A single-cell multi-omic atlas of extrahepatic cholangiocarcinoma progression with ST6GAL1-enriched preinvasive states.

176

Background: Extrahepatic cholangiocarcinoma (eCCA) is frequently diagnosed at advanced stages, limiting opportunities for early intervention. Chronic biliary inflammation is a recognized risk context, however, the cellular and epigenetic changes linking inflammatory injury to preinvasive lesions and invasive eCCA remain incompletely defined. We generated a stage-resolved single-cell multi-omic atlas to characterize this progression and prioritize candidates associated with preinvasive remodeling. Methods: Fresh human biliary tissues spanning the disease continuum (normal bile duct (n=2), inflammatory lesions (n=7), intraductal papillary neoplasm of the bile duct (IPNB; n=3), and invasive eCCA including perihilar (n=8) and distal (n=4) subtypes) were profiled using integrated single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq). Analyses included cross-sample integration, cell-type annotation, epithelial trajectory inference, copy-number inference, chromatin accessibility dynamics, and cross-modal candidate prioritization. Candidate genes were further evaluated using donor-level pseudo-bulk expression profiling across pathological stages. Results: We resolved an epithelial progression continuum characterized by increasing transcriptional reprogramming and genomic instability, with copy-number abnormalities detectable before overt invasion. IPNB exhibited substantial epithelial heterogeneity, consistent with branching preinvasive states. Cross-modal prioritization highlighted ST6GAL1 as a candidate associated with preinvasive remodeling. Donor-level pseudo-bulk profiling showed stage-dependent ST6GAL1 expression, with enrichment from inflammatory lesions/EBD to IPNB and sustained expression across invasive eCCA subtypes, consistent with preinvasive-enriched, stage-dynamic remodeling. Conclusions: This study provides a stage-resolved single-cell multi-omic map of eCCA progression from biliary inflammation to invasion and defines ST6GAL1-enriched preinvasive states as a translationally relevant feature for biomarker development and early interception studies.