Abstract
The presence of circulating tumor cells (CTCs) in peripheral blood is associated with metastasis and prognosis in hepatocellular carcinoma (HCC) patients. The epithelial–mesenchymal transition (EMT) has a pivotal role in tumor invasion and dissemination. To identify more sensitive biomarkers for evaluating metastasis and prognosis, we investigated the expression of EMT markers, including vimentin, twist, ZEB1, ZEB2, snail, slug and E-cadherin in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues. After isolating viable CTCs from the peripheral blood of HCC patients using asialoglycoprotein receptors (ASGPRs), the CTCs were identified with immunofluorescence staining. CTCs were detected in the peripheral blood obtained from 46 of 60 (76.7%) HCC patients. Triple-immunofluorescence staining showed that twist and vimentin expression could be detected in CTCs obtained from 39 (84.8%) and 37 (80.4%) of the 46 patients, respectively. The expression of both twist and vimentin in CTCs was significantly correlated with portal vein tumor thrombus. Coexpression of twist and vimentin in CTCs could be detected in 32 (69.6%) of the 46 patients and was highly correlated with portal vein tumor thrombus, TNM classification and tumor size. Quantitative fluorescence western blot analysis revealed that the expression levels of E-cadherin, vimentin and twist in HCC tumors were significantly associated with the positivity of isolated CTCs (P=0.013, P=0.012, P=0.009, respectively). However, there was no significant difference in ZEB1, ZEB2, snail and slug expression levels in CTCs, primary HCC tumors and adjacent non-tumoral liver tissues across samples with regard to the clinicopathological parameters. Our results demonstrate that the EMT has a role in promoting the blood-borne dissemination of primary HCC cells, and the twist and vimentin expression levels in CTCs could serve as promising biomarkers for evaluating metastasis and prognosis in HCC patients.
Highlights
The epithelial–mesenchymal transition (EMT) is a complicated process that endows epithelial cells with enhanced metastatic and invasive potential.[11]
It has been reported that the EMT markers snail, twist and slug are expressed in Hepatocellular carcinoma (HCC), and the independent and collaborative effects of snail and twist on HCC metastasis have been confirmed.[18]
It has been reported that asialoglycoprotein receptor (ASGPR) is exclusively expressed in human hepatoma cell lines.[27]
Summary
The epithelial–mesenchymal transition (EMT) is a complicated process that endows epithelial cells with enhanced metastatic and invasive potential.[11]. CTCs spreading into the circulation appear to be a very heterogeneous population of cells with variable potential to establish distant metastasis.[20,21] CTCs disseminate from the primary tumor by undergoing phenotypic changes that allow the cells to penetrate blood vessels.[6,22] Aberrant activation of EMT has been implicated in this process, based on studies with a mouse model and human cancer cell lines.[23,24] A recent report addressing the correlation between EMT-marker expression in CTCs and breast cancer progression encourages future studies regarding the expression of EMT-related markers in CTCs and cancer progression.[25] recent studies have demonstrated that vimentin is expressed in the CTCs of breast and advanced prostate cancer patients.[11,26] it remains largely unclear whether EMT-related markers are expressed in CTCs or are involved in progression in the context of HCC. It would be extremely significant to investigate whether EMT-related markers are expressed in CTCs and whether their expression levels may serve as prognostic factors in HCC patients
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