Email this article EXPRESSION OF TUMOR ASSOSIATED AND EPITHELIAL-MESENCHYMAL TRANSITION MARKERS IN 2D AND 3D CELL CULTURES OF MCF-7
Abstract
The target effects on the expression of epithelial-mesenchymal transition regulation molecules are promising for cancer therapy, including breast cancer. 3D cell culture is a model for studying epithelial-mesenchymal transition in vitro and may become a test system for anticancer therapy.
Aim of research. The aim of this research was to evaluate and compare the expression of tumor associated and epithelial-mesenchymal transition markers in tumor cells of breast adenocarcinoma (MCF-7 cell line) in 2D and 3D cell culture.
Methods. For realization of the aim MCF-7 cell line (breast adenocarcinoma) was chosen as an experimental model in vitro. The monolayer cell culture was cultured in standard conditions (37 0C, 5 % CO2, humidity 95 %). The initial density of inoculated cells was 2 x 104 cells/cm2. The cells were incubated for two days before their use in the experiment. For the initial generation of spheroids the monolayer cell culture was removed off the substrate after the four days of incubation, using 0,25 % Trypsin-EDTA, and placed in nutrient medium with 5 % carboxymethyl cellulose (Bio-Rad, USA) at concentration of 5 x 105 cells/ml. Then the plates were incubated on an orbital shaker (Orbital shaker, PSU-10i, Biosan, Latvia) at 50 rpm for 3–5 hours. Half of culture medium was replenished every 3 days. A spheroid culture was maintained for 14 days. Detection of markers (ER, p53, EpCAM, vim, AE1/AE3, panCK, EGFR) in 2D and 3D cell culture was performed using immunohistochemistry method with primary monoclonal antibodies. Histological samples of cells were photographed to compare the morphological characteristics and the expression of proteins in monolayer and spheroid culture
Results. The results demonstrated that the percentage of tumor marker positive cells (ER+, EGFR+, EpCAM+, panCK+, AE1/AE3+) in monolayer culture is 1.25–2 times than more in spheroid culture. In contrast, tumor spheroids consist of fewer cells with the expression of epithelial markers such as EpCAM and AE1/AE3, but they contain a large number of cells that expressed mesenchymal marker vimentin by 5 % and p53 by 10 %. This may indicate that the cells acquire a mesenchymal phenotype. However, tumor cells of monolayer cell culture were not expressed vimentin.
Conclusions. Our results demonstrated the differences of expression of tumor associated and epithelial-mesenchymal transition markers in 2D and 3D breast cancer cell cultures. Thus, the percentage of epithelial markers (Cytokeratines and epithelial cell adhesion molecule) in tumor spheroids is less than in cells of monolayer however spheroids cells begin expressing a mesenchymal marker – vimentin. In 3D cell culture only the outer cell layers expressed tumor associated proteins unlike 2D cell culture in which all of cells showed equally expression. Reduced of manifestation of tumor associated markers in 3D cell culture may indicate an increase of stem properties. These data showed that 3D cell culture more than 2D cell culture characterized processes of epithelial-mesenchymal transition.Keywords
Full Text:
PDFReferences
Khaitan, D., Dwarakanath, B. S. (2006). Multicellular spheroids as an in vitro model in experimental oncology: applications in translational medicine . Expert Opinion on Drug Discovery, 1 (7), 663–675. doi: 10.1517/17460441.1.7.663
Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., Kunz-Schughart, L. A. (2010). Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of Biotechnology, 148 (1), 3–15. doi: 10.1016/j.jbiotec.2010.01.012
Yamada, K. M., Cukierman, E. (2007). Modeling Tissue Morphogenesis and Cancer in 3D. Cell, 130 (4), 601–610. doi: 10.1016/j.cell.2007.08.006
Gurski, L., Petrelli, N., Jia, X., Farach-Carson, M. (2010). 3D matrices for Anticancer Drug Testing and Development. Oncology Issues, 25, 20–25
Dufau, I., Frongia, C., Sicard, F., Dedieu, L., Cordelier, P., Ausseil, F. et. al. (2012). Multicellular tumor spheroid model to evaluate spatio-temporal dynamics effect of chemotherapeutics: application to the gemcitabine/CHK1 inhibitor combination in pancreatic cancer. BMC Cancer, 12 (1). doi: 10.1186/1471-2407-12-15
