Single-cell and Spatial Transcriptomics Reveal Cholesterol Metabolism Heterogeneity in Pancreatic Ductal Adenocarcinoma and Identify NSDHL as a Key Regulatory Gene

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and reprogramming of cholesterol metabolism plays a critical role in its malignant progression. However, the heterogeneity of cholesterol metabolism at the single-cell level and its key regulatory genes remain unclear. Methods This study integrated single-cell RNA sequencing, spatial transcriptomics, and bulk transcriptomic data (TCGA, GEO, ICGC) to systematically dissect the cellular landscape of cholesterol metabolism in PDAC. Malignant ductal epithelial cells were scored using five independent algorithms and divided into low (LCM), intermediate (ICM), and high (HCM) cholesterol metabolic subgroups. Cell communication, pseudotime trajectory, hdWGCNA, and XGBoost feature importance ranking were performed. Functional experiments including CCK-8, colony formation, EdU, wound healing, Transwell, and nude mouse tumor models were used to validate the role of NSDHL. Results Malignant ductal epithelial cells exhibited the highest cholesterol metabolic activity. The HCM subgroup showed enhanced communication with CAFs and endothelial cells, increased stemness, and coordinated activation of pro-tumor pathways (hypoxia, TNFα, NF-κB) and metabolic pathways (glycolysis, fatty acid synthesis). Pseudotime analysis revealed that cholesterol metabolism gradually increased along the LCM→ICM→HCM trajectory. Through hdWGCNA combined with XGBoost, NSDHL was identified as the most critical gene for the HCM phenotype. NSDHL was significantly overexpressed in PDAC tissues, enriched in high-malignancy regions, and correlated with poor prognosis. Knockdown of NSDHL significantly inhibited PDAC cell proliferation, migration, and invasion, and delayed tumor growth in nude mice. Conclusion This study reveals the heterogeneity landscape of cholesterol metabolism in PDAC and identifies NSDHL as a key gene driving the HCM phenotype and malignant progression, providing a new theoretical basis for prognosis evaluation and targeted therapy of PDAC.