Phosphatidylserine on Hypoxic Tumor-Derived sEVs Regulates ADAM17 Activity and T-Cell Fate 2310261

Abstract

Tumor hypoxia is a hallmark of head and neck squamous cell carcinoma (HNSCC) and promotes tumor prgression. Tumor-derived small extracellular vesicles (sEVs) are mediators of hypoxia-driven signaling and contribute to immune suppression within the tumor microenvironment. ADAM17, a membrane-associated sheddase regulating inflammatory and immune pathways, is sensitive to membrane lipid composition, particularly phosphatidylserine (PS). We investigated how hypoxia-induced lipidomic remodeling of tumor-derived sEVs affects ADAM17 activity and CD8+ T-cell apoptosis. HNSCC cells (PCI-30) and normal keratinocytes (HaCaT) were cultured under normoxia (21% O₂) or hypoxia (1% O₂). sEVs were isolated by size-exclusion chromatography and characterized by nanoparticle tracking analysis, transmission electron microscopy, immunoblotting, and high-resolution untargeted lipidomics. Surface PS and ADAM17 expression were assessed by bead-based flow cytometry, and ADAM17 activity was measured using fluorogenic assays. Human CD8+ T cells were incubated with hypoxic or normoxic tumor-derived sEVs. PS-mediated interactions were blocked using Annexin V, and CD8+ T-cell apoptosis was quantified by flow cytometry. sEVs (125—135 nm) expressed Alix, CD63 and CD9 but not Grp94. Hypoxia selectively enriched PS species with mono-, polyunsaturated and very long acyl chains, reducing surface-exposed PS. Despite decreased PS exposure, hypoxic TEX displayed significantly increased ADAM17 expression and enzymatic activity, resulting in an elevated ADAM17/PS ratio. Functionally, hypoxic TEX induced higher levels of CD8+ T-cell apoptosis compared with normoxic TEX. Blockade of PS with annexin V significanlty reduced ADAM17 activity and attenuated TEX-induced CD8+ T-cell apoptosis. Hypoxia alters phosphatidylserine organization in tumor-derived sEVs, enhancing ADAM17 activation, promoting PS-dependent CD8+ T-cell apoptosis, and facilitating immune evasion in HNSCC. n/a Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)