Isolation And Characterization Of Cancer Stem Cells In Colorectal Cancer Cell Line

PART I PRELIMINARY STUDY ON CANCER STEM-LIKE CELLS (SIDE POPULATION) IN HUMAN COLORECTAL CANCER CELL LINESObjective:To investigate the content of cancer stem-like cells in different human colorectal cancer ce11 lines and explore the methods for enriching cancer stem-like cells in SW480.Methods:The percentage of side population cells(SP) in human colorectal cancer cell lines LOVO,SW480,HCT116 were detected by flow cytometry . SW480 cell line was cultivated in serum-free medium (SFM) supplemented with growth factors and cancer stem-like cells reforming into floating spheres were isolated. The isolated cancer stem-like cells were identified by limiting-dilution assay, differentiation assay, self-renewal assay, alternative cultivation assay.Results:The percentage of SP cells was 1.0% in LOVO, 1.2% in SW480 and 1.3% in HCT116.In the absence of serum, a minority (0.54%~0.62%) of cancer stem-like cells in SW480 cells survived, proliferated and assembled into the suspended tumor cell spheres. SW480 cancer stem-like cells possessed proliferative,self-renewal and differentiation potential, which were responsible for the floating tumor clone. 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 maintained their characters.Conclusion: LOVO,SW480 and HCT116 cell lines contain a tiny minority of SP cells with stem cell properties .The cancer stem-like cells in SW480 line can be maintained in SFM using a floating culture method . PART II ISOLATION,CULTURE AND CHARACTERIZATION OF COLON CANCER STEM CELLS IN LOVO CELL LINEObjective: To isolate,culture and Characterize the colon cancer Stem Cells with CD44+/EPCAMhigh phenotype in LOVO cell line , observe their biological behaviors and testify the existing of cancer stem cells in LOVO cell line.Methods: Flow cytometry was used to detect and isolate the colon cancer Stem Cells with CD44+/EPCAMhigh phenotype , which were cultured in serum-free medium added with growth factors(SFM) , next observed the proliferation capacity and finally seduced into differentiation with serum . The proliferative capability of these cells were estimated by MTT method,and the apoptosis ,cell cycle distribution was detected by flow cytometry. In the end , compare the neoplastic rates of CD44/EPCAM positive cells (Positive Group PG),EPCAM negative cells (Negative Group NG ) and unisolated cells (Control Group CG ) through transplanting the three groups cells in the immuno-deficient mice . The secondary tumors were determined the expression of CD44/EPCAM by Immunofluorescence technique.Results: There are 17.4percentage of CD44+/EPCAMhigh phenotype cells in the LOVO cell line , which can proliferate steadily,assemble into the suspended tumor cell spheres in SFM and be seduced to differentiate into adherent bulk cells in Serum Supplemented Medium (SSM). Compared with NG cells and CG cells, NG cells demonstrate increased proliferation capacity and have a group of cells major in G0/G1 cell cycle stage. Moreover , the neoplastic rate is 90%(9/10) transplanting 500 PG cells in immuno-deficient mice, with the comparison of 0 (0/10) with 105 NG cells transplanted . Meanwhile the expression of CD44/EPCAM also can be detected under the Fluorescence microscope.Conclusion: The colon cancer stem cells with CD44/EPCAM phenotype , maintaining proliferation steadily in SSF and being seduced to differentiation in SSF , can be isolated from LOVO cell line ,which makes a basis for future .