CD133(+)EpCAM(+) phenotype possesses more characteristics of tumor initiating cells in hepatocellular carcinoma Huh7 cells

Abstract

Background: EpCAM or CD133 has been used as the tumor initiating cells (TICs) marker in hepatocellular carcinoma (HCC). We investigated whether cells expressing with both EpCAM and CD133 surface marker were more representative for TICs in hepatocellular carcinoma Huh7 cells.

Methods: Four different phenotypes of CD133(+)EpCAM(+), CD133(+)EpCAM(-), CD133(-)EpCAM(+) and CD133(-)EpCAM(-) in Huh7 cells were sorted by flow cytometry. Then cell differentiation, self-renewal, drug-resistance, spheroid formation and the levels of stem cell-related genes were detected to compare the characteristics of TICs. The ability of tumorigenicity was measured in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice to verify TICs.

Results: CD133(+)EpCAM(+) cells have many characteristics of TICs in Huh7 cells compared with CD133(+)EpCAM(-), CD133(-)EpCAM(+), CD133(-)EpCAM(-) cells, including enrichment in side population cells, higher differentiation capacity, increased colony-formation ability, preferential expression of stem cell-related genes, appearance of drug-resistant to some chemotherapeutics, more spheroid formation of culture cells and stronger tumorigenicity in NOD/SCID mice.

Conclusion: CD133(+)EpCAM(+) phenotype is precisely represented TICs in Huh7 cells. It might be useful for studying biology mechanism of TICs in hepatocellular carcinoma and screening new targets for cancer therapy.

Keywords: CD133; EpCAM.; Hepatocellular carcinoma; Tumor initiating cells.

Figure 1

Expression of CD133 and EpCAM in hepatocellular carcinoma (HCC) cells. A. The surface expression of CD133 and EpCAM were detected by flow cytometry in various HCC cells. B. The protein levels of CD133 and EpCAM were detected by western blot. β-tublin was used as an internal control.

Figure 2

Expression of CD133 and EpCAM in normal and SP Huh7 cells. A. In immunofluorescence analysis, Huh7 cells were stained with anti-CD133 (red) and anti-EpCAM (green) antibodies. Nuclei (blue) are labeled with Hoechst 33358 dye. B. SP cells were identified as the poorly staining cell population (black triangle P2) that largely disappeared when verapamil was used. Then expression of CD133 and EpCAM were detected by flow cytometry in enriched SP and non-SP cells. SP, side population.

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.

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^-.

Figure 5

Enhanced proliferation and drug-resistance were observed in CD133⁺EpCAM⁺ cells. A and B. Various phenotypes were treated without (A) or with (B) doxorubicin (DOX) or 5-fluorouracil (5-Fu) for 48 h, and then cell proliferation was detected by SRB assay. Data were from triple independent experiments. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, **P<0.01 to CD133^-EpCAM^-, ††P<0.01 to CD133^-EpCAM⁺.

Figure 6

CD133⁺EpCAM⁺ cells possessed other characteristics of TICs. A. Various phenotypes were collected, and then the expression of Nanog, Sox2 and Oct4 were measured by real-time PCR. Data were from triple independent experiments. B. Cell spheres were imaged by microscope (x200 fields) after cultured in modified medium for 7 days. Data were shown as mean ± SD of three independent experiments. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, #P<0.05 to CD133⁺EpCAM^-.

Figure 7

Raised tumorigenicity of CD133⁺EpCAM⁺ cells was detected in NOD/SCID mice. A. The indicated numbers of various phenotypes were injected subcutaneously in NOD/SCID mice for 90 days. The incidence of tumors was examined bi-weekly. B. 1,000 CD133⁺EpCAM⁺ or CD133^-EpCAM^- cells were injected in the indicated place for 90 days. C. The tumors were stained with hematoxylin-eosin. *P<0.05 to CD133^-EpCAM^-, †P<0.05 to CD133^-EpCAM⁺, #P<0.05 to CD133⁺EpCAM^-.