predict response to therapy. 12,16 the cmx assay is distinguished by its ability to harvest live cells for analysis via gentle airfoam release, without damaging these cells. 18 this facilitates downstream analyses of ctcs comprising enumeration, gene expression, methylation, and mutations. although ctcs have been extensively studied for more than two decades, there are few reliable methods of detecting and isolating ctcs in the early stages of cancer. 19–21 experimental in vivo studies have suggested that ctcs are present early in the natural history of solid tumor growth, before the development of metastasis. 22 more recently, ctcs have been detected in the blood at early stages and at recurrence in women with breast cancer, 14,23 and in premalignant stages of prostate cancer tumor progression. 24 the problem of isolating rare ctcs is technologically challenging and complex. 25 ctcs exist in frequencies in the range of one in one billion blood cells. 11,26 numerous research and commercial efforts have failed to isolate ctcs in early stage cancer, utilizing techniques ranging from gradient centrifugation, 27 affinity separation 24,28 to filtration. 29–33 currently, there are at least three clinical trials registered with the us national institutes of health that use ctcs for cancer screening. they include lung ca ncer 3 4 (https://clinicaltrials.gov/ct2/show/ nct02500693), breast cancer 35 (https://clinicaltrials.gov/ ct2/show/nct01322750), and crc trials (https://clinical trials.gov/ct2/show/nct02005913). in studies utilizing the cellsearch ® fda-cleared ctc test with an epcam cell enrichment strategy, authors concluded that multiple sample processing steps resulted in the loss of ctcs 36 and suggested that gentler methods could reduce this loss and enable the detection of ctcs at earlier stages of disease. 21 while isolating ctcs in patients with precancerous lesions or early stage crc has been a formidable challenge, a recent prospective study correlating ctcs to outcomes in nonmetastatic crc concluded that preoperative ctc detection is a powerful prognostic marker in nonmetastatic crc. 37,38 technologies that increase sensitivity have the potential to enable early cancer detection. 21 peripheral blood ctcs can be detected in patients with precancerous colorectal polyps as well as crc. 3 9 a significant difference in peripheral ctc counts has been observed between benign and malignant disease; a ctc count >3/3 ml has been correlated with the presence of a primary tumor. ctc counts also vary with respect to anatomical location and degree of tissue differentiation of the primary tumor. 39 the cellmax (cmx ® ) platform is uniquely suited for ctc detection in early stage disease due to technological aspects that include (i) a biomimetic surface coating on a microfluidic chip that reduces non-specific binding and enables capture of ctcs with threefold greater sensitivity and sixfold greater purity (less contamination than white blood cells) than conventional coating, (ii) proprietary high-affinity antibodies with sixfold greater affinity for cancer cells than conventional antibodies, and (iii) a gentle airfoam release mechanism that enables the capture and collection of low epcam expressing cancer cells that can be further used for several downstream applications. 18,37,40 this study validated the accuracy, linearity, limits of blank and detection, and the reproducibility of the cmx platform. it also explored the possibility of a cmx test for the early detection of crc with the inclusion of a clinical feasibility study. methods cmx ctc assay the cmx ctc assay (figure 1(a)) utilizes a microfluidic chip (figure 1(b)) consisting of a top layer of poly(methyl methacrylate) with a proprietary etched pattern and a glass bottom coverslip sandwiched together with double-sided 3m tape. the inner surface of the glass layer is coated with a supported lipid bilayer (slb). this slb mimics the cell 2 journal of circulating biomarkers https://clinicaltrials.gov/ct2/show/nct02500693 https://clinicaltrials.gov/ct2/show/nct02500693 https://clinicaltrials.gov/ct2/show/nct01322750 https://clinicaltrials.gov/ct2/show/nct01322750 https://clinicaltrials.gov/ct2/show/nct02005913 https://clinicaltrials.gov/ct2/show/nct02005913 membrane and provides an antifouling surface property that reduces nonspecific binding to the chip surface (figure 1(a), a1). the epithelial cell adhesion molecule (epcam) is a glycosylated membrane protein that is common to epithelial cells and often overexpressed on ctcs derived from solid tumors. the cmx platform uses a highaffinity, monoclonal, anti-epcam antibody conjugated to the slb to capture ctcs from the peripheral blood (figure 1(a), a2). this conjugation is based on the neutravidin-biotin chemistry. whole blood and reagents enter the chip at the inlet port and exit the chip at the outlet port (figure 1(b)). the etched pattern on the chip produces an optimal flow disturbance that results in maximal contact 4 1 between ctcs and the capture antibody (figure 1(a), a3). the fluidic nature of the slb enhances ctc binding to the epcam antibody molecules by allowing additional proximal antibody molecules to migrate toward the cell. this “clustering effect” further strengthens the binding force and increases capture efficiency (figure 1(a), a4). 18 residual white blood cells (wbcs) and red blood cells are efficiently removed from the adhered ctcs with a phosphate-buffered saline (pbs) buffer wash. 18 then the unique airfoam generation and release mechanism recovers captured cells from the chip at the inlet port. the airfoam is generated by mixing cell culture medium and air from two syringes and applied to the chip with a syringe pump. the hydrophilic outside and hydrophobic core property of the airfoam facilitates the interaction with the hydrophobic side of the slb layer. this allows for the gentle release of the ctc from the slb surface (figure 1(a), a5). 41 this unique release method causes minimum stress to the ctc and improves recovery while keeping the cells intact. these released cells are collected in an eppendorf tube for further analysis. for ctc enumeration, the cell solution is transferred to a slide equipped with millipore filter membrane for immunofluorescent staining. 18 the final steps in the assay involve ctc counting using proprietary artificial intelligence (ai)-based software to screen and identify ctcs on images and quantify stain parameters and cellular features using cellmax developed proprietary software—cellreviewer (figure 1(c)). figure 1. cellmax life’s cmx ctc assay platform. the cellmax life ctc assay is performed on the cmx platform (a) using a microfluidic chip (b) that includes several distinctive features to isolate rare circulating tumor cells from peripheral blood and employs specific antibodies to identify the origin of these cells. the cmx technology (a) includes chip surface design and treatment (a1), antibody modification (a2), microfluidic chip processing (a3), purification (a4), and target cell release by air foam (a5). a diagram of the chip is represented in (b). the final step in the assay is the software-assisted enumeration of ctcs using ai-based software and in-house developed imaging software—cellreviewer (c). ctc: circulating tumor cell. gupta et al. 3 clc preparation, sample collection, processing, and ctc detection peripheral blood (4–8 ml) is drawn from the median decubitus vein by a trained phlebotomist and collected in a bd vacutainer tube containing k2edta as the anticoagulant. streck cell preservative (streck inc., omaha, nebraska, usa) is added to the tube, typically within 2–4 h at a ratio of 4:1 (blood:preservative), then gently inverted 5 to 10 times to mix, and stored at room temperature until delivery. the sample tubes are delivered in ambient conditions to the cellmax laboratories in sunnyvale (clia#: 05d2119031, cap #: 9478056) or taiwan (cap #: 9258554) for processing. the standard operating procedures for microfluidic chip fabrication, micropattern etching, chip surface modification (lipid and epcam coating), and airfoam generation and application originate from published research studies at academia sinica. 18,40 whole blood (2 ml mixed with 0.5 ml preservative) is loaded by syringe into the inlet port and pulled through a microfluidic channel by a syringe pump connected to the outlet port at a flow rate of 1.5 ml/h. the unbound cells are washed out of the chip with three washes of pbs buffer (0.2 ml at 3 ml/h). the bound cells are fixed on the chip with 4% paraformaldehyde and are recovered using the airfoam mechanism connected to the outlet port. released cells are collected in an eppendorf tube via the inlet port. a small volume of ethanol is added to de-bubble the foam–cell mixture in the tube. these cells are then transferred to the membrane chip for counting. contrived samples for analytical validation included both donor blood and cell culture medium (dulbecco’s modified eagle’s medium, thermo scientific 11965084 with 10% fetal bovine serum, thermo scientific 26140079) spiked with prestained cancer cell line cells (clcs) ht29 (htb-38, atcc). prestaining was performed by adding celltracker™ green cmfda or deep red dye (thermofisher c2925 or c34565) to the cell culture prior to harvesting, following the manufacturer’s recommendation. the stained cells were fixed with 4% paraformaldehyde and stored in the refrigerator for use. the prestained cell concentration was first estimated with a scepter 2.0 automated cell counter (emd millipore phcc20060) and then diluted to appropriate concentrations. aliquots of prestained cells for the final working concentrations were mounted on membranes, imaged with the microscope, and counted by two separate operators to determine precise counts for spiking. post-capture and release, the cell solution was carefully placed on a membrane, and liquid was wicked away with a blotting pad. the sample was then mounted for imaging and counting. for samples collected for the clinical feasibility analysis, the released cells were incubated with goat serum (10%) for 60 min at room temperature. primary antibodies were incubated overnight at 4�c in a refrigerator. the cells were washed with pbs and incubated with fluorescently tagged secondary antibodies. after final washes, the cells were mounted with mounting medium containing 40,6diamidino-2-phenylindole (dapi) antifade (thermo fisher p36931) onto a glass slide for image capture. figure 2 provides a schematic depiction of the cmx assay workflow. the samples are accessioned on day 1 and blood processing commences on day 2. for contrived samples, captured cell release, slide mounting and image capture are performed on day 2, followed by clc counting. for clinical samples, immunostaining, slide mounting, and ctc enumeration are performed on day 3. clc enumeration a clc is defined as a cell that is round to oval in shape and that demonstrates both a nucleus stain (dapi, blue color) and a prestain (green color). cells with a single stain were not counted. counting of prestained ht29 cells was performed with the commercial software metamorph (version 7.8, molecular device). the las-x stitched images (from step i described below) were loaded into metamorph, assigned colors (dapi-blue, prestain-green), and merged into a single image. this image was then magnified and reviewed by an operator using a raster scanning mechanism with the aid of location marks on the monitor. ctc enumeration step i: image capture. for each ctc sample (cells on 10-mm diameter membrane), 100 (10 � 10) frames of images are captured in each of three channels: red, green, and blue, corresponding to tritc for cytokeratin 20 (ck20), fitc for lymphocyte common antigen (cd45), and dapi for nucleus counterstain. the leica autofocus mapping mechanism is applied to ensure images are in focus. using the built-in stitching function of leica las-x software, these 100 image frames are stitched together as one image volume for each of the three channels. step ii: identification of candidate cells. we use custom software based on an ai algorithm developed specifically to search the stitched images for regions that have the characteristics of a cell. the coordinates of each target of interest on the image are recorded. the ai algorithm is repeatedly trained with confirmed ctcs and wbcs to improve its sensitivity and specificity. each of the candidate cells is assigned a confidence index, which allows identification for further morphology-based review and confirmation. step iii: reviewing candidates to enumerate ctcs. for ctc confirmation and enumeration, cellmax life has developed stringent criteria to avoid false positivity using a custom software—cellreviewer. the ctc candidate cell’s morphology is reviewed by a trained technician. ctcs must be round-to-oval shaped, have a cell size between 8 mm and 40 mm and be ck20þ, cd45�, and dapiþ (have 4 journal of circulating biomarkers a genuine dapi-stained nucleus). additionally, wbcs identified by the characteristic multi-segmented (multilobed) nucleus are further excluded. ctcs are further reviewed by a technical expert or an experienced pathologist to confirm the absence of wbcs as needed. final ctc counts are recorded. patient cohort in the clinical feasibility study, we enrolled nine patients with crc and one patient with colorectal polyp. among the patients diagnosed with crc, two were at stage 1, one was at stage 2, three were at stage 3, and three were at stage 4 cancer based on tumor–node–metastasis staging. the patient with colorectal polyp had a tubular adenoma 2 mm in size. results analytical validation design we validated the following ctc assay characteristics: accuracy, linearity, limit of blank (lob), limit of detection (lod), specificity, and precision (reproducibility). to more closely mimic the cells expected from patients, ht29—a crc cell line was chosen as a ctc surrogate for this assay validation. ht29 is an epithelial cancer cell line derived from human colorectal adenocarcinomas and has stable expression of both epcam and ck20, the two markers used to identify a colorectal ctc. to measure assay performance characteristics, the ht29 clcs were prestained (see “methods” section) and each dilution is carefully counted under a microscope prior to spiking into healthy donor blood or culture medium. sample processing figure 2. the cmx ctc assay workflow. the entire ctc process from sample collection to image analysis is summarized in this diagram. step 1 is performed on day 1, while steps 2, 3 and 5 are performed on day 2 for contrived clc samples. for clinical samples, steps 2, 3 and 4 up to primary antibody (1 ab) staining are performed on day 2, while secondary antibody (2 ab) staining to step 5 are performed on day 3. ctc: circulating tumor cell; clc: cancer cell line cell. gupta et al. 5 and clc enumeration of spiked samples followed the protocol described in the “methods” section. table 1 lists the analytical performance characteristics validated in this study. assay accuracy the assay accuracy is defined as percentage recovery of spiked ht29 cells across a range that covers meaningful concentrations of ctcs. for this test, 33 dilutions of cells in four concentration groups: high (101–200 cells), medium (51–100 cells), low (11–50 cells), and rare (1–10 cells) were counted under the microscope prior to spiking into 2 ml medium or healthy donor blood. the overall efficiency was defined as the cell numbers (recovered cells from the process) observed under a microscope as a proportion of the spiked cell numbers. the average overall efficiency for the spiked medium was 78.1%, with relatively low standard deviation (6%) (figure 3 and table 2). the average overall efficiency was 64.2% + 11% in blood across the entire spiked cell range (figure 3 and table 3). inherently, donor blood can interact with spiked cells and increase variability in the overall efficiency. for this reason, we also quantified the overall recovery efficiency by spiking ht29 into cell culture medium to factor out the variability of blood from the system performance. note that at the “rare” concentration level, numbers as low as 2 and 3 cells were counted prior to spiking into 2 ml (assay volume) medium and blood. instead of estimating cell counts in serial dilutions, we selected this precise counting technique to correctly contrive samples and thus determine the assay accuracy at a very low spiked concentration, thus simulating rare ctc events in early stage disease. assay linearity the assay linearity was measured by plotting observed cell counts as a function of spiked in cell counts across various concentrations. thirty three dilutions of cells with concentrations ranging from 2 to 189 cells were spiked into 2 ml medium or healthy donor blood. the assay was shown to be linear across all spiked concentrations from 2 to 189 cells with a slope of 0.7521 and r 2 ¼ 0.9765 in medium (figure 4(a)), and with a slope of 0.7166 and r 2 ¼ 0.9822 in blood (figure 4(b)). lob and lod the lob is defined as the highest ctc count expected to be found when replicates of a blank sample containing no ctcs are tested. 42 in the current study, we tested replicates of eight non-spiked medium samples and found no ctcs. therefore, the lob is estimated to be zero ctcs. there are no acceptance criteria for the lob. the lod is defined as the ctc count for which the probability of falsely claiming the absence of a ctc is 5%, given a 5% (or lower) probability of falsely claiming the presence of a ctc. 42 figure 5 displays the (natural logarithm of) observed ctc counts (plus 1) versus the (natural logarithm of) known ctc counts (plus 1) shown as the blue line along with two-sided 90% confidence intervals for 52 donor blood samples spiked with 2 to 281 ht29 cells. note that the known ctc count is the number of table 1. performance characteristics measured in the analytical validation of cmx ctc assay. performance characteristics definition sample composition measured parameter 1 accuracy (recovery efficiency) percent (recovered cells/spiked-in cells) 33 spiked samples in 4 concentrations in medium and blood percent recovery efficiency 2 linearity linearity plot of percent (recovered cells/ spiked-in cells) 33 spiked samples of 2 to 189 cells in medium and blood regression curve in linear plot for recovered versus spiked cells 3 lod limit of detection 52 spiked blood samples containing 2 to 281 cells lowest measurable and quantifiable clc count per 2 ml blood 4 lob and specificity limit of blank and rate of falsepositive detection 8 nonspiked medium and 32 healthy control blood samples ctc detection in nonspiked medium and blood 5 precision (reproducibility) intra-assay variability (3 tests) spiked blood samples in 3 concentrations: high (96–181 cells), medium (12–21 cells), and low (2–11 cells) percent coefficient of variation of recovery efficiency for 3 concentrations (high, medium, low) in 3 test conditionsinter-assay variability (3 days) inter-operator variability (3 operators) ctc: circulating tumor cell; clc: cancer cell line cell. 6 journal of circulating biomarkers clcs counted and spiked into each blood sample. the intersection of the lower one-sided 95% confidence interval for the least squares fitted mean with the lob (i.e. 0) was 0.71 ctcs. therefore, the estimated theoretical lod of the cmx assay is 0.71 ctcs. there are no acceptance criteria for the lod. assay specificity assay specificity was demonstrated via analysis of donor samples from 32 young healthy adults (<35 years of age); it has been observed previously that young adults (aged 20– 40 years) have a very low (1–2%) prevalence of adenomas. 43 the cmx ctc assay was performed on 4 ml blood from each donor and run in duplicate (two chips with 2 ml for each donor). ctcs were counted for each chip, and average counts are reported in table 4. zero ctcs were found in 18 of 32 samples, 9 of 32 samples had 1 ctc, and 5 of 32 samples had 1.5 ctcs. using a cutoff of 2 ctcs/2 ml (as described in “clinical feasibility” section below), all of the samples were negative, resulting in 100% analytical specificity (table 4). figure 3. overall recovery efficiency of clcs in medium and blood. the bar plot displays the overall efficiency, defined as the cell count (recovered from the process) observed under a microscope as a proportion of the spiked cell count, in both medium (blue) and blood (gray). the error bars represent the standard deviations. clc: cancer cell line cell. table 2. overall recovery efficiency in medium. clc dilution range mean number of cells spiked mean number of cells recovered average recovery (%) standard deviation (%) coefficient of variation (%) high (101–200) 146.7 109.6 74.7 9.76 13.1 medium (51–100) 66.4 53.4 80.4 4.73 5.9 low (11–50) 19.9 16.1 80.8 9.53 11.8 rare (1–10) 7.4 5.7 76.5 24.1 31.5 clc: cancer cell line cell table 3. overall recovery efficiency in blood. clc dilution range mean number of cells spiked mean number of cells recovered average recovery (%) standard deviation (%) coefficient of variation (%) high (101–200) 160.4 96.8 60.3 15.37 25.5 medium (51–100) 88.8 60.6 68.2 7.56 11.1 low (11–50) 18.9 13.2 69.6 10.94 15.7 rare (1–10) 6.3 3.7 58.7 26.43 45.0 gupta et al. 7 precision (reproducibility) the precision of the cmx platform was assessed by evaluating variation in overall recovery efficiency for triplicate samples of ht29 cells spiked in blood. to better characterize assay precision, recovery efficiency was further evaluated across multiple days and among multiple operators. intra-assay, inter-assay, and inter-perator reproducibility. triplicate (3) contrived samples of spiked ht29 cells in blood for three concentrations (high, medium, and low) with known ctc counts were processed, and recovered cells were recounted to determine the overall recovery efficiency (table 5). intra-assay variability was measured for one operator who ran triplicate samples in three concentrations for a total of nine samples (n ¼ 9), whereas inter-assay variability was measured using triplicate samples across three concentrations for three different days for a total of 27 samples (n ¼ 27). inter-operator repeatability was measured for three operators; each operator ran triplicate samples across three concentrations for a total of 27 samples (n ¼ 27). results for the precision analyses are listed in table 5. for intra-assay precision, coefficient of variation (cv) for overall efficiency is reported for the triplicate samples in three concentrations. for inter-assay precision, cv for the overall efficiency is reported for 3 days, with triplicate samples run in three concentrations on each day. for inter-operator precision, cv for overall efficiency is reported for three operators, with each operator processing triplicate samples in three concentrations. it is worth noting that precision studies at very low spike concentrations are challenging, with high inherent variability likely at these cell concentrations due to difficulty in controlling spiked cell counts. however, we were able to visualize and count spiked cells at concentrations as low as 2–11 cells per 2 ml of blood and thus demonstrate the reproducibility of rare cell recovery. clinical feasibility to establish clinical feasibility for the cmx test, we enrolled 47 study subjects, consisting of 15 subjects with known colonoscopy results (nine crc patients, one adenoma, five negative) and 32 self-declared young healthy subjects under 35 years of age. the 15 colonoscopy verified samples were collected in taiwan and processed both in taiwan and the united states. the samples from young healthy subjects were collected in the united states and processed only in the united states. ctc counting for all samples was conducted with cellmax life’s proprietary ai-based software and figure 4. cmx assay linearity in medium (a) and blood (b). linearity was characterized by plotting the observed cells (y-axis) versus the spiked in number of cells (x-axis) for 33 cell dilutions (ranging from 2–189 cells) and calculating the linear regression. figure 5. cmx assay limit of detection. the plot displays the natural logarithm of observed ctc counts (plus 1) versus the natural logarithm of known ctc counts (plus 1) shown as the blue line along with two-sided 90% confidence intervals for 52 donor blood samples spiked with 2 to 281 ht29 cells. the intersection of the lower one-sided 95% confidence interval for the least squares fitted mean with the lob (i.e. 0) was 0.71 ctcs. the red line represents 100% recovery efficiency. ctc: circulating tumor cell; lob: limit of blank. 8 journal of circulating biomarkers cellreviewer. the clinical feasibility study had two goals: (1) to compare ctc counts for the same samples processed at two different sites, taiwan versus the united states and (2) to compare ctc counts in colonoscopy-negative subjects and young self-declared healthy subjects. the cohort, mean subject age, and mean ctc counts for samples processed at two sites are listed in table 6. the colonoscopy-verified subjects’ samples were processed in cellmax’s cap accredited laboratories in taipei, taiwan, and sunnyvale, california, usa. the young healthy adults’ samples were processed only in the united states. ctc counts for the same samples processed in united states were generally lower than those processed in taiwan, likely due to transportation to the united states. although preservative was added to each sample collected in the hospital in taiwan upon blood draw, transition time and transportation impact may have contributed to the decrease in ctc counts. the average across sites of the mean ctc counts for taiwan and the united states were 11.1 for cancer, 6.0 for polyp (adenoma), and 2.1 for colonoscopy-negative subjects. for the 32 young self-declared healthy donors processed in the united states only, the mean ctc count was 0.5. more clinical data need to be collected for this comparison, but the cmx test with its ai-based image pattern recognition shows promise for ctc identification. since a ctc count of greater than three ctcs/3 ml has been correlated with the presence of a primary tumor, 39 we chose a threshold of 2 ctcs/2 ml (three or more ctcs as positive). using this cutoff, for the samples processed in the united states, the cmx test identified 8 of 10 diseased (cancer and adenoma) samples as positive (80% clinical sensitivity) and 4 of 5 controls as negative (80% clinical specificity). discussion this study addresses the analytical validation of the cmx ctc assay platform by evaluating its overall recovery accuracy, assay linearity, lob and lod, specificity, and precision. performance characteristics were evaluated with precise counting of spiked clcs at concentrations as low as two cells in 2 ml of blood or medium, with the aim of quantifying the performance for detecting true rare events. spiking error, including dilution, pipetting, and aliquoting can contribute significantly to the variation at low cell concentrations. the purpose of the assay accuracy analysis was to determine the true overall recovery of the cmx process, independent of this error. hence, we chose to prestain clcs and perform precise cell counting under a fluorescent microscope prior to spiking, unlike previous validation studies in which cells were diluted without further counting prior to spiking and recovered cell counts sometimes exceeded 100% of spiked in cells.44,45 at table 4. summary of ctc counts in young healthy (<35 years old) donor blood samples to demonstrate specificity.a patient number sample number patient age average ctc counts patient 1 crc232 26 0 patient 2 crc243 33 0.5 patient 3 crc244 33 0 patient 4 crc245 33 0 patient 5 crc246 24 0 patient 6 crc247 29 0 patient 7 crc231 24 1.5 patient 8 crc235 20 0 patient 9 crc236 29 1 patient 10 crc248 26 0 patient 11 crc255 34 2 patient 12 crc256 33 2 patient 13 crc233 30 0 patient 14 crc234 19 0.5 patient 15 crc257 22 0 patient 16 crc258 22 1.5 patient 17 crc259 34 0.5 patient 18 crc267 20 0 patient 19 crc268 17 1 patient 20 crc283 19 0 patient 21 crc284 20 1 patient 22 crc269 28 0 patient 23 crc270 29 0.5 patient 24 crc271 31 0 patient 25 crc272 24 0 patient 26 crc280 29 0 patient 27 crc291 31 0 patient 28 crc292 28 1.5 patient 29 crc293 29 1 patient 30 crc294 31 0 patient 31 crc295 30 0.5 patient 32 crc296 24 0 ctc: circulating tumor cell; crc: colorectal cancer. a eighteen of 32 samples had zero ctcs detected, 9 of 32 had one ctc detected, and only 5 had more than one (but fewer than 2) ctcs detected. table 5. precision analyses of cmx assay showed the percentage cv of overall recovery efficiencies for triplicate blood samples spiked with ht29 cells. precision parameter cv, overall efficiency high concentration (96–181 cells) (%) medium concentration (12–21 cells) (%) low concentration (2–11 cells) (%) intra-assay (n ¼ 9) 8.8 21.9 37.0 inter-assay (3 days, n ¼ 27) 9.9 15.8 36.6 inter-operator (3 operators, n ¼ 27) 13.7 11.0 35.3 cv: coefficient of variation gupta et al. 9 64.2% recovery when spiked in blood, the assay achieved 11% standard deviation across all spiked concentrations, and without the interference of blood components mean recovery was 78.1% with a relatively low standard deviation (6%) across all dilutions (tables 2 and 3; figure 3). assay specificity was determined to be 100%, derived from 32 young healthy donor blood samples (table 4). moreover, using 8 unspiked medium samples (blank), we demonstrated lob of zero ctcs. the cmx platform also demonstrated excellent assay linearity (r 2 ¼ 0.98) (figure 4) across a wide range of cell dilutions from 2 cells to 189 cells per 2 ml of blood. at low, medium, and high concentrations (11–50, 51–100, and 101–200) of spiked cells, the assay is reproducible with low variation in overall recovery efficiency (11.1–25.5% cv). at an extremely rare concentration (1–10 cells/ 2 ml blood), the cmx assay still demonstrated a variability of only 45% cv. assuming a poisson distribution for the detected ctcs/sample, the theoretical cv for a set of measurements carried out on the same samples is 44.7% at a comparable concentration of 5 ctcs/7.5 ml. 46 without the interference of donor blood, variability was further reduced to 31.5% when ht29 cells were spiked in medium at this rare concentration. we were able to quantify the system accuracy at such low concentrations with a combination of prestaining and precise counting. dilutions and/or counting errors likely led to previous attempts resulting in 120–200% recovery of spiked cells when concentrations were below 10 cells per sample. 45 from the analysis of 52 donor blood samples spiked with 2 to 281 ht29 cells, we were able to establish a theoretical lod of 0.71 cells per 2 ml of blood (figure 5). with the ability to precisely control for spiking and counting, we further proved reproducibility by studying intra-assay, inter-assay, and inter-operator variability. at high spike concentrations (96–181 cells/2 ml), wherein mixing and aliquoting are less likely to contribute to variation, the cvs for triplicate samples remained at a low level (<15%). at medium concentrations (12–21 cells/2 ml), the cv for triplicate samples was in the range of 11.1–21.9% and at very low concentrations (2–11 cells/2 ml), the cvs were between 35% and 37%. the higher variability at the lowest concentration is expected as error for quantification in each step is greater at these concentrations. the ability to maintain a similar, narrow range of precision across multiple days and among multiple operators is important for a reproducible test, and we were able to demonstrate it with spiked cells across relevant concentrations as low as 2–11 cells/sample (table 5). a recent study of another ctc detection platform 44 demonstrated 88% accuracy in ctc recovery, but at dilution rates of 6 to 300 clcs/slide, rather than this study’s more realistic measured range of 2 to 189 clcs/2 ml blood. linearity and specificity were comparable, while the precision achieved by the cmx assay was superior for the same number of clcs/slide. with regard to the cellsearch ® system, cummings et al. state that the exact determination of low ctc counts (<10 cells/7.5 ml of blood) is crucial for clinical significance and assigned patients to groups with a relatively favorable (<5 ctcs/ 7.5 ml of blood) or unfavorable prognosis (� 5 ctcs/7.5 ml of blood) based on ctc count. 46 however, major limitations have been reported with regard to the heterogeneity of ctc populations. 19 results from controls and spiked blood confirmed a threeto fourfold higher degree of imprecision at low cell counts, and individual analysts introduced a highly specific error into the interpretation of ctc images, correlated to their level of training and experience. 46 although our data demonstrated the ability to precisely count and characterize cmx assay performance at clinically relevant ctc count of <5 ctcs, we only provide limited clinical data as part of a clinical feasibility study. we hope to address some of these challenges by performing clinical validation in future studies. the core strength of our assay is that the cmx platform enables the harvesting of live cells for analysis via the gentle air foam release of the underlying lipid layer from the chip, without breaking antibody–antigen bonds. unlike other ctc platforms in which cells cannot be separated from substrates such as ferromagnetic beads without disrupting or negatively influencing the cells for further analysis, 47 the cmx platform was designed to allow gentle release of cells using airfoam. this allows for downstream analyses of ctcs, including (but not limited to) enumeration, gene expression, methylation, mutations, and so on, with these cells conveniently contained in an eppendorf tube. we have developed the assay keeping in mind that several epithelial-derived cancer types (e.g. renal cell carcinoma) do not express epcam; however, some epcam expressing cancers (e.g. breast cancer) can lose epcam table 6. ctc counts in healthy and diseased subpopulations. subject category (total ¼ 47 patients) mean ctc counts type number of subjects mean age processed in the united states processed in taiwan average cancer 9 51 6.6 15.7 11.1 adenoma 1 66 3.0 9.0 6.0 colonoscopy negative 5 59 1.2 3.0 2.1 young healthy 32 26 0.5 n/a n/a ctc: circulating tumor cell. 10 journal of circulating biomarkers expression when they are treated and will display cancer stem cell phenotype. the cmx assay can be easily adapted to other capture markers, including an antibody cocktail, as the microfluidic chip coating and manufacture process is based on the well-established neutravidin-biotin chemistry. however, the assay does have some limitations. the reproducibility of the current manual process is highly dependent on the skills of the trained technical staff. we anticipate that the automation of the cell capture and release will greatly reduce variability among operators. therefore, a prototype ctc instrument is being employed for this purpose. another limitation is the result of immunostaining on the millipore filter membrane; this membrane retains captured cells while allowing liquid to pass through. the membrane also reduces wash efficiency due to the small amount of buffer that can pass through. a revision to this process is currently being implemented by staining cells while they are retained in-chip and then applying airfoam to release stained cells. finally, the clinical feasibility study results demonstrate sensitivity and specificity of the cmx assay for crc and adenoma detection, despite being derived from a limited data set. based on the samples processed in the united states, the mean ctc counts were 6.6 for cancer, 3.0 for adenomas, and 1.2 for colonoscopy-negative subjects (table 6). since colonoscopy can fail to detect polyps at a rate of 6–27%,48 the odds are that some of the ctcs detected in “colonoscopy-negative” subjects may not be truly “false positive.” in a necropsy analysis, young adults (20–40 years old) were found to have only a 1–2% adenoma prevalence rate. 43 we thereby enrolled young adults aged 18–35 years to test the ctc detection specificity by the cmx process. on average, only 0.5 ctcs were found in 32 young self-declared healthy young adults (table 4), thus providing stronger evidence for the specificity of the cmx assay. there are possibilities that the ctcs detected in young healthy donors and colonoscopy-negative patients could be tumor cells (of colorectal or other origin) or non-neoplastic cells (epcam and ck20 positive epithelial cells in circulation from benign diseases) or simply false positive for other reasons. hence, further studies of a larger cohort of asymptomatic subjects are necessary and already underway to validate the clinical performance of the cmx assay. conclusions this validation study indicates the potential of the cmx assay for diagnosing individuals at risk for crc. identifying, isolating, and enumerating ctcs in peripheral blood could be an important new tool for cancer detection and management, while liquid biopsy is an effective test modality that drives compliance for routine testing. further validation of the cmx ctc assay with a multi-analyte approach is underway, and studies focused on its clinical utility will identify the range of indications for which it may be suitable. acknowledgements the authors would like to acknowledge dr wen-sy tsai of chang gung memorial hospital who provided clinical samples for this study. we would also like to thank sumedha sinha and rebecca suttmann for writing assistance. declaration of conflicting interests the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: all authors are employees of cellmax life, which provided funding for this study. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: cellmax life provided funding for this study. orcid id rui mei https://orcid.org/0000-0001-7903-2846 research ethics and patient consent analytical control cell lines were acquired from a commercial vendor. institutional review board approval and informed consent were obtained from chang gung memorial 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102(10): 1495–1502. 37. tsai ws, chen js, shao hj, et al. circulating tumor cell count correlates with colorectal neoplasm progression and is a prognostic marker for distant metastasis in nonmetastatic patients. sci rep 2016; 6: 24517. 38. bork u, rahbari nn, schölch s, et al. circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study. br j cancer 2015; 112(8): 1306–1313. 39. yang c, zhuang w, hu y, et al. clinical significance of peripheral circulating tumor cell counts in colorectal polyps 12 journal of circulating biomarkers and non-metastatic colorectal cancer. world j surg oncol 2018; 16(1): 13. 40. lai jm, shao hj, wu jc, et al. efficient elusion of viable adhesive cells from a microfluidic system by air foam. biomicrofluidics 2014; 8(5): 052001. 41. chen jy, tsai ws, shao hj, et al. sensitive and specific biomimetic lipid coated microfluidics to isolate viable circulating tumor cells and microemboli for cancer detection. plos one 2016; 11(3): e0149633. 42. armbruster da and pry t. limit of blank, limit of detection and limit of quantitation. clin biochem rev 2008; 29(suppl 1): s49–52. 43. pendergrass cj, edelstein dl, hylind lm, et al. occurrence of colorectal adenomas in younger adults: an epidemiologic necropsy study. clin gastroenterol hepatol 2008; 6(9): 1011–1015. 44. werner sl, graf rp, landers m, et al. analytical validation and capabilities of the epic ctc platform: enrichment-free circulating tumour cell detection and characterization. j circ biomark 2015; 4:3. 45. veridex_llc_510 k, cellsearch 510 k summary k071729. 2003. 46. cummings j, morris k, zhou c, et al. method validation of circulating tumour cell enumeration at low cell counts. bmc cancer 2013; 13: 415–415. 47. lv sw, wang j, xie m, et al. photoresponsive immunomagnetic nanocarrier for capture and release of rare circulating tumor cells. chem sci 2015; 6(11): 6432–6438. 48. ahn sb, han ds, bae jh, et al. the miss rate for colorectal adenoma determined by quality-adjusted, back-to-back colonoscopies. gut liver 2012; 6(1): 64–70. gupta et al. 13 << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams true /maxsubsetpct 100 /optimize true /opm 1 /parsedsccomments true /parsedsccommentsfordocinfo true /preservecopypage true 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i announce, journal of circulating biomarkers (jcb) is now indexed in pubmed central (pubmed). i want to take this opportunity to thank the contributions from editors in chief, shidong jia, and shu-wing ng for past contributions, as well as our associate editors and editorial board members for their enduring support. indexing in pubmed central has been a major focus of the journal up to now, so we are delighted to have reached this milestone. indexing will improve the visibility and discoverability of our content and is therefore be a great benefit to our authors, both past and future. our next challenge will be to obtain indexing in the web of science and science citation index expanded database and so receive an impact factor. this ambitious task will require continued support from our editorial board, authors, and readers, so i thank you now in advance. jcb will continue to maintain a high standard, to publish the best science-driven and implementation studies of any circulating biomarker, that will move the basic and clinical research field forward. in doing so, we update our editorial roles, by recruiting new editors in chief, associate editors, and editorial board members, to assist me in achieving our next milestone. for those who are interested, feel free to contact me directly. jcb is positioned to broadly publish, but not exclusively, the fast growing interest in liquid biopsy across many disease indications, as well as the therapeutic aspect for immunotherapy and car-t cells, technology development of new methods for isolation, characterization and tracking, and the basic science research to provide a foundation for the former to be possible. topics related to circulating biomarkers (found in any biofluid), not limited, but including extracellular vesicles (exosomes, microvesicles, ectosomes, apoptotic bodies, platelet dust, etc.) cellfree dna, circulating tumor dna, circulating tumor cells, platelets, free floating nucleic acids, and others, as it is related to health and disease. this will include studies that involve animal models that will provide insight to the function, detection, implementation of a method for human benefit, evaluating delivery methods for therapeutics, and so on. i encourage our associate editors and editorial members to submit manuscripts too. jcb will continue to accept original and review articles as well as perspectives, short research reports, protocols and methods, clinical trial reports, notes to the editor, letters to the editor, and conference/meeting dispatch reports. manuscripts selected for publication should contain highquality, reproducible data that advance the circulating biomarker field both at the basic and clinical aspects and contribute to a broader understanding of the underlying processes for each study. as part of our continued growth and efforts to support the field, jcb will look to establish partnerships with scientific societies, and academic and industry leaders to advance the field and to allow free access to knowledge in the circulating biomarker space. i want to conclude by sending my sincere gratitude to my fellow editors in chief, editorial team, and our authors and readers for your continuous support, and i look forward to working with everyone as we move into the next phase of the journal. winston patrick kuo cloudhealth genomics ltd, shanghai, china journal of circulating biomarkers volume 6: 1 ª the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417745757 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417745757 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice cbx670782 1..8 research article x-rays and metformin cause increased urinary excretion of cell-free nuclear and mitochondrial dna in aged rats azhub gaziev, serazhutdin abdullaev, gulchachak minkabirova, and kristina kamenskikh abstract activation of cell death in mammals can be assessed by an increase of an amount of cell-free dna (cf-dna) in urine or plasma. we investigated the excretion of cf nuclear dna (ndna) and cf mitochondrial dna (mtdna) in the urine of rats 3 and 24 months in age after x-irradiation and metformin administration. analyses showed that prior to treatment, the amount of cf-ndna was 40% higher and cf-mtdna was 50% higher in the urine of aged rats compared to that of young animals. at 12 h after irradiation, the content of cf-ndna and cf-mtdna in the urine of young rats was increased by 200% and 460%, respectively, relative to the control, whereas in the urine of aged rats, it was 250% and 720% higher. after 6 h following metformin administration, the amount of cf-ndna and cf-mtdna in the urine of young rats was elevated by 25% and 55% and by 50% and 160% in the urine of aged rats. thus, these preliminary data suggest that x-rays and metformin cause a significant increase of cf-dna in the urine of older rats caused by the active cell death in tissues. these results also suggest that metformin possibly initiates the death of the cells containing structural and functional abnormalities. keywords cell-free urine dna, old and young rats, x-irradiation, metformin administration date received: 20 april 2016; accepted: 1 september 2016 introduction the death of cells with structural and functional abnormalities in the tissues of organisms is an active process that supports the renewal of cell populations and the functional integrity and homeostasis of tissues. 1 the presence of circulating cell-free dna (cf-dna) in mammalian biological fluids, such as blood and urine, is due to continuous cell death in the tissues. according to the results of a number of studies, cell death accompanied by a release of cf-dna fragments can occur not only through apoptosis and necrosis but also via mechanisms of autophagy. the process of autophagy is generally assumed as a mechanism of survival or a cytoprotective mechanism that removes damaged organelles, proteins, and other macromolecules. 2,3 however, in the events of excessive increase in the amount of organelles and macromolecules with structurally functional disorders, the cell may be subjected to death by autophagic degradation. 1,3 an increase in the content of cf-dna in plasma (serum), which is caused by an activation of cell death in tissues, occurs not only under external damaging influences 4,5 but also with aging of the organism. 6,7 the increase of cf-dna in plasma with aging of the organism and in the absence of external influences may possibly happen largely due to the enhancement of programmed cell death. 8 it should be noted that autophagic cell death in tissues, most likely, does not contribute significantly to the institute of theoretical and experimental biophysics, ras, pushino, moscow region, russia corresponding author: azhub gaziev, institute of theoretical and experimental biophysics, ras, 142290 pushino, moscow region, russia. email: gaziev.iteb@gmail.com journal of circulating biomarkers volume 5: 1–8 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416670782 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 3.0 license (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:gaziev.iteb@gmail.com https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454416670782 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage age-dependent increase in the content of cf-dna in biological fluids, since the autophagic activity in tissues decreases along with the aging of the organism. 9 nevertheless, the activity of autophagic processes increases in response to such signals as starvation, hypoxia, oxidative stress, ionizing radiation (ir), infection, and other types, which are accompanied by a gain of the amp/atp ratio and an upregulation of 50-adenosine monophosphateactivated protein kinase (ampk) in cells. 10,2 autophagy in tissues can also be stimulated by the introduction of some pharmacological agents, as well as other compounds obtained from natural sources. 11 metformin, which is widely used for the treatment of diabetes mellitus type 2, can be regarded as one of these drugs (activators of autophagy). although the antidiabetic mechanisms of actions of metformin are not completely understood, its indirect participation in autophagy via activation of ampk is generally accepted. 12 perhaps the introduction of metformin in old animals, with suppressed autophagic activity and increased level of damage of cellular structures, will cause autophagy enhancement accompanied by the destruction of these dysfunctional cells. it is known that ir is also a potent activator of ampk 13,14 and an inducer of various cell death pathways, including apoptosis, necrosis, and autophagy. 15,16 it can be presumed that activation of autophagy provides a significant contribution to the elimination of damaged cells from the tissues after exposure to ir. we have previously demonstrated a significant increase in the excretion of fragments of cell-free nuclear dna (cfndna) and cell-free mitochondrial dna (cf-mtdna) in the urine of rats exposed to ir. notably, the cf-mtdna increase is much higher than that of cf-ndna, which suggests the involvement of mitophagy (by autophagic mechanism) in postirradiation cell death. 17 metformin, as mentioned above, initiates the death of cells containing structural and functional abnormalities by the mechanism of autophagy. the intensification of cell death at the level of a whole mammalian organism can be estimated by a raise in the content of cf-dna in urine or plasma. since urinalysis is more preferable as a noninvasive approach, we conducted a comparative study for the excretion of cf-ndna and cf-mtdna in the urine of male rats (3 and 24 months age) after x-irradiation and administration of metformin. materials and methods animals and their treatment the present study involved fisher 344 male rats aged between 3 and 24 months, obtained from the nursery of the branch of institute of bioorganic chemistry ras (pushchino, moscow region). during the experiment, the animals were kept under standard conditions in the vivarium of the institute of theoretical and experimental biophysics ras. the experiments were conducted in accordance with all the requirements established by the institutional committee for the control of animal use in biomedical experiments. the animals were acclimatized for 1 week before the start of experiments. rats were fed a special diet for mice and rats ad libitum, with free access to clean drinking water, and were housed three animals per cage under standard 12-h light/12-h dark cycle at a temperature of 22 + 2�c and 45 + 5% humidity. irradiation of rats was performed using a commercially available x-ray apparatus rtu-12 (340 kvp, 20 ma; medrent, russia) with 1 mm aluminum and copper filters. an expert for radiation dosimetry controlled the absorbed dose using an ionization chamber and a clinical x-ray dosimeter (vacudap, ntc-amplitude, moscow, russia). rats were irradiated by a sublethal dose of 5 gy at a dose rate of 1 gy/min. for the period of rat whole-body irradiation, the animal was placed in an individual plexiglass container to prevent from moving in the process of x-ray irradiation. metformin (1,1-dimethylbiguanide hydrochloride; merck, darmstadt, germany) was introduced in rats at 300 mg/kg body weight perorally with drinking water twice during 1 day with an interval of 3 h. this dose does not exert genotoxic effect (in vivo) 18 and even mice injected daily with 600 mg/kg body weight for 9 months did not exhibit significant kidney damage. 19 urine collection and dna extraction all chemicals were purchased from sigma-aldrich (st louis, missouri, usa) unless stated otherwise. for urine collection, rats were placed in individual metabolic cages (hatteras instruments, cary, north carolina, usa) equipped with glass containers. to the bottom of the container, 0.5 ml of 0.1 m ethylenediaminetetraacetic acid (edta; ph 8.0) solution was added and coated with a layer of paraffin oil. such cages allow collection of urine separately from feces. in all cases, the urine of rats was collected (before and after irradiation or treatment with metformin) in the evening, starting at 6:00 p.m. for urine collection, rats were placed into metabolic cages before irradiation and before administration of metformin and at certain time points (6, 12, 24, 72 h) after these treatments. in all cases, the rats were housed in metabolic cages at 22 + 2�c for 5.5–6.0 h until 5 ml of liquid (urine þ edta) was collected in the container. urine samples were centrifuged (5000 r/min, 10 min), and supernatant was poured into another test tube and frozen at �20�c for subsequent dna extraction. to isolate total urine dna (mtdna and ndna), a frozen urine sample was thawed at room temperature, then placed on ice immediately prior to dna isolation, and used for dna extraction within 5 min. dna was extracted using the wizard plus minipreps dna purification system (promega, madison, wisconsin, usa) according to the manufacturer’s instructions and as described in the study by su et al 20 and tsui et al. 21 three milliliters of urine sample 2 journal of circulating biomarkers were mixed with 1.5 volumes of 6 m guanidine thiocyanate (sigma-aldrich) by inverting eight times. one milliliter of resin (wizard dna isolation kit, promega) was added into the urine lysate and incubated for 3 h at room temperature under gentle stirring. the resin–dna complex was centrifuged, transferred to a minicolumn (provided in the kit), and washed with a buffer provided by the manufacturer; then, the dna was eluted with tris-edta (te) buffer. each dna sample was dissolved in 0.1 ml of millipore q water. dna concentration was determined by reaction with picogreen, according to the manufacturer’s protocol, after which fluorescence detection on a tecan infinite-unit 200 (austria) followed. samples of total dna from rat urine were subjected to separation by electrophoresis on a 1% agarose gel stained with ethidium bromide to estimate the length of dna fragments. marker 100 base-pair dna ladder (new england biolabs, canada) was used as a standard. electrophoresis was carried out on equipment manufactured by helicon (moscow, russia). for comparison, dna samples isolated from the urine of rats collected prior to and 6 h after irradiation, as well as of aged rats 6 h after administration of metformin, were loaded on agarose gels for electrophoresis. quantitative analysis of nuclear and mtdna by real-time polymerase chain reaction the quantitative analyses of ndna and mtdna were carried out by real-time polymerase chain reaction (pcr; qpcr) using the taqman technology and the qpcr thermal cycler prism 7500 (applied biosystems, foster city, california, usa). the change in the relative mtdna copy number was determined as the ratio between the copy number of the mitochondrial trna gene and that of the nuclear housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (gapdh) which was amplified in the same tube. 20 the efficiency of the assay for amplifying both ndna and mtdna was measured using standard curves generated by a dilution series with 20, 10, 5, 2, 1, and 0.1 ng of total rat liver dna per reaction. to determine the quantities of mtdna and ndna present in the tested samples, the average threshold cycle (ct) values for ndna and mtdna were obtained from each reaction. the cycle number (ct) at which the fluorescent signal of a given reaction crossed the threshold value was used as a basis for quantification of mtdna and ndna copy numbers. 22 pcr assays were performed in duplicate or triplicate for each dna sample. the following primers were used for trna gene amplification (73 bp) from mtdna: forward—50-aat ggt tcg ttt gtt caa cga tt-30; reverse—50-aga aac cga cct gga ttg ctc-30; and a probe—r6gaag tcc tac gtg atc tga gtt-rhq1. for amplification of the gapdh gene (80 bp) from ndna, the following primers were used: forward—50-tgg cct cca agg agt aag aaa c-30; reverse—50-ggc ctc tct ctt gct ctc agt atc-30; and a probe—famctg gac cac cca gcc cag caa-rtq1. primers and probes were chosen using basic local alignment search tool database (http:// blast.ncbi.nlm.nih.gov/blast.cgi), so that the amplicon from mtdna had no significant homology with the nuclear genome. 23 primers and probes for pcr were synthesized by syntol, inc. (moscow, russia). pcrs were performed in a 20 ml volume containing 10 ml of taqman universal pcr master mix 29 buffer (applied biosystems), 2 ml of dna solution, and 250 nm of each primer and probe. pcr cycles were as follows: 5 min at 95�c followed by 40 amplification cycles (95�c for 30 s, annealing and elongation at 60�c for 1 min). fluorescence values were analyzed and calculated by the abi prism 7500 software. statistical analysis statistical differences between the data obtained before and after treatment of rats were analyzed by the standard student’s t-test using graphpad prism software (version 5.0). the results are calculated and presented as a mean + se of the mean (sem, n ¼ 8). a p value of <0.05 was considered to be statistically significant. results the results of electrophoretic separation of total cf-dna (ndna and mtdna) samples isolated from the urine of rats are presented in figure 1. it demonstrates an electropherogram of urine cf-dna collected from 3-month rats figure 1. electrophoresis of dna extracted from rat urine, 1% agarose gel. (1) dna from young rat urine collected before irradiation; (2) dna from young rat urine collected 6 h after irradiation (5 gy); (3) dna from old rat urine collected 6 h after administration of metformin; m: 100 base-pair dna ladder. gaziev et al. 3 http://blast.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.nlm.nih.gov/blast.cgi prior to irradiation and administration of metformin and 6 h after irradiation as well as cf-dna from the urine of 24-month rats collected after 6 h following metformin introduction. these results show that in all cases, the urine of rats contains degraded fragments of cf-dna. the prevalent fraction of cf-dna fragments is sized 300–600 bp. it is also seen that the fragments of cf-dna do not differ significantly in size between rat urine samples collected before and after irradiation. these data also demonstrate that the size of transrenal cf-dna fragments is not influenced by the age of rats and the introduction of metformin. figure 2 represents examples of kinetic curves of qpcr for cf-mtdna and cf-ndna from rat urine collected before irradiation. it is obvious that the exponential increase in the product amplification level of mtdna occurs earlier than that of ndna. the lower value of the threshold cycle indicates a greater copy number of mtdna compared to ndna. the reaction efficiency and the threshold fluorescence level were equal for all the reactions to be compared. first of all, we carried out a comparative analysis for estimation of the contents of cf-mtdna and cf-ndna fragments in the urine of rats collected before x-ray irradiation and administration of metformin. the results testify that the quantity of cf-mtdna in the urine of older rats is higher in comparison to that in the urine of 3-month rats. the copy numbers for fragments of both types of cf-dna is much higher (cf-ndna by 40% and cf-mtdna by 50%) in the urine of 24-month rats, compared with the data obtained for their content in the urine of 3-month rats (figure 3). summarized results of analysis of the content of cf-mtdna and cf-ndna in the urine of rats of different ages collected before and after exposure to ir at different times following irradiation (6, 12, 24, and 72 h) are presented in figure 4. these results indicate that the quantitative content of cf-mtdna and cf-ndna in the urine of 3-month and aged rats change significantly depending on the duration of time between irradiation and urine collection. most notably, we observed a sharp rise in the levels of cf-mtdna and cf-ndna in urine in both the groups of rats after 6, 12, and 24 h following irradiation. a significant reduction of cf-dna was registered in rat urine samples figure 2. qpcr amplification curves for mtdna and ndna. reactions were set up with dna samples isolated from urine of different rats prior to irradiation. qpcr: quantitative polymerase chain reaction; mtdna: mitochondrial dna; ndna: nuclear dna. figure 3. contents of cf-dna fragment copies in the urine of rats belonging to different age groups: (a) 3 months and (b) 24 months. statistical significance was set at: *p < 0.05; **p < 0.01. cf-dna: cell-free dna. figure 4. percentage of cf-dna from urine of rats belonging to different age groups, collected prior to irradiation (100%, c), and after 6, 12, 24, and 72 h following irradiation. (a) 3 months and (b) 24 months. data are presented as mean + sem (n ¼ 8). statistical significance was set at: *p < 0.05; **p < 0.01; ***p < 0.001; and p < 0.05 was considered statistically significant. cf-dna: cell-free dna; c: control. 4 journal of circulating biomarkers collected 72 h after irradiation. comparison of analysis data obtained from 3and 24-month-old rats shows that the percentage increase in the content of cf-dna in urine is more pronounced in aged rats after irradiation. it can be seen that the urine of young rats following 6 and 12 h after irradiation contains 150–200% more cf-ndna and 375–460% more cf-mtdna (relative to the control data obtained from the same rats prior to irradiation). by the same time points following irradiation, we registered an increase in the urine of 24-month rats in the content of cf-ndna and cf-mtdna by 200–250% and 550–720%, respectively. fragments of cf-mtdna in the urine of aged rats exhibit the most prominent increase, as identified in these analyses. experimental data for determining cf-dna in the urine of rats of different ages treated with metformin are presented in figure 5. the obtained results show that metformin administration in rats also results in an increase of cf-dna content in the urine of these animals. however, the results of these analyses differ from the data obtained from irradiated rats, as may be expected. the increase in the content of cf-ndna and cf-mtdna fragments in the urine of these rats is registered only after 6 and 12 h following metformin administration. that is, the amount of cf-ndna and cf-mtdna fragments in the urine of 3-month-old rats after 6 and 12 h following metformin administration is increased by 25% and 55%, respectively. the urine collected after 12 h from the same rats exhibited elevated levels of cf-dna only by the content of cf-mtdna (by 30%). the percentage increase in the number of cf-ndna and cf-mtdna fragments in the urine of older rats after administration of metformin is significantly higher than that in 3-month-old rats (figure 5). in particular, the levels of cf-ndna and cf-mtdna in the urine of aged rats are increased by 50% and 160%, respectively, after 6 h following administration of metformin. after 12 h following metformin administration, the levels of cf-ndna and cf-mtdna in the urine of the same animals exceed the control levels by 30% and 120%, respectively. the demonstrated results show that regardless of the time point of cf-dna analysis after irradiation or metformin administration, the quantitative content of cf-mtdna fragments in the urine of young and aged rats is higher than that of cf-ndna. these results preeminently indicate that mtdna is represented by a larger number of copies containing pcr-amplified regions than in ndna, in the composition of the total cf-dna isolated from the urine of rats. we conducted a comparative analysis of changes of the content ratio cf-mtdna/cf-ndna in the urine of rats after x-irradiation and administration of metformin. the results are presented in table 1, demonstrating that in most cases, the ratio cf-mtdna/cf-ndna has similar values (equal to 2.1–2.4; table 1). however, the values for the ratio cf-mtdna/cf-ndna obtained from the analysis of cf-dna contents in the urine of aged rats after 6 and 12 h following irradiation and treatment with metformin are significantly higher. in any case, the values of this ratio (cf-mtdna/cf-ndna) seem to be understated, given that mammalian somatic cells contain thousands of copies of mtdna, even though they make up only about 1.0% of the total dna content in the cell. thus, the results indicate that urine samples of rats collected after 6–12 h following ir irradiation and administration of metformin contain elevated levels of cf-ndna and cf-mtdna. elevated levels of cf-ndna and cf-mtdna induced by ir and metformin in the urine of 24-month-old rats were significantly higher compared to those in the urine of 3-month-old rats. discussion excessive increase of cf-dna contents in rat urine after ir irradiation indicates the induction of an active cell death process in the tissues of these animals. irradiation of aged rats contributes to an even more significant increase in figure 5. percentage of cf-dna from urine of rats belonging to different age groups, collected prior to administration of metformin (100%, c), and after 6, 12, 24, and 72 h following metformin treatment. (a) 3 months and (b) 24 months. data are presented as mean + sem (n ¼ 8). statistical significance was set at: *p < 0.05; **p < 0.01; ***p < 0.001; and p < 0.05 was considered statistically significant. cf-dna: cell-free dna; c: control. gaziev et al. 5 cf-ndna and cf-mtdna in their urine than the one observed in young rats. high levels of cf-dna in the urine of aged rats subjected to ir is obviously associated with the presence of defective cells (damaged organelles and dna) having low activity of reparation systems in the tissues of these rats prior to irradiation. 24 it should be noted that due to the reduction of activity in the functioning of protective and reparative systems, higher dna damage and cell death rates can be observed in the kidneys, similarly to other tissues of aged animals, as compared to young animals. 25 it can also be assumed that elevation of cf-dna level in the urine of irradiated rats is likely contributed by the development of radiation-induced nephropathy in these animals. 26 the process of autophagy can be stimulated under various cell disorders, upon oxidative stress, endoplasmic reticulum damage, imbalance of calcium homeostasis, and mitochondrial potential changes. such and more severe disorders also occur in cells under the action of ir. 16,27 radiation-mediated induction of cell death via autophagy is less studied than programmed cell death. however, a number of studies indicate a possibility for radiationinduced cell death through the mechanism of autophagy. radiation damage to mitochondria and post-irradiation energy expenditure lead to an increase in the ratio amp/ atp. perhaps it is the basic mechanism of ampk activation in irradiated cells, which can stimulate autophagy. 13,14 as mentioned previously, metformin belongs to the group of compounds which can indirectly induce autophagy through ampk activation. 11,12 over 35 years ago it was established that antidiabetic biguanides are capable to reduce tumor induction and increase the life span in mice. 28 currently, the research in this area gained a wide development, and antidiabetic drug metformin is regarded as an efficient tool for the prevention of carcinogenesis, aging, and combination therapy of tumors. 29,30 it has been established that metformin exhibits the highest accumulation in mitochondria, inhibiting electron transport chain complex i and mitochondrial glycerophosphate dehydrogenase. this leads to the blockade of atp synthesis and depletion of its intracellular pool, increasing the molecule ratio amp/atp. 31 an elevated level results in activation of ampk as a key regulator of energy homeostasis. 11 the activation of ampk stimulates catabolic pathways which lead to atp generation and suppression of anabolic processes that consume atp. ampk activation also contributes to inhibition of mammalian target of rapamycin (mtor) complex 1 (torc1) and activation of uncoordinated-51-like kinase, which directly initiates autophagy. 3,32 this is probably the most important mechanism of action of metformin. nevertheless, its effects are not limited only to this mechanism. for instance, it has been shown recently that biguanides promote accumulation of amp and related nucleotides in hepatocytes which inhibit glucagon signaling, suppress adenylate cyclase, and reduce camp level and activity of protein kinase a. 33 the results of our experiments demonstrated that in metformin-treated rats higher amounts of cfndna and cf-mtdna are excreted with urine. however, unlike the data obtained from irradiated rats, this increase is manifested not so dramatically, and is registered only after 6 and 12 h following metformin administration. increased cf-dna in the urine of x-irradiated rats may be due to cell death in tissues by various pathways as a result of cytotoxic and genotoxic effects of ir. 15,16,27 since metformin does not exert genotoxic or cytotoxic effects and does not have nephropathic effects, 18,19 on the contrary possessing a protective action, the observed increase of cf-dna in urine might possibly be associated with cell death involving autophagy mechanisms. in older rats, metformin apparently activates the clearance of organs from higher amounts of damaged cells which can represent a potential risk for malignant transformation or development of a particular disease. 29 the analysis of the obtained data shows that in all experiments, the number of cf-mtdna pcr copies, including the control data, is twice higher than the number of amplified cf-ndna fragments (table 1). however, the values of cf-mtdna/cf-ndna ratio obtained by analyzing the content of cf-dna in the urine of aged rats after irradiation and administration of metformin (after 6 and 12 h) are significantly higher, compared with the data for urine samples from young rats. since aging is accompanied by progressing induction and accumulation of damage in dna, rna, protein, and lipid macromolecules, additional damage caused by ir irradiation or autophagy activation in the table 1. ratio cf-mtdna/cf-ndna in the urine of rats of different age prior to irradiation and metformin administration and after treatment. treatment age, months control, prior to treatment time after irradiation and metformin administration (h) 6 12 24 72 x-irradiation 3 2.1 + 0.1 2.3 + 0.6 2.4 + 0.3 2.2 + 0.3 2.3 + 0.2 24 2.3 + 0.2 2.7 + 0.3* 2.8 + 0.2* 2.3 + 0.1 2.4 + 0.4 metformin administration 3 2.1 + 0.1 2.4 + 0.3 2.3 + 0.1 2.4 + 0.2 2.2 + 0.3 24 2.3 + 0.2 3.8 + 0.4* 3.3 + 0.2* 2.3 + 0.4 2.4 + 0.2 cfdna: cell-free dna; mtdna: mitochondrial dna; ndna: nuclear dna. *values with significant difference from control for p < 0.05. 6 journal of circulating biomarkers tissues of older animals results in enhancement of cell death and urinary excretion of cf-dna. upon additional damage of cells and dna, autophagy may be involved in a comprehensive dna damage response system. 14 therefore, it can be assumed that increased urinary excretion of cf-dna in aged rats, following administration of metformin, is caused by the death of cells with structural and functional disorders. at least elevated excretion of cf-mtdna with urine in aged rats after metformin administration results from selective removal of defective mitochondria (mitophagy) by autophagy mechanisms. 2,3 previously it has been demonstrated that activation of autophagy in aged rats caused by calorie restriction or administration of an antilipolytic agent or everolimus (inhibitor of mtor) results in a sharp increase in 8-oxy dg due to preferential degradation of damaged mtdna copies. 34,35 it is possible that other mechanisms also promote cell death under these conditions. it is known that although autophagy and programmed cell death occur in different ways, they are closely related, and their regulatory components may interact, contributing to the development of these processes and determining whether a cell lives or dies. 36 in conclusion, it can be mentioned that menendez and coauthors have earlier expressed a hypothesis, according to which metformin can function in the tissues as a ‘‘sweeper’’ of dysfunctional precancerous cells preventing them from piling up and completely transforming into malignant cells. 37 the results of our experiments seem to demonstrate the real mode of action of metformin as a sweeper contributing to elimination of dysfunctional cells from the tissues in aged animals. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work has been supported by the russian foundation for basic research (grant 12-04-31070 and grant 16-34-00832) and by the institute of theoretical and experimental biophysics ras. references 1. green dr and llambi f. cell death signaling. cold spring 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice jcb j circ biomark 2023; 12: 1-11issn 1849-4544 | doi: 10.33393/jcb.2023.2479original research article journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb © 2023 the authors. this article is published by aboutscience and licensed under creative commons attribution-noncommercial 4.0 international (cc by-nc 4.0). commercial use is not permitted and is subject to publisher’s permissions. full information is available at www.aboutscience.eu is still one of the main ophthalmic public health conditions in developed and developing countries (3). cataracts are mainly divided into nuclear cataract (nc), cortical cataract (cc), posterior sub capsular (psc) cataract, acquired cataract and congenital cataract (4). senile cataract is one of the common types of acquired cataract which occurs as a consequence of the aging process. it is characterized by initial opacity in the lens with subsequent swelling of lens and final shrinkage with complete loss of transparency (5). cataract is detected by an eye examination that includes a visual activity test, slit lamp exam (sle) and dilated eye exam (6). worldwide, cataract has caused >50% vision loss including 33.4% blind people and 18.4% people with moderate to severe visual impairment. globally 10.8 million people were blind and 35.1 million people were visually impaired from cataract in 2010 (7). additionally, data from the world health organization (who) has estimated that this number will increase to 40 million in 2025 due to the aging populations with greater life expectancies (8). up to 50 million people in impact of clinico-biochemical variations on the etiopathogenesis of cataract: a case-control study tabassum rashid1, syed sadaf altaf2, shabhat rasool1, rabiya iliyas1, sabia rashid2, sabhiya majid1, mosin saleem khan1,3 1 department of biochemistry, government medical college and associated smhs and super speciality hospital, karan nagar, srinagar, jammu & kashmir india 2 department of ophthalmology, government medical college srinagar and associated smhs and super speciality hospital, karan nagar, srinagar, jammu & kashmir india 3 department of biochemistry, government medical college baramulla and associated hospitals, kanth bagh, baramulla, jammu & kashmir india abstract purpose: cataract is a major cause of blindness worldwide with a greater prevalence in developing countries like india. owing to speculations about the relationship of various biochemical markers and cataract formation this case-control study was designed with the aim to know the impact of serum blood sugar, serum electrolytes and serum calcium on the etiopathogenesis of cataract in kashmiri population. methods: a total of 300 cases diagnosed with cataract and 360 healthy controls were taken for the study. serum of all the cases and controls was analyzed for blood sugar and calcium using spectrometric techniques. sodium and potassium were analyzed using ion-selective electrode technology. all the investigations were done on abbott c4000 fully automatic clinical chemistry analyzer. results: most of the patients in our study were ≥50 years of age having posterior subcapsular cataract. the mean levels of serum fasting blood sugar (mg/dl), serum sodium (mmol/l), serum potassium (mmol/l) and serum calcium (mg/dl) were 99.4 ± 7.7; 140.4 ± 2.5; 4.2 ± 0.5; and 8.9 ± 0.5, respectively, in cases compared to 107.7 ± 12.3; 142.9 ± 5.0; 3.8 ± 0.5; and 8.3 ± 1.7, respectively, in healthy controls. a significantly higher number of cataract cases had elevated serum glucose and sodium levels, low serum potassium and calcium levels compared to healthy controls. conclusions: hyperglycemia, hypernatremia, hypokalemia and hypocalcemia can independently increase the patients’ risk to cataracts. corrections in these biochemical parameters may reduce cataract incidence. keywords: blood pressure, blood sugar, calcium, cataract, intraocular pressure, potassium, sodium received: august 3, 2022 accepted: december 21, 2022 published online: january 16, 2023 corresponding author: dr. mosin saleem khan assistant professor department of biochemistry government medical college baramulla & associated hospitals kanth bagh 193101, baramulla, jammu & kashmir india mosinsaleemkhan@gmail.com introduction the international agency for the prevention of blindness has defined cataract as the clouding or opacification of the normally clear lens of the eye or its capsule that obscures the passage of light through the lens to the retina (1,2). this disease, which can significantly reduce patient’s quality of life, https://doi.org/10.33393/jcb.2023.2479 https://creativecommons.org/licenses/by-nc/4.0/legalcode mailto:mosinsaleemkhan@gmail.com biochemical markers in cataract2 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb the world suffer from senile cataract (9,10), and its prevalence in developing countries is much more than in developed ones (11). in india, the prevalence of blindness due to cataract was reported to be 8% in the age group of >50 years, as per the national blindness survey (12). an estimated 20 lakh new cases of cataract are being added to the burden every year in this country, showing a steep rise ranging from 0.5% above 30 years to 94.5% above 70 years (13). cataract accounts for 62.6% of all blindness, affecting 9-12 million bilaterally blind persons (14). the who/npcb (national programme for control of blindness) survey has shown that there are over 22 million blind in india and 80.1% of these are blind due to cataract (15). cataract is caused by degeneration and opacification of the lens fibers already formed. any factor that disturbs the critical intraand extracellular equilibrium of water and electrolytes or deranges the colloid system within the fibers tends to bring about opacification (16). several studies have been carried out to elucidate risk factors which are responsible for development of cataract. extensive research has established age, ion imbalance, altered calcium levels, diabetes and uv light exposure as causative risk factors for cataract, while recent studies have identified other potential risk factors like exogenous estrogen, nutrition, dietary fat and genetics which might play a role in the development of cataract (17). although cataract affects all age groups its incidence increases with advancing age (18). senile cataract affects equally persons of either gender, usually above the age of 50 years (19). the association between diabetes and cataract formation has been shown in clinical, epidemiological and basic research studies. due to increasing numbers of type 1 and type 2 diabetics worldwide, the incidence of diabetic cataracts steadily rises (20). several clinical studies have shown that cataract development occurs more frequently and at an earlier age in diabetic compared to nondiabetic patients (20). many studies have shown that serum electrolyte (potassium and sodium) concentration directly affects the concentration of electrolytes in aqueous humor and thereby induces cataract formation. concentration of sodium in lens is less compared to serum concentrations whereas it is vice versa in case of potassium concentrations, and this cationic balance is maintained by the osmotic pressure and thus water balance by the action of enzyme na+/k+ atpase. any imbalance in between the electrolytes leads to cataract formation (21). calcium is of particular concern in cataract. this cation is essential for various lens fiber cell metabolism processes (22). it has been shown that lens calcium content correlates with opacity in cataractous human lenses (23) and subsequent changes in serum calcium concentration might be an important factor in the development of cataract (24). demographic and clinico-biochemical biomarkers have been previously linked with the development of cataract from other parts of the world, but so far only few studies have been reported from the indian subcontinent regarding the interrelationship of cataract (25,26). keeping in view the ethnicity and relatively conserved genetic pool of kashmiri population, this case-control study was designed to elucidate the role of demographic and clinico-biochemical parameters in the etiopathogenesis and severity of cataract in kashmiri population. materials and methods ethics this study was performed in line with the principles of the declaration of helsinki. ethical clearance for the study was sought from institutional review board, government of medical college srinagar vide no. 2022t/eth/gmc. all the patients included in the study were informed about the study and informed consent both in vernacular as well as in english language was taken before eliciting history and sample collection from study subjects. standard questionnaire or patient proforma was properly recorded and drafted as per socio-demographic and clinico-biochemical parameters. the authors affirm that human research participants provided informed consent for publication of their details. study design this was a case-control study conducted by the department of biochemistry in collaboration with the department of ophthalmology, government medical college srinagar and associated smhs hospital srinagar, j&k, from march 2020 to march 2022. study subjects and sample size the study was conducted in ethnic population of kashmir; no restrictions were made among patients with respect to gender and dwelling. a total of 300 patients diagnosed with cataract and 360 healthy controls were enrolled for the study. keeping the power of study as 80% and allocation ratio of 1.2 and effect size of 0.4, the sample size was calculated by g power 23.0.1 version. inclusion and exclusion criteria “cases” included all those individuals who were >18 years of age and diagnosed with cataract. “cases” excluded all those individuals with any other eye ailment and symptomatic disease (liver, kidney, heart or other), trauma, infection, inflammation of eye, cancer or any other genetic abnormality. “controls” included healthy individuals >18 years of age. ophthalmic examination each patient was subjected to various ophthalmic measurements. uncorrected visual acuity was measured with snellen chart. refraction was done and the best corrected visual acuity was noted. intraocular pressure (iop) measurement was done with non-contact tonometer. corrected iop was calculated after measuring central corneal thickness by ultrasonic pachymetry. detailed anterior segment examination using slit lamp was done to rule glaucoma and associated ocular pathology. detailed fundus examination under full mydriasis obtained by 0.8% tropicamide and 5% phenylephrine was done with direct ophthalmoscopy, 78d and indirect ophthalmoscopy. rashid et al j circ biomark 2023; 12: 3 © 2023 the authors. published by aboutscience www.aboutscience.eu sample collection a total of 04 ml of venous blood was collected from each cataract patient and healthy control in a coagulation activating red top vial. blood was collected from each individual after 8-10 hours of fasting. blood was immediately centrifuged for separation of serum. serum was properly stored at ‒20°c till biochemical analysis was done in the biochemistry diagnostic laboratory, smhs hospital srinagar. biochemical analysis for electrolytes serum electrolyte levels were estimated on abbott architect c4000 fully automated clinical chemistry analyzer using ion-selective electrode. the reference ranges of serum sodium levels were taken between 135 and 145 mmol/l and serum potassium levels as 3.5-4.5 mmol/l. biochemical analysis for calcium serum calcium levels were estimated spectrophotometrically on abbott architect c4000 fully automated clinical chemistry analyzer using arsenazo iii dye. the reference ranges of serum calcium were taken as: 8.5-10.2 mg/dl. biochemical analysis of glucose serum glucose levels were measured spectrophotometrically on abbott architect c4000 fully automated clinical chemistry analyzer. glucose is phosphorylated by hexokinase (hk) in the presence of adenosine triphosphate (atp) and magnesium ions to produce glucose-6-phosphate (g6p) and adenosine diphosphate (adp). glucose-6-phosphate dehydrogenase (g6pdh) specifically oxidizes g6p to 6-phosphogluconate with the concurrent reduction of nicotinamide adenine dinucleotide (nad) and readily to nicotinamide adenine dinucleotide reduced (nadh). one micromole of nadh is produced for each micromole of glucose consumed. the nadh produced absorbs light at 340 nm and can be detected spectrophotometrically as an increased absorbance. the reference ranges of serum glucose were taken as: 99-100 mg/dl (normal) and >100 mg/dl (impaired). statistical analysis the data was analyzed using ibm statistical package for the social sciences (spss) software v. 25.0. descriptive statistics was performed and data was presented as frequency (n) and percentage (%). continuous data was presented as mean and standard deviation. chi-square test was used to compare proportions between groups as deemed proper by the statistical expert. p value <0.05 was considered statistically significant. results table i contains the socio-demographic and clinicopathological characteristics of cataract cases and healthy controls. cases and controls were matched with respect to age and gender; 17.5% (63 of 360) of the controls were <50 years of age as compared to 21% (63 of 300) of cases who were <50 years of age. with respect to gender, 47.5% (171 of 360) controls were men as compared to 52.0% (156 of 300) of men having cataract. accordingly, 90.0% (324 of 360) of the controls were inhabitants of rural areas as compared to 80% (240 of 300) of cases who belonged to rural areas. the cataract patients were evaluated for various clinical parameters. hypertension was present in 13.0% (39 of 300) of cataract patients. the family history of cataract was present in 4.0% (12 of 300) of cases. the history of any other eye disorder was present in 23.0% (69 of 300) of cataract cases. further the iop of all the patients was measured wherein 91.0% (273 of 300) had normal iop while 9.0% (27 of 300) had higher iop. table i socio-demographic and clinicopathological characteristics of cases having cataract and controls included in the study – controls n = 360 (%) cases n = 300 (%) χ2 p value age group <50 years ≥50 years 63 (17.5) 297 (82.5) 63 (21.0) 237 (79.0) 1.2 0.15 gender men women 171 (47.5) 189 (52.5) 156 (52.0) 144 (48.0) 1.3 0.15 dwelling rural urban 324 (90.0) 36 (10.0) 240 (80.0) 60 (20.0) 13.1 <0.0001 hypertension no yes – 261 (87.0) 39 (13.0) – – family history of cataract no yes – 288 (96.0) 12 (4.0) – – h/o any other eye disorder no yes – 231 (77.0) 69 (23.0) – – eyes affected one both – 177 (59.0) 123 (41.0) – – type of cataract nuclear cortical posterior subcapsular – 00 (0.0) 75 (25.0) 225 (75.0) – – grade i and ii iii and iv – 165 (55.0) 135 (45.0) – – iop normal high – 273 (91.0) 27 (9.0) – – h/o = history of; iop = intraocular pressure. biochemical markers in cataract4 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table ii depicts the biochemical parameters of cataract cases and healthy controls; 69.0% (207 of 300) of cases had impaired blood sugar level as compared to 22.5% (81 of 360) of controls having impaired blood sugar, and the association was found to be statistically significant (p<0.0001); 36.3% (109 of 300) of cases had hypernatremia as compared to only 3.0% (11 of 360) of controls with hypernatremia (p<0.0001). cataract cases had significantly low potassium levels compared to controls (17.5% vs. 6.1%; p<0.0001). in addition, 38.0% (114 of 300) of cases had hypocalcemia as compared to only 12.5% (45 of 360) of controls with hypocalcemia and the difference was statistically significant (p<0.0001). table ii biochemical parameters of cataract cases and healthy controls parameters controls n = 360 (%) cases n = 300 (%) or (95% ci) p value fasting blood sugar normal impaired 279 (77.5) 81 (22.5) 93 (31.0) 207 (69.0) 7.6 (5.4-10.8) <0.0001 sodium levels normal high 349 (97.0) 11 (3.0) 191 (63.7) 109 (36.3) 18.1 (9.5-34.4) <0.0001 potassium levels normal low 338 (93.9) 22 (6.1) 249 (83.0) 51 (17.0) 3.1 (1.9-5.3) <0.0001 calcium levels normal low 315 (87.5) 45 (12.5) 186 (62.0) 114 (38.0) 4.2 (2.9-6.3) <0.0001 ci = confidence interval; or = odds ratio. table iii shows the mean levels of various biochemical parameters in cases and healthy controls. a statistically significant difference was observed between cases and controls with respect to mean fasting blood sugar levels in mg/dl (107.7 ± 12.3 vs. 99.4 ± 7.7); mean sodium levels in mmol/l (142.9 ± 5.0 vs. 140.4 ± 2.5); mean potassium level in mmol/l (3.8 ± 0.5 vs. 4.2 ± 0.5); mean calcium level in mg/dl (8.3 ± 1.7 vs. 8.9 ± 0.5). table iii levels (mean ± sd) of various biochemical parameters in cataract cases and healthy controls parameters controls (mean ± sd) cases (mean ± sd) p value fasting blood sugar (mg/dl) 99.4 ± 7.7 107.7 ± 12.3 <0.0001 sodium levels (mmol/l) 140.4 ± 2.5 142.9 ± 5.0 <0.0001 potassium levels (mmol/l) 4.2 ± 0.5 3.8 ± 0.5 <0.0001 calcium levels (mg/dl) 8.9 ± 0.5 8.3 ± 1.7 <0.0001 sd = standard deviation. table iv shows the association of fasting blood sugar levels with various socio-demographic, clinico-pathological and biochemical parameters of cataract patients and controls. in each subgroup of age, gender, dwelling, sodium, potassium and calcium levels, a statistically significant difference in serum blood levels was noted between cases and controls (p<0.0001). among patients with psc cataract, 72.0% (162 of 225) had impaired blood sugar level as compared to cc wherein only 60% (45 of 75) had impaired blood sugar, and the difference was statistically significant (p = 0.05). no association was found between blood sugar levels and any other parameter of cases and controls. table v describes the association of serum sodium levels with various socio-demographic, biochemical and clinicopathological parameters of cataract cases and controls. a statistically significant difference in serum sodium levels was noted between cases and controls in each subgroup of age, gender, dwelling, potassium and calcium levels (p<0.0001). among cataract patients with grade i and ii disease, 41.2% (68 of 165) had high sodium levels while among cataract patients with grade iii and iv disease, only 30.4% (41 of 135) had high sodium levels and the difference was statistically significant (p = 0.05). we did not observe any other parameter influencing the sodium levels in cataract patients. table vi displays the association of serum potassium levels with various socio-demographic, clinicopathological and biochemical parameters of cataract patients and controls. among cases, 20% (48 of 240) of rural inhabitants had low potassium levels as compared to 6.5% (21 of 324) of rural control subjects having low potassium levels (p<0.0001). with respect to calcium level, 31.6% (36 of 114) of hypocalcemia cases had hypokalemia as compared to only 2.2% (01 of 45) of hypocalcemia controls having hypokalemia, and the difference was statistically significant (p<0.0001). among hypertensive cases, 15.4% (18 of 39) had hypokalemia but among normotensive cases only 12.6% (21 of 261) had hypokalemia, and the difference was statistically significant (p<0.0001). importantly, 33.3% (09 of 27) cases having high iop were hypokalemic but among cases having normal iop, only 15.4% (42 of 273) had hypokalemia, and the difference was statistically significant (p<0.0001). no association was found between serum potassium levels and any other parameter of cases and controls. table vii depicts the association of serum calcium levels with various socio-demographic, clinicopathological and biochemical parameters of cataract cases and controls. a statistically significant difference in calcium levels was noted between cases and controls in each subgroup of age, gender and dwelling (p<0.0001). we did not observe any other parameter influencing the calcium levels in cataract patients. discussion we evaluated cataract patients with respect to various socio-demographic, clinicopathological and biochemical characteristics. as aging itself is a major risk factor for the development of cataract in both women and men (27), most of the cataract patients were from the age group of ≥50 years. it has been suggested that with aging the alteration rashid et al j circ biomark 2023; 12: 5 © 2023 the authors. published by aboutscience www.aboutscience.eu table iv association of fasting serum glucose levels with various socio-demographic, biochemical and clinicopathological parameters of cataract cases and controls socio-demographic/ clinicopathological/ biochemical parameters fasting serum glucose levels in controls fasting serum glucose levels in cases or (95% ci) p value n = 360 (%) normal 279 (77.5) impaired 81 (22.5) n = 300 (%) normal 93 (31.0) impaired 207 (69.0) 7.6 (5.4-10.8) <0.0001 age group <50 years ≥50 years 63 (17.5) 297 (82.5) 57 (90.5) 222 (74.7) 06 (9.5) 75 (25.3) 63 (21.0) 237 (79.0) 17 (27.0) 76 (32.1) 46 (73.0) 161 (67.9) 25.7 (9.3-70.4) 6.2 (4.2-9.1) <0.0001 <0.0001 gender men women 171 (47.5) 189 (52.5) 141 (82.5) 138 (73.0) 30 (17.5) 51 (27.0) 156 (52.0) 144 (48.0) 48 (30.8) 45 (31.3) 108 (69.2) 99 (68.8) 10.5 (6.2-17.7) 5.9 (3.6-9.5) <0.0001 <0.0001 dwelling rural urban 324 (90.0) 36 (10.0) 255 (78.7) 24 (66.7) 69 (21.3) 12 (33.3) 240 (80.0) 60 (20.0) 75 (31.3) 18 (30.0) 165 (68.8) 45 (70.0) 8.1 (5.5-11.9) 4.6 (1.9-11.3) <0.0001 <0.001 sodium levels normal elevated 349 (97.0) 11 (3.0) 270 (77.4) 09 (81.8) 79 (22.6) 02 (18.2) 191 (63.7) 109 (36.3) 58 (30.4) 35 932.1) 133 (69.6) 74 (67.9) 7.8 (5.2-11.6) 9.5 (1.9-46.3) <0.0001 0.002 potassium levels normal low 338 (93.9) 22 (6.1) 261 (77.2) 18 (81.8) 77 (22.8) 04 (18.2) 249 (83.0) 51 (17.0) 84 (33.7) 09 (17.6) 165 (66.3) 42 (82.4) 6.6 (4.6-9.5) 21 (5.7-77.1) <0.0001 <0.0001 calcium levels normal low 315 (87.5) 45 (12.5) 246 (78.1) 33 (73.3) 69 (21.9) 12 (26.7) 186 (62.0) 114 (38.0) 60 (32.3) 33 (28.9) 126 (67.7) 81 (71.1) 7.4 (4.9-11.2) 6.7 (3.1-14.6) <0.0001 <0.0001 hypertension no yes – – – 261 (87.0) 39 (13.0) 78 (29.9) 15 (38.5) 183 (70.1) 24 (61.5) 0.6 (0.3-1.3) 0.3 family history of cataract no yes – – – 288 (96.0) 12 (4.0) 84 (29.2) 009 (75.0) 204 (70.8) 03 (25.0) 0.13 (0.03-0.5) 0.002 h/o any other eye disorder no yes – – – 231 (77.0) 69 (23.0) 72 (31.2) 21 (30.4) 159 (68.8) 48 (69.6) 1.03 (0.5-1.8) 1.000 eyes affected one both – – – 177 (59.0) 123 (41.0) 57 (32.2) 36 (29.3) 120 (67.8) 87 (70.7) 1.1 (0.6-1.8) 0.6 type of cataract cortical posterior subcapsular – – – 75 (25.0) 225 (75.0) 30 (40.0) 63 (28.0) 45 (60.0) 162 (72.0) 1.7 (0.9-2.9) 0.05 grade i and ii iii and iv – – – 165 (55.0) 135 (45.0) 45 (27.3) 48 (35.6) 120 (72.7) 87 (64.4) 0.6 (0.4-1.1) 0.1 iop normal high – – – 273 (91.0) 27 (9.0) 81 (29.7) 12 (44.4) 192 (70.3) 15 (55.6) 0.5 (0.2-1.1) 0.12 ci = confidence interval; h/o = history of; iop = intraocular pressure; or = odds ratio. biochemical markers in cataract6 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table v association of serum sodium levels with various socio-demographic, biochemical and clinicopathological parameters of cataract cases and controls socio-demographic/ clinicopathological/ biochemical parameters serum sodium levels in controls serum sodium levels in cases or (95% ci) p value n = 360 (%) normal 349 (97.0) high 11 (3.0) n = 300 (%) normal 191 (63.7) high 109 (36.3) 18.1 (9.5 -34.4) <0.0001 age group <50 years ≥50 years 63 (17.5) 297 (82.5) 63 (100.0) 286 (96.3) 0 (0.0) 11 (3.7) 63 (21.0) 237 (79.0) 25 (39.7) 166 (70.0) 38 (60.3) 71 (30.0) – 11.1 (5.7-21.5) <0.0001 <0.0001 gender men women 171 (47.5) 189 (52.5) 162 (94.7) 187 (98.9) 09 (5.3) 02 (1.1) 156 (52.0) 144 (48.0) 99 (63.5) 92 (63.9) 57 (36.5) 52 (36.1) 10.3 (4.9-21.8) 52.8 (12.5-221.7) <0.0001 <0.0001 dwelling rural urban 324 (90.0) 36 (10.0) 314 (96.9) 35 (97.2) 10 (3.1) 01 (2.8) 240 (80.0) 60 (20.0) 150 (62.5) 41 (68.3) 90 (37.5) 19 (31.7) 18.8 (9.5-37.2) 16.2 (2.0-127.3) <0.0001 <0.0001 potassium levels normal low 338 (93.9) 22 (6.1) 329 (97.3) 20 (90.9) 09 (2.7) 02 (9.1) 249 (83.0) 51 (17.0) 162 (65.1) 29 (56.9) 87 (34.9) 22 (43.1) 19.6 (9.6-39.9) 7.1 (3.8-13.3) <0.0001 <0.0001 calcium levels normal low 315 (87.5) 45 (12.5) 304 (96.5) 45 (100.0) 11 (3.5) 00 (0.0) 186 (62.0) 114 (38.0) 121 (61.5) 70 (61.4) 65 (34.9) 44 (38.6) 14.8 (7.5-29.0) – <0.0001 <0.0001 hypertension no yes – – – 261 (87.0) 39 (13.0) 165 (63.2) 26 (66.7) 96 (36.8) 13 (33.3) 0.9 (0.4-1.7) 0.7 family history of cataract no yes – – – 288 (96.0) 12 (4.0) 184 (63.9) 07 (58.3) 104 (36.1) 05 (41.7) 1.2 (0.3-4.0) 0.7 h/o any other eye disorder no yes – – – 231 (77.0) 69 (23.0) 153 (66.2) 38 (55.1) 78 (33.8) 31 (44.9) 1.6 (0.9-2.7) 0.11 eyes affected one both – – – 177 (59.0) 123 (41.0) 119 (67.2) 72 (58.5) 58 (32.8) 51 (41.5) 1.4 (0.9-2.3) 0.14 type of cataract cortical posterior subcapsular – – – 75 (25.0) 225 (75.0) 53 (70.6) 138 (61.3) 22 (29.3) 87 (38.6) 1.5 (0.8-2.6) 0.16 grade i and ii iii and iv – – – 165 (55.0) 135 (45.0) 97 (58.8) 94 (69.6) 68 (41.2) 41 (30.4) 0.6 (0.3-1.0) 0.052 iop normal high – – – 273 (91.0) 27 (9.0) 175 (64.1) 16 (59.3) 98 (35.9) 11 (40.7) 1.2 (0.5-2.7) 0.6 ci = confidence interval; h/o = history of; iop = intraocular pressure; or = odds ratio. rashid et al j circ biomark 2023; 12: 7 © 2023 the authors. published by aboutscience www.aboutscience.eu table vi association of serum potassium levels with various socio-demographic, biochemical and clinicopathological parameters of cataract cases and controls socio-demographic/ clinicopathological/ biochemical parameters serum potassium levels in controls serum potassium levels in cases or (95% ci) p value n = 360 (%) normal 338 (93.9) low 22 (6.1) n = 300 (%) normal 249 (83.0) low 51 (17.0) 3.1 (1.9-5.3) <0.0001 age group <50 years ≥50 years 63 (17.5) 297 (82.5) 62 (98.4) 276 (92.9) 01 (1.6) 21 (7.1) 63 (21.0) 237 (79.0) 48 (76.2) 201 (84.8) 15 (23.8) 36 (15.2) 19.3 (2.4-151.8) 2.3 (1.3-4.1) <0.0001 <0.0003 gender men women 171 (47.5) 189 (52.5) 163 (95.3) 175 (92.6) 08 (4.7) 14 (7.4) 156 (52.0) 144 (48.0) 132 (84.6) 117 (7.4) 24 (15.4) 27 (18.8) 3.7 (1.6-8.5) 2.8 (1.4-5.7) <0.0001 <0.0002 dwelling rural urban 324 (90.0) 36 (10.0) 303 (93.5) 35 (97.2) 21 (6.5) 01 (2.8) 240 (80.0) 60 (20.0) 192 (80.0) 57 (95.0) 48 (20.0) 03 (5.0) 3.6 (2.0-6.2) 1.8 (0.1-18.4) <0.0001 1.000 calcium levels normal low 315 (87.5) 45 (12.5) 294 (93.3) 44 (97.8) 21 (6.7) 01 (2.2) 186 (62.0) 114 (38.0) 171 (91.9) 78 (68.4) 15 (8.1) 36 (31.6) 1.2 (0.6-2.4) 20.3 (2.6-153.2) 0.6 <0.0001 hypertension no yes – – – 261 (87.0) 39 (13.0) 228 (87.4) 33 (84.6) 21 (12.6) 18 (15.4) 5.9 (2.8-12.2) <0.0001 family history of cataract no yes – – – 288 (96.0) 12 (4.0) 240 (83.3) 09 (75.0) 48 (16.7) 03 (25.0) 1.6 (0.4-6.3) 0.4 h/o any other eye disorder no yes – – – 231 (77.0) 69 (23.0) 195 (84.4) 54 (78.3) 36 (15.6) 15 (21.7) 1.5 (0.7-2.9) 0.27 eyes affected one both – – – 177 (59.0) 123 (41.0) 150 (84.7) 99 (80.5) 27 (15.3) 24 (19.5) 1.3 (0.7-2.4) 0.35 type of cataract nuclear cortical posterior subcapsular – – – 75 (25.0) 225 (75.0) 57 (76.0) 192 (85.3) 18 (24.0) 33 (14.7) 0.5 (0.2-1.0) 0.07 grade i and ii iii and iv – – – 165 (55.0) 135 (45.0) 132 (80.0) 117 (86.7) 33 (20.0) 18 (13.3) 0.6 (0.3-1.1) 0.16 iop normal high – – – 273 (91.0) 27 (9.0) 231 (84.6) 18 (66.7) 42 (15.4) 09 (33.3) 2.7 (1.1-6.5) 0.051 ci = confidence interval; h/o = history of; iop = intraocular pressure; or = odds ratio. biochemical markers in cataract8 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb table vii association of serum calcium levels with various socio-demographic, clinicopathological and biochemical parameters of cataract patients and controls socio-demographic/ clinicopathological/ biochemical parameters serum calcium levels in controls serum calcium levels in cases or (95% ci) p value n = 360 (%) normal 315 (87.5) low 45 (12.5) n = 300 (%) normal 186 (62.0) low 114 (38.0) 4.2 (2.9-6.3) <0.0001 age group <50 years ≥50 years 63 (17.5) 297 (82.5) 54 (85.7) 261 (81.7) 09 (14.3) 36 (12.1) 63 (21.0) 237 (79.0) 40 (63.5) 146 (61.6) 23 (36.5) 91 (38.4) 3.4 (1.4-8.2) 4.5 (2.9-6.9) 0.007 <0.0001 gender men women 171 (47.5) 189 (52.5) 147 (86.0) 168 (88.9) 24 (14.0) 21 (11.1) 156 (52.0) 144 (48.0) 96 (61.5) 90 (62.5) 60 (38.5) 54 (37.5) 3.8 (2.2-6.5) 4.8 (2.7-8.4) <0.0001 <0.0001 dwelling rural urban 324 (90.0) 36 (10.0) 282 (87.0) 33 (91.7) 42 (13.0) 03 (8.3) 240 (80.0) 60 (20.0) 144 (60.0) 42 (70.0) 96 (40.0) 18 (30.0) 4.4 (2.9-6.7) 4.7 (1.2-17.3) <0.0001 0.02 hypertension no yes – – – 261 (87.0) 39 (13.0) 168 (64.4) 18 (46.2) 93 (35.6) 21 (53.8) 2.1 (1.0-4.1) 0.3 family history of cataract no yes – – – 288 (96.0) 12 (4.0) 180 (62.5) 06 (50.0) 108 (37.5) 06 (50.0) 1.6 (0.5-5.2) 0.3 h/o any other eye disorder no yes – – – 231 (77.0) 69 (23.0) 147 (63.6) 39 (56.5) 84 (36.4) 30 (43.5) 1.3 (0.7-2.3) 0.3 eyes affected one both – – – 177 (59.0) 123 (41.0) 114 (64.4) 72 (58.5) 63 (35.6) 51 (41.5) 1.2 (0.7-2.0) 0.3 type of cataract nuclear cortical posterior subcapsular – – – 75 (25.0) 225 (75.0) 51 (68.0) 135 (60.0) 24 (32.0) 90 (40.0) 1.4 (0.8-2.4) 0.2 grade i and ii iii and iv – – – 165 (55.0) 135 (45.0) 99 (60.0) 87 (64.4) 66 (40.0) 48 (35.6) 0.8 (0.5-1.3) 0.4 iop normal high – – – 273 (91.0) 27 (9.0) 171 (62.6) 15 (55.6) 102 (37.4) 12 (44.4) 1.3 (0.6-2.9) 0.5 ci = confidence interval; h/o = history of; iop = intraocular pressure; or = odds ratio. in membrane permeability of the lens epithelium coupled with the changes in sodium and potassium ion levels in aqueous humor may accentuate ionic imbalance within the lens and lead to the development of cataract (28). in our study, a slightly higher percentage of men population was affected compared to women. according to different studies, women are more prone to getting most types of cataract than men. this is most likely due to lower estrogen levels after menopause in women (29). in our study, proportion of rural participants diagnosed with cataract was much higher compared to urban participants, which may be due to the differential exposure to contextual factors (30). furthermore, we speculate that rural areas lack quality healthcare and hygiene, hence putting inhabitants at more risk of developing cataract and its related complications. our results are in line with the observations from other parts of the country reporting significantly higher incidence of cataract in rural areas (31). we observed only 13.0% of the cataract cases to be hypertensive, but according to lee et al hypertension could induce conformational changes to proteins in lens capsules, rashid et al j circ biomark 2023; 12: 9 © 2023 the authors. published by aboutscience www.aboutscience.eu thereby exacerbating the cataract formation (32). per previous reports, the risk of cataract increases with long-standing hypertension (33). furthermore, some studies suggest that antihypertensive medications (such as diuretics and betablockers) are related to cataract (34). in our study, only few cataract patients had a family history of cataract. our findings are consistent with epidemiological studies demonstrating more prevalent occurrence of age-related cataracts in close relatives of cataract patients than in the general population (35). in addition, genetic studies have shown the effect of specific genes in the development of cataractous lenses (35). only one eye was affected in most of the cataract patients, which supports the previous findings that cataract may be present in one or both eyes but cannot spread from one eye to another (36). most of the enrolled patients were having psc cataract, which is the most common morphological form of cataract (37) and commonly studied across the country (38). psc cataract produces more and faster vision deterioration than other forms of cataract, and patients report earlier for cataract surgery, which may be a potential explanation for its elevated prevalence (39). various studies are going on all over the world to clarify the relationship between biochemical elements and cataract formation (40), and most of the studies from different populations have not found a remarkable difference in blood biochemical elements between cataract patients and healthy controls (41-43). to further explore the biochemical intricacies of cataract we evaluated the cases as well as controls for various biochemical markers and deduced the relationship, if any, between the disease development and biochemical markers. as evident from the dataset, most of the cataract patients in our study were having significantly impaired fasting blood sugar levels compared to controls. a previous study has found a significant relationship between hyperglycemia and incidence of cataracts (44). in vivo or in vitro studies showed evidence that diabetes mellitus is the cause of cataract (45,46). cataracts have multiple etiologies, one of which is chronic hyperglycemia which has been related to many systemic and ocular complications such as loss of vision. it has been suggested that the polyol via which the enzyme aldose reductase catalyzes the reduction of glucose into sorbitol is a central part of the mechanism of cataract development. the increased intracellular accumulation of sorbitol leads to a hyperosmotic effect, resulting in hydropic lens fibers that degenerate and form cataract (47,48). in our study population, the frequency of cataract patients having hypernatremia was significantly higher compared to controls, which is in line with the study conducted by mathur and pai who reported significant hypernatremia in cataract patients compared to the controls (49). since the lens metabolism is associated with aqueous humor, which itself is produced from blood secretions, serum electrolyte concentration directly affects electrolytes of aqueous humor and in turn lens metabolism (26). studies have upheld that, in case of increased concentrations of sodium in aqueous humor, it is difficult for the sodium pumps to maintain a low intracellular sodium ion level. in turn, a higher sodium ion concentration of the aqueous humor, coupled with an altered membrane permeability of lens, increases the intracellular sodium ion concentration leading to hydration of the lens, thereby resulting in loss of its transparency and development of cataract (50). in the current study, we observed hypokalemia in significantly higher number of cataract patients compared to controls. several studies revealed strong association of low serum potassium levels with ccs (51,52). according to duncan and bushell, cortical and mature cataracts had very low serum potassium levels (53). the frequency of hypocalcemia in cataract cases was significantly higher compared to controls. low serum calcium levels in cataract patients were seen in other populations, clearly supporting our results (54). cataract is the most common ocular symptom of hypocalcemia (55). seemingly, because of deposition of calcium in soft tissues producing reduced vision/cataract or calcification of basal ganglia, calcium gets depleted in human serum (56). in the further part of the study we deciphered the effect of various factors on the relationship between biochemical parameters and cataract. surprisingly none of the factors significantly affected the relationship of serum glucose, serum sodium and serum calcium with cataract. in addition, we have also identified various factors modifying the association between hypokalemia and cataract. the frequency of hypokalemic rural cataract patients was significantly higher compared to rural hypokalemic controls. difference in mean serum potassium concentrations among kashmiri cataract cases and healthy controls residing in rural locations might be due to difference in the quality of diet among the two groups (56,57). interestingly we observed a significantly higher proportion of hypokalemic cataract patients coexistent with hypocalcemia. although hypokalemia and hypocalcemia exhibit multiple interrelated acid-base and electrolyte abnormalities such as hypophosphatemia, respiratory/metabolic alkalosis, mixed acid-base disorders (58), the coexistence of these in cataract has not yet been reported and debated upon. further, hypokalemia was significantly associated with hypertension in cataract patients. even though the association of hypokalemia and hypertension has been previously reported (33,34), the common causes of hypertension with hypokalemia have been well established, which include essential hypertension with diuretic use, primary aldosteronism, cushing’s syndrome, pheochromocytoma, renal vascular disease and malignant hypertension (59). therefore, patients with coexistent hypertension and hypokalemia need to be evaluated further to establish the reason for the development of cataract. conclusion in conclusion, serum glucose, sodium, potassium and calcium levels were identified as the potentially modifiable parameters deranged in cataract. in future, large-scale studies to enhance the battery of biochemical markers and to establish cause and effect relationship between biochemical markers and cataract need to be done. limitation of the study the sample size of the study is relatively modest. biochemical markers in cataract10 © 2023 the authors. journal of circulating biomarkers issn 1849-4544 www.aboutscience.eu/jcb disclosures conflict of interest: the authors disclose that there are no financial or non-financial interests that are directly or indirectly related to the work submitted for publication. financial support: the study was funded by government medical college srinagar, karan nagar, srinagar, jammu & kashmir, india, vide gmc/2022/17. authors’/contributors’ list: conceptualization: msk, tr, sr; data curation: msk, ssa; 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accepted: 22 april 2017 introduction a goal for the diagnostics field is the isolation of disease biomarkers from easily acquired biofluids such as blood, urine, and saliva. such biomarkers make it possible to replace invasive tissue biopsies with the so-called “liquid biopsies.” for blood, there has been much success in this endeavor. numerous studies have used circulating tumor cells, circulating dna, extracellular vesicles (evs), and mirna isolated from blood as biomarkers for many diseases. 1 progress in the discovery of urinary extracellular biomarkers was relatively slow. one reason is the lack of efficient methods for the isolation of evs, proteins, and nucleic acids from urine. ultracentrifugation (uc), considered the gold standard for vesicle isolation from many biofluids, is efficacious but laborious. given that any biomarker should be readily and robustly isolatable by clinical laboratories that typically do not have an ultracentrifuge, this technique is not ideal. to fill this void, several companies have introduced ev isolation kits that do not require uc. 2–4 these kits use binding columns or precipitation reagents such as polyethylene glycol to isolate evs from a variety of biofluids. ymir genomics llc, cambridge, ma, usa corresponding author: p shannon pendergrast, ymir genomics llc, 516 green st. 1a, cambridge, ma 02139, usa. email: shannon@ymirgenomics.com journal of circulating biomarkers volume 6: 1–7 ª the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417712666 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:shannon@ymirgenomics.com https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417712666 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage ymir genomics has developed a novel, proprietary, and rapid precipitation method that does not require polyethylene glycol. 5 here, we compare the method with uc and three other commercial precipitation methods for their ability to isolate ev protein markers and extracellular rna. materials and methods urine sample isolation urine samples were collected from healthy volunteers (members of ymir genomics’ staff and family). all samples were first-void and clean-catch collections. samples remained at room temperature (rt) and were processed within 2 h, where applicable creatinine and protein levels were assessed. ev isolation evs were isolated from urine samples using ymir genomics’ ev isolation kit (ymir genomics, cambridge, massachusetts, usa), mircury exosome isolation kit (exiqon, woburn, massachusetts, usa), exoquick-tc (sbi, mountain view, california, usa), and total exosome isolation reagent (life technologies, carlsbad, california, usa) following manufacturers’ directions. evs were also isolated by uc as follows: urine sample was sequentially centrifuged for 10 min at 2000 � g and at 17,000 � g to remove cells and cellular debris, and then the resulting supernatant was centrifuged at 200,000 � g for 60 min at 24�c to sediment exosomes. immunoblot analysis ev samples were combined with sds sample buffer (boston bioproducts, ashland, massachusetts, usa), separated by denaturing electrophoresis on precasting 4–12% novex or 4–12% bolt gels (life technologies, carlsbad, california, usa), and then transferred onto a nitrocellulose membrane (life technologies) by applying 20 v for 7 min at rt. the blots were blocked with 5% milk in tbst (tris buffered saline-tween 20; boston bioproducts, ashland, massachusetts, usa) for 1 h at rt, and incubated with rabbit anti-cd9 (1:1000; cell signaling technologies, danvers, massachusetts, usa), rabbit anti-rab5 (1:300; santa cruz biotechnology, dallas, texas, usa), rabbit anti-cd63 (1:200, santa cruz biotechnology, dallas, texas, usa), rabbit anti-aquaporin-2 (1:1000; abcam, cambridge, massachusetts, usa), and rabbit anti-hsp70 (1:1000; abcam) antibodies in blocking buffer overnight at 4�c. after incubation, the blots were washed with tbst and incubated for 1 h at rt with goat anti-rabbit hrp antibody (1:2000; cell signaling technologies) in blocking buffer. western bright ecl (bioexpress, kaysville, utah, usa) was used to develop the blots. the equivalent of 3 ml of urine evs was loaded per lane. in figure 3(b), the immunoblot band intensity for aq2, cd9, and rab5 is represented in a heat map (excel), with red being the most intense and blue being the least intense. nanoparticle tracking analysis evs, diluted in pbs, were analyzed by nanoparticle tracking using the nanosight ns300 system (malvern instruments, malvern, uk) equipped with 405 nm laser. videos were collected and analyzed using the nta software (version 3.0 0060). mirna isolation and profile a 36 ml urine sample from a single volunteer was used. the sample was split into two equal halves and evs were isolated by the ymir ev kit or differential uc. mirna was isolated from urinary evs with mirvana paris kit (life technologies). mirna quality and concentration were determined using agilent 2100 bioanalyzer (agilent technologies, santa clara, california, usa) and small rna kit (agilent technologies, santa clara, california, usa). the mirna profile was examined with an abcam circulating mirna oncology fixed panel version 1.0 (abcam, cambridge, massachusetts, usa) and analyzed with abcam analysis workbench. quantitative reverse transcription polymerase chain reaction complementary dna (cdna) was synthesized from urinary exosomal rna using the taqman microrna rt kit (life technologies) according to the manufacturer’s instructions. quantitative pcr for mature mirnas was performed using taqman mirna gene assays (life technologies) and gene expression master mix (life technologies). primers for hsa-mir-10b, hsa-mir-223, and hsa-mir200c were obtained from life technologies. transmission electron microscopy. cryogenic electron microscopy was performed at the university of pennsylvania, school of medicine’s electron microscopy resource figure 1. outline of ymir genomics’ ev isolation kit protocol. ev: extracellular vesicle. 2 journal of circulating biomarkers laboratory. briefly, 3–5 ml of ev preparations from a singlepooled sample (three individuals contributing 1 ml each) were adsorbed to a holey carbon transmission electron microscopic (tem) grid, blotted with filter paper, and plunged into liquid ethane cooled to �180�c. samples were held at cryogenic temperature in a gatan 626 (gatan inc, pleasanton, california, usa) holder while observed at 200 kev on an fei tecnai f20 microscope (gatan inc, pleasanton, california, usa). images were digitally collected on a 2k�2k gatan orius camera at the indicated magnification for each image (gatan inc, pleasanton, california, usa). cell survival assay. evs were isolated from urine by uc and ymir method. relative concentration of evs was determined by the method of bradford (biorad, hercules, california, usa). hela cells in complete media (dulbecco’s modified eagle medium (dmem), gibco, carlsbad, california, usa; 5% fetal bovine serum (fbs), gibco, carlsbad, california, usa) were treated with 10 mg of exosomes, phosphate buffered saline (pbs) (gibco, carlsbad, california, usa; negative control), or ph9.0 alkaline buffer (positive control), and then incubated at 37�c with 5% carbon dioxide for 18 h. cell survival determined by alamar blue (biorad, hercules, california, usa) assay was carried out following the manufacturers’ instructions. each treatment was performed in triplicate. results the ymir genomics ev isolation method has a simple and rapid protocol (figure 1).to determine its efficacy, a healthy volunteers sample was divided into two and processed by the ymir genomics protocol and by a standard uc protocol. 6 the resulting preparations were analyzed by tem and nanosight particle analysis. 7 the size, shape, and number of particles observed in each preparation were similar (figure 2). to determine whether the protocol isolates vesicle protein markers, multiple first-void urine samples of varying figure 2. a comparison of urine extracellular vesicle size, number, and shape between uc and the ymir genomics’ ev isolation kit. top: urine vesicle size and shape as measured by tem. top left (ymir): electron micrograph of urine vesicles isolated with the ymir genomics’ extracellular vesicle kit. top right (uc): electron micrograph of urine vesicles isolated by differential uc. bar represents 1 mm. bottom: nanoparticle tracking analysis using a nanosight device of urine vesicles isolated by the ymir genomics’ extracellular vesicle kit (ymir) and differential uc. uc: ultracentrifugation; ev: extracellular vesicle; tem: transmission electron microscopy. markowska et al. 3 protein concentrations from different donors were divided into 5 � 1 ml samples and subjected to ev isolation via standard uc and four commercial kits from life technologies, exiqon, sbi, and ymir genomics. the preparations were subjected to immunoblot analysis using antibodies specific for the known vesicle marker cd63, hsp70, 8 exosomal/endosomal markers such as rab5 and cd9, 9 and a urine-specific vesicle marker aquaporin 2. 6 as figure 3(a) shows for the more concentrated sample, all the methods isolate detectable amounts of hsp70, cd63, rab5, and aquaporin 2, although the ymir genomics method isolates significantly more of the aquaporin 2 marker. however, for the more diluted sample, only the ymir genomics method isolates all three of these markers. a repeat of this experiment with 10 different samples with a range of protein concentrations yielded similar results (figure 3(b); supplemental figure s1), suggesting the ymir genomics method is the best method for a variety of samples. to probe the concentration lower limit of this method, we took a separate sample and diluted it 2�, 4�, and 8� with pbs and then applied various vesicle isolation methods followed by immunoblot for aq2, cd9, and rab5. once again, the ymir genomics’ kit is the only method that isolates all three of these vesicle markers from the diluted sample (figure 4). strikingly, this method efficiently isolates all three markers even from the same sample diluted 4� while neither of the other commercial kits function at the 2� dilution. even uc does not efficiently isolate these markers at the 4� dilution. ymir genomics also makes a urine extracellular rna extraction kit that is based on the same precipitation figure 4. a comparison of ev protein markers over the course of multiple dilutions. a single relatively high concentration sample (above 0.1 mg/ml as judged by bradford assay) was sequentially diluted with pbs. each diluted sample was split into four equal parts and subjected to the indicated ev isolation method followed by immunoblotting with three markers for urine evs. ev: extracellular vesicle. figure 3. a comparison of yield for ev protein markers. (a) a composite immunoblot using antibodies specific for five published markers for urine evs is shown. two 9 ml urine samples, namely, one displaying a relatively high protein concentration (above 0.1 mg/ml as judged by bradford assay) and another displaying a relatively low protein concentration (below 0.05 mg/ml as judged by bradford assay) were split into three equal parts and processed via differential uc, the sbi exosome isolation kit, and the ymir genomics’ ev isolation kit. these data are representative of three similar experiments with different samples. (b) a heat map representing the yield of three published markers is shown for urine evs from 10 separate urine samples processed by the ymir genomics’ method, exiqon’s mircury exosome isolation kit, and the sbi exosome isolation kit. red/orange denotes strong signal, yellow denotes weak signal, and blue denotes little or no signal. ev: extracellular vesicle; uc: ultracentrifugation. 4 journal of circulating biomarkers reagent. bioanalyzer analysis of rna produced from a single sample by this protocol or uc followed by rneasy rna purification shows a similar yield and a similar size profile (figure 5(a)). interestingly, when quantitative reverse transcription pcr analysis was used to look for three mirnas known to be in the extracellular fraction of urine, then the yield was significantly higher using the ymir genomics’ kit compared to uc (figure 5(b)). to expand on this result, similar preparations were applied to the abcam oncology microrna array that simultaneously assesses the relative amounts of 69 micrornas implicated cancer. 10 figure 5(c) shows the raw data from that array. in general, both methods agree as to which mirnas are present in the sample. however, the ymir genomics preparation consistently yields a stronger signal for a majority of the mirnas detected (figure 5(c)). in all, 27 of the detected mirnas show a significantly stronger signal from the ymir genomics preparation compared to 3 having a stronger signal from uc and 5 being within error bars. these results strongly suggest that the ymir genomics preparation is more efficient at isolating extracellular mirnas compared to uc. given that the ymir genomics’ method uses a novel small molecule to induce precipitation, we wanted to make sure that the ymir genomics’ preparations would not be toxic to cells. to test this, we isolated evs from a single-pooled urine sample using the ymir method and differential uc and then added 10 mg of each preparation to hela cells in standard media for 18 h figure 5. a comparison of rna isolated from urine ev preparations. a single urine sample was divided into two equal portions and processed by differential uc and the ymir ev isolation kit (y or ymir). (a) bioanalyzer analysis of both preparations. (b) rt-pcr analysis of mirnas such as 223, 200c, and 10b. (c) firefly mirna array analysis of mirnas found in urine ev preparation using differential uc and ymir ev isolation kit (ymir). un-normalized arbitrary units describe relative amounts of each of the 69 mirnas. note: the values for mir-16 (au ¼ 4621.9 + 401.9), mir-26a (2887.0 + 388.8), and mir-223 (7769.6 + 633.0) were cutoff for the ymir genomics’ preparation for aesthetic reasons. ev: extracellular vesicle; uc: ultracentrifugation; rt-pcr: reverse transcription polymerase chain reaction. markowska et al. 5 followed by bromodeoxyuridine viability assay. eighteen hours is more than a typical time frame and 10 mg is a typical amount for ev delivery studies. 11 figure 6 shows that the ev preparation from ymir genomics does not adversely affect these cells compared to uc and the negative control pbs while the positive control basic wash does. discussion despite its easy, noninvasive collection, relative sterility, and richness in a variety of biomolecules, currently, only about 3% of discovery research for extracellular biofluid biomarkers is performed in urine. 12 one reason for this is that the concentration of biomarkers is significantly less in urine when compared to blood. for instance, evs are approximately 1000� more concentrated in blood than in urine. 13 this makes the isolation of evs more challenging, particularly when using precipitation methods. urine is also a more variable medium than blood in terms of ph and osmolality. unlike blood which is almost always ph 7, urine varies daily from ph 5 to ph 8. depending on what an individual eats and drinks and alcohol or drugs taken, the osmolality, protein, and creatinine levels can also vary widely. these challenges lead to significant sample-tosample variability in the efficiency of extracellular biomolecule isolation. unlike other commercial kits, the ymir genomics’ urine ev and rna isolation kits use a sample treatment buffer to ameliorate initial sample ph differences. furthermore, the method uses a novel precipitation reagent that is less dependent on concentration (as judged by protein; figure 5). interestingly, consistent with a recent report 14 that looked at density, we see that uc is not as effective for less concentrated samples. only the ymir genomics method worked on diluted samples. the method is the most rapid of the methods attempted (30 min for the ev preparation) and does not require special equipment. furthermore, ev preparations from the method are not toxic to cells (figure 6). thus, the method should make biomarker isolation from urine more accessible to research and clinical laboratories. acknowledgments we thank dewight williams of biomedical research core facilities at the perelman school of medicine at the university of pennsylvania for his technical assistance with the tem. we thank kevin riley of malvern for technical assistance with the nanosight particle analysis. we thank tom volpe for important discussions. declaration of conflicting interests the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: the authors are all employees of ymir genomics llc and benefit from the sale of the ymir genomics urine ev isolation kit. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was funded by ymir genomics llc. supplemental material the online data supplements/etc. are available at http://journals. sagepub.com/doi/suppl/10.1177/1849454417712666. references 1. hyun ka, kim j, gwak h, et al. isolation and enrichment of circulating biomarkers for cancer screening, detection, and diagnostics. analyst 2016; 141: 382–392. 2. system biosciences inc. https://www.systembio.com/ m i c r o r n a r e s e a r c h / e x o q u i c k e x o s o m e s / o v e r v i e w ? gclid¼cmm_gz-7qsocfyqxhwodv_oknq (cat no. exo tc 10a-1, accessed 2016). 3. exiqon inc. http://www.exiqon.com/exosome-isolationkits?gclid¼co6unfs7qsocfyerhwodymqhpg (cat no. 300102, accessed 2016). 4. thermofisher scientific. total exosome isolation reagent (from urine). https://www.thermofisher.com/order/catalog/ product/4484452 (cat no. 4484452, accessed 2016). 5. ymir genomics llc. https://www.ymirgenomics.com/ (cat no. y001, accessed 2016). 6. pisitkun t, shen rf and knepper ma. identification and proteomic profiling of exosomes in human urine. proc natl acad sci usa 2014; 101: 13368–13373. 7. varga z, yuana y, grootemat ae, et al. towards traceable size determination of extracellular vesicles. j extracell vesicles 2014; 3: 23298–23305. 8. mathew a, bell a and johnstone rm. hsp-70 is closely associated with the transferrin receptor in exosomes of maturing reticulocytes. biochem j 1995; 308: 823–830. 9. chaput n and thery c. exosomes: immune properties and potential clinical implementations. semin immunopathol 2011; 33(5): 419–440. 10. abcam multiplex circulating mirna array. http://www.abcam. com/nav/multiplex-mirna-assays/circulating (accessed 2015). figure 6. ymir genomics’ ev preparations are not toxic to cells. a single-pooled urine sample was divided into two equal portions and processed by differential uc and the ymir ev isolation kit (ymir). a bradford protein assay determined yield. ten micrograms of each preparation were applied to hela cells. after 18 h, the cell survival was determined by alamar blue assay. ev: extracellular vesicle; uc: ultracentrifugation. 6 journal of circulating biomarkers http://journals.sagepub.com/doi/suppl/10.1177/1849454417712666 http://journals.sagepub.com/doi/suppl/10.1177/1849454417712666 https://www.systembio.com/microrna-research/exoquick-exosomes/overview?gclid=cmm_gz-7qsocfyqxhwodv_oknq https://www.systembio.com/microrna-research/exoquick-exosomes/overview?gclid=cmm_gz-7qsocfyqxhwodv_oknq https://www.systembio.com/microrna-research/exoquick-exosomes/overview?gclid=cmm_gz-7qsocfyqxhwodv_oknq https://www.systembio.com/microrna-research/exoquick-exosomes/overview?gclid=cmm_gz-7qsocfyqxhwodv_oknq http://www.exiqon.com/exosome-isolation-kits?gclid=co6unfs7qsocfyerhwodymqhpg http://www.exiqon.com/exosome-isolation-kits?gclid=co6unfs7qsocfyerhwodymqhpg http://www.exiqon.com/exosome-isolation-kits?gclid=co6unfs7qsocfyerhwodymqhpg https://www.thermofisher.com/order/catalog/product/4484452 https://www.thermofisher.com/order/catalog/product/4484452 https://www.ymirgenomics.com/ http://www.abcam.com/nav/multiplex-mirna-assays/circulating http://www.abcam.com/nav/multiplex-mirna-assays/circulating 11. lasser c, alikhani vs, ekstrom k, et al. human saliva, plasma, and breast milk exosomes contain rna: uptake by macrophages. j transl med 2011; 9: 9. 12. razvi, e. market report “trends and segments in the exosome marketplace” selectbio. 2014. enal@selectbio.us. 13. li m, zeringer e, barta t, et al. analysis of the rna content of the exosomes derived from blood serum and urine and its potential as biomarkers. philos trans r soc lond b biol sci 2014; 369: 20130502 14. royo f, zuniga-garcia p, sanchez-mosquera p, et al. different ev enrichment methods suitable for clinical settings yield different subpopulations of urinary extracellular vesicles from human samples. j extracell vesicles 2016; 5: 29497. markowska et al. 7 http://enal@selectbio.us << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false 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laghezza masci 1 , anna rita taddei 2 , gabriella gambellini 2 , franco giorgi 3 , and anna maria fausto 4 abstract this study shows that fibroblasts migrating into a collagen matrix release numerous microvesicles into the surrounding medium. by spreading in regions of the matrix far distant from cells of origin, microvesicles carry metalloproteinase 9 (mmp-9) to act upon the collagen fibrils. as a result, the collagen matrix is gradually transformed from a laminar to a fibrillar type of architecture. as shown by western blots and gelatin zymography, mmp-9 is secreted as a 92 kda precursor and activated upon release of 82 kda product into the culture medium. activation is more efficient under threedimensional than in two-dimensional culturing conditions. while mmp-9 labeling is associated with intraluminal vesicles clustered inside the microvesicles, the microvesicle’s integrin b1 marker is bound to the outer membrane. the intraluminal vesicles are recruited from the cortical cytoplasm and eventually released following uploading inside the microvesicle. here, we propose that fusion of the intraluminal vesicles with the outer microvesicle’s membrane could work as a mechanism controlling the extent to which mmp-9 is first activated and then released extracellularly. keywords microvesicles, exosome, ectosome, metalloproteinase, collagen matrix date received: 21 october 2015; accepted: 30 may 2016 introduction cell communication plays a key role in multicellular organisms. 1 cells communicate by either ligand–receptor interaction, surface recognition of specific cell adhesion molecules, or transfer of cytoplasmic components through junctional coupling. 2 in recent years, it has become apparent that cells may also interact through the release of extracellular vesicles. originally regarded as cellular debris with no apparent function, 3 microvesicles are now recognized as specific structures released by cells under a variety of physiological and pathological conditions. 4–8 they may either originate as exosomes upon fusion of the endosomal compartment with the plasma membrane or be directly released from the cell membrane in the form of shedding vesicles. 9,10 the role played by microvesicles in cell communication is largely dependent upon the molecular cargo they carry as well as on the mechanism(s) that target them to other cells. while cell destination is defined by the type of ligands or receptor(s) expressed on their limiting membrane, the variety of molecules—enzymes, messenger rna, microrna, or rna interference—carried by microvesicles will ultimately affect the developmental fate of the target cells. 11–13 1 department of innovation in biological, agrifood and forestry systems (dibaf), tuscia university, viterbo, italy 2 section of electron microscopy, great equipment center, tuscia university, viterbo, italy 3 university of pisa, pisa, italy 4 department of innovation in biological, agrifood and forestry systems, tuscia university, viterbo, italy corresponding author: valentina laghezza masci, universita degli studi della tuscia, viterbo 01100, italy. email: biovalentina@gmail.com journal of circulating biomarkers volume 5: 1–11 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416663660 cbx.sagepub.com creative commons cc-by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 3.0 license (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:biovalentina@gmail.com http://www.sagepub.co.uk/journalspermissions.nav https://doi.org/10.1177/1849454416663660 http://cbx.sagepub.com https://us.sagepub.com/en-us/nam/open-access-at-sage because of this capacity, microvesicles are involved in many pathologies, including cancer metastasis, besides having a beneficial role in a variety of cell responses under physiological conditions. 14–17 microvesicles are also involved in every step of the wound healing process, from blood clotting to neogenesis of the scar tissue. interestingly, in each of these circumstances, the proteolytic activity of the extracellular matrix has been shown to be associated with the release of microvesicles. through this mechanism, various cell types may contribute to generate an appropriate microenvironment for tissue repair. 5,18 the primary objective of wound healing is to reestablish the integrity of the injured tissues. 19 to this end, a variety of skin cells, including keratinocytes and dermal fibroblasts, is expected to migrate from the wound edges and to invade the underlying connective tissue. 20 the mechanism by which migrating cells interact with the extracellular matrix and eventually regulate collagen metabolism during tissue formation has been the subject of extensive investigations in recent years. 21–23 it is generally recognized that wound healing entails degradation of the damaged matrix through recruitment of inflammatory cells, followed by deposition of new collagen fibers by migrating fibroblasts. 20,24 a proper balance between degradation and synthesis of collagen is thus essential for a new granulation tissue to be formed on the wound bed and to be later transformed into a mature scar. 25–27 during this transition a number of specific growth factors are known to stimulate fibroblasts to synthesize and release a number of matrix metalloproteinases (mmps). 28–30 by acting upon the damaged extracellular matrix, these enzymes are known to degrade collagen and to allow nearby cells to take up the resulting peptides. 31,32 mmps constitute a class of collagen-degrading enzymes known to play key roles in many physiological and pathological tissue remodeling processes, including embryogenesis and cancer, besides wound repair. 33 expression of mmps can be regulated by either gene transcription, 34 pro-enzyme activation of the catalytic domain, 35 or inhibition through specific tissue matrix proteinases. 36 however, metalloproteinase activity may also be controlled through selective anchoring on specific membrane domains 37 or by confinement into membrane-enclosed compartments. 38 in each case, enzyme activation is delayed until release or detachment from the membrane compartment. in this study, we show that fibroblasts migrating in vitro into an accessible collagen matrix release a number of microvesicles into the culture medium. these vesicles act as carriers conveying mmp-9 to nearby collagen fibers. the primary objective of this study is thus to verify whether the release of microvesicles can actually account for (i) the diffusion of pro-metalloproteinases through the collagen matrix; (ii) the activation of metalloproteinase upon interaction with collagen fibers and eventually; and (iii) the extracellular processing of prosthetic collagen prior to cell uptake and intracellular digestion by fibroblasts. materials and methods cell culturing stabilized murine fibroblasts (nih 3t3) were used throughout this study. to collect enough cells for plating on collagen matrices, nih 3t3 fibroblasts were first cultured in 75 cm 2 flasks containing dulbecco’s modified eagle’s medium supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin/streptomycin, and maintained in a humidified incubator at 37�c supplied with a 5% carbon dioxide (co2) atmosphere. upon reaching confluence, cells were passed and the medium changed every 3 days. three-dimensional (3-d) collagen scaffolds were cut into blocks of approximately 1 cm 2 of surface area with a sterile razor blade and each block inserted into 24-well plates. aliquots of 2 � 105 cells were seeded on the upper surface of every collagen block and incubated at 37�c in 1.5 ml of culture medium under a continuous flow of 5% co2 for time intervals ranging from 4 h to 14 days. medium was then renewed every 3 days. scanning electron microscopy collagen scaffolds inclusive of their cellular loads were cut into a number of small blocks with a sterile razor blade and fixed overnight at 4�c with 3% glutaraldehyde in 0.05 m phosphate buffer (pb) at ph 7.2. following extensive rinsing with the same buffer at 4�c, blocks were soaked for 1 h in 0.5% tannic acid in pb at 4�c. then they were rinsed four times in the same buffer for 15 min at 4�c, and postfixed with 1% osmium tetroxide in pb for 1 h at 4�c. samples were washed in distilled water, dehydrated in a graded series of ethanol, and freeze-dried in a balzers union cpd 020 (balzers, liechtenstein) using the procedure of critical point drying. samples were eventually sputter coated with gold in a balzers med 010 unit and observed in a jeol jsm 6010la electron microscope (japan). transmission electron microscopy samples were cut into a number of small blocks with a sterile razor blade and fixed as described above. following postfixation in 1% osmium tetroxide, samples were washed in distilled water, block stained with 1% uranyl acetate in distilled water, and then dehydrated in a graded series of ethanol. subsequently, they were infiltrated for 2 days in graded mixtures of lr white resin/ethanol (london resin company, berkshire, england). by the end of this procedure, samples were embedded in fresh lr white resin and cut with a reichert ultracut ultramicrotome (depew, new york, usa) equipped with a diamond knife. ultrathin sections (60–80 nm thick) were collected on copper grids, stained with uranyl acetate and lead citrate, and observed in a jeol 1200 ex ii electron microscope. micrographs were acquired using an olympus sis veleta ccd camera (japan) equipped with an the tem imaging platform (item) software version, item 5.1 (build 1700). 2 journal of circulating biomarkers immunohistochemistry immunohistochemistry was performed using the abcperoxidase reaction followed by nickel enhancement. 39 collagen and 3t3 samples were first fixed in 4% paraformaldehyde in 0.05 m phosphate buffer saline (pbs) at 4�c and then embedded in paraffin wax. serial sections of 5–7 mm were incubated for 18 h at 4�c with a rabbit polyclonal antiserum anti-mmp-9 (ab 38898, abcam, uk) diluted 1:500. negative controls were run by omitting the primary antibody. subsequently, sections were incubated for 60 min with a biotinylated goat anti-rabbit immunoglobulin g (igg) serum (vector labs, burlingame, california, usa), diluted 1:1000 with pbs containing 0.1% sodium azide and 1% bovine serum albumin (bsa), followed by 60 min incubation in the avidin–biotin–peroxidase complex (abc elite vectastain, vector laboratories). following extensive rinsing in distilled water, sections were dehydrated, mounted, and examined with a zeiss light microscope (germany) under a bright-field illumination. images were acquired with a color camera (axio cam mrc, arese, milan, italy) equipped with an axiovision-ks 300 software version, axiovision 40v 4.1.1.0. immune gold labeling samples were fixed for 2 h at room temperature with a mixture of 4% paraformaldehyde and 0.25% glutaraldehyde in 0.05 m piperazine-n, n0-bis-2-ethanesulfonic acid at ph 7.3. after rinsing for 10 min in the same buffer, samples were dehydrated in a graded series of ethanol and embedded in medium grade lr white resin, following addition of the lr white accelerator. the resin was then polymerized for 20 min at room temperature in tightly capped gelatin capsules. ultrathin sections were obtained with a reichert ultracut ultramicrotome equipped with a diamond knife and collected on nickel grids. for immune gold labeling, 60–80 nm-thick sections were processed following the incubation protocol originally developed by aurion (vageningen, the netherlands). unspecific binding of antibody was prevented by treating sections for 20 min with 0.05 m glycine in pbs, at ph 7.4, followed by a 30 min blocking step in 5% bsa, 5% goat serum, and 0.1% fish gelatin in cold distilled water. sections were subsequently washed in an incubation buffer containing 0.1% aurion bsa-c™ in pbs and incubated overnight in a moist chamber in the presence of one of the following antibodies: anti-mmp-9 (ab38898 abcam) polyclonal rabbit antibody, anti-integrin b1 (ts2/16; ab119333 abcam) mouse monoclonal antibody or anti-type i collagen (ab34710, abcam) rabbit polyclonal antibody. antibodies were diluted 1:100 (if polyclonal) or 1:10 (if monoclonal) in the incubation buffer. following this labeling step, sections were thoroughly washed in incubation buffer (6 � 5 min) and incubated for an additional hour in a secondary antibody. mmp-9 and type i collagen were revealed with a goat anti-rabbit antibody conjugated to 10 nm gold particles (british biocell international, uk), whereas integrin b1 was revealed with a goat anti-mouse antibody conjugated to 20 nm gold particles (british biocell international), diluted 1:10 in incubation buffer. after rinsing in incubation buffer (6� 5 min), grids were washed in pbs (6 � 5 min). sections were hence stained with uranyl acetate and observed in a jeol jem exii transmission electron microscope (tem) set at 100 kv. micrographs were acquired by means of an olympus sis veleta ccd camera equipped with the item software. control samples were obtained by omitting the primary antibody from the labeling mixture. double immune labeling was carried out by first exposing sections to the primary antibody—either rabbit polyclonal or mouse monoclonal antibody—and subsequently to their respective goat secondary antibodies, sequentially applied for each of the antigen to be revealed. negative staining droplets of sample suspensions (10 ml) from either 2-d or 3-d culture media were placed on formvar–carbon-coated grids and allowed to be adsorbed for 60 s. excess liquid was gently removed from each droplet with a strip of filter paper. processing for negative staining was carried out by placing a drop of negative stain (2% uranyl acetate in distilled water) on the specimen grids, followed by quick blotting. this step was repeated a second time on a new drop of negative staining solution and prolonged for 60 s. samples were eventually observed at a jeol 1200 ex ii electron microscope and micrographs acquired by the olympus sis veleta ccd camera equipped with the item software. western blot by the end of every incubation period (1, 3, or 7 days from the time of seeding) samples consisting of either fibroblasts (2-d: 3t3) or collagen blocks containing migrating fibroblasts (3-d: coll3t3) were washed twice in sterile pbs. both samples were then frozen in liquid nitrogen for 2 min, pulverized, and incubated for 30 min at room temperature in ripa buffer (sigma-aldrich, st louis, missouri, usa) with protease inhibitors added. the protein concentration was determined in each extract using the bradford microassay method (sigma-aldrich) with bsa as a standard. samples were loaded on a 10% sodium dodecyl sulfate (sds)-polyacrylamide gel in the concentration of 50 mg of proteins per well and resolved by electrophoresis at 20 ma constant current until the dye front reaches the bottom line. to detect antibody-binding proteins, samples were transferred onto nitrocellulose membranes (millipore, burlington, massachusetts, usa) using a wet transfer system (bio-rad, hercules, california, usa). these membranes were then blocked in tris-buffer saline with 0.01% tween-20, ph 7.4 with 5% bsa (tbst) for 1 h at room temperature, washed, and incubated overnight at laghezza masci et al. 3 4�c with a rabbit polyclonal antiserum anti-mmp-9 (ab 38898, abcam) diluted 1:1000. nitrocellulose membranes were washed and incubated for 4 h with a horseradish peroxidase-conjugated goat anti-rabbit igg secondary antibody diluted 1:1000 with tbst. western blots (wb) were developed using the ecl chemiluminescence detection system (chemidoc, bio-rad). gelatin zymography to detect the mmp activity in 3-d (collþ3t3) or 2-d (3t3) culture samples, the protocol of toth and fridman 40 for gelatin zymography was routinely used. sample solutions were processed under native conditions in the absence of reducing agents and without boiling. they were then electrophoresed in a 10% sds-polyacrylamide gel containing 0.1 mg/ml of porcine gelatin (sigma-aldrich) and subsequently incubated for 30 min at room temperature in a 2.5% (v/v) triton x-100 solution to remove sds from the resolved polypeptides. following a thorough rinse in double distilled water for enzyme renaturation, gels were incubated at 37�c for 20 h in developing buffer (50 mm tris/ hydrochloric acid buffer, ph 7.8 containing 20 mm sodium chloride, 5 mm calcium chloride, and 0.02% brij-35), stained with 0.1% coomassie brilliant blue r-250 and eventually destained in 10% acetic acid and 30% methanol. gelatinolytic activity was detected as unstained bands on a blue background and revealed with the chemidoc instrument (bio-rad). acquired pictures were quantitatively evaluated using the imagej program version, imagej 1.48v – wayne rasband, national institute of health, usa. results fibroblasts cocultured under 3-d conditions until 7 days, that is, in the presence of a prosthetic collagen scaffold, can be shown to migrate rapidly inside the matrix and take close contact with the collagen fibers. 41 figure 1(a) to (c) depicts an initial condition (1 day) of this in vitro culture. at this stage, roundish fibroblasts appear firmly attached onto a collagen matrix, still characterized by a prevailing laminar structure. by the end of this culture period (7 days), cells had already become highly intermingled with collagen fibers, the matrix itself having lost most its laminar structure and acquired, in the meantime, a rather fibrillar appearance (figure 1(d)). the collagen fibers themselves are no longer aligned in continuous laminar sheets, but are rather scattered in the form of a fragmented network with no recognizable orientational order (figure 1(e)). to account for the laminar-to-fibrillar transition of the prosthetic collagen, two aspects of the in vitro culturing setup were further examined: (i) migrating fibroblasts were analyzed for their capacity to release metalloproteinases in the surrounding medium and, (ii) the enzymes released by fibroblasts were visualized in their structural relationship with collagen processing. of the numerous metalloproteinases identified so far, we chose to study the mmp-9 because it is already known to be constitutively expressed in many cell types, including fibroblasts, besides being endowed with the capacity to remodel the extracellular matrix and degrading a broad variety of collagen types. 35 figure 2 is a gelatin zymography of fibroblasts cocultured in a 3-d collagen matrix showing the presence of two major protein fractions. these are identifiable as bona fide pro-mmp-9 and active mmp-9 on the basis of their respective molecular weights and antibody reactivity, both data being highly indicative of a precursor–product relationship. 42 when detected by wb analysis (see figure 2, western blot (wb) line), the antimmp-9 antibody appears to bind preferentially to the 92 kd fraction, suggesting that the antibody may be more specific for the mmp-9 precursor epitope(s). however, only the lower molecular weight fraction should be expected to be enzymatically active by zymography. indeed, activation of the pro-mmp-9 is thought to result from an artificial enzyme exposure to the denaturing conditions of the gel electrophoresis. 43 results from gelatin zymography show that the relative concentration of the active mmp-9 form increases from 1 to 7 days of in vitro culture, while the pro-mmp-9 is gradually declining during the same time period. this temporal correlation is interpreted as indicating that coculture with the collagen matrix is a condition necessary, though not sufficient, for the mmp-9 to be released from the fibroblasts and figure 1. sem and tem micrographs showing how the collagen matrix is structurally modified in the presence of migrating fibroblasts. figures (a), (b), and (c) show the collagen matrix at day 1 following cell seeding. notice the laminar structural appearance of the collagen matrix (a) and the highly intertwined connections with fibroblasts’ filopodia (b, c). bars: (a) 10 mm; (b) 5 mm; (c) 1 mm. figures (d) and (e) depict fibroblasts after 7 days of in vitro culture. notice that the collagen matrix has acquired a prevailing fibrillar organization. bars: (d) 5 mm; (e) 2 mm. sem: scanning electron microscopic; tem: transmission electron microscopy. 4 journal of circulating biomarkers subsequently activated in the extracellular milieu. by contrast, fibroblasts cultured under 2-d conditions, that is, in the absence of any collagen interference, appear to have a much lower mmp-9 activity and a higher enzyme turn over, both mmp fractions having already disappeared following 3 days of in vitro culture (see right panel in figure 2). mmp-9 expression, as detected by gelatin zymography, was also extended to the analysis of culture media under 2-d and 3d conditions. interestingly, mmp-9 expression in 3-d culture media follows a kinetics similar to that already recorded for cell lysates with the proand active enzymes being temporally correlated in a precursor–product relationship. by comparison, mmp-9 expression in 2-d culture media was restricted to the sole 82 kda active form (figure 3). these observations are highly suggestive of the possibility that in vitro cultured fibroblasts may actually release ‘‘something’’ in the culture medium that is potentially capable of acting as a carrier for conveying the mmp-9 activity onto the collagen matrix. to verify whether that something spreading the enzyme activity in the culture medium could be accounted for by the release of microvesicles from fibroblasts, extracts from all culture media were routinely checked by negative staining. results show that vesicles recovered from these extracts fall within the size range of 30–300 nm (see figure 4), suggesting that these samples may be comprised of both intact microvesicles and their relative intraluminal vesicles figure 2. immune detection of pro-mmp-9 and relative enzyme activity in cell lysates following in vitro culture in the presence of a collagen matrix 3-d (collþ3t3) or in 2-d culture medium alone (3t3) for 1, 3, or 7 days. gz; wb with anti-mmp-9 antibody. zymograms were scanned with densitometer equipped with imagej program. mmp: matrix metalloproteinase; gz: gelatin zymography; wb: western blot. figure 3. immune detection of mmp-9 enzyme activity in the culture medium extracted from the 3-d collagen matrix (collþ3t3)) or following in vitro 2-d cell culturing (3t3) for 1, 3, or 7 days. gz; wb with anti-mmp-9 antibody reacting exclusively with the pro-active enzyme form. zymograms were scanned with densitometer equipped with imagej program. mmp: matrix metalloproteinase; gz: gelatin zymography; wb: western blot. laghezza masci et al. 5 that are likely to be released upon shedding from the fibroblasts or during extraction from the culture medium. an extensive ultrastructural analysis of 2-d or 3-d cultured fibroblasts has shown that microvesicles are composite organelles containing numerous smaller intraluminal vesicles ranging in size from 30 nm to 200 nm (figure 5(a)). to verify whether mmp-9 activity and the release of microvesicles are indeed causally related events, fibroblasts from both in vitro culturing conditions were examined by immune anti-mmp-9 labeling. the low magnification micrograph depicted in figure 5(b) shows that under these experimental conditions many of the microvesicles dispersed along the fibroblast cell periphery become heavily labeled with gold-tagged anti-mmp-9 antibodies. interestingly, anti-mmp-9 labeling is not associated with the entire microvesicle, but is instead restricted to some, if not all, of the intraluminal vesicles present inside it. the micrographs shown in figure 5(c) to (e) were selected to depict a hypothetical sequence of mmp-9 release through microvesicle shedding from the fibroblast cell surface. intraluminal vesicles are likely to be uploaded in the microvesicles following transfer from the cortical cytoplasm and docking to the cell plasma membrane (figure 5(c) and (d)). mmp-9 activity may be eventually released in the extracellular space through fusion of the intraluminal vesicles with the outer microvesicle membrane (figure 5(e)). to identify the fibroblast microvesicles described in this study as real intercellular organelles transferring mmp-9 in membrane-bound intraluminal vesicles, some specific cell markers had to be used. integrin b1 is a cell adhesion molecule recognized by many investigators as a reliable microvesicle marker for a variety of cell types. 44 figure 4. negative staining of representative samples taken from culture media under 2-d (a) or 3-d conditions; (b) showing the vesicular composition of the relative extracts. the size range of the vesicles detected through this technique is 30–300 nm. bars: 500 nm. figure 5. immune gold labeling of anti-mmp-9. (a) high-resolution tem micrograph showing a microvesicle continuous with the cell membrane loaded with numerous intraluminal vesicles, bar 500 nm; (b), low magnification micrograph of a 3-d cultured fibroblast showing numerous microvesicles heavily labeled with anti-mmp-9 gold-tagged antibody along the entire cell periphery, bar 1 mm; (c) to (e), a hypothetical sequence of an mmp-9 activity uploaded via intraluminal vesicles from the cortical cytoplasm and eventually released from a shed microvesicle. bars 500 nm. mmp: matrix metalloproteinase; tem: transmission electron microscopic. 6 journal of circulating biomarkers accordingly, 10 nm gold-tagged anti-mmp-9 antibodies were used to detect the mmp-9 activity as the intraluminal microvesicle content, while 20 nm gold-tagged antiintegrin b1 antibodies were used to identify the enclosing microvesicle as the respective enzyme container. as shown in figure 6(a), anti-integrin antibodies bind the microvesicle’s outer membrane, while anti-mmp-9 antibodies are restricted to the vesicle’s inner space. since the prolonged treatment required for double immune labeling is known to affect the binding efficiency of gold-tagged antibodies, 45 mmp-9 and integrin b1 were also colocalized on alternate thin sections. very briefly, this consisted in identifying the same microvesicle in subsequent grids and exposing it to different gold-tagged antibodies, followed by merging of the resulting images (figures 6(b) to (d)). vesicular shedding was also shown to occur in 2-d cultures; fibroblasts proved capable of synthesizing and releasing mmp-9 even in the absence of the collagen matrix. however, the extent to which microvesicles are released into the surrounding milieu under these conditions is by far much less pronounced than in 3-d cultures. a detailed analysis of 3-d cell cultures from 1 to 7 days has clearly shown that microvesicles can gain access to regions of the collagen matrix far away from their cells of origin. it is not clear whether microvesicles actually move away from their budding site or simply remain anchored to the collagen fibrils, while cells migrate along the collagen fibers. whatever mechanism(s) sustains their migration, microvesicles occur either as isolated clusters or in association with numerous frayed collagen fibrils (figure 7(a) and (b)). under both circumstances, microvesicles appear still loaded with a mmp-9 cargo, as shown by their binding capacity to mmp-9 antibody (figure 7(c)). however, when observed in close contact with collagen, gold label appears to spread onto frayed fibrils (figure 7(d)), indicating that the mmp-9 cargo has been released from microvesicles and dispersed in the surrounding extracellular environment. that the amorphous or fuzzy material contacting microvesicles should actually be identified as collagen, in spite of the difficulty of recognizing it in the absence of a native banding pattern, it is clearly demonstrated by its reactivity to the anticollagen antibody (see figure 7(e)). the ultimate result of mmp-9 release from microvesicles is the spread of the enzyme throughout the entire collagen matrix. as clearly indicated by the sequence of images depicted in figure 8(a) to (c), labeling with antimmp-9 antibody becomes progressively more pronounced figure 6. in vitro cultured fibroblasts subjected to double immune labeling with 10 nm gold-tagged anti-mmp-9 antibodies and 20 nm gold-tagged anti-integrin b1 antibodies. (a) microvesicles shed along the fibroblast cell periphery are internally labeled with 10 nm gold particles (arrows) and on the outer membrane with 20 nm gold particles (double arrows). bar 1 mm; (b) microvesicle labeled with antiintegrin b1 antibodies along the outer membrane; (c) the same vesicle as in (b) labeled with anti-mmp-9 antibodies; and (d), merging of (b) and (c) micrographs obtained by overlapping 50% transparent images. bars: 250 nm. mmp: matrix metalloproteinase. laghezza masci et al. 7 figure 7. (a) high-resolution tem micrograph showing microvesicles dispersed in the collagen matrix, bar 1 mm; (b) sem micrograph showing a cluster of microvesicles associated with collagen fibrils, bar 2,5 mm; (c) immune gold localization of anti-mmp-9 in association with a cluster of isolated microvesicles. note that gold particles are both inside the intraluminal vesicles as well as along the enclosing membrane of the microvesicles, bar 4 mm; (d) immune gold localization of anti-mmp-9 in a microvesicle associate with a bundle of frayed collagen fibrils; the arrowhead points to gold labeling released from the microvesicle onto the collagen fibrils, bar 200 nm; and (e) immune gold localization of anti-collagen antibody on a bundle of frayed collagen fibrils, bar 500 nm. tem: transmission electron microscopic; sem: scanning electron microscopy; mmp: matrix metalloproteinase. figure 8. immunohistochemical localization of mmp-9 on collagen matrix following 1, 3, or 7 days of in vitro 3-d culture in the presence of fibroblasts. (a to c) thick sections of formaldehyde-fixed and paraffin-embedded collagen matrix exposed to anti-mmp-9 antibody and stained with dab and nickel enhancement as described in material and methods. bars 10 mm. (d to f) thin sections of formaldehyde-fixed and glycol-methacrylate embedded collagen matrix exposed to anti-mmp-9 antibody and treated with a gold labeled secondary antibody. note how gold particles remain associated with the collagen fibrils even in case where the collagen banding pattern is lost. bars 500 nm. mmp: matrix metalloproteinase. 8 journal of circulating biomarkers on the collagen matrix as culturing times prolong from 1 to 7 days. this observation can also be verified at the ultrastructural level where collagen fibers exposed to the antimmp-9 antibody appear progressively more labeled with gold particles (figure 8(d) to (f)). discussion the main objective of this study was to find out what causal relationship holds between collagen degradation, microvesicle release, and metalloproteinase activities. when cocultured with fibroblasts in vitro, the collagen matrix is gradually transformed from a prevailing laminar to an exclusive fibrillar type of architecture, indicating that collagen fibers are gradually frayed, disassembled, and, possibly, degraded into smaller peptide fragments. given their temporal coincidence over the time period tested in this study, both microvesicle release and metalloproteinase activity could be potential candidates for causing this collagen transformation. indeed, the use of gelatin zymography on fibroblast cell lysates demonstrates that the 82 kda mmp-9 form increases gradually in activity as the relative 92 kda prommp-9 progressively declines. during the same time period, the extent to which microvesicles are released from the fibroblast plasma membrane is also significantly enhanced, as clearly documented by the numerous micrographs depicting fibroblast cell peripheries and various regions of the collagen matrix. however, temporally coincident occurrences can be meaningful only if causally related, for they could be independent of each other and act on collagen degradation through different mechanisms. the evidence that microvesicles and mmp secretion are causally related has come from the finding that microvesicles are labeled with anti-mmp-9 antibody, indicating that they may indeed act as vesicular carriers for this enzyme activity. the causal role played by microvesicles is also demonstrated by the progressive enhancement of mmp-9 expression as they spread in regions of the matrix far distant from their cells of origin. during this time period, mmp-9 expression becomes progressively more pronounced as the collagen matrix is gradually transformed from a laminar to a fibrillar type of fibril assembly. this finding is in line with the observation that the amount of collagen deposited in damaged tissues is directly correlated with the level of mmp-9 expression, suggesting that wound healing is somehow conditioned by the tissue capacity to remove any impaired collagen matrix. 46 the present finding that metalloproteinase and microvesicle shedding are causally related events is consonant with earlier studies demonstrating how microvesicles from various cell types affect persistence and stability of the extracellular matrix under a variety of normal and pathological conditions. a growing body of evidence provides additional support to the notion that many tissue-specific stem cells can manipulate their environment during wound healing and cancer progression through the release of microvesicles 47 and that, in all of these circumstances, microvesicles act as vehicles transferring metalloproteinases to modify the extracellular matrix and increase tumor cell mobility. 48 however, to the best of our knowledge, the evidence reported in this study is the first demonstrating such a high resolution localization of a specific metalloproteinase by immune labeling on an in vitro cultured collagen matrix. interestingly, mmp-9 is heterogeneously distributed inside the microvesicle, its localization being exclusively restricted to the intraluminal vesicles contained therein (see figure 5(c) to (e)). the ultrastructural evidence gathered in this study indicates that these intraluminal vesicles are recruited from the cortical cytoplasm, conveyed to the shedding site of the fibroblast cell membrane and eventually uploaded inside the microvesicle. we are not aware of whether similar structures have been reported in other cell types, except for the so-called multivesicular cargo that fertig et al. 49 have recently described in cardiac telocytes, a new cell type of mesenchymal origin, supposedly playing key roles in tissue regeneration and repair. 50,51 however, the presence of intraluminal vesicles inside microvesicles is at variance from what is currently known about their origin and role(s) in cell communication and information transfer. usually, intraluminal vesicles are sorted out in the endosomal compartment through specific tetraspanin interaction before they are actually clustered in multivesicular bodies and released as extracellular exosomes. 