Isolation and analysis of plasma-derived exosomes in patients with glioma

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. Mayo Clinic researchers 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.

Plasma exosome isolation by density gradient ultracentrifugation (DGU)

(A) Whole blood samples collected in EDTA tubes underwent brief centrifugation (3,000 RPM or 1,811 xg for 10 min) for plasma isolation. The isolated plasma was transferred to a fresh tube and spun for 15 min at 3,000 RPM (1,811 xg) to remove any remaining cellular debris and erythrocytes. (B). Density gradient ultracentrifugation to purify exosomes was performed by mixing 1 ml of plasma with 1 ml of 50% OptiPrep solution. Eleven ml of 10% OptiPrep was layered on top of the homogenized solution and this underwent ultracentrifugation for 90 min at 24,000 RPM or 102,445 xg (Spin 1). The top layer (10 ml) was collected. A portion was used for nanotracker particle analysis and nanoscale flow cytometry analysis while the remainder underwent ultracentrifugation at 24,000 RPM (102,445 xg) for 16 h (Spin 2). The supernatant was discarded and the pellet was resuspended in a total volume of 200 μl. Further analysis (nanoparticle tracking, western blots, protein arrays, and ELISAs) was performed using this final, concentrated solution.