Abstract
Objective To isolate colorectal cancer stem cells from human fresh carcinoma samples obtained at surgical operation and to study their biological characteristics.Then to provide foundation for the follow-up experiments. Methods Colorectal cancer tissues obtained from surgically resected specimens of colorectal cancer patients were fully chopped, trypsinized, and filtered to prepare single cell suspensions.The cells were cultured in serum-free DMEM/F12 medium, with LIF, EGF, bFGF and 2% fetal bovine serum weekly.The stem cell-markers AC133 and EpCAM on the isolated cells were detected by using flow cytometry.The tumorigenic potent of the isolated cells was evaluated via the transplantation into Balb/C nude mice.All the variables were tested with T-test.P<0.05 was considered to be statistically significant. Results AC133+ EpCAM+ colorectal cancer stem cells were isolated and purified from the human primary colorectal carcinoma.The percentage of AC133+ EpCAM+ colorectal cancer stem cells in primary colorectal cancer was 1.5%~35%(mean 4.8%). There were colon cancer stem cells in colon cancer by limited dilution assay.The percentage was 2.18±0.15%.The suspension spheres were generated by the isolated AC133+ EpCAM+ Co-CSCs in serum-free medium.Their differentiation were induced in medium containing 10% FBS.AC133+ EpCAM+ CCSCs were implanted into Balb/C nude mice, and observed stronger capacity of tumor initiation.The percentage of AC133+ EpCAM+ cells in xenografts was 4.9%~40.2%(mean 17.2%). Human CRC xenografts established in Balb/C nude mice grew as differentiated carcinomas, which formed gland-like structures and scored positive for expression of colon-specific differentiation markers, such as cytokeratin-20(CK20), and neutral epithelial mucins. Conclusions There are colon cancer stem cells in human colorectal carcinoma.These cells are characterized by AC133+ EpCAM+ surface marker, which possess apparent feature of stem cells and stronger capacity of initiating tumors, moreover, generated spheroid in serum-free medium.These findings could provide foundation for the follow-up experiments. Key words: AC 133; Epithelial cell adhesion molecule; Colorectal cancer stem cells
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