Characterisation of the tumour microenvironment and PD-L1 granularity reveals the prognostic value of cancer-associated myofibroblasts in non-invasive bladder cancer.

High-risk non-muscle-invasive bladder cancer (NMIBC) presents high recurrence and progression rates. Despite the use of Bacillus Calmette-Guérin gold-standard immunotherapy and the recent irruption of anti-PD-1/PD-L1 drugs, we are missing a comprehensive understanding of the tumor microenvironment (TME) that may help us find biomarkers associated to treatment outcome. Here, we prospectively analyzed TME composition and PD-L1 expression of tumor and non-tumoral tissue biopsies from 73 NMIBC patients and used scRNA-seq, transcriptomic cohorts and tissue micro-array to validate the prognostic value of cell types of interest. Compared to non-tumoral tissue, NMIBC presented microvascular alterations, increased cancer-associated fibroblast (CAF) and myofibroblast (myoCAF) presence, and varied immune cell distribution, such as increased macrophage infiltration. Heterogeneous PD-L1 expression was observed across subsets, with macrophages showing the highest expression levels, but cancer cells as the primary potential anti-PD-L1 binding targets. Unbiased analysis revealed that myoCAF and M2-like macrophages are specifically enriched in high-grade NMIBC tumors. The topological distribution of these two cell types changed as NMIBC progresses, as shown by immunofluorescence. Only myoCAFs were associated with higher rates of progression and recurrence in three independent cohorts (888 total patients), reaching prediction values comparable to transcriptomic classes, which we further validated using tissue micro-array. Our study provides a roadmap to establish the landscape of the NMIBC TME, highlighting myoCAFs as potential prognostic markers.