Establishment Of A Model To Enrich Colon Cancer Stem Cells

Objective:Determine the optimal growth factor concentration combination scheme and culture time for obtaining colon cancer stem cells by serum-free suspension culture in order to establish a technology model for enriching colon cancer stem cells,which is a basic research for colon cancer cancer stem cells,clinical research and targeted therapy research Provide important theoretical support.Methods:Suspension culture of colon cancer in serum-free medium(SFM)containing different concentrations of epidermal growth factor(EGF)and basic fibroblast growth factor(bFGF)DLD-1 cells to obtain spherical cells.The culture time gradient was set to 10 days,20 days,and 30 days,and the experiment was divided into 9 groups according to different concentrations of EGF and bFGF.Flow cytometry(FCM)was used to detect the expression ratio of CD44+,CD133+ and CD44+CD133+ double positive spheroid cells in the spheroids of each experimental group;Real-time PCR was used to detect tumor stemness-related genes KLF4,Lgr5,CD44,CD 133),epithelial-mesenchymal transition(EMT)genes(E-cadherin,Vimentin)and Wnt/β-catenine pathway genes(Wnt-3a)mRNA expression changes;ball formation experiment(Sphere-forming assay)detects the self-renewal ability of the cell sphere;the colony formation assay detects the tumor-forming ability of cells in vitro;the nude mouse subcutaneous tumor-forming experiment detects the tumor-forming ability of cells in vivo.Results:Flow cytometry(FCM)analysis:CD 133+and CD44+cells in the G9 group(20ng/ml EGF+20ng/ml bFGF)had the highest expression ratio at 30 days(F=123.554,99.528P<0.001),CD133+ CD44+ the expression ratio of double-positive cells was highest in G9 group at 30 days(except G3 group at 10 days and G6 group at 20 days)(F=57.897,P<0.001);Real-time PCR detected the relative mRNA expression results of each group of spherical cells:The expression of KLF4,Lgr5,CD44,CD 133,Vimentin and Wnt-3a was the highest in the G9 group at 30 days(F=22.682,25.401,3.272,7.852,13.331,17.445 P<0.001),and E-cadherin was expressed in the G9 group at 30 days The lowest amount(F=10.851,P<0.001);the Sphere-forming assay showed that the G9 group had the largest number of 30-day spheroid cells(F=19.147,P<0.001);the Colony formation assay also showed that the G9 group had the most 30d cells(F=60.767,P<0.01);The 30-day spheroid cells in the G9 group had the largest tumor volume in the nude mouse subcutaneous tumor formation experiment(F=12.539,P<0.01).Conclusions:20ng/ml EGF+20ng/ml bFGF can effectively enrich colon cancer cancer stem cells in suspension culture for 30 days,and has the highest efficiency compared with other combinations.