Glioblastoma biomarkers in urinary extracellular vesicles reveal the potential for a ‘liquid gold’ biopsy

Biomarkers that reflect glioblastoma tumour activity and treatment response are urgently needed to guide clinical management, particularly for recurrent disease. As the urinary system is a major clearance route of circulating extracellular vesicles (EVs; 30–1000 nm nanoparticles), we explored whether sampling urinary EVs (uEVs) could serve as a simple and non-invasive liquid biopsy approach for measuring glioblastoma-associated biomarkers. Fifty urine specimens (15–60 ml) were collected from 24 catheterised glioblastoma patients immediately prior to primary (n = 17) and recurrence (n = 7) surgeries, following gross total resection (n = 9), and from age/gender-matched healthy participants (n = 14). EVs isolated by differential ultracentrifugation were characterised and proteomes analysed by high-resolution data-independent acquisition liquid chromatography tandem mass spectrometry (DIA-LC-MS/MS). Overall, 6857 proteins were confidently identified in urinary EVs (p ≤ 0.01), including 94 EV marker proteins. Glioblastoma-specific proteomic signatures were determined, and putative uEV biomarkers corresponding to tumour burden and recurrence were identified (fold change ≥ 2, adjusted p ≤ 0.05, AUC > 0.9). DIA-LC-MS/MS characterisation of uEVs substantiates them as a viable source of glioblastoma biomarkers. The promising ‘liquid gold’ biomarker candidates described warrant further investigation.