Abstract PR010: High-dimensional spatial profiling identifies distinct and targetable tumor-infiltrating lymphocyte phenotypes in localized prostate cancer

Abstract Background: Prostate cancer (PCa) is considered immunologically “cold, ” however, we have previously identified an immunogenic subset of high-risk localized PCa with T-cell tumor infiltration within antigen-presenting-cell (APC) niches. Here, we used high-dimensional single-cell spatial proteomics to dissect tumor-infiltrating-lymphocyte in (TIL) -high (H), TIL-low (L), and TIL-excluded (X) PCas, to understand the tumor-specific and conserved mechanisms of immune dysregulation for targeted therapeutics. Methods: Treatment-naive prostatectomy specimens of all three tumor immune microenvironment (TIME) phenotypes, were assembled into tissue microarrays representing within-tumor and adjacent foci. TIL-X was exclusively observed in cribriform pattern 4 cases. A 40-plex antibody panel made of phenotyping and functional markers (e. g AR) was applied to the TMAs, enabling single-cell annotation of key immune cell types (e. g CD8+ T cells) and assessment of their functional states within distinct spatial microenvironments. Results: In TIL-X PCa, immunosuppressive macrophages and DCs were enriched in the tumor nest whereas T cells accumulated at the periphery with minimal tumor infiltration. Tumor adenosine 2A receptor (A2AR) expression was uniquely enriched within tumor, suggesting a metabolically suppressive microenvironment that reinforces immune exclusion. TIL-H tumors had comparable T cell proportions inside and outside the tumor, accompanied by the enrichment of myeloid cells within the tumor. CD8+ T cells expressing high levels of AR and B7H3 frequently co-localized with tumor and APCs, forming APC niches and tertiary lymphoid-like structures. CD8+ARhi and CD8+B7H3hi subsets with elevated exhaustion scores preferentially infiltrated tumor, whereas CD8+ARlo and CD8+B7H3lo subsets with moderate exhaustion levels remained peripheral; defining TIL-H PCa as tumors harboring antigen-experienced but exhausted CD8 T cells in a TIME that supports immune cell engagement but restrains anti-tumor activity. TIL-L PCa had sparse T cell infiltration and enrichment of myeloid cells with moderate AR expression within tumor, suggesting a role of androgen signaling and myeloid cells in shaping the TIME, marked by high PD-1+CD8+ cells and PD-L1+ tumor cells. Thus, TIL-L PCa shows low immune recruitment, yet strong tumor-mediated inhibitory signaling that constrains anti-tumor immunity. Conclusion: TILs in TIL-H PCa are phenotypically, functionally, and spatially distinct from those in TIL-L or TIL-X tumors, reflecting tumor-intrinsic immune programs. High-dimensional spatial proteomics reveal differential molecular modules and signaling pathways that define unique and potentially targetable mechanisms of immunosuppression across the three TIME phenotypes; additional spatial transcriptomics validation and dissection experiments are underway. These findings demonstrate that the PCa TIME is diverse yet spatially organized, and that comprehensive understanding of baseline spatial and transcriptional immune states may guide personalization of immunotherapy strategies in PCa. Citation Format: Precious Cramer, Yang Wang, Wenrui Wu, Huaying Qiu, Lindsay Parmelee, Berkay Simsek, Gabriel Roberti. De Oliveira, Seifeldin Awad, Yao Yu Yeo, Stephanie Pei Tung. Yiu, Hendrik Michel, Sabina Signoretti, Yue Sun, Steven Balk, Sizun Jiang, David Einstein Einstein. High-dimensional spatial profiling identifies distinct and targetable tumor-infiltrating lymphocyte phenotypes in localized prostate cancer abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Prostate Cancer Research and Treatment; 2026 Jan 20-22; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86 (2Suppl): Abstract nr PR010.