Background: Cancer stem cells are subpopulation of cancer cells that show self-renewal potential and the capacity to differentiate into diverse populations comprising a tumor. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free media. The aim of this study was isolation of colon cancer stem cells by sphere formation assay and characterization of them in human colonic adenocarcinoma HT-29.

Materials and Methods: In this experimental study, colon CSCs markers including CD44 and EPCAM in spheroid and HT-29 cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and HT-29 cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated using in vivo xenografts assay.

Results: Our data showed over 92% of spheroids were CD44+/EpCAM+, while HT-29 cells only have expressed 37% of CD44/EpCAM markers. In compared with the HT-29 cells, expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells (p< 0.05). Further, As little as 2500 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x106 of HT-29 cells was needed to form tumor.

Conclusion: Our data suggest that spheroid formed by colon cancer cell lines highly enriched in CSCs and showed increasing expression of stemness genes and tumorigenic in nude mice.