Background: Non-muscle-invasive bladder cancer (NMIBC) is challenging due to high recurrence and progression rates. Bacillus Calmette-Guerin (BCG) is the standard treatment for high-risk NMIBC, but emergence of anti-PD-1/PD-L1 drugs necessitates a deeper understanding of the tumour microenvironment (TME) for improved prognostic markers and therapies. Objective: To extensively analyse NMIBC's TME, focusing on cellular composition, PD-L1 expression, and the role of cancer-associated fibroblasts (CAFs) in prognosis. Design, Setting, and Participants: A prospective study between December 2019 and December 2022 collected 98 NMIBC and non-pathological tissue (NPT) samples from cytology-positive/suspicious patients (66 patients in final analysis). Intervention(s): Assessment of immune and non-immune cell subsets and PD-L1 expression using flow cytometry. Transcriptomic data and histology validated findings and evaluated prognostic markers. Outcome Measurements and Statistical Analysis: We assessed the distribution of 11 cell types and PD-L1 expression in NMIBC, comparing them to NPT and across pathological stage and grade. Statistical methods evaluated the association of myofibroblasts (myoCAFs) and other CAF subsets with progression-free and recurrence-free survival. Results and Limitations: Compared to NPT, NMIBC's TME exhibited microvascular alterations, increased fibroblast and myoCAF presence, and varying immune cell distribution. Heterogeneous PD-L1 expression was observed across subsets, with cancer cells as primary potential anti-PD-L1 binding targets. MyoCAFs were associated with high grade tumours and poor prognosis, while other CAF subsets were not. Study limitations included a modest sample size and a relatively short follow-up period. Conclusions: This comprehensive analysis provides a roadmap to establish the full NMIBC's TME, highlighting myoCAFs as potential prognostic markers. Understanding its complexity may enhance therapeutic strategies and risk stratification for NMIBC patients. Further research is essential to validate findings and explore myoCAF implications in NMIBC therapy. Patient Summary: We described the composition of non-muscle invasive bladder cancer, identified cellular components associated with aggressive tumors, and found markers to predict outcome.
