P08.05.A LIQUID BIOPSY FOR GLIOBLASTOMA: UTILIZING PLASMA EXTRACELLULAR VESICLES FOR TUMOR DIAGNOSIS AND MONITORING

Abstract BACKGROUND Glioblastoma (GBM), the deadliest brain tumor in adults, is traditionally diagnosed and characterized using neuroimaging and brain biopsies. These methods, however, suffer from limited specificity and sensitivity. The surgical risks associated with tumor sampling further complicate the monitoring of GBM’s molecular progression. Consequently, the identification of circulating GBM biomarkers is crucial for non-invasive tracking of the disease from diagnosis to relapse. Extracellular vesicles (EVs) secreted by GBM cells, which cross the blood-brain barrier and carry tumor-derived molecules, present a promising avenue for the continuous evaluation of GBM presence and status. MATERIAL AND METHODS We isolated extracellular vesicles (EVs) using Size Exclusion Chromatography (SEC) from 2 mL of plasma and characterized them via immunoblotting, flow cytometry, and transmission electron microscopy. The concentration and size of EVs were measured using Tunable Resistive Pulse Sensing (TRPS). We compared EV levels in 50 glioblastoma (GBM) patients pre-surgery with those in non-GBM controls, which included 100 healthy individuals and 50 patients with brain malignancies that present similarly to GBM in neuroimaging studies. Longitudinal assessments of plasma EVs were performed in 44 GBM patients by comparing pre-operative and post-operative samples taken 72 hours after surgery. Additionally, we analyzed surface markers on GBM and non-GBM plasma EVs using MACSPlex and conducted proteomic analyses via mass spectrometry in 27 GBM patients and 38 healthy individuals. RESULTS We found higher concentrations and mean sizes of plasma EVs in GBM patients compared to controls, with a noticeable decrease post-operatively. Multiplex flow cytometry indicated universal expression of CD9, CD63, and CD81 across all samples, with significant enrichment of CD63 and CD81 in GBM plasma EVs. The expression profiles of the other 34 markers were similar across all samples, suggesting dilution of tumor-derived EVs among non-tumor EVs. However, T-cell markers CD8 and HLA-DRDPDQ were more prevalent on GBM-derived EVs. Proteomic analysis identified over 2,000 proteins in all samples, with 117 being upregulated in GBM samples, predominantly within the complement cascade pathways. CONCLUSION Our findings suggest that monitoring circulating EV levels provides a reliable, non-invasive method for the differential diagnosis and follow-up of GBM patients. The combined analysis of EV concentration, size, and proteome offers potential as promising biomarkers for longitudinal monitoring of GBM, supporting the implementation of liquid biopsy in GBM care.