Abstract 154: Single-cell spatial transcriptomics unravels cell states and ecosystems associated with clinical response to immunotherapy

Abstract The tumor microenvironment (TME) is a highly dynamic and complex ecosystem, where the diversity of cell types, their functional states, and spatial interactions determine responses to immunotherapy. Despite its critical role, the precise mechanisms through which these factors influence clinical outcomes remain poorly understood. In this study, we leveraged single-cell spatial transcriptomics to systematically dissect the TME and identify determinants of immunotherapeutic response in vulvar high-grade squamous intraepithelial lesions (vHSIL). We analyzed 20 treatment-naïve vHSIL lesions, stratified based on clinical response to tumor-specific peptide vaccination into: complete responders (CR, 100% lesion clearance, n=6), partial responders (PR, >50% lesion clearance, n=7), and non-responders (NR, <50% lesion clearance, n=7). Using a 1000-gene panel, we mapped over 274, 000 single cells in situ, revealing 18 cell clusters that were further delineated into 99 distinct non-epithelial cell states. Validation against public single-cell transcriptomic datasets confirmed the broader relevance of these findings across tumor types. Our findings demonstrated profound heterogeneity within the TME across response groups. Notably, CR lesions exhibited a significantly higher ratio of immune-supportive to immune-suppressive cells compared to PR and NR lesions—a pattern mirrored in other solid tumors following neoadjuvant checkpoint blockade therapy (anti-PD1, anti-PDL1, anti-CTLA4). Key immune populations enriched in CR lesions included CD4+CD161+ effector T cells and chemotactic CD4+ and CD8+ T cells, implicating these populations as critical drivers of favorable therapeutic outcomes. Conversely, PR lesions were characterized by increased proportions of T helper 2 cells and CCL18-expressing macrophages, which are associated with the recruitment of type 2 T cells and regulatory T cells. NR lesions displayed preferential infiltration with immunosuppressive fibroblasts. Distinct spatial immune ecosystems further defined response groups: type 1 effector cells dominated interactions in CR lesions, type 2 cells were prominently interacting in PR lesions, while NR lesions lacked organized immune cell interactions. Thus, these findings emphasize the dual importance of cellular composition and spatial organization in steering response to immunotherapy. By elucidating the mechanisms underlying clinical responses to immunotherapeutic vaccination, this study provides actionable insights to advance precision immunotherapy. Citation Format: Ziena Abdulrahman, Roderick C. Slieker, Daniel McGuire, Marij J.P. Welters, Mariette I.E. van Poelgeest, Sjoerd H. van der Burg. Single-cell spatial transcriptomics unravels cell states and ecosystems associated with clinical response to immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 154