Tumor immune microenvironment and immune phenotypes in PD-L1-tested canine urothelial carcinoma

Immune checkpoint inhibition (ICI) is a promising therapeutic strategy for counteracting tumor immune evasion. The therapeutic response largely depends on interactions between cancer cells and the tumor immune microenvironment (TIME). This study aimed to characterize the TIME and its relationship with the immune checkpoint ligand Programmed Death-Ligand 1 (PD-L1) in canine urothelial carcinomas (UCs). UCs were retrospectively selected and tested for PD-L1 using single-antibody immunohistochemistry. Multiplex immunohistochemistry was performed using anti-CD3, -CD20, and -IBA1 antibodies, to co-localize the immune cells (ICs). Both ICs and PD-L1 expression were quantified with computer-assisted image analysis (QuPath software). Based on the spatial distribution and density of ICs, tumors were classified in three distinct immune phenotypes: immune-inflamed, immune-excluded, and immune-desert. Among the 49 UCs analyzed, 11 (22%) were PD-L1+. Forty carcinomas were classified as immune-inflamed (9 PD-L1+; 31 PD-L1-), 7 as immune-excluded (2 PD-L1+; 5 PD-L1-), and 2 as immune-desert (PD-L1-). Macrophages and T-cells were the most numerous ICs, while B-cells were significantly fewer (p < 0.0001). PDL1 + tumors exhibited a significantly higher number of macrophages compared to PD-L1- tumors (p = 0.003). Immuno-inflamed tumors showed a higher density of T cells (p = 0.01) and a lower macrophages-to-T lymphocytes ratio (p = 0.02) compared to immune-excluded and immune-desert phenotypes. In summary, most UCs were immune-inflamed and T-cell rich; a subset of tumors was PDL1 + and associated with a higher number of macrophages. Further characterization of T lymphocytes and macrophages polarization is necessary to better stratify the immune response.