Abstract
<jats:title>Abstract</jats:title> <jats:p>Neutrophils display marked functional plasticity in cancer; however, it remains poorly understood how soluble factors derived from hypoxic and irradiated head and neck squamous cell carcinoma (HNSCC) cells reprogram neutrophil phenotype and function.</jats:p> <jats:p> Here, we employed a well characterized and controlled <jats:italic>in vitro</jats:italic> model to examine how tumor-conditioned media (TCM) from HNSCC cell lines cultured under ambient (21% O <jats:sub>2</jats:sub> ) or hypoxic (1% O <jats:sub>2</jats:sub> ) conditions, with or without 6 Gy gamma irradiation, modulate human neutrophil phenotype, and functional and transcriptional responses. Initial analyses were performed using TCM from three different HNSCC cell lines, whereas subsequent mechanistic characterization focused on FaDu-derived TCM. </jats:p> <jats:p>We show that TCM prolongs neutrophil survival in a cell line–dependent manner. Among the tested cell lines, hypoxia-conditioned FaDu-derived TCM promoted immunomodulatory neutrophil state characterized by enhanced survival, selective priming of ROS production, and elevated TRAIL-R3/TRAIL-R2 ratio. Induction of classical activation markers (CD11b, CD62L) was not evident. Transcriptomic analysis revealed minimal effects of normoxic TCM. Hypoxia-conditioned TCM induced a pronounced transcriptional program enriched in hypoxia- and stress-associated pathways. In contrast, irradiation of tumor cells had a limited additional impact on neutrophil reprogramming.</jats:p> <jats:p> Together, these findings indicate hypoxia-conditioned tumor secretomes as important drivers of neutrophil functional adaptation <jats:italic>in vitro</jats:italic> , supporting a model in which soluble factors alone are sufficient to induce a persistent, immunomodulatory neutrophil phenotype. This work provides mechanistic insight into tumor–neutrophil crosstalk, highlighting hypoxia-driven signaling as a potential therapeutic target in radioresistant HNSCC and supporting a role for neutrophil reprogramming in this context. </jats:p>