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

Abstract The selective protein cargoes carried within small EVs create a distinctive proteome that mirrors the condition of tumour cells, making them an invaluable source of biomarkers for head and neck cancer (HNC). Liquid biopsies utilising EVs from biofluids offer a non‐invasive alternative to tissue biopsies for cancer diagnosis and monitoring. However, whether small EVs enriched from biofluids can accurately reflect tumour‐derived small EVs remains unknown. In this study, we first optimised small EV isolation from tumour tissue of HNC patients. Subsequently, we evaluated the protein overlap of small EVs from tumour, saliva and plasma and characterised their potential application in liquid biopsy. Small EVs were isolated from tumour and saliva using ultracentrifugation and plasma samples using size exclusion chromatography. Cryogenic Transmission electron microscopy (cryo‐TEM) and nanoparticle tracking analysis (NTA) were employed to assess the morphology, size and concentration of small EVs. 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 tumour tissue, saliva and plasma displayed the expected lipid bilayer morphology, with a size range of 40 to 200 nm. Over 60% of the protein cargoes identified in small EVs were shared among tumour tissue, saliva and plasma. 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. The high degree of similarity in protein cargoes among small EVs from tumour tissue, saliva and plasma underscores the promise of liquid biopsies for cancer detection and monitoring. Saliva and plasma, being easily accessible, represent viable, non‐invasive sources for biomarker discovery and clinical application. These findings provide a strong foundation for further research to validate liquid biopsy approaches in clinical settings.