52,53 whether the same or similar mechanism(s) is responsible for the vesicular recruitment in both types of organelles is yet to be established, although their functional similarity suggests that they may be largely analogous and likely to make use of similar molecular interactions. in spite of their morphological differences, they could in fact avail of the same endosomal sorting complexes required for transport (escrt) to segregate endocytic membrane domains destined to become intraluminal vesicles. for this very reason, they have recently been termed ectosomes, rather than being generically referred to as microvesicles. 54 their identity as ectosomes was further confirmed in this study through the use of specific vesicular markers. integrin b1 is cell adhesion molecule frequently used as a reliable marker for distinguishing ectosomes from exosomes. 6,54 the observation that anti-integrin b1 antibody binds the microvesicle’s outer membrane, while anti-mmp-9 antibody localization remains confined to the intraluminar vesicles is a clear-cut evidence that these microvesicles are indeed ectosomes acting as carriers delivering their metalloproteinase cargo onto the extracellular matrix. in wound healing, ectosomes are primarily addressed to sustain cell-to-matrix relationship, rather than cell-to-cell communication. in a cell-to-matrix relationship they may break directly in the extracellular environment and release their content upon shedding from the cell of origin or at some distance from it. 8 under these conditions, cells may activate their metalloproteinase cargo by controlling the extent to which intraluminal vesicles are either allowed or laghezza masci et al. 9 impeded to fuse with the outer membrane. since metalloproteinases remain inactive as long as they are confined within these multivesicular compartments, the possibility to be released in the surrounding milieu, and consequently to act on the nearby collagen substrate, can only occur when the two membranes—the outer membrane and the intraluminal membrane—are transiently fused by exocytosis. how this could occur in vivo is as yet to be established, although a number of plausible mechanisms may be at work in different tissues. as mmp-9 is released extracellularly in the form of a stable, inactive pro-mmp-9 zymogen, a number of factors could potentially convert it to an active form. on the whole, many of the factors triggering this conversion could act in combination at different levels, from gene transcription, to zymogen activation or endogenous inhibition. 34 binding to specific cell surface antigens, such as cd44 or integrin receptors, could also facilitate the proenzyme activation by allosteric interference with its catalytic site. 38 it is well known that adhesive interactions with the extracellular matrix play important roles in tissue remodeling, especially if mediated by mmp activation. 55 it is also likely that activation of metalloproteinase may eventually result in the release of a number of collagen breakdown products that, by themselves, may have some chemotactic effect on cells migrating along the wound bed during formation of the granulation tissue. 56 in conclusion, the experimental evidence reported in this study demonstrates that collagen breakdown in vitro, and perhaps wound healing in vivo, are causally dependent on the release of metalloproteinases via microvesicle shedding. given this relationship, what remains to be verified is how pro-mmp-9 come to be uploaded inside the shedding microvesicles, and eventually how they are targeted to specific matrix substrates following release in the extracellular milieu. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) received no financial support for the research, authorship, and/or publication of this article. references 1. gilbert sf. cell-cell comunication in development. 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datta-chaudhuri1, pragney deme1 , alex dickens2, raha dastgheyb1, pavan bhargava1, honghao bi1, and norman j haughey1,3 abstract there is a wide variety of extracellular vesicles (evs) that differ in size and cargo composition. evs isolated from human plasma or serum carry lipid, protein, and rna cargo that provides insights to the regulation of normal physiological processes, and to pathological states. specific populations of evs have been proposed to contain protein and rna cargo that are biomarkers for neurologic and systemic diseases. although there is a considerable amount of evidence that circulating lipids are biomarkers for multiple disease states, it not clear if these lipid biomarkers are enriched in evs, or if specific populations of evs are enriched for particular classes of lipid. a highly reproducible workflow for the analysis of lipid content in evs isolated from human plasma or serum would facilitate this area of research. here we optimized an ms/msall workflow for the untargeted analysis of the lipid content in evs isolated from human serum. a simple sequential ultracentrifugation protocol isolated three distinct types of serum evs that were identified based on size, targeted protein, and untargeted lipidomic analyses. evs in the upper and middle fractions were approximately 140 nm in diameter, while evs in the pellet were approximately 110 nm in diameter. evs in the upper most buoyant fractions contained the highest concentration of lipids, were enriched with phospholipids, and immunopositive for the cytoskeletal markers actin, a-actinin, and the mitochondrial protein mitofillin, but negative for the typical ev markers cd63, tsg101, and flotillin. a central fraction of evs was devoid of cytoskeletal and mitochondrial markers, and positive for cd63, and tsg101, but negative for flotillin. the ev pellet contained no cytoskeletal or mitochondrial markers, but was positive for cd63, tsg101, and flotillin. the ev pellet contained the lowest concentration of most lipids, but was enriched with ceramide. these results provided new insights into the lipid composition of evs isolated from serum using a simple ultracentrifugation isolation method suitable for lipidomic analysis by mass spectrometry. keywords extracellular vesicle, exosome, lipidomics, lipid, serum, blood date received: 27 september 2018; accepted: 3 september 2019 introduction communication between cells and organs involving the secretion of membrane vesicles (collectively called extracellular vesicles or evs) has attracted a great deal of interest, 1–3 and our understanding of roles for this form of cellular communication in normal physiology and pathology is rapidly increasing. there are multiple types of evs that are released by distinct mechanisms, 4 and there is evidence that the stimulus used to evoke ev shedding modifies the cargo. 5,6 evs are composed of a lipid bilayer, 1 department of neurology, richard t johnson division of neuroimmunology and neurological infections, the johns hopkins university school of medicine, baltimore, md, usa 2 turku centre for biotechnology, turku university, turku, finland 3 department of psychiatry, the johns hopkins university school of medicine, baltimore, md, usa corresponding author: norman j haughey, department of neurology, richard t johnson division of neuroimmunology and neurological infections, the johns hopkins university school of medicine, 600 north wolfe street, pathology 517, baltimore, md 21287, usa. email: nhaughe1@jhmi.edu journal of circulating biomarkers volume 8: 1–12 ª the author(s) 2019 article reuse guidelines: sagepub.com/journals-permissions doi: 10.1177/1849454419879848 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). https://orcid.org/0000-0001-9117-891x https://orcid.org/0000-0001-9117-891x https://orcid.org/0000-0001-5194-4122 https://orcid.org/0000-0001-5194-4122 mailto:nhaughe1@jhmi.edu https://sagepub.com/journals-permissions https://doi.org/10.1177/1849454419879848 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage transmembrane proteins, cytosolic proteins, and rna. 1 surface proteins of evs appear to regulate targeting and capture by recipient cells, which can then incorporate ev cargo, resulting in modifications of the recipient cells’ physiological state. 7,8 the possibility of using evs, or subpopulations of evs as biomarkers for disease, or as molecular targets to be therapeutically regulated are active areas of research. 9,10 various “omic” and targeted techniques have been applied to evs to determine the composition of protein and mirna cargo. 11,12 this information has been broadly applied to understand regulatory and pathological roles for evs, and particular cargo have been proposed as surrogate measures for a wide variety of disease activity 13–17 (also see roy et al. 18 for a recent review). compared with protein and mirna, the lipid components of evs have been relatively less studied, especially in biological fluids. the vast majority of ev lipidomic studies have been conducted on evs isolated from tissue culture. 19–21 the concentration and protein/mirna composition of circulating evs has been associated with various physiological and pathological conditions, and there is increasing evidence that the cargo evs originating from abnormal tissues may serve as biomarkers for disease status. 9,22,23 human blood contains a complex mixture of evs originating from multiple tissues, making the isolation of well-defined ev fractions a challenging task. 24 circulating evs are gaining popularity as a source for biomarkers that could be used to diagnose and/or track the trajectory of diseases including alzheimer’s, 25 hiv-associated neurocognitive disorders, 26 parkinson’s disease, 27 cancers, 9,28 and kidney disease. 29 very little is currently known about the lipidomic composition of circulating evs. as many of these lipid components are biologically active, a better understanding of the lipid components of evs isolated from serum samples will be beneficial for the discovery of novel biomarkers. the study of pathways and networks for lipid metabolism and signaling is a rapidly expanding research field. 30,31 the essential roles of lipidomics in identifying the biochemical mechanisms of lipid metabolism, investigating the functions of genes of interest, discovering novel biomarkers, and evaluating drug efficacy are becoming increasingly recognized. 32 mass spectrometry (ms)-based lipidomics has become one of the predominant approaches because of its high throughput, qualitative and quantitative abilities in lipid detection. 33 ms-based lipidomics strategies can be divided into untargeted and targeted approaches, each with their own advantages and limitations. targeted lipidomics focuses on the analysis of a priori defined lipid species. although these methods can be fully quantitative, these approaches are limited in scope. direct infusion 34 or liquid chromatography (lc)-based information-dependent acquisition (ida) protocols have been developed for untargeted approaches. these methods preselect a list of candidate precursor ions in real time based on survey ms data and user-defined informationdependent criteria to identify all lipid species that have these defined precursors. although more comprehensive than targeted approaches, ida approaches can be compromised by reproducibility and ms/ms data quality. 35 recently, alternative data-independent ms/ms acquisition (dia) methods for untargeted lipidomics analyses have received considerable attention. 36,37 in contrast to ida, dia methods conduct ms/ms for all precursors simultaneously, thereby increasing the coverage of observable molecules and reducing the identification of false negatives. 38 among available dia methods, the sequential precursor ion fragmentation (ms/ms all ) technique of stepping through a predefined mass range in small increments is rather promising owing to its speed, simplicity, and high-quality ms/ms data. this ms/ms all approach is bias-free and delivers high-quality product ion spectra, even in the absence of an ms precursor ion signal. the generated ms/ms data can further be directly applied toward batch library searching or spectral ms2-level algorithms for lipid molecular profiling workflow. we believe the ms/ms all method is well suited to deconvolute the complex lipid compositions of evs. in this study, we optimized this untargeted lipidomic strategy for a highly reproducible characterization of lipids in evs fractionated from human serum. methods human serum human serum samples were collected from 10 healthy volunteers. subject demographics are shown in online supplemental table s1. five men and five women were enrolled with average age of 37.3 years. seven of the subjects were caucasian and three were asian. the collection and use of human samples was approved by the johns hopkins school of medicine institutional review board, in accordance with the wma declaration of helsinki. serum samples were individually analyzed. isolation of evs evs were isolated from normal human serum by differential ultracentrifugation as described previously. 39,40 serum (200 ml) was diluted to 600 ml with phosphate buffered saline (pbs) and centrifuged at 2700 � g for 15 min at 20�c to remove large debris, then ultracentrifuged at 10,000 � g for 30 min at 4�c to remove larger microvesicles and apoptotic bodies. the resulting supernatant was ultracentrifuged at 100,000 � g for 3 h at 4�c. a total of seven fractions were collected in a top-down fashion (f1– f6) ending with the bottom pellet (f7) (figure 1(a)). the size and number of evs in all seven fractions were determined by nanoparticle tracking analysis (described in more detail below). the top (f1), middle (f5), and bottom (pellet) were extracted for lipidomic analyses. 2 journal of circulating biomarkers nanoparticle tracking analysis size and number of evs were quantified using a zetaview nanoparticle tracker (particle metrix gmbh, meerbusch, germany), and corresponding zetaveiw software (8.03.04.01). a nanosphere size standard (100 nm diameter; thermo scientific; waltham, ma) was used to calibrate the instrument prior to readings. instrument preacquisition parameters were set to a temperature of 23�c, sensitivity of 65, frame rate of 30 frames per second (fps), shutter speed of 100, and a laser pulse duration equal to that of shutter duration. post-acquisition parameters were set to a minimum brightness of 25, maximum size of 200 pixels, and a minimum size of 10 pixels. for each sample 1 ml of diluted evs were injected into the sample-carrier cell and the particle count was measured at five positions, with two cycles of reading per position. the cell was washed with pbs after every sample. the mean size and concentration of evs/ml (+sem) was calculated from four replicate experiments. the coefficient of variation (cv) as determined from a pooled sample was 4.8% for size and 4.4% for concentration of evs. ev and serum extraction evs and serum samples were extracted to obtain a crude lipid fraction. in brief, 200 ml of the pellet enriched with evs, or 45 ml of an evs-depleted serum fraction (the remained serum after removal of exosomes by ultracentrifugation) or 15 ml of original serum were gently mixed in a glass vial with appropriate volume of ultrapure water to 1 ml. then 2.9 ml methanol:dichloromethane (2:0.9, v/v) that included internal standards to identify 12 classes of figure 1. isolation and characterization of ev fractions isolated from human serum. (a) a differential ultracentrifugation procedure was used to isolate six fractions and a high-density pellet of evs from human serum. (b) representative western blot showing the immunoreactivity of isolated serum fractions for the indicated proteins that include the lipid raft-associated protein flotillin, the tetraspan protein cd63, the escrt-1 protein tsg101, the cytoskeletal proteins actin, a-actinin, and the mitochondrial protein mitofillin. (c) ev size and (d) number measured by nanoparticle tracking analysis for each fraction. data are mean + sd. evs: extracellular vesicles; sd: standard deviation. chen et al. 3 lipids was added to form a monophasic solution. to obtain a biphasic mixture, 1 ml of ultrapure water and 0.9 ml dichloromethane were added, and the resultant mixture incubated at 4�c for 30 min then centrifuged (10 min, 3000 � g, 4�c) to separate the organic and aqueous phases. then, 1 ml of the organic phase containing a crude lipid extract was transferred to a 2 ml glass vial and stored at �20�c. prior to analysis, 0.5 ml of the organic layer extract was dried using a nitrogen evaporator (organomation associates, inc., berlin, massachusetts, usa) then resuspended in 120 ml of running solvent (dichloromethane:methanol (1:1) with 5 mm ammonium acetate) containing 5 mg/ml of the ceramide (c17:0) as an internal standard. materials internal standards included (online supplemental table s2) 1,2-dilauroyl-sn-glycero-3-phosphocholine (pc 12:0/ 12:0), 1,2-dilauroyl-sn-glycero-3-phospho-l-serine (sodium salt) (ps 12:0/12:0, ps c12), 1,2-dilauroyl-snglycero-3-phosphoethanolamine (pe 12:0/12:0, pe c12), 1,2-dilauroyl-sn-glycero-3-phospho-(10-rac-glycerol) (sodium salt) (pg 12:0/12:0, pg c12), 1,2-dilauroyl-snglycero-3-phosphate (sodium salt) (pa 12:0/12:0, pa c12), cholesteryl-d7 palmitate (cholesterol-d7 ester 16:0, cholesterol ester d7), n-lauroyl-d-erythro-sphingosine (c12 ceramide d18:1/12:0, cer c12), n-heptadecanoyl-derythro-sphingosine (c17 ceramide d18:1/17:0, cer c17), n-lauroyl-d-erythro-sphingosylphosphorylcholine (12:0 sm d18:1/12:0 sm c12), 1,3-dihexadecanoyl glycerol (d5) [1,3-16:0 dg (d5), dg d5], 1,3(d5)-dihexadecanoyl2-octadecanoyl-glycerol [tg d5-(16:0/18:0/16:0), tg d5], d-galactosyl-b-1,10 n-lauroyl-d-erythro-sphingosine [c12 galactosyl(b) ceramide (d18:1/12:0), glccer c12], d-lactosyl-b-1,10 n-dodecanoyl-d-erythro-sphingosine [lactosyl (b) c12 ceramide, laccer c12] were supplied by avanti polar lipids (alabaster, alabama, usa). atmospheric pressure chemical ionization (apci) positive calibration solution (p/n: 4460131) was purchased from ab sciex (concord, ontario, canada). standard stock solutions of cer c12:0 (5 mg/ml), cer c17:0 (5 mg/ml), pe c12:0 (12.5 mg/ml), pa c12:0 (7.25 mg/ml), pg c12:0 (12.5 mg/ml), dg d5 (0.5 mg/ml), tg d5 (0.5 mg/ml), laccer c12:0 (1.25 mg/ml), and glccer c12:0 (2.5 mg/ml) were dissolved in dichloromethane:methanol 1:1 (v/v), sm c12:0 (10 mg/ml), pc c12:0 (25 mg/ml), and ps c12:0 (10 mg/ml) were dissolved in methanol, cholesterol ester d7 was dissolved in dichloromethane and stored at �20�c. ultrapure water was used in all experiments (resistivity >18 mo cm). untargeted lipid analysis by ms/ms all crude lipid extracts were analyzed by ms/ms all using a tripletof ™ 5600 (ab sciex, redwood city, california, usa) mass spectrometer. samples (50 ml) were introduced into the mass spectrometer system by direct infusion using a duospray electrospray ionization source and autosampler (shimadzu; kyoto, japan) at the flow rate of 7 ml/min and run in duplicate in the positive ion mode. the running solvent is the mixture of the dichloromethane and methanol (1:1, v/v) with 5 mm ammonium acetate. the instrument was operated at a mass resolution of 30,000 for time of flight (tof) ms scan and 15,000 for product ion scan in the high sensitivity mode, and automatically calibrated every 10-sample injections using apci positive calibration solution delivered via a calibration delivery system (ab sciex). the source parameters include ion source gases 15 lbf/in 2 (gs1), 20 lbf/in 2 (gs2), curtain gas 30 lbf/in 2 , temperature 150�c, positive ion spray voltage þ5200 v, declustering potential at 80 v, and collision energy at 10 v. an initial tof ms scan provided an overview of the total lipid content at an accumulation time of 5 s. precursor ions were selected by sequential 1 da mass steps from 200.050 to 1200.050 m/z. the analytes in each 1 da step were then introduced into the collision chamber and fragments were produced by collision-induced dissociation and identified by tof with a scan range of 100–1500 m/z (accumulation time of 300 ms). the collision energy for each ms/ms step was 40 ev. a pooled sample containing extracts from all groups was repeatedly run eight times for lipid identification and subsequent selection of targeted lipids. all data were acquired using analyst 1.7 tf (ab sciex, concord, ontario, canada). data processing and analysis the tof ms and ms/ms all data obtained from each sample run and pooled samples were post-aligned to the internal standards using analyst 1.7 tf with mass error less than 5 ppm. broad lipid assignments and identifications were conducted on the aligned data of pooled samples with lipidview ™ software (v1.3 beta, ab sciex, concord, ontario, canada). the identification of the lipid species is based on the matched pairs of precursor and fragment to the in silico tandem ms database of lipidview ™ . to ensure that the identified lipids were reliable and reproducible we only included lipids whose peaks of ms/ms fragments appeared in seven of the eight pooled runs, and had a cv value less than 20%. lipids meeting these criteria were included in the targeted list to identify these lipids in experimental samples. the calculation of cv values and the identification of lipids meeting criteria were conducted using a customized program in matlab (version r2017a; mathworks). a targeted processing method was generated based on the targeted lipid list, and used to probe the experimental sample data set using multiquant software (version 3.0, ab sciex, concord, ontario, canada). for the relative quantification, the resultant peak intensities of each characteristic fragment were corrected by the internal standard for the corresponding class of lipid, and each 4 journal of circulating biomarkers sample duplicate was averaged for further analysis. the intensities of isomeric triacylglyceride (tag) species were calculated individually based on the each neutral loss of fatty acid (fa) moieties, instead of the sum of all the fa fragment ion intensities. peak intensities of 0 were replaced with the minimum detectable intensity that was calculated by dividing 0.01 by the average intensity value of the internal standard for the corresponding class of lipid. average molar ratios were calculated as follows: the relative intensity of each lipid species was normalized to the intensity of the internal standard for the respective class of lipid in the f1, f5, and pellet fractions. the relative intensities for each lipid in f1 and f5 were compared to the corresponding lipid in the pellet fraction and expressed as a ratio. the resulting values obtained for each lipid species were then expressed as a fraction of the total lipid content in the respective class of lipid to obtain the average molar ratio for each lipid species by class. this is an estimate of the molar ratio, as the relative intensity for each lipid species is proportional to its molar concentration. effects of the cv threshold on precision in exploratory studies, we examined the relationship of the cv to instrument precision by investigating the intraday and interday precision of the selected features during repeated runs with three different cv thresholds. intraday precision was determined by measuring the relative standard deviation (rsd) of the relative intensity of each targeted lipid species in six injections during an 8-h ms run. the interday precision was determined by measuring the rsd of the relative intensity for each targeted lipid species in six injections conducted over three consecutive days (two injections/day). we used these data to identify a cv threshold that provided the most robust lipid identifications with optimal precision. nonparametric analysis the relative peak intensity for each lipid species across all fractions was subjected to z-score normalization by subtracting the mean intensity of the lipid from all values, and dividing this value by the standard deviation. this produces scaled intensity values with a mean of 0 and a standard deviation of 1. z-score normalization was necessary for visualization and comparison due to large differences in lipid intensity depending on the class and species of lipid. the z-scored values for each fraction were then averaged and converted into a heat map using graphpad prism 7 without hierarchical clustering. western blotting the protein amount in each fraction and pellet were measured by bicinchoninic acid protein assay. equal amount of protein (20 mg) from each fraction and pellet were resolved by 10% sds–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (bio-rad; hercules, ca). nonspecific binding sites were blocked with 5% (w/v) milk in tbs containing 0.1% tween 20 (tbs-t). after blocking, blots were incubated overnight with the primary polyclonal antibody flotillin 1 (1:1000; abcam), cd63 (1:200; santa cruz biotechnology), tsg101 (1:1000; bd biosciences), actinin-4 (1:1000; gentex), and mitofilin (1:5000; thermo fisher scientific). after washes with tbs-t, blots were incubated for 2 h with the appropriate igg horseradish peroxidase–linked secondary antibody (1:1000; cell signaling technology) and developed by enhanced chemiluminescence. image analysis was performed using a g: box imaging system (syngene). annotation of lipid species lipid species were annotated as previously defined. 41 for example, glycerophospholipid and glycerolipid species were annotated by “sum composition,” and denoted as <lipid class><total number of c in fa moieties>:<total number of double bonds in fa moieties> (e.g. pc 34:1). sphingolipid species were annotated by “sum composition,” and denoted as <lipid class><total number of c in the long-chain base and fa moiety>:<total number of double bonds in the long-chain base and fa moiety>; <total number of oh groups in the long-chain base and fa moiety> (e.g. cer 34:1;2, sm 35:1;2). plasmalogen species were annotated by “sum composition,” and denoted as <lipid class><o-><total number of c in fa moieties>: <total number of double bonds in fa moieties> (e.g. pc o-36:1). molecular species glycerophospholipid were annotated by “molecular species composition,” and denoted as <lipid class><number of c in the first fa moiety>:<number of double bonds in the second fa moiety>/ <number of c in the second fa moiety>:<number of double bonds in the second fa moiety> (e.g. pg 16:0/16:0). molecular species sphingolipid annotated by “molecular species composition” were denoted as <lipid class><d/ t><total number of c in the long-chain base><total number of double bonds in the long-chain base>/<total number of c in fa moiety><total number of double bonds in fa moiety> (e.g. cer d18:1/16:0, the “d” and “t” designations used in shorthand notation of sphingolipids refer to 1,3 dihydroxy and 1,3,4-trihydroxy long-chain bases, respectively). results isolation of evs a stepwise ultracentrifugation technique was used to isolate ev fractions. western blotting was first applied to analyze the characteristic proteins in each fraction. fraction 1 was immunopositive for the cytoskeletal protein actin, and the mitochondrial protein mitofillin. fractions 2 and chen et al. 5 3 were positive for actin and mitofillin. fraction 3 was immunopositive for typical ev markers that included the tetraspan protein cd63, the esct-1 complex protein tsg101, and were negative for the cytoskeletal protein a-actinin, and the mitochondrial protein mitofillin, suggesting an absence of contaminating cellular debris (with the potential exception of fraction 4 that was immunopositive for actin). fractions 5, 6 were immunopositive for cd63, tsg101 and negative for actin, a-actinin and mitofillin. the lipid raft-associated protein flotillin was only detected in the pellet, and this fraction was also positive for cd63, tsg101, but negative for actin, a-actinin, and mitofillin (figure 1(b)). the number of particles in fractions 1–5 were similar at approximately 1 � 109 particles, the particle number increased in fraction 6, and was highest at 2.3 � 109 + 3.2 � 108 particles in the pellet (figure 1(c)). particle size was strikingly similar in fractions 1–5 at approximately 140 nm, and smallest in the pellet with a particle size of 116.3 + 12.8 nm (figure 1(d)). based on these findings, we conducted a lipidomic analysis on the top fraction (f1) that did not contain any of the typical ev markers, the middle fraction (f5) that contained typical ev markers, but did not contain markers of cellular contamination, and the bottom fraction (pellet) that contained the typical ev markers and the lipid raft marker flotillin. validation of reproducible lipid features we first extracted a pooled serum sample consisting of small aliquots of each experimental sample (f1, f5, and the pellet) to identify features that are highly reproducible for the subsequent analysis of individual samples. a pooled extract from each of f1, f5, and the pellet were subjected to ms analysis with eight sequential injections using the identical direct infusion ms/ms all dia method applied to experimental samples. a broad lipid assignment and identification was conducted on the aligned data of pooled samples with lipidview ™ software. this initial method identifies all features that appear at least once in the eight runs. to ensure that we are only conducting further analysis on consistently identified features, only those features present in at least seven of the eight pooled runs, with a cv value of less than 20% are included for further analysis. using these criteria, 4024 of the initially identified features were reduced to 597 highly reproducible features. these highly reproducible features were used to create a targeted lipids list in lipidview ™ , and this targeted list was used to probe the experimental data set. these targeted lipid lists were used to analyze the lipid data of each fraction of serum. to further confirm the cv threshold, we explored the relationship of cv to instrument precision by investigating the intraday and interday precision of the selected features during repeated runs. a cv threshold of 10% showed the best precision with 88% of the interday, and 91% of the intraday features showing an rsd less than 20%. however, only 219 features met these criteria. increasing the cv threshold to 20% only slightly reduced precision, with 78% of the interday, and 80% of the intraday features showing an rsd <20%, but the number of feature meeting criteria increased to 597. increasing the cv threshold to 30% resulted in a large reduction of precision, with 48% of the interday and 55% of intraday features showing an rsd <20%, with 1400 features meeting criteria. without any threshold for the selection of features 55% of interday and 48% of intraday features had an rsd <20% (online supplemental table s3), with 4024 identified features. we used a 20% cv threshold for this study, as it provided optimal precision and feature coverage. from an initial 597 features that met criteria, a total of 422 of lipid features were confirmed using the lipidview ™ database after removal of unlikely lipid species (i.e. fa side chain with a carbon number less than 12, and additional fragments that each identified the same lipid). the identified lipid classes included glycerophospholipid/lysoglycerophospholipid species (pa/pa o/lpa/lpa o, pc/ pc o/lpc/lpc o, pe/pe o/lpe/lpe o, pg/pg o, ps/ps o/lps/lps o), sphingolipid species (cer, hexcer/hex2cer, sm), cholesteryl ester (ce), and glycerolipid species (dag, tag). the detailed information of each lipid feature including the sum composition, molecular composition, precursor and product ions are listed in online supplemental table s4. lipidomic analysis of ev fractions we next conducted a lipidomic analysis of fractions 1 (f1), 5 (f5), and the pellet using equal numbers of particles from each fraction to normalize the amount of input material. the total number of lipid features meeting criteria for selection in f1 (n ¼ 370), f2 (n ¼ 341), and the pellet (n ¼ 375) were similar. fractions f1, f5, and the pellet contained 177 shared lipid species (figure 2(a)), and there were 106 lipid features unique to f1, 85 unique to f5, and 110 unique to the pellet (figure 2(a)). the pellet contained the largest number of unique ceramides (n ¼ 12), compared with f1 (n ¼ 4), or f5 (n ¼ 6). in contrast, f1 contained the largest number of unique phospholipids (n ¼ 41), compared with f5 (n ¼ 19), and the pellet (n ¼ 21). the fa composition of tags in f1 was largely unsaturated, f5 contained a mixture of saturated and unsaturated species, and the pellet contained largely saturated fas (table 1). comparing lipids that were consistently detected in f1, f5, and the pellet, there was a clear visual separation where f1 contained the highest concentration of nearly all lipid species, f5 was intermediate, and the pellet contained the lowest concentration of most lipid species (figure 2(b)). a total of 53 (out of 422) independent lipid species were significantly different between the f1, f5, and the pellet (table 2). the lipid content of f1 was generally higher than f5 or the pellet, consistent with the buoyancy of this fraction. comparing the average molar ratios for all lipid species 6 journal of circulating biomarkers by class we found that f1/pellet contained the highest concentration of all lipid classes followed by f5/pellet (with the exception of ceramides) (figure 2(c)). these data demonstrate that f1 and f5 are enriched for all lipid classes with the exception of ceramides that are enriched in the pellet. discussion disease-associated modifications in plasma lipid content have been reported in alzheimer’s disease, hiv infection, parkinson’s disease, cerebrovascular damage, and a number of different metabolic disorders. 42–59 although the direct infusion ms/ms all approach has been applied in the lipidomics analysis of clinical studies, there are potential issues with reproducibility that need to be optimized. one potential issue is the reliable identification of lipid species. after ms/ms analysis, lipid identification and quantification software such as lipidview ™ identify thousands of potential lipid species from samples. however, a fairly large proportion of the lipid species identified and/or the peak intensity assigned to any particular identification are not reproducible when samples are repeatedly analyzed several times. presumably, these variations result from inconsistencies in the ionization process of sample with complex matrices. the identification of a reliable lipid species list for further data analysis is critical for obtaining accurate and reproducible experimental results in studies using clinical samples. one of the commonly used solutions in lc-based lipidomics is the “80% rule,”60,61 in which a lipid feature will be maintained if it has a nonzero measurement value in at least 80% of the sample data set. using this rule, missing values caused by slight shifts in analyte peaks are reduced. however, this strategy does not consider inconsistencies in the ionization efficiency of lipid species contained in a complex matrix. these inconsistencies can produce variable peak intensities that are used for quantitation. to increase the consistency of peak intensities for quantitative analyses we defined a cv for each of the identified lipid species and removed ones that were not reliably quantified. briefly, a pooled sample is subjected to direct infusion ms/ms all analysis for eight consecutive runs, and the data applied to the lipidview ™ for lipid identification. only the lipid species that appeared with figure 2. lipid composition of f1, f5, and the pellet isolated from serum fractions. (a) venn diagram showing the distribution of common and unique lipids identified in the indicated fraction. (b) z-score normalized heat map showing the relative concentrations of shared lipids in the three fractions. the relative intensities of each lipid in specific class of lipid were normalized to the corresponding internal standard for each lipid class. (c) average molar ratios for the indicated classes of lipid in f1 and f5 compared with the pellet. chen et al. 7 nonzero values seven of eight runs, with a calculated cv of <20% were retained for further analysis. here we developed and optimized an untargeted msbased lipidomic method for the analysis of ev lipid content in human serum. a simple commonly used differential table 1. the unique lipids features identified from the pooled f1, f5, and pellet fractions of the serum exosomes isolation by ultracentrifugation. f1 f5 pellet ce 20:1 ce 22:2 ce 18:4 ce 22:6 cer d18:1/22:0 ce 24:2 cer d18:0/14:1 dag 30:0 (-fa 14:0) ce 32:6 cer d18:1/18:0 dag 32:0 (-fa 16:0) ce 34:4 cer d18:1/24:1 dag 32:2 (-fa 16:1) ce 34:5 dag 32:1 (-fa 16:0) dag 36:4 (-fa 18:2) cer d18:1/14:0 dag 34:2 (-fa 14:1) dag 40:5 (-fa 20:1) cer d18:0/16:0 dag 34:2 (-fa 16:1) hexcer d18:1/16:0 cer d18:0/16:3 dag 40:4 (-fa 20:1) hexcer d18:1/24:0 dag 32:0 (-fa 14:0) hexcer d18:2/24:1 hexcer d18:1/26:0 dag 34:0 (-fa 14:0) lpc 20:4 lpc 20:4 dag 38:2 (-fa 18:1) lpc 20:5 lpc o-26:0 dag 38:3 (-fa 18:1) lpc 30:0 lpe o-24:0 dag 38:3 (-fa 18:3) lpc o-16:0 pa 44:3 dag 38:4 (-fa 18:2) lpc o-16:1 pa o-16:0/16:0 dag 40:1 (-fa 20:0) lpe 18:1 pc 32:3 dag 40:4 (-fa 20:0) lps 28:1 pc o-20:3 dag 40:5 (-fa 20:0) lps o-30:3 pc o-20:4 hexcer d18:1/16:3 pa 40:4 pc o-22:6 hexcer d18:1/18:0 pa o-40:4 pc o-24:0 lpc o-20:0 pa o-48:2 pc o-44:6 lpe 26:0 pa o-48:3 pe 34:0 lpe o-30:4 pc 30:0 pe o-36:1 lpg 24:0 pc 30:3 pe o-36:3 lpg 24:1 pc 34:5 pg 36:1 lps 16:3 pc 36:1 pg 36:5 lps 20:3 pc 40:1 pg o-36:6 lps o-18:0 pc 40:4 pg o-38:1 lps o-18:2 pc 42:5 pg o-38:6 lps o-30:0 pc o-32:2 pg o-40:2 pa 38:5 pc o-34:4 ps 36:4 pa o-48:1 pc o-36:1 ps 48:3 pa o-48:2 pc o-38:0 sm 32:3;2 pa o-48:3 pe 36:5 sm 40:1;2 pc o-42:5 pe 38:0 sm 42:3;2 pc o-44:3 pe 40:4 tag 42:2 (-fa 16:0) pe 38:1 pe 40:5 tag 44:1 (-fa 14:1) pe o-38:0 pe o-34:1 tag 44:4 (-fa 14:0) pe o-46:6 pe o-40:4 tag 46:3 (-fa 14:1) pg 20:2/20:2 pe o-40:5 tag 50:5 (-fa 16:0) pg o-20:0/20:0 pe o-42:6 tag 50:6 (-fa 12:0) ps 32:2 pe o-46:5 tag 50:8 (-fa 18:2) ps 36:4 pg 38:6 tag 52:6 (-fa 18:1) ps 36:6 pg 48:5 tag 52:7 (-fa 16:2) ps 42:6 pg 48:6 tag 54:0 (-fa 16:0) ps o-28:1 pg o-18:1/18:1 tag 54:0 (-fa 20:0) ps o-36:1 pg o-38:0 tag 54:2 (-fa 20:2) ps o-38:0 ps 28:0 tag 54:4 (-fa 20:1) ps o-38:1 ps 36:5 tag 54:6 (-fa 16:2) ps o-46:0 ps o-38:6 tag 54:8 (-fa 20:5) ps o-48:6 ps o-48:6 tag 54:9 (-fa 20:5) sm 34:1;2 sm 44:1;2 tag 56:1 (-fa 20:0) sm 46:1;2 sm 44:2;2 tag 56:2 (-fa 16:0) sm 48:1;2 sm 46:2;2 tag 56:2 (-fa 22:1) tag 36:1 (-fa 12:1) sm 48:1;2 tag 56:4 (-fa 18:3) tag 38:0 (-fa 18:0) (continued) table 1. (continued) f1 f5 pellet tag 42:3 (-fa 18:3) tag 56:6 (-fa 22:6) tag 38:2 (-fa 16:0) tag 44:1 (-fa 12:1) tag 56:7 (-fa 18:3) tag 40:0 (-fa 12:0) tag 44:2 (-fa 14:1) tag 56:7 (-fa 20:3) tag 40:1 (-fa 16:1) tag 44:2 (-fa 16:1) tag 58:10 (-fa 20:5) tag 40:2 (-fa 14:0) tag 44:3 (-fa 12:0) tag 58:4 (-fa 18:1) tag 40:2 (-fa 18:2) tag 44:3 (-fa 16:1) tag 58:4 (-fa 18:2) tag 42:0 (-fa 12:0) tag 44:4 (-fa 18:2) tag 58:5 (-fa 18:1) tag 42:0 (-fa 18:0) tag 46:2 (-fa 12:1) tag 58:6 (-fa 18:1) tag 42:1 (-fa 14:0) tag 46:2 (-fa 14:1) tag 58:7 (-fa 18:3) tag 42:2 (-fa 12:0) tag 46:3 (-fa 16:1) tag 58:8 (-fa 22:6) tag 42:2 (-fa 18:1) tag 48:5 (-fa 12:1) tag 60:10 (-fa 20:4) tag 42:6 (-fa 16:0) tag 48:5 (-fa 20:4) tag 60:10 (-fa 22:6) tag 44:1 (-fa 12:0) tag 48:5 (-fa 16:3) tag 60:3 (-fa 24:0) tag 44:2 (-fa 14:0) tag 50:5 (-fa 20:3) tag 60:9 (-fa 20:4) tag 44:3 (-fa 14:1) tag 50:6 (-fa 18:2) tag 60:9 (-fa 22:6) tag 44:3 (-fa 16:0) tag 50:6 (-fa 22:6) tag 44:5 (-fa 14:0) tag 50:7 (-fa 18:1) tag 46:3 (-fa 12:1) tag 52:6 (-fa 22:6) tag 46:3 (-fa 14:0) tag 52:8 (-fa 22:6) tag 48:0 (-fa 24:0) tag 54:3 (-fa 18:3) tag 48:4 (-fa 14:1) tag 54:4 (-fa 22:1) tag 48:4 (-fa 16:1) tag 54:7 (-fa 20:3) tag 48:4 (-fa 18:1) tag 54:8 (-fa 22:6) tag 48:5 (-fa 12:0) tag 56:0 (-fa 20:0) tag 48:5 (-fa 16:0) tag 56:10 (-fa 22:6) tag 48:5 (-fa 18:2) tag 56:3 (-fa 16:0) tag 50:5 (-fa 20:1) tag 56:3 (-fa 22:1) tag 54:6 (-fa 20:4) tag 56:4 (-fa 20:0) tag 56:1 (-fa 16:0) tag 56:6 (-fa 20:5) tag 56:3 (-fa 20:0) tag 58:1 (-fa 16:0) tag 56:3 (-fa 20:2) tag 58:1 (-fa 20:0) tag 56:4 (-fa 16:0) tag 58:5 (-fa 18:2) tag 56:4 (-fa 20:4) tag 58:7 (-fa 20:4) tag 56:7 (-fa 20:2) tag 58:8 (-fa 20:2) tag 58:11 (-fa 20:5) tag 58:8 (-fa 20:4) tag 58:3 (-fa 18:2) tag 60:3 (-fa 18:2) tag 58:5 (-fa 16:0) tag 58:6 (-fa 16:0) tag 58:7 (-fa 20:2) tag 60:4 (-fa 20:0) tag 60:5 (-fa 20:5) ce: cholesterol ester; cer: ceramide; dag: diacylglyceride; hexcer: hexosylceramide; hex2cer: dihexosylceramide; pa: phosphatidic acid; lpa: lysophosphatidic acid; pc: phosphatidylcholine; lpc: lysophosphatidylcholine; pe phosphatidyletanolamine; lpe: lysophosphatidyletanolamine; pg phosphatidylglycerol; lpg: lysophosphatidylglycerol; ps: phosphatidylserine; lps: lysophosphatidylserine; sm: sphingomyelin; tag: triacylglyceride; fa: fatty acid; o-: ether-linked lipids. 8 journal of circulating biomarkers table 2. shared lipid features that were significantly different in abundance between ev fractions. a lipid name f1 avg (sd) f5 avg (sd) ev avg (sd) p values f1 � ev f5 � ev f1 � f5 ce 30:3 12.54 (2.65) 8.27 (1.74) 1.44 (0.71) <0.0001 <0.0001 <0.0001 cer d18:1/16:2 0.80 (0.07) 0.47 (0.04) 0.33 (0.04) <0.0001 <0.0001 <0.0001 cer d18:1/24:0 0.13 (0.04) 0.08 (0.04) 0.01 (0.01) <0.0001 <0.001 <0.05 dag 16:0/20:0/0:0 8.69 (2.09) 4.70 (1.52) 0.95 (0.49 <0.001 0.09264 0.2094 dag 18:2/20:0/0:0 106.64 (22.2) 62.12 (19.93) 10.65 (6.45) <0.0001 <0.0001 <0.0001 hexcer d18:0/18:0 0.03 (0.01) 0.01 (0.01) 0.03 (0.01) 0.547458 <0.001 <0.001 lpc 16:0 1.06 (0.23) 0.67 (0.11) 0.11 (0.06) <0.0001 <0.0001 <0.0001 lpc 18:0 0.59 (0.12) 0.37 (0.05) 0.06 (0.04) <0.0001 <0.0001 <0.0001 lpc 18:1 0.36 (0.09) 0.23 (0.05) 0.04 (0.03) <0.0001 <0.0001 <0.05 lpc 18:2 0.69 (0.24) 0.45 (0.15) 0.07 (0.05) <0.0001 <0.0001 <0.0001 pc 34:1 17.05 (2.95) 10.92 (2.35) 1.79 (1.32) <0.0001 <0.0001 <0.0001 pc 36:2 20.24 (3.59) 13.00 (2.39) 2.61 (1.85) <0.0001 <0.0001 <0.0001 pc 36:3 13.52 (3.27) 8.34 (2.21) 1.10 (0.80) <0.0001 <0.0001 <0.0001 pc 36:4 15.09 (3.51) 9.70 (2.57) 1.28 (0.90) <0.0001 <0.0001 <0.0001 pc 38:3 3.37 (0.69) 2.01 (0.49) 0.29 (0.16) <0.0001 <0.0001 <0.05 pc 38:4 6.93 (1.75) 4.39 (1.12) 0.54 (0.31) <0.0001 <0.0001 <0.01 pc 38:5 2.45 (0.98) 1.66 (0.54) 0.20 (0.12) <0.001 <0.001 0.24255 pc 38:6 2.25 (0.98) 1.59 (0.69) 0.18 (0.14) <0.001 <0.01 0.32440 pc o-32:0 0.17 (0.03) 0.11 (0.02) 0.02 (0.01) <0.001 <0.01 0.26278 pc o-32:1 0.18 (0.04) 0.11 (0.03) 0.02 (0.01) <0.001 <0.01 0.24071 pc o-34:1 0.54 (0.10) 0.33 (0.07) 0.05 (0.03) <0.0001 <0.0001 <0.001 pc o-34:2 0.77 (0.20) 0.46 (0.12) 0.07 (0.03) <0.0001 <0.0001 <0.0001 pc o-34:3 0.63 (0.21) 0.38 (0.13) 0.06 (0.03) <0.0001 <0.0001 <0.0001 pc o-36:3 0.61 (0.15) 0.36 (0.09) 0.05 (0.03) <0.0001 <0.0001 <0.0001 pc o-36:4 1.24 (0.29) 0.74 (0.20) 0.10 (0.05) <0.0001 <0.0001 <0.0001 pc o-36:5 0.82 (0.25) 0.50 (0.17) 0.07 (0.03) <0.0001 <0.0001 <0.0001 pc o-38:4 0.72 (0.12) 0.46 (0.09) 0.06 (0.04) <0.0001 <0.0001 <0.0001 pc o-38:5 1.04 (0.21) 0.66 (0.15) 0.09 (0.06) <0.0001 <0.0001 <0.0001 pc o-38:6 0.46 (0.14) 0.30 (0.10) 0.04 (0.02) <0.0001 <0.0001 <0.01 pc o-40:5 0.18 (0.03) 0.11 (0.02) 0.02 (0.01) <0.01 <0.01 0.287674 pe 34:2 0.03 (0.01) 0.02 (0.01) 0.001 (0.01) <0.0001 <0.001 0.07888 pe 36:1 0.02 (0.01) 0.01 (0.01) 0.001 (0.01) <0.01 <0.05 0.09374 pe 36:2 0.10 (0.05) 0.06 (0.02) 0.02 (0.01) <0.0001 <0.0001 <0.0001 pe 36:3 0.04 (0.02) 0.02 (0.01) 0.01 (0.01) <0.0001 <0.0001 <0.05 pe 36:4 0.03 (0.01) 0.02 (0.01) 0.01 (0.01) <0.001 <0.001 0.178920 pe 38:4 0.05 (0.02) 0.03 (0.01) 0.001 (0.01) <0.0001 <0.0001 <0.01 pe o-38:5 0.01 (0.01) 0.01 (0.01) 0.001 (0.01) <0.0001 <0.0001 <0.01 pe o-38:6 0.01 (0.01) 0.00 (0.01) 0.001 (0.01) <0.001 <0.001 0.2377739 sm 34:1;2 10.76 (2.26) 6.55 (1.70) 0.92 (0.63) <0.0001 <0.0001 <0.0001 sm 36:1;2 1.82 (0.46) 1.10 (0.36) 0.18 (0.12) <0.0001 <0.0001 <0.05 sm 38:2;2 6.11 (1.83) 2.67 (1.56) 0.56 (0.42) <0.0001 <0.0001 <0.0001 sm 42:1;2 1.99 (0.42) 1.18 (0.33) 0.19 (0.12) <0.0001 <0.0001 <0.05 sm 42:2;2 5.37 (1.00) 3.16 (0.72) 0.47 (0.28) <0.0001 <0.0001 <0.0001 tag 54:2 (-fa 18:1) 2.10 (0.68) 1.03 (0.33) 0.64 (0.59) <0.0001 0.05789 <0.0001 tag 54:2 (-fa 18:2) 0.17 (0.04) 0.08 (0.03) 0.05 (0.04) 0.693309 0.87924 0.737423 tag 54:3 (-fa 18:0) 1.52 (0.38) 0.74 (0.25) 0.43 (0.42) <0.001 0.13283 <0.01 tag 54:3 (-fa 18:1) 10.05 (4.53) 4.57 (1.85) 2.77 (3.39) <0.0001 <0.0001 <0.0001 tag 54:3 (-fa 18:2) 1.58 (0.40) 0.75 (0.23) 0.44 (0.43) <0.0001 0.1276712 <0.001 tag 54:4 (-fa 18:1) 10.32 (4.87) 4.68 (2.04) 3.01 (4.07) <0.0001 <0.0001 <0.0001 tag 54:4 (-fa 18:2) 5.84 (2.40) 2.68 (1.05) 1.70 (2.10) <0.0001 <0.0001 <0.0001 tag 54:5 (-fa 18:1) 4.07 (1.91) 1.91 (0.81) 1.23 (1.51) <0.0001 <0.01 <0.0001 tag 54:5 (-fa 18:2) 5.73 (3.02) 2.68 (1.21) 1.77 (2.22) <0.0001 <0.001 <0.0001 tag 54:6 (-fa 20:5) 2.58 (1.70) 1.31 (0.72) 0.03 (0.02) <0.0001 <0.0001 <0.0001 ce: cholesterol ester; cer: ceramide; dag: diacylglyceride; hexcer: hexosylceramide; hex2cer: dihexosylceramide; pa: phosphatidic acid; lpa: lysophosphatidic acid; pc: phosphatidylcholine; lpc: lysophosphatidylcholine; pe phosphatidyletanolamine; lpe: lysophosphatidyletanolamine; pg phosphatidylglycerol; lpg: lysophosphatidylglycerol; ps: phosphatidylserine; lps: lysophosphatidylserine; sm: sphingomyelin; tag: triacylglyceride; fa: fatty acid; o-: ether-linked lipids; sd: standard deviation; anova: analysis of variance; ev: extracellular vesicle. a data are mean + sd. anova with bonferroni correction for multiple comparisons. chen et al. 9 ultracentrifugation technique was used to separate serum that produced several distinct ev fractions. the upper three fractions contained larger evs that were negative for the typical ev markers, cd63 and tsg101, but were positive for the cytoskeletal protein actin, and contained the highest lipid content. these data are consistent with a more buoyant ev that likely originated from a membrane bubbling or an apoptotic event. fraction 4 appears to be a transitional fraction, and contains cd63, tsg101, and actin. the lower fractions 5 and 6 contained cd63, tsg101, but were negative for actin, and mitofillin. they contained an intermediate lipid content compared with fraction 1 and the pellet. these evs are likely a mixture of cellular blebs, apoptotic bodies, and some exosomes. the pellet contained cd63, tsg101 and was the only fraction immunopositive for flotillin. the pellet contained the lowest concentration of lipids, and the highest concentration of ceramide, consistent with the high protein/lipid ratio found in exosomes. 62 there are several caveats to this method that should be noted. although ultracentrifugation seems to greatly enrich the evs content, the composition of plasma or serum is complex and contains multiple types of lipoproteins with different sizes and densities. high-density (d) lipoproteins (hdl, d ¼ 1.063–1.21 g/ml) have been reported to co-isolate with evs in blood samples (d ¼ 1.13–1.19) subjected to ultracentrifugation. 30,40 since evs may themselves carry apolipoproteins, 63–65 it is not possible to definitively conclude if apolipopoteins are present or absent from the ev fraction using this isolation method. this may explain the high content of neutral lipids such as ce and glycerolipids we observed in the pellet of fractionated serum, or it is possible that these particular lipid classes are enriched in evs isolated from serum. this method is also not ideal for high throughput isolation of evs from clinical samples. ultracentrifugation takes hours to complete and the number of samples that can be isolated is limited by the rotor capacity (typically 4–6 samples at a time can be isolated). lastly, although we defined a method to enhance the reproducibility of data produced by the ms/ms all approach, this method is not an absolute quantitation of lipid concentration. although between group comparisons are accurate, it is not possible to compare lipids within a given class, as the ionization efficiency varies with chain lengths. this study reports an optimized workflow for untargeted lipidomic analysis of serum evs. a simple ultracentrifugation technique isolates at least three clear populations of evs. the upper three fractions contain more buoyant evs that are phospholipid rich, but lack cd63, tsg101, or flotillin. fractions 5 and 6 are of medium buoyancy, and contain cd63, tsg101, but are negative for flotillin. the pellet is dense, ceramide rich, and contains cd63, tsg101, and flotillin. these results provided new insights into the classical ultracentrifugation method used to isolate serum evs. future studies will determine if the lipid composition from subfractions of evs can be used for diagnostic or prognostic measures of disease activity. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the national institutes of health awards (ag057420, da040390, mh077542, mh075673, mh110246, mh105280, and mh096636). orcid ids pragney deme https://orcid.org/0000-0001-9117-891x norman j haughey https://orcid.org/0000-0001-5194-4122 supplemental material supplemental material for this article is available online. references 1. yanez-mo m, siljander prm, andreu z, et al. biological properties of extracellular vesicles and 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article the signature of circulating microparticles in heart failure patients with metabolic syndrome alexander e berezin 1 , alexander kremzer 1 , tatyana berezina 2 , and yu martovitskaya 3 abstract the role of pattern of circulating endothelial cell-derived microparticles, platelet-derived microparticles (pmps), and monocyte-derived microparticles (mmps) in metabolic syndrome (mets) patients with chronic heart failure (chf) is not still understood. the aim of the study was to investigate a pattern of circulating microparticles (mps) in mets patients with chf in relation to neurohumoral and inflammatory activation. the study retrospectively involved 101 patients with mets and 35 healthy volunteers. biomarkers were measured at baseline of the study. the results of the study have shown that numerous circulating pmpsand mmps in subjects with mets (with or without chf) insufficiently distinguished from level obtained in healthy volunteers. we found elevated level of cd31þ/annexin vþ mps in association with lower level of cd62eþ mps. therefore, we found that biomarkers of biomechanical stress serum n-terminal brain natriuretic peptide and inflammation (high-sensitive c-reactive protein , osteoprotegerin) remain statistically significant predictors for decreased cd62eþ to cd31þ/annexin vþ ratio in mets patients with chf. in conclusion, decreased cd62eþ to cd31þ/annexin vþ ratio reflected that impaired immune phenotype of mps may be discussed as a surrogate marker of chf development in mets population. keywords chronic heart failure, metabolic syndrome, circulating microparticles, cardiovascular risk factors inflammation, neurohumoral activation date received: 7 september 2015; accepted: 15 july 2016 introduction the traditionally recognized metabolic syndrome (mets) is defined as risk-factor clustering related to the development of type 2 diabetes mellitus (t2dm) and cardiovascular disease (cvd). 1 mets includes abdominal obesity, insulin resistance, dyslipidemia, and elevated blood pressure and associates with other comorbidities including the prothrombotic and pro-inflammatory states. 2 accumulating evidence has shown that mets is a powerful risk factor for cvd event as well as all cause and cvd mortality in total population. 3–5 the underlying pathophysiological mechanisms resulting in the mets, that is, insulin resistance (ir), associate with activation of neurohumoral mechanisms, immunity, cytokine production, systemic pro-inflammatory response, and oxidative stress. 6–8 all these factors may have effect on the development of cvd through inducing endothelial dysfunction 9,10 and microvascular inflammation. 11 1 consultant of the therapeutic unit, internal medicine department, state medical university, zaporozhye, ukraine 2 private center vita-center, zaporozhye, ukraine 3 pathology department, dia-service ltd, zaporozhye, ukraine corresponding author: alexander e berezin, state medical university, mayakovsky av., 26, zaporozhye 69035, ukraine. email: dr_berezin@mail.ru journal of circulating biomarkers volume 5: 1–10 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416663659 cbx.sagepub.com creative commons cc-by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 3.0 license (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:dr_berezin@mail.ru http://www.sagepub.co.uk/journalspermissions.nav https://doi.org/10.1177/1849454416663659 http://cbx.sagepub.com https://us.sagepub.com/en-us/nam/open-access-at-sage recent studies have shown a controversial role of mets in patients at high risk of chronic heart failure (chf) and in subjects with documented chf. although mets associates with cardiovascular risk factors and cvd outcomes, 12–15 prognostic impact of mets on chf progression is not fully confirmed and widely discussed. 16,17 therefore, it is still unclear whether mets may induce development and progression of cardiac failure through imbalance between endothelial injuries and repair. 18,19 probably, microparticles (mps) corresponding cell-to-cell cooperation, immunity, tissue reparation, and vascular function are key factors that coordinate microvascular integrity and function. 20 extracellular mps are microvesicles with sizes ranging between 50 nm and 1000 nm, released from plasma membrane of wide variety of cells, including endothelial cells, mononuclear cells, platelets, and by specific (cytokine stimulation, apoptotic agents, mononuclear cooperation, coagulation, and so on) and nonspecific (shear stress) stimuli. 21 circulating endothelial-derived microparticles (emps) depending on their origin (apoptotic-derived or activatedendothelial cell production) are capable of transferring biological information (regulating peptides and hormones) or even genetic material, as well as proteins and lipid components, from one cell to another without direct cell-to-cell contact to maintain cell homeostasis. 22,23 emps derived from activated endothelial cells may have pro-angiogenic and cardioprotective properties. 24 in opposite, apoptotic-derived emps originated from damaged endothelial cells are discussed as a marker of endothelial cell injury and vascular aging. 25 platelet-derived microparticles (pmps) are heterogeneous population of microvesicles that are secreted from chemokine and cytokine activated platelets. pmps mediate multiple cellular responses that predominantly affected protein and lipid metabolism, coagulation, and inflammation. 26 elevated pmps show a relation to clinical outcomes and mortality in several patient populations. 27 numerous studies have shown that monocyte-derived microparticles (mmps) are realized from activated and/or apoptotic monocytes in response to various stimuli, that is, antigen stimulation, growth factors, inflammatory interleukins, chemokines and cytokines, and so on. 28–30 elevated level of circulating mmps is documented in almost all thrombotic diseases, infective, rheumatic and autoimmune diseases, stroke, myocardial infarction, atrial fibrillation as well as in metabolic, ischemia/hypoxia states, and critical conditions. 31–33 however, the significance of mps in mets patients as an inductor of development and progression of chf remains controversial. an example of this controversy is that it is still unknown if circulating mps found in peripheral blood cause injury of endothelium and worsening of chf whether they are the result of disease progression in response to endothelial dysfunction and vascular dysintegrity. 34,35 the aim of the study was to investigate the pattern of circulating emps, pmps, and mmps in mets patients with chf in relation to neurohumoral and inflammatory activation. methods the study retrospectively involved 101 patients with mets (54 subjects with chf and 47 patients without chf) without documented coronary artery stenosis >50% at least of one artery and 35 healthy volunteers who were examined between february 2013 and november 2013. the study was approved by the local ethics committee of state medical university, zaporozhye, ukraine. the study was performed in conformity with the declaration of helsinki. all the patients have given their informed written consent for participation in the study. mets was diagnosed based on the national cholesterol education program adult treatment panel iii criteria. 36 patients were enrolled in the mets cohort when at least three of the following components were defined: waist circumference �90 cm or �80 cm in men and women, respectively; high-density lipoprotein (hdl) cholesterol <1.03 mmol/l or <1.3 mmol/l in men and women, respectively; triglycerides (tg) �1.7 mmol/l; blood pressure �130/85 mmhg or current exposure of antihypertensive drugs; fasting plasma glucose �5.6 mmol/l. subjects with defined t2dm or treatment with oral antidiabetic agents or insulin were not enrolled in the study. current smoking was defined as consumption of one cigarette daily for 3 months. anthropometric measurements were made using standard procedures. methods for visualization of coronary arteries contrast-enhanced multispiral computed tomography angiography was performed for all the patients with dysmetabolic disorder prior to their inclusion in the study on optima ct660 scanner (ge healthcare, milwaukee, usa) using non-ionic contrast omnipaque (amersham health, ireland). 37 subjects with atherosclerotic lesions >50% of diameter at least of one coronary artery were excluded for further enrollment in the study. transthoracic echocardiography transthoracic echocardiography was performed according to a conventional procedure on ultrasound scanner acuson (siemens, germany) in b-mode and tissue doppler imaging with phased probe of 2.5–5 mhz. left ventricular (lv) end-diastolic and end-systolic volumes and lv ejection fraction (lvef) were measured by modified simpson’s method. 38 calculation of glomerular filtration rate glomerular filtration rate (gfr) was calculated using chronic kidney disease epidemiology collaboration formula. 39 2 journal of circulating biomarkers measurement of circulating biomarkers to determine circulating biomarkers, blood samples were collected at baseline in the morning (at 7–8 a.m.) into cooled silicone test tubes wherein 2 ml of 5% trilon b solution were added. then they were centrifuged upon permanent cooling at 6000 r/min for 3 min. plasma was collected and refrigerated immediately to be stored at a temperature of �70�c. serum n-terminal brain natriuretic peptide (nt-probnp), adiponectin, serum receptor activator of nf-kb ligand (rankl), and osteoprotegerin (opg) were measured by high-sensitive enzyme-linked immunosorbent assays using commercial kits (r&d systems gmbh, wiesbaden-nordenstadt, germany) according to the manufacturers’ recommendations. the inter-assay coefficients of variation were as follows: nt-probnp: 4.5%, adiponectin: 5%, rankl: 7.0%; and opg: 8.2%. high-sensitive c-reactive protein (hs-crp) was measured by commercially available standard kit (r&d systems gmbh, wiesbaden-nordenstadt, germany). the intra-assay and inter-assay coefficients of variation were <5%. fasting insulin level was measured by a double-antibody sandwich immunoassay (elecsys 1010 analyzer; f. hoffmann-la roche diagnostics, mannheim, germany). the intra-assay and inter-assay coefficients of variation were <5%. the lower detection limit of insulin level was 1.39 pmol/l. ir was assessed by the homeostasis model assessment for ir (homa-ir) 40 using the following formula: homa � ir ðmmol=l �m u=mlþ ¼ fasting glucoseðmmol=lþ� fasting insulinðm u=mlþ 22:5 concentrations of total cholesterol , cholesterol of lowdensity lipoproteins (ldls-c), and cholesterol of hdls-c were measured by enzymatic method. assay of circulating emps circulating mps were isolated from 5 ml of venous citrated blood drawn from the fistula-free arm. no hemolysis in the samples was found. all samples were not frozen before analysis. to prevent the contamination of samples plateletfree plasma (pfp) was separated from whole blood. pfp was centrifuged at 70,476 � g for 70 min. mp pellets were washed with dmem (sigma-aldrich chemie gmbh munich, germany) (supplemented with 10 mg/ml polymyxin b, 100 ui of streptomycin, and 100 u/ml penicillin) and centrifuged again (70,476 � g for 90 min).41 the obtained supernatant was extracted, and mp pellets were re-suspended into the remaining 200 ml of supernatant. pfp, mps, and supernatant were diluted 5-, 10-, and 5-fold in phosphate-buffered saline, respectively. only 100 ml of supernatant was prepared for further analysis through incubation with different fluorochrome-labeled antibodies or their respective isotypic immunoglobulins (beckman coulter, pasadena, california, usa). mps were labeled and characterized by flow cytometry by phycoerythrin-conjugated monoclonal antibody against cd31 (platelet endothelial cell adhesion molecule-1), cd41a, cd64, cd105, cd144 (vascular endothelialcadherin), cd62e (e-selectin), and annexin v (bd biosciences, san jose, ca, usa) followed by incubation with fluorescein isothiocyanate-conjugated annexin v (bd biosciences) per high-definition fluorescence activated cell sorter (hd-facs) methodology independently after supernatant diluted without freeze. 42 the samples were incubated in the dark for 15 min at room temperature according to the manufacturer’s instructions. the analysis of area, height, and width forward scatter (fsc) and side scatter (ssc) parameters as well as side scatter width (ssc-w) was performed. particle sizing by dynamic light scattering revealed a characteristic size of the mps (sigma, st louis, missouri, usa). a mps’ gate was established on the facs aria instrument (bd biosciences, san jose, ca, usa) by preliminary standardization experiments using a blend of size-calibrated fluorescent beads, with sizes ranging from 0.1 mm to 1.0 mm. two size gates were defined based on forward angle light scattering from polystyrene microsphere (0.5–0.9 mm) accordingly to the standard protocol. the upper and the outer limit of the mp gate was established just above the size distribution of the 0.9-mm beads in a fsc-a and ssc-a setting (log scale) using the ‘auto-gate’ function. accordingly, mps’ gate was defined less than a 0.4 mm polystyrene microsphere extending down to the noise threshold level, that is, equivalent to cell-derived mps <1 mm diameter. the lower limit was the noise threshold of the instrument, and an absolute minimum threshold of 200 was set at the ssc-a parameter (instead of fsc-a) to avoid exclusion of the smallest events. in order to separate true events from background noise, we defined mps as particles that were less than 1.0 mm in diameter and expressed cell specific markers. for each sample, 500,000 events have been analyzed. compensation tubes were used with similar reagents as were used in the sample tubes. data were constructed as numerous mps depending on marker presentation (positive or negative) and determination of mp populations (figure 1). calculation of the number of mps per liter plasma was based upon the particle count per unit time, the flow rate of the flow cytometer, and the net dilution during sample preparation of the analyzed mp suspension. mp-exposed antigen concentrations were calculated in each sample by multiplying the total concentration of positive mps by the mean fluorescence intensity of the antigen exposure of the total positive mp population. the reproducibility of epcs using standard protocol was 4.5% determination of mp populations cd41aþ was used as a more specific marker of platelets, and cd64þ was considered as a more specific marker of monocytes. cd31 antigen was determined as an essential berezin et al. 3 marker for endothelial cells, platelets, and leukocytes. cd144þ was used to identify a pure population of endothelial cells. cd31þ/annexin vþ and cd144þ/cd31þ/ annexin vþ mps were defined as apoptotic emps, mps labeled for cd105þ or cd62eþ were determined as mps produced due to activation of endothelial cells. 43 statistical analysis statistical analysis of the results obtained was performed in spss system for windows, version 22 (spss inc, chicago, illinois, usa). the data were presented as mean (m) and standard deviation (+sd) as well as median (me) and 25–75% interquartile range (iqr). to compare the main parameters of patient cohorts, two-tailed student t test or mann-whitney u test were used. to compare categorical variables between groups, w2test and fisher exact test were used. univariable and multivariable regression analysis was used for determining the predictors of decreased cd62eþ to cd31þ/annexin vþ ratio. all sufficient predictors with p <0.2 obtained by univariable regression analysis were included in the multivariate regression model. a two-tailed probability value of <0.05 was considered as significant. results general characteristic of patients participating in the study was reported in table 1. there was a significant difference between healthy volunteers and entire patient cohort in body index mass (bmi), waist circumference, cardiovascular risk factors (hypertension, dyslipidemia, and adherence to smoking), chf class, blood pressure levels, heart rate, lvef, homa-ir, lipid abnormalities, and framingham risk score. however, mets patients without chf have demonstrated lower incidence of dyslipidemia, lower concentrations of ldl-c, hs-crp, srankl, opg, nt-probnp compared with mets subjects with chf. therefore, higher lvef, tg, hdl-c, and homa-ir were found in mets patients without chf in comparison to mets patients with chf. table 2 reports the numbers of circulating mps in patients participating in the study. numerous pmps and mmps with immune phenotypes labeled as cd41aþ and cd64þ were similar in healthy volunteers and entire patient cohort. controversially, there is lower circulating level of activated emps with phenotype cd62eþ and cd105eþ in mets patients compared with healthy volunteers (p < 0.001 for all cases). there were no significant differences between numbers of circulating mps labeled as cd144þ and cd144þ/cd31þ originated from endothelial cells obtained from healthy volunteers and mets patients. although circulating levels of annexin vþ, cd144þ/annexin vþ, and cd144þ/cd31þ/annexin vþ mps derived from apoptotic cells including endothelial cells were similar in both cohorts, cd31þ/annexin vþ mps were significantly elevated in mets patient (p < 0.001). cd62eþ to cd31þ/annexin vþ ratio was significantly elevated in healthy persons when compared with mets patients (p < 0.001), while cd105eþ to cd31þ/annexin vþ ratio was not. interestingly, similarities of circulating levels of mps different origin were determined in both mets patient cohorts apart from cd31þ/ annexin vþ mps. therefore, cd62eþ to cd31þ/annexin vþ ratio was found to be higher in the mets patients without chf compared with mets patients with chf. there was weak correlation between numerous cd31þ/ annexin vþ mps and bmi (r ¼ 0.27, p ¼ 0.001), nt-probnp (r ¼ 0.26, p ¼ 0.001), opg (r ¼ 0.26, p ¼ 0.001), hs-crp (r ¼ 0.25, p ¼ 0.001), and framingham risk score (r ¼ 0.21, p ¼ 0.001). numerous cd62eþ mps correlated positively with bmi (r ¼ 0.22, p ¼ 0.001), waist circumference (r ¼ 0.22, p < 0.001), and negatively with opg (r ¼ �0.23, p ¼ 0.001), hs-crp (r ¼ �0.21, p ¼ 0.001), smoking (r ¼�0.20, p ¼ 0.001). there was correlation between cd62eþ to cd31þ/ annexin vþ ratio, cardiovascular risk factors, hemodynamic performances, and other biomarkers. we found that cd62eþ to cd31þ/annexin vþ ratio was directly related with nt-probnp (r ¼�0.512, p ¼ 0.001), bmi (r ¼ 0.46, p ¼ 0.001), opg (r ¼ �0.412, p ¼ 0.001), hs-crp (r ¼ �0.445, p ¼ 0.001), homa-ir (r ¼ �0.414, p ¼ 0.001), egfr (r ¼ 0.312, p ¼ 0.001), tg (r ¼ �0.304, p ¼ 0.001), dyslipidemia (r ¼ �0.248, p ¼ 0.001), creatinine (r ¼�0.242, p ¼ 0.001), framingham risk score (r ¼�0.23, p ¼ 0.001), waist circumference (r ¼ 0.23, p < 0.001), gender (r ¼ 0.228, p < 0.001 for male), age (r ¼ �0.225, p ¼ 0.001), and smoking (r ¼ �0.212, p ¼ 0.001). therefore, cd62eþ to cd31þ/annexin vþ ratio is associated positively with numerous of mets components (r ¼ 0.42, p ¼ 0.003). figure 1. distribution of mps according presentation of annexin v fits and cd31 pe. the results of flow cytometry analysis. mps: microparticles 4 journal of circulating biomarkers no significant association cd62eþ to cd31þ/annexin vþ ratio with fasting plasma glucose, hba1c, means of systolic and diastolic bp was found. we did not find possible ageand gender-related correlation between metabolic status and the presence of emps. by multivariate regression analyses, nt-probnp (b coefficient ¼ �0.42, p ¼ 0.012), opg (b coefficient ¼ �0.32, p ¼ 0.026), hs-crp (b coefficient ¼ �0.21, p ¼ 0.044), and bmi (b coefficient ¼ 0.142, p ¼ 0.036) were found as independent factors to decrease of cd62eþ to cd31þ/annexin vþ ratio (table 3). discussion the results of the study have shown that circulating numerous of pmps and mmps in subjects with mets (with or without chf) insufficiently distinguished from level obtained in healthy volunteers. we found elevated level of apoptotic emps labeled cd31þ/annexin vþ mps in association with lower level of activated endothelial cellderived mps phenotyped as cd62eþ mps. all these lead to decreased cd62eþ to cd31þ/annexin vþ ratio among patients with mets in comparison with healthy volunteers as well as in mets patients with chf compared with those who did not demonstrate chf. thus, development of chf in mets patients was closely related to altered balance between activated emps and apoptotic endothelial cellderived mps. this phenomenon was described as impaired phenotype of circulating mps that might probably preexist chf and appeared to be clinically significant endothelial dysfunction. 20 whether impaired phenotype of emps is the result of early stages of endothelial injury due to neurohumoral and inflammatory activation associated with dysmetabolic states or chf development or circulating mps that table 1. general characteristic of patients participating in the study a . healthy volunteers (n ¼ 35) entire cohort of enrolled mets patients (n ¼ 101) mets patients without chf (n ¼ 47) mets patients with chf (n ¼ 54) age (years) 46.12 + 4.22 48.34 + 7.80 48.30 + 3.94 48.42 + 6.10 males (n (%)) 23 (65.7%) 64 (63.3%) 30 (63.8%) 34 (63.0%) bmi (kg/m2) 21.5 (16.1–23.5) 28.4 (16.5–32.4)b 28.2 (16.7–31.0) 28.5 (16.8–32.1) waist circumference (sm) 78 (63–89) 93 (76–103) b 92 (77–105) 95 (90–104) hypertension (n (%)) – 68 (67.3%)b 32 (68.0%) 36 (66.7%) i nyha class chf – 17 (16.8%) b – 17 (31.5%) c ii nyha class chf – 22 (21.9%)b – 22 (40.7%)c iii nyha class chf – 15 (14.9%) b – 15 (27.8%) c dyslipidemia (n (%)) – 59 (58.4%)b 26 (55.3%) 33 (61.1%)c adherence to smoking (n (%)) 6 (17.1%) 31 (30.7%)b 16 (34.0%) 15 (27.7%) framingham risk score (%) 2.55 + 1.05 8.12 + 2.88b 8.09 + 2.12 9.28 + 2.32 systolic bp (mm hg) 122 + 5 138 + 6b 137 + 4 139 + 5 diastolic bp (mm hg) 72 + 4 87 + 6b 87 + 5 88 + 4 heart rate, beats per 1 min 66 + 6 75 + 7b 71 + 6 78 + 5 lvef (%) 66.8 (61.2–73.5) 50.6 (42.5–55.3)b 52.4 (48.3–57.5) 44.2 (40.3–48.1)c gfr (ml/min/1.73 m2) 102.1 (91.4–113.2) 93.1 (79.5–109.7) 92.5 (83.1–107.4) 93.8 (80.4–106.8) hba1c (%) 4.75 (4.36–5.12) 6.7 (5.3–8.2)b 6.82 (5.61–8.37) 6.64 (5.53–8.31) fasting blood glucose (mmol/l) 4.52 (4.43–4.76) 6.50 (5.8–7.0)b 6.46 (5.73-6.86) 6.54 (5.69–6.98) insulin (mu/ml) 4.98 (1.5–14.1) 15.45 (13.69–16.62)b 15.2 (12.5–15.7) 15.6 (12.9–16.8) homa-ir (mmol/l � mu/ml) 1.01 (0.91–1.07) 4.46 (4.17–5.20)b 4.36 (4.12–5.18) 4.53 (4.11–5.12) creatinine (mmol/l) 62.1 (55.7–82.4) 71.2 (59.9–87.2) 70.5 (59.6–88.3) 72.3 (56.1–86.9) total cholesterol (mmol/l) 4.76 (4.21–5.05) 5.3 (4.6–6.0)b 5.3 (4.5–5.9) 5.4 (4.8–5.8) ldl-c (mmol/l) 3.10 (2.78–3.21) 3.60 (3.20–4.18)b 3.48 (3.30–4.07) 3.80 (3.20–4.20)c hdl-c (mmol/l) 1.13 (1.05–1.17) 0.94 (0.92–1.06)b 1.01 (0.90–1.13) 0.94 (0.88–1.04) tg (mmol/l) 1.18 (1.07–1.30) 1.68 (1.44–1.98) b 1.77 (1.62–1.95) 1.45 (1.42–1.51) c hs-crp (mg/l) 4.11 (0.97–5.03) 7.96 (4.72–9.34)b 7.80 (4.92–9.43) 8.13 (5.90–10.85)c srankl (pg/ml) 16.10 (2.1–30.1) 29.10 (15.2–56.7) b 24.10 (14.7–36.9) 34.20 (20.1–55.2) c opg, (pg/ml) 88.3 (37.5–136.6) 804.5 (579.9–1055.3)b 718.5 (572.1–846.2) 882.5 (697.1–1046.2)c adiponectin (mg/l) 6.17 (3.44–10.15) 13.65 (10.12–24.93) b 13.61 (9.74–22.35) 14.12 (10.12–23.10) nt-probnp (pg/ml) 96.1 (64.5–125.8) 687.5 (84.7–1244.5)b 92.2 (55.8–133.2) 1475.3 (584.7–2293.5)c se: standard error; iqr: inter quartile range; bmi: body mass index: tg: triglycerides, bp: blood pressure; bmi: body mass index: chf: chronic heart failure; lvef: left ventricular ejection fraction; gfr: glomerular filtration rate; hdl-c: high-density lipoprotein cholesterol; ldl-c: low-density lipoprotein cholesterol; hs-crp: high-sensitive c reactive protein; srankl: serum receptor activator of nf-kb ligand; mets: metabolic syndrome; opg: osteoprotegerin. adata are presented as mean and + se; median and 25–75% iqr. categorical variables are expressed as numerous (n) and percentages (%). p-value is a comparison of mean or median variables (anova test). bsignificant difference between healthy subjects and entire cohort of enrolled mets patients. csignificant difference between mets subjects with and without chf. berezin et al. 5 are able to directly induce endothelial dysintegrity is still not fully clear. 35,44 indeed, the ability of endothelium to release activated emps with pro-angiogenic capacity may have a causality role in improving the clinical outcomes in chf subjects with known mets in comparison to none mets subjects. 44 interestingly, circulating numbers of mps that are phenotypically nearly identical to cd31þ/annexin vþ mps were closely associated with cardiovascular risk factors, while they were not elevated in dysmetabolic disorders without known atherosclerosis or/and cardiovascular diseases. 43–47 probably, subpopulations of mps labeled as annexin vþ are not sensitive marker of early endothelial injury and this requires performing measurements of doubleand triplelabeled annexin vþ mps, such as cd31þ/annexin vþ mps. the results of the study report that numerous cd31þ/annexin vþ mps are not only elevated in mets patients but also they increase sufficiently in chf development in mets population. therefore, nt-probnp, opg, hs-crp, and bmi independently predicted decrease of cd62eþ to cd31þ/annexin vþ ratio reflected impaired immune phenotype in mets with and without chf. we suggested that decreased cd62eþ to cd31þ/annexin vþ ratio and probably elevated apoptotic emps level may discuss surrogate markers of vascular dysfunction at early stages in mets patients with high risk of chf development. in fact, apoptotic emps play a pivotal role in the development of vascular complications in mets and diabetes through promoting various processes, that is, coagulation, thrombosis, and angiogenesis. 47,48 in contrast, activated emps may avoid inducing tissue injury and worsening vasomotion via genome involved mechanisms, and they are able to protect the endothelium from damage. therefore, pmps and leukocyte-derived mps have probably not sufficient effect on vascular integrity and vascular complications among mets. 49 these findings support our hypothesis that imbalance between activated and apoptotic emps may predict cv diseases and events in general population and patients with known t2dm and mets. 50,51 surprisingly, in our study, independent associations of cd62eþ to cd31þ/annexin vþ ratio with cardiovascular risk factors were not found, while association of tg and lipid abnormality with cd62eþ to cd31þ/annexin vþ ratio was shown. a recent study has shown that dyslipidemia and especially increased tg level in mets patient populations may have a negative effect on ability of table 2. numbers of microparticles in participators of the study a . immune phenotype of mps healthy volunteers (n ¼ 35) entire cohort of enrolled mets patients (n ¼ 101) mets patients without chf (n ¼ 47) mets patients with chf (n ¼ 54) cd41aþ mps (n/ml) 23 (19–28) 25 (16–33) 23 (15–31) 27 (19–36) cd64þ mps (n/ml) 3.9 (3.5–4.6) 4.2 (3.2–5.1) 4.0 (3.4–4.8) 4.3 (3.6–5.2) cd62eþ mps (n/ml) 1.35 (0.95–1.68) 1.03 (0.86–1.13)b 1.05 (0.88–1.18) 0.98 (0.89–1.12) cd105eþ mps (n/ml) 2.32 (1.92–2.56) 2.24 (1.85–2.41)b 2.37 (1.92–2.68) 2.09 (1.58–2.50) cd144þ mps (n/ml) 0.29 (0.22–0.36) 0.33 (0.24–0.39) 0.30 (0.22–0.37) 0.35 (0.21–0.40) cd144þ/cd31þ mps (n/ml) 0.87 (0.27–1.25) 0.92 (0.36–1.32) 0.89 (0.32–1.29) 0.93 (0.41–1.33) annexin vþ mps (n/ml) 4655 (3724–6237) 5495 (3988–6957) 5114 (3695–6547) 5844 (4213–7167) cd144þ/annexin vþ mps (n/ml) 0.95 (0.11–1.78) 1.15 (0.13–2.41) 1.08 (0.13–2.39) 1.17 (0.15–2.55) cd144þ/cd31þ/annexin vþmps (n/ml) 0.82 (0.27–1.55) 1.01 (0.39–1.70) 0.94 (0.38–1.52) 1.12 (0.40–1.67) cd31þ/annexin vþ mps (n/ml) 0.154 (0.03–0.21) 0.316 (0.261–0.374)b 0.285 (0.253–0.318) 0.355 (0.294–0.382)c cd62eþ to cd31þ/annexin vþ ratio, unit 8.77 (7.95–9.18) 3.26 (3.23–3.30)b 3.68 (3.47–3.81) 2.76 (2.42–3.04)c cd105eþ to cd31þ/annexin vþ ratio, unit 15.1 (8.59–23.4) 7.07 (4.85–10.90) 8.31 (6.02–10.65) 5.89 (4.11–7.67) iqr: inter quartile range; mps: microparticles; mets: metabolic syndrome; chf: chronic heart failure; adata are presented as median and 25–75% iqr. p-value is a comparison of mean or median variables between both cohorts (anova test). bsignificant difference between healthy subjects and entire cohort of enrolled patients. csignificant difference between mets subjects with and without chf. table 3. univariable and multivariable associations with decrease of cd62eþ to cd31þ/annexin vþ ratio.a univariable analysis multivariable analysis b coefficient p value b coefficient p value framingham risk score (%) �0.014 0.34 – – egfr 0.012 0.22 – – homa-ir 0.018 0.26 – – waist circumference 0.052 0.38 – – bmi 0.16 0.046 0.142 0.036 nt-probnp �0.46 0.001 �0.42 0.012 opg �0.36 0.001 �0.32 0.026 hs-crp �0.28 0.001 �0.21 0.044 adiponectin �0.015 0.22 – – tg �0.032 0.42 – – creatinine �0.025 0.36 – – bmi: body mass index; egfr: estimated glomerular filtration rate; homa-ir: homeostasis model assessment for insulin resistance; nt: probnp: n-terminal pro-brain natriuretic peptide; hs-crp: highsensitive c-reactive protein; tg: triglycerides; opg: osteoprotegerin athe multivariate regression model included all variables with p < 0.2. 6 journal of circulating biomarkers endothelium to produce activated macrovesicles with angiogenic capacities and secreted apoptotic-derived mps. 52,53 therefore, it is discussed the question regarding dyslipidemia-induced apoptotic-related emps production. 54 in fact, infiltration of subintima by ldl may induce production of free radicals, oxidation of cytockeleton, and membrane vesiculation of endothelial cells. 55 the oxidative-driven vesiculation of endothelial cells may relate to low intensity inflammation in vasculature, which associates with over production of cytokines, that is, hscrp, adiponectin, and opg. 56 moreover, membrane vesiculation may enhance inflammatory cytokines in convey of biomechanical stress. 57 as well-known hs-crp and opg appear to be sufficiently increased in mets and they may be compensatory up-regulated in the atherosclerosis and microvascular inflammation. 58 therefore, there was ntprobnp-dependent regulation of microvesiculation in endocardial endothelium. 59 the clinical significance of this phenomenon is still not clear and planned/ongoing clinical studies with large sample population are absent. 60 although initially there was skepticism regarding origin of imbalance of activated and apoptotic emp in patients with impaired glucose metabolism and dyslipidemia, we suppose that inflammatory cytokine over production and probably lipid abnormalities may consider a possible cause of predominantly immune phenotype of mps not directly related with glucose impairment and other parameters of mets. obviously, patients with different types of dysmetabolic disorders might have different patterns of mps, 61 which contribute the development of chf. 62,63 thus, pattern of mps correlates with parameters usually used in the characterization of hf, including bnps, and authors have believed that it presents any advantages over the currently used biomarkers to stratify the patients at the risk of negative clinical outcomes. importantly, decreased cd62eþ to cd31þ/annexin vþ ratio reflected impaired immune phenotype of mps beyond mets parameters and other traditional cv risk factors. finally, determination of impaired phenotype of emps appears to be as novel biological marker of chf development in mets population. study limitations this study has some limitations. the first limitation is the lack of standardization of mp measurements, while commercial flow cytometers are existed. this study is specifically assessing mps between 50 and 1000 nm that might also include exosomes (50–>100 nm), small apoptotic bodies (<1000 nm) and other microvesicles originated from various cells. at the same time, exosomes are not able to express annexin antigen, while they are defined as cd 63þ cd9þ microvesicles. because exosomes and mps are often released concomitantly, differentiation of these two microvesicular species might be difficult and this is a study limitation. it is necessary to note that a large pool of mps might be produced after blood sampling due to destruction of platelets and blood cells. in this study, we used platelet free plasma to prevent the contamination of samples with mps originated from platelets. therefore, preparation of mp isolates from samples is the most sophisticated step for further examination. the next limitation might relate to complicated assay and suffers from resolution of mp detection technique that is worth considering. indeed, there were several technical-related difficulties in the measurement of mps affected centrifugation of samples, labeling of mps, using hd-facs methodology and final assay of results obtained. although hd-facs methodology is widely used, theoretically overlap between two or more fluorochromes might reflect some obstacles for further interpretation of obtained results, especially including size gating in mp determination. therefore, rotor type and centrifugation time theoretically may influence on purity of extracellular vesicles. overall, the definition of a blood mp using flow cytometry is still an area of great debate. however, flow cytometry is commonly used procedure, while it is not standardized and is difficult for use. additionally, we cannot discuss whether cd62eþ to cd31þ/annexin vþ ratio is better than traditional measures (bnp, and so on.) at predicting patient outcomes, because the design of clinical study does not correspond with clinical events’ evaluation. the advantage of cd62eþ to cd31þ/annexin vþ ratio over the currently used biomarkers requires to be reassayed in the large clinical trial in the future. another limitation of this study is that a specific role of mps is also possible and has not been characterized in depth in mets patients. however, the authors suppose that these optionally technically restrictions might have no significant impact on the study data interpretation. additionally, retrospective, relatively small sample size may limit the significance of this study. in conclusion, decreased cd62eþ to cd31þ/annexin vþ ratio reflected impaired immune phenotype of mps might discuss a surrogate marker of chf development in mets population. biomarkers of biomechanical stress (nt-probnp) and inflammation (hs-crp, opg) were found as significant predictors for decreased cd62eþ to cd31þ/annexin vþ ratio in mets patients especially with chf. acknowledgments we thank all patients for their participation in the investigation, staff of the regional zaporozhye hospital (ukraine), and the doctors, nurses, and administrative staff in regional center of cardiovascular diseases (zaporozhye, ukraine) and city hospital # 6 (zaporozhye, ukraine), general practices, and site-managed organizations that assisted with the study. authors’ contributions alexander e berezin initiated the hypothesis and designed the study protocol, contributed to collect, analyze, and interpret the data, performed statistical analysis, and wrote the manuscript. alexander a kremzer contributed to enroll the patients, collected berezin et al. 7 and analyzed the data, reviewed the source documents, and drafted the article. tatyana a berezina contributed to the study protocol design, enrolled the patients in the study, collected the data, analyzed and interpreted the data obtained, and drafted the article. yulia v martovitskaya performed biomarker measurements, including determination of mps, and analyzed and interpreted the data received by flow cytometry. all authors revised the manuscript critically, had consolidated agreement to be accountable for all aspects of the work, and finally approved the version to be published. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) received no financial support for the research, authorship, and/or publication of this article. references 1. ogbera ao. prevalence and gender distribution of the metabolic syndrome. diabetol metab syndr 2010; 2: 1. 2. alberti kgmm, eckel rh, grundy sm, et al. harmonizing the metabolic syndrome: a joint interim statement of the international diabetes federation task 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extracell vesicles 2014; 3: 23111. 42. orozco af and lewis de. flow cytometric analysis of circulating microparticles in plasma. cytometry a 2010; 77(6): 502–514. 43. lacroix r, judicone c, mooberry m, et al. the isth ssc workshop. standardization of pre-analytical variables in plasma microparticle determination: results of the international society on thrombosis and haemostasis ssc collaborative workshop. j thromb haemost. epub ahead of print 2 april 2013. doi: 10.1111/jth.12207. 44. leroyer as, tedgui a and boulanger cm. microparticles and type 2 diabetes. diabetes metab 2008; 34(1): s27–s32. 45. amabile n, cheng s, renard jm, et al. association of circulating endothelial microparticles with cardiometabolic risk factors in the framingham heart study. eur heart j 2014; 35(42): 2972–2979. 46. nomura s. dynamic role of microparticles in type 2 diabetes mellitus. curr diabetes rev 2009; 5(4): 245–251. 47. puddu p, puddu gm, cravero e, et al. the involvement of circulating microparticles in inflammation, coagulation and cardiovascular diseases. can j cardiol 2010; 26(4): 140–145. 48. jimenez jj, jy w, mauro lm, et al. endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis. thromb res 2003; 109(4): 175–180. 49. arteaga rb, chirinos ja, soriano ao, et al. endothelial microparticles and platelet and leukocyte activation in patients with the metabolic syndrome. am j cardiol 2006; 98(1): 70–74. 50. berezin ae. circulating endothelial-derived apoptotic microparticles as novel perspective biomarker for diabetes. diabetes res treat 2014; 1(4): 117–120. 51. berezin ae, kremzer aa, samura ta, et al. inflammatory phenotype of circulating endothelial-derived microparticles in chronic heart failure patients with metabolic syndrome. j mol pathophysiol 2015; 4(2): 51–58. 52. chironi gn, boulanger cm, simon a, et al. endothelial microparticles in diseases. cell tissue res 2009; 335(1): 143–151. 53. shantsila e. endothelial microparticles: a universal marker of vascular health? j hum hypertens 2009; 23: 359–361. 54. müller g, schneider m, biemer-daub g, et al. microvesicles released from rat adipocytes and harboring glycosylphosphatidylinositol-anchored proteins transfer berezin et al. 9 rna stimulating lipid synthesis. cell signal 2011; 23(7): 1207–1223. 55. camussi g, deregibus mc, bruno s, et al. exosome / microvesiclemediated epigenetic reprogramming of cells. am j canc res 2011; 1: 98–110. 56. poulsen mk, nybo m, dahl j, et al. plasma osteoprotegerin is related to carotid and peripheral arterial disease, but not to myocardial ischemia in type 2 diabetes mellitus. cardiovas diabetol 2011; 10: 76–84. 57. wang y, chen lm and liu ml. microvesicles and diabetic complications novel mediators, potential biomarkers and therapeutic targets. acta pharmacol sin 2014; 35(4): 433–443. 58. mause sf and weber c. microparticles: protagonists of a novel communication network for intercellular information exchange. circ res 2010; 107(9): 1047–1057. 59. montoro-garcı́a s, shantsila e, marı́n f, et al. circulating microparticles: new insights into the biochemical basis of microparticle release and activity. basic res cardiol 2011; 106(6): 911–923. 60. berezin a, zulli a, kerrigan s, et al. predictive role of circulating endothelial-derived microparticles in cardiovascular diseases. clin biochem 2015; 48: 562–568. 61. agouni a, andriantsitohaina r and martinez mc. microparticles as biomarkers of vascular dysfunction in metabolic syndrome and its individual components. curr vasc pharmacol 2014; 12(3): 483–492. 62. berezin ae, kremzer aa, samura ta, et al. immune phenotypes of endothelial-derived microparticles in dysmetabolic patients. j proteomics bioinformatics 2015; 8: 060–066. 63. berezin ae, kremzer aa, samura ta, et al. apoptotic microparticles to progenitor mononuclear cells ratio in heart failure: relevance of clinical status and outcomes. jcvd 2014; 2(2): 50–57. 10 journal of circulating 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primary tumor resection mohamed abdouh1, dana hamam1,2, vincenzo arena3, manuel arena4, hussam alamri1,5, and goffredo orazio arena1,6 abstract we reported that single oncosuppressor-mutated (som) cells turn malignant when exposed to cancer patients’ sera. we tested the possibility to incorporate this discovery into a biological platform able to detect cancer in healthy individuals and to predict metastases after tumor resection. blood was drawn prior to tumor resection and within a year after surgery. blood samples from healthy individuals or metastatic patients were used as negative and positive controls, respectively. patients at risk for cancer were included in the screening cohort. once treated, cells were injected into nonobese diabetic/severe combined immunodeficiency mice to monitor tumor growth. all samples of sera coming from metastatic patients transformed som cells into malignant cells. four samples from screened patients transformed som cells. further clinical tests done on these patients showed the presence of early cancerous lesions despite normal tumor markers. based on the xenotransplants size, we were able to predict metastasis in three patients before diagnostic tests confirmed the presence of the metastatic lesions. these data show that this serum-based platform has potentials to be used for cancer screening and for identification of patients at risks to develop metastases regardless of the tumor node metastasis (tnm) stage or tumor markers level. keywords in vitro platform, transformation, metastasis, screening, prediction, patient serum, cell culture date received: 9 february 2016; accepted: 13 july 2016 introduction despite progress in the understanding of the molecular and genetic basis of cancers, cure or even the 5-year survival rate has remained very low due to metastatic disease, which is recognized as the prominent cause of cancer-related death. 1 understanding the mechanisms underlying the metastatic process is the cornerstone to improve cancer patient survival, and such knowledge is needed to develop new prognostic and diagnostic tools. traditionally, metastasis is described as a multistage process initiated by cancer cells detachment from the primary tumor site, circulation of the cells in the blood flow, with subsequent homing in distant sites for the establishment of secondary foci of disease. 2 in this context, research 1 cancer research program, mcgill university health centre-research institute, montreal, quebec, canada 2 department of experimental surgery, faculty of medicine, mcgill university, montreal, quebec, canada 3 department of obstetrics and gynecology, santo bambino hospital, catania, italy 4 department of surgical sciences, organ transplantation and advances technologies, university of catania, catania, italy. 5 department of surgery, king saud university, riyadh, saudi arabia 6 department of surgery, mcgill university, st mary hospital, montreal, quebec, canada corresponding author: goffredo orazio arena, cancer research program, mcgill university health centre-research institute, 1001 decarie boulevard, montreal, quebec h4a 3j1, canada. email: goffredo.arena@mcgill.ca journal of circulating biomarkers volume 5: 1–10 ª the author(s) 2016 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454416663661 cbx.sagepub.com creative commons cc-by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 3.0 license (http://www.creativecommons.org/licenses/by-nc/3.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:goffredo.arena@mcgill.ca http://www.sagepub.co.uk/journalspermissions.nav https://doi.org/10.1177/1849454416663661 http://cbx.sagepub.com https://us.sagepub.com/en-us/nam/open-access-at-sage has mainly been focused on the determination of the identity of these circulating tumor cells (ctcs). nowadays, the detection and molecular characterization of ctcs are one of the most active areas of translational cancer research. 3 if on one hand, tremendous increase in the amount of research, examining the potential clinical utility of ctcs in the management of cancer has been accomplished, on the other hand, the analytical specificity and clinical utility of these detection methods have not been demonstrated unequivocally. 4 controversies have arisen since reports from different investigators have shown conflicting results regarding the prognostic relevance of ctcs, and their exploitation, as a prognostic marker, is still a subject of many ongoing investigations. 5–9 furthermore, the lack of correlation between the presence of ctcs and development of metastatic disease has triggered questions regarding the undisputed validity of the ‘‘seed and soil’’ theory. 10 recent studies have reported that human cancer cells could transfer signaling molecules to target cells predisposing them to malignant transformation. 11–13 this suggests that metastases might occur via transfer of biologically active circulating factors, derived from the primary tumor, to susceptible target cells located in distant organs. this alternative theory has been strengthened by the discovery that blood-circulating factors (i.e. cell-free nucleic acids) or factors carried in circulating microvesicles (such as mrna, micro-rna, mutated and amplified oncogene sequences and retrotransposon elements) are indeed shed from several types of human tumors and have different biological effects on distinct types of cells. 14–21 the oncogenic potential of circulating factors has been first described in murine immortalized fibroblasts and was called ‘‘genometastasis.’’ 22–24 more recently, we observed that exposing immortalized or single oncosuppressor-mutated (som) human cells (i.e. human embryonic kidney 293 (hek293) cells and breast cancer 1 (brca1)-deficient fibroblasts) to metastatic cancer patients’ serum induced their transformation into malignant cells, confirming the validity of the genometastasis theory in human cells. the above effect was not seen if these cells were exposed to sera coming from healthy patients. 25,26 cells transformed even when they were exposed to conditioned media obtained from colon cancer cell cultures proving that the oncotransforming factors were actually produced by cancer cells. based on these observations, we hypothesize that the ability of som cells to incorporate cancer factors could constitute the basis of a novel in vitro serum-based platform that could function both as a cancer screening test for healthy patients and as a predictor of metastatic recurrence after primary tumor resection in cancer patients. materials and methods blood collection and serum preparation blood samples were collected in vacutainer tubes containing clot-activation additive and a barrier gel to isolate serum (becton dickinson, franklin lakes, new jersey, usa). after 60 min clotting at room temperature, tubes were centrifugation at 1500 � g for 15 min. serum was collected and subjected to a second centrifugation at 2000 � g for 10 min to clear it from contaminating cells. serum was aliquoted and stored at �80�c until use. patient categorization recruitment for this study was conducted at the department of general surgery at the royal victoria hospital and st mary’s hospital (montreal, canada), in accordance to an approved ethics protocol by the ethics committee of our institution (sdr-10-057). thirty-seven patients were studied (table 1). the test was conducted using, as a detecting biological platform, hek293 cells and brca1 knockout (brca1-ko) fibroblasts. 26 in the hek293 group, five patients who had undergone resection of primary cancer and readmitted for treatment of metastatic disease served as positive controls (cases m1–m5). eight healthy individuals (cases c1–c8) were enrolled in the study as a negative control group. the inclusion criteria considered to be enrolled in the healthy cohort were (i) age (30–60-year old), (ii) no signs and symptoms or personal history of cancer, and (iii) family history negative for cancer. eight patients with clinical suspicion for cancer were enrolled in the screening cohort (cases s1–s8). sixteen patients admitted for resection of different cancers were recruited and monitored for metastatic recurrence (cases f1–f16). in the brca1-ko fibroblast group (table 2), 3 healthy individuals were used as negative controls (cases c1–c3), 10 patients with metastatic disease served as positive controls (cases m6–m15), 2 patients (cases s8 and s9) were enrolled in the screening group, and 2 patients (cases f17 and f18) in the monitoring group. patient medical statuses and follow-up durations (mean ¼ 22 months; range 3–60 months) are summarized in tables 1 and 2. blood samples were obtained with written consent from all participants. cell culture conditions hek293 cells were from atcc (manassas, virginia, usa). brca1-ko fibroblasts were established as described previously. 26 cells were maintained in recommended culture medium until 30% confluence, at which point, they were treated with dmem-f12 medium (wisent, québec, canada) supplemented with 10% v/v of either cancer patient or control serum filtered through 0.2-mm filters. half of the media was changed every second day. when the cells reached 80% confluence, they were passaged (one in six) using trypsin/ethylenediaminetetraacetic acid (wisent). to confirm that there was no contamination or carryover of blood-derived cells, aliquots of the culture medium were placed in a culture plate and incubated at 37�c, 5% carbon dioxide. 2 journal of circulating biomarkers population doubling level calculation cells were considered at population doubling zero at the first time they were exposed to patient serum-containing culture medium. at every passage, cell number was determined and population doubling level (pdl) was calculated using the following formula: pdl ¼ log(nh/ni)/log2, where nh is the number of cells harvested at the end of the incubation time and ni is the number of cells inoculated at the beginning of the incubation time. cumulative pdl was calculated by adding the previously calculated pdl. in vivo tumor growth five-week-old female nonobese diabetic/severe combined immunodeficiency (nod/scid) mice (jackson laboratory, bar harbor, maine, usa) were used in compliance with mcgill university health centre animal compliance office (protocol 2012–7280). cells growing in log phase were harvested and washed twice with hank’s balanced salt solution (hbss). mice were injected subcutaneously with 2.10 6 cells in 200 ml hbss/matrigel mixture. mice were followed-up daily for any sign of discomfort and tumor table 1. clinical profiles of the patients enrolled (hek293 cells group). cases id blood collection disease tnm status transforming potential metastases follow-up (months) cea (ng/ml) c1 – healthy – no – – – c2 – healthy – no – – – c3 – healthy – no – – – c4 – healthy – no – – – c5 – healthy – no – – – c6 – healthy – no – – – c7 – healthy – no – – – c8 – healthy – no – – – m1 post-op (visit 1) crc lm yes yes – – m1-1 post-op (visit 2) crc lm yes yes – – m2 post-op pcc lm yes yes – – m3 post-op crc lm yes yes – – m4 post-op bc lm yes yes – – m5 post-op crc lm yes yes – – s1 – healthy – no – – – s2 – healthy – no – – – s3 – healthy – no – – – s4 – thyroid cyst benign no no 6 2.0 s5 pre-op panc. cyst. carcinoma in situ yes no 3 1.2 s6 pre-op crc t1n1 yes no 8 1.0 s7 pre-op liver mass benign no no 3 2.0 s8 pre-op sigmoid cancer t3n2 yes no 3 2 f1* pre-op crc t3n0 yes no 7 3 f1 post-op crc t3n0 yes no 7 3.9 f2* pre-op crc t3n0 yes no 7 12 f2 post-op crc t3n0 yes yes 7 11 f3* pre-op crc t3n0 yes no 6 6.7 f3 post-op crc t3n0 yes no 6 1.4 f4 post-op crc t3n2 yes yes 3 22 f5 post-op crc t3n0 yes no 4 0.4 f6 post-op crc t4n1c yes no 20 3.7 f7 post-op crc t4n0 yes no 16 1.6 f8 post-op crc t3n0 yes no 12 1.8 f9 post-op crc t2n1 no no 48 1.5 f10 post-op crc t4n0 yes no 60 1.3 f11 post-op crc t3n0 yes no 36 0.7 f12 a post-op crc t2n0 yes no 60 1.4 f13 post-op sbc – yes no 60 1.6 f14 post-op crc t3n0 yes yes 60 6.7 f15 post-op lymphoma – yes no 12 – f16 post-op lung cancer t1n0 yes no 6 2.1 bc: breast cancer; crc: colorectal cancer; lm: liver metastasis; pcc: pancreatic cancer; panc. cyst.: pancreatic cystic cancer; sbc: small bowel cancer; hek293: human embryonic kidney 293; cea: carcinoembryonic antigen. a patient f12 was suspected to have a metastatic lesion, but the biopsy revealed a nonmalignant lesion. *blood collected pre-op. abdouh et al. 3 growth monitoring. four weeks postinjection, mice were euthanized and tumor size recorded with a caliper. histological analysis mice xenotransplants were collected, fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin according to standard protocols or processed for immunohistochemistry. briefly, 5-mm tissue sections were dewaxed in xylene and rehydrated with distilled water. after antigen unmasking, and blocking of endogenous peroxidase (3% hydrogen peroxide), the slides were incubated with primary antibodies specific for tumor markers as described previously. 25,26 labeling was performed using iview dab detection kit (ventana, oro valley, arizona, usa) on the ventana automated immunostainer. sections were counterstained lightly with hematoxylin before mounting. histological analyses were performed by a certified pathologist who was blinded to the type of cells from which the cancerous masses, which formed in mice, had been derived. statistical analysis for in vitro cell growth and viability, statistical differences were analyzed using an analysis of variance followed by the scheffé post hoc test for multiple comparisons. given that samples distribution was not normal, we applied the nonparametric wilcoxon rank sum significance test to compare xenotumor sizes obtained with the sera of metastatic and nonmetastatic patients (regardless of groups; screening, control, or confirmed metastatic). to analyze data of the screening test, we ranked all data (tumor volumes) and determined the 0.95 percentile, which helped, set a cutoff. this was set at 0.13 cm 3 , and this value was used as a threshold for metastasis prediction in the screening group. for this purpose, we used 2 � 2 table and fisher’s exact test. for all these tests, a p value <0.05 was considered statistically significant. results cancer patient sera did not affect either cell proliferation or cell viability during in vitro exposure our previous studies had shown that serum from metastatic cancer patients increased the proliferation and induced the transformation of primed human cells. 25,26 in the present study, we tested the effectiveness of this discovery to be used to develop a biological platform that could be used as a cancer screening tool in healthy individuals and as a way to predict which patients might develop metastases after primary tumor resection. we verified that treated cells had the same growth potential and the same viability at the time when they were injected in nod/scid mice to test for their tumorigenic potential. for this purpose, we treated som cells for 2 weeks with daily medium refreshment. every 5 days, cells were collected, counted, and passaged. at every passage, we calculated the pdl in each condition (figure 1(a) and (b)). in addition, we determined cell viability (figure 1(c)). independently of the serum used, neither the cumulative pdl nor cell viability varied significantly. these data suggest that treated cells had the same proliferation potential at the end of the in vitro exposure regimen. table 2. clinical profiles of the patients enrolled (brca 1-ko fibroblasts group). cases id blood collection disease site of metastasis transforming potential metastases follow-up (months) cea (ng/ml) c1 – healthy – no – – – c2 – healthy – no – – – c3 – healthy – no – – – m6 post-op adrenal cancer lung yes yes – – m7 post-op bc lung and liver yes yes – – m8 post-op net liver yes yes – – m9 post-op bc liver yes yes – – m10 post-op crc liver yes yes – – m11 post-op anal scc liver yes yes – – m12 post-op crc liver yes yes – – m13 post-op crc liver yes yes – – m14 post-op crc liver yes yes – – m15 post-op crc liver yes yes – – s9 pre-op pcc peritoneal carcinomatosis yes yes 3 40 s8 pre-op crc – yes no 3 2 f17 post-op crc liver yes yes 5 6 f18 post-op lung cancer – yes no 3 2 bc: breast cancer; crc: colorectal cancer; pcc: pancreatic cancer; scc: squamous cell carcinoma; net: neuroendocrine tumor; brca1-ko: breast cancer 1 knockout; cea: carcinoembryonic antigen. 4 journal of circulating biomarkers validation of the efficacy of the som cell platform as cancer screening test to validate the accuracy of our som cell-based blood test, we recruited nine patients with clinical suspicion of cancer and we enrolled them in the screening group. eight cases were tested on hek293 cells (cases s1–s8), and two patients (cases s8 and s9) were tested on brca1-ko fibroblasts. one patient was tested with both types of cells (case s8). fifteen patients (cases m1–m5 in the hek293 group and cases m6–m15 in the brca1-ko fibroblast group) who had undergone resection of primary cancer and readmitted for treatment of metastatic disease were used as positive controls. one patient was tested twice at an interval of about 3 years (case m1 and m1-1 in the hek293 group). at the end of the in vitro treatments, cells were injected subcutaneously in nod/scid mice and tumor growth was monitored. once tumors were palpable, their diameters were measured (figure 2(b) and (c)). in addition, tumor volumes were calculated at euthanasia (figure 3). four of nine patients that were screened with the som cell platform transformed the som cells into cancer (cases s5, s6, s8, and s9). upon further investigations, case s9 was found to have elevated carbohydrate antigen 19-9 (ca 19-9) and pancreatic cancer. case s6 was found to have a large sessile polyp in the colon and normal carcinoembryonic antigen (cea). pathological analysis of the resected colon showed a t1n1 colon cancer. case s5 on computed tomography (ct) scan was found to have large pancreatic cysts and normal ca 19-9. because of the size of the cyst, despite normal tumor markers, the patient underwent a subtotal pancreatectomy, whose pathological analysis showed pancreatic cancer in situ. case s8 upon further investigations was found to have a mass in the sigmoid colon and normal cea. the pathology analysis of the resected specimen showed a t3n2 colon cancer. in summary, four of four patients that were positive at the som cell platform test were found to have cancer despite normal tumor markers. the other five patients who were negative at the som cell test were still investigated to rule out neoplastic disease. while three of them (cases s1–s3) were also negative when screened with conventional tests (serum values, imaging tests), the other two patients were found to have a thyroid nodule concerning for cancer (case s4) and a liver mass (case s7). upon further investigations, the thyroid nodule was found to be benign as predicted from the som platform. the concerning features of the liver mass prompted a surgical resection of the liver whose pathology showed a benign atypical hemangioma, confirming the accuracy of the som platform results. altogether, these findings suggest that som cells are capable to sense cancer-produced factors even when tumor markers are negative and suggest that this in vitro blood serum-based platform might be used as a screening test to rule out neoplastic disorders. som cells accurately respond to the presence of circulating cancer factors in metastatic disease and transform into cancer all sera from patients with metastases transformed the som cells as confirmed by tumor formation following transplantation in nod/scid mice (fisher’s exact test, p < 0.01; tables 1 and 2, figures 2(b), 3, and 4). histopathological analyses of the hek293 cells derived tumors showed that they had identical histological appearances and the types of tumors grown were not dependent of the patient’s type of cancer. the histology confirmed that most tumors were poorly differentiated carcinomas with a high mitotic index (over 90%) and small foci of necrosis (figure 2(e)). attempts to characterize these tumors 0 20 40 60 80 100 (c) c el l vi ab il it y (% ) 0 2 4 6 8 10 12 14 16 18 0 5 10 15 screening metastatic control monitoring (a) c u m u la ti ve p op u la ti on d ou b li n gs culture duration (days) 0 2 4 6 8 10 12 14 16 18 (b) c u m u la ti ve p op u la ti on d ou b li n gs figure 1. cell growth and viability were not affected during in vitro treatment duration. cells were cultured for 2 weeks in human serum as stated in the legend. (a) and (b) cell growth was analyzed by counting the number of viable cells at every passage (5 days duration for every passage). (a) line graph shows the population doublings capability and (b) column graph represents cumulative population doublings at the end of the in vitro treatment periods. the ordinate axis are the same in (a) and (b). (c) cell viability was calculated as the percentage of viable cells over total counted cells using trypan blue exclusion dye. data are mean + sd (p > 0.05). abdouh et al. 5 immunohistochemically failed to show any more differentiating features. on the other hand and strikingly enough, histopathological analyses of the brca1-ko fibroblasts showed full differentiation of the cells into colon and pancreatic cancer when they were exposed to colon cancer serum and pancreatic cancer serum, respectively, proving the hypothesis that som cells are able to integrate cancer factors in their genome. 26 the in vitro blood test was able to predict patients at risk for metastases based on the evidence that the sera of metastatic patients gave rise to tumor significantly greater than those obtained with the sera of nonmetastatic patients (wilcoxon rank sum test, p < 0.01; figure 3(a)) and given the fact that the same trend was observed when we analyzed the xenotransplants obtained with cells treated with the sera of patients enrolled for the monitoring study (wilcoxon rank sum test, p < 0.01; figure 3(b)), we asked whether the size of the xenotransplants could be utilized as a predictor for future metastatic recurrence. we ranked all the xenotransplants according to the tumor volumes and determined the 0.95 percentile. this allowed the determination of a cutoff value that was set at 0.13 cm 3 . this value was used as a threshold for metastasis prediction in patients enrolled in the monitoring group. in this cohort of patients, we tested our in vitro cellbased platform with sera of 18 patients (cases f1–f18; tables 1 and 2). three of 15 patients (cases f1–f3) underwent the hek293 test with blood drawn prior and after surgical resection of the tumor. the sera of all tested (a) control days after inoculation days after inoculation days after inoculation (c) screening(b) metastatic (e). metastatic monitoringscreening 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 5 10 15 20 25 30 c1 c2 c3 c4 c5 c6 c7 c8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 5 10 15 20 25 30 s1 s2 s3 s4 s5 s6 s7 s8 (d) monitoring t u m or d ia m et er ( cm ) t u m or d ia m et er ( cm ) t u m or d ia m et er ( cm ) t u m or d ia m et er ( cm ) days after inoculation 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 5 10 15 20 25 30 f1* f2* f3* f4 f6 f8 f10 f12 f14 f16 f1 f2 f3 f5 f7 f9 f11 f13 f15 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 5 10 15 20 25 30 m1 m2 m3 m4 m5 m1-1 figure 2. time course of xenotransplants growth. (a) to (c) hek293 cells were cultured for 2 weeks in human serum as stated in the figure. cells were injected subcutaneously in nod/scid mice and tumor growth was monitored every second day. once tumors were palpable, their diameters were measured (a) to (d), and their volumes at euthanasia were calculated (see figure 3). values are mean + sd (n ¼ 2–3 mice per group). representative pictures of tumors obtained are shown. (e) formalin-fixed paraffin-embedded xenotransplant samples were processed for h&e staining. hek293: human embryonic kidney 293; nod/scid: nonobese diabetic/severe combined immunodeficiency; h&e: hematoxylin and eosin. 6 journal of circulating biomarkers patients, but one (case f9), still retained their oncotransforming abilities even after 5 years from primary tumor resection. when we measured the size of the xenotransplants, we noticed that sera of patients who had no signs of metastasis generated xenotransplants with sizes smaller than 0.13 cm 3 (0.04 + 0.02 cm3; range 0.01–0.08 cm3; figure 3(b) and (c)). in contrast, in the hek293 group, the sera of five patients (cases f1–f4 and f14) gave rise to xenotransplants larger than 0.13 cm 3 (0.18 + 0.12 cm3; range 0.14–0.47 cm3; figure 3(b) and (c)). four of them displayed metastatic disease (cases f1, f2, f4, and f14). statistical analysis showed that xenotransplants volume and metastasis are dependent variables (figure 3(b), fisher’s exact test, p < 0.01). three of the five patients had serum drawn also prior to surgical resection (cases f1–f3). when we compared the size of the xenotransplants, in these patients, we noticed that the size of the xenotransplants had not decreased after surgical resection but it had actually increased in two of them (cases f1 and f2). one of them (case f2) developed metastases at 7 months after the resection. the other patient (case f1) has been found to have cea values higher than normal and is currently monitored every 3 months with ct scans to rule out metastatic recurrence (table 1). case f3 had the test (c) (a) (b) t u m or v ol u m e (c m 3 ) no metastasis (n = 32) 0.6 metastasis (n = 9) 0.4 0.2 0.0 0.8 1.0 t u m or v ol u m e (c m 3 ) no metastasis (n = 24) 0.3 metastasis (n = 3) 0.2 0.1 0.0 0.4 control metastatic screening t u m or v ol u m e (c m 3 ) 0.9 c1 0.6 0.3 0.0 monitoring patients who developed metastasis c2 c3 c4 c5 c6 c7 c8 m1-1m1 m2 m3 m5m4 s1 1.0 s2 s3 s4 s5 s6 s7 s8 f1 * f1 f2 * f2 f3 * f3 f4 f5 f6 f7 f8 f9 f10 f11 f12 f13 f14 f15 f16 figure 3. effect of patient serum on the tumorigenicity of hek293 cells. hek293 cells were cultured for 2 weeks in human serum as stated in the figure. cells were injected subcutaneously in nod/scid mice. four weeks after injection, growing tumors were excised and their volumes were calculated. (a) whisker plot for the size of the xenotransplants obtained with cells treated with the sera of patients who developed metastasis and those who did not, regardless of the groups (control, metastatic, screening, or monitoring). note that metastatic patients’ sera gave rise to tumor significantly greater than those obtained with the sera of nonmetastatic patients (p < 0.01). (b) whisker plot for the size of the xenotransplants obtained when the analysis was done using only xenotransplants obtained with cells treated with the sera of patients in the monitoring group. note that metastatic patients’ sera gave rise to tumor significantly greater than those obtained with the sera of nonmetastatic patients (p < 0.01). (c) barres plot showing xenotransplants obtained for each individual involved in the study. values are mean + sd (n ¼ tumors obtained with 2–3 mice per group). the line represents the threshold for metastasis prediction (set at 0.13 cm3). *blood collected pre-op. hek293: human embryonic kidney 293; nod/scid: nonobese diabetic/severe combined immunodeficiency. abdouh et al. 7 repeated 6 months after surgery and it was noted that the size of the xenotransplants obtained with the serum this time was below the metastatic risk threshold compatible with her t3n0 stage. interestingly, the serum of one patient (case f12) who was diagnosed with metastatic lesions in the liver induced only a slight transformation of hek293 cells with size of the xenotransplants not typical for metastatic disease but more in keeping with nonactive neoplastic disease. after surgical resection, the pathology of the liver revealed that the lesion in the liver was not malignant as predicted by the hek293 test (table 1 and figure 3(c)). together, these data indicate that som cells are capable to sense cancer-produced factors even after the primary tumors have been removed and suggest a possible use of this biological platform as a way to identify patients at risks to develop metastases after primary cancer resection. discussion recently, our group reported that som cells turn into malignant cells when exposed to sera of patients with metastatic cancer. the above effect was not seen if som cells were exposed to sera coming from healthy patients. 25,26 in the present study, our goal was to test the hypothesis that cells with a single oncosuppressor mutation might be used to identify cancer factors circulating in the blood and explore the potential to incorporate this discovery into a serum-based platform that could be used to both screen healthy patients for cancer disease and to predict which patients might develop metastases after primary tumor resection. to confirm the validity of this hypothesis and the accuracy of this in vitro blood test, we used the sera of four different cohorts of patients: patients with established metastatic diagnosis, healthy donors with no history or sign of cancer, patients with clinical suspicion of cancer, and patients with resected cancer disease. the results of the tests in the screening group proved the effectiveness of this som cell platform to detect cancercirculating material even at early stages and regardless of the presence of positive tumor markers. the ability of the test to predict pancreatic cancer in situ and early colon cancer seems promising since none of the blood tests available nowadays has shown such sensitivity. the negative pathology results shown on the two patients who were taken to surgery for strongly suspicious lesions strengthen the evidence that this test has great potentials to be also highly specific. for these reasons, it seems that the ability of som cells to ‘‘sense’’ neoplastic factors in the blood might help redefining new diagnostic approaches to cancer disease incorporating high sensitivity and high specificity in a single test effective at detecting different types of cancers, in different organs. we called this novel test metastatic and transforming elements released discovery platform (mater-d platform) to highlight the newly found evidence that som cells are truly able to detect cancer factors circulating in the serum of patients, integrate them in their genome, and undergo malignant transformation. 25,26 the observation that sera of patients who had cancer resection and are cancer free still retains its oncotransforming ability on the som cells, even after 5 years, paves the way to fascinating hypotheses. speculations can be made that cancer cells or circulating mutated oncogenes are still present in the body even after resection of the primary tumor, as already shown in other studies 27,28 but the gnineercscitatsatemlortnoc t u m or v ol u m e (c m 3 ) 3 c1 2 1 0 monitoring patients who developed metastasis c2 c3 m6 m7 m8 m9 m10 m11 m12 m13 m15m14 4 s8s9 f17f18 figure 4. effect of patient serum on the tumorigenicity of brca1-deficient fibroblasts. cells were cultured for 2 weeks in human serum as stated in the figure. cells were injected subcutaneously in nod/scid mice. four weeks after injection, growing tumors were excised and their volumes were calculated. barres plot display xenotransplants obtained for each individual involved in the study. values are mean + sd (n ¼ tumors obtained with 2–3 mice per group). nod/scid: nonobese diabetic/severe combined immunodeficiency; brca1: breast cancer 1. 8 journal of circulating biomarkers oncogenic potential of these factors is counterbalanced by homeostatic processes such as the immune system, with mechanisms still unknown to us. the size of the cancerous xenografts could be viewed as a reflection of the efficiency of these mechanisms, with larger xenografts representing failure of the protective mechanisms to control the proliferation of the cancer cells. in our small study, the formation in the hek293 group of malignant xenografts larger than 0.13 cm 3 was correlated with a higher risk to develop metastases after surgical resection regardless of the tnm stage of the tumor. the evidence that in one patient (case f3) the size of the xenografts were above the metastatic threshold to then fall down below it after surgery, strengthen our belief that the size of the xenografts might be a valid representative of the effectiveness of the treatment and an indicator of the interaction between cancer and the body defensive mechanisms. we couldn’t extrapolate any metastatic risk value in the brca1-ko fibroblast group due to the small number of patients enrolled in this cohort. we are currently running parallel experiments with both groups of som cells to verify the presence of any statistically significant value in the brca1-ko fibroblast group. our recent discovery that brca1-ko fibroblasts fully differentiate into colon and pancreatic cancers when exposed to sera of patients with colon and pancreatic cancer, respectively, strengthens the notion proved herein that som cells are able to incorporate genetic material in their genome and undergo malignant transformation. identification of the oncogenic factors responsible for the malignant transformation of the som cells would be the next natural step to better understand the role of these hypothetical factors in cancer disease and metastatic recurrence. putative factors that might be implicated in the observed effects and which are being currently investigated, in order to develop new diagnostic tests, are circulating cell-free dna, or molecules packed in circulating microvesicles. 19,22–24 a growing body of evidence demonstrates that cancer cells are capable of generating microvesicles in vivo, whose number and production increase with cancer stages. 11,29– 33 in keeping with that, high levels of circulating microvesicles have been associated with poorly differentiated tumors and shorter disease overall survival in patients with colorectal cancer and the degree of malignancy in ovarian cancer. 33,34 we reported recently that oncosuppressordeficient target cells were significantly more prone to internalize cancer patients’ serum-derived exosomes, when compared to wild-type target cells. 26 this finding suggests that the efficacy of our platform seems to be dependent on the same principle behind the liquid biopsy tests: the detection of mutated genes carried in the blood either as free circulating material or packed in exosomes or microvesicles. 11,12,20,21,35–39 although the results of our experiments are striking, several limitations can be seen in this study such as the small number of patients enrolled so far and the prevalence of mainly colorectal cancer cases. these limitations can be overcome by a proper designed clinical trial, with an adequate sample size and more heterogeneous cohorts of cancer patients. if the validity of the mater-d platform to detect transfecting material in the serum, of both healthy patients and primary cancer-treated patients, is proven with a larger and properly designed trial, primary and tertiary prevention might be incorporated in a single test, making the secondary prevention efficacious at its best. furthermore, this cell-based platform might have strong potential to help identifying those circulating biomarkers that can be detected early during cancer formation and possibly during metastatic recurrence. the discovery of these factors would eventually lead to the creation of antibody-based laboratory tests, which might hold strong promise for early detection of cancer in healthy patients and for supporting cancer patient management and monitoring. acknowledgment we are grateful to ayat salman for her assistance with the ethical committee approvals. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was financially supported by giuseppe monticciolo. the funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. references 1. jemal a, bray f, center mm, et al. global cancer statistics. ca cancer j clin 2011; 61: 69–90. 2. nguyen dx, bos pd and massagué j. metastasis: from dissemination to organ specific colonization. nat rev cancer 2009; 9: 274–284. 3. danova m, torchio m and mazzini g. isolation of rare circulating tumor cells in cancer patients: technical aspects and clinical implications. expert rev mol diagn 2011; 11(5): 473–485. 4. alix-panabières c and pantel k. circulating tumor cells: liquid biopsy of cancer. clin chem 2013; 59(1): 110–118. 5. cohen sj, punt cj, iannotti n, et al. prognostic 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review article alexander e. berezin1* 1 state medical university, zaporozhye, ukraine *corresponding author(s) e-mail: dr_berezin@mail.ru received 06 november 2015; accepted 02 march 2016 doi: 10.5772/62797 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract heart failure (hf) is multifactorial syndrome with high cardiovascular (cv) morbidity and mortality rates associ‐ ated with an increasing prevalence worldwide. measuring plasma levels of circulating biomarkers, i.e., natriuretic peptides, cardiac-specific troponins, metabolomic inter‐ mediates, galectin-3, st2, cardiotrophin-1, soluble endo‐ glin and growth differentiation factor 15, may assist in the prognostication of hf development. however, the role of biomarker models in the prediction of an early stage of hf with a preserved ejection fraction (hfpef) and hf with a reduced ejection fraction (hfref) is not still understood. this review explores the knowledge regarding the utility of cardiac biomarkers, aiming to reclassify patients with different phenotypes of hf. the review reports that several biomarkers reflected on subsequently alter collagen turnover, cardiac fibrosis and inflammation, which might have diagnostic and predictive value in hfpef and hfref. the best candidates for determining the early stage of hf development were sst2, galectin-3, ct-1 and gdf-15. however, increased plasma concentrations of these biomarkers were not specific to a distinct disease group of hfpef and hfref. finally, more investigations are required to determine the role of novel biomarkers in the prediction of hf and the determination of the early stages of hfpef and hfref development. keywords heart failure phenotypes, biomarkers, prog‐ nostication, risk stratification abbreviations bnp – brain natriuretic peptide cad – coronary artery disease cabg – coronary artery bypass grafting gdf – growth differentiation factor ct-1 – cardiotrophin-1 cv – cardiovascular hf – heart failure hfpef – heart failure with preserved ejection fraction hfref – heart failure with reduced ejection fraction lv – left ventricle pci – percutant coronary angioplasty procedure 1j circ biomark, 2016, 5:6 | doi: 10.5772/62797 1. introduction heart failure (hf) remains an important clinical entity that has increased in prevalence worldwide due to improved survival after a hf diagnosis [1, 2]. recent studies have shown sufficient differences in the aetiology, pathophysi‐ ology, clinical presentation and outcomes, as well as the prognosis, between hf with a preserved ejection fraction (hfpef) and hf with a reduced ejection fraction (hfref) [3-5]. hfpef is a phenotypic and heterogeneous clinical syn‐ drome characterized by cardiovascular (cv) disease and dysmetabolic and inflammatory states associated with both advanced age and various non-cv co-morbidities, which finally lead to the impairment of myocardial structure and function, unless under the condition of declining global ef <45% [6, 7]. although global left ventricular ef >50% is currently used to differentiate between reduced and preserved cardiac pump function, this cut-off point is widely discussed as a likely inadequate criterion [8, 9] however, old age, being female, suffering from diabetes mellitus, hypertension, atrial fibrillation and chronic kidney disease are strong predictors of hfpef’s development [10-12]. based on evidence from endomyocardial biopsies, some of the specific cardiac structural phenotypes to be targeted in hfpef may be represented by myocyte hypertrophy and interstitial fibrosis [13, 14]. hfref has been described as a disease of aged elderly subjects, with a male predomi‐ nance that is frequently associated with dilation cardiomy‐ opathy, ischaemia, inflammatory and diabetic aetiology, and rarely with arterial and pulmonary hypertension [15, 16]. cell loss due to ischaemia, apoptosis and necrosis, myocardial inflammation associated with oxidative stress, expanded interstitial fibrosis leading to the disintegrity of the cardiac wall, increased passive myocardial stiffness, the worsening of cardiac configuration and contractile func‐ tion are common in hfref’s development [17]. many questions remain unanswered regarding differences in the molecular signals that initiate the development of hfpef and hfref [18]. in this context, it might be possible to appropriately stratify at risk hfpef and hfref patients by using biomarkers. recently, brain natriuretic peptides, cardiac specific troponins, metabolomic intermediates, galectin-3, st2, cardiotrophin-1, soluble endoglin, growth differentiation factor 15 and other new biological markers associated with hf’s development have been widely investigated [5, 6, 12, 13, 17, 19]. however, the current data on the interrelationship of these biomarkers and pheno‐ types of hf are limited. the aim of the review is devoted to the accumulation of knowledge regarding the utility of cardiac biomarkers, aiming to reclassify patients with different phenotypes of hf. 2. biomarkers in hf risk stratification the routine use of biomarkers might help to stratify the patients with hfref and hfpef at higher risk of death and clinical outcomes. the current guidelines — the 2012 european society of cardiology (esc) guidelines for the diagnosis and treatment of acute and chronic heart failure and the 2013 american college of cardiology foundation/american heart association (accf/aha) guideline for the management of heart failure — are well accepted by many clinicians regarding hfref’s prognosti‐ cation. indeed, hfpef is the one that really requires the improvement of biomarkers for diagnosis and prognosis. in this context, many biological markers, which reflect several faces of the pathogenesis of hf, have been investi‐ gated in detail, but only natriuretic peptides, soluble st2, galectin-3 and highly sensitive cardiac-specific troponins have been validated thus far. table 1 offers summarized evidence regarding the predictive role of biomarkers in patients with different hf phenotypes. 3. brain natriuretic peptides within the last two decades, cardiac natriuretic peptides (bnp and nt-probnp) have been defined as biomarkers that we may use to screen for lv systolic dysfunction in patients with symptoms suggestive of hf. bnp and ntprobnp are now included in the current guidelines for hf diagnosis, management and risk assessment because of their high specificity and sensitivity [19]. despite bnp and nt-probnp improving discrimination modestly for hf above and beyond conventional risk factors, and substan‐ tially improving the risk classification for hf, peak con‐ centrations of bnp and nt-probnp and serial measurements of nt-probnp levels in longitude are not able to allow the differentiation of hf phenotypes [20, 21]. however, there were important differences in the prog‐ nostic value of nt-probnp in hfpef versus hfref in the ntprobnp-guided arm of the time-chf study [22]. ntprobnp has demonstrated less prognostic value in hfpef as compared to hfref, and has not predicted a develop‐ ment of hfpef or hfref. nt-probnp lost significance as a risk stratifier in ambulatory patients with stable hf and probably also in those who have hfpef. there are attampts to use of sing sample measurement of mid-regional atrial natriuretic peptide (mr-anp) and nt-proanp in order to screen hfpef and hfref in individuals, when the diag‐ nosis of hf is not obvious. in this setting, the diagnostic value and prognostic ability for hf-related mortality and cv hospitalization for both mr-anp and nt-proanp were not superior to those of nt-probnp [23]. 4. cardiac troponins recent studies have shown that elevated levels of highly sensitive cardiac troponin i (hs-ctni) and t (hs-ctnt) as biomarkers of subclinical myocardial injury may provide to be clinically useful prognostic information, concerning both the future risk of hf’s manifestation in asymptomatic subjects and the risk of fatal events and primary/readmissions in the hospital in those with already established symptomatic acute, acutely decompensated and chronic 2 j circ biomark, 2016, 5:6 | doi: 10.5772/62797 stable hf related to ischaemic and non-ischaemic causes [24-27]. moreover, cardiac troponin mutations are consid‐ ered a cause of impaired relaxation in the mutant cardiac myocytes due to myofibril hypersensitivity to ca2+ [28]. cardiac-specific troponins exhibited the strongest associa‐ tions with hospitalization, survival and outcomes in cases of hf; there are expectations regarding the ability of troponins to emerge as an aetiology-dependent relation to phenotypes of hf. seliger et al. [29] hypothesized that hsctnt would identify hf risk among older adults with left ventricular hypertrophy (lvh). in the cardiovascular health study, its authors found that the adjusted risk of hfref was 7.8 times higher among those with the highest tertile of hs-ctnt and lvh (hr=7.83; 95% ci: 4.43-13.83). patients with lvh and longitudinal increases in hs-ctnt or nt-probnp were approximately three times more likely to develop hf (primarily hfref), compared with those without lvh and with stable biomarkers. thus, in this study, the authors were not able to find sufficient advan‐ tages regarding hs-ctnt compared nt-probnp in order to characterize sub-phenotypes of hf. in another study, neeland et al. [30] reported that identifying a malignant sub-phenotype of lvh was the better predictive surrogate marker than a limited elevated level of hs-ctnt, and even increased nt-probnp among asymptomatic individuals with a high risk of progression to hf and cv death in the general population. therefore, there was evidence that the higher levels of ctnt and nt-probnp correlated well with the risk of hf in older adults, but were not associated with phenotypes of hf [31]. overall, the circulating level of the cell injury biomarker is not a powerful tool for hf-pheno‐ type detection. 5. systematic metabolomic biomarkers zordoky et al. [32] suggested that a systematic metabolo‐ mic analysis would reveal a novel metabolomic fingerprint of hfpef that will help us to understand its pathophysiol‐ ogy and assist us in establishing new biomarkers for its diagnosis. compared to non-hf control, hfpef patients demonstrated higher serum concentrations of acylcarni‐ tines, carnitine, creatinine, betaine and amino acids, and lower levels of phosphatidylcholines, lysophosphatidyl‐ cholines and sphingomyelins. mediumand long-chain acylcarnitines and ketone bodies were higher in hfpef than in hfref patients. the authors suggested that this abovementioned metabolomic fingerprint has been utilized to identify two novel panels of metabolites that can separate hfpef patients from both non-hf controls and hfref patients. however, this assumption requires further investigation. biomarkers patient population the most important findings references natriuretic peptides exerted dyspnoea predictor of hf risk manifestation, risk of admission in the hospital and hf-related deaths [19] known hfpef and hfref biomarkers independently predicted hf-related outcomes, cv mortality, all-cause death, admission in the hospital, but they did not predict a development of hfpef or hfref [19, 20, 21] cardiac troponins ischaemia-induced hf predictors of hf manifestation risk in asymptomatic subjects predictors of death in hfpef and hfref [24, 25] ischaemia-induced and nonischaemia-related hf predictors of death, primary/re-admissions in the hospital [25-27] galectin-3 general population prognosticator of hf risk, risk of death from any cause [33] known hf patients predictor of cv death, hf-related deaths, primary and re-admission in the hospital [35-37] soluble st2 general population predictor of higher risk of all-cause mortality, hf manifestation [42] known hf patients independent predictor of cv deaths, hf-related deaths, admission in the hospital [44] cardiotrophin-1 known ischaemia-induced hf patients predictor of cv clinical outcomes [53, 54] endoglin patients at higher risk of cv disease predictor of cv events/ outcomes, hf manifestation [58-60] growth differentiation factor 15 patients with known cv disease predictor of hf manifestation [64, 65] known hf patients predictor of hf-related outcomes [68] abbreviation: cv, cardiovascular; hf, heart failure; hfpef, hf with preserved ejection fraction; hfref, hf with reduced ejection fraction. table 1. predictive role of biomarkers in patients with different hf phenotypes 3alexander e. berezin: prognostication in different heart failure phenotypes: the role of circulating biomarkers 5.1 galectin-3 it has been suggested that various alternative biomarkers might offer insight into the different pathways of hf’s pathophysiology, and that they might help to identify individuals in the general population at higher risk of developing hf and patients with known chronic hf with poor outcomes [33]. galectin-3 is a soluble beta galactosidebinding lectin produced by activated macrophages that bind and activate the fibroblasts [34]. currently, galectin-3 is considered a biomarker that mediates an important link between inflammation and fibrosis, which plays a pivotal role in cv remodelling. the pathogenetic role of galectin-3 in the various settings of pressure overload, neuroendocrine activation, hypertension, coronary artery disease/myocardial infarction, atrial fibrillation and hf has been established. galectin-3 has emerged as a predictive value for the onset of hf in apparently healthy patients and has been found to be a surrogate marker of a worse prognosis, mortality and re-admission in hf [35, 36]. however, serial measurements of galectin-3 levels in ambulatory hf patients might not be of benefit [37]. in the context of determining the different phenotypes of hf, the measurement of circulating galectin-3 might have a significant value because elevated levels of galectin-3 were found in patients with impaired lv diastolic function, but without symptomatic hf [38]. gurel et al. [39] reported that galectin-3 could be a promising biomarker for the detection of lv diastolic dysfunction in patients undergo‐ ing maintenance haemodialysis. it has been suggested that this biomarker could be a useful surrogate for structural and functional abnormality of the myocardium among individuals at higher risk of hfpef development, espe‐ cially that associated with hypertension, coronary artery disease and diabetes [40, 41]. however, there is no irrefut‐ able evidence regarding the clinically significant advan‐ tages of galectin-3 in predicting hfpef’s evolution compared with hfref’s development. 5.2 st2 soluble st2 (sst2), a peptide belonging to the interleukin-1 receptor family, is secreted by cardiomyocytes and cardiac fibroblasts under mechanical strain, and is thus regarded as a biomarker of myocardial fibrosis, cardiac stretching and cv remodelling [42, 43]. measurement of sst2 levels is useful for death risk stratification and prognosis predic‐ tion in hf patients, beyond other cv risk factors [44]. the sst2 concentration showed a weak correlation with the nyha functional class, lfef, other cardiac performances and renal function [45, 46]. recent studies have shown that sst2 may have a special superiority as a risk predictor in hfpef and hfref as compared with natriuretic peptides and galectin-3 [47, 48]. however, there are no current data on the predictive value of sst2 concentrations for hfpef or hfref development. 5.3 cardiotrophin-1 cardiotrophin-1 (ct-1) is a member of the interleukin 6 cytokine superfamily and one of the endogenous ligands for gp130 signalling pathways in the heart, with controver‐ sial biological effects. ct-1 is able to induce hypertrophic growth and contractile dysfunction in cardiomyocytes, as well as having potent hypertrophic and survival effects on cardiac myocytes [49]. ct-1 is closely associated with many cv diseases, i.e., hypertension, myocardial infarction and hf, and exhibits a cardioprotective effect in ischaemiareperfusion injury during cabg and angioplasty [50]. recent clinical studies have shown that ct-1 levels are increased in hf patients, and that it is significantly corre‐ lated with the lv mass index, suggesting that ct-1 plays an important role in structural lv remodelling [51, 52]. increased cardiotrophin-1 plasma levels might predict the presence of an inappropriate lv mass merge in hyperten‐ sive subjects [53], and the development and progression of hf [54]. moreover, ct-1 is elevated in patients with hfpef and is associated with nt-probnp and estimated lv filling pressures [55]. whether increased serum ct-1 may provide additional information to aid risk stratification in the development of hfref or hfpef is not completely clear. 5.4 soluble endoglin endoglin (also known as cd105) is a membrane co-receptor for transforming growth factor-β, which is released into the circulation in a soluble form and which disrupts tgfβ1 signalling in the endothelium, thereby promoting inflam‐ mation, endothelial dysfunction, cardiac fibrosis and vascular remodelling [56, 57]. endoglin is required for vascular barrier function, endothelial survival and homeo‐ stasis of the adult microvasculature, although endoglin is expressed in cardiac fibroblasts and may modulate profi‐ brogenic actions of angiotensin ii [58]. a recent clinical study has revealed that the expression of endoglin is increased in patients with atherosclerosis and that the endoglin level is thought to predict cv events in patients with chronic coronary artery disease after pci [59]. there is evidence regarding the predictive role of elevated serum endoglin in patients with pre-eclampsia [60]. in patients with hfref, elevated soluble endoglin levels predicted elevated lv end-diastolic pressures, and correlated well with the new york heart association class, irrespective of lvef, as well as with both atrial and brain natriuretic peptides [56, 61]. the ability of soluble endoglin in predic‐ tion of hfpef and hfref is not understood, while there are expectations regarding the role of this biomarker for prognostication in lv dysfunction at early onset. however, extended scrutiny is required to receive more information for testing this assumption. 5.5 growth differentiation factor 15 growth differentiation factor 15 (gdf-15) is a stressresponsive cytokine, which belongs to the super family of 4 j circ biomark, 2016, 5:6 | doi: 10.5772/62797 the transforming growth factor beta [62]. gdf-15 is widely presented in the wide spectrum of various cells and plays a pivotal role in inflammation, cell growth and differentia‐ tion. elevated gdf-15 was found in patients with established cv diseases (hypertension, stable coronary artery disease, acute coronary syndrome, myocardial infarction, ischaemic and non-ischaemic-induced cardio‐ myopathies, hf, atrial fibrillation), type-two diabetes mellitus, chronic renal disease, infection and liver cirrho‐ sis and malignancy [63]. recent studies have revealed that gdf-15 was associated with the nyha class, nt-probnp and exercise capacity, suggesting that the marker has diagnostic and potentially prognostic value in hf [64-66]. it has been suggested that gdf-15 might categorize hfref and predict major hfrelated clinical outcomes [67]. chan et al. [68] reported that the plasma levels of gdf15 in hfpef and hfref were similar. therefore, there was an independent verification of the prognostic utility of gdf15 in hfref and hfref. the authors have shown that gdf15 was a significant inde‐ pendent predictor for composite outcome, even after adjusting for important clinical predictors including hstnt and nt-probnp [68]. overall, gdf15 was not able to assist in detecting the early stage of hfpef, and this biomarker has produced very limited evidence regarding the deter‐ mination of diastolic dysfunction. 6. conclusion several reports have shown that biomarkers reflecting the differentiation of fibroblasts into myofibroblasts, subse‐ quently altering collagen turnover, cardiac fibrosis and inflammation, might have diagnostic and predictive value in hfpef and hfref. the biomarkers with most predictive value in determining the early stage of hf’s development were sst2, galectin-3, ct-1 and gdf-15. however, increased plasma concentrations of these biomarkers were not specific for a distinct disease group of hfpef and hfref. finally, more investigations are required to determine the role of novel biomarkers in predicting hf and the determination of the early stage of hfpef and hfref’s development. 7. conflict of interest the authors declare no conflict of interest. 8. acknowledgements this article received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. 9. references [1] go as, mozaffarian d, roger vl, benjamin ej, berry jd, blaha mj, et al. heart disease and stroke statistics — 2014 update: a report from the ameri‐ can heart association. circulation. 2014; 129(3): e28–e292 [2] dunlay sm, roger vl. understanding the epidemic of heart failure: past, present, and future. curr heart fail rep. 2014; 11(4): 404–15. 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[epub ahead of print] 8 j circ biomark, 2016, 5:6 | doi: 10.5772/62797 article journal of circulating biomarkers characterization of a cell-culturing system for the study of contact-independent extracellular vesicle communication original research article anne louise schacht revenfeld1*, evo kristina lindersson søndergaard1, allan stensballe2, rikke bæk1, malene møller jørgensen1 and kim varming1 1 department of clinical immunology, aalborg university hospital, aalborg, denmark 2 department of health science and technology, laboratory for medical mass spectrometry fredrik bajersvej, aalborg university ,aalborg, denmark *corresponding author(s) e-mail: anlor@rn.dk received 27 november 2015; accepted 18 february 2016 doi: 10.5772/62580 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract appropriate and well-documented in vitro cell-culturing systems are necessary to study the activity and biological function of extracellular vesicles (evs). the aim of this study was to describe an experimental system, in which dynamic, vesicle-based cell communication can be investi‐ gated. a commercially available cell-culturing system was applied to study contact-independent cell communication, which separated two cell populations using a membrane with a pore size of 0.4 μm. the ev exchange characteristics between the two compartments in the culture set-up was preliminarily investigated in a cell-free set-up, and ana‐ lysed using the extracellular vesicle (ev) array and nanoparticle tracking analysis. the application of the cellculturing set-up was demonstrated using co-cultures of human primary cells. the effects of the relative placement of the two cell populations on the phenotype of evs found in the cell supernatant were investigated. the results indicate that this placement can be important for the biological hypothesis that is being investigated. these observations are relevant for short (<24h) as well as long (several days) studies of vesicle-based cell communication. moreover, the introduced cell-culturing set-up and analyt‐ ical strategy can be used to study contact-independent vesicle communication in a reproducible manner. keywords extracellular vesicles, cell communication, contact-independent, ev array, phenotype, transwell 1. introduction it is currently well accepted that extracellular vesicles (evs) are released from a plethora of cell types in many biological systems [1]. furthermore, these vesicular entities can be used as mediators of intercellular communication by a cargo of proteins and rnas [2]. in line with this, evs play an important role in many cellular processes in humans, both in physiological and pathophysiological scenarios [1-3]. however, uncovering the specific biological func‐ tions of evs necessitates well-documented approaches before the carrying out of valid functional studies. many in vitro experiments carried out to answer these biological questions use two different approaches: i) evs from one cell population/condition are isolated and added to another cell population [4-7]. subsequently, the effect of the evs on the 1j circ biomark, 2016, 5:3 | doi: 10.5772/62580 second cell population is investigated. ii) the two cell populations are co-cultured, but are separated by a membrane with a pore size that allows for the transport of vesicles of a defined size range, as well as soluble factors [4, 8, 9]. the effects of the evand signal molecule-based communication can thereafter be determined for either one or two cell populations. the latter set-up represents the most dynamic of the two approaches for studying vesiclebased communication, since it is based on co-cultures. consequently, it incorporates the continuous communica‐ tion between the studied cells, with a consequent greater resemblance to the in vivo conditions. the cell-culturing set-up described in the current study is based on the co-culture approach introduced above. the principle of this set-up is depicted in figure 1. several factors need to be taken into account when designing such a study. a key factor, which this study investigates, is the importance of which cell populations are placed in the upper compartment (uc) and the lower compartment (lc), relative to the biological hypothesis being tested. this is relevant, since we were able to demonstrate that this placement can affect the experimental outcomes, both for short (<24h) and long studies (several days) of vesiclebased cell communication. additionally, the subsequent analysis of the evs must be reliable, reproducible and provide as much information as possible. the aim of this report is to present a combined cell culturing and analytical set-up, which allows for an easy and reproducible detection of differences in ev phenotypes caused by contactindependent cellular communication. 2. materials and methods 2.1 transmembrane exchange of evs cell culture conditions and ev collection: cell-free and evenriched conditioned cell media were obtained from the human colon-cancer cell line ls180 (atcc® cl-187™; atcc, manassas, va, usa) and cultured in a growth medium containing rpmi 1640 (gibco, life technologies, carlsbad, ca, usa), 10 % ultracentrifuged (100.000 xg, 24 h, 4 °c; ti45 rotor, beckman coulter, brea, usa) heatinactivated foetal bovine serum (fbs) (gibco), 100 u/ml penicillin and 10 μg/ml streptomycin (ampliqon, odense, dk)). to remove the cells, the conditioned media were centrifuged at 500 xg, 5 min, rt, after collection. to the cellfree supernatant, a protease inhibitor cocktail was added (edta-free, roche, basel, switzerland, diluted 1:50 in pbs). subsequently, the ev-rich supernatant was reduced in volume by using a 15 ml amicon® ultra filter unit with a 100k mwco (merck millipore, darmstadt, germany). the volume-reduced, ev-rich supernatant was washed twice with pbs prior to use. the final volume of the cell supernatant was approximately 1/20 of the original volume. dilution series to investigate transmembrane exchange of evs: the following dilutions of the ev-rich supernatant from ls180 were included: undiluted, 1:10, 1:50, 1:100, 1:500 and 1:1000. all dilutions were made with the growth medium. initially, 800 μl of the supernatant was placed in a well in a 24-well plate (nunc, thermo scientific, waltham, ma, usa). this compartment was designated as the lc (figure 1). subsequently, 400 μl of growth medium was put in the uc, constituted by a millicell® hanging cell culture insert (#piht 12r 48, merck millipore). with this set-up, the exchange of evs from the lc to the uc was investigated. to describe the transmembrane ev exchange from the uc to the lc, the reverse set-up was made (i.e., growth medium in the lc; ev-rich supernatant in the uc). the culture plate holding the inserts was placed in a co2incubator (temperature: 37 °c; co2-concentration: 5 %; relative humidity: 90 %) for 24 hours with no agitation. after this, the contents of each compartment was harvested into separate tubes and stored at -40 °c until semi-quanti‐ fication of the vesicles by the ev array. no further isolation of the evs was performed. 2.2 contact-independent cell communication isolation of cells: buffy coats were obtained from healthy blood donors at the aalborg university hospital blood bank. each blood donor had signed a written consent form, allowing the use of his or her blood for research purposes. the procedure was approved by the local ethics legislation. figure 1. principle of the cell-culturing system for contact-independent cellular communication. the use of cell culture inserts was applied in order to create a two-compartment cell-culturing system. the insert was placed within a well of a standard 24-well culture plate, creating an upper compartment (uc) and a lower compartment (lc). multiple flanges at the top of the insert suspended it onto the edge of the culture plate well, ensuring the lack of direct contact between the insert and the well. an incorporated membrane in the bottom of the insert facilitated the separation of the compartments. the membrane pore size of the applied inserts was 0.4 μm in diameter, thus allowing for the passage of smaller vesicle subsets, as well as soluble factors (indicated by the arrows). the pore density was 1 x 108 pores/cm2, while the effective membrane area was 33 mm2. 2 j circ biomark, 2016, 5:3 | doi: 10.5772/62580 isolation of peripheral blood mononuclear cells (pbmcs) was accomplished by using gradient centrifugation with a lymphoprep™ (axis-shield, oslo, no). a final washing step (350 xg, 7 min, rt) was performed to reduce the number of platelets in the final cell suspension. the pbmcs were either used directly after the isolation or stored at -140 °c in a storage medium (rpmi 1640, 40 % heat-inactivated fbs, 10 % dimethyl sulphoxide (merck millipore), 100 u/ml penicillin and 10 μg/ml streptomycin). cell culture set-up and ev collection: a co-culture was created with pbmcs from two donors with known hla serotypes. the pbmcs from one donor were designated the stimulator cells, while the cells from the second donor were entitled the responder cells. prior to the co-culture, the stimulator cells were irradiated (1700 rad) to inhibit their proliferation. a millicell® hanging cell culture insert separated the stimulator cells and responder cells (figure 1). the uc of the cell-culturing system contained 2.5x105 cells in a total volume of 400 μl, while the lower chamber contained 5x105 cells in 800 μl. control samples with monocultures of both stimulator and responder cells were also included. here, 5x104 of either responder or stimulator cells were seeded in a 96-well plate in a total of 150 μl culture medium. the cells were placed in a co2-incubator (tem‐ perature: 37 °c; co2-concentration: 5 %; relative humidity: 90 %) for six days. on day 6, the conditioned cell media were harvested from each compartment separately and centrifuged once at 500 xg, 10 min, rt to pellet cells. a protease inhibitor cocktail (edta-free, diluted 1:50 in pbs) was added to the cell-free supernatants prior to storage at -40 °c until vesicle phenotyping by the ev array or size determination with nanoparticle tracking analysis (nta) was carried out. no further isolation of the evs was performed. 2.3 ev array analysis production of microarrays: microarray printing was per‐ formed on a spotbot® extreme protein edition microarray printer (sunnyvale, arrayit, ca, us), as previously described [10]. antibodies/proteins for the phenotyping of vesicles: for the phenotyping, a total of 10 anti-human antibodies and one protein were used. they are listed in the following with the corresponding product number (#) or clone. from r&d systems (minneapolis, mn, usa): cd82 (#423524) and tnfri (#dy225). from biolegend: cd63 (mem-259) and hla-dr (l243). from lifespan biosciences, inc. (seattle, wa, usa): cd9 (#ls-c35418) and cd81 (#ls-b7347). from abcam (cambridge, ma, usa): flotilin-1 (#ab41927). from haematologic technologies, inc. (essex juncton, vt, usa): lactadherin (#blac-1200) (protein). from bd biosciences: cd3 (hit3a). from abbiotec (san diego, ca, usa): cd11a (hi111). from ebioscience (san diego, ca, usa): icam-1 (r6.5). all antibodies/proteins for the phenotyping were printed in triplicate at 200 μg/ml diluted in pbs containing 5 % glycerol. antibodies for the semi-quantification of vesicles: for the semiquantification of vesicles, anti-cd9, anti-cd63 and anticd81 were printed on the microarray slides, as previously described [11]. in short, 18 repeated spots were printed using a cocktail of the three antibodies, each in a concen‐ tration of 100 μg/ml. catching and visualization: the entire procedure was performed as described previously [11]. in brief, the printed slides were blocked, incubated with the evcontaining sample, followed by the detection of bound evs with biotinylated anti-cd9, -cd63 and -cd81 and, subse‐ quently, cy5-labelled streptavidin. data analysis: data analysis and the creation of graphs were carried out using sigmaplot (version 11, systat software inc, san jose, ca, usa) and excel (version 2013, microsoft, redmond, wa, usa). for a given antibody spot, the signal intensity was calculated as the mean signal of the triplicate spots (18 spots for semi-quantification) in relation to the sample signal of the negative spot (pbs) in triplicate. for each spot, the signal intensity was calculated by subtracting the mean of the background (no sample/blank, washing buffer) from the mean of the foreground (spot signal). before visualization and the calculation of linearity, the antibody signal intensities were converted to log space by log2 transformation. the transmembrane ev exchange, evaluated from the semi-quantitative data (figure 2), was calculated as: ev array signal in compartment with growth medium/ ev array signal in compartment with added evrich cell supernatant. 2.4 nanoparticle tracking analysis (nta) instrument details: for ev-size determination, nta was performed with a nanosight lm10-hs system equipped with a finely tuned 405 nm laser (nanosight ltd., ames‐ bury, uk), supplied with the nta 3.0 0060 analytical software, which was used for capturing and analysing the data. the camera type was emccd and the camera level was set to 11. size determination: the nanosight was calibrated with 100 nm polystyrene latex microbeads (thermo scientific, fremont, usa) prior to analysis. dulbecco's phosphate buffered saline (dpbs) without ca2 + and mg+, and filtered using a 0.22 μm filter prior to use (lonza, verviers, belgium), was used to dilute the microbeads (1:1000 dilution) and the ev-rich supernatants (1:40 dilution). the samples were manually injected into the sample chamber and a temperature-measuring device inserted directly into the sample chamber was applied to record the temperature of the sample for each run. samples were measured with a slide shutter of 600 and with a slider gain of 300 for 60 s. the applied dilutions yielded between 20-100 particles/ frame and each sample was measured in triplicate. subse‐ quently, the integrated software automatically processed the data, yielding values such as the mean, the median, the 3anne louise schacht revenfeld, evo kristina lindersson søndergaard, allan stensballe, rikke bæk, malene møller jørgensen and kim varming: characterization of a cell-culturing system for the study of contact-independent extracellular vesicle communication mode particle size, the value of the highest point of the peak and the corresponding standard deviations. the detection threshold was set to three and the blur setting was 9 x 9. 3. results 3.1 transmembrane ev exchange between the two compartments of the cell-culturing set-up initially, the exchange characteristics of evs between the two compartments of the cell-culturing system (figure 1) were investigated using a cell-free approach. this was done to evaluate whether the experimental outcome was affected by the relative position of the cells in the two compart‐ ments. for this purpose, a volume-reduced, ev-rich supernatant from the human colon-cancer cell line ls180 was placed in one of the compartments and a growth medium was placed in the opposite compartment. conse‐ quently, the transport of evs from the uc to the lc, and in the reciprocal direction, was studied. after 24h, the extent of transmembrane ev exchange was evaluated in each compartment by using the ev array to semi-quantify the contents of cd9-, cd63and/or cd81-containing evs. the results from this analysis can be seen in figure 2. here, the transmembrane ev exchange illustrated in figure 2a designates the ratio between the ev array signal from the compartment, which initially only contained growth medium, and the signal from the ev starting compartment. from figure 2a, it can be deduced that the transmembrane ev exchange was greater from the uc to the lc in the investigated time frame, as compared to the reciprocal direction. this difference was already noticeable with the first dilution. here, the signal from the uc of the lower to upper set-up (orange) was reduced to roughly 50 % of that from the lc, while the signals from both uc and lc of the reversed set-up were almost similar (blue). furthermore, with the 1:50 dilution, practically no signal could be detected in the uc for the lower to the upper combination. figure 2. transmembrane exchange of evs between the two compartments of the cell-culturing set-up. the exchange of evs from the uc to lc of the cellculturing set-up, and vice versa, was evaluated. a) an ev-rich, cell-free supernatant from the human cell line ls180 was placed in one of the two compartments, while growth medium was placed in the opposite compartment. this was done with six dilutions of the supernatant, as indicated on the x-axis. after 24h, the contents of each compartment were harvested, and the amount of cd9, cd63 and/or cd81 was semi-quantified using the ev array for the evs that had bound to the array. the transmembrane ev exchange shows the relationship between the ev array signals from the compartment initially containing growth medium and the compartment, to which the ev-rich supernatant was originally added. b) the size distribution of the evs in the uc and lc of the 1:50 dilution was determined by nta. here, the upper to lower samples are shown, while a summary of the remaining samples can be found in table 1. in addition, the applied growth medium was also analysed separately. for each sample, the histogram indicates the mean of the triplicates. the numbers in blue indicate the particle size at the peak measurements in the histogram, while the red bars indicate +/1 standard error of the mean. the x-axis has been truncated from 1000 nm to 700 nm, since no particles were detected in the largest size range. 4 j circ biomark, 2016, 5:3 | doi: 10.5772/62580 for the reversed combination, this tendency was observed later, at the 1:500 dilution. as an additional investigation, the size distributions of the evs present in each compartment were determined by nanoparticle tracking analysis (nta) after 24h of trans‐ membrane ev exchange. with this investigation, it was possible to evaluate whether both small and large evs were present in the ev-rich supernatants. the analysed samples included those from the 1:50 dilution (figure 2). the histograms of the ev size distribution for the upper to lower combination can be seen in figure 2b. the remaining results from the nta are summarized in table 1, in which they can be compared to the corresponding ev array signals. the samples presented in figure 2b contained smaller vesicles, with 90 % being smaller than approxi‐ mately 100 nm and 150 nm for the lc and uc, respectively. moreover, no larger vesicles/particles (>400 nm) were present in any of the samples (figure 2b), including the growth medium analysed prior to the investigation of the transmembrane ev exchange. from the mean and mode values found in table 1, it is not possible to accurately distinguish the transferred evs from those inherently present in the growth medium. however, two points can be deduced when comparing the nta data with the matching ev array signals. first, it appears that even though vesicles were present in the growth medium, despite using uc fcs, these evs do not elicit a specific and interfering ev array signal. this consequently leads to the second point, indicating that the ev array signals detected in the compartments initially only holding growth medium come from a transmembrane exchange of ls180 evs. the ev array signal from that compartment was 8.6 fold higher in the upper to lower scenario than in the opposite direction (table 1). this accordingly supports the tendency deduced from figure 2a, in which the transmembrane ev exchange was seemingly greatest from the uc to the lc. ev array signal mean size (sem) [nm] mode (sem) [nm] ev transport: upper to lower uc 7.8 105.9 (5.5) 88.6 (2.6) lc 4.3 79.3 (2.7) 87.3 (9.1) ev transport: lower to upper lc 7.7 102.6 (0.6) 91.8 (5.8) uc 0.5 97.1 (3.3) 85.3 (5.1) growth medium 0 103.9 (3.1) 102.1 (3.7) table 1. size distribution of the vesicles after 24h of transmembrane ev exchange. an ev-rich, cell-free supernatant was placed in either the uc or lc of the cell-culturing set-up, while growth medium was placed in the opposite compartment. after 24h, the size of the evs present in each compartment was determined by nanoparticle tracking analysis (nta). the data presented are from the 1:50 dilution shown in figure 2. for each compartment, the signal from the ev array is given along with the results from the nta. the mean and the mode, and the corresponding standard error of the mean (sem), are given for triplicate measurements of each sample. 