Primary Study On The Characterization And Surface Marker Of Cancer Stem-like Cells In Hepatocellular Carcinoma Cell Lines

Object i ve:To establish an effective method for enriching hepatocarcinoma stem cells and to analysis the characteristics and surface marker of hepatocarcinoma stem cells. Theses research will facilitate the elucidation of essence of hepatocarcinoma stem cells.Methods:1. To confirm the existence of hepatocarcinoma stem cells, the percentage of side population cells (SP) in human hepatocellular carcinoma cell lines Hep3B, HepG2and SK-Hep-1were detected by Flow Cytometry. SK-Hep-1cell line was cultivated in serum-free medium(SFM) supplemented with growth factors and cancer stem-like cells reforming into floating spheres were isolated.2. Cell proliferation and cell cycle of spheroid cells were detected by MTS and Flow Cytometry. The expression of cell cycle regulator was detected by Western Blot.3. Colony formation assay was conducted to identify the self-renewal and propagation abilities of both spheroid cells and SK-Hep-1cells in vitro. Spheroid cells were induced to differentiate in the medium supplemented with10%FBS and extensive differentiation ability of spheroid cells was observed from cellular morphology.4. MTS and Flow Cytometry assay were conduced to detect drug resistance and anti-apoptosis of spheroid cells treated with DDP. To explore the resistance mechanism, the mRNA and protein levels of ABCG2、MDR1and MRP1in spheroid cells were detected by Real-time PCR and Flow Cytometry.5. The hTERT mRNA level was detected by Real-time PCR and telomerase activity was detected by TRAP-PCR argentation. The mRNA and protein levels of Oct4, Nanog, BMI-1, Notchl, B-catenin and SMO in spheroid cells were detected by Real-time PCR and Western Blot.6. Tumor formation assay was conducted to identify tumorigenicity of both spheroid cells and SK-Hep-1cells in NOD/SCID mice.7. MTS and transwell invasion assay were conducted to detect the adhesion and invasiveness of spheroid cells.8. The expression of CK7, CK19, CK8and CK18in spheroid cells were detected by immunohistochemisty.9. Flow Cytometry was used to detected the expression of cell surface marker (CD133, CD117, CD90, CD34, CD44and EpCAM) in spheroid cells. Western Blot and immunohistochemisty were detected the expression of CD117in spheroid and SK-Hep-1cells. Flow Cytometry was used to detected the expression of CD117in the process of spheroid cell differentiation.Results:1. SP cells in SK-Hep-1cell were detected in1.20%, and not detected in HepG2and Hep3B. Human hepatocarcinoma SK-Hep-1cell could be maintained in serum-free medium with floating-culture methods with successive generations.2. Most spheroid cells were in silent period, and using Western Blot analyses, we found that spheroid cells isolated from SK-Hep-1cells expressed higher protein levels of p21and p27, and lower protein levels of cyclinD.3. Spheroid cells possessed the capability for maintenance of high clonogenicity. Cloning efficiency of spheroid cells (51.63%) was higher than SK-Hep-1cells (17.54%). Spheroid cells could be induced to differentiate and adhere to the bottom of the culture plate, which morphology was the same as SK-Hep-1cells.4. After treated with various chemotherapeutics, cell viability of spheroid cells is higher than SK-Hep-1cells, with a dose-dependent effect. Apoptosis rate of spheroid cells treated with20μg/ml DDP for48hrs (22.92%) is lower than SK-Hep-1cells (63.55%). Using Western Blot analyses, we found that spheroid cells highly expressed the mRNA and protein levels of ABCG2, MDR1and MRP1, when compared with SK-Hep-1cells.5. Spheroid cells possessed higher hTERT mRNA level and telomerase activity compared with SK-Hep-1cells, and highly expressed mRNA and protein levels of Oct4, Nanog, BMI-1, Notch1and SMO. However, B-catenin protein level of spheroid cells is not higher than SK-Hep-1cell, even though its mRNA level is higher.6. NOD/SCID mice injected1000spheroid cells could generate tumors and4/4mice injected7000spheroid cells developed tumors. Only more than10000SK-Hep-1cells could generate tumors by subcutaneous injection. These results indicated the high capability for tumor formation of spheroid cells.7. Spheroid cells had higher adhesion and invasiveness than SK-Hep-1cells.8. Spheroid cells could differentiate into CK8-CK18+/CK7-CK19+cells from CK8+CK18+/CK7-CK19-cells, which was the same as SK-Hep-1cells. These results also indicated the extensive differentiation ability of spheroid cells.9. Flow Cytometry experiment revealed that the expression of CD117in spheroid cells is only about1%, and CD117in SK-Hep-1cells is95%. The change is greatly significant. Western Blot and immunohistochemisty results were consistent with the result of Flow Cytometry. And CD117increased gradually along with the cultivation of differentiated spheroid cells. Other markers in spheroid cells were not different, when compared with SK-Hep-1cells.Conclusion:SK-Hep-1cell was heterogeneity and could be maintained in serum-free medium with floating-culture methods. Spheroid cells isolated from SK-Hep-1cells possessed unique properties of self-renewal, longevity, extensive differentiation, higher expression of sternness genes, stronger drug resistance and metastasis in vitro and much stronger tumorigenicity in vivo. Spheroid cells were proved to be the cells with characteristics of cancer stem-like cells in this experiment. Therefore, we presume that spheroid cells may be cell population enriching hepatocarcinoma stem cell, also called hepatocarcinoma stem/progenitor-like cells, and whether CK7-CK19-CD117-is the surface marker of hepatocarcinoma stem cell will be studied in future.