Small extracellular vesicles as a novel liquid biopsy approach for glioblastoma.
Authors: Gagan Deep, Yangen He, Ashish Kumar, Sangeeta Singh, Susy Kim, Yixin Su, Denise Herpai, Ashley Fansler, Christopher T Whitlow, Roy E. Strowd, Waldemar Debinski, Glenn Jay Lesser
Journal: Journal of Clinical Oncology (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 (sEV glioma ) 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, sEV glioma 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). sEV glioma 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, sEV glioma were assessed by digital PCR for IDH1 status (wild type or mutated). Results: The average size of isolated sEV glioma was less than 200nm. Importantly, compared to TE, sEV glioma 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, sEV glioma , 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 sEV glioma was higher in glioma patients with shorter survival (&lt;12 months) compared to patients with longer survival (&gt;20 months). Lastly, we successfully detected the existence of wild type IDH1 and absence of mutated IDH1 R132H in sEV glioma 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 sEV glioma 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.