Cultivation and Characterization of Tumor Spheres Generated from Human Colorectal Cancer Cell Lines

Objective To study on the cultivation method for tumor spheres from colorectal cancer cell lines and identify whether resulting Colo205 spheroid cells display cancer stem cell characteristics. Methods Lovo,Colo205 and SW480 cells were seeded in serum free medium (SFM) with EGF and bFGF. Flow cytometry analysis,cell invasion assay and xenograft experiment were applied to examine the cell surface marker expression pattern,cell invasive ability and in vivo tumorigenicity of both Colo205 spheres and parental cells. CD44 expression of tumor spheroid cells was also analyzed after cultured with serum supplemented medium by flow cytometry. CD44,Musashi-1 and Oct4 mRNA were detected in these two cells by RT-PCR. Results Tumor spheres could be generated from three colorectal cancer cell lines in SFM. The formation and proliferation of tumor spheres were benefited from fresh SFM,cell dissociation reagent Accutase and the floating status of cancer cells. The overwhelming majority of spheroid cells were CD44+ cells. But CD44+ cells were gradually decreased when spheres cultured with serum supplemented medium. Colo205 spheres have higher Musashi-1 and Oct4 mRNA expression,tumor-initiating capability and invasive ability compared with those of parental cells. Conclusion Tumor spheres in which enrich cancer stem cells can be generated and matained from colorectal cancer cell lines in SFM on floating-culture condition.