ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. EXPLORING EPITHELIAL COMMUNICATION TO THE MESENCHYME AND ITS IMPACT ON THE EXPRES- SION OF GENES RELATED TO TUMORIGENESIS JAY PATEL, MICHAEL VERZI (FACULTY ADVISOR) ✵ ABSTRACT Homeostasis of the epithelium is depend- ent on the Wnt/beta-catenin pathway, which regu- lates the proliferation of intestinal stem cells. The gain of function mutations in the beta-catenin gene leads to rapid cell proliferation and malignant growth in the epithelium. In addition, the mainte- nance of these stem cells appears to be linked to mesenchymal-derived factors. Although the com- munication between epithelial and mesenchyme cell populations remains uncharacterized, under- standing this mechanism will help us further under- stand the various pathways involved in tumor initia- tion processes. Our results show that the communi- cation between the mesenchyme and epithelium during transformation is influenced by varying levels of protein-expressing genes including Wnt2b, Grem1, and Bmp6. 1 INTRODUCTION The Wnt/beta-catenin pathway regulates the transcription of Wnt target genes—genes which are highly deregulated in solid tumors (Fevr et al., 2007). In the absence of the Wnt ligand, beta- catenin, a protein normally sequestered in the cyto- plasm, is constantly degraded and is prevented from entering the nucleus. In the presence of the Wnt ligand, Wnt is able to bind to its receptor Frizzled, which activates downstream signaling and prevents the destruction of beta-catenin, thus enabling its entry into the nu- cleus. In the nucleus, beta-catenin promotes the transcription of Wnt target genes, causing rapid cell proliferation (Cleavers and Nusse, 2012). Cells di- viding at an accelerated rate can become cancerous and eventually lead to the formation of a tumor. The intestinal stem cell niche is primarily made up of several cell types, including: (1) differ- entiated cells responsible for the transport and ab- sorption of nutrients, and (2) mesenchymal cells which supply signals to the epithelial cells to main- tain homeostasis. Increased Wnt signaling can be induced by activating the beta-catenin gene, which allows for abnormal epithelial growth. Due to the expression of tamoxifen inducible Cre recom- binase, Villin-CreERT2 transgenic mice have the ability to delete exon 3 of the beta-catenin allele in the in- testinal epithelium (Marjou et al., 2004). Mice ex- pressing Villin-CreERT2 can delete a portion of the beta-catenin gene in the presence of tamoxifen, re- sulting in a more transcriptionally active form of the gene. Mouse models have shown that mice with Vil- lin-CreERT2 and injected with tamoxifen show visible malignant tissue transformation in the duodenum. But in organoid models, exposed to tamoxifen, ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV rapid cell proliferation can be seen almost immedi- ately. However, epithelial transformation only be- comes visible around Day 13 in mouse models. Alt- hough organoids mimic intestinal stem cells (crypts) in vitro, they do not provide a holistic view of a bio- logical system. For example, they lack the presence of the mesenchyme. This suggests that the mesen- chyme must play some role in the suppression of mutant growth. However, the way mesenchyme and epithelial cells influence one another is unknown. Yet, it is known that mesenchymal-derived factors are essential to maintain intestinal epithelial stem cells, so the reduction of these factors may be con- tributing to the suppression of intestinal transfor- mation in the transgenic mouse (Stzepourginski et al., 2017). AREG, a ligand of EGFR (epidermal growth factor receptor), can trigger signaling cascades that mediate cell survival, proliferation, and motility. Thus, AREG may be a candidate ligand that allows the epithelium to communicate with the mesen- chyme (Wang et al., 2020). Transformed epithelium expresses high levels of AREG, which may be sensed by mesenchyme cells. Mesenchyme cells like PDGFRAhi are enriched near the crypt-villus junction where stem cells reside and promote BMP ligands (promotes differentiation/blocks prolifera- tion). A subset of PDGFRAlo cells can be found at the bottom of crypts and provide growth factors for stem cells (McCarthy et al., 2020). If AREG is com- municating with PDGFRAlo cells in the mesenchyme, the PDGFRAlo cells would express decreased levels of GREM1, a protein that functions to suppress BMP and permits proliferation. Subsequently, this may lead to an upregulation in expression