Isolation And Initial Identification Of Tumor Stem-like Cells From Human Prostatic Carcinoma Cell Line PC-3 Cells

Objective:.To isolate and identify prostate cancer stem-like cells from human prostatic carcinoma cell line PC-3 by Maitland's culture of serum-free, to set up the foundation for the further investigation of human prostate cancer stem cells (hPCSC).Methods:1. Prostatic carcinoma cell line PC-3 cells were cultivated in serum-free medium (SFM) supplemented with growth factor, while cells cultivated in serum-supplemented medium (SSM) served as the control.2. The proportion of prostate cancer stem-like cells that cultured in SFM and SSM were detected by flow cytometry.3. Prostate cancer stem-like cells were identified by cell immunofluorescence.4. The proliferation potentiality and doubling time of prostate cancer stem-like cells was calculated and compared with the common prostatic carcinoma cells PC-3 by MTT.5. The inducing differentiation process of prostate cancer stem-like cells was observed in serum-supplemented medium (SSM).6. The cell surface markers of the prostate cancer stem-like cells after differentiated were detected by cell dual immunofluorescence.Results:1.In serum-free medium (SFM) consisting of epidermal growth factor(EGF), basic fibroblast growth factor (bFGF)and human leukaemia inhibitory factor (LIF), the minority PC-3 cells could survive and form the suspended prostatic carcinoma cell spheres.2.The proportion of prostate cancer stem-like cells cultured in SFM was 1.43%, while the proportion of prostate cancer stem-like cells cultured in SSM was 0.41%, there was significant difference between them(P<0.05).3.The reproductive activity of prostate cancer stem-like cells was stronger than common prostatic carcinoma PC-3 cells ( 9.6×105 pieces vs 3×105 pieces ,P<0.05)and the doubling time of the former was shorter than the latter ( 21.90 h vs 28.38 h, P<0.05).4. The experiment of cell immunofluorescence showed that prostate cancer stem-like cells expressed CD133, which belongs to the surface antigen of hPCSC, but the common prostatic carcinoma PC-3 cells didn't express CD133. 5. Prostate cancer stem-like cell spheres could differentiate in SSM, and hypo-expressed the cell surface antigens of hPCSC CD44 and CD133 after differentiated.Conclusions:Prostatic carcinoma cell line PC-3 cells that cultured in vitro include a minority of prostate cancer stem-like cells, which express cell surface makers of hPCSC, possess characteristics of tumor stem cells, and could be enriched by serum-free medium (SFM).