Intrahepatic Cell Lines Research Articles

Galectin-9 expression and decreased survival in advanced biliary tract cancers.

465 Background: Biliary tract cancers (BTCs) are a group of rare and aggressive malignancies often refractory to chemotherapeutic or targeted therapies, carrying an overall poor prognosis and high mortality. Identifying prognostic biomarkers and effective therapeutic targets for this disease therefore remains a high priority. T-cell immunoglobulin mucin domain-containing protein 3 (TIM-3), a negative regulatory immune checkpoint receptor, is known to induce immune tolerance and inhibit T-cell antitumor immunity. Its ligand, galectin-9 (Gal-9), has been shown to play a paradoxical role in tumorigenesis in a number of disease states. However, the role of Gal-9 in the setting of BTCs is not completely understood. Methods: We examined expression patterns of total cellular Gal-9 and TIM-3 in a panel of human BTC cell lines with diverse molecular profiles via immunoblot. Soluble Gal-9 (sGal-9) was measured by enzyme-linked immunoassay (ELISA). Tetrazolium-based MTT assay was utilized to evaluate cell viability following Ab-mediated Gal-9 neutralization. Pre-treatment peripheral blood was obtained from patients with metastatic BTCs enrolled in a randomized phase II clinical trial (NCI10139) and sGal-9 levels were measured. ROC curve analysis was employed to identify an optimal concentration cut-point value. Kaplan-Meier models were used to assess the association between sGal-9 levels and overall survival. Results: Human BTC cells demonstrate detectable and differential expression levels of total Gal-9 and TIM-3 proteins in vitro, with greater expression among intrahepatic cell lines (HUCCT-1, HUH28, SNU478). TIM-3 expression was not detected among extrahepatic (MzChA-1) and gallbladder cancer (WITT) cell lines. sGal-9 is secreted at variable levels between cell lines and its neutralization does not affect viability. Pre-treatment peripheral blood was obtained from 73 patients with metastatic BTCs (53% (n = 39) intrahepatic cholangiocarcinoma, 22% (n = 16) extrahepatic cholangiocarcinoma, 25% (n = 18) gallbladder cancer). There was no significant difference in sGal-9 levels between disease sites (p = 0.08). Patients were dichotomized by low and high sGal-9 levels. High baseline plasma levels of sGal-9 were associated with worse overall survival among patients with advanced BTCs (p = 0.03). Conclusions: Biliary tract cancer cells differentially express galectin-9 and its ligand, TIM-3, in vitro. Higher plasma levels of soluble galectin-9 are associated with worse overall survival, suggesting galectin-9 expression may be related to a more aggressive disease state. Additional work is needed to better inform the mechanistic role of the galectin-9/TIM-3 axis on disease progression and how best to leverage this pathway as a therapeutic target in the management of biliary tract cancers.

Preparation of mouse anti-human asialoglycoprotein receptor monoclonal antibody and its preliminary application

Objective To prepare a mouse monoclonal antibody against human asialoglycoprotein receptor(ASGPR),and to apply it for detecting ASGPR expression in cell lines and tissues.Methods The structure of ASGPR H1major subunit was analyzed and the full length of ASGPR1was selected to synthesize immunizing peptide.cDNA was amplified by RT-PCR and then subcloned into prokaryotic vector pGEX-4T-1.The recombinant protein was expressed by E.coli BL21and purified for subsequent immunization.The conventional hybridoma technique was used to generate mouse monoclonal antibody.The isotype and the titer were regularly tested.Inhibition experiment was conducted to identify the specific binding of the antibody to ASGPR.Finally,the expression of ASGPR was detected in various intra-hepatic and extra-hepatic cell lines by flow cytometry and in different liver tissues by immunohistochemistry method.Results Monoclonal antibody against human ASGPR was successfully prepared and was identified as IgG1,with the titer reaching 112 800.Inhibition experiment indicated a satisfactory specific binding of the antibody to ASGPR and that the recognition epitope was located in the extracellular domain of ASGPR.Flow cytometric analysis showed various levels of ASGPR expression in intra-hepatic cell lines,but not in extra-hepatic cell lines.Immunohistochemistry detection showed that ASGPR was specifically expressed in the normal liver tissues and hepatocellular carcinoma(HCC)tissues,and the expression in HCC tissues was associated with the differentiation degree,with the expression being significantly higher in well-differentiated HCC than that in the poorly-differentiated HCC(75.0% vs 28.6%,P0.05).Conclusion We have successfully prepared the monoclonal antibody against human ASGPR with high specificity;the antibody can be used for flow cytometric analysis and immunohistochemistry detection of ASGPR and for clinical distinguish of primary or metastatic liver cancer.