levels of BMP. Early intestinal tumorigenesis reflects the deregula- tion of Wnt and BMP signals (Davis et al; 2015). It has also been recognized that BMP signaling restricts in- testinal epithelium hyperproliferation (Qi et al., 2017). If we could understand how the mesenchyme is communicating with the epithelium, it would give insight to how it is able to suppress the abnormal growth, allowing for novel therapeutic interventions during initial tumor development. The goal of this project is to learn how epithelial cells are communi- cating with mesenchyme cells in an elevated Wnt activity background. Elevated levels of Wnt prevents beta-catenin from being degraded, which pro- motes expression of Wnt target genes, resulting in the mutant phenotype. Through signals (e.g. AREG) sent by the epithelium, the mesenchyme may detect the cellular transformation and respond in hopes of preventing the abnormal growth. If mesenchyme cells are treated with conditioned media from mu- tant organoids (crypts), then the genes which are al- tered are communicating with the mesenchyme. If epithelial cells undergo abnormal growth, the lig- ands produced could be sensed by PDGFRAlo cells in the mesenchyme compartment, which may re- spond by increasing Bmp genes and decreasing Grem1 and Wnt2b production. 2 MATERIALS AND METHODS ANIMAL AND TISSUE PROCESSING Animal experiments are conducted in ac- cordance with Rutgers University Institutional Ani- mal Care and Use Committee (IACUC).. The trans- genic mice in this study are engineered to condi- tionally express the beta-catenin exon3 deletion. To induce recombination, tamoxifen (1mg/20g) is in- jected intraperitoneally for 4 days daily. Day 1 is considered the beginning of treatment, and mice are sacrificed and investigated at Day 10. This model is ideal for studying tumorigenesis in-vivo be- cause of similarities in mouse and human gastroin- testinal tracts. At Day 10, mouse intestines (duode- num) are collected and fixed in 4% paraformalde- hyde solution overnight at 4°C. Tissues are then washed with phosphate-buffered saline (PBS) and dehydrated in increasing concentrations of ethanol. Tissues are then transferred to xylene and paraffin mixture for one hour, then 100% paraffin for another hour. Tissues are embedded for sectioning. ORGANOID FORMING ASSAY Crypt-derived organoids are isolated from mouse duodenum on Day 10 after 4 consecutive treatment days of tamoxifen administered at 1mg/20g mice. Collected duodenum samples are washed in PBS, cut into ¼ inch pieces, and rotated in 3mM EDTA for 5, 10, and 20 minutes (each time ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV replace the EDTA). Tissues are then agitated and fil- tered through a 70μm filter for crypt enrichment. Crypts are then washed in PBS and the pellet is re- suspended in BME-R1. Media for the crypts include advanced DMEM supplemented with GlutaMAX (auxiliary energy source for rapidly dividing cells), HEPES (used to maintain pH), EGF (epidermal growth factor: involved with cell signaling pathway controlling cell division), Noggin (prevents cell dif- ferentiation), NAC (N-acetylcysteine: protects against rise of internal oxidant levels), N2 (promotes in vitro differentiation of stem cells), B27 (promotes growth and proliferation, but not differentiation), RSPO (positively regulates Wnt/beta-catenin signal- ing, which induces proliferation), and Pen-Strep (prevents bacterial contamination). After 3 days of organoids being treated with this media, it becomes conditioned media (contains ligands excreted by epithelium). GENOTYPING Mice toes are clipped and used as a source for DNA genotyping analysis. DNA is extracted us- ing the Kappa Buffer QIAgen Kit followed by PCR for amplification. Genotypes are confirmed by DNA electrophoresis. HEMATOXYLIN AND EOSIN STAINING To understand the changes in the epithelial architecture in terms of the formation of crypt pro- genitor cell phenotype (CPC), we conducted H&E histological analysis. Tissues are initially washed in xylene twice (5 min each). Then tissues go through two consecutive 100% EtOH washes (5 min each), followed by a 3 min 95% EtOH wash, a 3 min 85% EtOH wash, a 3 min 70% EtOH wash, and a 5 min double-distilled water wash. Unstained sections of mouse duodenum are stained with Hematoxylin (30 seconds) and dehydrated by dipping in water, fol- lowed by increasing alcohol percentages. Tissues are then counterstained with eosin for 1 minute (eo- sin in counterstain, which distinguishes between cy- toplasm and nuclei). RNA/DNA