3.2 demonstration of the applicability of the cell-culturing setup and analysis platform in addition to the cell-free set-up, the presented cellculturing system was further investigated using a coculture of human peripheral blood mononuclear cells (pbmcs). in this co-culture, one cell population was designated as the stimulator cells, since these cells could induce an immunological response in the responder cells, constituted by the second cell population. the stimulator cells were not subject to a reciprocal activation, since they had been irradiated, making the cell communication oneway. both combinations of the stimulator and the respond‐ er cells in the uc and lc were made and, after six days of co-culturing, the phenotype of the cell-derived evs was determined, as shown in figure 3. it can be seen for both combinations of the co-culture that all the reported protein markers were present on evs above control levels (grey bars). moreover, the presence of the more ev-specific markers, including cd9, cd81 and cd82, were the most abundant, when compared to the remaining markers included in the ev phenotyping. one exception to this related to cd63, which was only marginally detected in comparison to cd9, cd81 and cd82. the ev array signals for the majority of the more cell-specific markers were at the lower level of detection for the applied array, with the exception of cd11a and tnfri. nonetheless, the relative placement of the two cell populations in the co-culturing set-up affected the obtained ev array signals for both the vesicleand cell-specific markers, although to a varying degree. from the responder cell compartments (figure 3, top panel), some of the most pronounced differences were observed for icam-1, with an almost 3.5 fold higher signal for this marker from the evs harvested from the uc (orange) than for the lc (blue) of the responder cells. moreover, cd3 could only be detected from evs in the responder cell compartment in one of the settings. finally, the detection of cd81 was almost 1.5 times higher in the uc sample, when compared to the lc sample. in contrast, comparable signals for cd81 were observed in both stimulator cell compartments (figure 3, bottom panel). for these stimulator cells, a differential ev array signal was particularly observed for hla-dr. this marker was preferentially present, when analysing the evs in the uc sample (blue), with an approximate 2.5 fold enrichment. a similar observation was demonstrated for cd63. as a final notion, the reproducibility of the presented cellculturing set-up and analysis platform was evaluated. accordingly, the contact-independent co-culture was repeated several weeks apart, using cells from the same individuals. in figure 4, the results of three selected markers are presented for these two technical replicates. it can be seen that, for cd9 and cd81, the detected ev signals predominantly correlated from replicate to replicate, with 10 of the 12 obtained %cv values ranging from 1.7%-15.6% (figure 4). the last two %cv values for cd9 and cd81 were 30% and 36.9%, and they were both calculated from the 5anne louise schacht revenfeld, evo kristina lindersson søndergaard, allan stensballe, rikke bæk, malene møller jørgensen and kim varming: characterization of a cell-culturing system for the study of contact-independent extracellular vesicle communication stimulator cells in the lower compartment. for cd63, four of the six %cv values could not be determined, as either one or both ev array signals from the two technical replicates were below the lower limit of detection (lod). the last two %cv values for cd63 were 6.9 and 100.7. for the latter sample, the detected log2 signals were very close to the lower lod (log2 values: 0.13 and 0.75). 4. discussion and conclusion in the research field of evs, much effort is put into deci‐ phering the biological functions of these vesicular entities. consequently, useful experimental and analytical plat‐ forms are of great interest. in this technical report, we have investigated a commonly used cell-culturing system for the study of dynamic, contact-independent cell communica‐ tion, focusing on the ability of the evs involved to enter each compartment (depicted in figure 1). in the applied setup, a membrane with 0.4 μm pores separated the cells, but other pore sizes between 1-8 μm are also available. initially, we wanted to investigate whether the transmembrane exchange of evs were similar for both compartments. with the results presented in figure 2, it is apparent that for this short study (<24h), the exchange of evs was greater from the uc to the lc than in the opposite direction. the observed differential ev exchange is relevant because there are many studies investigating selected features of contactindependent cellular communication that have applied short incubation times [4, 8, 9, 12, 13]. our study also suggests that this is important, since for short studies it may be advantageous to place the primary ev donor cells in the uc, while the primary recipient cells should be placed in the lc. these suggestions are based exclusively on the observations derived from figure 2 and table 1, since it was not determined which contributing factors could have affected this differential transmembrane ev exchange, such as diffusion, sedimentation or hydrostatic pressure. moreover, the transmembrane ev exchange could have been investigated using a cell-containing set-up. neverthe‐ less, a cell-free approach was chosen since it incorporated the possibility of demonstrating any concentration depend‐ ency of the transmembrane ev exchange by making precise dilutions of the ev-rich supernatant. this option was not given when using cells, as it has not been confirmed that there is a linear correlation between the number of cells and the amount of evs produced. one element that may alleviate any possible effects of the observed differential ev transport is agitation of the culturing set-up. however, this was not investigated in the current study, as agitation has not been employed in several other studies investigating contact-independent cell communication [4, 8, 9, 12, 13]. consequently, the presented data relate to the practice of figure 3. ev phenotype after the co-culture of human peripheral blood mononuclear cells. a six-day, contact-independent co-culture between peripheral blood mononuclear cells (pbmcs) from two different individuals was created. the phenotypes of the cell-derived evs were evaluated using the ev array to detect differences caused by the relative placement of the two cell populations. consequently, both combinations of responder (resp) and stimulator (stim) cells in the lower compartment (lc) and upper compartment (uc) were included. for non-specific reactions, monocultures of both stimulator (stim ctrl) and responder cells (resp ctrl) were included. antibodies targeting the listed markers on the x-axis were used for the capturing of the evs. the panel of antibodies targeted both immunological markers, which were related to the different leukocyte subsets within the pbmcs, and more general vesicle-related markers. the observed signal for each of the markers implies the simultaneous presence of cd9, cd63 and/or cd81, since a cocktail of antibodies against these three ev markers was used for detection. for each combination of responder and stimulator cells in the uc and the lc, the bars show a mean value ± sem from two independent experiments using cells from the same two donors. ev array measurements were performed in triplicate. 6 j circ biomark, 2016, 5:3 | doi: 10.5772/62580 many relevant studies. as a technical note, it can be seen in table 1 that the mean size of vesicles detected in the compartment, which initially only contained growth medium, was variable (79.3 nm versus 97.1 nm, respective‐ ly). when addressing the corresponding ev array signals (4.3 versus 0.5, respectively), it has previously been demonstrated that vesicles that bind to the array are primarily <100 nm [11]. however, despite this reported size maximum, the mean size of the eluted evs from the array in reality had a mean size <85 nm. hence, for the present study, this correlates with the observation that the smaller sized vesicles yielded a higher amount of detected cd9, cd63 and cd81. the differential transport of evs across the insert mem‐ brane was observed in a simplified version of the cellculturing system, which does not incorporate the dynamics of a cell-based set-up, where evs are continuously pro‐ duced and taken up by cells. however, the observed difference may be a contributing factor in the cell-based system as well. this might possibly be connected to the differences detected for the phenotype of the cell-derived evs, which was affected by the relative placement of the two cell populations (figure 3). biological variations alone could, most likely, not account for this phenotypic differ‐ ence, since the set-up was reproducible (figure 4). hence, this points to other determining factors, such as a differen‐ tial transmembrane ev exchange. moreover, the induced ev phenotypes in the co-cultures were not artefactual, as all investigated markers were detected above control levels. the cellular phenotype, which was investigated for a subset of the responder cells, was not affected to the same degree as the vesicular phenotype by the relative placement of the cell populations (data not shown). any biological relevance of these observations has not yet been investi‐ gated in more detail. however, the ev phenotype may be more sensitive to the experimental design, with a possible importance of the functional consequences of the ev-based communication between the involved cell populations. consequently, this stresses the point that it may not be trivial how the cell populations are placed relative to each other, both for short and long studies of contact-independ‐ ent cell communication. several studies do not account for their choice of the relative placement of the cells in the two compartments [4, 8, 9, 12, 13], which our study indicates as being relevant. as a final note, the recommendations, which are made using the presented data, are based on a co-culture of pbmcs. this cellular population consists predominantly of suspension cells with a smaller fraction of adherent cells. therefore, applying other cell types with different characteristics could entail other affecting factors, such as an increased blocking of the membrane pores. nevertheless, this yet again underlines the significance for investigating any influence that the relative placement of the cells has on the experimental outcome, regardless of the cell types used. as mentioned previously, it is essential to have reliable assays to delineate the biology and functionality of evs. therefore, the reproducibility of the cell-based experiments was evaluated by repeating the co-cultures and the subsequent analysis after several weeks, using cells from the same individuals. as presented in figure 4, the ob‐ tained %cv values for cd9 and cd81 ranged from 1.7 to 36.9. however, 10 of these 12 samples had a %cv below 16, whereas the two highest values were calculated for the same sample, namely the stimulator cells in the lc, pointing to an isolated trend for this sample. it has been established that the ev array yields %cv values below 10 when working with plasma samples [10], and %cv values below 25 have been proposed as an acceptable limit for immunoassays [14]. however, this limit can be expanded if an experimental rationale is present [14], which may be the case when both technical and biological variations exist, as in this study. nonetheless, one of the %cv values for cd63 differed to a somewhat greater extent, amounting to 100.7 (figure 4). the log2 intensities from the relevant replicates, forming the basis of the %cv calculation, were 0.13 and 0.75. hence, the standard deviation was very large compared to the mean of the two intensity values, with a consequent large impact on the calculated %cv. this is an inherent issue for small intensities close to the lower lod, which is difficult to completely circumvent. a third technical replicate may serve to improve the %cv. how‐ ever, several other results from our work point to the fact that cd63 is a poor marker for evs in general since it occurs figure 4. reproducibility of the cell-culturing set-up and analytical platform. to demonstrate the reproducibility of the presented methodology, two independent experiments of the contact-independent co-culture were performed (using one biological replicate). the plots display the results from three selected ev markers; cd9, cd63 and cd81 (also shown in figure 3] from the ev array analysis of the resulting cell supernatants from the uc and the lc. the corresponding %cv values are noted below each sample, and were calculated based on the two technical replicates. the %cv values were not calculated for the samples with log2 signal intensities below the lower limit of detection (marked by a -). 7anne louise schacht revenfeld, evo kristina lindersson søndergaard, allan stensballe, rikke bæk, malene møller jørgensen and kim varming: characterization of a cell-culturing system for the study of contact-independent extracellular vesicle communication in relatively small amounts, as compared to cd9 and cd81 (unpublished data and [10, 11, 15-17]). as part of the combined experimental set-up presented here, the determination of the ev phenotype plays a major role. using the ev array to phenotype the vesicles provides the opportunity to gain much information about the system that is being studied. in the context of vesicle-based cell communication, the ev phenotype may be used for several purposes. first, the use of an extensive ev phenotype may aid in fine-tuning the biological hypothesis that is being investigated. in this study, we targeted 11 protein markers. however, 60 analytes (in this case, antibodies) can current‐ ly be used simultaneously for each sample when pheno‐ typing evs with the ev array [10]. consequently, the ev phenotype may not only provide a large amount of information but it can also be used to optimize the experi‐ mental design in an iterative fashion. furthermore, when applying the ev array, this can be achieved without having to resort to extensive ev isolation procedures. currently, the designed ev array does not provide direct information about which cells produce the evs and their absolute quantities. however, by linking the extensive ev pheno‐ type to a number of additional experimental outcomes, such as the cellular phenotype, or the ev rna and subproteome cargo, unravelling the biological functions of the ev-based communication becomes substantial and highly relevant. 5. compliance with ethical research standards the authors declare no conflicts of interest. all research on human subjects presented in this paper was conducted in accordance with the local ethics legislation. each human subject signed a written consent form, allowing for the use of his or her blood for research purposes. 6. acknowledgements the authors thankfully acknowledge dr. shona pedersen and clinical professor søren risom kristensen (depart‐ ment of clinical biochemistry, aalborg university hospi‐ tal, aalborg, denmark) for their support in the nta experiments. the authors also acknowledge technician anne elbæk (department of clinical immunology, aal‐ borg university hospital, aalborg, denmark) for excellent technical assistance. 7. references [1] revenfeld al, baek r, nielsen mh, stensballe a, varming k, jorgensen m. diagnostic and prognos‐ tic potential of extracellular vesicles in peripheral blood. clin ther. 2014 jun 1;36(6):830-46. 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[17] basu s, bhattacharyya sn. insulin-like growth factor-1 prevents mir-122 production in neighbour‐ ing cells to curtail its intercellular transfer to ensure proliferation of human hepatoma cells. nucleic acids res. 2014 jun;42(11):7170-85. 9anne louise schacht revenfeld, evo kristina lindersson søndergaard, allan stensballe, rikke bæk, malene møller jørgensen and kim varming: characterization of a cell-culturing system for the study of contact-independent extracellular vesicle communication article journal of circulating biomarkers elevated adiponectin antibody levels in sera of patients with atherosclerosisrelated coronary artery disease, cerebral infarction and diabetes mellitus original research article takaki hiwasa1*, xiao-meng zhang1, risa kimura1, mikiko ohno2, po-min chen2, eiichiro nishi2, koh ono2, takeshi kimura2, ikuo kamitsukasa3, takeshi wada4, akiyo aotsuka4, seiichiro mine5,6, hirotaka takizawa7, koichi kashiwado8, minoru takemoto9, kazuki kobayashi9, harukiyo kawamura9, ryoichi ishibashi9, koutaro yokote9, rika nakamura1,10, go tomiyoshi1,10, natsuko shinmen1,10 and hideyuki kuroda10 1 department of biochemistry and genetics, graduate school of medicine, chiba university, chiba, japan 2 department of cardiovascular medicine, graduate school of medicine, kyoto university, kyoto, japan 3 department of neurology, chiba rosai hospital, chiba, japan 4 department of internal medicine, chiba aoba municipal hospital, chiba, japan 5 department of neurological surgery, chiba prefectural sawara hospital, chiba, japan 6 department of neurological surgery, graduate school of medicine, chiba university, chiba, japan 7 port square kashiwado clinic, kashiwado memorial foundation, chiba, japan 8 department of neurology, kashiwado hospital, chiba, japan 9 department of clinical cell biology and medicine, graduate school of medicine, chiba university, chiba, japan 10 medical project division, research development center, fujikura kasei co., saitama, japan *corresponding author(s) e-mail: hiwasa_takaki@faculty.chiba-u.jp received 15 october 2015; accepted 15 march 2016 doi: 10.5772/63218 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract adiponectin secreted from the adipocytes plays pleiotrop‐ ic, anti-atherosclerotic roles, such as enhancement of insulin secretion and an increase in energy expenditure. the measurement of levels of circulating adiponectin is useful to evaluate the progression of atherosclerosisrelated diseases, such as coronary artery disease (cad), cerebral infarction (ci) and diabetes mellitus (dm). we examined the serum antibody levels against recombinant adiponectin protein via the amplified luminescent proxim‐ ity homogeneous assay-linked immunosorbent assay (alphalisa) method. the results revealed that the anti‐ body levels were significantly higher in patients with cad, ci and type 2 dm, than in healthy donors. receiver operating curve analysis showed that the sensitivity was in a range of 41–48% for cad, ci and dm. thus, the serum anti-adiponectin antibody levels could be a common marker for atherosclerosis-related diseases. keywords atherosclerosis, cardiovascular disease, cerebral infarction, diabetes mellitus, antibody biomark‐ er 1j circ biomark, 2016, 5:8 | doi: 10.5772/63218 abbreviations aci: acute cerebral infarction ami: acute myocardial infarction auc: area under the curve cad: coronary artery disease dm: diabetes mellitus hd: healthy donor roc: receiver operating curve. 1. introduction adiponectin is a peptide hormone that plays a variety of roles in glucose and lipid metabolism, diabetes (dm) and metabolic syndrome [1–4]. the circulating adiponectin level is negatively correlated with obesity, coronary artery disease (cad) and metabolic disorders [5,6]. adiponectin reduces atherosclerosis in apolipoprotein e-deficient mice [7]. furthermore, exacerbation of heart failure was ob‐ served in adiponectin-deficient mice [8]. adipor1 and adipor2 are the major typical receptors of adiponectin, and depletion of both receptors leads to insulin resistance and glucose intolerance [9]. these results support the idea that adiponectin has a causal role in suppressing the development of diabetes mellitus (dm), cad and athero‐ sclerosis. we have recently reported novel atherosclerosis-related antibody markers, such as antibodies against rpa2 for stroke [10], antibodies against sostdc1 and tubb2c for ci and dm [11,12], and antibodies against atp2b4 and bmp-1 for atherosclerosis-related diseases, such as ci, cad, dm and chronic kidney disease [13]. abnormality of the blood vessels may result in the leaking out of the antigenic proteins that can produce these antibodies. in the present study, the levels of autoantibody against those of adiponectin were examined. our results showed that the antibody levels were associated with cad, ci and dm. 2. methods 2.1 sera of patients and healthy donor (hd) subjects the local ethical review board of chiba university, graduate school of medicine (chiba, japan) as well as those of co-operating hospitals approved the study. sera were collected from patients after they had provided written informed consent. each serum sample was centrifuged at 3,000 × g for 10 min, and supernatants were stored at -80˚c until use. the serum samples of cad, including acute myocardial infarction (ami), were obtained from the kyoto university hospital. the samples of acute cerebral infarc‐ tion (aci) were obtained from chiba rosai hospital and chiba aoba municipal hospital. the samples of type 2 dm were obtained from chiba university hospital. sera from hd subjects were obtained from chiba university, chiba prefectural sawara hospital and port square kashiwado clinic. 2.2 amplified luminescence proximity homogeneous assay (alphalisa) the antigen used for the analysis of cad and aci speci‐ mens was recombinant his-tag-conjugated, full-length adiponectin protein (prospec-tany technogene ltd., ness ziona, israel). the antigen used for the analysis of agematched ami and dm specimens was his-tag-conjugated adiponectin peptide (amino acids 108-244) (atgen co. ltd., seongnam, south korea). alphalisa was performed using 384-well microtitre plates (white opaque optiplate™, perkin elmer, waltham, ma) containing 2.5 µl of 1/100diluted sera and 2.5 µl of his-tag adiponectin (10 µg/ml) in alphalisa buffer (25 mm hepes, ph 7.4, 0.1% casein, 0.5% triton x-100, 1 mg/ml dextran-500 and 0.05% proclin-300). the reaction mixture was incubated at room temperature for 6–10 h. nickel-chelate donor beads (2.5 µl of 40 µg/ml) and anti-human igg-conjugated acceptor beads (2.5 µl of 40 µg/ml) were then added and incubated further for seven to 21 days at room temperature in the dark. the chemical emission was read on an enspire alpha microplate reader (perkinelmer) as previously described [11–13]. specific reactions were calculated by subtracting alpha values without antigens from the values of adipo‐ nectin proteins. 2.3 statistical analyses student's t test and the mann-whitney u test were used to determine the significance of the differences between the two groups. the correlation was examined via spearman’s correlation analysis. all statistical analyses were carried out using graphpad prism 5 (graphpad software, la jolla, ca). the predictive values of markers for diseases were assessed by receiver operating curve (roc) analysis, and the cut-off values were set at the values that maximize the sums of the sensitivity and specificity. all tests were twotailed and a p value below 0.05 was considered significant. 3. results 3.1 levels of adiponectin antibodies (adiponectin-abs) are associated with cad we examined the relationship between adiponectin-abs and cad, which included sera from ami and unstable angina patients obtained from kyoto university hospital. hd subjects from kashiwado clinic and chiba prefectural sawara hospital were selected as those who had no apparent abnormality on regular health check-ups, includ‐ ing those based on mri examination. the average ages of the hd subjects and patients were 49.8 and 66.6, respec‐ tively. the results of alphalisa showed that the levels of 2 j circ biomark, 2016, 5:8 | doi: 10.5772/63218 serum adiponectin-abs were significantly higher in patients with cad than those in the hd subjects (figure 1a). when the cut-off value was determined as the average + 2sd of the hd specimens, the positive rates of adiponec‐ tin-abs in hd subjects and patients with cad were 6.8% and 17.7%, respectively (table 1). receiver operating curve (roc) analysis was carried out to evaluate the ability of these markers to detect cad. the areas under the curve (aucs) of adiponectin-abs for cad were 0.649 (95% ci: 0.603-0.695) (figure 1b). when the cut-off value of the adiponectin-ab level was determined to be 3,020, the sensitivity and specificity of the antibody level for the diagnosis of cad were calculated to be 41.0% and 83.3%, respectively. 0 0.5 1.0 0 0.5 1.0 a lp ha c ou nt hd cvd 2000 4000 6000 8000 10000 a b p ＜ 0.0001 1 specificity s en si tiv ity 3,020 (0.410. 0.833) p < 0.0001 auc = 0.649 95% ci: 0.603 0.695 figure 1. comparison of serum adiponectin antibodies (adiponectin-ab) levels between the healthy donor (hd) subjects and patients with coronary artery disease (cad). serum antibody levels examined by alphallisa are shown by a box-whisker plot (a). the box plots display the 10th, 20th, 50th, 80th and 90th percentiles. p values as compared to the hd specimens are shown. receiver operating curve (roc) analysis was carried out for assessing the ability of adiponectin-abs to detect cad (b). numbers in the curves indicate cut-off values of marker levels and those in parentheses indicate sensitivity (left) and specificity (right). p values of roc analysis, areas under the curve (auc) and a 95% confidence interval (ci) are also shown. we then compared the antibody levels between the hd subjects and age-matched patients with ami. the average ages of the hd subjects and the patients were 57.9 and 57.2, respectively. the levels of adiponectin-abs were also significantly higher in patients with ami as compared with age-matched hd subjects (table 1). the positive rates in the hd subjects and patients with ami were 4.7% and 14.1%, respectively. roc analysis revealed that the auc of adiponectin-abs in patients with ami was 0.641 (95% ci: 0.574-0.709). the sensitivity and specificity of the antibody level for diagnosis of ami were calculated to be 47.7% and 74.2%, respectively. 3.2 levels of adiponectin-abs are increased in patients with aci we next examined adiponectin-ab levels in the sera of the hd subjects obtained from chiba university and chiba prefectural sawara hospital, and in the sera of patients with aci obtained from chiba rosai hospital and chiba aoba municipal hospital. the average ages of the hd subjects and the patients were 44.3 and 69.3, respectively. (figure 2a). the positive rates of adiponectin-abs in the hd subjects and patients with aci were 2.8% and 30.4%, respectively (table 1). roc analysis revealed that the auc of adiponectin-abs for aci was 0.652 (95% ci: 0.595-0.710; figure 2b). when the cut-off value of the adiponectin-ab level was determined to be 2,933, the sensitivity and specificity of the antibody level for the diagnosis of aci were 46.2% and 86.1%, respectively. because the average ages of the hd subjects and the patients were different, the antibody levels of age-matched specimens were compared after depletion of the appropri‐ ate samples. the average ages of the hd subjects and the patients selected were 50.8 and 51.0, respectively. despite the decrease in sample numbers examined, the levels of cad ami, age-matched aci aci, age-matched dm, age-matched hd average 2,526 1,310 2,535 2,492 959 sd 640 455 368 383 673 cut-off value 3,805 2,219 3,272 3,257 2,306 total no. 191 127 72 44 128 positive no. 13 6 2 2 6 positive rate (%) 6.8% 4.7% 2.8% 4.5% 4.7% patient average 3,087 1,679 3,045 2,813 1,392 sd 1,368 550 1,078 896 1,076 total no. 378 128 286 54 128 positive no. 67 18 87 11 12 positive rate (%) 17.7% 14.1% 30.4% 20.4% 9.4% p value (patient vs. hd) 6.2e-11 1.6e-08 1.5e-10 2.4e-02 1.5e-04 table 1. comparison of serum anti-adiponectin antibody levels between the hd subjects and aci, age-matched aci, cad, age-matched ami and age-matched type 2 dm patients, examined by alphalisa. the table shows averages, sds, cut-off values (average + 2sd), total sample numbers, the numbers of positive sera of which antibody levels were higher than the cut-off value and the positive rates (%) of the hd subjects; averages, sds, total sample numbers, numbers of positive sera of which the antibody levels were higher than the cut-off value and the positive rates (%) of patients; and p values of the statistical comparison between the hd subjects and patients. 3takaki hiwasa, xiao-meng zhang, risa kimura, mikiko ohno, po-min chen, eiichiro nishi, koh ono, takeshi kimura, ikuo kamitsukasa, takeshi wada, akiyo aotsuka, seiichiro mine, hirotaka takizawa, koichi kashiwado, minoru takemoto, kazuki kobayashi, harukiyo kawamura, ryoichi ishibashi, koutaro yokote, rika nakamura, go tomiyoshi, natsuko shinmen and hideyuki kuroda: elevated adiponectin antibody levels in sera of patients with atherosclerosis-related coronary artery disease, cerebral infarction and diabetes mellitus adiponectin-abs were still significantly higher in patients with aci than in the hd subjects (p = 0.024; table 1). the positive rates of adiponectin-abs in the hd subjects and patients with aci were 4.5% and 20.4%, respectively. a lp ha c ou nt hd aci 2000 4000 6000 8000 a b p ＜ 0.0001 0 0.5 1.0 0 0.5 1.0 1 specificity s en si tiv ity 2,933 (0.462. 0.861) p < 0.0001 auc = 0.652 95% ci: 0.595 0.710 figure 2. comparison of serum adiponectin-ab levels between the hd subjects and patients with acute cerebral infarction (aci). serum antibody levels examined by alphallisa are shown by a box-whisker plot as described in the legend to figure 1. the results were also evaluated by roc analysis (b). 3.3 levels of adiponectin-abs are related to dm because atherosclerosis is closely related to type 2 dm, we then compared the specimens of the hd subjects and agematched patients with type 2 dm obtained from kashiwa‐ do clinic and chiba university hospital, respectively. the average ages of the hd subjects and the patients were 57.8 and 58.5, respectively. the levels of adiponectin-abs were higher in patients with dm than in the hd subjects (figure 3a). the positive rates of adiponectin-abs in the hd subjects and patients with dm were 4.7% and 9.4%, respectively (table 1). roc analysis revealed that the auc of adiponectin-abs for dm was 0.646 (95% ci: 0.579-0.713) (figure 3b). the sensitivity and specificity of the antibody levels for the diagnosis of dm were calculated to be 46.9% and 75.0%, respectively. a b p ＜ 0.0001 1,231 (0.469. 0.750) p < 0.0001 auc = 0.646 95% ci: 0.579 0.713 1 specificity s en si tiv ity 0 0.5 1.0 0 0.5 1.0 a lp ha c ou nt hd dm 2000 4000 6000 figure 3. comparison of serum adiponectin-ab levels between the hd subjects and patients with type 2 diabetes mellitus (dm). serum adiponec‐ tin-ab levels examined by alphallisa are shown by a box-whisker plot as described in the legends of figure 1 (a). the results were also evaluated by roc analysis (b). 4. discussion recent studies have shown that autoantibodies develop and increase, not only in instances of autoimmune diseases and cancer but also in other metabolic and vascular diseases, such as autoantibodies to oxidized low-density lipoprotein and β2-glycoprotein i in atherosclerosis [14,15], heat shock proteins (hsps) in acute cardiovascular diseases [16], hsp60 in stroke [17] and gad in dm [18,19]. we have reported that rpa2 antibodies increase in stroke [10]. sostdc1 and tubb2c antibodies are associated with ci and dm [11,12]. atp2b4 and bmp-1 antibodies increase in atherosclerosis-related diseases, such as ci, cad, dm and chronic kidney disease [13]. adiponectin is a protein that is closely and inversely associated with glucose tolerance and atherosclerosis [1–9]. thus, we examined the presence of autoantibodies against adiponectin by the highly sensitive alphalisa method, which produces highly reproducible and stable results because it makes plate washing unnec‐ essary. this is the first report that confirms the presence of adiponectin-abs in sera. the levels of adiponectin-abs were significantly higher in patients with aci, ami and dm than in the hd subjects (figures 1–2, table 1). when the cut-off values were determined as the average + 2sd of the hd specimens, the positive rates of aci were somewhat higher than those of cad (table 1). however, when the age-matched specimens of hd and patients were compared, the positive rates were not apparently different. furthermore, auc values and percentages of sensitivity and specificity calculated by roc analysis were quite similar among cad, aci and dm (figures 1–3). taken together, this suggests that adiponectin-abs are almost equally associated with these atherosclerosis-related diseases. spearman’s correlation analysis was performed between the adiponectin-ab levels and the patients' data, including age, gender, height, weight, bmi and blood pressure, using the sera of patients with aci from rosai hospital. unex‐ pectedly, the antibody levels failed to show a significant correlation with the patients' data (data not shown). therefore, the adiponectin-ab levels may simply reflect whether or not the patients suffer from atherosclerosisrelated diseases, such as cad, aci and dm. in most cases, the development of autoantibodies is caused by the overexpression of the particular corresponding antigens [20–22]. the expression level of adiponectin itself is negatively correlated with obesity, cad and metabolic disorders [5,6]. namely, the adiponectin level decreases during the progression of these diseases. on the other hand, the autoantibodies increased in patients with atherosclerosis-related diseases, such as aci, ami and dm (figures 1–3). this implies that the development of adipo‐ nectin-abs did not simply result from antigen overexpres‐ sion but may play a causal and suppressive role in the progression of atherosclerosis. 4 j circ biomark, 2016, 5:8 | doi: 10.5772/63218 the concentration of adiponectin in the sera of healthy individuals ranges from 5–30 µg/ml [23], whereas the concentration of adiponectin autoantibodies may be much lower. adiponectin can form a variety of multimer com‐ plexes, among which high-molecular weight multimers, consisting of 12–18 monomers, cause anti-inflammatory, anti-atherogenic and anti-diabetic effects [24,25]. if only multimers consisting entirely of active monomers can cause such effects, the function might be disturbed by the binding of the antibody to a single monomer subunit. thus, a low autoantibody level may be able to affect adiponectin function. because adiponectin has a causal role in the progression of metabolic syndrome, dm, cad and atherosclerosis, several trials using adiponectin as a therapeutic agent have been undertaken [26–28]. it may be necessary to take into account the presence of anti-adiponectin-abs, not only in the diagnosis but also in the therapy of atherosclerosisrelated diseases. 5. competing interests this work was performed in collaboration with fujikura kasei co., ltd. and celish fd inc. rn, gt, ns and hk are employees of fujikura kasei co., ltd. 6. acknowledgements this work was supported, in part, by a research grant from the japan agency for medical research and development (amed) (practical research project for life-style related diseases including cardiovascular diseases and diabetes mellitus), grants-in-aid of japan science and technology agency (jst) and by the ministry of education, culture, sports, science and technology (mext) in japan. 7. references [1] kadowaki t, yamauchi t, kubota n, hara k, ueki k, tobe k. adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome. j clin invest 2006;116:1784–92. 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[28] turer a, scherer p. adiponectin: mechanistic insights and clinical implications. diabetologia 2012;55:2319–26. 6 j circ biomark, 2016, 5:8 | doi: 10.5772/63218 cbx763725 1..6 research article case report: whole exome sequencing of circulating cell-free tumor dna in a follicular thyroid carcinoma patient with lung and bone metastases jianlu song and zhili yang abstract metastatic follicular thyroid carcinoma (ftc), unresectable or resistance to radioactive iodine, is associated with poor survival. it is believed that this kind of ftc is driven by mutated genes. however, what kind of changes of genome and underlying mechanisms are elusive. the aim of this article is to understand whether there are somatic mutations in circulating cell-free tumor dna (cfdna) in a ftc patient with lung and bone metastases. a 55-year-old woman was diagnosed with ftc with bone and lung metastases. appropriate amounts of dna were extracted from formalin-fixed, paraffin-embedded thyroid tumor, peripheral cell-free plasma, and peripheral blood leukocytes and then sequenced. the significance of dna sequencing was evaluated. there were 13,519 common variants in both tissue dna and cfdna. fifty-five somatic mutations were identified in tumor, with 5 of them nonsynonymous. seventy-two somatic mutations were found in cfdna, with 2 of them causing amino acid change. sixteen common alterations existed in both samples, that is, 31.3% of all the tissue somatic mutations. this pilot study provided proof that cfdna represents the genomic characteristics of ftc primary tissue dna well, but also metastatic tumors. further studies are needed to better prove the effectiveness of cfdna in the field of thyroid cancer metastatic mechanism research and real-time monitoring. keywords follicular thyroid carcinoma, metastasis, circulating cell-free dna, whole exome sequencing date received: 11 december 2017; accepted: 2 february 2018 introduction most follicular thyroid carcinomas (ftcs) grow slowly after they are first identified at an early stage. it is frequently cured with adequate surgical management and radioactive iodine ablation therapy. however, few cases develop fast with lung and bone metastases. most metastatic ftcs are unresectable and refractory to radioactive iodine and associated with poor survival. 1 analysis of carcinogenesis has revealed that gene mutations play a very important role in the progression and development of tumors. the genetic profile of solid tumors is often obtained from surgical or biopsy specimens. however, tumor samples may not be obtained due to the confirmation of metastasis. in that situation, blood samples or biopsy can be used for analysis. several studies of circulating cell-free dna (cfdna) in plasma have been used for analyzing individual loci, genes, or structural variants to quantify tumor burden and detect previously characterized resistance-conferring mutations. 2–4 individual mutations of cfdna and slc5a8 and slc26a4 hypermethylation department of general surgery, shanghai jiao tong university affiliated sixth people’s hospital, shanghai, china corresponding author: zhili yang, department of general surgery, shanghai jiao tong university affiliated sixth people’s hospital, 600 yi-shan road, shanghai 200233, china. email: yangzhililaoshi@126.com journal of circulating biomarkers volume 7: 1–6 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454418763725 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:yangzhililaoshi@126.com https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454418763725 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage of thyroid carcinoma including follicular carcinoma have been reported. 5 given that sequencing of entire genes to detect ftc mutations in circulating dna has not been demonstrated, we report a somatic mutational analysis of cfdna in a ftc patient with lung and bone metastases by whole exome sequencing (wes). the results demonstrate that the detection of cfdna may reflect the mutation of ftc and further develop to be a better way for monitoring the progression of thyroid cancer. patient and methods patient a 55-year-old woman was diagnosed with ftc with synchronous bone and lung metastases and then performed total thyroidectomy at shanghai jiao tong university affiliated sixth people’s hospital (figure 1). the informed consent for dna sequencing was obtained from the patient. preoperative peripheral cell-free plasma and peripheral blood leukocytes and postoperative formalinfixed, paraffin-embedded (ffpe) thyroid tumor of this patient were obtained. procedure and protocol were reviewed and approved by the ethics committee of the sixth people’s hospital. dna extraction three different types of dna were obtained from this present patient, including peripheral blood leukocytes, ffpe thyroid tumor tissue, and cfdna. total amount of dna were extracted according to the manufacturer’s instructions using qiaamp dna blood mini kit (cat# 51106), qiaamp dna ffpe tissue kit (cat# 56404), and cfdna qiaamp circulating nucleic acid kit (cat# 5514), respectively. dna concentrations were 81 ng/ml, 85 ng/ml, and 81 ng/ml, respectively, and the optical density 260/280 is 1.86, 1.87, and 1.89, respectively. dna samples were then subjected to agilent 2100 for more accurate tests. all three dna samples passed a strict quality test. whole exome sequencing the three different samples above were proceeded with wes. briefly, first, dna libraries were established with agilent sureselect human all exonv5 kits, covering about 23,000 genes. after the quality test, qualified libraries were sequenced as 100-bp paired-end reads on illumina hiseq 2000 platform (illumina, san diego, california, usa) according to the manufacturer’s instruction. all of the experiments were carried out in zhangjiang center for translational medicine, shanghai, china. figure 1. imaging of primary and metastasis lesions and pathological examination of primary thyroid lesion. (a) imaging ct of primary thyroid lesion. black arrow indicates tumor location. (b) histological analysis of primary lesion of ftc (�100). (c) lung metastasis as indicated by white arrow. (d) rib metastasis as indicated by black arrow. ct: computed tomography; ftc: follicular thyroid carcinoma. 2 journal of circulating biomarkers data analysis clean data were achieved using fastqc and low quality reads were filtered. burrows–wheeler alignment (0.7.12) methods were adopted to map the clean reads to reference genome (ucsc hg19). then, picard (http://picard.source forge.net/) and genome analysis toolkit (gatk) methods were used for duplicate removal, local realignment, and base quality recalibration. gatk unified genotyper was used for variants calling. annovar (2015-03-22) software was used to annotate variants for function (exonic, tr, intronic) reference gene, exonic function (synonymous, nonsynonymous, frameshift, stopgain, unknown), amino acid change, allele frequency (af) 1000 genomes project, and dbsnp reference number. somatic mutations were analyzed by subtracting the variants of the peripheral blood dna from the tumor dna or cfdna. t-test was used to compare the af between different types of samples. pearson correlation coefficient was adopted to calculate the relationship between different types of samples. results exome sequencing, sequence alignment, and variant calling in this study, we totally sequenced three different types of dnas from one patient. more than 10 gb sequencing data were generated per sample. the average sequencing depth for peripheral blood dna, tissue dna, and cfdna is 139�, 140�, and 133�, respectively. for three samples, more than 98.7% of the exome was covered at least 10�. the coverage rate for the above three samples was larger than 99.6%. more details are shown in table 1. the mutation types in different samples were nearly the same. variations identified in different samples we compared variations (both snv and indel) in different dna samples. generally, a large concordance between the three types of samples was observed (figure 2(a)). there were 13,519 common variants in both tissue dna and cfdna. they shared more than 98.6% common variants compared with peripheral blood sample. the coefficient rate was 0.95 (p value < 10 �15 ) and 0.96 (p value < 10 �15 ), respectively; 1.39% of variants identified in tissue dna were not found in peripheral blood and 1.13% of variants identified in cfdna did not exist in peripheral blood. comparing tissue dna with cfdna showed that 99.2% variants were identical (figure 2(b) to (d)). the pearson coefficient between cfdna and tissue dna is 0.95 (figure 2(e)). somatic mutations were determined by subtracting the variants of the peripheral blood leukocytes dna from the tumor dna or cfdna. fifty-five somatic mutations were found in tissue dna, in which 5 mutations were nonsynonymous. seventy-two somatic mutations were found in cfdna, in which 2 of them changed amino acid. sixteen common alterations existed in both samples, that is, 31.3% in all the tissue somatic mutations (table 2). most of the common mutations were synonymous. in cfdna,only glud2 (c.g103a: p.g35r) and hla-b (c.a652g: p.i218 v) were nonsynonymous mutations, of which glud2 (c.g103a: p.g35r) was also found in the tumor dna. discussion genome-wide sequencing of plasma dna was first used in prenatal diagnostics, demonstrating comprehensive genome characteristics. 6 recent studies showed that sequencing of circulating cfdna from plasma is a potential tool for monitoring advanced cancer. compared with traditional biopsy, cfdna sequencing is noninvasive, easy to get samples repeatedly, and to large extent reflects the comprehensive genomic characteristic of the tumor progress. 2–4,7 in our data, cfdna from ftc was for the first time sequenced together with primary tumor in the advanced thyroid cancer. the results demonstrate that snv and mutation rate of cfdna are characterized by the features of ftc, and both tissue dna and cfdna shared more than 95% common alterations. it will be helpful for doctors or researchers to use cfdna as a new method for the diagnosis and monitoring of thyroid carcinoma. recent studies showed that cancers arise through a process of somatic evolution that can result in substantial subclonal heterogeneity within tumors, and a large fraction of polyclonal tumors and a larger sub-clonal mutation fraction may be associated with relapse and metastasis. 8,9 it has been established that there were different mutation variants between primary and metastatic tumors. in our results, there were different variants identified in tissue dna and cfdna except for common alterations. one of the reasons may be the existence of distinct mutational genes of bone and lung metastatic tumors in cfdna. 7 in addition, the detection of genomic alteration by sequencing of cfdna is to some extent dependent on the af of the mutant alleles in the tissue dna. af value of variants which existed in both cfdna and tissue dna is significantly larger than that of variants only table 1. outline of the wes of cfdna, tumor dna, and peripheral blood dna. peripheral blood dna tumor dna cfdna total effective sequence data (gb) 11.0 10.0 11.2 coverage rate on targeted region 99.76 99.67 99.8 mean depth 140.04 139.59 133.21 no. of snvs 41,410 41,874 41,206 no. of indels 3259 3490 3486 cfdna: cell-free dna; wes: whole exome sequencing. song and yang 3 http://picard.sourceforge.net/ http://picard.sourceforge.net/ f ig u r e 2 . r e la ti o n sh ip b e tw e e n th e th re e ty p e s o f d n a sa m p le s. (a ) m u ta ti o n ty p e s o f th re e d if fe re n t d n a sa m p le s; (b ) an d (c ) co m m o n va ri an ts ar e sh o w n b y ve n n y d ia gr am ; (d ) p e rc e n ta ge s o f co m m o n o r d if fe re n t va ri an ts sh o w n in h is to gr am (g re e n , co m m o n va ri an ts ; re d , d if fe re n t va ri an ts ); (e ) co rr e la ti o n am o n g va ri an ts in d if fe re n t sa m p le s. 4 journal of circulating biomarkers existed in tissue dna, indicating that the higher the af is in tissue dna, the easier the cfdna is to be detected. thirdly, the sensitivity of cfdna sequencing was dependent on sequencing depth. 10 high sequencing depth will help to find rare mutations in tissue dna. in this study, we acquired 100 sequencing depth, so the mutation with less than 1% af is unable to be identified theoretically. except for fam69a, rapgef4-as1, loc100287072, and cdrt15l2, most of these common mutation genes are related to many functions of cancer cells, and unc5d gene inhibits thyroid cancer cell behaviors. 11 as a common nonsynonymous mutant gene glud2, its activity supports cancer cell proliferation under glutamine depletion. 12 therefore, these gene mutations may be involved in the metastatic process of ftc. in summary, we first performed wes from cfdna of an advanced thyroid cancer patient. this pilot study provided preliminary evidence that cfdna represents the genomic characteristics of primary ftc, implying the risk of metastasis. further studies guarantee sequencing of metastatic tumors, increase more cases, and determine the biomarker of cfdna for metastasis of thyroid cancer. acknowledgements the authors thank dr bo peng and dr yanbing qi (zhangjiang center for translational medicine, shanghai, china) for sequencing analysis. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the national natural science foundation of china (# 81472499). table 2. list of genes with somatic mutations in tumor and cfdna. tissue somatic mutation genes cfdna somatic mutation genes intersection (31%) fam69a ube2l3 ttc39a cilp2 fam69a rpl5 tfap2e fam69a znf431 rpl5 ankrd45 fbxo28 rpl5 defb124 ankrd45 slc9a2 rhoa tmem56 loc101927631 rapgef4-as1 rapgef4-as1 loc101243545 ankrd45 ube2l3 eomes atf2 tapt1-as1 nucks1 fam9b twf2 eomes brd9 diexf sh3kbp1 ncald twf2 neurod6 cnrip1 efnb1 mpv17l ruvbl1 tmem229a ermn pja1 ube2l3 tm4sf19-as1 unc5d rapgef4-as1 mospd1 unc5d march6 dpy19l4 eomes mir4424 fjx1 kctd20 glipr2 twf2 map6d1 snx20 tmem248 hras golim4 hla-b cdrt15l2 dnajc2 fjx1 hes1 hla-drb1 loc100287072 ncald fam86c1 pdcl2 ppia eid2 has2-as1 rcor1 anxa5 ncf1c glud2 rab1b pdcd7 cpe unc5d cxcr5 chrna5 nudcd2 fam35bp lnx2 snx20 casc15 fam35dp ctage10p alox15p1 epb41l2 fjx1 emc7 cdrt15l2 fzd1 loc338797 snn loc100287072 xpo7 slitrk1 mpv17l vat1 ncald mir4511 dcxr eid2 msantd3-tmeff1 idh3a rwdd2b ncoa3 tmeff1 gnptg glud2 csgalnact2 snx20 ambra1 pafah1b1 naalad2 cdrt15l2 fgfr1op2 loc100287072 micu2 cers1 meis2 gdf1 mpv17l eid2 znf771 gng8 tom1l2 znf816-znf321p smarce1 glud2 cfdna: cell-free dna. song and yang 5 references 1. kapiteijn e, schneider tc, morreau h, et al. new treatment modalities in advanced thyroid cancer. ann oncol 2012; 23(1): 10–18. 2. murtaza m, dawson sj, tsui dw, et al. non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma dna. nature 2013; 497(7447): 108–112. 3. forshew t, murtaza m, parkinson c, et al. noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma dna. sci transl med 2012; 4(136): 136ra68. 4. bettegowda c, sausen m, leary rj, et al. detection of circulating tumor dna in earlyand late-stage human malignancies. sci transl med 2014; 6(224): 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one 2014; 9(8): e104417. 11. zhang mm, sun f, cui b, et al. tumor-suppressive function of unc5d in papillary thyroid cancer. oncotarget 2017; 8(56): 96126–96138. 12. takeuchi y, nakayama y, fukusaki e, et al. glutamate production from ammonia via glutamate dehydrogenase 2 activity supports cancer cell proliferation under glutamine depletion. biochem biophys res commun 2018; 495(1): 761–767. 6 journal of circulating biomarkers << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged 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/flattenerpreset << /clipcomplexregions true /convertstrokestooutlines false /converttexttooutlines false /gradientresolution 300 /linearttextresolution 1200 /presetname ([high resolution]) /presetselector /highresolution /rastervectorbalance 1 >> /formelements true /generatestructure false /includebookmarks false /includehyperlinks false /includeinteractive false /includelayers false /includeprofiles true /marksoffset 9 /marksweight 0.125000 /multimediahandling /useobjectsettings /namespace [ (adobe) (creativesuite) (2.0) ] /pdfxoutputintentprofileselector /documentcmyk /pagemarksfile /romandefault /preserveediting true /untaggedcmykhandling /usedocumentprofile /untaggedrgbhandling /usedocumentprofile /usedocumentbleed false >> ] /syntheticboldness 1.000000 >> setdistillerparams << /hwresolution [288 288] /pagesize [612.000 792.000] >> setpagedevice article journal of circulating biomarkers protein detection using the multiplexed proximity extension assay (pea) from plasma and vaginal fluid applied to the indicating fta elute micro card™ original research article malin berggrund1, daniel ekman2, inger gustavsson1, karin sundfeldt3, matts olovsson4, stefan enroth1 and ulf gyllensten1* 1 department of immunology, genetics and pathology, science for life laboratory, biomedical center, uppsala university, uppsala, sweden 2 olink bioscience ab, dag hammarskjölds väg, uppsala, sweden 3 department of obstetrics and gynecology, institute for clinical sciences, sahlgrenska cancer center, gothenburg university, gothenburg, sweden 4 department of women’s and children´s health, uppsala university, uppsala, sweden *corresponding author(s) e-mail: ulf.gyllensten@igp.uu.se received 17 march 2016; accepted 28 april 2016 doi: 10.5772/64000 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract the indicating fta elute micro card™ has been developed to collect and stabilize the nucleic acid in biological samples and is widely used in human and veterinary medicine and other disciplines. this card is not recommended for protein analyses, since surface treatment may denature proteins. we studied the ability to analyse proteins in human plasma and vaginal fluid as applied to the indicating fta elute micro card™ using the sensitive proximity extension assay (pea). among 92 proteins in the proseek multiplex oncology iv2 panel, 87 were above the limit of detection (lod) in liquid plasma and 56 among 92 above lod in plasma applied to fta cards. washing and protein elution protocols were compared to identify an optimal method. liquid-based cytology samples showed a lower number of proteins above lod than fta cards with vaginal fluid samples applied. our results demonstrate that samples applied to the indicating fta elute micro card™ are amendable to protein analyses, given that a sensitive protein detection assay is used. the results imply that biological samples applied to fta cards can be used for dna, rna and protein detection. keywords fta elute card, vaginal fluid, plasma, prox‐ imity extension assay 1. introduction a number of sampling devices have been developed to collect blood or plasma/serum for protein analyses, ranging from solid phase matrices to the recent use of nanocapillary technology [1]. the collection of blood spots, buccal cells and saliva on filter paper is a com‐ mon sampling method, used for the screening of newborns for inherited diseases, in forensic medicine and for many other purposes. specific surface treatments have 1j circ biomark, 2016, 5:9 | doi: 10.5772/64000 been developed to stabilize nucleic acids applied onto filter paper. whatman fta technology consists of two distinct chemistries, both of which have the ability to lyse cells on contact, denature proteins and protect dna and rna from degradation. the fta elute card™ (ge healthcare, united kingdom) contains a chaotropic salt and proteins remain tightly bound while dna is eluted from the matrix. the indicating fta elute micro card™ also includes a purple dye that turns white when a clear sample is applied. this colour change is useful for verifying that the biological sample has been deposited on the card surface, specifically when the card is used for self-collection of samples. the indicating fta elute micro card™ allows for a simple elution of dna and rna from the surface of the card, and is extremely suitable for genotyping, sequencing and real-time pcr applications [2, 3]. the indicating fta elute micro card™ is presently used for the self-collection of cervico-vaginal fluid (cvf) samples in cervical cancer screening programmes [4-10]. it will therefore be of interest to determine if samples collected onto the fta card™ can also be used for analyses of protein biomarkers. the fta elute card is not recommended for protein analyses, since surface treatment may denature proteins and to the best of our knowledge, no study has been performed to determine whether proteins applied to the fta elute micro card™ can be detected. the aim of this study was to examine the potential for using the indicating fta elute micro card™ for protein analyses by employing the very sensitive and highly specific protein extension assay (pea) [11, 12]. pea is based on the use of two antibodies towards the target protein, providing high specificity. each antibody has a dna tail attached and if the dna tails on the two antibodies are within reach of each other, they can act as a substrate for dna polymerase. protein detection is achieved through a real-time pcr, resulting in high analytical sensitivity. the pea has previously been used to measure protein in biomarker studies [13, 14]. if protein detection is indicated as feasible in samples applied to the indicating fta elute micro card™, it will offer a quick, cost-effective, easy-to-store option for sample collection, both for diagnostic purposes and population screening. 2. samples and methods 2.1 samples and preparation cvf samples were collected from women participating in the organized cervical cancer screening using the rover viba brush (rover medical devices b.v., oss, the nether‐ lands) and applied to the indicating fta elute micro card™ as described earlier [15]. all fta card samples were selected from hpv-negative women. one sample, which had been stored for two weeks, was selected for a first buffer-test. twenty-eight cvf samples were gathered representing a storage time on the card of 1-29 months prior to analysis. twelve samples had been stored for one month, six samples for seven months, six samples for 19 months and four samples were stored for 29 months. five additional samples, with storage times of less than two weeks, were selected for the testing protocols for washing. plasma samples were obtained from five women. for each woman, 10 μl plasma aliquots were applied to an indicating fta elute micro card™; the card surfaces were dried and stored at room temperature for either 24 h or 72 h. all samples applied to the fta cards were processed using the 3 mm ø harris micro punch (whatman, inc., clifton, nj) for collecting punches as previously described [15]. the following washing procedures were compared: i) no wash prior to elution; ii) 1 x 5 min. wash in 100 μl water; iii) 2 x 5 min. wash in 100 μl water. proteins were eluted from the indicating fta elute micro card™ by placing the punch in either a 20 μl radioimmunoprecipitation assay buffer (ripa) or 20 μl phosphate-buffered saline (pbs)-tween 20 (0.05 %) buffer for 60 to 90 minutes. we also investigated samples from 28 women that were collected using the procedure applied in liquid-based cytology. these samples were collected using a cytobrush and dissolved in a 20 ml thinprep storage solution (holo‐ gic, inc. san diego, usa), and stored at room temperature for 3-10 days prior to freezing. from this solution, 1 μl was used for the pea analysis. 2.2 protein analysis one μl of either liquid plasma, elution from fta card, or liquid-based cytology sample was analysed using the proximity extension assay (pea) and the proseek multiplex oncology i v2 panel (olink bioscience ab, uppsala, sweden). this panel includes 92 proteins selected on the basis of their reported associations with cancer. the protein analysis is reported as normalized protein expression levels (npx), which are ct values normalized by the subtraction of values for extension control, as well as an interplate control; the scale is shifted using a correction factor (normal background noise) and reported in log 2 scale. all assay characteristics including detection limits and measure‐ ments of assay performance and validations are available from the manufacturer's webpage (http://www.olink.com/ products/proseek-multiplex/downloads/). 2.3 statistical analysis all analyses and illustrations were performed in r [16]. spearman correlations were used throughout. the signifi‐ cance of differences in distributions was calculated using the double-sided wilcoxon signed-rank test. resulting pvalues were corrected for multiple testing using either bonferroni or fdr correction and considered significant if the q-value < 0.05. linear modelling for estimating the correlation between the npx measurement and storage time was performed with the lm function and the anova function. 2 j circ biomark, 2016, 5:9 | doi: 10.5772/64000 http://www.olink.com/products/proseek-multiplex/downloads/ http://www.olink.com/products/proseek-multiplex/downloads/ 3. results 3.1 elution buffer optimization we first compared two elution buffers, pbs-tween 20 (0.05 %) and ripa, for their efficiency in terms of recovering proteins from the indicating fta elute micro card™. a single punch from one fta sample was eluted in 20 μl pbstween 20 (0.05 %) or ripa buffer and analysed using the proseek multiplex oncology i v2 panel. for both buffers, 80 of the 92 proteins in the panel had levels above the assay background (spearman’s rho, r2=0.92, p-value < 2.2e-16) (figure 1). with a cut-off of >1 npx above background, the pbs-tween 20 buffer resulted in the detection of 43 proteins and the ripa buffer in 33 proteins. additionally, 60 proteins showed higher npx values for the sample fta elution card than a blank fta elution card for pbs-tween 20 buffer and 52 proteins for the sample eluted in ripa buffer. thus, the pbs-tween 20 buffer performed better than the ripa buffer and was therefore used in the following analyses. figure 1. comparison of the npx values for the 92 proteins in the proseek multiplex oncology iv2 panel from analysis of a sample applied to the indicating fta elute micro card™ and eluted with either ripa (y-axis) or pbs-tween 20 buffer (x-axis). pbs-tween reports slightly higher npxvalues for the majority of the 92 proteins. 3.2 proteins are measurable from the fta cards we next compared liquid plasma samples with plasma applied to the indicating fta elute micro card™ and stored for either 24 h or 72 h. one punch from each fta card was eluted in 20 μl pbs-tween 20 buffer and analysed using the proseek multiplex oncology i v2 panel. as expected from the elution protocol, the plasma sampled and applied to the fta showed generally lower protein amounts than the liquid plasma samples. in total, 87 of the 92 proteins in the panel were detected in all liquid plasma samples and 56 and 52 proteins were detected in the samples on fta cards stored for 24 h and 72 h, respectively (table 1). protein plasma (% of samples over lod) 24 h (% of samples over lod) 72 h (% of samples over lod) il-8 100% 100% 100% vegf-a 100% 100% 100% am 100% 100% 100% gdf-15 100% 100% 100% plgf 100% 100% 100% csf-1 100% 100% 100% cstb 100% 100% 100% mcp-1 100% 100% 100% klk6 100% 100% 100% trail-r2 100% 100% 100% lap-tgf-beta-1 100% 100% 100% tf 100% 100% 100% tnf-r1 100% 100% 100% pdgf-subunit-b 100% 100% 100% park7 100% 100% 100% cxcl11 100% 100% 100% ve-statin 100% 100% 100% il-7 100% 100% 100% scf 100% 100% 100% cxcl9 100% 100% 100% tnf-r2 100% 100% 100% tnfsf14 100% 100% 100% gh 100% 100% 100% fasl 100% 100% 100% fas 100% 100% 100% ccl19 100% 100% 100% emmprin 100% 100% 100% cxcl10 100% 100% 100% ep-cam 100% 100% 100% hgf 100% 100% 100% litaf 100% 100% 100% cxcl5 100% 100% 100% mk 100% 100% 100% u-par 100% 100% 100% cdh3 100% 100% 100% lyn 100% 100% 100% flt3l 100% 100% 100% hb-egf 100% 100% 100% cd69 100% 100% 100% tr-ap 100% 100% 100% cdkn1a 100% 100% 100% reg-4 100% 100% 100% 3malin berggrund, daniel ekman, inger gustavsson, karin sundfeldt, matts olovsson, stefan enroth and ulf gyllensten: protein detection using the multiplexed proximity extension assay (pea) from plasma and vaginal fluid applied to the indicating fta elute micro card™ protein plasma (% of samples over lod) 24 h (% of samples over lod) 72 h (% of samples over lod) vegf-d 100% 100% 100% he4 100% 100% 100% cxcl13 100% 100% 100% ca-125 100% 100% 100% prss8 100% 100% 100% pecam-1 100% 100% 100% fr-alpha 100% 100% 100% tie2 100% 100% 80% mic-a 100% 100% 80% il-6 100% 100% 60% icoslg 100% 100% 60% fs 100% 100% 60% ntrk3 100% 100% 20% ilt-3 100% 80% 80% vegfr-2 100% 80% 80% itga1 100% 80% 80% erbb3-her3 100% 60% 80% mia 100% 60% 80% ptpn22 100% 60% 60% sele 100% 60% 40% fur 100% 60% 40% ar 100% 60% 20% eif-4b 100% 40% 40% ezr 100% 40% 20% prl 100% 40% 20% il-12 100% 20% 40% mmp-1 100% 20% 40% erbb2-her2 100% 20% 40% tnfrsf4 100% 20% 40% il-6ra 100% 20% 20% baff 100% 20% 20% erbb4-her4 100% 20% 20% nemo 100% 20% 20% fadd 100% 20% 0% ifn-gamma 100% 20% 0% vim 100% 0% 40% casp-3 100% 0% 20% cd40-l 100% 0% 0% il-1ra 100% 0% 0% hk11 100% 0% 0% caix 100% 0% 0% tgf-alpha 100% 0% 0% protein plasma (% of samples over lod) 24 h (% of samples over lod) 72 h (% of samples over lod) egfr 100% 0% 0% thpo 100% 0% 0% il-17rb 100% 0% 0% cea 80% 20% 20% myd88 80% 0% 20% epo 80% 0% 0% il-2 60% 80% 100% tnf 40% 100% 100% table 1. list of proteins and percentage of samples over limit of detection (lod) for each protein when using liquid plasma (left column) plasma on indicating fta elute micro card™ stored for 24h (middle column) and 72h (right column) the correlation between all observations for all proteins between 24 h and 72 h was high (spearman’s rho, r2=0.89, p-value < 2.2e-16) (figure 2). in a per-protein analysis, replacing values below the limit of detection with protein specific lod-values, five proteins (csf-1, ntrk3, icoslg, scf and pecam-1) were found to have nomi‐ nally significant differences between the two time-points (p < 0.05, wilcox test). none of these associations remained significant after multiple hypothesis corrections. only two proteins showed a mean difference of more than 1 npx unit between samples stored for 24 h and 72 h (csf-1 and tnfr1, both of which had lower npx values at 72 h). figure 2. correlation between npx values for all observations with measurements above the limit of detection at two time-points from cervicovaginal fluid samples stored on fta cards for 24 h (x-axis) and 72 h (y-axis), spearman’s rho, r2=0.89, p-value < 2.2e-16 3.3 washing optimization there was a large overlap between the proteins that were detectable in liquid plasma and on the fta cards. only six 4 j circ biomark, 2016, 5:9 | doi: 10.5772/64000 proteins were detectable in liquid plasma and on fta cards stored for 24 h, but not on fta cards stored for 72 h (figure 3). twelve out of 15 plasma samples on the fta cards did not pass the proseek internal controls for incubation, extension and detection. nonetheless, 56 of 92 proteins were detected over lod in all samples for fta cards stored 24 h and 51 of 92 proteins were detected in all samples for fta cards stored for 72 h. we reasoned that components in the surface chemistry of the fta elute cards™ had a negative effect on the assay. figure 3. the overlap in proteins above lod in 100% of the samples when analysing liquid plasma (plasma), plasma samples applied to the indicating fta elute micro card™ and stored for 24 h (t24) and 72 h (t72) prior to analysis in order to reduce the problem regarding failed internal controls, we tested three different protocols for washing punches prior to protein elution. one punch from each of 28 indicating fta elute micro cards™ with a vaginal fluid sample was analysed using one of three different protocols: i) no wash prior to elution; ii) 1 x 5 min. wash in 100 μl water; iii) 2 x 5 min. wash in 100 μl water. samples were then eluted in 20 μl pbs-tween 20 buffer and analysed using the proseek multiplex oncology i v2 panel. with method i (no wash), eight samples showed signs of inhibition of internal controls, compared to one sample with method ii and zero samples with method iii. in total, 57 of 92 proteins had levels over lod in all 28 samples using method i and 73 proteins were detected in 80% of the samples. with method ii, 29 of 92 proteins were detectable in all 28 samples and 56 proteins were detected in 80% of the samples. with method iii, 21 of 92 proteins had levels over lod in all 28 samples and 45 proteins were detected in 80% of the samples. the number of proteins detected using the different extraction protocols as a function of the fraction of individuals with protein levels over lod is shown in figure 4. 3.4 detectable proteins remain stable on the fta-cards the 28 samples were stored for up to 29 months. no significant effect of storage time on protein amounts was observed using bonferroni correction (p > 0.065). with fdr correction, 19 proteins showed an effect due to storage time (p < 0.05). the urokinase plasminogen activator surface receptor (upar) protein had the largest difference between samples stored for one month and samples stored for 29 months, with a mean difference of 3.59 npx (supplemen‐ tary table s1). 3.5 adding more material does not improve detectability the additional wash improved the analysis, but may also have resulted in loss of protein. we therefore investigated whether increasing the input material, i.e., the number of punches from a sample, would result in higher protein amounts. to this end one, two, three or four punches were obtained from an fta card with cvf sample from five women and washed for 2 x 5 min. in 100 μl water (method iii), eluted in a 20 μl pbs-tween 20 buffer and analysed using the proseek multiplex oncology i v2 panel. the samples using one punch passed all the assay controls, while the samples using two punches or more did not pass the incubation control. extension and detection controls were not affected, regardless of the number of punches used. independent of the number of punches used, roughly 50% of proteins in the panel showed levels above the lod in 100% of the women (47/92 with one punch, 47/92 with two punches, 46/92 with three punches and 46/92 with four punches). when considering proteins that were measura‐ ble in more than 50% of individuals, 62 and 58 proteins were above lod, using one and four punches, respectively. thus, increasing the number of punches did not increase the number of proteins to a level above the lod when using the presently used wash and elusion protocol. figure 4. the number of proteins detected over limit of detection (lod) for the three washing methods as a function of the percentage of samples studied with a npx value over the lod 5malin berggrund, daniel ekman, inger gustavsson, karin sundfeldt, matts olovsson, stefan enroth and ulf gyllensten: protein detection using the multiplexed proximity extension assay (pea) from plasma and vaginal fluid applied to the indicating fta elute micro card™ 3.6 comparison with liquid cytology samples for comparison we also investigated samples collected with the procedure used in liquid-based cytology (thin‐ prep transport solution). a total of 28 cytology samples were analysed using the proseek multiplex oncology i v2 panel. two of the samples did not pass the internal assay controls and were removed from the analysis. only seven proteins were above the lod in 100% of the 26 cytology samples, compared to 21 proteins that were above the lod in 100% of the fta samples (method iii) (table 2). protein fta elute micro cardtm (% of samples over lod) protein liquid cytology samples (% of samples over lod) il-8 100% il-8 100% vegf-a 100% vegf-a 100% am 100% il-1ra 100% plgf 100% cstb 100% ezr 100% u-par 100% il-1ra 100% he4 100% cstb 100% prss8 100% mcp-1 100% mcp-1 96% tnf-r1 100% tnf-r1 96% park7 100% cea 96% fadd 100% ezr 92% fas 100% hk11 92% emmprin 100% ep-cam 92% litaf 100% cxcl5 92% casp-3 100% tf 88% cdkn1a 100% fas 88% tgf-alpha 100% emmprin 88% he4 100% casp-3 88% eif-4b 100% fr-alpha 88% ca-125 100% gdf-15 85% prss8 100% mk 85% gdf-15 96% tgf-alpha 85% tf 96% cxcl13 85% cxcl9 96% vim 85% ptpn22 96% plgf 81% cxcl10 96% il-6 81% cxcl5 96% cxcl10 77% mk 96% hgf 77% u-par 96% ar 77% lyn 96% cxcl9 73% cxcl13 96% erbb3-her3 73% protein fta elute micro cardtm (% of samples over lod) protein liquid cytology samples (% of samples over lod) cea 96% tnf-r2 65% fur 93% tr-ap 65% ep-cam 93% klk6 62% ar 93% trail-r2 62% nemo 93% fadd 62% klk6 89% litaf 62% hk11 89% nemo 62% erbb3-her3 89% eif-4b 62% il-6 82% ca-125 58% lap-tgfbeta-1 82% lap-tgfbeta-1 50% fasl 82% csf-1 46% cd69 82% park7 46% vim 82% fur 46% fr-alpha 82% caix 42% hgf 79% erbb4-her4 42% icoslg 79% cd69 31% csf-1 71% mic-a 31% erbb2-her2 71% am 27% mic-a 71% erbb2-her2 27% ve-statin 68% flt3l 27% cdh3 64% ccl19 23% flt3l 57% ptpn22 23% pecam-1 57% lyn 23% ccl19 54% pecam-1 23% myd88 54% scf 15% ilt-3 46% hb-egf 15% tnf-r2 46% cxcl11 12% pdgfsubunit-b 39% baff 12% tnf 39% tnfrsf4 12% il-7 36% il-12 8% tnfsf14 36% fasl 8% erbb4-her4 36% myd88 8% itga1 36% mia 8% trail-r2 32% cdkn1a 8% cxcl11 32% egfr 8% scf 21% itga1 8% fs 21% fs 8% reg-4 18% ilt-3 4% hb-egf 14% tie2 4% 6 j circ biomark, 2016, 5:9 | doi: 10.5772/64000 protein fta elute micro cardtm (% of samples over lod) protein liquid cytology samples (% of samples over lod) mmp-1 11% pdgfsubunit-b 4% tie2 7% il-2 4% baff 7% il-7 4% mia 7% il-6ra 4% tnfrsf4 7% mmp-1 4% tr-ap 7% tnfsf14 4% thpo 7% gh 4% ntrk3 7% cdh3 4% sele 4% reg-4 4% il-12 4% vegf-d 4% prl 4% il-17rb 4% gh 4% cd40-l 0% caix 4% sele 0% egfr 4% epo 0% cd40-l 0% ve-statin 0% epo 0% prl 0% il-2 0% tnf 0% il-6ra 0% icoslg 0% vegfr-2 0% vegfr-2 0% ifn-gamma 0% ifn-gamma 0% vegf-d 0% thpo 0% il-17rb 0% ntrk3 0% table 2. list of proteins and percentage of samples over the limit of detection (lod) among the indicating fta elute micro card™ samples (left column) and the liguid-based cytology samples (right column); 2 of the 28 liguidbased cytology samples did not pass the quality controls and were excluded. 7 proteins were detected in all 26 liguid-based cytology samples, compared to 21 proteins that were detected in the 28 fta samples. 4. discussion this study shows that plasma and cervico-vaginal fluid samples applied to the indicating fta elute micro card™ are amendable to protein analysis. the pbs-tween (0.05 %) elution is the preferable elution buffer. the majority of proteins in the proseek multiplex oncology i v2 panel that were detectable in liquid plasma were also above lod in samples applied to the indicating fta elute micro card™. the npx values were generally lower in the fta card samples, although some proteins showed high npx values. this was to some extent expected because of the elution process, since for fta card samples, the 10 μl of plasma in a punch was eluted in a 20 μl of buffer, resulting in half of the concentration of proteins entering the assay, compared to the liquid plasma samples. our comparison of washing methods showed that al‐ though extensive washing may results in a loss of protein, adding a second wash to the punches from fta cards reduced the number of samples that did not pass the internal controls. the results showed that the proteins on the fta card appear stable. the protein profile in the vaginal fluid samples on fta cards from 28 women stored for up to two years showed an effect on 19 proteins, with fdr correction (p < 0.05); however, this effect was not seen using bonferroni correction (p > 0.065). we also studied whether the number of proteins above lod detected in the fta card samples could be increased by adding more starting material, i.e., more punches in the washing and elution steps. however, this did not result in additional proteins appearing above lod, but only increased the number of samples failing internal control. an alternative would be to use a protocol that selectively removes inhibitors from the fta cards' surface prior to the elution of the proteins. the liquid cytology samples showed a slightly higher abundance of some proteins compared to samples from the indicating fta elute micro card™, but the number of proteins with levels above lod was higher for the fta cards. the lower number of detectable proteins in the liquid cytology samples may have been due to the procedure for sample collection and storage, and in particular, the substantial dilution of the original sample in the transport solution. our study demonstrates the feasibility of measuring proteins from vaginal fluid and plasma collected onto the indicating fta elute micro card™. this opens up the possibility of studying both dna, rna and protein biomarkers in a single sample applied to the indicating fta elute micro card™. the indicating fta elute micro card™ is extremely suitable where biobanking is concerned, since these cards can be stored at room temperature and do not require large storage space or the subzero conditions needed for liquid cytology samples. 5. compliance with ethical research standards daniel ekman is employed at olink bioscience ab, uppsala. all other authors declare no conflicts of interest. 6. acknowledgements this study was supported by the swedish cancer founda‐ tion and the swedish foundation for strategic research (ssf). biocare is a national strategic cancer research program at the university of gothenburg. the pea measurements were performed by olink bioscience ab, uppsala, sweden. 7. references [1] chen jq, wakefield lm, goldstein dj. capillary nano-immunoassays: advancing quantitative proteomics analysis, biomarker assessment, and molecular diagnostics. j transl med. 2015;13:182. 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[10] wang sm, hu sy, chen w, chen f, zhao fh, he w, et al. feasibility and accuracy evaluation of three human papillomavirus assays for fta card-based sampling: a pilot study in cervical cancer screening. bmc cancer. 2015;15:848. [11] lundberg m, eriksson a, tran b, assarsson e, fredriksson s. homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood. nucleic acids res. 2011;39(15):e102. [12] assarsson e, lundberg m, holmquist g, bjorkesten j, thorsen sb, ekman d, et al. homogenous 96-plex pea immunoassay exhibiting high sensitivity, specificity, and excellent scalability. plos one. 2014;9(4):e95192. [13] enroth s, bosdotter enroth s, johansson a, gyllens‐ ten u. effect of genetic and environmental factors on protein biomarkers for common non-communi‐ cable disease and use of personally normalized plasma protein profiles (pnppp). biomarkers. 2015;20(6-7):355-64. 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[16] r core team. r: a language and environment for statistical computing.: r foundation for statistical computing; 2014. 8 j circ biomark, 2016, 5:9 | doi: 10.5772/64000 article journal of circulating biomarkers oxygen-related differences in cellular and vesicular phenotypes observed for ovarian cell cancer lines original research article evo k. lindersson søndergaard1*, lotte hatting pugholm1, rikke bæk1, malene møller jørgensen1, anne louise schacht revenfeld 1 and kim varming1 1 department of clinical immunology, aalborg university hospital, aalborg, denmark *corresponding author(s) e-mail: e.soendergaard@rn.dk received 20 august 2015; accepted 10 december 2015 doi: 10.5772/62219 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract extracellular vesicles (evs) are one of several tools that cells use to communicate with each other. this communication is facilitated by a number of surface-associated proteins and the cargo of the vesicles. for several cancer types, the amount of evs is observed to be up-regulated in patients com‐ pared to healthy individuals, possibly signifying the presence of an aberrant process. the hypoxia-induced release of evs from cancer cells has been hypothesized to cause the malignant transformation of healthy recipient cells. in this study, the phenotype of cells and evs from the ovarian cancer cell lines, cov504, skov3, and pt4, were quantified and analysed under normoxic and hypoxic conditions. it was shown that both cells and evs express common markers and that the ev phenotype varies more than the cellular phenotype. additionally, cells subjected to 24 hours of hypoxia compared to normoxia produced more evs. the phenotyping of evs from cancer cell lines provides information about their molecular composition. this information may be translated to knowledge regarding the functionality of evs and lead to a better understanding of their role in cancer. keywords extracellular vesicles, ovarian cancer cell line, ev array, phenotype, hypoxia 1. introduction the interest in extracellular vesicles (evs) has increased immensely over the last few years. evs are believed to be an important component of extracellular communication [1], although, their exact functions are not fully understood. in humans, evs are a heterogeneous population of mem‐ brane-enclosed vesicles that are released into the extracel‐ lular space by most cell types. they have been shown to be involved in a variety of important physiological and immunological processes [2–4]. they may also be involved in the progression of pathological conditions such as cancer [5,6]. evs are often divided into three major subpopula‐ tions based on their size, biogenesis and molecular compo‐ sition, namely exosomes, microvesicles and apoptotic bodies [1,7–11]. even though the molecular composition of these three subsets of evs is different, several markers overlap. these markers are not ubiquitously expressed on evs but are found on a majority of them [7,9,12]. so far, 1j circ biomark, 2016, 5:1 | doi: 10.5772/62219 identification of a specific marker that, with certainty, can distinguish between the three subsets has not been discov‐ ered. along with these proteins, the molecular composition depends on the cellular source, often mirroring the parent cell [13,14]. notably, recent data suggest that the phenotype of evs is specific and well-regulated, and not just the result of a casual sampling of molecules from the parent cell [15]. the current study investigates the subset of evs that presents cd9, cd63 and cd81. a large number of studies have reported that evs are involved in the development and progression of cancer. their role in cancer is emphasized by the fact that the release of evs is accelerated in tumour cells, as demon‐ strated by in vitro studies, as well as by the large amount of evs or ev-like structures that can be purified from plasma, ascites and pleural effusions of cancer patients [16–18]. tumour-derived evs have been reported to both stimulate and suppress tumour-specific and non-specific immune responses. this capacity may be explained by the similarity of the protein composition of evs and the parent cell type, which suggests that the tumour-derived evs contain tumour-specific antigens that can stimulate or inhibit an anti-tumour response [17–22]. hypoxia is one of many factors that are believed to be important for the maintenance of the tumour milieu. hypoxic regions are observed in most solid tumours [23,24] and numerous factors involved in the promotion of metastasis have been described to be induced by hypoxia [25–28]. furthermore, it has been reported that patients with hypoxic primary tumours developed more metastasis than patients with less hypoxic tumours [29,30]. hence, hypoxia is an important factor for the tumour milieu, as well as for the metastatic processes. taken together, it seems that both hypoxia and tumour-derived evs can play important roles at multiple stages of tumour pathogenesis, ranging from suppressing the anti-tumour responses to facilitating the formation of a suitable microenvironment in distant metastatic sites [31–35]. in this study, the cellular and ev phenotypes from the three ovarian cancer cell lines, cov504, skov3 and pt4, were analysed. these three cell lines were chosen as they all originally derive from ovarian cancer patients with different tumour forms. additionally, it was investigated whether hypoxia could affect the phenotypes of cells and evs. for the cellular phenotype, a flow cytometric analysis was used and five different markers were chosen on behalf of their functional differences. carboxic anhydrase ix (caix) and carboxic anhydrase xii (caxii) are known as hypoxic markers [36]. cd151 is known as an exosomal protein associated with tumour progression. it enhances cell motility, invasion and metastasis of cancer cells and is over-expressed in many tumour types [37]. cd9 and cd81 are considered as general exosomal markers [9,12]. cd9 is a cell surface glycoprotein known to complex with, e.g., integrins and other tetraspanins. it can modulate cell adhesion and migration and trigger platelet activation and aggregation [38]. cd81 is a surface glycoprotein that is known to complex with integrins, and it is involved in activation, co-simulation and differentiation [39]. for the ev characterization, the protein microarray based ev array technique was applied to analyse and phenotype a subset of these cell-derived vesicles, which carries the general ev markers, cd9, cd63 and cd81 [40]. the extensive phenotyping involved 31 markers that are related to general ev proteins, cell-specific markers and a number of cancer markers. 