Abstract B048: Spatiotemporal analysis during tumor rejection reveals coordinated cellular dynamics underlying successful immune-checkpoint therapy
Authors: Medrano Ruan FV , Sukhov Vladimir , Artyomov Maxim , Schreiber Robert 
Published: February 2025

Abstract
There is a growing appreciation for the role that the spatial organization of immune cells within the tumor microenvironment (TME) plays in shaping cellular function and response to therapy. However, the majority of studies have been limited to analyzing immune responses at a single time point, and so, little is known about the spatiotemporal changes that underlie successful tumor rejection after cancer immunotherapy. In this study, we used the CODEX multiplex imaging system with a 32-plex antibody panel to longitudinally characterize the spatial changes in the lymphoid and myeloid cell populations that were previously identified by scRNA-Seq and CyTOF to be associated with complete tumor rejection in mice treated with the combination of α-PD-1 and α-CTLA-4 (combo ICT) in our well-characterized mouse T3 MCA sarcoma model. T3 tumor-bearing mice were treated with control antibody or combo ICT, tumors were harvested at multiple time points (day 7, 9, 10, 11 and 13), fresh frozen, sectioned, and subjected to CODEX multiplex imaging. A total of 42 tumors were imaged with the full range of the tumor-immune boundary. A hierarchical cell clustering approach developed in-house was used to profile 13,051,156 cells resulting in the identification of 13 distinct cell types. Our results show that after ICT the decrease in T3 cells in the tumor tissue starts on day 10 and culminates in the complete elimination of tumor cells by day 13. This immune-mediated response is driven by an increase in CD4+ T cells, which allows them to more frequently co-localize with CD8+ T cells and type 1 conventional dendritic cells (cDC1), forming three-cell clusters with increased cell contacts. This change in cell-to-cell interactions is followed by a broader reorganization of the TME as noted by the expansion of a lymphoid-rich cellular neighborhood (CN) that harbors the majority of CD4+ and CD8+ T cells. Interestingly, this CN becomes a hub for T cell effector functions, characterized by an enrichment of GZM-B+ and KI67+ T cells, and exhibits spatial dynamics throughout tumor regression. While both GZM-B+ and KI67+ T cells are observed in proximity to blood vessels on days 7 and 9, they begin to spatially separate into distinct regions after day 10, indicating that cytotoxic T cells move towards areas with tumor cells, while proliferating T cells are more frequently observed at the periphery. This dynamic behavior was further confirmed by assessing the communication rules between CNs, which showed that the lymphoid-rich CN acts by eliminating layers of tumor cells at the tumor boundary and by modulating nearby myeloid populations. Additionally, integration of CODEX dataset with scRNA-seq revealed CD4+ T cells produce Interferon-gamma and a change in the molecular chemotactic pathways orchestrating lymphoid and myeloid communication. Overall, this study provides a detailed spatial view of the cellular events that result in successful ICT and allows us to propose a computational model of the actions of T cells in coordinating the antitumor response after α-CTLA-4/α-PD1.
Citation Format: Ruan FV Medrano, Vladimir Sukhov, Maxim Artyomov, Robert Schreiber. Spatiotemporal analysis during tumor rejection reveals coordinated cellular dynamics underlying successful immune-checkpoint therapy [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B048.