Characterization of stemness properties of colonospheres in colon adenocarcinoma patients

Proliferation and expansion of cancer stem cells as spheroids were proved in previous studies. But, capability of primary tumor-derived stem cells to keep their unique properties in vitro is still disputed. So, the goal of this study was to isolate, expand and characterize of colon cancer-derived stem cells.In the present work, colon cancer stem cells markers including CD44 and EPCAM in spheroid and parental cells were analyzed by flow cytometry. The expression levels of stemness genes in both spheroid and parental cells were investigated using real-time PCR. Tumorigenic potential of spheroid cells was evaluated and used implantation of tumor xenografts into nude mice. Our data shows 79% of spheroids were CD44+/EpCAM+, while parental cells only expressed 20% of CD44/EpCAM markers (p< 0.01). In compared with the parental cells, the expression levels of ‘‘stemness’’ genes, like Sox2, Oct4, Nanog, C-myc, and Klf4 were significantly increased in spheroid cells (p< 0.05). Furthermore, as little as 1000 spheroid cells were sufficient to obtain tumor growth in nude mice, while 1x106 of parental cells was needed to generate tumor.Sphere formation assay is a useful method to enrich cancer stem cells. Spheroid cells showed increasing expression of stemness genes and tumorigenic activity in nude mice.