Hypoxia regulates small extracellular vesicle biogenesis and cargo sorting through HIF-1α/HRS signaling pathway in head and neck squamous cell carcinoma.

Small extracellular vesicles (sEVs) act as crucial messengers that transmit biological signals in hypoxic tumor microenvironment (TME), significantly impacting cancer progression. However, the precise mechanism by which hypoxia influences sEV biogenesis remains poorly understood. In this study, we observed increased sEV secretion and alterations in cargo composition in head and neck squamous cell carcinoma (HNSCC) cells under hypoxic conditions. We found that hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a key component of the endosomal sorting complexes required for transport (ESCRT), was upregulated during hypoxia. This upregulation activated the endosomal system and reduced degradation of multivesicular bodies (MVBs). HRS depletion altered the packaging of protein cargoes such as mitochondria-related proteins into sEVs under hypoxia, and these cargoes promoted a pro-tumorigenic phenotype of macrophages. Importantly, we demonstrated that HRS is transcriptionally activated by hypoxia inducible factor-1α (HIF-1α). Spatial transcriptomics and immunohistochemistry revealed a positive correlation between HRS and HIF-1α. These findings establish a link between the hypoxic response, sEV biogenesis, and cargo packaging, enhancing our understanding of how the hypoxic TME influences sEV biogenesis in HNSCC cells.