Biom-48. a novel liquid biopsy-based approach to isolate and characterize glioblastoma-derived small extracellular vesicles from blood

Abstract Glioblastoma multiforme (GBM) is a highly aggressive brain cancer characterized by a poor prognosis and limited treatment options. The absence of early diagnostic measures, prognostic biomarkers, and reliable methods for monitoring disease progression contribute to the high mortality rate among GBM patients. In recent years, liquid biopsy has emerged as a non-invasive tool for assessing cancer onset and therapeutic responses. Among the various liquid biopsy approaches, extracellular vesicles (EVs) have gained attention; as lipid bilayer membrane-delimited 30-150nm sized small extracellular vesicles (sEV) are released by all cell types. These vesicles carry molecular cargo reflecting the cell of origin and its pathophysiological state, making them a promising alternative to other liquid biopsy methods, such as circulating tumor cells and circulating nucleotides. However, the application of sEV-based liquid biopsy is hindered by the lack of methods to enrich GBM tumor-specific sEV in the blood. Our team has developed unique tools and expertise in isolating cell/tissue-specific sEV from biofluids. We have previously reported the isolation of sEV derived from specific brain cells in the blood, characterizing them for diagnosing neurodegeneration and predicting treatment response. In this study, we first isolated total sEV (TE) from the plasma of GBM patients (n = 11). Subsequently, we enriched GBM-specific sEV (sEVGBM) from TE using the astrocyte-specific surface marker glutamate aspartate transporter (GLAST). To achieve this, we utilized biotin-tagged GLAST antibody and streptavidin-coated magnetic beads for sEVGBM isolation. We then extensively characterized the size and concentration (number/ml) of sEVGBM by nanoparticle tracking analyses, while evaluating their purity through nanoflow cytometry for GLAST expression. Furthermore, we confirmed the expression of GBM-specific markers EGFRvIII, EPHB2 and IL-13Rα2 on the surface of sEVGBM (n = 3). Importantly, the expression of these markers was significantly enriched on sEVGBM compared to TE, underscoring the significance of our strategy as a potential liquid biopsy for GBM.