Abstract 2089: Spatial profiling of exhausted T cells using high-plex imaging mass cytometry

Abstract The spatial organization and cellular composition of the tumor microenvironment (TME) has the potential to inform clinical and translational researchers about mechanisms of disease progression and therapeutic success. Of particular interest are immune cells, especially T cells, which can become exhausted due to chronic stimulation. This poses a significant challenge in cancer therapy as exhausted T cells have reduced effector functions and sustained expression of inhibitory receptors such as PD-1 and CTLA-4, resulting in failure to effectively eliminate tumor cells. Imaging Mass Cytometry™ (IMC™) technology is a high-plex imaging technique that enables deep characterization of the heterogeneity and complexity of the TME. The Hyperion™ XTi Imaging System utilizes IMC technology to provide signal intensities over a wide dynamic range and entails one-step detection of 40-plus markers without issues of tissue autofluorescence, making it ideally suited for spatial biology applications. Whole slide imaging modes and an automated slide loader function enable a streamlined, versatile scalable workflow for high-throughput analysis. We used IMC technology to characterize immune cell populations and the spatial distribution of T cell exhaustion markers across various cancer types. We applied a 40-plus-marker IMC panel by integrating the Human Immuno-Oncology IMC Panel with the Human T Cell Exhaustion IMC Panel to study the TME of multiple human tissues. IMC analysis revealed striking heterogeneity with distinct tumor and immune-rich niches in the TME. In colon adenocarcinoma, we detected functional effector T cells and multiple tertiary lymphoid structures (TLSs). In contrast, we observed fewer TLSs and more T cells expressing PD-1 and TIM-3 at the tumor periphery in the urothelial carcinoma tissue, suggesting that they are in advanced exhaustion stages. We also observed expression of PD-L1, IDO and VISTA in the tumor cells. t-SNE and PhenoGraph clustering analysis of Cell Mode data segregated and spatially resolved the T cells into effector and exhausted T cell subpopulations, which were mapped back to the segmented cell masks. Unsupervised pixel clustering and analyses of Tissue Mode data further delineated distinct tumor areas based on the presence of infiltrating immune cells, tumor replicative activity and spatial proximity to stromal components. This work characterizing T cell exhaustion markers in multiple cancers showcases the capabilities of IMC technology and establishes it as a reliable high-plex, high-throughput spatial biology imaging platform. IMC technology is ideally suited for developing future translational and clinical applications and has the potential to help guide personalized therapeutic strategies for cancer treatment. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Thomas D. Pfister, Jyh Yun Chwee, Qanber Raza, Nikesh Parsotam, David Howell, Liang Lim, Christina Loh. Spatial profiling of exhausted T cells using high-plex imaging mass cytometry [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 2089.