Integrative single-cell and computational pathology analyses reveal a C11orf24-associated stem-like epithelial population linked to tumor microenvironment remodeling and immune evasion in bladder cancer.

BACKGROUND: Cancer stem-like cells drive tumor progression and therapeutic resistance, yet the epithelial stemness hierarchy in bladder cancer (BLCA) remains poorly defined. We aimed to characterize stemness-associated epithelial populations and their clinical significance. METHODS: We analyzed single-cell RNA sequencing data from 92,218 cells across 13 treatment-naïve BLCA samples to characterize epithelial stemness and tumor-microenvironment interactions. Spatial transcriptomics and independent bulk cohorts were used for orthogonal and clinical validation. An HStem-associated histopathology-based prognostic model was developed by integrating quantitative image features with deep-learning representations, followed by functional validation of the candidate gene C11orf24. RESULTS: We identified a highly stem-like epithelial population (HStem) at the apex of the differentiation hierarchy, exhibiting MYC activation, oxidative phosphorylation, and DNA repair enrichment, with preferential presence in advanced-stage tumors. HStem cells were inferred to serve as major communication hubs within the TME, interacting extensively with fibroblasts and endothelial cells via MIF-CD74/CXCR4 and MIF-CD74/CD44 axes. An 86-gene HStem signature enabled development of a robust image-based prognostic model. High-risk tumors showed immune exclusion, immunosuppressive enrichment, T-cell dysfunction, and predicted checkpoint blockade resistance. C11orf24 emerged as a candidate stemness-associated regulator; C11orf24-positive cells displayed enhanced stemness and stromal interactions. Functional experiments demonstrated that C11orf24 perturbation altered clonogenic growth, migration, and invasion in BLCA cells. Pharmacogenomic analyses identified predicted therapeutic vulnerabilities that warrant experimental validation. CONCLUSIONS: We identify an HStem population associated with BLCA progression, microenvironmental remodeling, and immune evasion. Integrating single-cell and spatial transcriptomics with computational pathology, we establish a clinically relevant stemness framework and identify C11orf24 as a potential therapeutic target, offering opportunities for stemness-directed precision oncology.