Objective:To generate cancer spheres from five different colorectal cancer cell lines in serum-free medium(SFM)

stably subculture them in vitro

and study their biological characteristics of proliferation

differentiation and metastasis.Methods:Human colorectal cells SW480

SW620

LOVO

HT29 and HCT116 were cultured in SFM with supplemented cell growth factors;stable subculture was induced to differentiate in serum supplemented medium(SSM).The expression of CD133+ in HCT116 monolayer cells and spheres were investigated by flow cytometry.Cell metastasis assay

immunofluorescence and RT-PCR were applied to examine cell metastasis ability in vitro and the expression of mesenchymal markers N-cadherin and Vimentin of both HCT116 monolayer cells and spheres.Results:Cancer spheres could be generated from five colorectal cancer cell lines in SFM.These spheres were stably subcultured and induced to differentiate by serum supplemented medium

and these differentiated cells were in adherent growth and kept the same morphology with the parental cells.HCT116 spheres

compared with parental cells

had higher CD133+

N-cadherin and Vimentin expressions as well as metastasis ability.Conclusion:Colorectal cancer spheres can be generated in SFM culture

and these cancer spheres are rich in cancer stem-like cells.