Isolation And Initial Identification Of Human Ovarian Cancer Stem Cells

To isolate and identify ovarian cancer stem cells from human ovarian cancer cell line SKOV-3 and to identify their biological properties.Methods SKOV-3 cells were cultured in serum free medium (SFM) with recombinant human epidermal growth factor (EGF) and basic fibroblast growth factor(bFGF)to derive tumor spheres. Flow cytometry analysis and cell invasion assay were applied to examine the cell surface marker expression pattern(CD44, CD117)and cell invasive ability of both tumor spheres and parental cells; Tumor spheres were then expanded and cultured in serum-supplemented medium (SSM) to permit their differentiation. The isolated cancer stem-like cells were identified by self-renewal assay and alternative cultivation assay. The proliferative capacity and drug resistance of tumor sphere-forming cells were tested by cell counting kit-8 (CCK-8) assay.Results (1) Small number of floating tumor spheres were isolated and expanded in serum free medium(SFM). These tumor spheres attached to the bottom of culture plates and began to differentiate in serum-supplemented medium (SSM). Serum addition into SFM resulted in the proliferation of cancer stem-like cells; after several generations and alternated cultivation in SSM and SFM, cancer stem-like cells still maintained their characters. (2) CD44 and CD117 antigens were significantly higher in tumor spheres than those in the common SKOV-3 cells, and invasive ability compared with those of parental cells (P<0.05). (3)By contrast with the differentiated cells under standard serum-containing culture conditions, these sphere-forming cells were more resistant to cisplatin after 24,48 and 72 hours of treatment (2.3%VS 9.1%,8.6%VS 22.8%and 18.7% VS 36.8%,P<0.05)Conclusion (1) In this study, it was found that there do exist a small sub-population of cells in ovarian cancer SKOV-3 cell line that could self-renew and proliferate continuously. The cells could form tumor spheres and passage in serum free medium (SFM). Moreover, Ovarian cancer stem cells (OCSCs) can express significantly CD44 and CD117 antigens. CD44 may be a specific antigen marker for the human ovarian cancer stem cells. The cancer stem-like cells in SKOV-3 line can be maintained in SFM using a floating culture method. (2) By contrast with the differentiated cells under standard serum-containing culture conditions, these sphere-forming cells were more invasive and resistant.