AIM:To investigate the expression of key biomarkers in hepatoma cell lines,tumor cells from patients’blood samples,and tumor tissues.METHODS:We performed the biomarker tests in two steps.First,cells plated on coverslips were used to assess biomarkers,and fluorescence intensities were calculated using the NIH Image J software.The measured values were analyzed using the SPSS19.0 software to make comparisons among eight cell lines.Second,eighty-four individual samples were used to assess the biomarkers’expression.Negative enrichment of the blood samples was performed,and karyocytes were isolated and dropped onto pretreated glass slides for further analysis by immunofluorescence staining.Fluorescence intensities were compared among hepatocellular carcinoma(HCC)patients,chronic HBV-infected patients,and healthy controls following methods similar to those used for cell lines.The relationships between the expression of biomarkers and clinical pathological parameters were analyzed by Spearman rank correlation tests.In addition,we studied the distinct biomarkers’expression with three-dimensional laser confocal microscopy reconstructions,and Kaplan-Meier survival analysis was performed to understand the clinical significance of these biomarkers.RESULTS:Microscopic examination and fluorescence intensity calculations indicated that cytokeratin 8/18/19(CK)expression was significantly higher in six of the seven HCC cell lines examined than in the control cells,and the expression levels of asialoglycoprotein receptor(ASGPR)and glypican-3(GPC3)were higher in all seven HCC cell lines than in the control.Cells obtained from HCC patients’blood samples also displayed significantly higher expression levels of ASGPR,GPC3,and CK than cells from chronic HBV-infected patients or healthy controls;these proteins may be valuable surface biomarkers for identifying HCC circulating tumor cells isolated and enriched from the blood samples.The stem cell-like and epithelial-mesenchymal transition-related biomarkers could be detected on the karyocyte slides.ASGPR and GPC3 were expressed at high levels,and thus three-dimensional reconstructions were used to observe their expression in detail.This analysis indicated that GPC3 was localized in the cytoplasm and membrane,but that ASGPR had a polar localization.Survival analyses showed that expression of GPC3 and ASGPR is associated with a patient’s overall survival(OS).CONCLUSION:ASGPR,GPC3,and CK may be valuable HCC biomarkers for CTC detection;the expression of ASGPR and GPC3 might be helpful for understanding patients’OS. AIM: To investigate the expression of key biomarkers in hepatoma cell lines, tumor cells from patients’ blood samples, and tumor tissues. METHODS: We performed the biomarker tests in two steps. First, cells plated on coverslips were used to assess biomarkers, and The fluorescence intensities were calculated using the NIH Image J software. The measured values ​​were analyzed using the SPSS 19.0 software to make comparisons among eight cell lines. Second, eighty-four individual samples were used to assess the biomarkers’expression.Negative enrichment of the blood samples was performed, and karyocytes were isolated and dropped onto pretreated glass slides for further analysis by immunofluorescence staining. Fluorescence intensities were compared among hepatocellular carcinoma (HCC) patients, chronic HBV-infected patients, and healthy controls following methods similar to those used for cell lines. The relationships between the expression of biomarkers and clinical pathological parameters were analyzed by Spear man rank correlation tests. In addition, we studied the distinct biomarkers’expression with three-dimensional laser confocal microscopy reconstructions, and Kaplan-Meier survival analysis was performed to understand the clinical significance of these biomarkers .RESULTS: Microscopic examination and fluorescence intensity calculations that cytokeratin 8/18/19 (CK) expression was significantly higher in six of the seven HCC cell lines examined than in the control cells, and the expression levels of asialoglycoprotein receptor (ASGPR) and glypican-3 (GPC3) were higher in all Seven HCC cell lines than in the control. Cells acquired from HCC patients’ blood samples also displayed significantly higher expression levels of ASGPR, GPC3, and CK than cells from chronic HBV-infected patients or healthy controls; these proteins may be valuable surface biomarkers for identifying HCC circulating tumor cells isolated and enriched from the blood samples. stem cell-like and epithelial-mesenchymal trans ition-related biomarkers could be detected on the karyocyte slides. ASGPR and GPC3 were expressed at high levels, and thus three-dimensional reconstructions were used to observe their expression in detail. This analysis shows that GPC3 was localized in the cytoplasm and membrane, but that ASGPR had a polar localization. Survival analyzes showed that expression of GPC3 and ASGPR is associated with a patient’s overall survival (OS) .CONCLUSION: ASGPR, GPC3, and CK may be valuable HCC biomarkers for CTC detection; the expression of ASGPR and GPC3 might be helpful for understanding patients’OS.