CD39/CD73-mediated immunosuppression and tumor aggressiveness in bladder cancer

Abstract Urothelial bladder cancer (BCa) is marked by high recurrence and mortality, and the efficacy of PD-1/PD-L1 immunotherapy remains limited because of immune evasion. The adenosinergic pathway (AP), mediated by ectonucleotidases CD39 and CD73, is a key immunosuppressive mechanism, but its role in BCa remains unclear. We conducted an integrated immunophenotypic analysis of peripheral blood (PB) and the tumor microenvironment (TME) from 39 patients with BCa and 14 healthy controls using multicolor flow cytometry and immunohistochemistry. High-risk (HR) patients exhibited systemic immunosuppression, characterized by an elevated neutrophil-to-lymphocyte ratio and increased circulating regulatory T cells (Tregs), along with reduced cytotoxic γδ T cells and diminished Th1/Tc1 functional subtypes. In the TME, we observed reduced CD8 + T cell infiltration accompanied by increased Tregs, and phenotypes characterized by poor immune infiltration in the tumor core predominated across the cohort. In both PB and the TME, CD39 and CD73 expression on T cells strongly correlated with an immunosuppressive environment, marked by increased M2-like macrophages and decreased effector T cells. Circulating double-positive T cells (CD4 + CD39 + CD73 + and CD8 + CD39 + CD73 + ) mirrored the intratumoral T-cell composition, suggesting their potential as non-invasive biomarkers. Elevated frequencies of circulating single-positive CD8 + CD39 + and CD8 + CD73 + T-cells were significantly associated with higher pathological grade and distinguished high-grade tumors with moderate accuracy (AUC > 0.70). This study demonstrates that BCa is characterized by extensive AP-linked immunosuppression, and that specific ectonucleotidase-expressing circulating T-cell subsets may serve as non-invasive biomarkers for assessing tumor infiltration and grade.