Epigenetic subtypes of high-grade T1 bladder cancer reveal intra-tumor heterogeneity and distinct interactions with tumor microenvironment

Rationale: High-grade T1 (HGT1) Non-Muscle Invasive Bladder Cancer (NMIBC) is a clinically heterogeneous disease characterized by unpredictable treatment responses and limited tools for recurrence prediction. Despite advances in molecular classification, patient stratification still relies mainly on clinicopathological features, providing limited precision. Methods: In this study, we integrated bulk chromatin profiling with single-nuclei RNA-seq, immunohistochemistry, and spatial transcriptomics to define epigenetic subtypes of HGT1, characterize their heterogeneity, and explore tumor-microenvironment interactions. Results: We identified distinct chromatin landscapes distinguishing urothelial (URO) and micropapillary (MP) histological variants of high-risk HGT1. Three main epigenetic states emerged: luminal-like inflammatory (LLI) and basal-like (BL) subtypes within URO tumors, and a separate signature unique to MP tumors. Single-cell and spatial analyses confirmed intratumoral heterogeneity and revealed subtype-specific microenvironmental contexts.Approximately 40% of URO tumors exhibited spatially distinct coexisting LLI and BL components. BL regions, enriched for angiogenesis and hypoxia pathways, were preferentially located near vascular stroma, whereas LLI regions were situated at the tumor core. MP tumors displayed a markedly different microenvironment with abundant cancer-associated fibroblasts (CAFs) and M2-polarized macrophages intermingled with tumor cells, suggesting an immunosuppressive niche that may underlie their poor prognosis. In contrast, URO tumors showed a more immune-excluded phenotype. Conclusion: These findings provide a detailed molecular and spatial map of HGT1 heterogeneity, linking epigenetic states to tumor architecture and microenvironmental interactions. They underscore the need for subtype-specific therapeutic strategies to effectively address the biological diversity already present in HGT1 bladder cancer.