Head and neck tumor cells treated with hypofractionated irradiation die via apoptosis and are better taken up by M1-like macrophages

The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2 × 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. Human monocytes were differentiated with the hematopoietic growth factors M-CSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-$\gamma$ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2 × 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.