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Topalidou, Irini, and Martin Chal!e. “Shared gene expression in distinct neurons expressing common selector genes.” Proceedings of the National Academy of Sciences 108.48 (2011): 1-6. PNAS. 25 Feb. 2012. Way, J.C. and Chal!e, M. 1988. mec-3, a homeobox-containing gene that speci!es di$erentiation of the touch receptor neurons in C. elegans. Cell 54, 5–16. Way, J. C., & Chal!e, M. 1989. #e mec-3 gene of Caenorhab- ditis elegans requires its own product for maintained expression and is expressed in three neuronal cell types. Genes Dev. 3, 1823 – 1833. Wu J, Duggan A, Chal!e M. Inhibition of touch cell fate by egl- 44 and egl-46 in C.elegans. Genes Dev 2001; 15, 789-802. Zhang S, Arnadottir J, Keller C, Caldwell GA, Yao CA, Chal!e M (2004) MEC-2 is recruited to the putative mechanosensory com- plex in C. elegans touch receptor neurons through its stomatin-like domain. Curr Biol 14, 1888 –1896. Zhang, Y., Ma, C., Delohery, T., Nasipak, B., Foat, B.C., Bounoutas, A., Bussemaker, H.J., Kim, S.K., Chal!e, M., 2002. Identi!cation of genes expressed in C. elegans touch receptor neu- rons. Nature 418, 331–335. Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 15 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 /2 * < References Breast cancer affects one in eight women per year, and 70% of patients with stage IV breast cancer develop metastases in bone, FDXVLQJ�OLIH�WKUHDWHQLQJ�VLGH�HIIHFWV��:H�H[DPLQH�WKH�XVH�RI�DQ�H[SODQW�V\VWHP�WR�PLPLF�EUHDVW�FDQFHU�PHWDVWDVLV�WR�ERQH�ZLWKRXW� confounding cell types and to determine the role that osteocytes, the bone mechanosensing cells, may have in the development of metastasis. Using the explant system, a custom cell seeder and sterile cell culture techniques, we introduced metastatic MDA-MB-231/ *)3�FHOOV�WR�D�WKUHH�GLPHQVLRQDO�ERQH�PDWUL[�ZLWK�RVWHRF\WHV�RQO\��&RQIRFDO�LPDJLQJ�FRQ¿UPV�WKDW�EUHDVW�FDQFHU�FHOOV�ZHUH��LQ�IDFW�� successfully seeded onto bone cores, mimicking metastasis. Though additional experiments will be necessary to determine the im- portance of breast cancer-osteocyte interactions, this study shows that the explant system is a viable methodology for studying breast cancer in bone. Cancer is a devastating disease that is responsible for thir- teen percent of deaths worldwide and has a$ected count- less families and individuals throughout the world (Cancer, World Health Organization). It a$ects 1 in 8 women every year (U.S. breast cancer statistics). Breast cancer originates from the inner lining of the lobules that supply the milk ducts in the breast (Wolf et al., 2003). Stage IV breast can- cer is metastatic, meaning that it is violent and transcends the host organ (the breast) and spreads to a secondary site. #e cancer that metastasizes is still considered breast can- cer. It has been reported that up to 70% of stage IV breast cancer patients will experience some form of metastasis of breast cancer to bone (Roth et al., 2009). Patients often experience pathological fractures, intense pain, hypercal- cemia, and various nervous compression complications (Zhang et al., 2010). #ese devastating e$ects are caused by an imbalance of bone remodeling, which involved the interactions of the three main bone cell types. Bone is comprised of three types of cells: osteocytes (OCY), osteoblasts (OB) and osteoclasts (OCL). Osteo- cytes are the primary mechanosensing cells in bones (Burg- er et al., 1995). #ey regulate the activity of osteoblasts and osteoclasts. Osteocytes are “trapped” in the mineralized bone matrix, and are thus they are thought to have only sig- naling functions, both intercellular and intracellular. After osteocytes sense a mechanical load, that load is transduced into a chemical signal is sensed by the cells. #is stress is translated into a biochemical signal that is communicated to the osteoblasts and osteoclasts, the bone forming and bone resorbing cells (Burger et al., 1995). Osteoblasts syn- thesize the bone matrix, which is subsequently de- posited and calci!ed to become bone mineral. When osteoblasts secrete too much matrix, they become stuck in the bone, and as a result they completely di$erentiate into osteocytes (Saladin, 2007; Buck- walter et al. 1995). Osteoclasts, on the other hand, resorb bone. To accomplish this, they use their “ruf- "ed” membrane (as shown in Figure 1) to create a seal around bone and then pump enzymes and hy- drochloric acid to degrade the matrix (Saladin, 2007; Buckwalter et al. 1995). #ere are two types of metastasis: osteolytic and os- teoblastic. Osteolytic metastases break down bone and are the most common type of metastasis for breast cancer. Os- teoblastic lesions, characterized by excess bone formation, a$ect 