Single-cell and spatial transcriptomics define an R-loop-associated malignant state in intrahepatic cholangiocarcinoma and prioritize MTHFD1L

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with substantial cellular heterogeneity and poor outcomes. R-loops link transcription-replication stress to genomic instability, but their roles across ICC cell states remain unclear. We integrated three ICC single-cell transcriptomic cohorts, spatial transcriptomic data, and two bulk transcriptomic cohorts to characterize an R-loop-associated malignant program and its prognostic relevance. A composite score derived from R-loop regulators identified R-loop-high malignant epithelial cells with enhanced proliferative signaling, reduced differentiation features, metabolic adaptation, and stronger interactions with macrophages, endothelial cells, and fibroblasts. These cells preferentially localized to specific intratumoral niches. An R-loop-related DeepSurv model stratified overall survival in training and validation cohorts. MTHFD1L emerged as a candidate molecular node; functional inference, virtual knockout, metabolite-mediated communication, and drug-response analyses linked MTHFD1L-positive malignant cells to mitochondrial one-carbon metabolism, redox homeostasis, metabolic plasticity, and proteostasis vulnerabilities. These findings identify an R-loop-associated malignant program in ICC and nominate MTHFD1L for further mechanistic and therapeutic investigation.