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Gallbladder cancer (GBC), a rare but lethal disease, is often diagnosed at advanced stages. So far, molecular characterization of GBC is insufficient, and a comprehensive molecular portrait is warranted to uncover new targets and classify GBC. We performed a transcriptome analysis of both coding and non-coding RNAs from 36 GBC fresh-frozen samples. The results were integrated with those of comprehensive mutation profiling based on whole-genome or exome sequencing. The clustering analysis of RNA-seq data facilitated the classification of GBCs into two subclasses, characterized by high or low expression levels of TME (tumor microenvironment) genes. A correlation was observed between gene expression and pathological immunostaining. TME-rich tumors showed significantly poor prognosis and higher recurrence rate than TME-poor tumors. TME-rich tumors showed overexpression of genes involved in epithelial-to-mesenchymal transition (EMT) and inflammation or immune suppression, which was validated by immunostaining. One non-coding RNA, miR125B1, exhibited elevated expression in stroma-rich tumors, and miR125B1 knockout in GBC cell lines decreased its invasion ability and altered the EMT pathway. Mutation profiles revealed TP53 (47%) as the most commonly mutated gene, followed by ELF3 (13%) and ARID1A (11%). Mutations of ARID1A, ERBB3, and the genes related to the TGF-β signaling pathway were enriched in TME-rich tumors. This comprehensive analysis demonstrated that TME, EMT, and TGF-β pathway alterations are the main drivers of GBC and provides a new classification of GBCs that may be useful for therapeutic decision-making.

The binding of Shiga toxin (Stx) to Gb3Cer in detergent-insoluble microdomains (DIM)/raft of the ACHN human renal tubular cell line causes the temporal activation of the Src-family kinase Yes [1]. As a strategy for examining signaling mechanisms in DIM/raft, monoclonal antibodies (MAbs) are reliable tools for characterizing the constituent molecules in these microdomains. Thus, we employed DIM/raft suspensions of ACHN cells as an immunogen to develop MAbs. Simply subcutaneous injections of ACHN DIM/raft could elevate the serum titer after several boosts. The first screening was performed using dot-blot immunostaining with culture supernatants on a polyvinylidene difluoride (PVDF) membrane, on which DIM/raft or their chloroform/methanol (C/M) (2:1, v/v) extracts were dot-blotted. The next screening was performed by flowcytometric analysis of ACHN cells treated with or without a permeabilizing reagent. Many of the clones (21/31 clones=68%) thus obtained were also found to recognize to lipid fractions of the DIM/raft. Strikingly, all of the 21 clones that reacted to the lipid fraction were found to recognize monosialosyl galactosylgloboside (MSGG) or GL7, which carries the SSEA-4 epitope. Using DIM/raft as immunogens may enable us to easily obtain MAbs for glycolipids.