15-20% of patients. Mixed types also exist, wherein the patient experiences unnecessary bone excess as well as dearth (Zhang et al., 2010). #e large majority of stage IV breast cancer cases end in metastasis to bone because bone has high levels of growth factors that breast cancer uses to survive. We can see that bone is a likely candidate for breast cancer metastasis due to the presence of “transforming growth factor B (TGFB), insulin-like growth factors I and II (IGF), !broblast growth factors (FGFs), platelet-derived !e Veri"cation of a Novel Explant System Used to De- termine the Role of Osteocytes in the Breast Cancer Vicious Cycle Sonia Bansal*, Genevieve N. Brown, X. Edward Guo %RQH�%LRHQJLQHHULQJ�/DERUDWRU\��'HSDUWPHQW�RI�%LRPHGLFDO�(QJLQHHULQJ��&ROXPELD�8QLYHUVLW\��1HZ�0LOOHU������@� Figure 4&OHDQHG�ERQH�FRUH��PHDVXULQJ���PP�LQ�KHLJKW�DQG�GLDPHWHU� [Adapted from Chan, ME, Lu, XL, Huo, B, Baik, AD, Chiang, V, HW�DO����������$�7UDEHFXODU�%RQH�([SODQW�0RGHO�RI�2VWHRF\WH� 2VWHREODVW�&R�&XOWXUH�IRU�%RQH�@ % ,2 ( 1 * ,1 ( ( 5 ,1 * Fetal Bovine Serum (FBS), and 1% Penicillin-Streptomycin (Pen-Strep). Creation of Experimental Groups !e cores were randomly assigned to four experi- mental groups, “Osteocytes and Breast Cancer” (OB), “Osteocytes” (O), “Dead Osteocytes and Breast Can- cer” (DB) and “Dead Osteocytes” (D) (See Figure 5). Half the cores were put through a repeated freeze-thaw technique in order to kill the osteocytes still in the cores. !is was done to see if mineralized bone, but not necessarily active osteocytes, a"ects breast cancer cell activity. Half of the dead osteocyte and live osteocyte groups were seeded with MDA-MB-231/GFP stage IV breast cancer cells via a custom cell seeder created in the lab on Day 0. !e bone cores were stuck onto needles and submerged in a solution containing 5x104 cells for each core. !e loader was placed on a stir plate with a magnetic stir #y for an hour to facilitate uniform adheration of the cancer cells. Figure 6 shows a schematic of the cell seeder. !is process simulated breast cancer in bone. Confocal Microscopy !e cores were initially stained with Cell Tracker Red and incubated for 45 minutes in order to stain the viable cells in the bone cores before being seeded. !e cores were imaged using a Leica confocal microscope on Day 0 after initial seeding, and on Day 4 using confocal microscopy. !e confocal imaging on Day 0 indicates that the cancer- ous cells do exist on the bone matrix. Figures 7.1 and 7.2 depict cores that were seeded with breast cancer cells, and the green #uorescence indicates that the cells are on the core. In contrast, Figures 7.3 and 7.4 only show the osteo- cytes in the lacunae of the bone core. !e subsequent set of images on Day 4 shows a much higher number of cells on the bone core, as indicated by Figures 8.1 and 8.2. Figures 8.3 and 8.4 again indicate that there are no breast cancer cells in these two experimental groups. !e confocal imaging on Day 0 indicates that the cancer- ous cells do exist on the bone matrix. Figures 7.1 and 7.2 depict cores that were seeded with breast cancer cells, and the green #uorescence indicates that the cells are on the core. In contrast, Figures 7.3 and 7.4 only show the osteo- cytes in the lacunae of the bone core. !e subsequent set of images on Day 4 shows a much higher number of cells on the bone core, as indicated by Figures 8.1 and 8.2. Figures 8.3 and 8.4 again indicate that there are no breast cancer cells in these two experimental groups. Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 19 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 ( 1 * ,1 ( ( 5 ,1 * Confocal Imaging of the Bone Cores Confocal imaging of the bone cores on Days 0 and 4 indicate a simulated metastatic process. !e $rst step to me- tastasis is the adheration of foreign, cancerous, metastatic cells into a new environment. !e images, taken on Day 0, indicate that the cells were added in an e"ective amount that attaches to the bone core appropriately. Figures 7.1 and 7.2 show this adherence and verify that we were able to force adheration of cancerous cells onto the matrix. Addi- tionally, the negative controls are veri$ed in Figures 7.3 and 7.4, which do not have green #uorescence. After this, Day 4 imaging indicates that the breast cancer cells were sustained on the bone cores as they maintained viability and also ex- panded in quantity on the bone cores. Figures 8.1 and 8.2 show the proliferation of the cells, as there are many more cells that e"ectively take over the lacunae. When looked at in conjunction with Figures 7.1 and 7.2, it is clear that the cancerous cells are thriving on Day 4. !ese images suggest the proliferation of the cancerous cells, indicating that