Establishment of six new human biliary tract carcinoma cell lines and identification of MAGEH1 as a candidate biomarker for predicting the efficacy of gemcitabine treatment

Abstract

The aim of this study was to establish new biliary tract carcinoma (BTC) cell lines and identify predictive biomarkers for the potential effectiveness of gemcitabine therapy. Surgical specimens of BTC were transplanted directly into immunodeficient mice to establish xenografts, then subjected to in vitro cell culture. The gemcitabine sensitivity of each cell line was determined and compared with the genome-wide gene expression profile. A new predictive biomarker candidate was validated using an additional cohort of gemcitabine-treated BTC cases. From 55 BTC cases, we established 19 xenografts and six new cell lines. Based on their gemcitabine sensitivity, 10 BTC cell lines (including six new and four publicly available ones) were clearly categorized into two groups, and MAGEH1 mRNA expression in the tumor cells showed a significant negative correlation with their sensitivity to gemcitabine. Immunohistochemically, MAGEH1 protein was detected in three (50%) out of six sensitive cell lines, and four (100%) out of four resistant cell lines. In the validation cohort of gemcitabine-treated recurrence cases, patients were categorized into "effective" and "non-effective" groups according to the RECIST guidelines for assessment of chemotherapeutic effects. MAGEH1 protein expression was detected in two (40%) out of five "effective" cases and all four (100%) "non-effective" cases. We have established a new BTC bioresource that covers a wide range of biological features, including drug sensitivity, and is linked with clinical information. Negative expression of MAGEH1 protein serves as a potential predictive marker for the effectiveness of gemcitabine therapy in BTC.

Figure 1

Cell morphology and tumor histology of primary specimen/xenograft of established new biliary tract carcinoma cell lines. In vitro cell morphology and tumor histology (H&E staining) of resected primary specimens, xenografts of primary tumor samples and xenografts of cell lines are shown. Scale line = 200 μm.

Figure 2

Sensitivity to gemcitabine (Gem) in 10 biliary tract carcinoma cell lines. Ratio of cell proliferation compared to the control (treated with DMSO) at each concentration (μM) of Gem was plotted. Note that 10 cell lines are clearly segregated into two groups (Gem‐sensitive and Gem‐resistant) with distinct Gem sensitivity.

Figure 3

(A) MAGEH1 mRNA expression in 10 biliary tract carcinoma cell lines. Relative expression of MAGEH1 mRNA compared to GAPDH expression in each cell line was quantified by microarray (blue columns) and quantitative RT‐PCR (red columns). MAGEH1 expression was significantly different between gemcitabine (Gem)‐sensitive and Gem‐resistant groups. (B,C) Immunohistochemical analysis of MAGEH1 protein in xenograft specimens of 10 biliary tract carcinoma cell lines. Tumor histology (H&E staining) of xenograft specimens of cell lines, split into Gem‐sensitive (B) and Gem‐resistant (C) groups, and MAGEH1 protein expression detected by anti‐MAGEH1 antibody in the same area are shown. All three cell lines that lacked MAGEH1 expression belong to the Gem‐sensitive group. Scale line = 200 μm.

Figure 4

Immunohistochemical analysis of MAGEH1 protein in primary tumor specimens of gemcitabine (Gem)‐effective and non‐effective groups. Tumor histology (H&E staining) of primary tumor specimens, split into Gem‐effective (A) and Gem‐non‐effective (B) groups and MAGEH1 protein expression detected by anti‐MAGEH1 antibody in the same area are shown. All three cases that lacked MAGEH1 expression belong to the Gem‐effective group. Scale line = 200 μm.