Abstract 4621: Beyond tumor biomarkers: Multiparametric biophysical assessment of T cells from peripheral blood to inform checkpoint blockade therapy

Abstract Clinical decisions for immune checkpoint blockade (ICB) treatment currently rely on tumor-based biomarkers such as mutational burden, microsatellite instability, and PD-L1 expression. While these markers serve as valuable proxies for tumor antigenicity and the immune status of the tumor microenvironment, they fail to account for the fitness of a patient’s T cells, a critical factor in anti-tumor immunity. Direct assessment of T cell functional capacity could complement existing biomarkers to improve patient stratification and efficacy prediction for ICB therapies. Our previous work showed that single-cell mass measurements serve as a label-free means of assessing immune cell activation and ICB response. T cells from patients with various malignancies displayed significant variability in both their activation capacity and ICB response ex vivo, indicating that mass measurements capture key functional differences in T cells across patients.Here we present a next-generation platform that measures cell mass alongside linked measurements of volume, density, and morphological features. This platform incorporates fluorescence exclusion measurements to determine single-cell volume which, when combined with cell mass, enables precise determination of single-cell density. Additionally, we utilize inline brightfield imaging to capture images from individual cells and extract morphological features using an autoencoder-based reconstruction model.Our results show that multiparametric measurements significantly enhance the ability to characterize T cell functional states. In activation experiments using healthy donor T cells, these combined signatures reveal notable phenotypic changes as early as six hours after activation—changes that remain undetectable with mass measurements alone.In a cohort of peripheral blood samples from patients with advanced melanoma, we observed substantial heterogeneity in T cell biophysical signatures at baseline and following stimulation, with and without immune checkpoint blockade. These signatures were distinct from those observed in T cells derived from healthy donor PBMCs.These findings underscore the potential of a blood-based functional precision medicine assay to assess patients' baseline immune fitness. When integrated with existing clinical biomarkers, these unique functional signatures offer orthogonal insights that could improve patient stratification for immune checkpoint inhibitor therapies. Moreover, the multiparametric nature of these measurements aligns well with emerging multimodal AI approaches, paving the way for more effective identification of patients most likely to benefit from these treatments. Citation Format: Robert Kimmerling, Selim Olcum, Mark Stevens, Rachel LaBella, Madeleine Vacha, Katelin Katsis, Reginald Aikins, Steven Wasserman, Tatyana Sharova, Aleigha Lawless, Sonia Cohen, Genevieve Boland. Beyond tumor biomarkers: Multiparametric biophysical assessment of T cells from peripheral blood to inform checkpoint blockade therapy [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 4621.