Small extracellular vesicles as a novel liquid biopsy approach for glioblastoma.
Authors: Deep Gagan , He Yangen , Kumar Ashish , Singh Sangeeta , Kim Susy , Su Yixin , Herpai Denise , Fansler Ashley , Whitlow Christopher T , Strowd Roy E. , Debinski Waldemar , Lesser Glenn Jay 
Published: June 2024

2073

Background: Glioblastoma (GBM) remains the most common and aggressive primary malignant brain tumor, and afflicted patients have limited options and poor overall survival. Diagnosis, prognosis, and assessment of treatment effectiveness is hampered by the absence of readily available sensitive, tumor specific, non-invasive blood-based assays which could be followed serially. Small extracellular vesicles (sEV) are nano-sized (≤200 nm) membrane bound bodies secreted by all cells, encapsulating cargo reflective of their parent cells. sEV have emerged as promising molecular indicator of a disease condition. Here, we report the feasibility of isolating glioma specific sEV (sEVglioma) from the plasma and characterizing those for GBM specific molecular biomarkers. Methods: Plasma samples were collected from patients (n=31) diagnosed with adult gliomas (including grade 3 and 4 GBM, grade 2 and 3 astrocytoma) and healthy individuals (n=9). Total sEV (TE) population was isolated from plasma by a modified precipitation (ExoQuick) method. Next, sEVglioma were isolated from the TE by an immunoprecipitation approach employing specific surface markers targeting cellular origin of gliomas, including astrocyte (GLAST and EAAT2), oligodendrocyte precursor cell (OSP and MOG), and neural stem cell (CD133). sEVglioma were characterized for size and concentration by nanoparticle tracking analyses; surface expression of glioma specific biomarkers by nano-flow cytometry and confocal microscopy; and the expression of a panel of specific miRNAs by RT-PCR. Lastly, sEVglioma were assessed by digital PCR for IDH1 status (wild type or mutated). Results: The average size of isolated sEVglioma was less than 200nm. Importantly, compared to TE, sEVglioma showed significant enrichment for glioma specific biomarkers such as ephrin type-A receptor 2 (14.7-fold), tenascin C (22.7-fold), and glial fibrillary acidic protein (8.4-fold) by nano-flow cytometry. Similarly, sEVglioma, but not TE, demonstrated high expression of EGFRvIII (3.6-fold), a known biomarker for GBM. These results were confirmed by confocal microscopy. Interestingly, expression of specific miRNAs (miR-9a-5p, miR-16-5p, miR-21-5p) in sEVglioma was higher in glioma patients with shorter survival (<12 months) compared to patients with longer survival (>20 months). Lastly, we successfully detected the existence of wild type IDH1 and absence of mutated IDH1 R132H in sEVglioma from GBM patients. This approach was validated in sEV isolated from the conditioned media of IDH1-wild type GBM cell lines (A172 and T98G) and a IDH1-mutated cell line (BT54). Conclusions: We present a novel approach to isolate sEVglioma from blood that could serve as a liquid biopsy, offering valuable molecular and genetic information. This approach promises early detection, potential to distinguish pseudo-progression, and assessment of treatment effectiveness with remarkable precision.