Single-cell and bulk transcriptomic analyses define an IFI27-centered interferon-response epithelial state in bladder cancer and its transcriptional response to irradiation.

BACKGROUND: Interferon-responsive tumor-cell states can simultaneously increase immune visibility and induce immune-regulatory programs, yet their epithelial-cell distribution and treatment responsiveness in bladder cancer remain incompletely defined. We sought to resolve a reproducible epithelial interferon-response state across patients, characterize its immune context, and determine whether its transcriptional components respond to ionizing radiation in vitro. METHODS: Single-cell RNA sequencing data from seven primary bladder tumors and one adjacent normal sample were reanalyzed with uniform quality control and doublet removal. After broad-lineage annotation, 29,777 identity-gated tumor epithelial cells were reclustered and analyzed by repeated non-negative matrix factorization across ranks and random seeds. An NMF-informed three-gene interferon-response score based on IFI27, ISG15, and IFI6 was used to define patient-internal upper- and lower-quartile states. Differential expression was assessed by patient-blocked pseudobulk edgeR models, followed by Hallmark, Gene Ontology, and Reactome enrichment, leave-one-patient-out analyses, immune-feature scoring, balanced CellChat analysis, and TCGA-BLCA corroboration. T24 bladder cancer cells were exposed to 0, 2, 4, or 8 Gy, and selected transcripts were quantified by qRT-PCR 24 h later. RESULTS: The final atlas comprised 39,224 cells assigned to six broad lineages. Repeated NMF reproducibly identified an IFI27-centered epithelial program, and the corresponding score showed marked interpatient variation while remaining detectable across all seven tumors. Patient-blocked analysis identified 179 genes with false-discovery rate <0.05 and absolute log2FC ≥ 0.5; IFIT1, CXCL10, IFI44L, IFITM1, HLA-B, B2M, CD74, IFI6, and ISG15 were representative upregulated genes. IFN-response-score-high epithelial cells showed strong enrichment of interferon-gamma response, interferon-alpha response, inflammatory response, IL6-JAK-STAT3 signaling, antigen processing and presentation, and chemokine-response programs. MHC-I, MHC-II, CXCL9/10/11-related, and immune-regulatory ligand scores increased in all seven patient-paired comparisons, whereas CD274 itself remained sparsely detected and was not stably associated with the continuous score. Balanced pooled CellChat analyses prioritized relatively enriched MDK-SDC1, MDK-SDC2, and MDK-ITGA6/ITGB1 candidate interactions. TCGA-BLCA analyses corroborated the broader interferon and antigen-presentation context at gene and pathway levels. Irradiation induced dose-associated increases in IFI27, ISG15, IFI6, IFIT1, CXCL10, and IFITM1 transcripts, accompanied by increases in CD274, VIM, and SNAI1 and a decrease in CDH1. CONCLUSION: Bladder tumors contain a reproducible IFI27-centered epithelial interferon-response state with coordinated antigen-presentation, chemokine-related, and selected immune-regulatory transcriptional features. Its molecular context is supported across patients and in bulk transcriptomes, while irradiation-associated qRT-PCR changes suggest that components of the program are treatment responsive. These findings define a hypothesis-generating epithelial immune state and candidate communication axes for future spatial, protein-level, and functional validation.