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Objective Side population (SP) cells may play a crucial role in tumorigenesis and the recurrence of cancer.Many kinds of cell lines and tissues have demonstrated the presence of SP cells, including colon cancer cell lines.This study is aimed to identify the cancer stem cells in the SP of colon cancer cell line SW480.Methods Side population (SP) cells were isolated by fluorescence activated cell sorting(FACS), followed by serum-free medium (SFM) culture, using colon cancer cell line.The self-renewal, differentiated progeny, clone formation, proliferation, invasion ability, cell cycle, chemosensitivity, and tumorigenic properties in SP and non-SP (NSP) cells were investigated through in vitro culture and in vivo serial transplantation.The expression profiles of ATP-binding cassette (ABC) protein transporters and stem cell related genes were examined by RT-PCR and Western blot.Results Human colon cancer cell lines LOVO, HCT-116, SW480 contain 0.93% ±0.11, 1.33% ± 0.05 and 1.1%-± 0.10 side population (SP) cells, respectively.We further investigated cancer stem cell characteristics with human colon cell line SW480.Flow cytometry analysis revealed that side population(SP) cells could differentiate into side population and non-side population (NSP)cells and could exhibit differentiation potential.SP cells were shown to have a higher proliferation potency and CFE than NSP cells.Compared to NSP cells, SP cells were also more resistant to CDDP and 5-FU, and were more invasive, and displayed increased tumorigenic ability.Moreover, SP cells showed higher mRNA and protein expression of ABCG2, MDR1,OCT-4, NANOG, SOX-2, NOTCH-1 ,CD44 and CD133.Conclusion Side population cells isolated from human colon cancer cell lines harbor cancer stem cell properties that may be related to the invasive potential and therapeutic-resistance of colon cancer.