Investigation of intrahepatic cholangiocarcinoma evolution by using single-cell RNA sequencing data

Abstract

Background: Intrahepatic cholangiocarcinoma (iCCA) is the second most common liver malignancy with two main divisions, large-duct and small-duct iCCA, according to the World Health Organization. Unlike large-duct iCCA, there is currently a few studies working on the origin of small-duct type. Thus, the cells of small-duct iCCA origin should be intensively explored to give evidence for further research on its early diagnosis and disease treatment. Methods: In this study, we used the raw data, fastq files, generated by droplet-based single-cell RNA sequencing 10X Genomics from small-duct iCCA (recognized by HE staining) on public database for analysis. Then, the bioinformatics tools (Cellranger and Seurat) were applied for data preprocessing and Malignant cells separation. From the gene expression and copy number variations (CNVs) profiles (detected from gene expression data by CopyKAT) of Malignant cells, trajectories were built by Slingshot. From the constructed trajectories, the GSEA software was employed to trace the origin. Results: First, dataset of 28559 single cells from 5 iCCA samples of 5 patients were collected to process in which 7800 cells were small-duct iCCA malignancy. Second, the bioinformatics pipeline for analyzing 10X Genomics data was constructed by integrating the tools and packages mentioned in Methods part. Besides, the pipeline was automatically processed (by Snakemake tool) for data investigation. From the pipeline result, the trajectories constructed from gene expression profile were partially consistent with the ones constructed from CNVs, and they were all used for reflecting the cancerous differentiation. Third, after analyzing the gene set enrichment between start site and end site of trajectories, the cells of origin for small-duct iCCA were indicated. Conclusion: The result of algorithm suggests that Hepatoblasts and Hepatocytes could be the origin of small-duct iCCA.