Marcucci, F., Stassi, G., De Maria, R. (2016). Epithelial–mesenchymal transition: a new target in anticancer drug discovery. Nature Reviews Drug Discovery, 15 (5), 311–325. doi: 10.1038/nrd.2015.13
Thiery, J. P., Lim, C. T. (2013). Tumor Dissemination: An EMT Affair. Cancer Cell, 23 (3), 272–273. doi: 10.1016/j.ccr.2013.03.004
Bill, R., Christofori, G. (2015). The relevance of EMT in breast cancer metastasis: Correlation or causality? FEBS Letters, 589 (14), 1577–1587. doi: 10.1016/j.febslet.2015.05.002
Edmondson, R., Broglie, J. J., Adcock, A. F., Yang, L. (2014). Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. ASSAY and Drug Development Technologies, 12 (4), 207–218. doi: 10.1089/adt.2014.573
Dave, B., Mittal, V., Tan, N. M., Chang, J. C. (2011). Epithelial-mesenchymal transition, cancer stem cells and treatment resistance. Breast Cancer Research, 14 (1), 202. doi: 10.1186/bcr2938
Lindsey, S., Langhans, S. A. (2014). Crosstalk of Oncogenic Signaling Pathways during Epithelial–Mesenchymal Transition. Frontiers in Oncology, 4. doi: 10.3389/fonc.2014.00358
Lamouille, S., Xu, J., Derynck, R. (2014). Molecular mechanisms of epithelial–mesenchymal transition. Nature Reviews Molecular Cell Biology, 15 (3), 178–196. doi: 10.1038/nrm3758
Gostner, J. M., Fong, D., Wrulich, O. A., Lehne, F., Zitt, M., Hermann, M. et. al. (2011). Effects of EpCAM overexpression on human breast cancer cell lines. BMC Cancer, 11 (1). doi: 10.1186/1471-2407-11-45
Spizzo, G., Fong, D., Wurm, M., Ensinger, C., Obrist, P., Hofer, C. et. al. (2011). EpCAM expression in primary tumour tissues and metastases: an immunohistochemical analysis. Journal of Clinical Pathology, 64 (5), 415–420. doi: 10.1136/jcp.2011.090274
Gires, O., Klein, C. A., Baeuerle, P. A. (2009). On the abundance of EpCAM on cancer stem cells. Nature Reviews Cancer, 9 (2), 143–143. doi: 10.1038/nrc2499-c1
Kalluri, R., Weinberg, R. A. (2009). The basics of epithelial-mesenchymal transition. Journal of Clinical Investigation, 119 (6), 1420–1428. doi: 10.1172/jci39104
Hardy, K. M., Booth, B. W., Hendrix, M. J. C., Salomon, D. S., Strizzi, L. (2010). ErbB/EGF Signaling and EMT in Mammary Development and Breast Cancer. Journal of Mammary Gland Biology and Neoplasia, 15 (2), 191–199. doi: 10.1007/s10911-010-9172-2
Misra, A., Pandey, C., Sze, S. K., Thanabalu, T. (2012). Hypoxia Activated EGFR Signaling Induces Epithelial to Mesenchymal Transition (EMT). PLoS ONE, 7 (11), e49766. doi: 10.1371/journal.pone.0049766
Chang, Z.-G., Wei, J.-M., Qin, C.-F., Hao, K., Tian, X.-D., Xie, K. et. al. (2012). Suppression of the Epidermal Growth Factor Receptor Inhibits Epithelial–Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells. Digestive Diseases and Sciences, 57 (5), 1181–1189. doi: 10.1007/s10620-012-2036-4
Serrano, M. J., Ortega, F. G., Alvarez-Cubero, M. J., Nadal, R., Sanchez-Rovira, P., Salido, M. et. al. (2014). EMT and EGFR in CTCs cytokeratin negative non-metastatic breast cancer. Oncotarget, 5 (17), 7486–7497. doi: 10.18632/oncotarget.2217
Goh, A. M., Coffill, C. R., Lane, D. P. (2010). The role of mutant p53 in human cancer. The Journal of Pathology, 223 (2), 116–126. doi: 10.1002/path.2784
Chang, C.-J., Chao, C.-H., Xia, W., Yang, J.-Y., Xiong, Y., Li, C.-W. et. al. (2011). p53 regulates epithelial–mesenchymal transition and stem cell properties through modulating miRNAs. Nature Cell Biology, 13 (3), 317–323. doi: 10.1038/ncb2173
Gergeliuk, T., Perepelytsina, О., Sidorenko, М. (2014). Comparing the impact of conditioned medium from human mesenchymal stem cells and interferon-population and the development of cancer cells MCF-7. Bukovina Medical Journal, 18 (2), 18–23.
DOI: http://dx.doi.org/10.21303/2504-5679.2016.00231
Refbacks
- There are currently no refbacks.
Copyright (c) 2016 Tetiana Herheliuk, Olena Perepelytsina, Natalia Yurchenko, Mykhailo Sydorenko, Lyudmila Ostapchenko
This work is licensed under a Creative Commons Attribution 4.0 International License.
ISSN 2504-5679 (Online), ISSN 2504-5660 (Print)