2. materials and methods 2.1 cell cultures skov3 (atcc® htb-77tm; atcc, manassas, va, usa) and pt4 (primary cell line from ascites from an ovarian cancer patient) were cultured in rpmi-1640 (gibco, life technologies, ca, usa) and supplied with 10% heatinactivated foetal calf serum (fcs) (gibco), 100u/ml penicillin and 0.1mg/ml streptomycin (amplicon, odense, dk). cov504 (07071902-1vl, sigma-aldrich, st. louis, mo, va) was cultured in dulbecco´s modified eagle´s medium (dmem) (gibco) supplemented with 10% fcs, 100u/ml penicillin and 0.1mg/ml streptomycin. the cells were cultured in 96-well plates in triplicates, with a concentration of 1.25 x 105 cells/well in a total volume of 250µl. media controls were included for each cell line. before supplementing the media with fcs, the fcs was centrifuged at 100.000 x g for 24h, at 4°c (ti45 rotor, beckman coulter, brea, usa) to deplete the evs present in the fcs. under normoxic conditions, the cells were cultured at 37°c in 5% (v/v) co2 and with an atmospheric o2 concentration. to induce hypoxia, the cells were cultured at 37°c in 5% (v/v) co2 and 1% (v/v) o2. to determine the cell density, images of the different cell lines were captured for each experimental condition, using a floid imaging station (life technologies). 2.2 phenotyping cells by flow cytometry the adherent cells were detached with trypsin (0.25%/ edta) (gibco) for 10 min. for each cell line, cells from the triplicate wells were pooled and washed once in cell media and then twice in pbs (500 x g, 5min, room temperature (rt)). the following antibodies and reagents were used for flow cytometry: anti-cd151 (210127) and anti-caxii (315602) (r&d systems, minneapolis, mn, usa); 7-aminoactino‐ mycin d (7aad), anti-cd9-percp cy5.5 (m-l13), control mouse igg1(k)-percp-cy 5.5 (mopc-21) and mouse igg1(k)-pe (mopc-21) (bd bioscience, san jose, ca, usa); anti-caix (2d3) (abcam, cambridge, ma, usa); anticd81-pe (1.3.3.22) (ancell corporation, mn, usa); and, as 2 j circ biomark, 2016, 5:1 | doi: 10.5772/62219 a secondary antibody, goat anti-mouse igg1(k)-pe (dako‐ cytomation, glostrup, dk). for antibody staining, each sample contained 1.2 x 105 cells. the cells were mixed with the relevant antibodies and incubated for 30 min at rt in the dark. the cells were then washed twice with pbs-bsa (0.5%, w/v) (500 x g, 5min, rt). the cells that had incubated with unconjugated antibodies were mixed with goat anti-mouse-pe and subsequently incubated for 30 min at rt in the dark. afterwards, the cells were washed, resuspended in sheath fluid with 1% paraformaldehyde (bd bioscience) and analysed. the acquisition of the stained cells was performed on a facscanto ii using facsdivtm software (version 6.1.3, bd biosciences). the analysis of the data was carried out with the flowjo software (version 10.0.7, flowjo llc, ashland, or, usa). the cellular phenotype for each marker was determined for all events except cell debris by applying a fsc/ssc gate that excludes debris. the negative isotype controls were utilized to identify the positive events. for all populations, the median fluorescence intensity (mfi) was the statistical value of choice. 2.3 preparation of evs from cell cultures for preparation of the evs, the harvested cell culture supernatant was centrifuged at rt for 10 min at 700 x g to pellet the cells. the cell-free ev supernatants were supple‐ mented with protease inhibitors (complete, edta-free, roche, de, usa) and stored at -40°c until an analysis on the ev-array was carried out. there was no further purification of the evs prior to measuring on the ev array. 2.4 phenotyping vesicles by the ev array microarray printing was performed on a spotbot® extreme protein edition microarray printer with a 946mp4 pin (arrayit, ca, us). as positive and negative control, 100 µg/ ml of biotinylated human igg and pbs with 5 % glycerol was printed, respectively. epoxy coated slides (75.6 mm x 25.0 mm; schott nexterion, de) were used and then left to dry at rt overnight prior to further analysis. for the ev array, the following antibodies and protein were used for capture: annexin v (polyclonal), caxii (315602), cd13 (498001), cd82 (423524), cd142 (323514), cd151 (210127), lamp2 (h4a3), muc1 (604804), tnf ri (16803), tnf rii (22210), tspan 8 (458811) (r&d systems); akap3 (c-20), epcam (o.n.277), mucin 16 (x306), nyeso-1 (e978), plap (8b6), tlr3 (tlr3.7) (santa cruz biotechnologies, tx, usa); alix (3a9), cd63 (mem-259), cd309 (7d4-6), hla abc (w6/32) (biolegend, ca, usa); caix (2d3), flotilin 1 (polyclonal), tsg101 (5b7), stn (219) (abcam); cd9, and cd81 (lifespan biosciences, inc. seattle, wa, usa); egfr (polyclonal), egfrviii (polyclo‐ nal) (antibodies online.com (de)); cd171 (polyclonal) (sigma-aldrich); icam-1 (r6.5) (ebiosciences, ca, usa). the bovine protein lachtadhedrin (haematologic technol‐ ogies inc, vermont, usa) was used for capture along with the listed antibodies. the antibodies and the protein were printed in triplicates at 90 – 200 µg/ml diluted in pbs containing 5% glycerol. for the semi-quantification of evs, the antibodies, cd9, cd81 (lifespan biosciences, inc.) and cd63 (mem-259) (biolegend), were printed in 18 repeated spots in a mixture/cocktail of 100 µg/ml of each antibody diluted in pbs and containing 5% glycerol. for detection, a cocktail of the following biotinylated antibodies were used: anti-cd9, -cd63 and -cd81 (life‐ span biosciences, inc.). the catching and visualization of evs, as well as the data analysis, were performed as previously described in [40] with minor modifications. in short, the slides were blocked (50 mm ethanolamine, 100 mm tris, 0.1% sds ph 9.0) prior to incubation with ev-containing cell supernatants (100µl for phenotyping and 75µl for the semi-quantification). the incubation was performed in multi-well hybridization cassettes (arrayit, ca, usa) at rt for two hours, followed by overnight incubation at 4 °c. after washing (pbs with 0.05% tween®20), the slides were incubated with biotiny‐ lated detection antibodies (anti-cd9, -cd63, and -cd81) (1:1500 in wash buffer). after washing, 30 min incubation with cy5-labelled streptavidin (life technologies) (1:1500 in wash buffer) was performed for detection. prior to scanning, the slides were first washed in washing buffer, followed by milliq water, and dried using a microarray high-speed centrifuge (arrayit). scanning and spot detection were performed as previously described [40]. briefly, the intensity of the antibody signal was calculated by subtracting the mean of the background (without sample/blank) from the mean of the triplicate antibody spots. this signal was then divided by the signal from the mean of the triplicate negative spots (without capture antibody, with sample). the relative fluorescence intensity was subsequently log2 transformed. graphs and statistics were made in graphpad prism (ver. 6.04, graphpad software, inc. ca, usa) and excel (ver. 2013, microsoft, usa) and heatmaps were generated in genesis (ver. 1.7.6, igb tu graz, austria). an unpaired ttest was applied to test for differences between the semiquantitative measurements of evs from the same cell line subjected to two different conditions. differences between groups were considered statistically significant when p < 0.05. unless otherwise specified, the data are presented as mean ± sd. 3. results 3.1 cell proliferation and viability following hypoxic incubation the three cancer cell lines, skov3, pt4 and cov504, were incubated under normoxic or hypoxic conditions. the cell number and viability were determined for each cell line 3evo k. lindersson søndergaard, lotte hatting pugholm, rikke bæk, malene møller jørgensen, anne louise schacht revenfeld and kim varming: oxygen-related differences in cellular and vesicular phenotypes observed for ovarian cell cancer lines directly after thawing, and after 12 and 24 hours of cultur‐ ing (table i). to investigate the oxygenic state of the cell cultures, the cell lines, skov3 and cov504, were used as examples and the expression of the glucose transporter, glut1, and hypoxia-inducible factor, 1α (hif-1α), were analysed after culturing the cells under hypoxic or nor‐ moxic conditions for 24 hours. data are presented in the supplementary (s1). the proliferation and the viability of skov3 and pt4 did not change much over time or between normoxic and hypoxic conditions. on the contrary, cov504 showed a decrease in the viability under hypoxic conditions, whereas the normoxic population retained a higher viability after 24 hours. furthermore, the cell count of cov504 following 24 hours of incubation under hypoxic conditions was only half the cell count observed in nor‐ moxic conditions. 3.2 cellular expression of selected surface markers the expression of cd9, cd81, cd151, caix and caxii were evaluated for the three cell lines, cov504, skov3 and pt4, followed by incubation at normoxic or hypoxic conditions. the specific surface expression was determined by flow cytometry directly following thawing after 12 and after 24 hours. the expression of the five markers changed marginally over time but, since most cultured cells need time to adapt to changes and to a new environment, the time point 24 hours is illustrated (figure 1). the flow cytometric analysis showed that the three cell lines clearly expressed cd9, cd81 and cd151. skov3 also expressed caix and caxii (> 3 times the isotype mfi), whereas cov504 and pt4 expressed caix and caxii (< 2 times the isotype mfi). in addition, the effects of hypoxia on the expression of the five cell surface markers were investigat‐ ed (figure 1). after 24 hours of hypoxic conditions, the median cd9 expression by cov504 was only half the mfi of cells cultured under normoxic conditions, indicating that cd9 expression by cov504 was affected by hypoxic conditions. for all other markers, the expression on cov504 remained unaffected by hypoxic conditions. similarly, the expression of the five markers on skov3 and pt4 were unaffected by hypoxic conditions. 3.3 phenotyping of cell-derived evs in addition to the cellular phenotypes, the phenotypes of the cell-derived evs found in the cell free supernatants were determined. the evs captured and measured by the ev array had a size of 30-300 nm (supplementary figure s2). this extensive phenotyping involved 31 markers comprising general ev proteins, cell-specific markers and a number of cancer markers. the effect of the oxygen levels of the ev phenotype was evaluated over time. a summary of the ev phenotypes can be seen in figure 2. the heatmaps demonstrate the phenotype of evs from the three ovarian cancer cell lines, subjected to either normoxic or hypoxic conditions for 12 or 24 hours. phenotypically, the evs from the three cell lines resembled each other and displayed more or less the same markers under normoxic conditions. the evs derived from all three cell lines grown under normoxic conditions expressed cd9, cd81, cd151, cd63, tspan8, cd82, icam-1, caxii and cd142, whereas 16 markers were not detected. only six markers (lamp2, tnf rii, plap, cd13, akap3 and alix) showed a variable expression. on the contrary, there were phenotypical differences between the evs derived from cells subjected to hypoxic conditions. the evs from cov504 and pt4 had a tendency to express their markers most strongly after 24 hours in normoxic conditions, whereas the evs from skov3 expressed their markers more strongly during hypoxic conditions. in particular, this was observed with cd9, cd81, cd151, cd63 and cd82. furthermore, the evs from skov3 expressed considerably more markers following 24 hours of hypoxia compared to the evs from cov504 and pt4. when comparing the expression level after only 12 hours, cov504 and pt4 expressed their markers most strongly under hypoxic conditions, whereas skov3 expressed most of the markers most strongly under normoxic conditions. notably, when looking at the three tetraspanins, cd9, cd63 and cd81, the evs derived from cell line tissue time point 7aad normoxia 7aad hypoxia cell density normoxia cell density hypoxia cell count normoxia cell count hypoxia skov3 adenocarcinoma ovary, ascites, epithelial 0h 8% 8% --------125000 125000 12h 8% 13% 50% 50% 103750 115000 24h 11% 8% 100% 100% 140000 130000 48h 10% 12% 100% 100% 163750 141250 cov504 epithelial serous carcinoma, pleural effusion, epithelial ovary 0h 1% 1% --------125000 125000 12h 20% 25% 30-40% 30-40% 62500 60000 24h 11% 29% 70-85% 70-80% 107500 52500 pt4 serous papillary carcinoma ovary, stage 2, ascites 0h 4% 4% --------125000 125000 12h 13% 19% 50-60% 60-70% 117500 97500 24h 14% 14% 60-80% 70-80% 132500 122500 table 1. listed are the percentages of cell death (positive for 7aad), percentages of cell density and cell count following harvest at the different time points and conditions (normoxia and hypoxia) for the three ovarian cancer cell lines skov3, cov504 and pt4 4 j circ biomark, 2016, 5:1 | doi: 10.5772/62219 all three cell lines did not express cd63 as strongly as the other two known ev markers. 3.4 comparison of ev and cellular phenotypes in order to investigate whether the ev phenotype mirrored the cellular phenotype, a comparison of the protein composition was made. skov3 is used here as an example but the other cell lines showed similar results (data not shown). figure 3 illustrates the relation between the cellular and ev phenotypes (for the three markers, cd9, cd81 and cd151) under normoxic and hypoxic conditions. the amount of cd9, cd81 and cd151 presented on the evs varied if the cells were subjected to normoxic compared to hypoxic conditions. the evs displayed more pronounced cd9 and cd81 after 12 hours of cell incubation in normoxic figure 1. the influence of hypoxia on the cellular phenotype of ovarian cancer cell lines. the different cell subsets were stained with antibodies against caix, caxii, cd151, cd81, cd9 and matched isotype controls and were analysed by flow cytometry. the overlays of the expression displayed by the cells subjected to either normoxic or hypoxic conditions for 24 hours for the three cell lines, cov504, skov3 and pt4, are displayed. figure 2. phenotyping of evs from ovarian cancer cell lines. the ev phenotypes were profiled using the ev array printed with 31 different capture antibodies and detected with a cocktail of antibodies against cd9, cd63 and cd81. the heatmaps of the phenotyping of evs derived from cov504, skov3 and pt4 subjected to either normoxic or hypoxic conditions for 12 or 24 hours. 5evo k. lindersson søndergaard, lotte hatting pugholm, rikke bæk, malene møller jørgensen, anne louise schacht revenfeld and kim varming: oxygen-related differences in cellular and vesicular phenotypes observed for ovarian cell cancer lines conditions compared to hypoxic conditions, whereas, after 24 hours, the presentation of cd9 was almost the same under both conditions and cd81 was slightly more expressed under hypoxic conditions. cd151 was more presented on the evs under normoxic compared to hypoxic conditions for both 12 and 24 hours. on the contrary, there were small differences in the cellular expression of cd9, cd81 and cd151 observed over time with the two condi‐ tions, but the differences were not as defined as the ones observed for the corresponding evs. figure 3. variations in ev and cellular phenotypes. the cellular phenotype was analysed by flow cytometry, whereas the phenotype of the evs was determined with ev array. the graphs display the ratio of the phenotypical variations of the three markers, cd9, cd81 and cd151, between the evs (mean values) and cells (median values) from the cell line, skov3, after either 12 or 24 hours under normoxic (n) or hypoxic (h) conditions. there is one mean value (evs) or median value (cells) for each of the three markers. therefore, no statistical measurements were performed. 3.5 the production of evs is influenced by various factors the evs from the cell culture supernatants were harvested at different time points and the relative amount of evs were estimated with the ev array. this was achieved by measuring the total fluorescence signal when antibodies against the exosomal markers (cd9, cd63 and cd81) were used both for the capture and detection of the evs. figure 4 displays the semi-quantitative measurement of the relative amount of evs derived from cells after 12 hours (a) and 24 hours (b) for all three cell lines subjected to either normoxic or hypoxic conditions. after 12 hours, the highest amount of evs was detected from the cells grown under normoxic conditions, which was observed for all three cell lines. in contrast, after 24 hours, the relative amount of evs considerably increased under hypoxic conditions. from 12 hours to 24 hours, the relative amount of evs released by cov504 increased by 8% under normoxic conditions, whereas, for the hypoxic conditions, the increase was 46%. the corresponding values for pt4 were 10% for normoxic conditions and 34% for hypoxic condi‐ tions. finally, the relative amount of evs from skov3 grown under normoxic conditions decreased by 8% and increased by 28% under hypoxic conditions. the results displayed in figure 4 only include one cell concentration. however, the same pattern was also observed for a lower cellular concentration (data not shown). as expected, a higher concentration of cells produced more evs than a lower concentration of cells, even though a two-fold increase in cell concentration did not result in a two-fold increase in the amount of cellderived evs for any of the three cell lines. the influence that the cell concentration had on the ev production was also consistent for all three cell lines tested here (data not shown). 4. discussion analysing the protein composition of both evs and their parent cells is helpful to further understand the mechanism of the evs’ biogenesis and the functional roles that they may play in the development of cancer [41]. furthermore, figure 4. the ev production is influenced by several factors. the amount of evs was semi-quantitatively estimated with the ev array measuring the total fluorescence signal when the evs were captured, as well as detected with a cocktail of antibodies against cd9, cd63, and cd81. each bar represents the relative fluorescence intensity (mean ± sd) of each triplicate measurement on the ev array. the relative amount of evs produced by cov504, skov3 and pt4 under either normoxic (n) or hypoxic (h) conditions for 12 hours (a) and for 24 hours (b) is displayed. 6 j circ biomark, 2016, 5:1 | doi: 10.5772/62219 the proteomic profile of the cancer-derived evs is impor‐ tant for discovering novel diagnostic biomarkers and therapeutic targets. cell culture supernatants have previ‐ ously been shown to contain evs [42–44]. analysing evs that are released from cell lines from a particular cancer type offers the potential for the identification of ev biomarkers that might be of diagnostic and prognostic value, without the risk of mistaking the ev origin. this is one advantage of using cell lines when looking into cancer research. the ev phenotypes presented here can only reflect the actual cell that they derive from, which makes it easier to interpret the data. in an ev population obtained from a heterogeneous cell population, there are several cells that can act both as producers and recipients of evs. prior to relating specific ev features to cancer or to other pathological conditions, it is of great importance to gain knowledge about evs in general and in cancer-like conditions. several studies prior to this have phenotyped evs but only with one or a few protein markers and with extensive purification steps, as reviewed in [7]. this has mostly been done with western blotting [45,46] and flow cytometry [47,48]. the ev array used for the investigation of the phenotypes and quantification of evs from the three ovarian cancer cell lines is a microarray technique that was developed in our laboratory. this technique is possible to perform without prior purification of the samples. in the present setup, the ev array was optimized to analyse the ev subset that carry the general exosomal markers, cd9, cd63 and cd81, and have a size of approximately 30-100 nm. additionally, the ev array provides an estimated relative quantification of evs in the samples by measuring the total fluorescence signal when a cocktail of anti-cd9,-cd63 and -cd81 is used to both capture and detect the evs. our data indicate that cells from all three cell lines subjected to hypoxic conditions for 24 hours have a tendency to produce more evs than cells subjected to normoxic conditions, independent of cellular concentration and level of conflu‐ ence (figure 3). nevertheless, with these data, it could not be decided whether a relatively higher amount of evs or a relatively higher amount of the markers, cd9, cd63 and cd81, were produced under these conditions. however, the measurement here is believed to be a semi-quantitative measurement of the amount of evs. this is based on the fact that the phenotyping of evs from both cov504 and pt4 display an increase in the level of cd9, cd63 and cd81 after 24 hours of cell incubation under normoxic conditions compared to hypoxic conditions (figure 2). thereby, the phenotyping displays the opposite situation of the semiquantification, which points to a higher amount of evs produced under hypoxia. this observation was not surprising as it has earlier been shown for rat osteoblasts [42], breast cancer cell lines [43,49] and lung cancer cell lines [44]. notably, there were more cells after 24 hours of incubation in the cells subjected to normoxic versus hypoxic conditions and, in the case of cov504, even as much as double the amount of cells (table i). moreover, the viability of most of the cells was the same for both normoxic and hypoxic conditions. this could indicate that the cells subjected to normoxia mostly focused on proliferation, whereas the cells subjected to hypoxia focused on ev release. this is in-line with another study that reported that breast cancer cells exposed to hypoxia increased their production of microvesicles, which stimu‐ lated invasion and metastasis of recipient breast cancer cells [49]. skov3 already reached confluence before 24 hours, without altering their viability (see table i) but, despite this, they continued to produce evs. even though these data do not conclude that the level of confluence is without influence on the ev production, they may indicate that cells do not stop producing evs when they reach confluence. the fact that more evs are produced per hour in the early cultures, which are less confluent, might be explained by the fact that cells produce evs to communicate with each other and the distance between cells are smaller in the cell cultures with high confluence than in the ones with low confluence. therefore, they need more communicational agents to communicate. it could also be because, as cell lines are copies of themselves, the cells in such cultures only communicate with themselves. surely, the picture in vivo is different and much more complex. this is probably the case as all three cell lines are viable and grow just as well after 12 hours as after 24 hours. the point that more evs can be measured after 24 hours than after 12 hours only suggests that evs are not quickly degraded or taken up by the cells. instead, they stay in the culture for at least 24 hours. previously, evs have been referred to as small copies of the cells that they derive from but more and more evidence show that this is not always the case. proteins that are highly expressed on evs are not always expressed by the cells (own observations for cd9, unpublished data). as evs often reflect the cells that they derive from, it is of interest to see how different the expression levels are between evs and their parental cells. the data presented in this study clearly show that the evs are not exact copies of their parental cells, even though the evs reflect the cells that they derive from. this is also consistent with data from im h et al., 2014, who show a phenotypical comparison of evs and cells from different ovarian cancer cell lines using flow cytometry and a nano-plasmonic exosome assay [50]. the overall ev phenotype from the three ovarian cancer cell lines show resemblances to each other, especially when comparing the overall ev phenotype to other cancer cell lines (data not shown). even though all three cell lines show resemblances to each other, they do so with variable degrees. different conditions influence the cell lines differently and, therefore, must be said to express different ev subsets. the different ev subsets that are seen between 7evo k. lindersson søndergaard, lotte hatting pugholm, rikke bæk, malene møller jørgensen, anne louise schacht revenfeld and kim varming: oxygen-related differences in cellular and vesicular phenotypes observed for ovarian cell cancer lines the three cell lines might be explained by the fact that the cells that the evs derive from come from different origins. furthermore, the different ev subsets that are seen and how they vary due to hypoxic conditions and time could indicate that they have different functions depending on which cell type they derive from. these data clearly show that hypoxia, in particular, has an effect on the expression of proteins on evs, even though there is no clear correlation between the three cell lines. in particular, skov3 is highly affected by hypoxia as it expresses considerably more markers after 24 hours of hypoxic conditions compared to normoxic conditions. our data are supported by another study that showed that vesicles released under hypoxia, compared to vesicles released under normoxia, were loaded with unique proteins that could enhance invasive‐ ness and induce microenvironment changes of the recipient cells [51]. furthermore, the expression level of the general ev markers, cd9, cd63 and cd81, on evs were clearly affected by hypoxia. these data still support cd9 and cd81 as useful general ev markers but it should be noted that the expression level of cd9 and cd81 can be affected by several factors. other interesting markers are muc1, egfr and cd142, which are all highly related to cancer, particu‐ larly ovarian cancer. firstly, many cancers, including ovarian, overexpress muc1 [52]. egfr is reported to be both increased in copy number and overexpressed in serious ovarian carcinoma and is associated with a high tumour grade, large residual tumour size, high prolifera‐ tion index and poor patient outcome [53]. finally, cd142 regulates tumour cell proliferation and apoptosis, and can promote tumour angiogenesis and metastasis in several different cancers, as reviewed by han x, et al. [54]. detected with the ev array, all three markers were up-regulated in evs containing supernatant from skov3 cells subjected to 24 hours of hypoxia compared to normoxia. the multi‐ plexed protein profiling of evs provides simultaneous information about numerous possible biomarkers, which increase the power of discrimination [55,56]. thus, if several of the proteins investigated in the present study were combined, the likelihood of finding biomarkers, which are of clinical relevance for early detection of ovarian cancer, is greater. in general, when looking at the five markers, caix, caxii, cd151, cd81 and cd9, the cellular phenotype was mostly unaffected by time and oxygen level, whereas their corresponding evs were much more affected. the only difference in the cellular expression of markers was seen for cd9 on cov504 after 24 hours of either normoxic or hypoxic conditions. this might be explained by the fact that the hypoxic cell population had a considerable decreased viability compared to the normoxic cell population. the phenotyical differences seen between the cells and their corresponding evs cannot be explained by only one of the factors (time, cellular concentration or oxygen level) but rather, a combination of them. even though there were more evs in the highest cellular concentration in the hypoxic population after 24 hours, both cov504 and pt4 displayed an increase in the level of most markers in the normoxic population, whereas evs from skov3 showed the opposite. this might support the idea that evs are used by cells for fast and effective communication with their surroundings. one study has even reported that evs can deliver a biologically active transcriptional factor, hif-1α, to a recipient cancer cell line [57]. to further stress the importance of investigating evs and their phenotypes for their potential role in cancer, a recently published study showed that plasma exosomes from cancer cells present a tumour-related proteomic profile. furthermore, the levels of bioactive molecules contained in the exosomes of patients with solid tumours before surgery are significantly higher than in exosomes from the same patients after surgery [58]. learning more about the role that evs play in cellular communication in healthy, and particularly under patho‐ logical conditions, will hopefully help to understand the way that cancers develop and metastasize. the tumour milieu has long been recognized as being important in the way that cancers develop. others have also shown that ph and hypoxia can influence cell migration [35,49,51]. the presented data emphasize the complexity of factors that might influence the cellular and ev phenotype and thereby the metastatic process. 6. conflict of interest and funding the authors declare no conflict of interest and have not received any funding or benefits from industry or any for profit organization for this work. 7. acknowledgements the authors thankfully acknowledge dr. shona pedersen and ph.d-student, thøger nielsen (department of clinical biochemistry, aalborg university hospital, denmark), for their support in the nta experiments. the authors also thank laboratory technician, anne elbæk (department of clinical immunology, aalborg university hospital), for her technical assistance. 8. references [1] théry c, ostrowski m, segura e. membrane vesicles as conveyors of immune responses. nat rev immunol [internet]. 2009 aug [cited 2014 jul 11];9(8):581–93. available from: http:// www.ncbi.nlm.nih.gov/pubmed/19498381 [2] bhatnagar s, shinagawa k, castellino fj, schorey js. exosomes released from macrophages infected with intracellular pathogens stimulate a proinflam‐ matory response in vitro and in vivo. blood [internet]. 2007 nov 1 [cited 2014 oct 28];110(9): 3234–44. available from: http://www.pubmedcen‐ tral.nih.gov/articlerender.fcgi?ar‐ 8 j circ biomark, 2016, 5:1 | doi: 10.5772/62219 tid=2200902&tool=pmcentrez&rendertype=abstrac t [3] abusamra aj, zhong z, zheng x, li m, ichim te, chin jl, et al. tumor exosomes expressing fas ligand mediate cd8+ t-cell apoptosis. blood cells mol dis [internet]. 2005 jan [cited 2014 oct 9];35(2): 169–73. available from: http://www.sciencedir‐ ect.com/science/article/pii/s1079979605001105 [4] szajnik m, czystowska m, szczepanski mj, man‐ dapathil m, whiteside tl. tumor-derived micro‐ vesicles induce, expand and up-regulate biological activities of human regulatory t cells (treg). plos one [internet]. 2010 jan [cited 2014 oct 21]; 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review article sivappriyan nagarajah1* 1 university of oxford, oxford, uk *corresponding author(s) e-mail: siva.nagarajah@st-hughs.ox.ac.uk received 05 october 2015; accepted 09 march 2016 doi: 10.5772/62975 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract less than 100 nm in size and spherical in form exosomes – vesicles expelled and taken up by cells, have ignited a new-found fascination. one which is derived from the sheer variety of exosomal content, ranging from micro‐ rnas to transcription factors, capable of affecting a multitude of processes and pathways simultaneously within a target cell. initially dismissed in 1983 as a waste disposal mechanism, today they form an entire field of research, being documented thus far in invertebrates, mammals, pathogens and potentially some plants. many studies have suggested these spherical enigmas may possess a function, being implicated in processes ranging from animal behaviour to viral infection. this review will evaluate the evidence for the role of exosomes in physiol‐ ogy and pathophysiology, as well as their potential for application in the diagnosis and treatment of disease. keywords exosomes, physiology, disease, diagnostics, diagnosis, therapeutics, treatment 1. introduction within our bodies, there exist minute space shuttles, making contact with unknown lands and, in the process, reshaping them. this is the world of exosomes bubble like spheres that bud off from cells containing a concoction of lipids, proteins and genetic material as their cargo. less than 100 nm in size, these vesicles possess the potential upon landing to not only alter a target cell's metabolism but ultimately function [1]. initially reported in red blood cells [2] and once dismissed as mere ejection pods removing cellular waste, today they stand at the forefront of research into intercellular, interorganismal and interspecies com‐ munication [1, 3, 4]. documented thus far in mammals [5-7], invertebrates [8-9], pathogens [10] and potentially some plants [11], exosomes offer a way for the world around us and ourselves to interact, both in health and disease [12]. indeed, the processes in which they are implicated reflect this, ranging from animal behaviour [13] to viral infection [14]. their presence in a variety of bodily fluids, from cerebrospinal fluid to urine, make them readily accessible, be it for study or as diagnostic tools [15]. as decoding of exosomal signalling advances, what does this hold for our understanding of disease and our attempts to alleviate it? exosomes form one part of a larger universe – that of extracellular microvesicles (mvs), which are broadly divided into exosomes, ectosomes and apoptotic vesicles. ectosomes are vesicles that directly budd off from the plasma membrane without involving the endocytic 1j circ biomark, 2016, 5:7 | doi: 10.5772/62975 pathway, whilst apoptotic vesicles are remnants of cells that have undergone programmed cell death [12]. this review will primarily focus on exosomes, as many studies have indicated that they may possess a biological function [10, 13, 14, 16-21], as well as being useful in diagnosis [15] and treating disease [22]. importantly, many exosome studies rely on vesicle size and biomarkers to fractionate and identify exosomes – a crude technique at best, which may mean that investigators are often studying a much more complex mixture of extracel‐ lular vesicles. however, the findings from these studies, the questions they raise and their implications, if true, make them highly significant. one must first look at exosomal biogenesis [12, 23-28] (fig. 1) in order to recognize the normal state of affairs and how this provides avenues for exploitation, both therapeutically and by pathogens. within a cell, as part of the endocytic pathway, a region of the plasma membrane invaginates, forming a vesicle that enters the cytoplasmic space, carrying with it cell surface receptors and extracellular fluid – an early endosome. as endosomes mature, they become more acidified and act as a sorting site, recycling certain proteins whilst targeting others for degradation. in these late endosomes, known as multivesicular bodies (mvbs), invaginations of the endosomal membrane also occur, forming numerous intraluminal vesicles (ilvs) that contain cytoplasmic content, such as short interfering rna (sirna). mvbs can then fuse with a lysosome, where proteins are degraded, or with the cell’s plasma membrane, releasing the ilvs – now known as exosomes – into the extracellular space. the power of exosomes as signalling agents arise from the sheer variety of the contents they carry – capable of affecting a multitude of processes and pathways simultaneously within a target cell: from micrornas, which prevent translation of mrnas [29], to proteins, which can act as transcription factors [30] or even emit oncogenic phenotypes [31]. 2. exosomes in physiology and pathophysiology 2.1 cancer exosomes appear to act as vehicles of transmission when hijacked. perhaps the most well-known case is cancer, in which exosomes are thought to contribute to the creation of a microenviromental niche that promotes cancer cell survival (fig.2), as well as reprogramming distant tissue for invasion [33]. take, for example, exosomes from epstein barr virus (ebv) transformed lymphoblastoid b cells, containing mirna: when exposed to dendritic cells (dcs), they lead to dose-dependent suppression of the immunor‐ egulatory gene cxcl11/itac known to be a target of ebv in promoting lymphomas. the peripheral blood of patients with ebv reveals ebv mirna to be present in non–b cell populations, unlike ebv-dna, which further suggests that mirna transfer occurs in vivo [34]. moreover, we are beginning to unveil how exosomes may mediate metastasis. injection of the fluorescently labelled pancreatic cancer cell line pan02derived exosomes, has confirmed their ability to increase the metastatic load in the liver of mice [35]. other pancreatic cell-line-derived exosomes, including human bxpc-3 exosomes, also display a preference for the liver. subsequent experiments have demonstrated that exosomal uptake by kupffer cells leads to tgf-β synthesis and release, activating hepatos‐ tellate cells that go onto express fibronectin. this, then, attracts bone marrow-derived macrophages and granulo‐ cytes, which are now determined to be a prerequisite for metastasis, opening an avenue for future work. figure 1. this depicts early endosome invagination from the plasma membrane of a cell. as the endosome matures to become a late endosome, endosomal membranes invaginate forming ilvs, giving rise to an mvb. proteins from the golgi complex can join the endosome prior to ilv formation as well as after. mvbs can then fuse with a lysosome, which enables proteasomal degradation, or with the plasma membrane releasing ilvs – now termed exosomes – into extracellular space. adapted from[32],this figure was produced using servier medical art, available from www.servier.com/powerpoint-image-bank. 2 j circ biomark, 2016, 5:7 | doi: 10.5772/62975 www.servier.com/powerpoint-image-bank. however, in both cases it is important to note that arbitrary exosomal concentrations were used, sometimes in vitro and further research is needed to establish what truly occurs in vivo. nonetheless, when considered with studies illustrating exosomal explusion of cancer drugs [36-39], exosomeinduced agiogenesis [40-43] and promotion of fibroblast differentiation and fibroblast-like cell formation, which sheaths the caner in a fibrous layer acting as a barrier (e.g., to drugs) [44-46] – exosomes add to the darwinian para‐ digm of cancer cells as an evolving agent, with those capable of manipulating the cell machinery gaining a clear advantage in selfpropagation. 2.2 hiv hiv dissemination also involves rerouting of cellular behaviour. in which case, could exosomes play a similar role here? conventionally, infected immature dcs are thought to travel to the lymph node, where they mature to interact with cd4+t cells, transferring the infection and resulting in cd4+ t-cell depletion with accompanying catastrophic immunodeficiency [48]. recent work, which pulsed hiv-1 with dcs, showed not only increased exosomal release from infected dcs, but also that these exosomes promoted apoptosis when exposed to cd4+ tcells [14]. how infection can be maintained, when given the short half-lives of cd4+ t-cells (less than two days), remains a puzzle. hiv is known to be able to enter dcs via endocytosis (transinfection), rather than direct fusion with the plasma membrane. the trojan exosome hypothesis, therefore, proposes that hiv enters dcs in this alternative fashion, becoming part of the endosome and being later released into the extracellular space with exosomes. this means that cd4+ t-cells, which are rapidly transcribing and translating hiv, and are soon likely to die, can transfer hiv to dcs, which frequently interact with naïve cd4+ tcells, thereby maintaining the infection. confocal micro‐ scopy and various other imaging techniques have shown, via visualization, that the above is certainly possible in vitro, while in vivo effects remain to be established. 2.3 physiology just as exosomes can become agents of dissemination, they may otherwise act as agents of control within the body when unaffected. presentation of mhc-peptide complexes or antigens by dc-derived exosomes is capable of eliciting antigen-specific immune responses from other dcs [1]. the effect of exosomes appears to be dependent on the cell type of origin and the physiological state of the said cell type. whilst exosomes from dcs can be immunostimulatory, cancer cell exosomes contain both cancer cell antigens, that could potentially be used to activate the immune system, and immunosuppressive molecules in vivo. establishing which of the aforementioned effects is more dominant remains to be determined. when dcs are exposed to immunosuppressive chemicals or altered in order to transcribe immunosuppressive cytokines, this change in their physiology appears to promote tolerance by the exosomes secreted instead. whilst roles in immune regulation emerge, exosomes are also known to be expelled by neurons [49], microglia [20] and possibly adipose tissue [50] – could any regulatory role extend beyond immunity to territories, such as neuroen‐ docrine physiology? in unravelling exosomal physiology and pathophysiology, we currently lack an established model to study exosomes in vivo. this is further complicat‐ figure 2. some emerging roles of exosomes in cancer. top left: promotion of nearby cells to act like fibroblasts secreting and sheathing the cancer in a fibrous layer that acts as a barrier, e.g., to drugs. top right: promotion of new blood vessel growth and metastasis. bottom left: aiding epithelial-mesenchymal transition (emt) – with cells becoming capable of expressing a variety of proteins, as well as leaving the original site. bottom right: enabling survival from immune response. exosomal mirna may be an important contributor to all of the above. adapted from [33].this figure was produced using servier medical art, available from www.servier.com/powerpoint-image-bank. 3sivappriyan nagarajah: exosome secretion – more than simple waste disposal? implications for physiology, diagnostics and therapeutics www.servier.com/powerpoint-image-bank. ed by our current techniques for selectively enriching and studying exosomes in vitro being too reliant on size and biomarkers features no longer accepted as effective in completely removing other microvesicles from the sample [12]. are any of the effects seen in vitro truly due to exo‐ somes, or even reflective of physiological concentrations and their effect? one way of overcoming this hurdle would be to knockout genes known to be involved in exosomal regulation in a simple invertebrate model, such as c. elegans. these knockouts can be both systemic and tissuespecific in order to determine any functional consequence. one group of candidates for this would be the escrt proteins, which are known to regulate mvb and ilv formation [12, 51]. however, as they are also involved in degradation of ubiquitylated proteins, when a mvb fuses with a lysosome, protein turnover could be affected. experimental models, therefore, would need to take this into account and distinguish any effects due to changes in exosomal biogenesis from the latter. despite the concerns raised above, perhaps the strongest evidence for exosomal functionality in vivo would be work done on drosophila melanogaster [13]. secondary cells of male accessory glands, equivalent to the prostate, were shown to release gfp-tagged exosomes in to the seminal fluid, which not only “fuse with sperm in vivo” but also interact with the epithelium of the female genital tract, specifically reducing remating in females and, thus, providing an evolutionary advantage to the male drosophi‐ la. when exosomal production is blocked in secondary cells, via removing proteins required for exosomal biogen‐ esis, either by rna interference or dominant negative rab11 expression, 50-60% of males were unable to prevent remating in females, compared to 18% in the control group. it is remarkable to consider that something as minute as an exosome may bring about a change in something as biologically complex as behaviour. furthermore, the observation of exosomal fusion with sperm is concordant with in vitro studies on human sperm, where prostatederived exosomes (prostasomes) contribute to sperm motility [16]. 3. exosomes as diagnostic tools from a diagnostic point of view, it does not matter whether exosomes have a function or not – only that a significant difference exists, either in composition or presence in both sickness and health, with these differences then being specific and attributable to different ailments. indeed, this appears to be the case for many pathological conditions, with both exosomal content and presence varying [12] (table 1). one interesting study concerns exosomes fractioned from the blood of pregnant women with no history of any previous pregnancies at 28-30 weeks of gestation [60]. significant differences existed between those going on to deliver at term and those delivering prematurely: specifi‐ cally, lower levels of fas l, hla-dr and reduced inhibi‐ tion of jak3 and cd3 zeta in premature births. could an exosomal screening programme be the future? given the small sample size, more work is needed before any clinical translation can occur. in cancer, however, the use of exosomes in diagnostics appears more imminent. exosome diagnostics have developed a “urine-based liquid biopsy” which looked at three specific exosomal rnas prior to prostate biopsy in 195 men. it was found that high grade cancer could be predicted based on a score derived from rna levels alone, with a sensitivity of 95.2% and a negative predictive value of 97.5% [65]. exosome diagnostics points to the use of a first catch urine sample and a single score that can prevent unnecessary biopsies in low grade tumours as key advan‐ tages. additional research looking at economics and outcomes is underway, as well as expansion into “blood plasma-based liquid biopsy” [66]. further excitement in this field arrived with a recent attempt to detect cancer exosome specific biomarkers. primary work on cancer cells and other non-cancer lineages using mass spectrometry suggested 48 proteins [57]. of these, glycoprotein glypican – 1 (gpc1), which, although found circulating at low levels in healthy patients sera, was higher in patients with breast cancer (n = 32, 75%) and pancreatic ductal adenocarcinoma table 1. examples of body fluid exosomal markers (specifically, some of the proteins and rnas) associated with pathology. modified from [15, 52] 4 j circ biomark, 2016, 5:7 | doi: 10.5772/62975 (pdac, n = 190, 100%). analysis of a small group of patients revealed that gpc1 levels distinguished precursor lesions (n=5) from benign pancreatic disease (pancreatitis & cystic adenomas, n = 18) something that could not be done by the current tumour marker for pancreatic cancer, ca-19-9. roc curve comparison of ca-19-9 and gpc-1 showed the latter to be superior at all stages of pdac with 100% sensitivity and specificity, as well as positive and negative predictive value. whilst the prospect of an early exosomal marker for pancreatic cancer is joyous, the nature of the small cohorts in this work means that further confirmation is required. exosomes offer three key advantages as a diagnostic tool: they are less invasive compared to a biopsy, they can be used where a biopsy is not possible (e.g., brain tumour) and may become cost-effective and time-efficient [67, 68]. although the most common techniques of exosomal fractioning, which are ultracentrifugation and density gradient separation can require over five hours [69], work is already underway to overcome this. one promising avenue is microfluidic-based exosomal detection, which is essentially a “lab on a chip” [68]. the prototype consists of various chambers, inlets and a microchannel within a polydimethylsiloxane chip, enabling integration of several processes. this includes exosomal isolation and enrich‐ ment, as well as the detection of biomarkers from minute volumes of plasma (30 μl). initially, a plasma sample is mixed with magnetic beads coated in antibodies to desired surface markers, and injected via an inlet. a magnetic field is then applied, trapping the exosomes bound to the beads, with subsequent washing in a pbs solution. following this separation and enrichment, incubation with a lysis buffer, which is inserted via another inlet, releases exosomal content. next, the lysate moves through a winding channel, with magnetic beads coated in antibodies flowing from the two side channels, targeting inter-exosomal content of interest. bead-inter-exosomal content is then trapped in a chamber via a magnetic field, where chemifluorescence detection can be carried out, meaning levels of specific inter-exosomal content can be measured. this technique not only takes one and a half hours, but was also successful in identifying the insulin-like growth factor 1r (igf-1r) – a potential biomarker for non-small cell lung cancer (nsclc), in plasma of patients with nsclc. it is also possible to embed exosomes extracted in this manner and view them under transmission electron microscopy, thereby enabling higher levels of characterization. with greater mapping of disease-specific exosomal markers, the use of microfluidic technology, such as this routinely, may become a reality. 4. exosomes as therapeutic tools one of the features that makes exosomes useful in diag‐ nostics also means they can be useful therapeutically. essentially, exosomal surface markers can be used to target specific cell types. be it for drug delivery or gene therapy, exosomes engineered and secreted by a chosen cell type can be packaged with desired components [22] and adminis‐ tered by a simple injection [70]. another appealing feature is the use of patients’ own cells to generate exosomes in order to enable biocompatibility [22]. although therapy is the most ambitious and fledgling aspect in exosomal research, its untapped potential is worth considering. take the brain for instance one of the most difficult places to deliver any kind of therapy within the human body. this is due to the blood-brain barrier, restricting what may and may not enter, in an attempt to protect the neural cavity [71]. however, exosomes are known to be released by neurons within the brain parenchyma [34], as well as transport pathological agents, such as alpha–synuclein between neurons in parkinson’s [17]. recently, rat choroid plexus-derived exosomes were shown to distribute folate vital for dna synthesis via cerebrospinal fluid [19]. work on c57bl/6 mice removed their bone marrow, selecting immature dcs known to produce copious amounts of exosomes that are non-t-cell stimulatory [70]. these cells were then transfected with plasmids expressing targeting peptides; in the case of the brain, these were rabies virus glycoprotein (rvg). this led to formation of exosomes with desired surface markers. next, electroporation was adapt‐ ed and applied at the nanoscale in order to enable exosomal uptake of exogenous sirna. rvg9r exosomes were also created with the addition of nine d-arginines to the rvg, which interacted electrostatically with loaded sirna. sirna to bace1 (a key gene in alzheimer’s) containing rvg exosomes were injected into c57bl/6 mice. controls included untreated mice and mice injected with rvg9r– bace1. three days later, cortical tissue analysis revealed significant protein knockdown (45%, p < 0.05, versus 62%, p < 0.01) when rvg9r-bace1 and rvg-bace1 were used, respectively. β-amyloid 1-42 levels, an important constitu‐ ent of amyloid plaques, fell by 55% (p <0.01) in mice treated with rvg exosomes, whilst serum markers of an inflam‐ matory response (ifn α, tnf – α, il 6, ip-10) were not increased. the ability to cross the blood-brain barrier is perhaps a pharmacological holy grail; like the holy grail, however, whether one should obtain it remains a question. can we treat in the vicinity of such a delicate and vital organ? although the same question could have been asked at the advent of bypass surgery, which is now almost routine. moreover, in situations such as immune-mediated loss of beta cells of the pancreas (type i diabetes) [72] or scarring of cardiac tissue post-myocardial infarction (mi) [73], a promising regenerative strategy has been stem cell implan‐ tation [74]. however, in some cases at least, it may be the exosomes released by stem cells that deserve credit. for example, a recent study showed that cardiosphere-derived stem cells (cdcs), when infused via coronary circulation in post-mi patients, led to decreased scar formation and increased viable mass at six months [75]. more interesting‐ ly, when cdcs were inhibited from releasing exosomes in a murine model of mi, they were unable to reproduce the 5sivappriyan nagarajah: exosome secretion – more than simple waste disposal? implications for physiology, diagnostics and therapeutics above effects. furthermore, cdc-derived exosomes alone could produce the regenerative effects displayed by cdcs [18]. this offers a potential cell-free approach to regenera‐ tive medicine, if applicable to other stem cells. another avenue is the use of exosomes as vaccines [76-78]. here, the ability of mature dcs to create immunostimula‐ tory exosomes could be exploited, via presenting dcs with pathologic antigens before injection of generated exo‐ somes. largely thus far explored in cancer, its application could be broader if successful. work using dcs, which are pulsed with tumour antigens as cancer vaccines, appears to be more potent at generating anti-tumour immunity than other techniques (e.g., viral vaccines). it remains to be seen whether exosomes can match, if not better, this. equally, in the treatment of autoimmune disease, the creation of immunosuppressive exosomes by altered dcs may be useful [1]. 5. conclusion what were once dismissed as waste disposal agents have now become an entire field of study [1]. with an evergrowing list of potential applications and investment opportunities, our knowledge regarding extracellular communication is likely to expand in the next decade. success will rely crucially on the development of an in vivo model for determining exosomal function. whilst the current work on therapeutics and diagnostics is promising, much remains to be seen as to how it will play out clinically. will it be safe? will we be able to transform concept into reality? despite the gathering interest, numerous un‐ knowns remain. however, this should not be a hindrance to progress – if anything these questions ought to dictate further research and exploration into a tantalizing field. 6. conflict of interest the author has no conflicts of interest to declare. 7. acknowledgements the author would like to thank professor clive wilson, for his advice and support during the creation of this manu‐ script. 8. references [1] théry c. exosomes: secreted vesicles and intercel‐ lular communications. f1000 biology reports. 2011;3:15. 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functional capacity. the study population consisted of 24 dcm patients and 8 control subjects. all dcm patients had normal coronary angiographic studies. plasma levels of gdf-15, matrix metalloproteinase-2 (mmp2), mmp3, mmp9, tissue inhibitor of mmp 1 (timp1), and soluble suppression of tumorigenicity-2 protein (sst2) were determined by enzyme-linked immunosorbent assays. brain natriuretic peptide (bnp) was measured as per core laboratory protocol assay at scott and white memorial hospital core laboratory. correlation analysis was performed between gdf-15 and each of the mmps—mmp2, mmp3, mmp9, and timp as well as new york heart association (nyha) class and echocardiographic parameters (left ventricular ejection fraction (lvef) and left ventricular internal dimension in diastole (lvidd)). lvef and lvidd were obtained by two-dimensional echocardiography. the protocol was approved by scott and white memorial hospital institutional review board (s&w irb). correlation analysis of control versus all dcm patients showed a strong correlation of gdf-15 with timp1 (r ¼ 0.83, p < 0.0001) and weaker correlation with mmp3 (r ¼ 0.41, p ¼ 0.011) and mmp2 (r ¼ 0.47, p ¼ 0.003). mmp9 showed poor correlation with gdf-15 (r ¼ 0.3036, p ¼ 0.046). gdf-15 correlated negatively with mmp2/timp1 ratio (r¼�0.47, p¼0.006). sst2 correlated strongly with gdf-15 (r¼0.7, p < 0.0001). gdf-15 correlated negatively with lvef (r¼�0.49, p¼ 0.004) and positively with lvidd (r¼ 0.58, p¼0.0006). gdf-15 showed significant positive correlation with nyha functional class (r ¼ 0.71, p < 0.00001) and bnp (r ¼ 0.86, p < 0.00001). significant associations of gdf-15 with mmps, sst2, lvidd, lvef, and nyha class reported here for the first time in nonischemic dilated hearts may open up new avenues of investigations to better understand molecular mechanisms controlling cardiac remodeling. this study is limited by its small size and needs validation in larger populations. keywords sst2, gdf-15, matrix metalloproteinases, lvidd, lvef date received: 7 june 2017; accepted: 8 december 2017 1 division of cardiology, scott and white memorial hospital, temple, tx, usa 2 division of cardiology, texas tech university health sciences center, lubbock, tx, usa 3 division of cardiothoracic surgery, scott and white memorial hospital, temple, tx, usa 4 memorial cardiac and vascular institutes, hollywood, fl, usa corresponding author: nandini nair, division of cardiology, texas tech university health sciences center, 3601 4th street, lubbock, tx 79430-0002, usa. email: nandini.nair@gmail.com journal of circulating biomarkers volume 7: 1–8 ª the author(s) 2018 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417751735 journals.sagepub.com/home/cbx creative commons non commercial cc by-nc: this article is distributed under the terms of the creative commons attribution-noncommercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:nandini.nair@gmail.com https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417751735 http://journals.sagepub.com/home/cbx introduction dilated cardiomyopathy (dcm) is characterized by absence of secondary causes. etiologies include hypertension, valvular dysfunction, coronary artery disease, myocarditis, and congenital pathophysiology. approximately 50% of dcm patients carry an idiopathic etiology. in the absence of familial causes, idiopathic dcm (idcm) is indistinguishable from the familial type. 1,2 growth and differentiation factor-15 (gdf-15) is a cytokine of the transforming growth factor (tgf)beta superfamily first cloned and sequenced in 1997 from an activated macrophage cell system. 3 gdf-15 has been implicated in fibrosis, inflammation, and ventricular remodeling. 4–6 soluble suppression of tumorigenicity-2 protein (sst2) is the circulating counterpart of the cellular receptor suppression of tumorigenity ligand (st2l). it is usually produced by cardiomyocytes and endothelial cells along with its ligand interleukin-33 (il-33) during cardiomyocyte injury. il-33 binds to st2l and promotes inhibition of myocardial hypertrophy, fibrosis, and remodeling under normal conditions. on the other hand in pathological conditions such as heart failure, there is a large increase in the circulating form (sst2) which competes with the cellular form st2l for the same ligand il33. this leads to pathogenesis as the protective role of st2l is disrupted as sst2 binding to il-33 does not result in inhibition of myocyte hypertrophy and remodeling. sst2 has therefore been postulated in prognosis and risk stratification of chronic heart failure. 7–9 disruptions in the myocardial extracellular matrix (ecm) is the hallmark of structural pathology seen in heart failure; ecm is a dynamic unit. the synthesis and breakdown of ecm occurs via the actions of matrix metalloproteinases (mmps) and their inhibitors (timps). it has been postulated that ecm plays an active role in myocardial remodeling and interstitial transport in addition to acting as a reservoir for growth factors and cytokines involved in remodeling making them important of therapeutic targets. 10–12 the role of gdf-15 in regulation of cardiac remodeling in idcm still remains poorly defined. this study was therefore undertaken to assess association of gdf-15 with metalloproteinases and sst2 which are molecular targets of fibrosis. the utility of these biomarkers in clinical evaluation of heart failure in idcm will be investigated in this study from their correlation with new york heart association (nyha) class. materials and methods the study population consisted of 24 dcm patients and 8 control subjects. this is a cross-sectional single center observational study. all dcm patients had normal angiographic studies. plasma levels of gdf-15, sst2, mmp2, mmp3, mmp9, and timp1 were measured by enzyme-linked immunosorbent assays (elisa). blood was collected from patients in edta tubes, and bnp was measured as per core laboratory protocol assay at scott and white memorial hospital core laboratory. blood samples (5 ml) were collected from patients in standard ethylenediaminetetraacetic acid (edta) tubes and centrifuged and plasma obtained was aliquoted into 100ml eppendorf tubes (fisher scientific, hampton, usa) frozen at �800�c. the commercial elisa kits used for gdf-15, mmp3, and sst2 were obtained from aviscera biosciences inc (santa clara, california, usa). mmp2, mmp9, and timp1 elisa kits were purchased from ray biotech inc( norcross, georgia, usa). all analytes were quantitated using commercially available elisa kits. they were all based on sandwich elisa principle wherein primary antibodies against the analyte were coated on a 96-well plate. analyte was captured from serum samples by these primary antibodies, unbound matrix was washed off, and the bound analyte quantitated using a tagged detection antibody. calibration curves were generated using serially diluted, pre-weighed analyte. the protocol followed was that obtained with the kit for use. left ventricular ejection fraction (lvef) and left ventricular internal dimension in diastole (lvidd) were obtained by twodimensional (2-d) echocardiography. all echocardiographic examinations were done in the s&w echo laboratory the interoperator variation for scanning and reading was estimated at around 5%. correlation analysis was performed between sst2 and gdf-15, mmp2, mmp3, mmp9, timp1, nyha class, lvef, and lvidd using microsoft excel as well as vassarstats online program. the study protocol was approved by s&w irb. spearman rank-order correlation analyses wereperformed between gdf-15 and each of the mmps—mmp2, mmp3, mmp9, timp1, and sst2 as well as nyha class and echocardiographic parameters (lvef and lvidd). lvef and lvidd were obtained by 2-d echocardiography. the protocol was approved by s&w irb. all statistical analyses were performed using the vassarstats online program. statistical significance was set at p < 0.05. tables show both the one-tailed and two-tailed p values. individual patient values used for correlations and the corresponding scatter plots have been provided in the online supplementary material. results characteristics of study population table 1 summarizes the characteristics of the study population. bnp levels, lvidd, and ejection fraction were table 1. characteristics of study population. baseline characteristic control (n ¼ 9) dcm (n ¼ 23) p value age (years) 58.3 + 10.3 50.4 + 34 0.18 gender male 56% 84% nd female 44% 16% nd bnp (pg/ml) 50 + 38 1653 + 45 0.0006* lvidd (cm) 4.5 + 0.3 6.5 + 0.5 <0.0001* lvef (%) 63.1 + 7.4 21.6 + 8.4 <0.0001* creatinine (mg/dl) 0.87 + 0.12 1.6 + 2.1 0.28 dcm: dilated cardiomyopathy; lvidd: left ventricular internal dimension in diastole; lvef: left ventricular ejection fraction; nd: not done. *p < 0.05 was considered significant. 2 journal of circulating biomarkers significantly different from the dcm versus controlled subjects. male comprised of 84% of the dcm group, while male and female were almost equally represented in the control group. this unequal gender representation in the two groups was not intentional. table 2 shows that all the five biomarkers analyzed such as gdf-15, mmp2, mmp3, mmp9, and timp1 were significantly upregulated in the dcm subjects. correlation of gdf-15 with mmps correlation of gdf-15 with mmps is shown in table 3. correlation analysis of control versus dcm patients showed a strong correlation of gdf-15 with timp1 (r ¼ 0.83, p < 0.0001) and weaker correlation with mmp3 (r ¼ 0.41, p ¼ 0.011) and mmp2 (r ¼ 0.47, p ¼ 0.003). correlation with mmp9 was only borderline significant (r ¼ 0.3036, p ¼ 0.046). gdf-15 correlated negatively with mmp2/timp1 ratio (r ¼ �0.24, p ¼ 0.09) but was not statistically significant at p < 0.05. figure 1(a) to (e) shows the scatter plots for the correlations between gdf-15 and timp1, mmp2, mmp3, mmp9, and mmp2/timp1 ratio, respectively. correlation of gdf-15 with worsening function and echocardiographic parameters table 4 shows gdf-15 correlated negatively with lvef (r ¼�0.49, p ¼ 0.003) and positively with lvidd (r ¼ 0.5, p ¼ 0.002). gdf-15 showed significant positive correlation with nyha functional class (r ¼ 0.71, p < 0.00001) and bnp (r ¼ 0.51, p ¼ 0.002). figure 2(a) to (d) shows the scatter plots for the correlations between gdf-15 and nyha class, lvidd, lvef, and bnp, respectively. correlation of sst2 with gdf-15, mmps, worsening function, and echocardiographic parameters table 5 shows correlation of sst2 with gdf-15 and mmps. plasma sst2 correlated strongly with gdf-15 (r ¼ 0.7, p ¼ 0.000005) and timp1 (r ¼ 0.68, p ¼ 0.00007). moderate correlation was noted with mmp2 (r ¼ 0.48, p ¼ 0.003) and weaker correlation with mmp3 (r ¼ 0.31, p ¼ 0.04). mmp9 showed poor correlation with no essential statistical significance (r ¼ 0.31, p ¼ 0.06). figure 3(a) to (e) shows the scatter plots for the correlations between sst2 and gdf-15, timp1, mmp2, mmp3, and mmp9, respectively. interestingly, sst2 correlated significantly with nyha class (r ¼ 0.31, p ¼ 0.000012) but not with lvef or lvidd as shown in table 6. figure 4(a) to (c) shows the scatter plots for the correlations between sst2 and nyha class, lvef, and lvidd, respectively. discussion gdf-15 has been implicated in fibrosis, inflammation, and ventricular remodeling. the role of gdf-15 in regulation of cardiac remodeling in idcm is not well-defined. this study has attempted to address the association of gdf-15 with sst2 and metalloproteinases, which are possible molecular targets of regulation in pathogenesis of dcm. pathophysiology of idcm at the molecular and cellular levels remains poorly understood. the correlations of sst2 with gdf-15 and mmps show significant associations between these molecules. in this study of idcm patients, gdf-15 appears to have significant associations with markers of fibrosis and remodeling such as soluble st2 and mmps. additionally, in our study, gdf-15 appears to significantly correlate with structural (echocardiographic) and functional (nyha class) parameters of cardiac function. increases in gdf15 in heart failure and its negative correlations with lvef as well as positive correlations with lvidd suggest that gdf-15 possibly influences structure/functional interactions affecting ventricular remodeling in these patients and that such influences could influence mortality. however, most of the existing studies on gdf-15 upregulation are in heart failure patient populations with heterogeneous etiologies. 13,14 a strong correlation between improved ejection fraction and decreased mortality has been demonstrated in a table 2. biomarker levels in control and dcm populations. gdf-15 (pg/ml) mmp2 (ng/ml) mmp9 (ng/ml) timp1 (ng/ml) mmp3 (ng/ml) control (n ¼ 8) 516 + 146 626 + 100 592 + 210 280 + 23 5.5 + 2 dcm (n ¼ 24) 3203 + 1354 848 + 167 976 + 238 565 + 173 9.7 + 2.8 one-tailed pvalue 0.01 0.01 0.006 0.001 0.006 two-tailed p value 0.02 0.02 0.013 0.002 0.013 gdf: growth and differentiation factor-15; mmp: matrix metalloproteinase; timp1: tissue inhibitor of mmp 1; dcm: dilated cardiomyopathy; sst2: soluble suppression of tumorigenicity-2 protein. table 3. correlation of gdf-15 with mmps. correlation coefficient (rs) p value (one-tailed) p value (two-tailed) timp1 0.83 p < 0.00001 p < 0.00001 mmp2 0.47 p ¼ 0.003 p ¼ 0.006 mmp3 0.41 p ¼ 0.011 p ¼ 0.022 mmp9 0.304 p ¼ 0.046 p ¼ 0.092 mmp2/timp1 �0.24 p ¼ 0.09 p ¼ 0.19 gdf: growth and differentiation factor-15; mmp: matrix metalloproteinase; timp1: tissue inhibitor of mmp 1. nair and gongora 3 recent meta-analysis. 15 in another study of an acute heart failure population of mixed etiologies, increases in gdf15 were associated with a greater risk of 60-day heart failure rehospitalizations/cardiovascular death as well as cardiac death at 180 days. 16 in a meta-analysis of a heterogeneous heart failure population, elevated gdf-15 levels were associated with increased risk of mortality. 17 further studies are needed for better characterization of gdf-15 use in prognostication in heart failure populations of specific etiologies. this study focuses on a single group of wellcharacterized idcm patients at a single center. patients with dcm in this study had no coronary artery disease as evidenced by coronary angiography. we present here some findings for the first time in idcm population which will possibly open up pathways of investigation to further knowledge on role of gdf-15 and markers of fibrosis such as soluble st2 and the mmps on ventricular remodeling in this population. gdf-15 correlates significantly and positively with nyha class and worsening functionality. gdf15 negatively correlates with mmp2/timp1. this finding sheds light on effect of gdf-15 on metalloproteinase metabolism, therefore suggesting a role in ventricular fibrosis and remodeling. it is also noteworthy that gdf-15 strongly correlates with bnp suggesting that gdf-15 is released analogous to the natriuretic peptides in response to cardiac injury signaling a possible protective response. figure 5 shows a possible effect of interactions of gdf15, sst2, and mmps on alterations in collagen i/iii ratios, mmp/timp ratios, and increased myosin heavy chain degradation; all of which could contribute to loss of myocytes/ventricular fibrosis/remodeling. 18 gdf-15 has also been implicated in anti-apoptosis as well as induction of figure 1. the scatter plots depicting the correlations between gdf-15 and timp1, mmp2, mmp3, mmp9, and mmp2/timp1 ratio. gdf: growth and differentiation factor-15; mmp: matrix metalloproteinase; timp1: tissue inhibitor of mmp 1. table 4. correlation of gdf-15 with nyha class, lvidd, lvef, and bnp. correlation coefficient (rs) p value (one-tailed) p value (two-tailed) nyha class 0.7 p ¼ 0.0001 p ¼ 0.0002 lvidd 0.5 p ¼ 0.002 p ¼ 0.004 lvef �0.49 p ¼ 0.003 p ¼ 0.007 bnp 0.51 p ¼ 0.002 p ¼ 0.004 gdf: growth and differentiation factor-15; nyha: new york heart association; lvidd: left ventricular internal dimension in diastole; lvef: left ventricular ejection fraction. 4 journal of circulating biomarkers hypertrophy in ventricular myocytes 19 further contributing to ventricular remodeling. a delicate balance between mmps and timps has always been postulated in preventing adverse remodeling, but circulating levels of timp1 have been shown in human dcm to be elevated. 20 the levels of circulating biomarkers appear to be different in their concentrations and trends as compared to all the literature on tissue samples where mmps and timps seem to hold an inverse relationship. 20,21 many factors such as cytokines, hormones and growth factors and stage of dcm as well as extent of upregulation of the neurohormonal system may operate synergistically to regulate the levels of mmps and timps in the myocardial tissue versus in circulation. it is unclear if the extent of inflammation also influences the level balance between mmps and timps in the remodeling processes. 20,21 hence, differences noted in this small study will need to be further investigated in different stages of dcm progression to determine whether changes in mmps/ timps would change with disease severity. bnp is known to counteract the hemodynamic stress by inducing vasodilation, natriuresis, and diuresis. the cardioprotective properties of st2 and il-33 are also well established. the protective effects in the heart of gdf-15 occur through inhibition of apoptosis, hypertrophy, and adverse remodeling in the injured heart. correlations between gdf-15, bnp, and sst2 serve to address the complicated interactions that may exist in myocardial stress. 22–25 it is however important to note that protective roles of these molecules may vary in efficiency as the disease progresses; hence, further studies are warranted in this domain. limitations the study is limited by its small population size and the fact that gdf-15 and all the biomarkers described here were only assayed in circulation. site of production of gdf-15 in heart failure of different etiologies still remains ambiguous. 25,26 conclusions this study shows for the first time that the strongest relationships were between gdf-15 and nyha class as well as between gdf15 and timp, sst2 with gdf-15, and with timp in idcm patients. this investigation opens the pathway for newer investigations on interactions between these molecular markers of fibrosis in the figure 2. the scatter plots depicting the correlations between gdf-15 and nyha class, lvidd, lvef, and bnp. gdf: growth and differentiation factor-15; nyha: new york heart association; lvidd: left ventricular internal dimension in diastole; lvef: left ventricular ejection fraction. table 5. correlation of sst2 with gdf-15 and mmps. correlation coefficient (rs) p value (one-tailed) p value (two-tailed) gdf-15 0.7 p ¼ 0.000005 p ¼ 0.00001 timp1 0.63 p ¼ 0.00007 p ¼ 0.00015 mmp2 0.48 p ¼ 0.003 p ¼ 0.006 mmp3 0.31 p ¼ 0.04 p ¼ 0.08 mmp9 0.31 p ¼ 0.06 p ¼ 0.12 gdf: growth and differentiation factor-15; mmp: matrix metalloproteinase; timp1: tissue inhibitor of mmp 1; sst2: soluble suppression of tumorigenicity-2 protein. nair and gongora 5 process of ventricular remodeling and dysfunction. further studies are required to precisely define the molecular interactions between gdf-15, sst2, and mmps in ventricular remodeling as well as influencing worsening functional capacity in dcm. future directions precise studies to elucidate the extent of changes in mmps, timps, and biomarkers of heart failure such as gdf-15 and soluble standard deviation in relation to bnp need to be undertaken. this is especially needed in the context of disease progression in order to help with prognostication and development of therapeutics. protective roles of bnp, gdf-15, and st2 should be studied in the context of worsening heart failure and disease progression. interactions between gdf-15, bnp, and soluble st2 need to be further investigated in order to define molecular mechanisms that work in synergy when subjected to myocardial strain and remodeling. another area of importance is to investigate the use of these biomarkers and heart failure to monitor therapy and progression of disease. further studies would help in developing a panel figure 3. the scatter plots depicting the correlations between sst2 and gdf-15, timp1, mmp2, mmp3, and mmp9. sst2: soluble suppression of tumorigenicity-2 protein; gdf: growth and differentiation factor-15; mmp: matrix metalloproteinase; timp1: tissue inhibitor of mmp 1. table 6. correlation of sst2 with nyha class, lvef, and lvidd. correlation coefficient (rs) p value (one-tailed) p value (two-tailed) nyha class 0.67 p ¼ 0.000012 p ¼ 0.000024 lvef 0.26 p ¼ 0.05 p ¼ 0.1 lvidd 0.32 p ¼ 0.03 p ¼�0.07 sst2: soluble suppression of tumorigenicity-2 protein; nyha: new york heart association; lvidd: left ventricular internal dimension in diastole; lvef: left ventricular ejection fraction. 6 journal of circulating biomarkers of biomarkers which can be used to risk stratify as well as for the development of therapeutics as interactions between biomarkers define the convergence divergence of complex biochemical pathways. this is especially important as heart failure is a complex syndrome where interactions between inflammation, myocardial strain, and remodeling contribute to the pathophysiology and progression of the disease. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this work was supported by the division of cardiology at scott and white memorial hospital, temple, texas, usa, under an internal grant. supplemental material supplementary material for this article is available online. references 1. elliott p, andersson b, arbustini e, et al. classification of the cardiomyopathies: a position statement from the european society of cardiology working group on myocardial and pericardial diseases. eur heart j 2008; 29: 270–276. 2. mestroni l, brun f, spezzacatene a, et al. genetic causes of dilated cardiomyopathy. prog pediatr cardiol 2014; 37: 13–81. 3. bootcov mr, bauskin ar, valenzuela sm, et al. mic-1, a novel macrophage inhibitory cytokine, is a divergent member of the tgf-beta superfamily. proc natl acad sci usa 1997; 94: 11514–11519. 4. jankovic-tomasevic r, pavlovic su, jevtovic-stoimenov t, et al. prognostic utility of biomarker growth differentiation factor-15 in patients with acute 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confounding factors among community-dwelling middle-aged and elderly persons ryuichi kawamoto 1,2 , daisuke ninomiya 1,2 , kensuke senzaki 1,2 , and teru kumagi 1 abstract abnormally high glycated hemoglobin (hb) (hba1c) is significantly associated with oxidative stress and an increased risk of cardiovascular disease (cvd). serum total bilirubin (t-b) may have a beneficial role in preventing oxidative changes and be a negative risk factor of cvd. limited information is available on whether serum t-b is an independent confounding factor of hba1c. the study subjects were 633 men aged 70 + 9 (mean + standard deviation (sd)) years and 878 women aged 70 + 8 years who were enrolled consecutively from among patients aged �40 years through a community-based annual check-up process. we evaluated the relationship between various confounding factors including serum t-b and hba1c in each gender. multiple linear regression analysis pertaining to hba1c showed that in men, serum t-b (b ¼�0.139) as well as waist circumference (b ¼ 0.099), exercise habit (b ¼ 0.137), systolic blood pressure (sbp) (b ¼ 0.076), triglycerides (b¼ 0.087), and uric acid (b¼�0.123) were significantly and independently associated with hba1c, and in women, serum t-b (b ¼�0.084) as well as body mass index (b ¼ 0.090), smoking status (b ¼�0.077), sbp (b ¼ 0.117), diastolic blood pressure (dbp) (b ¼�0.155), low-density lipoprotein cholesterol (b ¼ 0.074), prevalence of antidyslipidemic medication (b ¼ 0.174), and uric acid (b ¼ 0.090) were also significantly and independently associated with hba1c. multivariateadjusted serum hba1c levels were significantly high in subjects with the lowest serum t-b levels in both genders. serum t-b is an independent confounding factor for hba1c among community-dwelling middle-aged and elderly persons. keywords serum total bilirubin, hemoglobin a1c, community-dwelling persons, confounding factor, middle-aged and elderly persons date received: 09 march 2017; accepted: 14 july 2017 introduction glycated hemoglobin (hba1c) is a reliable indicator of mean blood glucose concentrations over the preceding 3 months and an important test for the management of diabetes. epidemiological studies have reported that high hba1c was significantly associated with oxidative stress 1 and an increased risk of chronic kidney disease (ckd), 2,3 cardiovascular disease (cvd), and all-cause mortality regardless of diabetes status. 