138 - Radiation-Enhanced CD24 Membrane Trafficking via GPI Anchoring Underlies Immune Evasion after Radiotherapy
Presenter(s)
L. Kong, M. Zhou, Y. Sun, X. Liu, W. Yuan, Y. Wang, and K. Yang; Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Purpose/Objective(s): Radiotherapy is pivotal in cancer treatment, yet radiation-induced immune evasion limits therapeutic synergy. This study interrogates radiation-induced immune evasion through the lens of innate immune checkpoints, specifically delineating CD24 as a radiation-responsive regulator and dissecting its post-irradiation regulatory mechanism, thereby advancing strategies to circumvent radioresistance.
Materials/Methods: Radiation-induced CD24 surface dynamics were assessed via flow cytometry, immunofluorescence and western blot. Proteomic profiling identified GPAA1 as a CD24 membrane trafficking regulator. Co-IP-MS and ubiquitination assays revealed the ANAPC5/GPAA1 regulatory axis. In vitro phagocytosis was quantified by FACS and confocal microscopy. In vivo therapeutic efficacy was validated in murine models (pancreatic, lung and liver carcinomas). Tumor immune microenvironment was profiled via flow cytometry and multispectral imaging.
Results: While radiation promoted immunogenic cell death and macrophage activation, phagocytosis assays revealed no significant increase in macrophage-mediated tumor cell clearance post-irradiation. Radiation triggered dose-dependent CD24 surface upregulation across tumor types, impairing macrophage phagocytosis. Combined CD24 blockade and radiotherapy significantly reversed phagocytic resistance, overcoming immune evasion. Mechanistically, molecular analyses demonstrated radiation augmented CD24 membrane presence without altering total protein levels. Proteomic profiling identified GPAA1—the catalytic subunit of glycosylphosphatidylinositol (GPI)-transamidase—as the key regulator of radiation-induced CD24 membrane localization. Specifically, radiation disrupted the Anaphase-Promoting Complex/Cyclosome (APC/C), suppressing ANAPC5-mediated ubiquitination of GPAA1 at lysine 111. The subsequent accumulation of GPAA1 facilitated GPI anchoring to substrate proteins, thereby enhancing CD24 membrane trafficking. Genetic ablation of GPAA1 or CD24 restored macrophage phagocytosis in vitro and synergistically enhanced radiotherapy efficacy in vivo. CD24 deficiency further elicited systemic abscopal effects, suppressing distal tumor progression in irradiated mice. Flow cytometric profiling of TIME demonstrated that combination therapy reversed macrophage dysfunction (M2-to-M1 polarization) and amplified T cell-mediated adaptive immunity (increased CD8+ T cell infiltration and IFN-? production). Antibody-mediated depletion of either T cells or macrophages abrogated therapeutic efficacy, confirming their indispensable roles.
Conclusion: This study establishes that radiation enhances CD24 membrane localization through GPI-anchoring governed by the ANAPC5/GPAA1 axis. Our findings redefine CD24 as a radiation-responsive innate immune checkpoint and propose a CD24-targeted combinatorial strategy to potentiate radiotherapy efficacy.