Single-Cell Phenotyping of CD73 Expression Reveals the Diversity of the Tumor Immune Microenvironment and Reflects the Prognosis of Bladder Cancer

The cutting edge of cancer immunotherapy extends to ecto-5'-nucleotidase (CD73), a cell membrane enzyme that targets the metabolism of extracellular adenosine. We herein focused on the expression of CD73 to clarify the state of CD73 positivity in cancer immunity and tumor microenvironment, thereby revealing a new survival predictor for patients with bladder cancer (BCa). We used clinical tissue microarrays of human BCa and simultaneously performed the fluorescent staining of cell type-specific markers (CD3, CD8, Foxp3, programmed cell death protein 1, and programmed death-ligand 1 [PD-L1]) and CD73 together with DAPI for nuclear staining. In total, 156 participants were included. Multiplexed cellular imaging revealed a unique interaction between CD73 expression and CD8⁺ cytotoxic T cells (CTLs) and Foxp3⁺ regulatory T (Treg) cells in human BCa, showing the high infiltration of CD8⁺CD73⁺ CTLs and Foxp3⁺CD73⁺ Treg cells in tumors to be associated with tumorigenesis and poor prognosis in BCa. Interestingly, from a biomarker perspective, the high infiltration of CD73⁺ Treg cells in tumors was identified as an independent risk factor for overall survival in addition to clinicopathologic features. Regarding the relationship between immune checkpoint molecules and CD73 expression, both CD73⁺ CTLs and CD73⁺ Treg cells tended to coexpress programmed cell death protein 1 as tumor invasiveness and nuclear grade increased. Additionally, they may occupy a spatial niche located distantly from PD-L1⁺ cells in tumors to interfere less with the cancerous effects of PD-L1⁺ cells. In conclusion, the present results on the status of CD73 in cancer immunity suggest that CD73 expression on specific T-cell types has a negative immunoregulatory function. These findings may provide further insights into the immunobiological landscape of BCa, which may be translationally linked to improvements in future immunotherapy practice.