HPV status and oxygen tension shape transcriptomic, inflammatory, and cell cycle responses in HNSCC treated with ionizing radiation

Head and neck squamous cell carcinoma (HNSCC) comprises biologically distinct subtypes defined by human papillomavirus (HPV) status, which may influence treatment outcomes. Hypoxia is a common feature of solid tumors, including HNSCC, and can reduce radiotherapy efficacy by modulating DNA repair, cell-cycle progression, and inflammatory signaling. However, the combined impact of hypoxia and HPV status on cellular radiosensitivity remains poorly defined. We investigated the influence of hypoxia (1% O2) and gamma-irradiation on proliferation, cell cycle, apoptosis, transcriptomic profiles, and cytokine/chemokine secretion in three HNSCC cell lines: HPV-negative FaDu and Detroit-562 (metastatic origin), and HPV-positive 2A3. Cells were cultured under chronic hypoxic or ambient conditions, with or without irradiation. Independent of HPV status or metastatic phenotype, hypoxia prolonged the cell doubling time, while only irradiated cells (under both oxygen conditions) exhibited features of an intermediate epithelial-mesenchymal transition phenotype. Treatment of HPV-positive cells significantly decreased the number of upregulated genes, reduced cytokine secretion (IL-8, SERPINE1), and affected all phases of the cell cycle compared with HPV-negative cells. Furthermore, only the metastatic cell line showed no change in cleaved caspase-3 levels after irradiation in hypoxia and produced higher levels of cytokines (IL-8, SERPINE1). Our findings highlight how tumor oxygenation and HPV status intersect to modulate the radiation response in HNSCC. These insights may guide the development of personalized, biomarker-driven radiotherapy strategies that account for both the tumor microenvironment and viral etiology.