Integrated multi-omics, single-cell and spatial transcriptomic analyses identify an HK2/S100A2/GALNT5-associated epithelial/ductal-like program in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked molecular and epithelial heterogeneity, but the cross-cohort and cellular context of candidate genes remains incompletely resolved. We integrated bulk transcriptomic and proteomic data with patient-grouped machine-learning assessment, independent-cohort expression replication, cross-cohort functional modelling, single-cell RNA sequencing, spatial transcriptomics, CNV-assisted analysis and cell-line qRT-PCR. Transcriptomic and proteomic screening identified 4,701 differentially expressed genes, 531 differentially expressed proteins and 238 genes with concordant directions. Four-algorithm integration prioritized a 21-gene candidate space. Repeated patient-grouped cross-validation yielded mean outer-fold AUCs of 0.990 for XGBoost and 0.993 for random forest. These values represent exploratory internal stability within a prespecified candidate space, rather than unbiased external predictive performance, because feature selection preceded the resampling procedure. In GSE62452, HK2, S100A2 and GALNT5 showed single-gene AUCs of 0.845, 0.745 and 0.891, respectively, while an equal-weight, within-cohort Z-score produced an AUC of 0.895 but did not outperform GALNT5. Across five RNA cohorts, HK2 was associated with glycolysis and hypoxia, S100A2 with a PDAC basal-like/squamous-aligned program, and GALNT5 with mucin O-glycosylation and O-glycan biosynthesis. Single-cell and spatial analyses supported heterogeneous epithelial localization and descriptive CNV-anchored concordance. CopyKAT classified 1,587 of 3,244 epithelial candidate cells as aneuploid, while 1,009 cells remained undefined. qRT-PCR showed higher relative HK2, S100A2 and GALNT5 mRNA levels in PDAC cell lines than in HPNE cells. These findings associate the three genes with complementary epithelial/ductal-like programs in PDAC, but do not establish causal regulation, tumor specificity, independent prognostic value or direct therapeutic targetability.