me- tastasis was e"ectively mimicked in this system. cusjcolumbia undergraduate science journal Research Articles 20cusj Volume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 ( 1 * ,1 ( ( 5 ,1 * Figure 5 Visual representation of the experimental design groups. First, we have cores with live osteocytes and no breast cancer cells, as a control. The variable condition is the core with live osteocytes with breast cancer cells. We also included two more groups, both with dead osteocytes. This was because we wanted to see if the mineralized bone, not necessarily the active osteocytes, affected the way breast cancer cells acted. Figure 6 Schematic of the cell seeding technique. Bone cores were stuck on the needles and suspended in the media. Adheration was facilitated by the stir bar.[Adapted from Chan, ME, Lu, XL, +XR��%��%DLN��$'��&KLDQJ��9��HW�DO����������$�7UDEHFXODU�%RQH� ([SODQW�0RGHO�RI�2VWHRF\WH�2VWHREODVW�&R�&XOWXUH�IRU�%RQH�@ Figure 7'D\���FRQIRFDO�LPDJLQJ�RI�ERQH�FRUHV��,PDJHV�WDNHQ�RQ�/HLFD�&RQIRFDO�PLFURVFRSH��5HG�ÀXRUHVFHQFH�LV�&HOO� 7UDFNHU�5HG�DQG�VKRZV�RVWHRF\WHV��GHDG�DQG�DOLYH���ZKLOH�JUHHQ�ÀXRUHVFHQFH�LQGLFDWHV�*)3�WDJJHG�0'$� 0%������7KHVH�LPDJHV�ZHUH�WDNHQ�WR�HQVXUH�WKDW�WKH�&HOO�7UDFNHU�5HG�VWDLQ�GLG�VWDLQ�RVWHRF\WHV��ZKLFK�LV�FOHDU� through the masses of red signals near the lacunae, and to ensure that the breast cancer cells were properly VHHGHG�RQWR�WKH�ERQH�FRUHV��6FDOH�EDU� �����P� Results Discussion Modi!cations to be made As the results indicate that the methodology is sound, we must look to future studies and the next step in the larger scale study. !e harvest technique, while sterile (given that there were no infections), was time consuming and would bene"t by becoming more e#cient, so instead of 8 cores being harvested at a time, we could harvest 32 at a time. Day 4 confocal imaging shows a decreased Cell Tracker Red signal, and a longer culture would require a more stable im- aging technique. A longer culture would provide for more complicated culture conditions involving all three bone cell types. Using more bone cell types would require more than two di$erent %uorescent stains, relying on the use of the lab’s four-color microscopy technology. Explant system as a viable in vitro model for study !e qualitative (confocal images) data suggests that the explant system is a good method to simulate metastasis of breast cancer, proving that it can be used to further study Columbia Undergraduate Science Journal Open-Access Publication | http://cusj.columbia.edu 21 cusjVolume 6Spring 2012 Columbia Undergraduate Sci J http://cusj.columbia.edu % ,2 ( 1 * ,1 ( ( 5 ,1 * Figure 8'D\���FRQIRFDO�LPDJLQJ�RI�ERQH�FRUHV��,PDJHV�WDNHQ�RQ�/HLFD�&RQIRFDO�PLFURVFRSH��5HG�ÀXRUHVFHQFH�LV�&HOO�7UDFNHU�5HG� DQG�VKRZV�RVWHRF\WHV��GHDG�DQG�DOLYH���ZKLOH�JUHHQ�ÀXRUHVFHQFH�LQGLFDWHV�*)3�WDJJHG�0'$�0%������7KH�&HOO�7UDFNHU� Red stain begins to fade at this point. These images were taken to ensure that the breast cancer cells were properly prolif- HUDWLQJ�DFURVV�WKH�ERQH�FRUH��ZKLFK�LV�FOHDU�JLYHQ�WKH�QXPEHU�RI�FHOOV�SUHVHQW�RQ�WKH�FRUHV��6FDOH�EDU� �����P� this topic. !e study presents a starting point for future experiments, as it demonstrates our methodology is viable and e#cient. Future studies would rely on the use of µCT tomography to determine the Bone Volume/Total Volume, also known as the Bone Volume Fraction (BVF). BVF is a good measure of the amount of bone mineral present in a sample as it determines the space of bone mineral versus total space of the core. Di$erences in BVF between seeded and nonseeded cores would indicate cancer-induced le- sions. !e incidence of these lesions would determine the extent of a variable cell’s role on the vicious cycle. In the future, the study would start to include other cell types, creating di$erent experimental groups such as a core with osteoclasts, osteoblasts and breast cancer cells, but no osteocytes. !is condition would simulate a bone without osteocytes, and the resulting BVF, as compared to a bone core with all three cell types and breast cancer, would indicate how osteocytes a$ect bone cell activity in the pres- ence of breast cancer cells. !e future implications of this experiment are promising, as it is the "rst step in determin- ing the individual role of each cell type; determining "nally how responsible osteocytes are for metastatic breast cancer spreading to bones. SB thanks XEG for allowing her to work in Bone Bioen- gineering lab and GNB for guidance and teaching her the cell techniques. SB was funded by the Columbia Depart- ment of Biological Sciences from the Summer Undergradu- ate Research Fellowship. Bonewald, LF (2011). !e Amazing Osteocyte. 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