Isolation and characterization of the primary epithelial breast cancer cells and the adjacent normal epithelial cells from Iranian women’s breast cancer tumors
As an experimental model, most studies rely on established human cancer cell lines; however, some genetical or phenotypical differences exist between these cells and their original tumor. Therefore, primary cells isolated directly from tissue are believed to be more biologically relevant tools for studying human and animal biology. Here, we aimed to isolate primary epithelial cancer and normal cells from breast tumors of Iranian women, for the first time. Thus, we isolated the epithelial and fibroblast cells from biopsy samples of patients with breast cancer based on differential centrifugation followed by culture in selective media. Normal epithelial cells obtained from the tissue biopsy away from the core of the tumor, based on the pathological diagnosis. Flow cytometry analysis indicated the positive immunoreactivity of the isolated epithelial cells against CD24 and Epithelial Specific Antigen (ESA/EpCAM), while they displayed a concomitant low expression of CD44 and CD49f. In contrat to fibroblasts, the qPCR data indicated the expression of luminal intracellular cytokeratin (Ck18) in both normal and cancer epithelial cells, but there was no expression of myoepithelial/basal markers, CK5 and vimentin. The epithelial cancer cells were reactive to cytokeratin 19 (CK19) antibody, whereas the normal epithelial cells were not. The expression of calmodulin-like protein (CLP) was also lower in the cancer epithelial cells than in the normal ones. In conclusion, primary epithelial normal and cancer cells, in addition to the fibroblasts were isolated and characterized from breast tumor of Iranian patients; and CLP expression is suggested as a susceptibility marker for breast cancer screening.
KeywordsPrimary breast cell CD24 CD44 EpCAM Cytokeratin CLP
This project was supported and funded by Iranian national Science Foundation (INSF) project No. 93036700. The authors are also thank full of the Research Console of Tarbiat Modares University for their support of this project. The authors are very thankful of Dr. Laleh Nikfarjam for her intellectual help during separation of the mentioned cells.
- Aigner S, Sthoeger ZM, Fogel M, Weber E, Zarn J, Ruppert M, Zeller Y, Vestweber D, Stahel R, Sammar M, Altevogt P (1997) CD24, a mucin-type glycoprotein, is a ligand for P-selectin on human tumor cells. Blood 89:3385–3395Google Scholar
- Bouzakri K, Roques M, Gual P, Espinosa S, Guebre-Egziabher F, Riou J-P, Laville M, Le Marchand-Brustel Y, Tanti J-F, Vidal H (2003) Reduced activation of phosphatidylinositol-3 kinase and increased serine 636 phosphorylation of insulin receptor substrate-1 in primary culture of skeletal muscle cells from patients with type 2 diabetes. Diabetes 52:1319–1325CrossRefGoogle Scholar
- Brooks MD, Bennett RD, Weaver AL, Sebo TJ, Eckert SE, Strehler EE and Carr AB (2013) Human calmodulin-like protein CALML3: a novel marker for normal oral squamous mucosa that is downregulated in malignant transformation. Int J Dent 2013:592843. https://doi.org/10.1115/2013/592843 CrossRefGoogle Scholar
- Ethier SP, Ammerman CA, Dziubinski ML (2000) Isolation and culture of human breast cancer cells from primary tumors and metastases. In: Ip MM, Asch BB (eds) Methods in mammary gland biology and breast cancer research. Springer, Boston, MAGoogle Scholar
- Freshney RI (1986) Animal cell culture: a practical approach. IRL Press, OxfordGoogle Scholar
- Martz E, Phillips H, Steinberg M (1974) Contact inhibition of overlapping and differential cell adhesion: a sufficient model for the control of certain cell culture morphologies. J Cell Sci 16:401–419Google Scholar
- Munz M, Fellinger K, Hofmann T, Schmitt B, Gires O (2007) Glycosylation is crucial for stability of tumour and cancer stem cell antigen EpCAM. Front Biosci J Virtual Libr 13:5195–5201Google Scholar
- Shah K (2015) Primary cells versus cell lines. https://sciencellonline.com/blog/primary-cells-versus-cell-lines/. Retrieved 27 Feb 2017
- Spizzo G, Went P, Dirnhofer S, Obrist P, Moch H, Baeuerle PA, Mueller-Holzner E, Marth C, Gastl G, Zeimet AG (2006) Overexpression of epithelial cell adhesion molecule (Ep-CAM) is an independent prognostic marker for reduced survival of patients with epithelial ovarian cancer. Gynecol Oncol 103:483–488CrossRefGoogle Scholar
- Taylor-Papadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane E, Leigh I (1989) Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J Cell Sci 94:403–413Google Scholar
- ZenBio Scientific Publications (2015) Primary cell culture: the game has changed! ZenBio Scientific Publications. http://www.zen-bio.com/pdf/PrimaryCells_whitepaper_ZenBio.pdf. Retrieved 27 Feb 2017