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.