4–8 bilirubin, which consists of an open chain of four pyrrolelike rings (tetrapyrrole) and is a natural end product of heme 1 department of community medicine, ehime university graduate school of medicine, toon-city, ehime, japan 2 department of internal medicine, seiyo municipal nomura hospital, seiyo-city, ehime, japan corresponding author: ryuichi kawamoto, department of internal medicine, seiyo municipal nomura hospital, 9-53 nomura, nomura-cho, seiyo-city, ehime 797-1212, japan. emails: rykawamo@m.ehime.ca.jp, rykawamo@m.ehime-u.ac.jp journal of circulating biomarkers volume 6: 1–7 ª the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417726609 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:rykawamo@m.ehime.ca.jp mailto:rykawamo@m.ehime-u.ac.jp https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417726609 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage catabolism, has been generally regarded as an important endogenous antioxidant 9 and anti-inflammatory molecule. 10 current studies demonstrate that mildly elevated serum bilirubin may provide important protection against metabolic syndrome, 11 diabetes, 12 cvd, and all-cause mortality in adults. 13,14 we also have demonstrated that low total bilirubin (t-b) was significantly associated with increased intimamedia thickness and/or plaque formation of the carotid artery among nondiabetic 15 and diabetic patients. 16 thus, some researchers hypothesize that the association of hba1c with bilirubin may at least partly mediate the association between hba1c and cvd and all-cause mortality. 17 oda and kawai 7 reported that bilirubin is negatively associated with hba1c independent of other cvd risk factors in apparently healthy japanese men and women. however, there are few reports on the relationship between serum t-b and hba1c in japanese middle-aged and elderly community-dwelling persons. firstly, this study investigated serum t-b and its relationship with potential confounding factors such as age, body mass index (bmi), habits, lipids, and glucose. secondly, this study investigated whether there is an independent association of serum t-b with hba1c. to examine these two issues, cross-sectional data from communitydwelling persons were used. methods subjects the study population aged �40 years was selected through a community-based annual check-up process from the nomura health and welfare center in a rural town located in ehime prefecture, japan. the physical activity level of subjects (e.g. exercise, drinking, and smoking habits), information on medical history, present conditions, and medications (e.g. antihypertensive, antidyslipidemic, and antidiabetic medication) were obtained by interview using a structured questionnaire. for all these individuals, overnight fasting plasma samples were made available. participants with serum t-b �2.0 mg/dl or alanine transaminase (alt) �100 iu/l or gamma glutamyl transpeptidase (ggt) �100 iu/l were excluded to avoid confounding factors due to the high possibility of potential gilbert syndrome and hepatobiliary disease. thus, 1511 (men 633 and women 878) patients were enrolled in the study. the study complies with the declaration of helsinki and was approved by the ethics committee of ehime university school of medicine with written informed consent obtained from each subject (institutional review board: 1402009). methods information on demographic characteristics and risk factors was collected using clinical files. bmi was calculated by dividing weight (in kilograms) by the square of the height (in meters). smoking status was defined as the number of cigarette packs per day multiplied by the number of years smoked (pack-year), and the participants were classified into never smokers, past smokers, light smokers (<20 pack-year), and heavy smokers (�20 pack-year). daily alcohol consumption was measured using the japanese liquor unit in which a unit corresponds to 22.9 g of ethanol, and the participants were classified into never drinkers, occasional drinkers, daily light drinkers (<2 unit/day), and daily heavy drinkers (�2 unit/day). triglycerides (tg), high-density lipoprotein cholesterol (hdl-c), low-density lipoprotein cholesterol (ldl-c), creatinine (enzymatic method), uric acid, hba1c, and serum t-b were measured when subjects were fasted. estimate glomerular filtration rate (egfr) was calculated using the chronic kidney disease epidemiology collaboration (ckd-epi) equations modified by a japanese coefficient (egfrckdepi): male—cr� 0.9 mg/dl, 141� (cr/ 0.9) –0.411� 0.993age� 0.813; cr > 0.9 mg/dl, 141� (cr/0.9)– 1.209�0.993age�0.813; female—cr�0.7 mg/dl, 144� (cr/ 0.7) –0.329� 0.993age� 0.813; cr > 0.7 mg/dl, 144� (cr/0.7)– 1.209 � 0.993age � 0.813.18 statistics all values are expressed as the mean + sd, unless otherwise specified, and for parameters with non-normal distribution (such as tg, hba1c, and t-b), the data are shown as median (interquartile range) values. in all the analyses, parameters with non-normal distributions were used after log-transformation. statistical analysis was performed using ibm spss statistics version 21 (statistical package for social science japan, inc., tokyo, japan). differences in means and prevalence among the groups were analyzed by student’s t-test for continuous data and w2 test for categorical data, respectively. pearson’s correlations were calculated in order to characterize the associations between various characteristics and serum t-b. forced entry and stepwise multiple linear regression analysis (p value for entry was <0.05 and for exit was >0.10) were used to evaluate the contribution of each confounding factor to hba1c. analysis of covariance (ancova) was performed using a general linear model approach to determine the association between confounding factors including that of serum t-b and hba1c. in these analyses, hba1c was the dependent variable, the four categories by quartile of serum t-b (men—first: 0.30–0.50; second: 0.51–0.70; third: 0.71–0.86; fourth: 0.87–1.90 and women—first: 0.20–0.49; second: 0.50–0.60; third: 0.61– 0.79; fourth: 0.80–1.78) were the fixed variables, and all confounding factors in model 2 of table 4 were added as covariates. a value of p < 0.05 was considered significant. results characteristics of subjects by gender table 1 shows the background characteristics by gender. the study subjects were 633 men aged 70 + 9 (mean + 2 journal of circulating biomarkers sd) years and 878 women aged 70 + 8 years. several characteristics differed between men and women. bmi, waist circumference, smoking status, alcohol consumption, history of cvd, dbp, tg, uric acid, and serum t-b were higher in men than in women. hdl-c, ldl-c, prevalence of antidyslipidemic medication, and egfrckdepi were higher in women than in men. there were no intergroup differences regarding age, exercise habit, sbp, prevalence of antihypertensive medication, and hba1c. simple relationships between confounding factors including serum t-b and hba1c within each gender table 2 shows the relationship between confounding factors including serum t-b and hba1c within each gender. in men, bmi, waist circumference, exercise habit, sbp, tg, and ldl-c correlated positively with hba1c, while hdl-c, uric acid, and serum t-b correlated negatively with hba1c. in women, age, bmi, waist circumference, prevalence of antihypertensive medication, tg, prevalence of antidyslipidemic medication, and uric acid correlated positively with hba1c, while smoking status, hdl-c, egfrckdepi, and serum t-b correlated negatively with hba1c. figure 1 shows the correlation between serum t-b and hba1c within each gender. the correlation coefficient between serum t-b and hba1c was significant in table 1. characteristics of subjects within each gender. a characteristics men, n ¼ 633 women, n ¼ 878 p value* age 70 + 9 70 + 8 0.824 body mass index 23.2 + 3.0 22.6 + 3.2 <0.001 waist circumference 82.4 + 8.3 80.4 + 9.0 <0.001 smoking statusb (%) 42.7/38.8/6.4/12.1 97.0/1.9/0.7/0.4 <0.001 alcohol habitc (%) 24.9/23.6/17.6/33.9 70.9/22.6/4.2/2.3 <0.001 exercise habit (%) 33.9 37.3 0.207 history of cvd (%) 9.2 4.3 <0.001 systolic blood pressure (mmhg) 135 + 17 136 + 17 0.220 diastolic blood pressure (mmhg) 80 + 10 77 + 9 <0.001 antihypertensive medication (%) 43.2 42.6 0.824 triglycerides (mg/dl) 89 (68–130) 87 (65–117) 0.004 hdl cholesterol (mg/dl) 61 + 16 68 + 17 <0.001 ldl cholesterol (mg/dl) 115 + 28 125 + 29 <0.001 antidyslipidemic medication (%) 13.0 27.7 <0.001 egfrckdepi (ml/min/1.73 m 2) 70.3 + 11.5 72.2 + 11.2 0.001 uric acid (mg/dl) 5.9 + 1.3 4.7 + 1.1 <0.001 serum total bilirubin (mg/dl) 0.7 (0.5–0.9) 0.6 (0.5–0.8) <0.001 hba1c (%) 5.7 (5.4–6.0) 5.7 (5.5–5.9) 0.112 cvd: cardiovascular disease; hdl: high-density lipoprotein; ldl: low-density lipoprotein; hb: hemoglobin. a data are presented as means + standard deviation. data for triglycerides, hemoglobin a1c, and serum total bilirubin were skewed, presented as median (interquartile range) values, and log-transformed for analysis. numbers in bold indicate significance (p < 0.05). bsmoking status was classified as never smokers, past smokers, light smokers (<20 pack-year), and heavy smokers (� 20 pack-year). calcohol habit was classified as never drinkers, occasional drinkers, daily light drinker (<2 unit/day), and daily heavy drinkers (�2 unit/day). *student’s t-test was used for the continuous data and w2 test for the categorical data. table 2. a simple relationship between variables including serum t-b and hba1c within each gender.a men, n ¼ 633 women, n ¼ 878 characteristics r (p value) r (p value) age 0.022 (0.585) 0.086 (0.011) body mass index 0.099 (0.013) 0.161 (<0.001) waist circumference 0.113 (0.005) 0.157 (<0.001) smoking status 0.047 (0.235) �0.074 (0.028) alcohol habit �0.046 (0.245) �0.050 (0.136) exercise habit 0.129 (0.001) 0.007 (0.847) history of cvd 0.038 (0.334) 0.042 (0.209) systolic blood pressure 0.109 (0.006) 0.064 (0.058) diastolic blood pressure 0.047 (0.238) �0.021 (0.536) antihypertensive medication 0.048 (0.224) 0.149 (<0.001) triglycerides 0.126 (0.001) 0.105 (0.002) hdl cholesterol �0.098 (0.014) �0.100 (0.003) ldl cholesterol 0.086 (0.030) 0.005 (0.873) antidyslipidemic medication 0.078 (0.050) 0.206 (<0.001) egfrckdepi 0.049 (0.221) �0.094 (0.005) uric acid �0.109 (0.006) 0.143 (<0.001) serum total bilirubin �0.126 (0.001) �0.083 (0.014) r: pearson’s correlation coefficient. adata for triglycerides, hba1c, and serum total bilirubin were skewed and log-transformed for analysis. numbers in bold indicate significance (p < 0.05). kawamoto et al. 3 both men (r ¼ �0.126, p ¼ 0.001) and women (r ¼ �0.083, p ¼ 0.014). multivariate relationships between confounding factors including of serum t-b and hba1c within each gender as presented in table 3, a multiple linear regression analysis performed to find independent confounding factors for hba1c showed that in men, serum t-b (b ¼ �0.139) as well as waist circumference (b ¼ 0.099), exercise habit (b ¼ 0.137), sbp (b ¼ 0.076), tg (b ¼ 0.087), and uric acid (b ¼ �0.123) were significantly and independently associated with hba1c, and in women, serum t-b (b ¼ �0.084) as well as bmi (b ¼ 0.090), smoking status (b ¼ �0.077), sbp (b ¼ 0,117), dbp (b ¼ �0.155), ldl-c (b ¼ 0.074), prevalence of antidyslipidemic medication (b ¼ 0.174), and uric acid (b ¼0.090) were also significantly and independently associated with hba1c. figure 1. relationship between serum total bilirubin and hba1c within each gender. table 3. multiple linear regression analysis of variables including t-b for hba1c within each gender.a men, n ¼ 633 women, n ¼ 878 multiple linear regression analysis multiple linear regression analysis model 1 model 2 model 1 model 2 characteristics b (p value) b (p value) b (p value) b (p value) age 0.018 (0.743) — �0.006 (0.890) — body mass index �0.018 (0.825) — 0.067 (0.269) 0.090 (0.011) waist circumference 0.105 (0.208) 0.099 (0.016) 0.018 (0.771) — smoking status 0.036 (0.376) — �0.071 (0.033) �0.077 (0.018) alcohol habit �0.030 (0.479) — �0.034 (0.337) — exercise habit 0.139 (<0.001) 0.137 (<0.001) 0.012 (0.724) — history of cvd 0.015 (0.705) — 0.004 (0.905) — systolic blood pressure 0.126 (0.040) 0.076 (0.049) 0.115 (0.032) 0.117 (0.020) diastolic blood pressure �0.056 (0.367) — �0.155 (0.003) �0.155 (0.002) antihypertensive medication 0.029 (0.504) — 0.066 (0.077) 0.066 (0.063) triglycerides 0.077 (0.077) 0.087 (0.032) 0.013 (0.737) — hdl cholesterol �0.018 (0.691) — �0.015 (0.700) — ldl cholesterol 0.078 (0.056) 0.071 (0.071) 0.068 (0.055) 0.074 (0.030) antidyslipidemic medication 0.047 (0.248) — 0.173 (<0.001) 0.174 (<0.001) egfrckdepi 0.090 (0.083) — 0.009 (0.834) — uric acid �0.088 (0.043) �0.123 (0.002) 0.095 (0.013) 0.090 (0.008) serum total bilirubin �0.128 (0.001) �0.139 (<0.001) �0.081 (0.014) �0.084 (0.010) r2 0.097 (<0.001) 0.075 (<0.001) 0.099 (<0.001) 0.097 (<0.001) b: standardized coefficient; r2: multiple coefficient of determination. model 1: forced entry method and model 2: stepwise method. a data for triglycerides, hba1c, and serum total bilirubin were skewed and log-transformed for analysis. numbers in bold indicate significance (p < 0.05). 4 journal of circulating biomarkers mean (95% ci) hba1c of the subjects categorized by quartile of serum t-b within each gender table 4 presents the levels of hba1c after adjustment for all confounding factors in model 2 of table 3. hba1c levels were significantly low in subjects with a high serum t-b level in both genders. discussion to examine any possible contribution of serum t-b to hba1c, we studied the relationship between confounding factors including serum t-b and hba1c. we found that serum t-b was independently and negatively related to hba1c. increased serum t-b occurred in parallel with the decrease in hba1c in both genders, and serum t-b quartiles were significantly and negatively associated with hba1c, independent of other confounding factors in both genders. to our knowledge, this is the first study to indicate a negative relationship between mildly elevated serum t-b and hba1c among japanese middle-aged and elderly community-dwelling persons. the precise mechanisms that lead to decreased hba1c in individuals with increased serum t-b are not completely understood. hba1c was significantly associated with an increase of oxidative stress. 1 numerous nonenzymatic antioxidants (e.g. vitamins c and e, glutathione (gsh), betacarotene, ubiquinone, uric acid, bilirubin, etc.) exist in cells and relate to oxidative stress. 19 zelenka et al. 20 demonstrated that mildly elevated serum bilirubin is generally associated with attenuation of oxidative stress and with better anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function. baranano et al. 21 and sedlak et al. 22 showed that bilirubin is an antioxidant that protects cells from a 10,000-fold excess of oxidants, suggesting that the highly protective properties of ho-1 may be mediated predominantly through the action of bilirubin derived from ho-1 by inhibition of vascular endothelial activation and dysfunction in response to pro-inflammatory stress. 23 an increase of oxidative stress and inflammation contribute as the most common causes of the pathogenesis of insulin resistance 24 and atherosclerosis. 25,26 moreover, serum t-b correlated with several confounding risk factors for cvd, such as gender, age, smoking, alcohol, blood pressure, hdl-c, tg, ldl-c, diabetes, and obesity, and correlated directly with hdl-c. 27 these contributions may appear to allow bilirubin to inhibit multiple steps in the pathogenesis of atherogenesis. in fact, current epidemiological studies have demonstrated that serum t-b is negatively correlated with risk of cvd. 15,27–29 therefore, mildly elevated serum t-b may inhibit the glycation of hemoglobin by reducing oxidative stress. serum bilirubin concentrations are affected by many factors including race, gender, age, smoking status, fasting, intake of numerous medications and/or plant products, and altitude. 30 these factors are likely to influence biological impact of bilirubin production on human body. since weight reduction is known to reduce several cvd risk factors, it is important to note that weight loss was associated with a linear increase in serum bilirubin level. 31 there are some limitations to this study. firstly, based on its cross-sectional study design, the present findings are inherently limited in the ability to eliminate causal relationships between confounding factors including serum t-b and hba1c. secondly, we could not eliminate the possible effects of underlying diseases (e.g. liver disease, gallstones, and excessive red cell destruction) and medications used for hypertension and dyslipidemia on the present findings. in this study, participants with serum t-b >2.0 mg/dl or alt � 100 iu/l or ggt � 100 iu/l were excluded, but individuals with gilbert’s syndrome could be included in the high t-b (1.00–2.00 mg/dl) group. thirdly, secondary prevention interventions in obesity, hypertension, dyslipidemia, and diabetes mellitus may be successful in reducing confounding factors, thus attenuating the observed association of confounding table 4. mean (95% ci) of hba1c of the subjects categorized by serum t-b within each gender. a characteristics men, n ¼ 633 women, n ¼ 878 n non-adjusted mean (95% ci) multivariate-adjusted mean (95%ci)b n non-adjusted mean (95%ci) multivariate-adjusted mean (95%ci)b quartile 1 153 5.86 (5.75–5.96) 5.85 (5.75–5.95) 259 5.80 (5.74–5.85) 5.79 (5.74–5.84) quartile 2 201 5.83 (5.74–5.92) 5.83 (5.74–5.92) 175 5.75 (5.69–5.82) 5.75 (5.69–5.82) quartile 3 125 5.78 (5.66–5.89) c 5.78 (5.67–5.89) c 211 5.70 (5.65–5.77) d 5.70 (5.65–5.75) d quartile 4 154 5.66 (5.56–5.75)e 5.66 (5.57–5.75)e 233 5.70 (5.65–5.76)d 5.71 (5.65–5.76)d ci: confidence interval. asubjects were divided into four groups based on quartile of serum total bilirubin within each gender (men—quartile 1, 0.30–0.50; quartile 2, 0.51–0.70; quartile 3, 0.71–0.86; quartile 4, 0.87–1.90 and women—quartile 1, 0.20–0.49; quartile 2, 0.50–0.60; quartile 3, 0.61–0.79; quartile 4, 0.80–1.78 mg/dl). badjusted for all confounding factors in model 2 of table 3. cp < 0.02 versus quartile 2. dp < 0.03 versus quartile 1. ep < 0.01. kawamoto et al. 5 factors with disease. therefore, the demographics and referral source may limit generalizability. in conclusions, the present study showed that serum t-b is strongly associated with hba1c in both genders. the underlying mechanism behind this relationship is unclear but seems to be independent of confounding factors such as age, bmi, smoking status, drinking status, exercise habit, lipids, glucose, and medication. further prospective population-based studies are needed to investigate the mechanism(s) underlying this association. declaration of conflicting interests the author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: this study was supported, in part, by a grant-in-aid from the foundation for the development of the community (2016). no additional external funding was received for this study. the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. references 1. ohara m, fukui t, ouchi m, et al. relationship between daily and day-to-day glycemic variability and increased oxidative stress in type 2 diabetes. diabetes res clin pract 2016; 122: 62–70. 2. bash ld, selvin e, steffes m, et al. poor glycemic control in diabetes and the risk of incident chronic kidney disease even in the absence of albuminuria and 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exosomes original research article jungreem woo1#, shivani sharma2*# and james gimzewski1,2# 1 department of chemistry and biochemistry, university of california, los angeles, usa 2 california nanosystems institute, university of california, los angeles, california, usa #these authors contributed equally to this work *corresponding author(s) e-mail: sharmas@cnsi.ucla.edu received 09 march 2016; accepted 09 may 2016 doi: 10.5772/64148 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract exosomes are ~100 nanometre diameter vesicles secreted by mammalian cells. these emerging disease biomarkers carry nucleic acids, proteins and lipids specific to the parental cells that secrete them. exosomes are typically isolated in bulk by ultracentrifugation, filtration or immu‐ noaffinity precipitation for downstream proteomic, genomic, or lipidomic analysis. however, the structural properties and heterogeneity of isolated exosomes at the single vesicle level are not well characterized due to their small size. in this paper, by using high-resolution atomic force microscope imaging, we show the nanoscale mor‐ phology and structural heterogeneity in exosomes derived from u87 cells. quantitative assessment of single exosomes reveals nanoscale variations in morphology, surface roughness and counts isolated by ultracentrifugation (uc) and immunoaffinity (ia) purification. both methods produce intact globular, 30-120 nm sized vesicles when imaged under fluid and in air. however, ia exosomes had higher surface roughness and bimodal size population compared to uc exosomes. the study highlights the differences in size and surface topography of exosomes purified from a single cell type using different isolation methods. keywords afm, exosome, nanoparticles, immunoaffini‐ ty, surface roughness, vesicles, isolation method abbreviations afm: atomic force microscopy; tem: transmission electron microscopy uc: ultracentrifugation ia: immunoaffinity 1. introduction cells utilize vesicles for the intercellular signalling, trans‐ porting and trafficking of metabolites [1-2]. a more collective term, “extracellular vesicle” (ev), is often used as a synonym for “membrane vesicles”, which includes microvesicles, exosomes, apoptic bodies and other vesicles. in this paper, we use the term “exosomes” due to the 1j circ biomark, 2016, 5:11 | doi: 10.5772/64148 characteristic size, morphology and specific surface markers present. exosomes, which are 30-120 nm-sized vesicles, are of particular interest for potential disease diagnostic markers. due to their small size, it was once believed that exosomes were random cell debris, but it is now known that they are actively secreted from cells with specific markers [3-6]. exosomes carry nucleic acids [7], proteins [8] and lipids [9], and can deliver these diverse components to distant recipient cells and thus hold potential as emerging bio‐ markers for diseases including cancers [10-12]. neverthe‐ less, the isolation and characterization of purified exosomes from bodily fluids, tissues, or cells of origin are crucial aspects for any downstream biomarker discovery research. many isolation procedures and commercially available kits are currently being actively researched in a bid to optimize the enrichment and isolation of purified exosomes in bulk [13]. for the isolation of exosomes, conventional methods utilize sequential centrifugation and ultracentrifugation (uc) to spin down exosomes in cell culture media or bodily fluids [14]. sucrose density gradient ultracentrifugation is required for additional purification and to minimize protein contamination [15]. size exclusion chromatography is also employed to purify exosomes based on their physical dimensions [16]. in addition, antibodies targeting exosome surface markers such as cd9, cd63, cd81 and epcam are attached to magnetic beads (immunoaffinity; ia) to capture and purify exosomes [15,17]. after isolation, the purified exosome samples are characterized using various imaging and biochemical techniques. it is difficult to characterize the 3d structure of single exosomes due to their nanometric dimensions. the particle size distribution of extracellular vesicles has been analysed with transmis‐ sion electron microscopy (tem), flow cytometry, resistive pulse sensing and nanoparticle tracking analysis [18]. tem provides standard size distribution information, but suffers from fixation artefacts and non-biocompatible conditions such as low temperature and high vacuum. flow cytometry is not optimal for exosomes, due to its poor resolution when particle diameters are less than 100 nm. resistive pulse sensing and nanoparticle tracking analysis resolves particles with diameters ranging from 10-1000 nm, but is limited in terms of resolving heterogeneous vesicle populations. the first structural characterization of exosomes was effected using tem with immunogold nanoparticle labelling [19]. based on em images, exosomes were thought to have 'cup-shaped structures'. however, atomic force microscope (afm) and field emission scan‐ ning electron microscope images subsequently revealed that exosomes are globular vesicles, similar to other vesicles present in cells [20]. one of the limitations of these bulk isolation methods is that they preclude identification of heterogeneity among exosome sub-populations. it is known that exosomes secreted by the same originating tissues, cell types and single cells, may vary in terms of composition, size and density [21], which can have significant implications for studies in exosome biology and their role as disease biomarkers in various pathologies [22]. the commonly used nanosight tracking analysis (nta) is a comparable non-microscopic method for measuring exosome sizes and distributions. many researchers have utilized and com‐ pared both microscopic and non-microscopic methods for obtaining exosome sizes [23]. however, nta does not provide quantitative surface information at sub-nm resolutions. recently, we reported high-resolution images of glioblastoma exosomes using peakforce afm imaging and showed that their nanofilament structure may influ‐ ence the delivery of exosomes into recipient cells [24]. here, using high-resolution afm imaging, we investigate the nanoscale morphology and structural heterogeneity in exosomes isolated from glioblastoma u87 cell lines, and the influence of particular isolation methods. quantitative assessment of exosomes at the single vesicle level reveal nanoscale variations in morphology, surface roughness and counts of exosomes isolated using two methods, i.e., ultracentrifugation (uc) and immunoaffinity (ia) bead purification with widely used exosomal markers (cd9, cd63, cd81 and epcam). our afm imaging data on single vesicles reveals differences in the size and surface topography of exosomes purified from the same cell type but using different isolation methods. we attribute the observed variations in size and surface roughness of isolated exosomes to the selection and/or reassembly of receptors on the surface of isolated exosomes. 2. results 2.1 single exosome images show differences in structure depending on purification method we investigated the structural differences among isolated exosomes at the single vesicle level between those purified using two different methods, i.e., uc and ia (fig. 1). the antibodies on the magnetic beads were against four different antigens: cd9, cd63, cd81 and epcam. each antibody is immobilized on separate bead solutions; then, the individual antibody-beads are mixed together. since the antibodies select exosomes according to their surface proteins, it is possible that exosomes with specific surface markers may result in the size selection of the entire exosome population. on the other hand, ultracentrifuge utilizes the density difference in the sample to purify exosomes. ultracentrifugation [14] is the most commonly used method for concentrating exosomes that include differential centrifugation steps up to 100 000 × g. though the method is time consuming (4-5 hours), requires an ultracentrifuge and is inefficient with regard to exosome yield (5-25% recovery), it nevertheless enables label-free isolation of exosomes. 2.1.1 the structural characterization of native exosomes in fluid: peak force imaging peak force mode is a recently developed afm imaging mode enabling very low imaging forces (less than 1 nn) for 2 j circ biomark, 2016, 5:11 | doi: 10.5772/64148 the detection of exosomes in fluid. figure 2 (a) shows ia exosomes imaged using afm in fluid. ia exosomes show globular structures of varying sizes with an average diameter of 39 ± 22 nm in width and an average height of 4.3 ± 3.7 nm. figure 2 (b) shows a close-up image of the smaller ia exosomes and cross-sections of the exosomes in figure 2 (c). the smaller ia exosomes have a diameter of ~30 nm and a height of ~2.5 nm. on the other hand, the bigger ia exosomes are shown in figure 2 (d) and their cross-sections are displayed in figure 2 (e). the bigger ia exosomes have a diameter of ~80 nm and a height of ~10-12 nm. both cross-sections from smaller and bigger exosomes confirm their globular shapes, as expected under native conditions. when compared to air afm imaging, the size distributions remain similar. uc exosomes also show a globular shape in afm images in fluid (figure 3 (a)). uc exosomes showed a single figure 1. (a) the scheme of exosome isolation by immunoaffinity magnetic beads kit method. for capturing exosomes, antibodies against four different surface markers (cd9, cd63, cd81 and epcam) were immobilized on the surface of 3 µm magnetic beads. each antibody was modified separately on the beads and the mixture of the antibody-beads used to capture antibodies from cell culture media (not drawn in scale). after capture and elution, isolated exosomes were diluted and dialyzed against pbs prior to characterization/storage; (b) flowchart of exosome isolation by ultracentri‐ fugation method. the exosome-containing media was centrifuged sequentially to remove particles denser than exosomes, then centrifuged to pellet down exosomes (exosome buoyant density of 1.10-1.24 g/ml). the flowchart indicates each centrifugation step and the location of exosomes. exosomes were pelleted following ultracentrifugation at 100 000 g and the pellet was washed with pbs, then ultra centrifuged again. the final exosome pellet was re-suspended in either deionized water or pbs and characterized/ stored. population with an average diameter of 49 ± 9 nm and an average height of 7.8 ± 2.8 nm. a smaller scan area of the uc exosomes are shown in figure 3 (b) and their crosssections are shown in figure 3 (c). the uc exosomes have a diameter of ~60 nm and a height of ~10 nm. 2.1.2 the structural characterization of native exosomes in air: tapping mode imaging exosomes were isolated using the ia method and their nanoscale structure was obtained using tapping mode afm imaging in air. figure 4 (a)-(c) shows the detailed structure of single exosomes isolated by the ia method. single exosomes show a globular structure with a diameter of 76 nm and a height of 9.3 nm. the dimensions of the exosomes are similar to the values previously reported [21]. the detailed structure of a single exosome is shown in figure 4 (d)-(e). surface roughness of the exosomes within figure 2. detection of ia exosomes by peak force afm imaging in fluid; (a) representative topographic image of exosomes showing varying vesicle size; (b) close-up of smaller ia exosomes. the smaller ia exosomes were 22.5 ± 3.5 nm in diameter and 1.8 ± 0.3 nm in height; (c) cross-section of (b) shows ia exosome dimensions to be approximately 30 nm in diameter and with a height of 2.3 nm; (d) close-up of larger ia exosomes. the dimensions of bigger ia exosomes are a diameter of 64.6 ± 9.2 nm and a height of 8.1 ± 3.1 nm; (e) the cross-section of (d) show ia exosome dimensions of approxi‐ mately 80 nm in diameter and a height of 12 nm. 3jungreem woo, shivani sharma and james gimzewski: the role of isolation methods on a nanoscale surface structure and its effect on the size of exosomes a 40 nm x 40 nm area, shown as a box in figure 4 (d), were measured as ra (arithmetic average of the absolute values, 1.48 nm) and rq (root mean squared, 1.17 nm). the roughness of the exosome surface is clearly evident in the figure 3. detection of uc exosomes by peak force afm imaging in fluid; (a) representative topographic image of uc exosomes. uc exosomes showed a single gaussian population in fluid imaging; (b) close-up of uc exosomes showing a diameter of 49 ± 9 nm and a height of 7.8 ± 2.8 nm; (c) the crosssection of (b) shows uc exosome dimensions of approximately 60 nm in diameter and with a height of 10 nm. phase channel in figure 4 (f). the phase image is deter‐ mined by the lag between incident resonant oscillations and the output signal oscillations, which are sensitive to surface properties such as elasticity, adhesion and friction. figure 5. high-resolution images of uc exosomes obtained by afm imaging in air; (a) representative topography, (b) amplitude and (c) phase images of exosomes. inset (a) is the cross-section of the exosome for the height channel. the dimensions of the exosome in (a) are a diameter of 79 nm and a height of 6.2 nm. the phase channel shows the smooth surface of the exosome. 2.2 exosomes show different size distributions depending on purification methods we measured the dimensions of ia and uc (fig. 5) exosomes to determine if their size distributions depended on the purification method. the diameter and height were measured for more than 100 exosomes. the average diameter was 68.1 ± 26.5 nm and 45.9 ± 10.3 nm for ia and uc, respectively. the average height was 9.6 ± 7.7 nm and 5.5 ± 2.4 nm for ia and uc, respectively. in the average values, the dimensions of the exosomes seemed to be similar, while ia exosomes were ~30% bigger than uc exosomes. figure 6 shows a histogram of (a) the diameter and (b) the height of ia and uc exosomes, which are markedly different. while the uc exosomes show normal distribution (solid lines) in diameters and heights, the ia figure 4. high-resolution images of ia exosomes obtained by afm imaging in air; (a) representative topography, (b) amplitude and (c) phase images of exosomes shown while the boxed exosome is magnified in (d) topography, (e) amplitude and (f) phase images. the diameter and height of the exosome in (d) is 76 nm and 9.3 nm, respectively. the phase channel shows the rough surface of the exosome. 4 j circ biomark, 2016, 5:11 | doi: 10.5772/64148 exosomes display bimodal distribution (dashed line). the ia exosomes show wider bimodal distributions. the first peak was observed at 23.9 ± 3.2 nm in width and 3.2 ± 0.5 nm in height, and the second peak at 54 ± 18 nm in width. in the case of uc exosomes, it shows relatively narrow singular distributions with a peak at 45.2 ± 0.1 nm in width and 3.2 ± 0.5 nm in height. the difference in size distribution may stem from the isolation mechanism employed. for the uc method, exosomes were isolated based on buoyant density and likelihood of resulting in a single gaussian population. however, the ia method selects exosomes based on surface antigens. as an ia kit contains four different antibodies, during purification, exosomes are affinity-purified based on surface markers, which may result in wider bimodal size distributions. figure 6. the size distributions of exosomes depending on isolation methods. the width distributions (a) and the height distributions (b) are shown with gaussian fits. the width distribution of exosomes isolated by ia show a bimodal population, with the first peak at 23.9 ± 3.2 nm and the second peak at 54 ± 18 nm. the width distribution of exosomes isolated by uc show a single gaussian population with the peak at 45.2 ± 0.1 nm. the height distribution of exosomes show a single gaussian population for both methods, with the peak at 3.2 ± 0.5 nm for ia and 5.0 ± 0.3 nm for uc. the width and the height of exosomes from ia and uc were significantly different at p<0.05. 3. discussion exosomes have the potential to be utilized as disease biomarkers that carry signature biomolecules (nucleic acids, proteins and lipids) from the secreting cells of origin [7, 9, 25-29]. however, due to their sub-100 nm size and low abundance (~106-9 vesicles per ml) in extracellular environ‐ ments, it is challenging to isolate and characterize single exosomes. during a special workshop presented by the international society for extracellular vesicles (isev), researchers and experts discussed the standardization of exosome isolation, handling and characterization. al‐ though a consensus was not reached [13], lötvall et al. later proposed basic experimental requirements for defining exosomes [30]. the proteomic and genomic contents of exosomes, depending on the isolation method, have since been compared and evaluated [15,26,31-33]. in addition, evaluation of the structural integrity and morphology of isolated exosomes is important for standardization and downstream biomolecular analysis. in this paper we focus on single exosome characterization using ultracentrifuga‐ tion and immunoaffinity capture to elucidate their nano‐ scale structure on an individual basis. this was achieved using high-resolution afm imaging of u87 cell-derived exosomes. we discovered that quantitative assessment of exosomes at the single vesicle level reveals nanoscale variations in the morphology, surface roughness and counts of exosomes isolated, which differ depending on the extraction method used – ultracentrifugation (uc) or immunoaffinity (ia) bead purification using exosomal markers (cd9, cd63, cd81 and epcam). our study is the first to reveal the high-resolution structural variations (morphology and size) and surface inhomogeneity (topo‐ graphic roughness) of exosome populations at the single vesicle level. our results indicate distinct differences between uc and ia exosomes purified at the single exosome level. although both methods yield globular vesicles, we found that uc exosomes have smooth surface, whereas ia exosomes exhibit a distinct roughness. secondly, the size distribu‐ tions were different. uc exosomes showed a single gaussian distribution, whereas ia exosomes showed a broader bimodal distribution. furthermore, peaks in ia exosome size histograms clearly indicate that ia exosomes are either smaller or larger than uc exosomes. this was observed under both afm imaging techniques and environments, i.e., tapping mode in air and peak force mode in fluid. we attribute the measured variations to the specific isolation protocol. table s 1 summarizes the differences in two exosome isolation methods, showing a side-by-side comparison and the pros and cons of each method, including the experimental details such as time, yield, sample starting/final volumes and equipment used. on the one hand, uc relies on the size and density (1.12-1.19 g/ml) of the vesicles to precipitate, at high centrifugal forces, intact unlabelled exosomes from the solution (figure 3 and 5). on the other hand, ia-isolated exosomes result from a combination of the selective sorting of vesicles enriched in either of the four targeted exosomal markers, varying in binding affinities, as well as the process of exosome elution from magnetic beads. affinity purifica‐ tion involves specific non-covalent binding interactions between immobilized target exosomal marker antibodies (cd9, cd63, cd81 and epcam) on beads with ligands on the exosome surface. addition of the cell culture superna‐ tant (a complex mixture containing exosomes) allows exosomes to bind according to their specific affinity to the immobilized antibody molecules. however, to elute exosomes from the beads, it is necessary to break the antigen-antibody binding. the binding force differs for each specific antibody-antigen, ranging from 50-500 pn [34-35] compared to the covalent binding force ranges of 1-2 nn [36-38]. although appropriate buffer conditions for binding in affinity purification vary, antibody-antigen binding is generally most efficient in aqueous buffers at a physiological ph and ionic strength, such as phosphatebuffered saline (pbs), which was employed in our protocol. after washing away the non-bound components of the complex mixture, the captured exosomes are released and recovered (i.e., eluted) from the “bead-immobilized antibody” using buffer conditions that disrupt the affinity 5jungreem woo, shivani sharma and james gimzewski: the role of isolation methods on a nanoscale surface structure and its effect on the size of exosomes interaction. methods such as raising or lowering the ph, adding a mildly active detergent, or altering the ionization state are commonly used. the proprietary exosome elution buffer (exocap, jsr life science) enabled the recovery of intact exosomes, as confirmed by afm images (figure 2 and 4). there are some possibilities that can explain the observed differences in exosome distribution and surface structure at the single vesicle level. first, the surfactant on the magnetic bead may transfer to the exosome surface. while this might explain the increase in roughness and size of the exosome, it cannot explain their shrinkage (width and height) as observed in the bimodal distribution (see figure 6). another possibility is that the ia exosomes are altered during the elution step (figure 7). when exosome-bead complexes are incubated with an elution buffer, not only is antigen-antibody interaction affected, but also the exosome itself, as well as antibody-bead linkers. to minimize such unwarranted effects, the elution buffer was diluted and immediately dialyzed against pbs. however, the antibodybead linker may nonetheless be disrupted and few anti‐ bodies will remain on the exosome resulting in the increased apparent size of the extracted exosome (figure 7(a)). additionally, it is possible for tetraspanins to be extracted from the exosome surface, resulting in the reassembly of exosomes by the active detergents in the elution buffer, which will lead to the underestimation of exosome dimensions (figure 7 (c)). furthermore, ucisolated exosomes showed a smoother surface (ra; 1.16 nm and rq; 0.95 nm, see also figure 5 (c)) and narrower size distributions. in contrast, in the case of ia exosomes, we observed a wider bimodal distribution of dimensions both in diameter and height (figure 6), and accompanied by a higher surface roughness compared to uc exosomes (ra; 1.48 nm and rq; 1.17 nm, see also figure 4 (c), (f)). all of these observed differences are consistent with our inter‐ pretation that using the ia method results in subtle changes in the exosome surface layer. figure 7. three possible ia exosomes eluted from beads; (a) antigenantibody interaction may remain intact, while the antibody-bead linkers are disrupted during elution. thus, antibodies are attached on the eluted ia exosomes, resulting in expanded sizes; (b) ideally, only the antigen-antibody interaction is affected and eluted ia exosomes will be the same as uc exosomes; (c) tetraspanins (cd9, cd63 and cd 81) are pulled out during the elution, causing ia exosomes' lost volume and partial contents. in summary, our afm images of iaand uc-isolated exosomes confirm in both cases high stability, nonaggregation and vesicle integrity, suggesting the retention of bio-functionality and suitability for downstream analysis, which is required for thorough investigation in future functional, microrna and/or protein assays. exosomes are heterogeneous vesicles with various surface markers [39]. since exosomes show sub-populations with different markers and sizes, it is desirable to perform more analysis on single vesicle levels than ensemble qualifica‐ tions. in the past few years, nanotechnology-based isola‐ tion techniques [40] have been developed for isolating exosomes. such advances make it further evident that detailed characterizations of exosomes are essential in order to advance a biological understanding and the biotechnological exploitation of exosomes, at the single vesicle and sub-vesicular level [41-42]. although the limitations of current technologies do not support the sorting of exosomes according to sizes, it nonetheless provides insight about the analysis of exosome ensemble data when sub-populations are acknowledged. both the uc and ia methods produce intact globular exosomes at the single vesicle level. the advantage of the ia method is speed and ease of use. in the current study, a mixture of four capture antibodies (anti-cd9, cd63, cd81 and epcam) was used for ia purification; hence, we cannot exclude the co-purification of various exosome subpopulations. on the other hand, purification by uc is based on the size and density of exosomes and as such, can be expected to yield a more homogeneous size distribution. in future, evaluating the size distribution of exosomes obtained from single capture antibodies will be addition‐ ally advantageous. however, this clearly indicates that the nanoscale characterization of isolated exosomes is advisa‐ ble, particularly in detailed proteomic investigations such as mass spectroscopy studies, where biological purity and the chemical properties of a sample greatly influence protein identification [43]. in addition, our data presents more implications pertaining to cell biological functions [44], as uc exosomes and ia exosomes display different sub-populations. for example, uc exosomes with smooth‐ er topographic surfaces may have distinct physical charac‐ teristics for cell binding and activation, whereas some of the epitopes of surface markers on ia exosomes may not be accessible, due to their rough surface. in addition, exo‐ somes isolated from each individual surface marker may have different biochemical, genomic, or proteomic proper‐ ties and may have the potential for being employed as diagnostic biomarkers. 4. materials and methods 4.1 u87 cell culture u87 cells were cultured in dulbecco's modified eagle medium (dmem) and supplemented with 10% heat inactivated fetal bovine serum (fbs), 100 units/ml penicil‐ lin g, and 100 µg/ml streptomycin. cells were incubated at 37°c and 5% carbon dioxide. at approximately 80% confluence, cells were washed with pbs and passaged using a 0.25% trypsin-edta treatment for dissociation. 4.2 exosome isolation u87 cells were cultured in six 60 mm petri dishes (falcon) with fbs-originated-exosome-free media (as instructed in 6 j circ biomark, 2016, 5:11 | doi: 10.5772/64148 the protocol by théry; fbs was ultracentrifuged at 100 000 g for 2 hours at 4ºc, then filtered with a 0.22 µm sterile filter) for 48 hours; after 48 hours, the media containing u87 exosomes was isolated. total cell count was 2x107 and 24 ml of u87-exosome-containing media was obtained. for the following isolation methods, the same batch of media was used. 4.3 isolation of exosomes using immunoaffinity (ia) magnetic beads kit method the procedure suggested in the manufacturer’s manual (jsr life science, tokyo, japan) was followed; 200 µl of u87 exosome-containing media was incubated with 100 µl of capture beads for 60 min at room temperature (rt) on a shaker. beads were separated from the supernatant by a magnet and washed with a 0.5ml wash buffer three times; 50 µl of elution buffer was added to the beads and the beads were gently re-suspended, then incubated without mixing for 3 min at rt. beads were separated and the supernatant was transferred to a slide-a-lyzer™ dialysis cassette (thermo fisher scientific, ma, usa), then dialyzed against pbs. purified exosomes were stored at 4°c until afm imaging. 4.4 isolation of exosomes using the ultracentrifugation (uc) method exosome isolation using ultracentrifugation was followed by a previously published protocol (figure 1) [14]. to remove cells/debris, the exosome-containing media was centrifuged at 2000g for 20 min at 4°c and supernatant 1 was isolated. then, supernatant 1 was centrifuged at 10 000g for 30 min at 4°c to remove microvesicles and again, supernatant 2 was carefully isolated. to isolate exosomes, supernatant 2 was ultracentrifuged at 100 000g for 2 hours at 4°c and supernatant 3 was discarded. to wash exo‐ somes, the pellet was re-suspended in 1 ml pbs and the mixture was ultracentrifuged at 100 000g for 1 hour at 4°c, and supernatant 4 was discarded. purified exosomes were re-suspended in 100 µl of pbs and stored at 4°c until afm imaging. 4.5 sample preparation and air imaging exosome samples purified from u87 were incubated on freshly cleaved mica for 5 min, washed with deionized water to remove any unbound exosomes and air-dried overnight. samples were imaged by dimension icon (bruker instruments, ca, usa) using the tapping mode via tesp cantilever (bruker instruments, ca, usa) and images were recorded at 1024 samples per line at 1 hz. image processing was done using spiptm software. for exosome yield calculation, images at a size of 1 µm x 1 µm and a resolution of 512 samples per line, at 1 hz, were used. for exosome surface roughness measurements, images were processed with spiptm to obtain ra (arithmetic average of the absolute values) and rq (root mean squared) values. 4.6 sample preparation and fluid imaging to anchor exosomes on the surface, (3-aminopropyl) triethoxysilane (aptes) modified mica was prepared; 10 µl of 10% aptes solution was incubated with clean, freshly cleaved mica discs in a vacuum chamber overnight. nitrogen gas was purged and the mica discs were stored in a nitrogen gas chamber; 50 µl of the exosome sample was incubated on an aptes modified mica disc for 10 min. to remove unbound exosomes, mica was washed with deionized water four times; 50 µl of deionized water was added on mica prior to imaging. samples were imaged using dimension icon (bruker instruments, ca, usa) with mlct-e cantilevers (bruker) for fluid imaging in qnm peak force mode [38,45]. afm probes were calibrated using a thermal method and images were taken at 256 samples per line, at 0.6 hz, then processed by spiptm. 5. conflict of interest the authors report no conflict of interest. 6. acknowledgements this work was supported by international center for materials nanoarchitectonics satellite (mana) at the national institute for materials science (nims), tsukuba, japan. we acknowledge the use of afms at the nano and pico characterization laboratory at california nanosys‐ tems institute. we also acknowledge jsr life science for providing an exocap kit for isolating exosomes. we thank to dr. k. das at ucla medical centre for providing the u87 cell line and dr. e. reisler at department of chemistry and biochemistry and molecular biology institute at ucla for using the ultracentrifugation facility. 7. references [1] colombo, m.; raposo, g.; thery, c. biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. annu rev cell dev biol 2014, 30, 255-89. 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radical prostatectomy for known high-risk disease were assayed for prostate-specific antigen (psa), free-psa, free-hk2, and prohk2. univariate cox regression and multivariate models were used to predict both gleason scores and progression-free survival (pfs). free-hk2 levels �80 ng/l were predictive of both gleason scores �7 (p ¼ 0.04) and pfs (p ¼ 0.03). psa �8.0 mg/l also was predictive of pfs (p ¼ 0.02). however, neither % free-psa nor pro-hk2, when treated as continuous or cutoff variables were associated with gleason score or pfs. multivariable models showed that clinical stage t1c versus t2/t3, gleason score �7, and psa �8.0 mg/l or clinical stage t1c versus t2/t3, gleason scores �7, and free-hk2 �80 ng/l were among the best models predicting pfs. both free-hk2 and psa in conjunction with clinical stage and gleason score are good predictors of pfs in prostate cancer. keywords free-hk2, pro-hk2, psa, prostate cancer, progression-free survival date received: 1 june 2016; accepted: 21 june 2017 introduction prostate cancer has been detected earlier and in most cases at a treatable stage following the introduction of psa as a noninvasive diagnostic test. 1 however, prostate cancer is still the second leading cause of cancer death in men in the united states. 2 risk group and staging, based on the gleason score, psa levels at biopsy, and other tests are the main factors predictive of prognosis in men diagnosed with prostate cancer. during recent years, there has been active investigation for new markers to complement the psa screening that would better differentiate high-risk cancers and identify those patients who are less likely to respond to conventional treatment and who could most benefit from multimodal or alternative therapies. several tissue-based gene signatures have been developed to predict biochemical relapse or metastasis after radical prostatectomy (rp). decipher ® (genomedx biosciences, vancouver, canada) is a 22 gene-tissue expression assay, which can be used to predict the 5-year risk of metastatic prostate cancer after rp. other reverse transcription polymerase chain reaction 1 mayo clinic, rochester, mn, usa 2 biomedical statistics and informatics, mayo clinic, rochester, mn, usa 3 department of urology, mayo clinic, rochester, mn, usa 4 department of radiation oncology, mayo clinic, rochester, mn, usa 5 beckman coulter, chaska, mn, usa 6 department of laboratory medicine and pathology, mayo clinic, rochester, mn, usa corresponding author: anatilde gonzalez guerrico, mayo clinic, 200 1st st sw, rochester, mn 55904, usa. email: gonzalezguerrico.anatilde@mayo.edu journal of circulating biomarkers volume 6: 1–8 ª the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417720151 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:gonzalezguerrico.anatilde@mayo.edu https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417720151 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage (rt-pcr) tissue-based gene signature assays for prediction of recurrence are myriad genetics prolaris score, oncotype dx ® genomic prostate score™ (gps), and nf-kappa b activated recurrence predictor 21 (narp21). 3 several proteins and a new generation of markers in blood and urine, such as micro-rna, circulating dna, and circulating tumor cells, are under investigation for prostate cancer diagnosis and prognosis. 3 hk2, a member of the kallikrein family of proteins, has been shown to be a predictive marker for prostate cancer progression, in some studies more so than serum psa. 4–6 several hk2 forms are detected in serum, including freehk2 and hk2-act. 4–5,7 free-hk2 is comprised of pro-hk2 and mature free-hk2, which are both increased in prostate cancer. 4,6 free-hk2 is associated with a higher gleason score. 4 an increase in free-hk2 after radiation therapy can predict recurrence. 8 in this study, we developed a sensitive semiautomatic immunoassay to specifically detect pro-hk2 in human serum and verified the analytical performance of a published immunoassay for free-hk2. 4 we evaluated the role of these markers for predicting prostate cancer progression using a cohort of 189 men with high-risk prostate cancer. material and methods analytic assays measurement of free-psa and psa. free-psa and psa were measured by hybritech psa and free-psa immunoassays (beckman coulter, brea, california, usa, access2). free-hk2 immunoassay. free-hk2 was measured by a twostep sequential immunoassay using a research version of the access2 immunoassay system (beckman coulter) as previously described with small modifications. 4 in the first step, 75 ml of serum reacted with 150 ng of biotinylated h499 antibody and 50 ml of 0.6 mg/ml of paramagnetic particles at 37�c for 30 min. after washing, the paramagnetic beads were incubated with alkaline phosphatase labelled h599 antibody for 30 min. six calibrators (0, 5, 15, 30, 300, and 3000 ng/l) were made by dilution of recombinant mature hk2 into psa zero diluent (beckman coulter). cross-reactivity with psa and prostate specific antigen-antichymotrypsin (psa-act) is negligible; cross-reactivity with hk2-act is 2%. pro-hk2 assay development. recombinant pro-hk2 a217v (hybritech, san diego, california, usa) was immunopurified from the condition medium of recombinant cell lines using hk1g586.1 immunoaffinity column (which recognizes pro-hk2 and mature hk2) as previously described. 9 the concentration of purified pro-hk2 a217v was determined by absorbance at 280 nm. 10 six calibrators (0, 8, 31, 125, 500, and 2000 ng/l) were made by addition of recombinant prohk2 a217v into human serum albumin (hsa) matrix (0.1 m hepes, 0.15 m sodium chloride, 7% hsa (sigma a3782), 0.3% plurafac a-38 (basf), 0.1% sodium azide). diluted standards were aliquoted and stored at �80�c. pro-hk2 was measured by an immunoassay similar to free-hk2 with two main modifications. in order to increase the sensitivity, the biotinylated capture antibody and alkaline phosphatase detection antibody were incubated off-line with serum for 18–20 h. to minimize interferences due to human anti-mouse and heterophilic antibodies in the serum, a preincubation with blockers was added to the process. two hundred microliters of serum or standards were incubated with 75 ml of blocking solution containing heat-inactivated mouse serum (hybritech), hbr-1, hbr-6, hbr-23 (scantibodies laboratories, santee, california, usa), and mab-33-igg polymer (roche diagnostics corp, indianapolis, indiana, usa) for 1 h at room temperature (rt) with rotation. biotinylated bt464.3 anti-pro-hk2 antibody and alkaline phosphatase hk1h 599.1 antibody were added and incubated with constant rotation at rt for 18–22 h. a onestep format on the beckman coulter access 2 system was used to capture and measure the antigen–antibody complexes; 200 ml of serum–blockers–antibodies mix was incubated with 50 ml of paramagnetic-streptavidin particles at 37�c for 20 min. the complex was washed and developed with substrate (dioxetane phosphate), which produced a chemiluminescent signal that was quantified in the research version of access 2 (beckman coulter). a grade 2 polynomial curve math model was used for standardization. the amount of free-hk2 or pro-hk2 in the sample was determined from the signal/background ratios (measured relative light units (rlu)/rlus of zero standard) by means of the calibration curve. the 2000 ng/l was excluded from the standardization curve when pro-hk2 values in the serum samples were lower than 500 ng/l. pro-hk2 assay and free-hk2 assay analytical performance. the detection limit was determined by assaying 20 replicates of hsa matrix zero diluent and 20 replicates of 4, 6, and 8 ng/l calibrators of pro-hk2, or 20 replicates of psa zero diluent and 20 replicates of 2, 3, and 4 ng/l of hk2. the limit of detection was taking 2.5 standard deviation (sd) of 20 replicates of the zero calibrator (in rlus) and using lineal regression with the zero and pro-hk2 or hk2 calibrators to calculate the limit of detection in nanograms per liter. the limit of quantification (loq) was the minimum concentration measured with imprecision <15%. the within run imprecision was calculated measuring 20 replicates of three different levels of serum pools within a run. the between run imprecision (coefficient of variation [cv] ¼ 100 � sd/mean) was assessed by measuring three serum pools in 20 independent analytical runs over approximately 2 months. we assessed linearity after dilution by serially diluting patient serums in standard diluent. cross-reactivity of relevant analytes was assessed by measuring psa, psa-act, hk2, and hk2-act diluted in hsa matrix zero diluent. the concentrations tested by 2 journal of circulating biomarkers pro-hk2 assay were as follows: psa, and psa-act, 150 mg/l; hk2, 9 ng/l; and hk2-act, 10 mg/l. to study the stability of pro-hk2 nine-patient samples kept at �80�c were defrosted and assayed within 4.5 h (time zero). we compared the initial values with readings of serum stored at rt for 1 day and at 40�c for 1 and 7 days. for reassessment of free-hk2 immunoassay, we compared the analytic performance characteristics with those previously published. 4 we also compared values with 100 serum samples previously measured for free-hk2 in 2010, and the correlation between both measurements was evaluated using the spearman test. human studies we used de-identified male and female serum for validation studies. one hundred twelve serum samples from men with negative biopsies for prostate cancer were used to evaluate the relation between pro-hk2 and age. we measured markers in a cohort consisted of 189 men who underwent open or robotic rp for prostate cancer at the mayo clinic from 2004 to 2008 and were considered high risk for recurrence. psa and free-psa were previously measured in all samples, and free-hk2 was previously measured in approximately half of the samples. free-hk2 and pro-hk2 were measured in all available samples (172 samples) in 2015 with prior measurements used for six patients who did not have adequate residual sample. high risk was defined as a psa >20 mg/l, a gleason score of 8 or higher, seminal vesicle invasion (svi), or a gps score of �10 at rp. all analyses were limited to the 178 men who had psa, % freepsa, and free-hk2 data to allow for a matched comparison of psa and hk2 data. these studies were approved by the mayo clinic institutional review board. statistical methods the primary objective was to evaluate whether free-hk2 and pro-hk2 are predictive of time to progression in highrisk prostate cancer patients undergoing rp. the primary endpoint was progression-free survival (pfs). progression was defined as the occurrence of psa progression as defined by american urology association/american society for radiation oncology (aua/astro) guidelines, 11 local recurrence, systemic progression or death. local recurrence or systemic progression was defined as demonstrable local or distant metastasis on imaging studies or via biopsy. time to progression is defined as the time to first occurrence of psa progression, local or regional progression or death. patients without an event were censored at the time of last contact. cox proportional hazard models were used to assess free-hk2, pro-hk2, psa, % free-psa, age, gleason score, gps, svi, and t stage to predict pfs. due to the skewness of the laboratory markers, the logarithmic values of these markers were determined and used in the cox proportional hazard modeling. in addition to assessing the association of the lab markers by taking the logarithmic value, dichotomous cut points were also investigated by first splitting each continuous covariant into quartiles and choosing the cut point that best maximized the difference between the progression and nonprogression groups. in addition, a few alternate cut points were explored. for the multivariate analysis, only factors thought to be clinically relevant or with a p value of �0.2 in univariate analysis were considered as candidates in the multivariate model. a secondary objective was to evaluate the association of the free-hk2 and pro-hk2 with gleason score. gleason score was dichotomized into two groups (<7 vs. �7). wilcoxon rank sum and w2 tests were used to assess the association of free-hk2, pro-hk2, psa, and % free-psa with gleason score. various cut points of the laboratory markers were also assessed using above mentioned methods. all tests were two-sided, with p values �0.05 considered statistically significant. correlation between assays was evaluated using spearman correlation test. statistical analyses were performed by using sas v9.3 (sas institute, cary, north carolina, usa). results hk2 and pro-hk2 immunoassay analytical performance we reanalyzed the analytical performance of the automated assay for free-hk2 and developed a semiautomatic assay for pro-hk2 using a research version of the beckman coulter access2 system. table 1 summarizes the analytical performance of freehk2 and pro-hk2 assays. because the concentration of pro-hk2 in circulation is very low, an immunoassay to measure it must have very low levels of detection and be highly reproducible. we table 1. assay performance. performance level free-hk2, ng/l pro-hk2, ng/l within run imprecision low mean ¼ 18 mean ¼ 26 cv ¼ 9.5% cv ¼ 3.1% medium mean ¼ 41 mean ¼ 82 cv ¼ 2.9% cv ¼ 2.3% high mean ¼ 236 mean ¼ 521 cv ¼ 3.2% cv ¼ 2.2% between run imprecision low mean ¼ 17 mean ¼ 18 cv ¼ 8.8% cv ¼ 11.6% medium mean ¼ 41 mean ¼ 91 cv ¼ 4.6% cv ¼ 4.1% high mean ¼ 226 mean ¼ 524 cv ¼ 7.2% cv ¼ 7.3% limit of detection lod 2.1 3.5 limit of quantitation loq 4.0 8.0 hk2: human kallikrein 2. guerrico et al. 3 developed a very sensitive semiautomatic immunoassay to measure pro-hk2, with a loq five times lower than that reported in an immunoassay described previously. 6 this increase in sensitivity was required to measure pro-hk2 in serum samples within the gray range of psa between 2 and 10 mg/l where the results of the pro-hk2 assay could have clinical relevance. twenty-eight percent of the samples in the range of psa of 2–10 mg/l had concentrations of pro-hk2 that were lower than the loq of 8 ng/l compared to 85% of the samples that would be under a loq previously described of 40 ng/l. 6 standard curves for free-hk2 and pro-hk2 are shown in figure 1. serum from females and males with and without known prostate disease was used to validate the pro-hk2 assay. pro-hk2 was under the loq in the 14 female serum samples tested. the mean value for pro-hk2 in male serums with unknown prostate cancer was 22 + 21 ng/l. six male samples were diluted with standard diluent (hsa matrix), and the dilutions were measured with pro-hk2 assay. the average recovery by dilution was 96%. three serum samples were diluted with psa zero diluent and measured with free-hk2 assay. the average recovery by dilution was 106%. cross-reactivity of pro-hk2 assay with psa, psa-act, and hk2-act was under the limit of detection and cross-reactivity with recombinant hk2 was of 0.07% in weight basis indicative that crossreactivity with potentially interfering proteins is negligible. stability studies showed that pro-hk2 is stable. the mean recovery for samples stored at rt for 24 h was 93 + 6%, f or samples s tored a t 4�c f or 1 da y was 97 + 5%, and for samples kept at 4�c for 1 week was 105 + 7%. y= 9e–05x2+ 0.008x + 0.984 0 5 10 15 20 25 30 35 0 100 200 300 400 500 600 pro-hk2 standard (ng/l) r l u s/ r l u s (s 0) y = 1e–05x2+ 0.040x + 0.973 0 50 100 150 200 250 0 1000 2000 3000 4000 free-hk2 standard (ng/l) r l u s/ r l u s (s 0) (a) (b) figure 1. average calibration curves for the free-hk2 (a) and prohk2 (b) access 2 research assays. the assays are calibrated with recombinant purified hk2 or pro-hk2. the y-axis represents the ratio of the rlu at various levels to the rlu for the zero calibrator. the solid lines represent the grade 2 polynomial regression curves. hk2: human kallikrein 2; rlu: relative light units. table 2. association of hk2 and psa with gleason score. gleason <7 (n ¼ 51) gleason �7 (n ¼ 126) p value psa (mg/l) 0.20 n 51 126 median 10.4 8.4 q1, q3 6.0, 17.7 5.8, 13.2 % free-psa 0.76 n 51 126 median 11.7 11.2 q1, q3 7.8, 15.6 8.0, 14.9 free-hk2 (ng/l) 0.07 n 51 126 median 42.0 51.2 q1, q3 20.7, 65.1 28.0, 96.0 pro-hk2 (ng/l) 0.22 n 50 120 median 20.0 22.0 q1, q3 7.0, 34.0 9.5, 47.0 free-hk2 �80 ng/l 16% 30% 0.04 hk2: human kallikrein 2. psa ≥ 8 μg/l free-hk2 ≥ 80 ng/l psa (μg/l) fr ee -h k 2 (n g/ l ) psa ≥ 8 μg/l free -hk2 ≥ 80 ng/l psa ≥ 8 μg/l and free-hk2 free-hk2 ≥ 80 ng/l psa ≥ 8 μg/l and/or ≥ 80 ng/l sensitivity 54 % 30 % 24 % 58 % specificity 41 % 84 % 86 % 39 % figure 2. categorical dot plot of free-hk2 versus psa discriminated by gleason score <7 and gleason score �7. the table underneath the graph shows the specificity and sensitivity for prediction of gleason score �7 by free-hk2 �80 ng/l and psa �8mg/l. the circled plus marks represent off-scale measurements. hk2: human kallikrein 2. 4 journal of circulating biomarkers free-hk2 assay equally detected recombinant hk2 and recombinant pro-hk2. free-hk2 values correlated well with previous measurements performed 5 years earlier with an immunoassay previously validated 9 (spearman correlation coefficient ¼ 0.92, p < 0.001). there was no significant correlation between free-hk2 and age (spearman coefficient ¼ 0.16, p ¼ 0.10) and a poor correlation between pro-hk2 and age (spearman coefficient ¼ 0.25, p ¼ 0.007) for men without prostate cancer (but including men with benign prostatic hypertrophy; n ¼ 111, age range ¼ 50–85 years). cohort study for prediction of pfs clinical-pathologic features of the cohort study are as follows: the median age was 63 years (interquartile range (iqr): 57–68) and predominantly caucasian (93%). fifty-one patients (29%) had gleason scores <7 and 76 (42%) patients had clinical t stage of t1c at initial diagnosis. the median psa value was 8.6 (iqr: 5.8–14.3). median follow-up for the entire cohort was 5.7 years (iqr: 2.1–7.8). free-hk2 and pro-hk2 correlated poorly with psa. the spearman correlation coefficient between free-hk2 and table 3. association of hk2 and psa with pfs. n events hr 95% ci p value age at surgery 178 84 1.00 (0.97, 1.03) 0.81 clinical stage t1c 76 25 0.44 (0.27, 0.70) <0.001 t2 or t3 102 59 gleason score �7 125 69 2.34 (1.34, 4.10) 0.003 <7 51 15 svi yes 55 34 2.00 (1.29, 3.09) 0.002 no 123 50 gpsm 178 84 1.21 (1.07, 1.36) 0.002 psa (log, mg/l) 178 84 1.07 (0.82, 1.40) 0.60 psa (mg/l) �8.0 100 54 1.72 (1.10, 2.68) 0.02 <8.0 78 30 % free-psa (log) 178 84 1.00 (0.66, 1.49) 0.98 free-hk2 (log, ng/l) 178 84 1.10 (0.89, 1.37) 0.38 free-hk2 (ng/l) �80 47 28 1.68 (1.07, 2.64) 0.03 <80 131 56 pro-hk2 (log, ng/l) 171 81 1.04 (0.86, 1.26) 0.69 multivariate model with clinical stage, gleason score, and psa clinical stage t1c 76 25 0.53 (0.32, 0.88) 0.01 t2 or t3 100 59 gleason score �7 125 69 1.90 (1.05, 3.44) 0.03 <7 51 15 psa �8.0 98 54 1.72 (1.10, 2.69) 0.02 <8.0 78 30 comparable multivariate model using free-hk2 instead of psa with clinical stage and gleason score clinical stage t1c 76 25 0.51 (0.31, 0.83) 0.006 t2 or t3 100 59 gleason score �7 125 69 1.77 (0.98, 3.18) 0.06 <7 51 15 free-hk2 (ng/l) �80 45 28 1.63 (1.03, 2.57) 0.04 <80 131 56 hk2: human kallikrein 2; pfs: progression-free survival; hr: hazard ratio; ci: confidence interval; svi: seminal vesicle invasion. guerrico et al. 5 psa was 0.24 and pro-hk2 and psa was 0.30. the hk2 and phk2 spearman correlation coefficient was 0.63. table 2 summarizes the association of free-hk2, prohk2, psa, and % free-psa with gleason score �7. patients with free-hk2 �80 ng/l were more likely to have a high gleason score (30% vs. 16%; p ¼ 0.04). it is interesting to note that the observed median psa was 10.4 mg/ l in gleason <7 group compared to 8.4 in the gleason �7 group (not statistically different; p ¼ 0.20). figure 2 shows the categorical dot plot of free-hk2 versus psa for gleason scores (gleason <7 and gleason �7), the table underneath the graph summarizes the specificity and sensitivity for the prediction of gleason score �7 by free-hk2 �80 ng/l and psa �8 mg/l. free-hk2 �80 is highly specific for prediction of high gleason score (84%) compared to psa �8 mg/l. overall univariate cox regression analysis show that clinical stage (t1c vs. t2/t3, hazard ratio (hr) ¼ 0.44; p � 0.001), psa �8 mg/l (hr ¼ 1.72; p ¼ 0.02), free-hk2 �80 ng/l (hr ¼ 1.68; p ¼ 0.03), gpsm (hr ¼ 1.21; p ¼ 0.002), gleason score �7 (hr ¼ 2.34; p ¼ 0.003), and svi (hr ¼ 2.00; p ¼ 0.002) were all significantly associated with pfs (table 3). figure 3 shows the categorical dot plot of free-hk2 vs. psa for prostate cancer progression. the table underneath the graph summarizes the specificity and sensitivity for the prediction of prostate cancer progression by free-hk2 �80 ng/l and psa �8 mg/l. free-hk2 �80 has a higher specificity but a reduced sensitivity for the prediction of progression respect to psa �8 mg/l. in the subgroup of patients with t1c disease, psa �8 mg/l had elevated sensitivity and specificity of 80% and 59%, respectively. kaplan–meier curves of pfs by psa �8 mg/l and freehk2 �80 ng/l are shown in figure 4(a) and (b), respectively. table 3 displays the final multivariate model which includes clinical stage (t1c vs. t2/t3, hr ¼ 0.53; p ¼ 0.01), gleason score �7 (hr ¼ 1.90; p ¼ 0.03), and psa �8.0 mg/l (hr ¼ 1.72; p ¼ 0.02). adding the free-hk2 �80 ng/l covariate to this model was not statistically significant (p ¼ 0.82). however, the overall significance of the three variable cox model with clinical stage, gleason score, and hk2 �80 ng/l was nearly identical to clinical stage, gleason score with psa model (�2 log likelihood ratio test p value < 0.001 for both models). clinical stage (t1c vs. t2/t3, hr ¼ 0.32; p ¼ 0.001), psa �8 mg/l (hr ¼ 2.51; p ¼ 0.003), and hk2 �80 ng/l (hr ¼ 2.38; p ¼ 0.01) were all significantly associated with pfs for patients with baseline psa <10 mg/l. the final multivariate model included clinical stage psa ≥ 8 μg/l free-hk2 ≥ 80 ng/l psa (μg/l) fr ee -h k 2 (n g/ l ) psa ≥ 8 μg/l free -hk2 ≥ 80 ng/l psa ≥ 8 μg/l and free-hk2 ≥ 80 ng/l psa ≥ 8 μg/l and/or free-hk2 ≥ 80 ng/l sensitivity 64 % 33 % 29 % 69 % specificity 51 % 80 % 84 % 47 % figure 3. categorical dot plot of free-hk2 versus psa discriminated by patients who did not have prostate cancer progression (no progression) and patients who progressed (progression). the table underneath the graph shows the specificity and sensitivity for prediction of prostate cancer progression by free-hk2 �80 ng/l and psa �8 mg/l. the circled plus marks represent offscale measurements. hk2: human kallikrein 2. fr ee o f p ro gr es si on (% ) psa < 8 μg/l psa ≥ 8μg/l free-hk2 < 80 ng/l free-hk2 ≥ 80 ng/l fr ee o f p ro gr es si on (% ) (a) (b) figure 4. (a) kaplan–meier curves of pfs by psa (psa �8 mg/l represented by the dotted line and psa <8 mg/l by the solid line). of 100 total patients with psa �8 mg/l, 54 progressed; and of 78 patients with psa <8 mg/l, 30 progressed. patients with psa �8 mg/l had shorter pfs time (log-rank p value ¼ 0.01). (b) kaplan–meier curves of pfs by free-hk2, with free-hk2 �80 ng/l (dotted line) and free-hk2 <80 (solid line). of 47 total patients with free-hk2 �80 ng/l, 28 progressed; and of 131 patients with free-hk2 <8 mg/l, 56 progressed. patients with free-hk2 �80 ng/l had shorter pfs time (log-rank p value ¼ 0.03). pfs: progressionfree survival; hk2: human kallikrein 2. 6 journal of circulating biomarkers (t1c vs. t2/t3, hr ¼ 0.36; p ¼ 0.004) and psa �8.0 (hr ¼ 2.08; p ¼ 0.02). as occurred with the whole cohort cox modeling, adding the free-hk2 �80 ng/l covariate to this model was not statistically significant (p ¼ 0.09). however, the overall significance of the two variables cox model with clinical stage and hk2 �80 ng/l was almost identical to clinical stage with psa model (�2 log likelihood ratio test p value <0.001 for both models). discussion improved blood and urine markers used alone or in combination that better differentiate more aggressive cancers and predict progression and response to therapy are needed. high rates of recurrence in certain subsets of patients (almost 50% in this retrospective cohort study) suggest the need for more intensive and multimodal therapies. markers that add predictive value for relapse after primary prostate cancer therapy may help select patients for clinical trials. in this study, we evaluated whether free-hk2 and pro-hk2 in serum could add information for clinical decision-making for prostate cancer after rp in a group of high-risk prostate cancer patients. it was previously shown that the correlation between free-hk2 and psa is low. 4 we also found that in our cohort study, both free-hk2 and pro-hk2 poorly correlate with psa, suggesting that free-hk2 and pro-hk2 might have independent clinical value and complement the information obtained by psa. immunoassay methods to measure kallikreins in serum could lead to major differences in the results due to antibody specificity and epitope recognition, matrix effect, and complex formation with other molecules. traceable methods to measure psa and hk2 using mass spectroscopy could help better improve and standardized immunoassays to measure markers in biological samples. 12 neither pro-hk2 nor % free-psa predicted progression. the lack of association between pro-hk2 and pfs could be due to a relative large proportion of samples under the limit of detection or with values in the lower end where the variability of the assay is higher and there is a potential for influence of interferences. also, we cannot rule out some degradation or auto processing of pro-hk2 due to storage of frozen serum samples at �80�c. univariate cox regression analysis shows high association of clinical stage and gleason score with relapse. we also found that both psa �8 mg/l and free-hk2 �80 ng/l significantly predicted recurrence. free-hk2 showed to be more specific but less sensitive than psa to predict prostate cancer progression. although free-hk2 was not an independent covariate in multivariate cox regression models, the significance of a three variable cox model with clinical stage, gleason score, and free-hk2 �80 ng/l was almost identical to a model of clinical stage, gleason score with psa �8 mg/l. thus, either psa or free-hk2 in conjunction with gleason score and clinical stage could predict prostate cancer progression. multivariable cox models including psa and clinical stage or free-hk2 and clinical stage also show good prediction of pfs in samples with psa <10 ng/ml. prospective studies should be performed to further validate the utility of free-hk2 as a predictive marker. additionally, whether an increase in free-hk2 after prostatectomy better predicts relapse than preoperative free-hk2 should also be evaluated. patients with free-hk2 �80 ng/l were more likely to have gleason score higher or equal to 7. none of the other variables evaluated showed statistically significant association with high gleason score. these results suggest that free-hk2 in serum could be a good marker for high-risk prostate tumors. it has been shown that the prostate cancer index is associated with gleason score �7 and early relapse.13–17 additionally, preoperative plasma transforming growth factor beta-1 (tgfb1) and serum interleukin (il)-6 and sil-6r have been associated with poor clinical outcome and disease progression. 18 whether free-hk2 adds to the prediction value of these markers to discriminate higher risk tumors and recurrence should be evaluated in future studies. free-hk2 in combination with other circulating markers together with sensitive imaging methods may increase the possibilities for curative success and reduce overtreatment of prostate cancer patients. declaration of conflicting interests the author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: this study was conducted in compliance with mayo clinic conflict of interest policies. mayo clinic and dr klee have received royalties of less than the federal threshold for significant financial interest from the licensing of technology to beckman coulter, inc. funding the author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: support for this study was provided by the mayo clinic prostate spore grant nih/nci ca091956 and beckman coulter, inc. to dr klee. references 1. schroder fh, hugosson j, roobol mj, et al. screening and prostate cancer mortality: results of the european randomised study of screening for prostate cancer (erspc) at 13 years of follow-up. lancet 2014; 384: 2027–2035. 2. siegel rl, miller kd, and jemal a. cancer statistics, 2015. ca cancer j clin 2015; 65: 5–29. 3. locke ja and black pc. next generation biomarkers in prostate cancer. front biosci (landmark ed) 2016; 21: 328–342. 4. grauer ls, finlay ja, mikolajczyk sd, et al. detection of human glandular kallikrein, hk2, as its precursor form and in complex with protease inhibitors in prostate carcinoma serum. j androl 1998; 19: 407–411. 5. klee gg, goodmanson mk, jacobsen sj, et al. highly sensitive automated chemiluminometric assay for measuring guerrico et al. 7 free human glandular kallikrein-2. clin chem 1999; 45: 800–806. 6. vaisanen v, eriksson s, ivaska kk, et al. development of sensitive immunoassays for free and total human glandular kallikrein 2. clin chem 2004; 50: 1607–1617. 7. saedi ms, hill tm, kuus-reichel k, et al. the precursor form of the human kallikrein 2, a kallikrein homologous to prostate-specific antigen, is present in human sera and is increased in prostate cancer and benign prostatic hyperplasia. clin chem 1998; 44: 2115–2119. 8. spratt de, evans mj, davis bj, et al. androgen receptor upregulation mediates radioresistance after ionizing radiation. cancer res 2015; 75: 4688–4696. 9. kumar a, goel as, hill tm, et al. expression of human glandular kallikrein, hk2, in mammalian cells. cancer res 1996; 56: 5397–5402. 10. mikolajczyk sd, millar ls, marker km, et al. ala217 is important for the catalytic function and autoactivation of prostatespecific human kallikrein 2. eur j biochem 1997; 246: 440–446. 11. valicenti rk, thompson i jr, albertsen p, et al. adjuvant and salvage radiation therapy after prostatectomy: american society for radiation oncology/american urological association guidelines. int j radiat oncol biol phys 2013; 86: 822–828. 12. klee ew, bondar op, goodmanson mk, et al. serum concentrations of prostate-specific antigen measured using immune extraction, trypsin digestion, and tandem mass spectrometry quantification of lsepaeltdavk peptide. arch pathol lab med 2014; 138: 1381–1386. 13. jansen fh, van schaik rh, kurstjens j, et al. prostatespecific antigen (psa) isoform p2psa in combination with total psa and free psa improves diagnostic accuracy in prostate cancer detection. eur urol 2010; 57: 921–927. 14. catalona wj, partin aw, sanda mg, et al. a multicenter study of [-2]pro-prostate specific antigen combined with prostate specific antigen and free prostate specific antigen for prostate cancer detection in the 2.0 to 10.0 ng/ml prostate specific antigen range. j urol 2011; 185: 1650–1655. 15. fossati n, buffi nm, haese a, et al. preoperative prostatespecific antigen isoform p2psa and its derivatives, %p2psa and prostate health index, predict pathologic outcomes in patients undergoing radical prostatectomy for prostate cancer: results from a multicentric european prospective study. eur urol 2015; 68: 132–138. 16. de la calle c, patil d, wei jt, et al. multicenter evaluation of the prostate health index to detect aggressive prostate cancer in biopsy naive men. j urol 2015; 194: 65–72. 17. lughezzani g, lazzeri m, buffi nm, et al. preoperative prostate health index is an independent predictor of early biochemical recurrence after radical prostatectomy: results from a prospective single-center study. urol oncol 2015; 33: 337:e7–e14. 18. shariat sf, kattan mw, traxel e, et al. association of preand postoperative plasma levels of transforming growth factor beta(1) and interleukin 6 and its soluble receptor with prostate cancer progression. clin cancer res 2004; 10: 1992–1999. 8 journal of circulating biomarkers << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false 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and in cad patients moran s saghiv1, d ben sira2, e goldhammer3, and m sagiv2 abstract different studies support the notion that chronic aerobic exercises training can influence the circulating levels of solubleklotho (s-klotho) and insulin-like growth factor 1 (igf-i). the effects of s-klotho include improving the quality of life, alleviating the negative impact of age on the body’s work capacity, and possibly increasing longevity. this review provides an overview of the latest findings in this field of research in humans. the different modes of dynamic exercise and their impact on circulating levels of s-klotho and igf-i in young adult athletes, untrained young adults, trained healthy older adults, untrained healthy older adults, and coronary artery disease (cad) patients are reviewed and discussed. together these findings suggest that long-lasting (chronic) aerobic exercise training is probably one of the antiaging factors that counteract the aging and cad process by increasing the circulating s-klotho and lowering the igf-i levels. however, following anaerobic exercise training the opposite occurs. the exact metabolic and physiological pathways involved in the activity of these well-trained young and master sportsmen should be further studied and elucidated. the purpose of this review was to provide a clarification regarding the roles of s-klotho and intensities and durations of different exercise on human health. keywords aerobic exercise, epigenetic, aging, anaerobic exercise, coronary artery disease, master athletes, elite athletes date received: 16 february 2017; accepted: 9 august 2017 introduction exercise training can alter gene expression patterns (epigenetic) and the physiological responses at rest and during exercise. 