%0 Online Multimedia %A Garcia, Luz M. Cumba %A Peterson, Timothy E. %A Cepeda, Mario A. %A Johnson, Aaron J. %A Parney, Ian F. %D 2019 %T Video_2_Isolation and Analysis of Plasma-Derived Exosomes in Patients With Glioma.MP4 %U https://frontiersin.figshare.com/articles/media/Video_2_Isolation_and_Analysis_of_Plasma-Derived_Exosomes_in_Patients_With_Glioma_MP4/8874305 %R 10.3389/fonc.2019.00651.s003 %2 https://frontiersin.figshare.com/ndownloader/files/16269938 %K glioblastoma %K plasma-derived exosomes %K isolation %K immunosuppression %K biomarkers %X

Gliomas including glioblastoma (GBM) are the most common primary malignant brain tumors. Glioma extracellular vesicles (EVs) including exosomes have biological effects (e.g., immunosuppression) and contain tumor-specific cargo that could facilitate liquid biopsies. We aimed to develop a simple, reproducible technique to isolate plasma exosomes in glioma patients. Glioma patients' and normal donors' plasma exosomes underwent brief centrifugation to remove cells/debris followed by serial density gradient ultracentrifugation (DGU). EV size/concentration was determined by nanoparticle tracking. Protein cargo was screened by array, western blot, and ELISA. Nanoscale flow cytometry analysis quantified exosome and microvesicle populations pre- and post-DGU. One-step DGU efficiently isolates exosomes for nanoparticle tracking. Wild type isocitrate dehydrogenase glioma patients' (i.e., more aggressive tumors) plasma exosomes are smaller but higher concentration than normal donors. A second DGU efficiently concentrates exosomes for subsequent cargo analysis but results in vesicle aggregation that skews nanoparticle tracking. Cytokines and co-stimulatory molecules are readily detected but appeared globally reduced in GBM patients' exosomes. Surprisingly, immunosuppressive programmed death-ligand 1 (PD-L1) is present in both patients' and normal donors' exosomes. Nanoscale flow cytometry confirms efficient exosome (<100 nm) isolation post-DGU but also demonstrates increase in microvesicles (>100 nm) in GBM patients' plasma pre-DGU. Serial DGU efficiently isolates plasma exosomes with distinct differences between GBM patients and normal donors, suggesting utility for non-invasive biomarker assessment. Initial results suggest global immunosuppression rather than increased circulating tumor-derived immunosuppressive exosomes, though further assessment is needed. Increased glioma patients' plasma microvesicles suggest these may also be a key source for biomarkers.

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