Untargeted Serum Metabolomics Reveals Differential Signatures in Gallstone-Associated and Gallstone-Free Gallbladder Cancer Variants

Gallbladder cancer (GBC) is an aggressive malignancy often associated with gallstones (GBCGS), a condition distinct from gallstone disease (GSD). Both GBC and GBCGS are rare, with unclear pathogenesis and no established biomarker-based diagnostics. This pilot study aimed to identify distinct metabolic signatures in GBC and GBCGS for early diagnosis and stratification of high-risk GSD patients. Comparative untargeted serum metabolomic profiling was performed across three groups: GBC (<i>n</i>₁ = 9), GBCGS (<i>n</i>₂ = 11), and GSD (<i>n</i>₃ = 10). A total of 35,385 mass features with MS/MS characteristics were detected and annotated into 736 biochemicals. Differential metabolome analyses relative to GSD identified 180 altered metabolites in GBC and 225 in GBCGS, with 138 shared by both. Correlation network and biomarker analyses subsequently identified 12 GBC-specific, 20 GBCGS-specific, and 30 shared metabolite signatures with high diagnostic efficiency, predominantly upregulated. Key metabolites identified included cholic acid, glycocholic acid, glycochenodeoxycholic acid, kynurenine, and glutamine, implicated in promoting metastasis and epithelial-to-mesenchymal transitions. Thus, serum metabolome reprogramming in GBC and GBCGS revealed a shared deregulation of metabolic pathways involving bile acids, amino acids, and their intermediates alongside distinct condition-specific biomarkers. These findings provide novel insights into the pathogenesis of GBC and GBCGS, advancing future diagnostic, prognostic, and therapeutic interventions.