Primary Research On Cancer Stem Cell Biological Characteristics In Hepatocellular Carcinoma And Its Clinical Significance

Objective:(1) To evaluate the expression of cancer stem cells(CSCs) biomarkers in patients with hepatocellular carcinoma (HCC) and its correlation with tumor microvessel density. To explore the role of liver cancer stem cells (LCSCs) in the process of tumor metastasis and recurrence, and its association with clinicopathological characteristics, including objectively assess the prognosis of HCC.(2) To sort side population cells from hepatocellular carcinoma cell line HepG2and to analysis the biological characteristics differents between SP cells and original cell line HepG2.Methods:(1) Immunohistochemistry was used to detect the expression of CD90, CD133and CD34in tumor tissues from61patients with HCC undergoing curative resection.(2) Fluorescence-activated cell sorting (FACS) was used to sort side population (SP) cells from HepG2cell lines. We defined SP cells as experimental group and same generation HepG2cells which didn’t go through FACS as control group. Soft agar cloning methods were applied to measure the tumor formation ability of SP cell in vitro and Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments was performed to test the tumorigenicity of the two groups. The two groups tumor cells adhesion ability were detected by cell-matrix adhesion experiment. The distinction between the two groups in proliferation growth curve, migration capability and matastatic potential were evaluated by MTT assay, Matrigel invasion assay and migration assay. Suspension sphere formation assay were used to test the ensphere capacity with Seru-free medium. Results:(1) The positive rates of CD90and CD133in HCC tissues were29.5%(18/61) and31.1%(19/61), yet the positive rates of CD90and/or CD133in HCC were45.9%(28/61) The positive rate of LCSCs biomarkers in patients with vascular invasion, tumor differentiation Ⅲ/Ⅳ, non-complete encapsulation, and TNM stage Ⅲ/Ⅳ was significantly higher than those with non-vascular invasion, tumor differentiation Ⅰ/Ⅱ, with complete encapsulation, and TNM stage Ⅰ/Ⅱ. There were a significant positive correlation between LCSCs biomarkers expression and MVD (r=0.5, P<0.05). As compared with negative LCSCs expression combine MVDlow group, patients with a positive LCSCs biomarkers expression and MVDhigh in tumours had significantly lower overall survival (OS) and relapse-free survival (RFS).(2) Soft agar cloning assay showed that SP cells had significantly higher clonogenicity. NOD/SCID xenograft transplant experiments demonstrated that2×104SP cells were sufficient to form tumor as to control group2×106cells couldn’t form tumor. There was no significantly difference in adhesion ability, invasive potential and cell proliferation growth curve between the two groups. Meanwhile migration assay and sphere formation assay give evidence that SP cells were more capable at migration and ensphere.Conclusions:(1) High expression levels of LCSCs biomarkers are related to markers of tumor microvessel density (MVD). Combine two of those we can classify patients and anticipate the prognosis after surgery.(2) SP cells sorted from HepG2cell lines have cancer stem cell-like property and function, indicating that they may rich in hepatocarcinoma stem cells.