1 recent studies show that even brief exercise alters gene expression, and the pattern of change involves diverse genetic pathways, consistent with a global dangertype response, for a range of physiological functions from inflammation to tissue repair that would be useful following a bout of physical activity. 2 physical exercise offers an epigenetic tendency with benefits in several health domains. yet, it is only recently that regular exercise has begun to be interpreted as a positive epigenetic mechanism to modify the genome-wide dna methylation patterns in humans. 1 this is achieved by helping to improve the work capacity and physical performance of humans in health and disease. 3 since aerobic exercise increases the circulating levels of soluble-klotho (s-klotho), the wnt-signaling transduction gets suppressed, and thus inhibiting cell senescence and preserving stem cells. 4 however, the precise mechanism underlying the aerobic exercise–induced 1 exercise physiology department, university of mary, bismarck, nd, usa 2 life sciences department, wingate college, wingate, israel 3 heart institute bnai-zion haifa medical center, technion institute, haifa, israel corresponding author: moran s. saghiv, exercise physiology department, casey center, room 141b, university of mary,7500 university drive, bismarck, nd 58504, usa. email: mssaghiv@umary.edu journal of circulating biomarkers volume 6: 1–8 ª the author(s) 2017 reprints and permissions: sagepub.co.uk/journalspermissions.nav doi: 10.1177/1849454417733388 journals.sagepub.com/home/cbx creative commons cc by-nc: this article is distributed under the terms of the creative commons attribution-non commercial 4.0 license (http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the sage and open access pages (https://us.sagepub.com/en-us/nam/open-access-at-sage). mailto:mssaghiv@umary.edu https://uk.sagepub.com/en-gb/journals-permissions https://doi.org/10.1177/1849454417733388 http://journals.sagepub.com/home/cbx https://us.sagepub.com/en-us/nam/open-access-at-sage increase in s-klotho secretion following 12 weeks of moderate aerobic exercise training or the elevation in insulin-like growth factor 1 (igf-i) following anaerobic training remains unclear. 5 the benefits of exercise intensity are positive epigenetic changes in terms of mitochondrial biogenesis. 6 in addition, exercise training is widely appreciated for its effects on the cardiovascular and musculoskeletal, endocrine, and immune systems. many of the benefits of exercise training, such as volume overload (such as aerobic exercise) and load overload (such as anaerobic activities), diminish within 2 weeks if physical activity is substantially reduced and disappear within 2–8 months if exercise training is not resumed. s-klotho klotho is a transmembrane protein that, in addition to other effects, provides some control over the sensitivity of the organism to insulin and appears to be involved in aging. 7 klotho is highly expressed in the brain, kidney, parathyroid, and pituitary glands but also serves as a circulating hormone by which shedding forms s-klotho which in turn can be detected in blood, cerebrospinal fluid, and urine. 8 the a-klotho gene is highly conserved forming s-klotho, a blood circulating protein to be highly conserved across species. in humans, the serum levels of s-klotho decrease after 40 years of age. age-related declines are manifested by a decreased ability for aged skeletal muscle to respond to physiological stimuli such as muscle loading or acute injury. indeed, older adults often exhibit an age-related reduction in the number and size of muscle fibers known as sarcopenia. 9 this decrease in blood s-klotho levels may be observed in patients with several aging-related diseases such as coronary artery disease (cad), cancer, hypertension, and kidney disease. 10,11 the klotho gene encodes a single-pass transmembrane protein that binds to multiple fibroblast growth factor (fgf) receptors and functions as a co-receptor for fgf23, a bone-derived hormone that suppresses phosphate reabsorption and vitamin d biosynthesis in the kidney. 12 in addition, the extracellular domain of s-klotho protein is shed, potentially functioning as a humoral factor of klotho protein. 13 the action mechanism of klotho is not fully understood, but it changes cellular calcium (ca) homeostasis, by increasing the expression and activity of transient receptor potential cation channels, vanilloid subfamily, member 5 (trpv5) which plays a key role in active ca þþ reabsorption in the kidney, and by decreasing the expression and activity of transient receptor potential canonical 6 (trpc6), a subtype of calcium-permeable channel. 14 additionally, klotho increases membrane expression of the inward rectifier renal outer medullary (potassium channel) kinase 1 (romk1) channels, that is, renal outer medullary k þ . 14,15 klotho-deficient mice show increased vitamin d production, and altered mineral–ion homeostasis is suggested to be a cause of premature aging-like phenotypes because the lowering of vitamin d activity by dietary restriction reverses the premature aging-like phenotypes and prolongs survival in these mutants. these results suggest that aginglike phenotypes were due to klotho-associated vitamin d metabolic abnormalities. 16,17 the klotho protein deficiency is known to cause premature aging. klotho is an important molecule in aging processes and its overexpression results in longevity. 18 the klotho gene encodes a transmembrane protein that after cleavage is also found as a secreted protein. importantly, its overexpression suppresses insulin/igf-i signaling and thus extends life span. in addition, klotho participates in the regulation of several other intracellular signaling pathways, including regulation of fgf23 signaling, cyclic adenosine monophosphate (camp), protein kinase c (pkc), transforming growth factor-b (tgf-b), p53/p21, and wnt signaling. 12,18 crasto et al. 19 in a population-based longitudinal study revealed that low plasma levels of klotho are associated with decreased activities of daily living in older individuals. in older community-dwelling adults, plasma klotho is an independent predictor of all-cause mortality. 20 in older, community-dwelling adults, low klotho levels in plasma have also been associated with a poor muscle strength. 21 igf-i igf-i, also called somatomedin c, is a cellular and secreted growth factor critical for normal body growth, development, and maintenance, and plays important roles in multiple biological systems. 22,23 igf-i is an endocrine and autocrine/ paracrine growth factor with major effects on development, cell growth and differentiation, and tissue repair, and it circulates at high levels in the plasma and is expressed in most cell types. 24 a variety of cellular responses are induced by igf-i, including cell proliferation, differentiation, migration, and survival. 25,26 these cellular responses have implicated igf-i in several conditions such as the pathophysiology of several cancers, 27 the mitogenic and myogenic processes during muscle development, regeneration, or hypertrophy, since, unlike other growth factors, igf-i acts as both a mitogen and a differentiation factor. 28 igf-i is an important factor that regulates a variety of cellular responses in multiple biological systems. the igfi gene comprises a highly conserved sequence and contains six exons, which give rise to heterogeneous mrna transcripts by a combination of multiple transcription initiation sites and alternative splicing. several protein synthesis and degradation pathways are mediated by igf-i, which act on the cellular and molecular levels to increase or reestablish the strength, and function of muscle fibers. 29 deficiency of igf-i in skeletal muscle may contribute to sarcopenia by severely influencing protein synthesis. igf-i has anabolic effects on muscle protein content by inhibiting protein degradation and promoting myogenesis. thus, igfi attrition in skeletal muscles is associated with less protein 2 journal of circulating biomarkers synthesis and muscle sarcopenia, 30 stress, and inflammation. 31,32 igf-i is generally thought to be associated with positive attributes such as growth, health, young look, and well-being, yet the bulk of the scientific evidence suggests that signaling through igf-i and insulin receptors is related to a shortened life span in adults. 33 indirect data have supported the concept that igf-i may be atherogenetic because it can induce vascular smooth muscle cell proliferation in vitro. 34 thus, igf-i has been considered a promoter of arterial obstructive lesions. 35 exercise training, s-klotho, and igf-i in healthy subjects regular participation in physical activity and/or exercise training programs can minimize the physiological alterations that occur during aging and may contribute to improvements in health and well-being. 36 when elite athletes engaging in various sports are analyzed, their mortality is lower than that of the general population. 37 thus, longterm moderately vigorous and vigorous exercise training are associated with increased survival rates in specific groups of athletes and cad patients. 38 there are several studies proving the definitive role of lifelong physical activity, which can be engaged in at any age, 39 even by those in their 80s or 90s. 40,41 . accordingly, regular aerobic exercise promotes older adult health and disease prevention. 36 endurance exercise like biking, walking, swimming, and running results in longer life expectancy compared to anaerobic exercise like power lifting. 42 the limited data on humans to explore the association between exercise and sklotho drove others to explore the relationship between exercise training and plasma s-klotho levels in mice. 43,44 as a first step to probe the connection between physical activity, age, and klotho expression, schefer et al. 43 quantified the effect of an acute exercise bout on circulating klotho levels in mice. it revealed that acute aerobic exercise significantly increased the circulating klotho levels. avin et al. 9 evaluated in young and older sedentary women the change in circulating klotho levels before and after completion of an acute aerobic exercise bout. it suggested that circulating klotho levels are upregulated in response to an acute exercise bout but the response may be dependent on fitness level. saghiv et al. 45 tested the hypothesis that long-lasting aerobic exercise training could prevent the age-associated reduction in s-klotho serum levels and increases igf-i levels, in 30 healthy sportsmen: 15 young aerobically well-trained elite athletes and 15 aerobically well-trained master athletes. this study demonstrated that circulating sklotho levels are similar for young healthy well-trained elite runners and elite master athletes. this suggests that the response of s-klotho depends on the aerobic fitness level. 9,42 in addition, levels of s-klotho were significantly higher in both groups, when compared with age-matched untrained subjects as reported earlier by lee et al., 46 suggesting that long-lasting aerobic training may be appropriate for mechanistically probing the role of physical activity on s-klotho expression. the human population from birth to 91 years screened previously by enzyme-linked immunosorbent assay (elisa) revealed that the level of s-klotho declines was with human physiological aging. 19 on the other hand, elderly with greater aerobic capacity have longer life expectancies compared to inactive people. 47 there are several studies proving the definitive role of lifelong physical activity, which can be initiated at any age. 39 compared to sedentary young and old subjects, in the elite aerobictrained young runners and master athletes, s-klotho levels are markedly elevated, while igf-i levels decreased. 48 igf-i is generally thought to be associated with anabolism and well-being, 49 yet, signaling through igf-i and insulin receptors is negatively related to adults. 50 a meta-analysis study indicated that increased circulating concentrations of igf-i are associated with increased risks of colorectal, prostate, and premenopausal breast cancers. 51 another study on the association between s-klotho serum levels and igf-i levels in regular young adults and elderly subjects trained aerobically for their health and well-being 45 demonstrated that following aerobic training in young and elderly subjects circulating s-klotho levels were found to be significantly higher compared to their untrained counterpartners. the cross-sectional study findings in young and aged individuals suggest that circulating s-klotho levels increase in response to long-lasting aerobic exercise training and that the response depends on fitness level. a similar increase in circulating s-klotho is also observed in response to an acute exercise in young and old mice, suggesting that this may be a good model for mechanistically probing the role of physical activity on klotho expression in mammals. 9 while in the aerobic-trained young runners and master athletes, s-klotho levels were markedly elevated, igf-i levels were decreased compared with sedentary young and old subjects. 48 the comparative analysis of biochemical indices measured showed that the long-lasting aerobic exercise training causes the significant decrease in igf-i concentrations, while no differences were noted in untrained subjects. this finding on the reduced igf-i clarifies the results of a previously reported study 50 that it was impossible to determine whether exercise affects igf levels. it seems that important methodological differences among studies, as well as concerns about study quality, limit the ability to draw firm conclusions in that abovementioned study. a metaanalysis indicated that increased circulating concentrations of igf-i binding protein 3 (igfbp-3) are associated with increased risks of colorectal, prostate, and premenopausal breast cancers. 51 thus, it appears that s-klotho is a protein that inhibits igf-i and insulin receptor and igf-ir signaling by inhibiting tyrosine phosphorylation of both receptors and their downstream signaling proteins. 52 saghiv et al. 3 anaerobic bouts can be limited by lactic acid levels in the blood and active muscles. it is characterized by exposing the subjects to a very high degree of sudden strenuous all-out exercise. thus, the increase of igf-i levels in the blood 53 was primarily due to a substantial major increase in plasma catecholamine concentrations. 54 in mice, anaerobic exercise bouts increase inhibition of weight gain and growth rate, which may result from exercise intensity and duration or frequency. 55 while an association between aerobic exercise and s-klotho expression has been previously suggested from longitudinal cohort studies, 19 a direct relationship between circulating s-klotho and anaerobic exercise training has been recently investigated. 45 it revealed that levels of s-klotho in aerobic-trained sportsmen were markedly higher compared to those measured in the anaerobic sprinters. these findings on long-lasting anaerobic exercise training suggest that circulating s-klotho levels in sprinters are similar to those of sedentary young adult males 56 and that the response depends on aerobic fitness level. 42 following long-lasting aerobic exercise training, s-klotho levels were markedly elevated and igf-i levels reduced in the aerobic-trained sportsmen, while in the anaerobically trained sprinters, s-klotho levels were significantly lower and had higher igf-i. in the anaerobically trained athletes, the levels of s-klotho and igf-i were similar to those reported previously for sedentary young adults. 57 the reduced s-klotho levels in the anaerobic sprinters may be associated in later life with increased mortality, increased rate of cardiovascular disease, and disability in daily living activities. 58,59 a recent study on well-trained elite anaerobic sprinters tested on an aerobic bout 60 revealed a positive relationship between circulating plasma s-klotho concentration and acute aerobic bout lasting 60 min, while the igf-i levels reduced. however, in long-lasting well-trained elite aerobic athletes with high levels of circulating s-klotho to begin with, an acute aerobic bout induced a slight increase in plasma s-klotho concentration and a significant decrease in igf-i levels. it seems that the increase in the plasmas s-klotho concentration, after 60 min of an aerobic bout in the anaerobictrained athletes, might be responsible for the decreased igf-i. exercise, s-klotho, and igf-i in aging the primary aging process occurs both independent of life style and in the absence or presence of disease. 51,61 aging is a complex multifactorial process that not only involves the natural processes of aging but also the increased risk of different diseases—coronary heart disease, diabetes, and cancer. 62 studies have demonstrated that when researchers adjust the genes in certain mice, yeast cells, and other organisms, they can almost double the life span of these mice. therefore, successful aging is a function of both genetic and environmental factors. 63,64 multiple age-related structural and functional changes are involved in skeletal, cardiac, and oxygen delivery-extraction ability during the human senescence, resulting in a significant decline in aerobic capacity and in the expression of a gene located on chromosome 13, klotho gene a suppressor of the aging phenomena as well as the circulation of s-klotho proteins. 65,66 the klotho protein deficiency causes premature aging. as an aging suppressor, klotho is an important molecule in aging processes and its overexpression results in longevity. due to many reasons, the insulin/igf-i has been considered as a key pathway in aging research. because klotho induces igf-i and insulin resistance, these findings appear to contradict the previous evidence of increased life span of dwarf mice with reduced igf-i and insulin levels and enhanced insulin sensitivity. however, activation of signaling molecules downstream from igf-i and insulin receptors is reduced in both klotho and dwarf mice, suggesting common mechanisms of delayed aging. 67 s-klotho inhibits igf-i and insulin receptor, igf-ir signaling, by inhibiting tyrosine phosphorylation of both receptors and their downstream signaling proteins, 68 thus, increasing longevity in mice. 69 moreover, it has been shown that the blockade of igf-i signaling induced by s-klotho increased resistance to oxidative stress, thereby improving survival. 70 because of genetic factors, work capacity decreases with aging regardless of the lifestyle. consequently, the maximal oxygen uptake decreases. such consequences contribute to the geriatric syndrome of frailty, thereby severely limiting the function, quality of life, and longevity. 71 apart from genetic endowment, an individual must also interact with environmental factors associated with longevity. one of these factors includes maintaining high level of physical activity. 72 chronic endurance training will attenuate the decline in maximal oxygen uptake associated with age. 61,73 in addition, antiaging effects have also been ascribed to aerobic exercise training. 67 an association between muscle function and s-klotho expression has been previously suggested from longitudinal cohort studies. 23 a direct relationship between circulating s-klotho and igf-i following aerobic exercise training has been recently investigated. 45 this study demonstrated that circulating s-klotho levels are similar for young healthy well-trained elite runners and master athletes. it seems that the response depends on aerobic fitness level. 42 in addition, levels of s-klotho were significantly higher in both groups when compared with untrained subjects as reported earlier. 46 since aging is an independent risk factor for age-related diseases and mortality, there are growing efforts in gerontology research to slow aging and extend healthy life span. the population aged from birth to 91 years screened previously by elisa revealed that the level of s-klotho declines with aging. 19 on the other hand, at any age, elderly individuals with aerobic capacity have longer life expectancies compared to inactive people. 39,47 4 journal of circulating biomarkers while in the aerobic-trained young runners and master athletes, the s-klotho levels were markedly elevated and the igf-i levels were decreased compared with sedentary young and old subjects. 48 the scientific evidence suggests that igf-i signaling through igf-i and insulin receptors is related to a shortened life span in adults. 50 in addition, a meta-analysis study indicated that increased circulating concentrations of igf-i are associated with increased risks of colorectal, prostate, and premenopausal breast cancers. 51 exercise, s-klotho, and igf-i in cad cad is a highly prevalent disease in the general adult population and is a major cause of death. several clinical studies have suggested that klotho gene exerts strong cardioprotective effects. s-klotho has been proposed as a key regulator of the development of cardiovascular disease. in the few published clinical studies, an association between low levels of s-klotho and the occurrence and severity of cardiovascular disease have been reported as well as a reduction in cardiovascular risk when levels were high. 74 earlier, a relationship between low levels of s-klotho and the occurrence and severity of cad as well as a reduction in cardiovascular risk when they are high are observed. 74 diverse studies suggest that alterations in the levels of this molecule may be associated with pathophysiological abnormalities that result in increased cardiovascular risk. 75 this protein is related to the attenuation of vascular calcification as well as prevention of cardiac hypertrophy. for instance, klotho gene has been shown to protect against vascular calcifications in rodent models of cad. while in humans without cad, higher s-klotho levels have been related to a lower incidence of mortality and cad, 68,75 while low s-klotho levels have been associated with increased arterial stiffness in chronic kidney disease patients. 63 additionally, several experimental studies indicate that s-klotho facilitates the maintenance of vascular homeostasis. s-klotho improves endothelial dysfunction through promotion of nitric oxide (no) production and mediates anti-inflammatory and antiaging effects such as suppression of adhesion molecules expression, attenuation of nuclear factor-k b or inhibition of wnt signaling, thus, limits wnt-mediated cellular senescence. 76 aerobic exercise training increases resistance vessel sensitivity and maximal responsiveness to adenosine. this has been confirmed in dogs and miniature swine in vivo. 77 aerobic exercise and klotho gene expression could reduce the risk of cardiovascular events in patients with prior cad, it may decrease the risk of mortality, 68,78 incidence, and severity of cardiac events. 79,80 furthermore, in patients with cad, exercise training improves endotheliumdependent vasodilatation both in epicardial coronary vessels and in resistance vessels. 81 patients with significant cad present lower concentrations of s-klotho as well as reduced expression of klotho gene in the vascular wall. the exonic variant kl-vs was associated with the incidence of atherosclerotic vascular disease and cad. 75 saghiv et al. 82 assessed the effect of chronic aerobic exercise training on s-klotho serum levels and igf-i levels in cad patients following long-lasting aerobic exercise training. the study demonstrated that in aerobically trained cad patients, circulating s-klotho levels were significantly higher, while igf-i were significantly lower compared to untrained cad patients and their healthy untrained counterpartners. in trained cad patients, these findings suggest that in cad patients, circulating s-klotho levels are augmented in response to long-lasting aerobic exercise training. the reduced s-klotho levels observed in the untrained cad patients 82 was similar to other premature vascular aging diseases, such as hypertension or diabetes mellitus. 75 further support for a direct role of klotho gene in vascular homeostasis comes from in vitro studies showing endogenous expression of klotho gene in human vascular smooth muscle cells. 83 conclusions inflection of klotho expression through aerobic exercise training represents an interesting relationship that may contribute to the explanation of the antiaging and anti-cad effects of long-lasting aerobic activity. both are factors that may promote upgrading capacities of the elderly, cad patients and healthy young adult subjects. accordingly, a long-lasting aerobically trained individual is associated with decreased risk factors and increased s-klotho that clearly counteracts the action of igf-i. following 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independently associated with cardiovascular disease in a population of dialysis patients. bmc nephrology 2014; 11: 197. 8 journal of circulating biomarkers << /ascii85encodepages false /allowtransparency false /autopositionepsfiles true /autorotatepages /none /binding /left /calgrayprofile (gray gamma 2.2) /calrgbprofile (srgb iec61966-2.1) /calcmykprofile (u.s. web coated \050swop\051 v2) /srgbprofile (srgb iec61966-2.1) /cannotembedfontpolicy /warning /compatibilitylevel 1.4 /compressobjects /off /compresspages true /convertimagestoindexed true /passthroughjpegimages false /createjobticket false /defaultrenderingintent /default /detectblends true /detectcurves 0.1000 /colorconversionstrategy /leavecolorunchanged /dothumbnails false /embedallfonts true /embedopentype false /parseiccprofilesincomments true /embedjoboptions true /dscreportinglevel 0 /emitdscwarnings false /endpage -1 /imagememory 1048576 /lockdistillerparams true /maxsubsetpct 100 /optimize true /opm 1 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healthy donors and cancer patients not detected in analysis of standard immune cell types original research article lauren m. lepone1#, renee n. donahue1#, italia grenga1, simon metenou1, jacob richards1, christopher r. heery1, ravi a. madan2, james l. gulley2 and jeffrey schlom1* 1 laboratory of tumor immunology and biology, center for cancer research, national cancer institute, national institutes of health, bethesda, md, usa 2 genitourinary malignancies branch, center for cancer research, national cancer institute, national institutes of health, bethesda, md, usa #these authors contributed equally to this work *corresponding author(s) e-mail: js141c@nih.gov received 09 october 2015; accepted 29 january 2016 doi: 10.5772/62322 © 2016 author(s). licensee intech. this is an open access article distributed under the terms of the creative commons attribution license (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. abstract recent advances in human immunology have led to the identification of novel immune cell subsets and the biological function of many of these subsets has now been identified. the recent us food and drug administration approval of several immunotherapeutics for the treatment of a variety of cancer types and the results of ongoing immunotherapy clinical studies requires a more thorough interrogation of the immune system. we report here the use of flow cytometry-based analyses to identify 123 immune cell subsets of peripheral blood mononuclear cells. the use of these panels defines multiple differences in younger (< 40 years) vs. older (≥ 40 years) individuals and between aged-matched apparently healthy individuals and meta‐ static cancer patients, aspects not seen in the analysis of the following standard immune cell types: cd8, cd4, natural killer, natural killer-t, regulatory t, myeloid derived suppressor cells, conventional dendritic cells (dcs), plasmacytoid dcs and b cells. the use of these panels identifying 123 immune cell subsets may aid in the identi‐ fication of patients who may benefit from immunotherapy, either prior to therapy or early in the immunotherapeutic regimen, for the treatment of cancer or other chronic or infectious diseases. keywords peripheral blood mononuclear cells, multicol‐ our flow cytometry, cancer, age 1. introduction with the recent us food and drug administration appro‐ vals of immunotherapeutics such as the checkpoint inhibitor anti-cytotoxic t lymphocyte-associated protein-4 1j circ biomark, 2016, 5:5 | doi: 10.5772/62322 (ctla-4) and anti-programmed cell death-1 (pd-1)/ programmed cell death ligand-1 (pd-l1) monoclonal antibodies (mabs), and the sipuleucel-t prostate cancer vaccine, as well as results emerging from ongoing clinical studies with other immunotherapeutics, immunotherapy is emerging as a modality for many cancer types and stages. it has long been believed that an individual’s immune system can play a role in both the development and control of cancer. it is known that the tumour itself can produce a spectrum of molecules such as immunomodulating cytokines, which can alter the patient’s immune system. [1] moreover, the incidence rates for many cancers increase with age and prior studies have shown that the immune system in older individuals (usually ≥ 65 years) differs from that of younger individuals. [2, 3] most prior studies have evaluated changes in an individu‐ al’s immune system by quantifying so-called “standard parental immune cell types” in the periphery such as cd4, cd8, regulatory t cells (tregs), b cells, natural killer (nk) cells, nk-t cells, conventional dendritic cells (cdcs) and plasmacytoid dcs (pdcs), and myeloid derived suppres‐ sor cells (mdscs). recent advances in cellular immunology have identified numerous subsets within each of the above immune cell types via the identification of new markers on immune cells and the use of polychromatic flow cytometry; prior studies have also identified the function(s) of many of these immune cell subsets. [4, 5] in the studies reported here, we employed mabs directed against immune cell markers and multi-laser flow cytom‐ etry analyses to identify 123 unique immune cell subsets in the peripheral blood of individuals. since the incidence of cancer rises sharply at the age of 40 and more cancers are being seen recently in younger individuals [6, 7], we have investigated whether changes exist in peripheral immune cell subsets between apparently healthy individuals < 40 and those ≥ 40 years of age; several immune cell subsets were found to be statistically different between these two age groups. we also found that numerous peripheral immune cell subsets can be differentiated between agematched healthy individuals and patients with a range of advanced human carcinomas. moreover, we have identi‐ fied differences among immune cell subsets expressing molecules on their surface that are the targets of existing checkpoint inhibitor therapies such as ctla-4, pd-1 and pd-l1, as well as other potential targets for which immu‐ notherapies are being developed. to further investigate the differences in peripheral immune cell subsets between healthy donors and advanced cancer patients, microarray analysis was performed on an additional small cohort of healthy donors and cancer patients. while gene pathway analysis demonstrated the regulation of multiple pathways previously associated with cancer, several genes implicat‐ ed in the regulation of immune cells, including mdscs and b cells, were also identified as differentially expressed between advanced cancer patients and healthy donors, corroborating the flow cytometry analysis. 2. materials and methods 2.1 healthy donors and cancer patients the flow cytometry analysis included 11 healthy donors under the age of 40, 15 healthy donors over the age of 40 and 30 patients with a variety of metastatic solid tumours over the age of 40. the median age of the healthy donor group < age 40 was 26 (range 18-31), with six males and five females. the median age of the healthy donor group > age 40 was 56 (range 46-78), with 12 males and three females. peripheral blood mononuclear cells (pbmcs) from the healthy donors were obtained from the nih clinical center blood bank (nct00001846), as previously described. [8] the median age of the cancer patients was 64 (range 42-77), with 17 males and 13 females. the cancer patients all had metastatic solid tumours and were enrolled in a phase i clinical trial (nct01772004), with pbmcs that were examined in this study obtained prior to the initiation of therapy. the national cancer institute institutional review board approved the trial procedures and informed consent was obtained in accordance with the declaration of helsinki. patients had 13 different types of cancer including adrenocortical (n=2), breast (n=3), chordoma (n=1), gastrointestinal (gi) (n=6), lung (n=1), medullary thyroid (n=1), mesothelioma (n=3), neuroendocrine (n=1), ovarian (n=1), pancreatic (n=6), prostate (n=1), renal cell (n=3) and spindle cell (n=1) cancer, and the median number of prior anti-cancer therapies was 3 (range 1-15). in an additional cohort of samples assessed by microarray, pbmcs were obtained from five healthy donors (median age: 54, three male, two female) from the nih clinical center blood bank, as well as four patients with advanced gi cancer (median age: 56.5, two male, two female) enrolled in a phase i study at the nci (nct00088413) [9]; pbmcs used in this study were isolated prior to treatment. 2.2 antibodies and flow cytometry multicolour flow cytometry was performed on frozen pbmcs as previously described. [10] one vial of pbmcs was thawed per cancer patient or healthy donor. cells were counted and plated in five different wells per subject in a 96-well plate with one million pbmcs per well. each of the five wells was used to stain a different panel with a maximum of 11 colours per panel in each well. staining was performed using five panels (supplemental table 1) to identify markers involved in pd-1 signalling (panel 1) and in subsets of cd4+ t cells, cd8+ t cells and b cells (panel 2), tregs (panel 3), nks, nk-t, cdcs and pdcs (panel 4), and mdscs (panel 5). using the outlined gating strategy (figure 1), these five staining panels with up to 11 antibod‐ ies per panel identified a total of 123 peripheral immune cell subsets (supplemental table 2), which included nine parental immune cell types and 114 subsets related to maturation and function within the parental types. opti‐ mal amounts of antibodies for staining were determined by 2 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 titration experiments. briefly, one million pbmcs per test were incubated for 15 minutes at 4°c with 2 µl of human trustain fcx (biolegend, san diego, ca) and live dead fixable stain blue (invitrogen, waltham, ma). surface antibodies were added for 30 minutes at 4° c. cells were then washed, fixed and permeabilized using the intracel‐ lular fixation and permeabilization buffer set (ebioscience, san diego, ca) and stained with intracellular antibodies for 30 minutes at room temperature. samples were ac‐ quired on a bd lsrii flow cytometer (bd biosciences, san jose, ca) equipped with four lasers (uv, violet, blue and red; configuration and filter sets of lsr ii listed in supple‐ mental figure 1) and analysed using flowjo v9.7 for macintosh (treestar, ashland, or). the gating strategy identified 123 peripheral immune cell subsets from the five staining panels that each contained up to 11 markers per panel (figure 1), with non-viable cells excluded and negative gates set based on fluorescence minus one controls. all values were reported as a percentage (%) of pbmcs in order to help eliminate the bias that could occur in the smaller populations with fluctuations in leukocyte subpopulations. [11] 2.3 microarray total rna was isolated from pbmcs using the qiagen rnaeasy plus minikit (valencia, ca) according to the manufacturer’s instructions and quality-checked on an agilent bioanalyzer (santa clara, ca). all samples used for microarray analysis had an rna integrity number >9; 100 ng of rna was reverse transcribed and amplified using an ambion wt expression kit (austin, tx), following the manufacturer’s suggested protocols. sense strand cdna was fragmented and labelled using an affymetrix wt terminal labelling kit (santa clara, ca). four replicates of markers icos+ cd4+ t cells: pd-l1, pd-1 markers in treg: cd45ra, cd49d, ctla-4, icos, pd-1, pd-l1 cd4+ t cells icos+ cd4+ t cells treg markers b cells: ctla-4, pd-l1, pd-1, tim-3 naive emraem cm naive emraem cm cd8+ t cells cd4+ t cells b cells markers in total and memory cd8: ctla-4, pd-l1, pd-1, tim-3 markers in total and memory cd4: ctla-4, pd-l1, pd-1, tim-3 cd8+ t cells cd4+ t cells markers in cd8: batf, eomes, tbet, tcr markers in cd4: batf, eomes, tbet, tcr figure 1. a. b. 1 c. continued on page 4. 3lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types each group were hybridized to affymetrix human gene st 2.0 genechip in an affymetrix hybridization oven at 45°c, at 60rpm for 16 hours. washing and staining were per‐ formed on an affymetrix fluidics station 450 and scanned on an affymetrix genechip scanner 3000. data were collected using affymetrix agcc software. 2.4 statistical analyses statistical analyses for flow cytometry data were per‐ formed using graphpad prism 6 (graphpad software, la jolla, ca). all p-values were calculated using the mannwhitney test. in view of the large number of tests per‐ formed, p-values were adjusted using holm’s method (step-down bonferroni) to account for the increased probability of type i errors (false positive) that occur when multiple outcome measures are assessed. [12] adjustment was made for the number of subsets with a frequency above 0.01% of pbmcs (n=9 for standard subsets, n=29 for subsets in cd4+ t cells, n=25 for cd8+ t cells, n=5 for tregs, n=14 for nk cells, n=3 for nk-t cells, n=4 for b cells, n=2 for cdcs, n=3 for pdcs and n=15 for mdscs). subsets with a poten‐ tially biologically relevant change were defined as subsets with an adjusted p<0.05, the median of groups showing a > 50% difference, and a frequency > 0.01% of pbmcs. statistical and clustering analysis for the microarray experiment was performed with partek genomics suite software (st. louis, mo) and employing a rma normali‐ zation algorithm. differentially expressed genes were identified via anova analysis. genes that were upor down-regulated more than 1.5-fold and with a p<0.05 were considered significant. significant genes were analysed for the enrichment for pathways using ingenuity pathway analysis software (redwood city, ca). 3. results 3.1 differences in peripheral immune cell subsets with age in healthy donors using multiparameter flow cytometry, a total of 123 peripheral immune cell subsets were examined, which included the nine standard parental immune cell types – cd4+ and cd8+ t cells, tregs, b cells, nk and nk-t cells, cdcs, pdcs and mdscs – and 114 subsets of these cell types relating to maturation and function (table 1). as the risk of cancer rises at age 40 [6, 7], donors were separated into younger and older groups, using this age as a cut-off. pbmcs were assayed from 11 healthy donors under the age of 40 and 15 healthy donors over the age of 40 (figure 2a). compared to the younger group, the older group had a significantly lower absolute lymphocyte count (alc) (p=0.0032, figure 2b). no statistical differences were figure 1 (continued). d. e. 2 markers in pdc: cd83, pd-l1, pd-1, tim-3 markers in cdc: cd83, pd-l1, pd-1, tim3 pdc cdc markers in nk-t: pd-l1, pd-1, tim-3 intermediate active immatureunconventional mature markers in total and nk subsets: pd-l1, pd-1, tim-3 nk-t nk lympho/ mono nonlympho/ mono monocytic lineage negative granulocytic mdsc markers in total and mdsc subsets: pd-l1, pd-1, cd16 figure 1. gating strategy of five staining panels to identity 123 peripheral immune cell subsets by flow cytometry. one vial of frozen peripheral blood mononuclear cells (pbmcs) per subject was thawed and then stained using 30 unique markers in five separate immune flow cytometry panels to identify a total of 123 peripheral immune cell subsets. each panel contained up to 11 markers. panel 1 (a) identified markers in cd8+ and cd4+ t cells involved in pd-1 signalling. panel 2 (b) identified the parental cell types of cd4+ and cd8+ t cells and b cells, as well as markers involved in function and maturation. panel 3 (c) identified icos+ cd4+ t cells and the parental cell type of tregs, as well as markers involved in function. panel 4 (d) identified the parental cell types of nk, nk-t, cdc and pdc, as well as markers involved in maturation and function. panel 5 (e) identified the parental cell type of mdsc, as well as markers involved in maturation and function. samples were collected on an lsr ii flow cytometer equipped with uv, red, blue and violet lasers, and analysed using flowjo, with gating set using fluorescence minus one controls. 4 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 evident when subsets were examined by age decade within the groups or by gender (data not shown). differences in peripheral immune cell subsets were identified as significant and relevant if the adjusted holm p-value was < 0.05, if medians of the group were at least 50% different and if the subset had a frequency of above 0.01% of pbmcs. using these criteria, of the nine standard immune cell types, seven were unchanged and two cell types (cd8+ t cells, nk cells) were significantly changed with age (table 2). healthy donors over the age of 40 had on average 60% less total cd8+ t cells compared to those under the age of 40 (median % of pbmcs = 8 in old and 19 in young, p=0.0009, figure 2c). older healthy donors, on the other hand, had on average 85% more nk cells com‐ pared to younger healthy donors (median % of pbmcs = 10 in older and 5 in younger, p=0.0009, figure 2d). of the 114 subsets relating to maturation and function within the standard cell types, 12 subsets were significantly different in older healthy donors compared to younger individuals (table 3a). the most notable decreases in subsets relating to maturation and function in healthy donors over age 40 were evident in cd8+ t cell subsets; these included on average 70% fewer pd-l1+cd8+ t cells (p=0.0080, figure 3a), 55% fewer ctla-4+cd8+ t cells (p=0.0480, figure 3b) and 60% fewer t cell immunoglobulin and mucin domain-3 (tim-3)+cd8+ t cells (p=0.0272, figure 3c). the markers pd-l1, ctla-4 and tim-3 are inhibitory receptors involved in immune checkpoint pathways and are increased upon t cell activation and differentiation. [13] donors over 40 also had decreases in subsets relating to maturation, with lower frequencies of naïve (p=0.0025, figure 3d) and central memory (cm) (p=0.0063, figure 1. cd4+ t cells: helper t lymphocytes (32 subsets) 2. cd8+ t cells: cytotoxic t lymphocytes (29 subsets) • markers of pd-1 pathway and t cell activation (in cd4 and cd8): – eomes: activation – tcr: activation – tbet: activation – batf: activation/exhaustion • maturation status of t cells (in cd4 and cd8): ‒ naïve: cd45ra+ ccr7+ ‒ central memory (cm): cd45raccr7+ ‒ effector memory (em): cd45raccr7‒ terminal (emra): cd45ra+ ccr7• t cell markers (in cd4 and cd8): – ctla-4: inhibition – pd-1: activation/inhibition – pd-l1: activation/cross-inhibition – tim-3: inhibition – icos: activation (only on cd4) 3. tregs: regulatory t lymphocytes (cd4+ cd25+ foxp3+ cd127-) (7 subsets) – cd45ra: tregs highly expandable in vitro – ctla-4: treg suppression – cd49d-: suppressive tregs – icos: treg suppression – pd-1: activation/inhibition – pd-l1: cross-inhibition 4. b cells: cd19+ (5 subsets) – ctla-4: inhibition – tim-3: inhibition – pd-1: activation/inhibition – pd-l1: cross-inhibition 5. nk: natural killer cells (cd56+ cd3-) (20 subsets) – cd16+ cd56dim: mature, lytic – cd16+ cd56br: functional intermediate, lytic and cytokine production – cd16cd56br: immature, cytokine production, abundant in placenta – cd16cd56dim: non-lytic, non-cytokine production – tim-3: activation – pd-1: activation/inhibition – pd-l1: cross-inhibition 6. nk-t: cd56+ cd3+ (4 subsets) – tim-3: activation – pd-1: activation/inhibition – pd-l1: cross-inhibition 7. cdcs: conventional dendritic cells (dcs) (cd3-cd56-cd1c+cd303-) (5 subsets) 8. pdcs: plasmacytoid dcs (cd3-cd56-cd1c-cd303+) (5 subsets) • markers of dc activation ‒ cd83: activation ‒ tim-3: inhibition ‒ pd-1: activation/inhibition ‒ pd-l1: cross-inhibition 9. mdscs: myeloid derived suppressor cells (cd11b+ hla-drlow/cd33+) (16 subsets) – cd14: common myeloid marker – cd15: granulocyte marker – cd16: immature mdscs – pd-1: activation/inhibition – pd-l1: cross-inhibition 1 frozen pbmcs were thawed then stained using 30 unique markers in 5 immune flow cytometry panels to identify a total of 123 peripheral immune cell subsets. samples were collected on an lsr ii flow cytometer equipped with uv, red, blue, and violet lasers, and analyzed using flowjo with gating set using fluorescence minus one controls. nine standard immune cell types as well as 114 additional subsets relating to maturation and function were compared between healthy donors under and over the age of 40, and between patients with metastatic cancer and age-matched healthy donors. -------batf, basic leucine zipper transcription factor atf-like; ctla-4, cytotoxic t lymphocyte-associated protein-4; eomes, eomesodermin; foxp3, forkhead box p3; hla, human leukocyte antigen; icos, inducible t cell costimulator; pbmcs, peripheral blood mononuclear cells; pd-1, programmed cell death-1; pd-l1, programmed cell death ligand-1; tbet, t box expressed in t cells; tcr, t cell receptor; tim-3, t cell immunoglobulin and mucin domain-3. table 1. flow cytometry analysis of parental immune cell types in pbmcs using 30 unique markers to identify 123 subsets 5lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types 3e) cd8+ t cells. the only subset that showed a trend (p=0.038) of increase in healthy donors over age 40 was tim-3+ b cells (figure 3f). tim-3 is an inhibitory marker often examined in t cells and nk cells, and only rarely figure 2. a. b. c. d. 1 age a g e age <40 age >40 0 20 40 60 80 100 p=0.0001 alc c o u n t x 1 0 3 p e r µ l age <40 age >40 0 1 2 3 4 p=0.0032 cd8 + t cells % p b m c age <40 age >40 0 10 20 30 p=0.0009 nk % p b m c age <40 age >40 0 5 10 15 20 p=0.0009 figure 2. standard parental immune cell types in healthy donors under and over the age of 40. a: healthy donors included in this analysis were separated as younger (age less than 40 years, n=11) and older (age greater than 40 years, n=15). b: absolute lymphocyte count (alc) of healthy donors under and over age 40. graphs display median age or alc with 25-75 percentiles. p-value was calculated using the mann-whitney test. cd8+ t cells (c) and natural killer (nk) cells (d) were different between healthy donors under and over age 40, as defined by an adjusted p<0.05, the median of groups showing a > 50% difference, and a frequency above 0.01% of peripheral blood mononuclear cells (pbmcs). graphs display median frequency as a percentage of pbmcs with 25-75 percentiles. p-value was calculated using the mann-whitney test and with holm adjustment made for multiple comparisons using the number of standard immune cell types with a frequency above 0.01% of pbmcs (n=9). table 2. standard parental immune cell types in healthy donors under and over age 40 1 a total of 9 standard parental immune cell types were analyzed. values are displayed as median % of pbmcs. pvalue was calculated with the mann whitney test with holm adjustment made for multiple comparisons using the number of standard immune cell types with frequency above 0.01% of pbmcs (n=9). differences were defined as subsets withan adjusted p<0.05, medians at least 50% different, and frequency above 0.01% of pbmcs. -------cdc, conventional dendritic cells; mdsc, myeloid derived suppressor cells; nk, natural killer; pbmcs, peripheral blood mononuclear cells; pdc, plasmacytoiddc; tregs, regulatory t cells. immune cell type age <40 age >40 p value direction of age >40 cd8+ t cells 19 8 0.0009 � nk 5 10 0.0040 � cd4+ t cells 34 29 0.8975 = tregs 1.4 1.5 0.9999 = b cells 11 16 0.1645 = nk-t 2.4 1.3 0.4116 = cdc 0.4 0.3 0.9999 = pdc 0.2 0.2 0.8975 = mdsc 5 4 0.9999 = table 2. standard parental immune cell types in healthy donors under and over age 40 6 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 described in minor populations of b cells [14]. total b cells were unchanged. other immune cell subsets also showed trends in differen‐ ces with age; trends were defined as those subsets having an unadjusted p-value < 0.01 (without a significant holm adjusted p-value), as well as at least a 50% difference in medians and a frequency of pbmcs above 0.01%. using these criteria, six additional subsets were found to have trends in differences with age (table 3b). while total cd4+ t cells did not change, the most notable trends were evident in several cd4+ t cell subsets expressing pd-1, which is an inhibitory receptor that is increased upon t cell activation [13]. healthy donors over the age of 40 had higher levels of pd-1 in total cd4+ t cells, inducible t cell co-stimulator (icos)+cd4+ t cells, effector memory (em) cd4+ t cells and cm cd4+ t cells (table 3b). thus, healthy donors over age 40 had lower levels of activation markers on cd8+ t cells, varied maturation status of cd8+ t cells and trends of higher activation markers in cd4+ t cells compared to healthy donors under age 40. 3.2 differences in peripheral immune cell subsets between patients with carcinoma and age-matched healthy donors pbmcs were evaluated for 30 patients with different types of metastatic solid tumours and 15 age-matched healthy donors, all over the age of 40 (p=0.1550, figure 4a). the alc was similar in both groups (p=0.1140, figure 4b). using the same criteria as described in the age analysis, seven of the standard parental immune cell types were similar and two (cd8+ t cells, b cells) were significantly different between cancer patients and healthy donors (table 4). surprisingly, cancer patients had on average 90% more cd8+ t cells compared to age-matched healthy donors (median % of pbmcs = 15 in patients and 8 in healthy donors, p=0.0168, figure 4c). on the other hand, cancer patients also had on average 50% less total b cells compared to healthy donors (median % of pbmcs = 8 in patients and 16 in healthy donors, p=0.0027, figure 4d). no notable differences were evident when subsets were compared between patients with different cancer types; however, this was likely due to the small number of patients within each indication that were examined (data not shown). of the 114 subsets related to maturation and function within the standard immune cell types, 23 were changed with cancer (table 5a). some of the notable increases seen in cancer patients included subsets of t cells expressing basic leucine zipper transcription factor, atf-like (batf), pd-l1 and ctla-4, which are inhibitory markers in‐ volved in immune checkpoint pathways and are in‐ creased upon t cell activation [13, 15]. while the percentage of cd4+ t cells was similar between cancer patients and healthy donors, cancer patients had 180% higher levels of batf+cd4+ t cells (p=0.0029, figure 5a). along with an increase in the percentage of cd8+ t cells, cancer patients also had 170% higher levels of pd-l1+cd8+ t cells (p=0.0025, figure 5b) and 165% higher levels of ctla-4+cd8+ t cells (p=0.0025, figure 5c). using the less stringent criteria as described above in the age analysis to identify subsets with trends, cancer patients showed naive cd8 + t cells % p b m c age <40 age >40 0 5 10 15 p=0.0025 cm cd8 + t cells % p b m c age <40 age >40 0.0 0.5 1.0 1.5 2.0 2.5 p=0.0063 tim-3 + b cells % p b m c age <40 age >40 0.00 0.05 0.10 0.15 p=0.0376 pd-l1 + cd8 + t cells % p b m c age <40 age >40 0.00 0.02 0.04 0.06 0.08 0.10 p=0.0080 ctla-4 + cd8 + t cells % p b m c age <40 age >40 0.00 0.05 0.10 0.15 0.20 0.4 0.5 p=0.0480 tim-3 + cd8 + t cells % p b m c age <40 age >40 0.0 0.2 0.4 0.6 0.8 p=0.0272 figure 3. a. b. c. d. e. 1 f. figure 3. differences in immune cell subsets related to maturation and function between healthy donors under and over the age of 40. a total of 114 immune cell subsets were analysed relating to maturation and function within the standard subsets. subsets were considered different if a subset had an adjusted p<0.05, the median of groups showing a > 50% difference, and a frequency above 0.01% for peripheral blood mononuclear cells (pbmcs). a-f: representative graphs are shown for notable subsets related to activation and maturation, with differences between healthy donors under and over the age of 40 indicated. graphs display median frequency as a percentage of pbmcs with 25-75 percentiles. p-value was calculated using the mann-whitney test, with holm adjustment made for multiple comparisons using the number of subsets within each standard subset with a frequency above 0.01% of pbmcs (n=29 for cd4+ t cells, 25 for cd8+ t cells, 5 for tregs, 14 for natural killer cells (nk), 3 for nk-t cells, 4 for b cells, 2 for conventional dendritic cells (cdcs), 3 for plasmacytoid dcs (pdcs) and 15 for myeloid derived suppressor cells (mdscs)). 7lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types increases in t box expressed in t cells (tbet+)cd8+ and eomesodermin (eomes)+cd8+, as well as tbet+cd4+ cells (table 5b). these subtypes are known to be increased upon activation and differentiation. [16] cancer patients also had notable increases in subsets involving maturation and function in suppressive immune cells. while the percentage of tregs and mdscs was similar between cancer patients and healthy donors, cancer patients had higher levels of a treg subset that expressed ctla-4 (p=0.0280, figure 5d), which is a suppressive marker for these regulatory cells [17], as well as increased levels of pd-l1+ mdscs (p=0.0088, figure 5e), a marker that has been implicated in suppressive function [18]. cancer patients also had higher levels of granulocytic mdscs (gmdscs) (p=0.0015, figure 5f), which have been found to be more suppressive than monocytic mdscs (mmdscs). [19] while the percentage of pdc levels was also similar between cancer patients and healthy donors, cancer patients also had higher levels of pd-l1+ pdcs (p=0.0003, figure 5g), which can have immunosuppressive and tolerogenic properties [20]. cancer patients, on the other hand, had lower levels of pd-l1+ b cells (p=0.0009, figure 5h), a marker that is upregulated upon b cell activation [21]. thus, patients with advanced cancer had elevated levels of activation markers involved in immune check‐ point pathways in both cd4+ and cd8+ t cells, higher tregs and mdscs with a suppressive phenotype, and differences in pd-l1 expression on antigen-presenting cells (apcs) compared to age-matched healthy donors. 3.3 differences in gene expression profiles between cancer patients and age-matched healthy donors to determine whether changes in immune cell subsets seen at the protein level could be corroborated at the gene level, a study was performed in which gene expression profiling was performed in pbmcs from patients with advanced gi table 3. differences in immune cell subsets relating to maturation and function between healthy donors under and over age 40 1 a. immune cell type age <40 age >40 p value direction of age >40 pd-l1 + cd8 + t cells 0.051 0.016 0.0080 � ctla-4 + cd8 + t cells 0.099 0.045 0.0480 � tim-3 + cd8 + t cells 0.380 0.152 0.0272 � tcr + cd8 + t cells 19 8 0.0025 � naïve cd8 + t cells 6.7 1.3 0.0025 � pd-l1 + naïve cd8 + t cells 0.021 0.004 0.0025 � ctla-4 + naïve cd8 + t cells 0.038 0.008 0.0080 � tim-3 + naïve cd8 + t cells 0.105 0.026 0.0090 � cm cd8 + t cells 0.862 0.417 0.0063 � tim-3 + cm cd8 + t cells 0.019 0.008 0.0025 � emracd4 + t cells 5 2 0.0145 � tim-3 + b cells 0.029 0.047 0.0376 � immune cell type age <40 age >40 unadjusted p value holm adjusted p value trend of age >40 pd-1 + cd4 + t cells 2 4 0.0054 0.1404 � pd-1 + icos + cd4 + t cells 0.550 0.829 0.0020 0.0560 � pd-1 + em cd4 + t cells 2 3 0.0077 0.1925 � pd-1 + cm cd4 + t cells 0.108 0.252 0.0025 0.0675 � mature nk 3 7 0.0064 0.0896 � emra cd8 + t cells 2.9 1.3 0.0077 0.1155 � b. a total of 114 subsets were analyzed relating to maturation and function within the standard subsets. a: table displays subsets that were different in healthy donors under and over age 40, as defined by an adjusted p<0.05, medians at least 50% different, and frequency above 0.01% of pbmcs. values represent median % of pbmcs, and p-value was calculated with the mann whitney test with holm adjustment made for multiple comparisons using the number of subsets within each standard subset with frequency above 0.01% of pbmcs (n=29 for cd4+ t cells, 25 for cd8+ t cells, 5 for tregs, 14 for nk cells, 3 for nk-t cells, 4 for b cells, 2 for cdcs, 3 for pdcs, 15 for mdscs). b: table displays subsets with notable trends between healthy donors under and over age 40, as defined by unadjusted p< 0.01, medians at least 50% different, and frequency above 0.01% of pbmcs. values represent median % of pbmcs. -------cdc, conventional dendritic cells; cm, central memory; ctla-4, cytotoxic t lymphocyteassociated protein-4; em, effector memory; emra, terminally differentiated effector memory; icos, inducible t cell co-stimulator; mdsc, myeloid derived suppressor cells; nk, natural killer; pbmcs, peripheral blood mononuclear cells; pdc, plasmacytoid dc; pd-1, programmed cell death-1; pd-l1, programmed cell death ligand-1; tcr, t cell receptor; tim-3, t cell immunoglobulin and mucin domain-3; tregs, regulatory t cells. table 3. differences in immune cell subsets relating to maturation and function between healthy donors under and over age 40 8 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 cancer (n=4) and age-matched healthy donors (n=5). a total of 157 genes were significantly different (p-value <0.05 and fold change > |1.5|) between the two groups (figure 6a). pathway analysis demonstrated that genes were localized figure 4. a. c. d. b. 1 age a ge patients healthy donors 0 20 40 60 80 100 p=0.1550 alc c ou nt x 10 3 pe r µ l patients healthy donors 0 1 2 3 4 6 8 p=0.1140 cd8+ t cells % p b m c patients healthy donors 0 10 20 30 40 p=0.0168 b cells % p b m c patients healthy donors 0 5 10 15 20 25 p=0.0027 figure 4. standard parental immune cell types in age-matched advanced cancer patients and healthy donors. a: patients with advanced cancer (n=30) and healthy donors (n=15) included in this analysis were age-matched above age 40. b: absolute lymphocyte count (alc) of cancer patients and healthy donors. graphs display median age or alc with 25-75 percentiles; cancer type indicated by shape. p-value was calculated using the mann-whitney test. cd8+ t cells (c) and b cells (d) were different in cancer patients and healthy donors, as defined by an adjusted p<0.05, the median of groups showing a > 50% difference, and a frequency above 0.01% for peripheral blood mononuclear cells (pbmcs). graphs display median frequency as a percentage of pbmcs with 25-75 percentiles; cancer type indicated by shape (square: gi (anal, colon, oesophageal); n=6; triangle: pancreatic, n=6; star: breast, n=3; plus sign: mesothelioma, n=3; diamond: renal cell, n=3; closed circle: other (adrenocortical, chordoma, lung, medullary thyroid, neuroendocrine, ovarian, prostate, spindle cell), n=9; open circle: healthy donors, n=15). p-value was calculated using the mann-whitney test, with holm adjustment made for multiple comparisons using the number of standard subsets with a frequency above 0.01% for pbmcs (n=9). table 4. standard parental immune cell types in age-matched advanced cancer patients and healthy donors 1 immune cell type cancer patients healthy donors p value direction of patients cd8+ t cells 15 8 0.0168 � b cells 8 16 0.0027 � cd4+ t cells 26 29 0.9999 = tregs 1.4 1.5 0.9999 = nk 6 10 0.0595 = nk-t 1.9 1.3 0.4650 = cdc 0.3 0.3 0.9999 = pdc 0.2 0.2 0.2142 = mdsc 4 4 0.9999 = a total of 9 standard parental immune cell types were analyzed. valuesare displayed as median % of pbmcs. p-value was calculated with the mann whitney test with holm adjustment made for multiple comparisons using the number of standard immune cell types with frequency above 0.01% of pbmcs (n=9). differences were defined as subsets with an adjusted p<0.05, medians at least 50% different, and frequencyabove 0.01%of pbmcs. -------cdc, conventional dendritic cells; mdsc, myeloid derived suppressor cells; nk, natural killer; pbmcs, peripheral blood mononuclear cells; pdc,plasmacytoid dc; tregs, regulatoryt cells. table 4. standard parental immune cell types in age-matched advanced cancer patients and healthy donors 9lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types to several pathways, including b cell receptor signalling and glutamate signalling pathways (figure 6b). several genes implicated in the regulation of immune cells includ‐ ing mdscs, b cells and t cells were identified as differen‐ tially expressed between the two groups (figure 6c). for mdscs, this included decreases in cd200, a ligand receptor expressed on myeloid cells involved in the inhibition of mdsc signalling [22], as well as increases in cd300lb, an immune receptor expressed in myeloid cells involved in the activation of suppressor function [23]. with regard to b cells, changes included decreases in the gene expression of cd19, cd22, cd72, ikzf3 and gcsam in cancer patients compared to healthy donors. cd19 is the hallmark differentiation antigen of b cell lineage, positively regulating antigen receptor signalling in these cells [24]; cd22 and cd72 are involved in b cell maturation [25, 26], ikzf3 is a transcription factor involved in b cell prolifera‐ tion and development [27], and gcsam, also known as hgal, has been shown to activate b cell receptor signalling by enhancing its kinase activity [28]. multiple t cell receptor genes were also decreased in both the alpha and beta families. 4. discussion with the expanded use of immunotherapeutics such as checkpoint inhibitor mabs, vaccines and immune modu‐ lators in the management of many cancer types, the identification (either prior to therapy and/or early in the therapeutic regimen) of patients most likely to benefit from immunotherapy becomes more important. for example, evidence has emerged from studies of patients with melanoma and other tumour types of prognostic factors derived from tumour biopsies such as: (a) the number of mutations, (b) the expression of pd-l1 on tumour cells and/ or (c) the presence of immune infiltrate. however, using these factors has not always provided a good surrogate of patient benefits with clear outliers (both positive and negative) for all three of these parameters. moreover, with the exception of melanoma and some metastatic lesions to the skin of other tumour types, biopsies of metastatic lesions of most solid tumours are not always feasible and numerous studies have shown that the phenotype of primary tumours often differs from metastatic lesions. analyses of immune cells in the periphery may very well aid – along with other parameters mentioned – in selecting a. b. c. d. e. f. g. h. figure 5. 1 batf+ cd4+ t cells % p b m c patients healthy donors 0.0 0.2 0.4 0.6 0.8 3 4 p=0.0029 pd-l1+ cd8+ t cells % p b m c patients healthy donors 0.00 0.05 0.10 0.15 0.20 p=0.0025 ctla-4+ cd8+ t cells % p b m c patients healthy donors 0.0 0.1 0.2 0.3 0.4 0.5 6 7 p=0.0025 ctla-4+ treg % p b m c patients healthy donors 0.00 0.05 0.10 0.15 p=0.0280 pd-l1+ mdsc % p b m c patients healthy donors 0 1 2 3 p=0.0088 gmdsc % p b m c patients healthy donors 0 1 2 3 4 p=0.0015 pd-l1+ pdc % p b m c patients healthy donors 0.00 0.02 0.04 0.06 0.08 0.10 0.15 0.35 p=0.0003 pd-l1+ b cells % p b m c patients healthy donors 0 1 2 3 4 p=0.0009 figure 5. differences in immune cell subsets related to maturation and function between age-matched advanced cancer patients and healthy donors. a total of 114 subsets were analysed related to maturation and function within the standard subsets. subsets were considered different if a subset had an adjusted p<0.05, the median of groups showing a > 50% difference, and a frequency above 0.01% for peripheral blood mononuclear cells (pbmcs). a-h: representative graphs are shown for notable subsets with differences between cancer patients and healthy donors. graphs display median frequency as a percentage of pbmcs with 25-75 percentiles; cancer type indicated by shape (square: gi (anal, colon, oesophageal), n=6; triangle: pancreatic, n=6; star: breast, n=3; plus sign: mesothelioma, n=3; diamond: renal cell, n=3; closed circle: other (adrenocortical, chordoma, lung, medullary thyroid, neuroendocrine, ovarian, prostate, spindle cell), n=9; open circle: healthy donors, n=15). p-value was calculated using the mann-whitney test, with holm adjustment made for multiple comparisons using the number of subsets within each standard subset with a frequency above 0.01% for pbmcs (n=29 for cd4+ t cells, 25 for cd8+ t cells, 5 for tregs, 14 for natural killer (nk) cells, 3 for nk-t cells, 4 for b cells, 2 for conventional dendritic cells (cdcs), 3 for plasmacytoid dcs (pdcs) and 15 for myeloid derived suppressor cells (mdscs)). 10 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 patients most likely to benefit from and/or in identifying patients early on in the agent regimen who will benefit from a specific immunotherapy. recent advances in human immunology using multicolour flow cytometry and high-throughput computation analysis (cytomics) now allow for the identification of numerous individual peripheral immune cell subsets [29-31]; more‐ over, the biological function and relevance of many of these subsets have been described [5]. to our knowledge, the study reported here is the most comprehensive to date, analysing 123 different peripheral immune cell subsets with a focus on subsets related to maturation and function (table 1), comparing healthy donors under vs. over the age of 40, and comparing metastatic cancer patients with agematched healthy donors. it should be noted that all comparisons between groups in this study employed the mann-whitney test and resulting p-values were adjusted using the step-down holm adjustment for multiple comparisons. this adjustment was applied to account for the increased probability of type i errors (false positives) that occur when multiple outcome measures (e.g., 123 subsets) are assessed. however, as p-value adjustments also increase the risk of type ii errors (false negatives), we additionally performed a less stringent analysis evaluating immune cell type cancer patients healthy donors p value direction of patients batf + cd4 + t cells 0.101 0.036 0.0029 � pd-l1 + cm cd4 + t cells 0.014 0.008 0.0448 � pd-l1 + cd8 + t cells 0.043 0.016 0.0025 � ctla-4 + cd8 + t cells 0.120 0.045 0.0025 � tcr + cd8 + t cells 14 8 0.0231 � pd-l1 + em cd8 + t cells 0.017 0.006 0.0025 � ctla-4 + em cd8 + t cells 0.048 0.021 0.0044 � ctla-4 + emra cd8 + t cells 0.022 0.009 0.0231 � ctla-4 + tregs 0.020 0.009 0.0280 � pd-l1 + mdsc 0.711 0.221 0.0088 � pd-1 + mdsc 0.708 0.115 0.0015 � cd16 + mdsc 1.004 0.189 0.0015 � gmdsc 0.903 0.006 0.0015 � pd-l1 + gmdsc 0.272 0.001 0.0015 � pd-1 + gmdsc 0.529 0.002 0.0015 � cd16 + gmdsc 0.761 0.002 0.0015 � pd-l1 + lin neg mdsc 0.183 0.041 0.0015 � pd-l1 + pdc 0.023 0.001 0.0003 � pd-1 + pdc 0.012 0.001 0.0003 � pd-l1 + b cells 0.797 1.754 0.0009 � pd-1 + b cells 0.354 0.726 0.0009 � immature nk 0.300 0.611 0.0014 � unconventional nk 0.316 0.785 0.0130 � a total of 114 subsets were analyzed relating to maturation and function within the standard subsets. a: table displays subsets that were different in cancer patients and age-matched healthy donors, as defined by an adjusted p<0.05, medians at least 50% different, and frequency above 0.01% of pbmcs. values represent median % of pbmcs, and p-value was calculated with the mann whitney test with holm adjustment made for multiple comparisons using the number of subsets within each standard subset with frequency above 0.01% of pbmcs (n=29 for cd4+ t cells, 25 for cd8+ t cells, 5 for tregs, 14 for nk cells, 3 for nk-t cells, 4 for b cells, 2 for cdcs, 3 for pdcs, 15 for mdscs). b: table displays subsets with notable trends between cancer patients and healthy donors as defined by unadjusted p< 0.01, medians at least 50% different, and frequency above 0.01% of pbmcs. values represent median % of pbmcs. -------batf, basic leucine zipper transcription factor atf-like; cdc, conventional dendritic cells; cm, central memory; ctla-4, cytotoxic t lymphocyte-associated protein-4; em, effector memory; emra, terminally differentiated effector memory; eomes, eomesodermin; gmdscs, granulocytic mononuclear derived suppressor cells; lin neg mdscs, lineage negative mdscs; nk, natural killer; pbmcs, peripheral blood mononuclear cells; pdc, plasmacytoid dc; pd-1, programmed cell death-1; pd-l1, programmed cell death ligand-1; tbet, t box expressed in t cells; tcr, t cell receptor; tim-3, t cell immunoglobulin and mucin domain-3; tregs, regulatory t cells. a. immune cell type cancer patients healthy donors unadjusted p value holm adjusted p value trend of patients tbet + cd4 + t cells 2.252 1.101 0.0053 0.1431 � tbet + cd8 + t cells 1.732 0.538 0.0035 0.0665 � eomes + cd8 + t cells 3 2 0.0098 0.1764 � b. table 5. differences in immune cell subsets relating to maturation and function between age-matched advanced cancer patients and healthy donors 11lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types unadjusted p-values and identified trends of changes in subsets with an unadjusted p<0.01. changes in the immune system with aging are evidenced by the observation that older individuals have a higher prevalence of autoimmunity, chronic diseases and cancer [32]. previous studies examining peripheral immune cells during aging have focused primarily on certain standard immune cell types and, in some cases, on their memory status. in addition, prior studies have examined pbmcs of individuals in a young group, defined as aged 18-34, vs. an older group, above the age of 65 [33, 34]; other studies have also used an older cut-off ranging between 60-65 years of age in order to separate older from younger individuals [35-37]. in the study reported here, age 40 [6, 7] was used as a demarcation to define younger and older healthy donor groups, as the risk of cancer rises substantially at age 40. with more cancers also arising earlier in life, it is also important to define the relationship of age on immune cell subsets evaluating a younger age population, rather than a. b. c. figure 6. 1 ingenuity canonical pathways -log (p value) pathogenesis of multiple sclerosis 3.09e00 primary immunodeficiency signaling 2.77e00 glutamate biosynthesis ii 1.96e00 glutamate degradation x 1.96e00 granulocyte adhesion and diapedesis 1.96e00 b cell receptor signaling 1.89e00 coagulation system 1.8e00 α-tocopherol degradation 1.78e00 systemic lupus erythematosus signaling 1.74e00 role of tissue factor in cancer 1.72e00 nad biosynthesis iii 1.66e00 role of pattern recognition receptors in recognition of bacteria and viruses 1.57e00 gene immune cell type fold change in patients p value cd200 mdsc -1.82 0.008 cd300lb mdsc 1.60 0.038 cd19 b cell -1.76 0.026 cd22 b cell -1.79 0.042 cd72 b cell -1.60 0.032 ikzf3 b cell -1.56 0.029 gcsam b cell -1.55 0.020 cd44 t cell 2.03 0.006 trbv7-8 t cell -2.18 0.024 trbv6-9 t cell -1.58 0.007 trbv6-6 t cell -1.87 0.018 trbv3-1 t cell -2.40 0.013 trbv20or9-2 t cell -1.77 0.027 trbv2 t cell -3.04 0.001 trav26-2 t cell -3.18 0.013 trav23dv6 t cell -2.01 0.040 trav16 t cell -2.18 0.046 trav13-1 t cell -1.80 0.014 traj9 t cell -2.21 0.037 traj45 t cell -1.82 0.050 traj43 t cell -2.42 0.003 traj42 t cell -2.02 0.004 figure 6. microarray analysis in pbmcs from metastatic patients with gi cancer and age-matched healthy donors. microarray analysis was performed on rna isolated from pbmcs of patients with advanced gi cancer (n=4) and age-matched healthy donors (n=5). a: total number of significantly affected genes (p < 0.05 and fold change > |1.5|). b: ingenuity pathway analysis of the 157 significantly affected genes. pathways are listed in the left column and p-value (fisher’s exact test) is listed in the right column. c: key immune related genes identified as differentially expressed (p < 0.05 and fold change > |1.5|), with known key immunological function between advanced cancer patients and age-matched healthy donors. includes gene name, known cell type, fold change and p-value. 12 j circ biomark, 2016, 5:5 | doi: 10.5772/62322 focusing on populations aged 65 and above. in this study, differences were not evident when subsets (albeit smaller number per group) were examined by age decade or gender; however, as described above, a number of changes were identified between healthy donors when age groups were defined as below or above the age of 40. we show here for the first time that healthy donors over the age of 40 had lower alc, lower cd8+ t cells, lower activation markers within cd8+ t cells, lower naïve and central memory cd8+ t cells and higher nk cells, com‐ pared to those under the age of 40 (figures 2 and 3). nk results are in agreement with those of others reporting an increase in total nk cells with age; however, this increase of nk cells in older individuals was reported when examining donors according to young and old extremes, and when using age 60 as a cut-off to define older individ‐ uals [38-40]. in prior studies, using age 60 as a cut-off for older individuals, it has been shown that the cd8+ t cell compartment was more affected by age than cd4+ t cells [32, 35]. the most notable changes in the studies reported here with regard to age (under vs. over 40) were evident in the t cell compartment and specifically involved cd8+ t cells. in addition to the standard immune cell types, we also examined many subsets related to maturation and function with known biological importance, including immune cells expressing immune checkpoints such as pd-1, pd-l1, ctla-4 and tim-3. with regards to maturation status, it is generally accepted that there is a decrease in naïve t cells and an increase in memory t cells with age [2]. it was shown here that healthy donors > 40 years had a decrease in both naïve and central memory cd8+ t cells. this substantiates and extends a prior report that elderly individuals have a decrease in both naïve and central memory cd8+ t cells in cases where younger donors with an average age of 30 were compared to older donors with an average age of 70 [35]. it has also been reported that t cell signalling can be altered with aging, involving the expression of t cell receptor (tcr) components, signal transduction and the expression of costimulatory receptors [41]. the most notable differences in our age analysis were related to activation markers in cd8+ t cells. we found (figure 3) that healthy donors over the age of 40 had decreases in cd8+ t cells that expressed pd-l1, ctla-4 and tim-3, which are inhibitory molecules involved in t cell immune checkpoints and can serve as markers of immune activation [13, 42]. many prior studies comparing healthy donors with cancer patients have focused mainly on standard immune cell types and maturation markers [43-48], while others [42, 49] have also examined several additional subsets. we report here that among the standard immune cell types, there were significantly higher levels of cd8+ t cells and lower levels of b cells in cancer patients compared to agematched healthy donors (figure 4). notable differences in immune cells related to maturation and function were also evident between cancer patients and healthy donors. we show here for the first time that patients with multiple indications of advanced cancer had higher activation markers involved in immune checkpoint pathways in cd4+ and cd8+ t cells, several higher suppressive treg and mdsc subsets, and altered expression of pd-l1 on apcs (figure 5). ctla-4 and pd-l1 are co-inhibitory molecules that are expressed on t cells upon activation [42, 50] and batf is a transcription factor involved in pd-1 signalling [15]. cancer patients had higher levels of batf+cd4+ t cells, pd-l1+cd8+ t cells and ctla-4+cd8+ t cells com‐ pared to healthy donors. expression of pd-l1 on apcs was also altered in cancer patients, with lower levels detected on b cells and higher levels on pdcs when compared to healthy individuals. prior studies [51, 52] have shown that cancer patients also had higher levels of tregs and mdscs compared to healthy donors. in the studies reported here, while levels of total tregs were similar to healthy donors, cancer patients had increased levels of ctla-4+ tregs, which is a phenotype of a biologically suppressive treg [17]. it has also been previously reported that while prostate cancer patients had a similar frequency of total tregs in peripheral blood as healthy donors, the ctla-4+ tregs from patients had greater suppressive functionality [53]. additionally, patients with head and neck squamous cell carcinomas have also been shown to have increased levels of the ctla-4+ on tregs when compared to healthy donors [54]. while we did not find an increase in mdscs in cancer patients in this study, cancer patients displayed an in‐ creased frequency in several subsets of mdscs with a suppressive phenotype, including pd-l1+ mdscs and gmdscs. in order to corroborate changes between cancer patients and healthy donors at the phenotypic level with those at the gene level, we performed genome-wide gene expres‐ sion profiling in an additional subset of advanced cancer patients and age-matched healthy donors. in the flow cytometry analysis, some subsets of mdscs were in‐ creased, while b cells were decreased in cancer patients relative to healthy donors. in the microarray data set, we observed a down-regulation of the mdsc suppressor cd200 and up-regulation of the mdsc activator cd300lb, as well as decreased expression of cd19 and several other markers of b cell maturation and activation. it has already been well-established that the identification of gene transcripts in pbmcs correlate to changes in survival, disease progression and other various clinical outcomes, especially in cancer [55-60]; however, these data demon‐ strate the potential for combining extensive phenotypical analysis with genome-wide gene expression analysis. while the study reported here identified 123 immune cell subsets using flow cytometry, additional subsets could provide important insight regarding the effect of age and cancer on the immune system. for example, up to 17 unique b cell subsets have been described in the peripheral blood [61]; however, due to limitations in both the quantity of 13lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types patient blood available, as well as the number of colours able to be run per panel on the lsr ii, we were able to include only five b cell subsets in our study. future studies might incorporate additional panels focusing on specific subsets such as b cells and nk cells – where additional phenotyp‐ ic markers have been described in the literature – in order to gain deeper insight into the standard subsets. in addi‐ tion, the utilization of new technologies such as cytof, the next generation of cell detection that allows for the simulta‐ neous detection of up to 50 antibodies in a single panel, may overcome these limitations and allow for the efficient detection of numerous immune subsets from small sam‐ ples [62, 63]. analyses of the 123 immune cell subsets reported here required only 1 x 107 pbmcs obtained from ~5-10 ml of peripheral blood. this is a minimally invasive procedure and analyses could be performed immediately prior to immunotherapy and/or early in the agent regimen. analyses of pbmc subsets can also identify changes in the patient’s immune constitution as a result of prior and/or more recent therapies. studies [64] have shown that some so-called “non-immune”-based therapies, such as certain chemotherapeutics and targeted therapies, can have appreciable effects (either positive or negative) on subse‐ quent immunotherapy regimens. recent hypothesisgenerating clinical studies have also provided evidence in different immunotherapy trials that analyses of immune cell subsets may correlate with patient benefits [8, 42, 65]. larger trials will need to be carried out to expand and confirm these findings. purified pbmcs in this study were frozen and stored for batch analyses to reduce any assay to assay variations. the studies reported here are meant to provide a founda‐ tion for the use of the 123 peripheral immune cell subset panels in future randomized clinical studies involving well-defined and homogeneous patient populations, as well as for use as an adjunct to other prognostic analyses such as the interrogation of patient biopsies, when availa‐ ble. in addition, to more carefully assess the changes in immune cell subsets as cancer progresses, longitudinal studies are planned that will include patients with different stages of cancer. using these panels, we were able to identify differences in peripheral immune cell subsets between younger and older healthy donors, and between advanced cancer patients and age-matched controls. while the studies here focused on differences in age and between healthy donors and cancer patients, the analysis of 123 peripheral immune cell subsets can potentially also be employed to provide valuable information in the prognosis and/or therapy of patients with autoimmunity and other chronic or infectious diseases. 5. compliance with ethical research standards all of the subjects gave informed consent. samples from healthy donors were obtained from the nih clinical center blood bank (nct00001846), and patients were enrolled in clinical trials approved by the nci irb (nct01772004, nct00088413). 6. conflict of interest the authors declare that they have no conflicts of interest. 7. funding grant support was provided by the intramural research program of the center for cancer research, 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17lauren m. lepone, renee n. donahue, italia grenga, simon metenou, jacob richards, christopher r. heery, ravi a. madan, james l. gulley and jeffrey schlom: analyses of 123 peripheral human immune cell subsets: defining differences with age and between healthy donors and cancer patients not detected in analysis of standard immune cell types