Abstract Background The study of cytotoxic T-lymphocytes (Tcy) has shown increased attention from the scientific community in the last years because of their predictive role in anti-cancer therapy with specific interest in immune check point blocking therapy. However, most of these studies have used bulk or dissociation-based single-cell technologies to draw their biological insights. Lately, new spatial -omics technologies are increasing our knowledge on this population by adding the spatial location of these cells in-situ in the native structure of the tissue. In this study, we have compared the activation of Tcy in reactive and cancerous conditions at single cell level and in the micro-anatomical context of the tissue. Experimental procedures One tissue micro array slide containing: reactive tonsil, inflammatory liver disease, breast cancer, glioblastoma and melanoma was stained using the COMET™ instrument (Lunaphore Technologies SA) for a multiplex panel of 20 markers. The derived multiplexed images were analyzed using the DISSCOVERY software platform developed by the MILAN Unit at KU Leuven (Belgium). 12/20 markers were specifically aimed at identifying the main cell populations in the tissue (CD11b, CD163, CD20, CD3, CD31, CD4, CD56, CD68, CD8, CK, FOXP3, S100) while the remaining 8 markers were aimed at adding functional information on immune cell types of interest (CD69, HLA-DR, Ki67, LAG3, OX40, PD-1, PD-L1, TIM3). Results The general activation levels of the Tcy in the reactive conditions were higher than in the three cancer subtypes. When looking at Tcy activation as a function of the distance from the vascular structures (identified via CD31+ endothelial cells), we observed an orderly distribution of activated Tcy around CD31+ endothelial cells with a gradient of increasing exhaustion levels with increasing distance from the vessel in the reactive conditions. In the cancer samples, on the contrary, a patchy disorderly organized pattern of exhausted and activated Tcy was observed around the vessels. Conclusion The spatial location of inflammatory cells plays a critical role to understand their functional behavior and thanks to technological progress it is now possible to start doing this at scale. The study of the dynamics of exhaustion of Tcy in the tissue will help clarify the interplay between cancer cells and immune environment. These results will be crucial to better select patients who may benefit the most of immune check point blockade. Citation Format: Maxim De Schepper, Asier Antoranz, Nikolina Dubroja, Giuseppe Floris, Frederik De Smet, Francesca Bosisio. Spatial dynamics of cytotoxic T lymphocyte exhaustion in reactive and tumoral tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6773.
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