Abstract 6439: Actin remodeling, inhibitory ligand polarization and small vesicle recruitment hinder effective anti-tumor immunity by shaping the tumor cell side of the immune synapse

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Abstract Cancer immune evasion is a major obstacle to effective anticancer immunotherapies. Recently, we unveiled a mechanism crucial in cancer escape from natural killer (NK)-mediated killing. The identified process is characterized by a rapid and massive accumulation of actin filaments at the cancer cell side of the immunological synapse (IS). We termed this process “actin response” (AR) and discovered that its inhibition is sufficient to restore cancer cell susceptibility to NK-mediated killing in vitro. Importantly, the AR is conserved across a wide range of malignancies, highlighting the broad translational potential of targeting this pathway. In this study, we investigated the molecular mechanisms underlying AR-mediated cancer cell immune evasion. Cancer-NK cell conjugates were analyzed by imaging flow cytometry (IFC) to investigate the polarization of inhibitory and activating ligands to the cancer cell side of the IS relative to the AR. We found that the AR is associated with and necessary for polarization of several inhibitory ligands. For instance, HLA-A,-B,-C molecules, which act as potent inhibitors of NK cells, were 2-fold increased at the IS of cancer cells with an AR when compared to cancer cells without an AR. Using confocal microscopy, we found that such polarization of inhibitory molecules is associated with the loss of MTOC and lytic granule polarization in conjugated human primary NK cells. In addition, correlative light and electron microscopy analysis revealed the presence of small vesicles (100 nm in size) and multivesicular bodies (MVBs) in the synaptic region of cancer cells with an AR. Quantification of vesicle markers at the IFC revealed a significant enrichment of CD63+/CD9+/CD81+ vesicles in the synaptic region of cancer cells with an AR. Interestingly, ultrastructure analysis showed that the AR is largely made of long actin-rich protrusions projecting into and altering the morphology of the synaptic cleft. Moreover, these protrusions were decorated with inhibitory molecules, such as PD-L1, and CD63. We identified a novel, highly conserved immune escape mechanism that exploits the fast remodeling of the actin cytoskeleton in cancer cells to induce the polarization of inhibitory ligands along with small tumor vesicles towards the IS. These findings suggest that actin remodeling in tumor cells at the IS actively promotes immune evasion. Further insights on the molecular and cellular mechanisms underpinning this pathway, such as the identification of selective targeting of linker proteins between the actin cytoskeleton and inhibitory ligands or small vesicles, represent a promising therapeutic strategy to improve the efficacy of anticancer immunotherapies. Nevertheless, additional research is needed in order to evaluate the translational potential of targeting the AR in a clinical setting. Citation Format: Andrea Michela Biolato, Liza Filali, Céline Hoffmann, Felix Kleine-Borgmann, Elena Ockfen, Max Krecke, Michel Mittelbronn, Clément Thomas. Actin remodeling, inhibitory ligand polarization and small vesicle recruitment hinder effective anti-tumor immunity by shaping the tumor cell side of the immune synapse [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 6439.