M1 macrophage-derived exosomal miR-20b promotes radiosensitization via CCND1 in HPV+ 1 HNSCC

Liu Huan^1,2SIwei Zhang²Zengchen Liu²Tingdan Gong^2,3Siyu Duan²Tianyang Liu²Fangjia Tong²Wanlin Li²Shuang Pan^1*Lanlan Wei^2*

• ¹First Affiliated Hospital of Harbin Medical University, Harbin, China
• ²Shenzhen Third People's Hospital, Shenzhen, China
• ³Harbin Medical University, Harbin, China

Background: Human papillomavirus (HPV) is a significant risk factor for head and neck squamous cell 13 carcinoma (HNSCC). HPV positive (HPV+) HNSCC is more sensitive to radiotherapy and has a better 14 prognosis than HPV negative (HPV-) HNSCC. M1 macrophages not only enhance the radiosensitivity of 15 HPV+ HNSCC, but the M1 macrophages derived exosmoes (M1 exos) also possess anti-tumor activity. 16 However, the role of M1 exos in the radiosensitivity of HNSCC remains unclear. 17 Materials and Methods: HPV status and macrophage infiltration levels in 25 HNSCC tissues were 18 evaluated by immunohistochemistry (IHC). M1 macrophages were induced and cultured in vitro, and 19 exosomes were extracted through differential ultracentrifugation. The effect of M1 exos on the radiosensitivity 20 of HPV+ HNSCC was assessed via an in vitro co-culture system. The expression level of γ-H2AX was 21 assessed by immunofluorescence. The levels of miR-20b in HNSCC were ananlyzed using multicenter data 22 (from TCGA and GEO databases), along with their correlation to radiosensitivity and prognosis. Cellular 23 experiments demonstrated that overexpressing miR-20b significantly enhanced radiosensitivity in HPV+ 24 HNSCC. Bioinformatic and experimental validation identified CCND1 as a target of miR-20b. 25 Results: In HPV+ HNSCC, M1 macrophages were highly infiltrated and played a crucial role in 26 improving the therapeutic effect of HPV+ HNSCC. M1 exos infiltrated HPV+ HNSCC, increasing their 27 sensitivity to radiation. Meanwhile, M1 macrophages exhibited higher miR-20b levels than M2 macrophages, 28 and the radiosensitivity of HPV+ HNSCC was significantly increased by transfecting them with a miR-20b 29 mimic. Functional analysis of target genes, CCND1 as a key gene through which miR-20b enhanced 30 radiosensitivity in HPV+ HNSCC. 31 Conclusion: In this study, our results suggest that M1 exos, enriched with miR-20b, regulate the DNA 32 damage repair pathway in tumor cells by targeting CCND1, enhancing the radiosensitivity of HPV+ HNSCC.