Single-cell profiling reveals systemic immune reprogramming in muscle-invasive urothelial carcinoma

Background Urothelial carcinoma (UC) is classified into non-muscle-invasive (NMI) and muscle-invasive (MI) subtypes, with the latter associated with poor prognosis. Although tumor-infiltrating immune responses have been extensively studied, how the peripheral immune system is reprogrammed during disease progression remains largely unexplored. Understanding systemic immune alterations may reveal mechanisms underlying disease progression. Unlike most previous studies focusing solely on bladder UC, our cohort also included upper tract UC cases, providing a broader view of systemic immune alterations in urothelial carcinoma. Methods We performed single-cell RNA sequencing of peripheral blood mononuclear cells from 20 UC patients (5 MI and 15 NMI), integrating publicly available healthy control and MI datasets. A total of 144,019 high-quality cells were analyzed to characterize immune cell composition, functional states, and intercellular communication. Results Seven major immune cell populations were identified, with T and NK cells predominating. MI samples exhibited upregulation of genes related to chromatin remodeling, mitochondrial metabolism, and protein translation, alongside downregulation of immune signaling pathways, indicating metabolic stress and immune suppression. Pseudotime analysis revealed an MI-specific CD4⁺T-cell differentiation trajectory enriched in genes such as SLC25A6 and H3F3B and regulated by YBX1. B cells and monocytes showed functional reprogramming, with MI B cells metabolically active and NMI B cells immune-active. MI classical monocytes exhibited pro-tumor phenotypes and suppressed CD8⁺T-cell function via TGF-β signaling, with downstream JUN and SLC7A5 correlating with poor survival. Intercellular communication among T cells, B cells, and monocytes was enhanced in MI. Conclusions These findings indicate that peripheral immune cells in MI UC undergo stage-specific functional reprogramming, combining immune suppression, metabolic adaptation, and enhanced intercellular crosstalk. This highlights potential targets for immunomodulatory therapy and provides new insights into systemic immune alterations underlying urothelial carcinoma progression.