Whole-slide multiplexed tissue imaging of prostate adenocarcinoma to investigate immune niches in high- versus low-grade tumors.

224 Background: The tumor-immune microenvironment (TME) is crucial in the progression of prostate cancer (PCa), yet little is known about its spatial organization and immune composition in treatment-naïve, localized PCa. While immunotherapy has had limited success in metastatic PCa, its efficacy in localized disease remains largely unknown. This study aims to dissect tumor-immune interactions in primary PCa and explore differences in immune landscape between low- and high-Gleason PCa, thus providing biologic insights for immunotherapy actionability. Methods: We performed spatial profiling of radical prostatectomy (RP) specimens from 28 treatment-naïve prostate cancer patients. These included 14 patients with low-grade disease (Gleason Grade Group 1–2) and 14 patients with high-grade disease (Gleason Grade Group 4–5). Using multiplex cyclic immunofluorescence (CyCIF) and a 27-marker panel, we obtained single-cell resolution across whole-slide images. Tumor and stromal compartments were manually annotated, and immune cell densities and neighborhood were analyzed and compared between low- and high-grade tumors. Spatial analysis focused on identifying immune clusters (IC -suggestive of tertiary lymphoid structures -TLS) and quantified the degree of immune infiltration along with characterization of T cell exhaustion markers such as CD8+PD1+TCF1+ cells. Results: High-grade tumors exhibited significantly higher infiltration of CD8+ T cells and CD20+ B cells compared to low-grade tumors. Spatial profiling revealed organized ICs, especially in high-grade tumors, which exhibited characteristics consistent with TLS. These ICs were enriched with precursor-exhausted CD8+PD1+TCF1+ T cells (TPEX), suggesting a state of immune exhaustion that may be therapeutically reactivatable. The presence of TLS and immune exhaustion markers in high-grade tumors highlights a more active immune microenvironment than previously appreciated in primary prostate cancer. Conclusions: Our findings reveal that a subset of high-grade prostate cancers harbor an organized, immune-infiltrated tumor microenvironment, including adaptive immune features such as TLS and CD8+ T cell exhaustion. These immune landscapes, marked by significant CD8+ and CD20+ cell infiltration and spatial clustering, suggest that high-grade prostate tumors may be amenable to immune-based therapies, despite the failure of immune checkpoint blockade in metastatic PCa. These results raise the hypothesis that immunotherapy might be effective in treatment-naïve high-grade prostate cancer. Future efforts should explore whether these immune profiles can be leveraged for risk stratification or as biomarkers for response to immunotherapy in advanced disease.