DNA-dependent protein kinase regulates cytosolic double-stranded DNA secretion from irradiated macrophages to increase radiosensitivity of tumors

• Published in: Radiotherapy and oncology 2024-04, Vol.193, p.110111, Article 110111
• Main Authors: Oh, Taerim, Kang, Gi-Sue, Jo, Hye-Ju, Park, Hye-Joon, Lee, Ye-Rim, Ahn, G-One
• Format: Article
• Language: English
• Subjects: cytosolic dsDNA; DNA repair; DNA-PK; Macrophages; Radiosensitivity
• ISSN: 0167-8140; 1879-0887; 1879-0887
• DOI: 10.1016/j.radonc.2024.110111

[Display omitted] •Radiotherapy is given to tumor bearing volume, which includes not only cancer cells but also tumor-associated macrophages.•Irradiated macrophages secrete cyotosolic dsDNA to increase radiosensitivity of cancer cells by inhibiting DNA repair.•Secretion of cytosolic dsDNA from irradiated macrophages is regulated by DNA-PK, which phosphorylates MLC.•Tumors grown in myeloid-specific Prkdc (gene encoding DNA-PK) knockout mice are more radioresistant than wild-type mice. To investigate the molecular mechanism by which irradiated macrophages secrete cytosolic double-stranded DNA (c-dsDNA) to increase radiosensitivity of tumors. Irradiated bone marrow-derived macrophages (BMDM) were co-incubated with irradiated EO771 or MC38 cancer cells to determine clonogenic survival. c-dsDNA were measured by agarose gel or enzyme-linked immunosorbent assay. BMDM or cancer cells were analyzed with immunostaining or western blot. Subcutaneously implanted MC38 cells in myeloid-specific Prkdc knockout (KO) mice or littermate control mice were irradiated with 8 Gy to determine radiosensitivity of tumors. We observed that irradiated BMDM significantly increased radiosensitivity of cancer cells. By performing immunostaining, we found that there was a dose-dependent increase in the formation of c-dsDNA and phosphorylation in DNA-dependent protein kinase (DNA-PK) in irradiated BMDM. Importantly, c-dsDNA in irradiated BMDM could be secreted to the extracellular milieu and this process required DNA-PK, which phosphorylated myosin light chain to regulate the secretion. The secreted c-dsDNA from irradiated BMDM then activated toll-like receptor-9 and subsequent nuclear factor kappa-light-chain-enhancer of activated B cells signaling in the adjacent cancer cells inhibiting radiation-induced DNA double strand break repair. Lastly, we observed that irradiated tumors in vivo had a significantly increased number of tumor-associated macrophages (TAM) with phosphorylated DNA-PK expression in the cytosol. Furthermore, tumors grown in myeloid-specific Prkdc KO mice, in which TAM lacked phosphorylated DNA-PK expression were significantly more radioresistant than those of the wild-type control mice. Irradiated macrophages can increase antitumor efficacy of radiotherapy through secretion of c-dsDNA under the regulation of DNA-PK.