A TMED3-governed disulfidptosis-related diagnostic signature reveals tumor microenvironment remodeling in intrahepatic cholangiocarcinoma
Background Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy with poor prognosis and limited treatment options. Disulfidptosis, a novel cell death pathway driven by disulfide bond accumulation, has emerged as a potential mechanism in cancer biology; however, its role in ICC remains unclear. Methods We integrated single−cell RNA sequencing (GSE138709) with bulk transcriptomic datasets (TCGA−CHOL, GSE107943, GSE32225) to systematically characterize the ICC cellular landscape. Analyses included CNV inference, stemness scoring, disulfidptosis activity assessment, and cell−cell communication profiling. A diagnostic model was constructed using LASSO−logistic regression with 10−fold cross−validation and validated in independent cohorts. TME characterization, survival analysis, and drug−target screening were also performed. Experimental validation included HPA immunohistochemistry, qRT−PCR, and functional assays following TMED3 knockdown. Results Seven major cell types were identified, with malignant cholangiocytes exhibiting high aneuploidy (74%), elevated stemness, upregulated disulfidptosis activity, and extensive communication via SPP1−CD44 and IGFBP3−TMEM219 networks. A five−gene signature (TMED3, TMEM184B, MAPK13, MFSD10, GRB7) demonstrated robust diagnostic performance. Survival analysis showed borderline prognostic value for TMED3 (adjusted HR = 2.37, P = 0.073), while TMEM184B emerged as an independent prognostic factor (adjusted HR = 4.79, P = 0.028). PPI and co−expression analyses established links between signature genes and disulfidptosis regulators. Functional experiments confirmed that TMED3 knockdown suppressed ICC cell proliferation, migration, and enhanced sensitivity to glucose deprivation−induced disulfidptosis. Network−based drug screening identified eight high−priority candidates for therapeutic repurposing. Conclusion This study provides a comprehensive single−cell atlas of ICC, identifies TMED3 as a key regulator of a disulfidptosis−related diagnostic signature, and demonstrates its functional role in promoting ICC malignancy. The five−gene signature shows diagnostic and prognostic promise, and the drug screening offers preliminary leads for therapeutic repurposing, providing a foundation for precision diagnosis and targeted therapy in ICC.