PREPARATION AND QPCR Mesenchyme cells are dissolved in Trizol. RNA is prepared according to the manufacturer’s protocols. The RNA is then reverse transcribed to cDNA using SuperScript III First-Strand Synthesis System. SYBR Green is used to amplify the cDNA through qPCR analysis. Vimentin is used for normal- ization. MESENCHYME TREATMENT WITH CONDITIONED MEDIA The main experiment consists of using mes- enchyme cells and treating them with (1) condi- tioned media from mutant organoids treated with CCM, (2) mutant organoids treated with mesen- chyme media, (3) wild type organoids treated with CCM, and (4) mesenchyme media as a control. To demonstrate the response by the mesenchyme, changes in ligand expression are examined among treatment groups. After passaging mesenchyme cells, they are plated, and are treated with mesen- chyme media on Day 1. Pictures of the cells are taken on Day 4, and the media is aspirated and re- placed with conditioned media or mesenchyme me- dia for control. The plate consists of 4 rows and 3 columns; rows 1 and 2 are made up of a confluent wild-type mesenchyme cells, and row 3 consists of sub-confluent wild type mesenchyme cells. Samples in the first column serve as controls and are treated with mesenchyme media, samples in the second column are treated with conditioned CCM from mu- tant organoids, samples in the third column are treated with conditioned mesenchyme media from mutant organoids, and samples in the fourth col- umn are treated with conditioned CCM from wild type organoids. The fourth column is a control be- cause the wild type organoids do not express mu- tant growth, so we expect there to be no change in Grem1 and Wnt2b expression. On Day 6, the cells are harvested with Trizol for RNA isolation, which is then converted to cDNA. For this experiment we measure 11 protein coding genes (Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Grem1, Wnt2b, Rspo1, Rspo3, Wnt5a, and Wnt4) using qPCR and compare their relative abundances. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV 3 RESULTS Tissue images are taken at Day 10 post- tamoxifen treatment and stained using hema- toxylin and eosin. FIGURE 1 shows the histology of the control and mutant epithelium. The control and mutant epithelium for replicates 1 and 2 show little difference in terms of transformation. This demonstrates the delay stated before, sug- gesting that the mesenchyme is playing a role to suppress mutant epithelial growth. To understand the mesenchyme re- sponse to ligands secreted by mutant epithe- lium, gene expression changes are examined following treatment with conditioned media de- rived from organoid cultures. qPCR (FIGURE 2A and FIGURE 2B) analysis demonstrated that conflu- ent and sub-confluent mesenchyme cells do not respond in the same way. For the confluent cells, Grem1 and Wnt2b tend to be downregu- lated. Grem1 is a Bmp antagonist; therefore, by downregulating the gene Bmp can have a larger effect on the epithelium which would lead to a decrease in activity of the Wnt/beta-catenin pathway. For the sub-confluent mesenchyme cells, Grem1 is downregulated in all experi- mental groups while Wnt2b is also downregu- lated except in cells treated with conditioned CCM from wild type organoids. Again, the con- ditioned media from mutant organoids are causing Grem1 and Wnt2b to be downregu- lated in order to suppress mutant epithelial growth. FIGURE 1: H&E staining of two independent replicates showing the difference between the control and mu- tant epithelium. FIGURE 2A: The image shows the density of the cells; cells are confluent. 647 is the confluent mesenchyme cell line. The leftmost bars represent Grem1 and Wnt2b expression in the control: mesenchyme cells treated with mesen- chyme media. To the right of that is mesenchyme cells treated with mesenchyme conditioned media from mutant organoids (647 MM-CM exon3f/f). To the right of that is mesenchyme cells treated with CCM condition media from wild type organoids (647 CCM-CM WT), and the rightmost graph shows mesenchyme cells treated with CCM con- ditioned media from mutant organoids (647 CCM-CM Exon3f/f). The graph shows relative gene expression (n=3), compared to cells treated with mesenchyme media. ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 2B: Image shows the density of cells; cells are sub-confluent. Graph shows relative gene expression of the three samples treated with conditioned media to the mesenchyme media for sub-confluent cells. Graph portrays same conditions as graph above except for sub-confluent cells. The graphs in FIGURE 3 demonstrate the relative gene expression seen across all 11 pro- tein coding genes that were tested. The graph focuses on the mesenchyme conditioned media from mutant organoids vs. the control. The Bmp profile between the confluent and sub-conflu- ent cells shows many similarities including Bmp2 and Bmp3 being downregulated, Bmp4 staying constant, and Bmp6 being upregulated. The upregulation in sub-confluent cells is much more profound. The Wnt profile in confluent and sub-confluent cells contain some differ- ences like Rspo3 being upregulated in conflu- ent cells while being downregulated in sub-con- fluent cells. While Wnt5a is upregulated in both confluent and sub-confluent, Wnt4 is downreg- ulated in confluent, while profoundly upregu- lated in sub-confluent cells. 4 DISCUSSION The goal of the experiment is to identify possible signaling pathways that mesenchymal cells could use to communicate with epithelial cells. While the multitude of cells in the small in- testine and their functions are known, like troph- ocytes, telocytes, and enterocytes (McCarthy et al., 2020), their ability to communicate with the microenvironment is understudied. The signal- ing crosstalk between the stroma cell popula- tion and the epithelium remains unknown. Un- derstanding how these two types of cells may communicate can provide novel therapeutic in- terventions during initial tumor development. Histological review of H&E stained slides re- ported a lack of altered or abnormal tissue structures, suggesting that the mesenchyme is responsible for suppressing mutant epithelial growth. Protein expressing genes such as Wnt2b, Grem1, and Bmp6 may be responsible for the cross talk between these two cell types. When conditioned media from mutant organ- oids is introduced to mesenchyme cells Grem1 and Wnt2b are downregulated. The downregu- lation of Wnt prevents the expression of Wnt tar- get genes, therefore controlling mutant trans- formation. Grem1 in these cell lines is seen to be downregulated, which directly and indirectly (by inhibiting Bmp genes) prevents epithelial transformation. What is unusual is why most of the Bmp genes are either downregulated or stay constant while Bmp6 is upregulated. Bmp signaling can be seen as the primary suppressor of dedifferentiation in the intestinal epithelium, and better understanding of its ligands will pro- vide important steps to how the epithelium pro- tects against oncogenesis (Perekatt et al., 2018). Although the Bmp gene expression is found to ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV FIGURE 3: Graphs show relative gene expression of the aforementioned 11 genes in sub-confluent and confluent mesenchyme cells. Graph compares the major experimental group to the control group. Because CCM is the pri- mary media used to grow and sustain organoids, its data would be most resembled in-vivo conditions. This group would have organoids function normally so that the conditioned media would mimic in-vivo conditions, and then the mesenchyme response could be observed. not increase, this could be because Grem1 ex- pression is downregulated, leading to the trans- lation of more Bmp proteins. The up and down regulation of Bmp, Grem1, and Wnt2b ligands may be, in part, how the mesenchyme is influ- encing epithelial cells, preventing early tumor- igenesis. The experiment demonstrated the dif- ferences between confluent and sub-confluent mesenchyme cells, as well as how different con- ditioned media affects mesenchyme cells. These differences are most profoundly seen in the genes Bmp6 and Wnt4. Confluent cells have the ability to sense when they are running out of space, which can cause the cells to become less proliferative. Sub-confluent cells are rather in a more active growth phase. This could be affect- ing the signals the mesenchyme releases to the ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV epithelium, and responsible for the difference seen in response between confluent and sub- confluent cells. These results come from a single experimental design and provide a basis for fu- ture experiments. Bmp, Grem1, and Wnt2b may be signals that allow the mesenchyme to communicate with the transformed epithelium. These genes could be responsible for early prevention of ini- tial tumorigenesis. These genes are potential ligands that suppress mutant epithelial growth. From here, geneticists could design experi- ments involving blocking these ligands with an- tibodies or treatment with