Isolation And Identification Of Cancer Stem-like Cells From Colorectal Cancer Cell Line HCT116

ObjectiveTo isolate clone spheres from colon cancer cell line HCT116 though the limited dilution and serum-free method.By detecting the expression of tumor stem cell markers and the distribution of cell cycle besides soft agar colony forming experiment to identify these spheres with cancer stem-like cells.To prove that colon cancer stem-like cells are more resistant to drugs than HCT116 cells through corresponding drugs intervention experiment.Methods1.Using the limited dilution and serum-free method to get single-cell-derived monoclonal cell clone spheres,and Self-renewness and differentiation of cancer stem-like cells were demonstrated by cultivating these cell clone spheres with serum-free medium or serum-supplemented medium from colon cancer cell line HCT116.2.Flow cytometry was conducted to detect the expression of CD133?CD44 in the clone spheres.To go a step further,soft agar colony forming experiments were performed to verify the self-renewness of cancer stem-like cells.3.Method CCK-8 is used to find out whether the growth inhibition of colorectal cancer stem-like cells can be induced by anti-cancer drugs such as 5-fluorouracil and Huaier cream,and to figure out the difference of drug resistance between stem-like cells and HCT116 cells.Results1.We isolated 14 cancer stem-like cell clone spheres fortunately from HCT116 using the limited dilution and serum-free method.The sphere-forming efficiency is 28.6%.These cancer stem-like cells of clone spheres showed us the characteristics of rapid proliferation,re-differentiation and higher repeat sphere-forming rate(43.2%).2.In flow cytometry,expressions of CD133+CD44+ in clone spheres and HCT116 are respectively 93.87%and 87.97%.The proportions of cells in G1 of clone spheres and HCT116 were77.18%and 49.00%respectively,and statistical difference between the two groups is significant.In soft agar colony forming experiments,ball formation rates of cells from two clone spheres were 51.97%and 39.54%,which were obviously higher than the original plant cell clone formation rate 5.67%,difference between the two groups with statistical significance.3.In drug resistance experiment,with the increasing of concentration of drugs,inhibition on the proliferation of cancer cells was reinforcing.Moreover,cancer stem-like cells showed stronger drug resistance than HCT116.Conclusion1.Cancer stem-like cells can be isolated from colorectal cancer cell line HCT116.2.Compared with HCT116 cells,cancer stem-like cells have both higher expression rate of CD133+CD44+ and proportion of cells in G1,showing higher ball formation rates,demonstrating a more resistance to drugs.