Song Hee Kim,1, Ji Won Kim,1, Min Seok Kim,1 Hee Jeong Cha,2 Seong Who Kim,3 Myung Woul Han1 1Department of Otorhinolaryngology-Head and Neck Surgery, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Korea; 2Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan, Republic of Korea; 3Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of KoreaThese authors contributed equally to this workCorrespondence: Myung Woul Han, Department of Otorhinolaryngology-Head and Neck Surgery, Ulsan University Hospital, University of Ulsan College of Medicine, 877 Bangeojinsunhwan-doro, Dong-gu, Ulsan, 44033, Republic of Korea, Email brightmoon@uuh.ulsan.kr Seong Who Kim, Department of Biochemistry and Molecular Biology, Asan Medical Center, University of Ulsan College of Medicine, 88 Olympic-Ro 43-Gil, Songpa-Gu, Seoul, 05505, Republic of Korea, Email swhokim@amc.seoul.krBackground: Head and neck squamous cell carcinoma (HNSCC) is characterized by a highly immunosuppressive tumor microenvironment (TME), with tumor-associated macrophages (TAMs) playing a central role in resistance to therapy. The immune checkpoint molecule CD47, known for its âdonât eat meâ signal, and EphA3, a receptor tyrosine kinase, are both upregulated in radiation-resistant HNSCC. However, their cooperative role in regulating TAMs and therapeutic resistance remains poorly understood.Methods: We established a radiation-resistant HNSCC model (MOC2R) using repeated irradiation and confirmed increased expression of CD47 and EphA3 in these cells. The effects of single and dual blockade of CD47 and EphA3 were evaluated through in vitro phagocytosis assays, Western blot, co-immunoprecipitation, and in vivo tumor models. Transcriptomic analysis and immunofluorescence staining were performed to elucidate molecular pathways involved in TAM modulation. We demonstrated in three independent experiments (n = 3) with statistical significance (P < 0.05).Results: CD47 and EphA3 were upregulated in radioresistant HNSCC and recurrent tumors. Dual blockade synergistically enhanced macrophage-mediated phagocytosis (P < 0.01) and significantly reduced clonogenic tumor cell survival compared to single blockade (P < 0.0001). EphA3 inhibition reprogrammed tumor-associated macrophages toward an M1-like phenotype, with increased expression of pro-inflammatory markers and decreased M2-associated markers. In vivo, combination therapy significantly reduced tumor volume and weight and increased M1 macrophage infiltration, while reducing M2 macrophages. Mechanistically, EphA3 regulated macrophage polarization via the β-catenin/FOSL2/ARID5A axis, enhancing the efficacy of CD47 blockade.Conclusion: Our findings demonstrate that EphA3, in cooperation with CD47, promotes an immunosuppressive TME by modulating β-catenin/FOSL2/ARID5A signaling in TAMs. Dual targeting of EphA3 and CD47 represents a promising strategy to reprogram macrophages and enhance anti-tumor immunity in radiation-resistant HNSCC.Keywords: head and neck cancer, recurrence, EphA3, CD47, tumor-associated macrophage, phagocytosis, macrophage
Kim et al. (Fri,) studied this question.