these ligands to see the epithelial response from the mesenchyme∎ 5 REFERENCES [1] Clevers, Hans, and Roel Nusse. “Wnt/β-Catenin Signaling and Disease.” Cell, Cell Press, 7 June 2012, HTTPS://WWW.SCIENCEDIRECT.COM/SCIENCE/AR- TICLE/PII/S0092867412005867. [2] McCarthy, Neil. “Distinct Mesenchymal Cell Pop- ulations Generate the Essential Intestinal BMP Signaling Gradient.” Define_me, Cell Press,. [3] Davis, Hayley, et al. “Aberrant Epithelial grem1 Expression Initiates Colonic Tumorigenesis from Cells Outside the Stem Cell Niche.” Nature Med- icine, U.S. National Library of Medicine, Jan. 2015, HTTPS://WWW.NCBI.NLM.NIH.GOV/PMC/ARTI- CLES/PMC4594755/. [4] Stzepourginski, Igor, et al. “CD34+ Mesenchymal Cells Are a Major Component of the Intestinal Stem Cells Niche at Homeostasis and after In- jury.” PNAS, National Academy of Sciences, 24 Jan. 2017, HTTPS://WWW.PNAS.ORG/CONTENT/114/4/E506. [5] Perekatt, Ansu O., et al. “SMAD4 Suppresses Wnt-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.” Cancer Re- search, American Association for Cancer Re- search, 1 Sept. 2018, HTTPS://CANCERRES.AACRJOUR- NALS.ORG/CONTENT/78/17/4878#. [6] El Marjou, Fatima. “Tissue-Specific and Inducible CRE-Mediated Recombination in the Gut Epithe- lium.” Genesis (New York, N.Y.: 2000), U.S. Na- tional Library of Medicine, July 2004, HTTPS://PUB- MED.NCBI.NLM.NIH.GOV/15282745/. [7] Qi, Zhen, et al. “BMP Restricts Stemness of Intes- tinal LGR5+ Stem Cells by Directly Suppressing Their Signature Genes.” Nature News, Nature Publishing Group, 6 Jan. 2017, HTTPS://WWW.NA- TURE.COM/ARTICLES/NCOMMS13824. [8] Zhang, Ya, and Xin Wang. “Targeting the WNT/β- Catenin Signaling Pathway in Cancer - Journal of Hematology & Oncology.” BioMed Central, Bio- Med Central, 4 Dec. 2020, HTTPS://JHOONLINE.BIO- MEDCENTRAL.COM/ARTICLES/10.1186/S13045-020-00990- 3. [9] Wang, Li, et al. “Areg Mediates the Epithelial- Mesenchymal Transition in Pancreatic Cancer Cells via the EGFR/ERK/NF-ΚB Signalling Path- way.” Oncology Reports, D.A. Spandidos, May 2020, HTTPS://WWW.NCBI.NLM.NIH.GOV/PMC/ARTI- CLES/PMC7107775/. [10] Fevr, Tea, et al. “Wnt/Beta-Catenin Is Essential for Intestinal Homeostasis and Maintenance of Intes- tinal Stem Cells.” Molecular and Cellular Biology, American Society for Microbiology (ASM), Nov. 2007, HTTPS://WWW.NCBI.NLM.NIH.GOV/PMC/ARTI- CLES/PMC2169070/. [11] Tissue‐Specific and Inducible Cre‐Mediated ... - Wiley Online Library. HTTPS://DOI.ORG/10.1002/GENE.20042. [12] Harada N;Tamai Y;Ishikawa T;Sauer B;Takaku K;Oshima M;Taketo MM; “Intestinal Polyposis in Mice with a Dominant Stable Mutation of the Beta-Catenin Gene.” The EMBO Journal, U.S. Na- tional Library of Medicine, HTTPS://PUBMED.NCBI.NLM.NIH.GOV/10545105/. https://www.sciencedirect.com/science/article/pii/S0092867412005867 https://www.sciencedirect.com/science/article/pii/S0092867412005867 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594755/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594755/ https://www.pnas.org/content/114/4/E506 https://cancerres.aacrjournals.org/content/78/17/4878 https://cancerres.aacrjournals.org/content/78/17/4878 https://pubmed.ncbi.nlm.nih.gov/15282745/ https://pubmed.ncbi.nlm.nih.gov/15282745/ https://www.nature.com/articles/ncomms13824 https://www.nature.com/articles/ncomms13824 https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-00990-3 https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-00990-3 https://jhoonline.biomedcentral.com/articles/10.1186/s13045-020-00990-3 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107775/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7107775/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2169070/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2169070/ https://doi.org/10.1002/gene.20042 https://pubmed.ncbi.nl/ https://pubmed.ncbi.nlm.nih.gov/10545105/ ARESTY RUTGERS UNDERGRADUATE RESEARCH JOURNAL, VOLUME I, ISSUE IV Jay Patel is a junior undergraduate student conducting research in the Ge- netics Department at Rutgers New Brunswick. His interest in scientific re- search was sparked by his experience with the Rutgers Waksman pro- gram, during his time in high school. He currently works with Dr. Verzi and his mentor, Oscar, where they investigate the pathway by which the intes- tinal epithelium and mesenchymal cell populations are communicating. Along with being a researcher, Jay also enjoys riding as an EMT and the Highland Park First Aid Squad, volunteering as a part of the Rutgers Red Cross, and playing basketball with his friends.