Plate primary colony formation assay showed that CD133⁺EpCAM⁺ cells markedly enhanced bigger and more tumor colonies by 1.8-fold, 1.9-fold and 7.9-fold than CD133⁺EpCAM^-, CD133^-EpCAM⁺ and CD133^-EpCAM^- cells respectively. Secondary colony formation assay also showed that CD133⁺EpCAM⁺ cells formed bigger and more tumor colonies than other three cells (Fig. 3A). In soft agar colony formation assays, CD133⁺EpCAM⁺ cells induced bigger and greater numbers of tumor colonies than CD133^-EpCAM⁺, CD133⁺EpCAM^-, CD133^-EpCAM^- cells. The reading of fluorescence of CD133⁺EpCAM⁺ cells was higher than that of other three cells (Fig. 3B). The data suggested that CD133⁺EpCAM⁺ cells possessed stronger clonogenic ability than other three cells.

 Figure 3 

CD133⁺ and EpCAM⁺ phenotypes enhanced the differentiation of Huh7 cells. A. CD133^-EpCAM^- (R1), CD133^-EpCAM⁺ (R2), CD133⁺EpCAM^- (R3) and CD133⁺EpCAM⁺ (R4) phenotypes of Huh7 cells were isolated by flow cytometry sorting. B. The sorting purity of R1, R2, R3 and R4 was detected respectively. C. R1, R2, R3 and R4 phenotypes were incubated for 7 days, and analyzed by flow cytometry. Data were from two independent experiments.

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 Figure 4 

Colony formation ability was increased in CD133⁺EpCAM⁺ Huh7 cells. A. In plate colony formation assay, various phenotypes were cultured for 14-21 days, and then the primary colonies were replanted for another 14-21 days. Each experiment was performed three times. B. In soft agar colony formation assay, various phenotypes were planted on soft agar and cultured for 8-10 days. The stained colonies were photographed, and measured by the fluorescence reader. Data were from triple separate experiments. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, #P<0.05 to CD133⁺EpCAM^-.

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