Potential clinical utility of small extracellular vesicles derived from head and neck tumors

Small extracellular vesicles (EVs) carry selective protein cargo that reflect the physiological state of their parent tumor cells, making them a promising source of biomarkers for head and neck cancer (HNC). HNC-derived small EVs are known to contribute to key pathological processes, including immunosuppression, therapy resistance, and metastatic potential. Saliva and plasma, as accessible and minimally invasive biofluids, represent surrogates for tumor-derived biomarkers to enhance clinical monitoring. However, it remains unclear whether small EVs enriched from these fluids accurately represent tumor-derived EVs. This study optimised small EV isolation from HNC tumor tissue and evaluated proteomic overlap between tumor-, saliva-, and plasma-derived EVs. Small EVs were isolated from tumor tissue and saliva by ultracentrifugation and from plasma using size exclusion chromatography. The morphology, size and concentration of small EVs were assessed by cryogenic Transmission electron microscopy (cryo-TEM) and nanoparticle tracking analysis (NTA). The proteome of small EVs was profiled and quantified using SWATH mass spectrometry. Functional analysis of overlapping and unique proteins was performed to determine their biological relevance. Small EVs isolated from tumor tissue, saliva and plasma exhibited typical lipid bilayer morphology with sizes ranging from 40 to 200 nm. More than 60% of the identified protein cargoes were shared across all three sources. These shared proteins were significantly enriched in pathways associated with cancer progression, including mTORC1 signalling, coagulation, complement, epithelial-mesenchymal transition, and PI3K/AKT/mTOR signalling. To support the reliability of the proteomic results, a subset of proteins was further validated by immunoblotting. The substantial proteomic overlap between small EVs from tumor tissue, saliva, and plasma supports the feasibility of using biofluid EVs as surrogates for tumor EVs. Saliva and plasma, being easily accessible, represent viable, non-invasive sources for biomarker discovery and clinical monitoring. These findings provide a strong foundation for advancing liquid biopsy approaches in HNC.