Single-cell atlas of diverse immune populations in the advanced biliary tract cancer microenvironment

Immunotherapies have been explored in treating solid tumors, albeit with disparate clinical effects in distinct cancer types. Systematic interrogation of immune cells in the tumor microenvironment (TME) is vital to the prediction of immunotherapy response and the development of innovative immunotherapeutics. To comprehensively characterize the immune microenvironment in advanced biliary tract cancer (BTC), we utilized single-cell RNA sequencing in unselected viable cells from 16 matched samples. We identified nineteen cell subsets from a total of 45,851 cells, in which exhausted CD8+ T cells, macrophages, and dendritic cells (DCs) in BTC were shown to augment and communicate within the TME. Transcriptional profiles coupled with T cell receptor (TCR) sequences revealed that exhausted CD8+ T cells retained clonal expansion and high proliferation in the TME, and some of them highly expressed the endoplasmic reticulum stress (ER) sensor XBP1, indicating its key role in remodeling TME. Functional assays demonstrated that XBP1 and common immune checkpoints (PD1, TIGIT) were significantly upregulated in CD8+ T cells cocultured within the TME of BTC cell lines (GBC-SD, HCCC-9810). When treating the coculture groups with the specific inhibitor of IRE1α-XBP1 (4μ8C), the downregulation of TIGIT was observed in the treatment group. Collectively, comprehensive transcriptome profiling provides deeper insights into the immune atlas in advanced BTC, which might be instrumental in exploring novel immunotherapy strategies. Overall design: Five patients diagnosed with cholangiocarcinoma in the Eastern Hepatobiliary Surgery Hospital were incorporated into the current study, including two intrahepatic cholangiocarcinoma (iCCA), two gallbladder carcinoma (GBC) and one dCCA patients. Of note, none of the patients have undergone any preoperative treatments (chemotherapy, radiation or anti-tumor medicines) prior to tumor resection. Tumor primary locus and metastasis as well as lymph node were consistently harvested for each participant, if available. Meanwhile, peripheral blood samples were collected prior to surgical procedures using anticoagulant tubes.

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