Abstract
BackgroundCholangiocarcinoma was a highly malignant liver cancer with poor prognosis, and immune infiltration status was considered an important factor in response to immunotherapy. In this investigation, we tried to locate immune infiltration related genes of cholangiocarcinoma through combination of bulk-sequencing and single-cell sequencing technology.MethodsSingle sample gene set enrichment analysis was used to annotate immune infiltration status in datasets of TCGA CHOL, GSE32225, and GSE26566. Differentially expressed genes between high- and low-infiltrated groups in TCGA dataset were yielded and further compressed in other two datasets through backward stepwise regression in R environment. Single-cell sequencing data of GSE138709 was loaded by Seurat software and was used to examined the expression of infiltration-related gene set. Pathway changes in malignant cell populations were analyzed through scTPA web tool.ResultsThere were 43 genes differentially expressed between high- and low-immune infiltrated patients, and after further compression, PNOC and LAIR2 were significantly correlated with high immune infiltration status in cholangiocarcinoma. Through analysis of single-cell sequencing data, PNOC was mainly expressed by infiltrated B cells in tumor microenvironment, while LAIR2 was expressed by Treg cells and partial GZMB+ CD8 T cells, which were survival related and increased in tumor tissues. High B cell infiltration levels were related to better overall survival. Also, malignant cell populations demonstrated functionally different roles in tumor progression.ConclusionPNOC and LAIR2 were biomarkers for immune infiltration evaluation in cholangiocarcinoma. PNOC, expressed by B cells, could predict better survival of patients, while LAIR2 was a potential marker for exhaustive T cell populations, correlating with worse survival of patients.
Highlights
Cholangiocarcinoma (CCA) has long been deemed as a malignancy with poor prognosis in liver cancer
We further examined the correlations between PNOC and scores for B cell infiltration in TCGA CHOL samples, calculated by single sample gene set enrichment analysis (ssGSEA) and MCPcounter methods, and results showed PNOC was highly correlated with B cells (Figures 5E–H)
We used bulk sequencing data of cholangiocarcinoma patients in TCGA database to calculate the immune infiltration scores of different immune cell populations, and we compared expression difference between groups, locating immune infiltration highly associated genes; we found PNOC was mainly expressed by infiltrated B cells, which was survival related, while LAIR2 was mainly expressed by Tregs and partial CD8+/GZMB+ T cells, indicating exhaustive immune status of T cells
Summary
Cholangiocarcinoma (CCA) has long been deemed as a malignancy with poor prognosis in liver cancer. Understanding tumor immune microenvironment (TIME) and infiltration status of CCA could better guide the clinical appliance of immunotherapy [7,8,9]. Characterization of CCA immune microenvironment is limited, so in this study to characterize immune cell components in TIME, we combined bulk sequencing data with scRNA-seq data, which could provide a better understanding of functional cell clusters related to disease severity. Cholangiocarcinoma was a highly malignant liver cancer with poor prognosis, and immune infiltration status was considered an important factor in response to immunotherapy. In this investigation, we tried to locate immune infiltration related genes of cholangiocarcinoma through combination of bulk-sequencing and singlecell sequencing technology
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