Inflammation- and regulated-cell-death–relevant transcriptomic signatures in bladder urothelial carcinoma: cross-cohort reproducibility, exploratory single-cell localization, and in vitro qRT-PCR validation

Background Bladder urothelial carcinoma develops within an immune-active, metabolically stressed, and tissue-remodeling microenvironment in which regulated cell-death pathways may interact with inflammatory signaling. We evaluated whether tumor-associated transcriptional alterations are reproducible across independent cohorts and whether selected candidates can be localized at single-cell resolution and supported by in vitro expression validation. Methods TCGA-BLCA (412 primary tumors and 19 solid-tissue normal samples) and GSE13507 (165 primary tumors and 9 normal bladder mucosa controls) were analyzed independently for differential expression. A 15-gene tumor-upregulated transcriptional signature derived in TCGA was evaluated in GSE13507 without gene re-selection. Five pure-urothelial-carcinoma tumor regions from three patients in GSE293189 were analyzed exploratorily by single-cell RNA sequencing. ULBP1, IL37, COL10A1, COL11A1, and IGF2BP3 were further assessed by qRT-PCR in SV-HUC-1, T24, 5637, and UM-UC-3 cell lines. Results Across 16,759 matched genes, cohort-specific effects were positively correlated (Spearman ρ = 0.58, P < 1 × 10 −300 ), and 4,102 genes were significant in both cohorts with concordant direction. The TCGA-derived signature was higher in tumors than normal tissues and varied across reported pathologic stages, although the highly unbalanced stage distribution precluded interpretation as a clinical stage classifier. Single-cell analysis retained 1,512 cells and resolved epithelial/tumor-associated, myeloid, T-cell, B-cell, NK-cell, and fibroblast compartments with distinct basal, luminal, EMT, and immune states. All five qRT-PCR candidates were consistently upregulated in bladder-cancer cell lines relative to SV-HUC-1. Conclusion The study identifies a cross-cohort reproducible tumor-associated transcriptional signal and places selected candidates within a heterogeneous cellular context. qRT-PCR provides orthogonal expression-level support for selected candidates, but neither transcriptomic nor qRT-PCR data alone establish functional execution of apoptosis, necroptosis, pyroptosis, or ferroptosis.