The Research Of Tumor Stem/progenitor-like Cell From Human Ovarian Cancer Cell Line Of SK-OV-3

Objective:Isolation and identification of the tumor stem/progenitor-like cells is one of the key points in the field of study of tumor stem cell. In the present study, we managed to isolate the tumor stem/progenitor-like cells from human ovarian cancer cell line of SK-OV-3 by sphere-formation method in SGM, then to identify these cells in vitro and in vivo. The results of the study will facilitate the elucidation of tomorigenicity and guide the therapeutic strategies of ovarian carcinoma.Study Design:1.Human ovarian cancer cell line of SK-OV-3 was cultured in serum-free DMEM/F12 containing EGF, bFGF, hydrocortisone, B27 and insulin to isolate the tumor stem/progenitor-like cells.2.Sphere-formation cells isolated by SGM were induced to differentiate in the medium with 10% FBS supplemented.3.Soft agar test was performed to study the capacity for anchorage-independent growth of sphere-formation cells in vitro.4. The distribution of cell cycle of sphere-formation cell with FCM.5.The apoptosis ratio of sphere-formation cells treated with DDP of 10μg/ml was measured by FCM.6.Side population and ALDH positive cells of sphere-formation cell were detected.7.Real-time RT-PCR was performed to detect the expression of Oct4 and Nanog of sphere-formation cells.8.Tumorigenicity of sphere-formation cells were examined in the nude mice.Result:1. SK-OV-3 cell line of ovarian can be maintained in serum-free medium with floating-culture methods for at least fifty passages.2.Sphere-formation cells can be induced to differentiate and adhere to the bottom of the culture plate in the CM, besides morphology is similar to SK-OV-3 cells. 3.The results of clone-formation ratio in soft agar show that sphere-formation cells have strong anchorage independence growth.4.The result of cell cycle reveals that sphere-formation cells mostly exist G0/G1 phase.S.After treated with DDP at the point of 0,12,24,36 hours, the apoptosis ratio of sphere-formation cells is lower than SK-OV-3 cells, so sphere-formation cells were strong resistant to DDP.6.Side population cells and ALDH positive cells can be found in the sphere-formation cells and SK-OV-3 cell line, moreover sphere-formation cells is higher than the SK-OV-3 cells significantly.7.The result of real-time RT-PCR reveals that spheres-formation cells highly express Oct4 and Nanog.8.Transplantation of sphere-formation cells in nude mice indicated that sphere-formation cells have much stronger tumorigenicity than SK-OV-3 cells.Conclusion:Cells isolated from human ovarian cancer cell line of SK-OV-3 by sphere-formation method in SGM are thought to possess characters of stem/progenitor-like cells in vitro and strong tumorigenicity in nude mice. These data provided evidence of sphere-formation cells that have unique properties of tumor stem/progenitor-like cell.