Study On Preliminary Enriching Human Colon Cancer Cell Line Caco-2Stem Cell By Using Serum-free Culture Method

Study BackgroundIn recent years, the morbidity of colon cancer has increased. Because the effect of treatment is limited, tumor metastasis and recurrence are common in clinical. Cancer stem cell is considered as the root of tumor occurrence、 development recurrence and metastasis, further clarify its generation mechanism and biological character will bring the new hope to colon cancer diagnosis、treatment、prevention and condition assessment. However, how to isolate and purify colon cancer stem cell effectively is the primary technical difficulty to face.ObjectivePreliminarily enrich human colon cancer cell line Caco-2stem cell by using serum-free culture method and investigate the feasibility, hoping to provide an idea of separating colon cancer cell line Caco-2stem cell effectively, also to build a new stem cell model for the study on colon cancer.MethodsCulture human colon cancer cell line Caco-2cell in serum-free medium, so as to enrich Caco-2stem cell preliminary, observe the process of growth and proliferation, seduce differentiation by alternative cultivation in serum supplemented medium and serum-free medium, verify the drug resistance by fluorouracil and oxaliplatin, detect the expression of colon cancer stem cell’s special marker—CD133by immunofluorescence staining.Results1. One part of Caco-2cells proliferated and formed into the suspended cancer stem cell spheres in serum-free medium,and the other part of the cells died due to can’t resist serum-free condition.2. Caco-2cancer stem cell spheres maintained their characters after several generations in serum-free medium,and after that, they growed fast,became regular,also had great quantity.3. Caco-2cancer stem cell spheres disintegrated gradually and differentiated into adherent monolayer cells in serum supplemented medium.4.Culture Caco-2cancer stem cell spheres alternately in serum supplemented medium and serum-free medium, they changed between adherent monolayer cells and cancer stem cell spheres.5.Digested Caco-2cancer stem cell spheres into single cells, cultured in serum-free medium combined chemotherapy drug intervention, one part of the cells were killed, and the other part of the cells survived and formed into spheres again. Compared with Non-drug intervention group which using simple serum-free culture, their spheres grew fast, had great quantity and big volume.6.The quantity of Caco-2cancer stem cell spheres in Non-drug intervention group、 fluorouracil intervention group、 oxaliplatin intervention group、 fluorouracil combined oxaliplatin intervention group were (10.0±1.15) spheres per well、(16.1±1.52) spheres per well、(17.5±1.43)spheres per well、(18.8±1.14)spheres per well. The quantity of spheres in Non-drug intervention group was less than three experimental groups (P<0.01) respectively. The quantity of spheres in fluorouracil intervention group was less than oxaliplatin intervention group (P<0.05) and fluorouracil combined oxaliplatin intervention group (P<0.01) respectively. The quantity of spheres in oxaliplatin intervention group was less than fluorouracil combined oxaliplatin intervention group (P<0.05)7. Immunofluorescence staining indicated that Caco-2cancer stem cell spheres showing the orange-red fluorescence, they were positive in CD133, and the positive cells were not found in negative control.Conclusions1. Human colon cancer cell line Caco-2cell can proliferate and form into the suspended cancer stem cell sphere in serum-free medium.2. Caco-2cancer stem cell sphere has the capability of self-renewal、 constantly proliferation、 differentiation and drug resistance. 3. Serum-free culture combined chemotherapy drug intervention is higher than pure serum-free culture in enrichment capability of Caco-2stem cell,and oxaliplatinis is higher than fluorouracilin in enrichment capability, fluorouracil combined oxaliplatin is higher than single-drug in enrichment capability.4. Caco-2cancer stem cell spheres can express CD133,which is the special marker of colon cancer stem cell.5. The stem cell-like cell in human colon cancer cell line Caco-2can be enriched by using serum-free culture method.