Isolation and Analysis of Plasma-Derived Exosomes in Patients With Glioma

Abstract

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.

Cytokine profiling in serum-derived exosomes isolated by different methods

“…MBC patients' exosomes had a low concentration of MIP-3 alpha, IL-23, M-CSF, Eotaxin-3, BLC, SDF-1 alpha, IL-2R, MDC, FGF-2, IL-22, and IL-31, suggesting that metastatic disease may be associated with immune suppression related to low exosomal cytokines. These results are consistent with previous reports that the levels of the cytokines IFN-γ, IL-10, and IL-3 were decreased in plasma exosomes from grade 4 GBM (glioblastoma multiforme) patients compared to normal donors 37 . Unlike exosomes, MBC patients' serum had a high concentration of MCP-1 and MIF, suggesting that high levels of MCP-1 and MIF play an important role in metastasis.…”

Differential plasma exosomal long non-coding RNAs expression profiles and their emerging role in E-cigarette users, cigarette, waterpipe, and dual smokers

“…We chose to use plasma-derived exosomes for this study as they are the most widely studied and extensively characterized exosomes [ 6 , 51 ]. Blood plasma is easy to collect as compared to other biofluids like BALF and the method of isolation of exosomes (with high purity and yield) from blood plasma is well characterized [ 6 , 52 ]. Furthermore, plasma and serum-derived exosomes have the potential to develop as biomarkers for various disease condition [ 51 , 53 , 54 ].…”

Uncovering the Exosomes Diversity: A Window of Opportunity for Tumor Progression Monitoring

“…TDEs are, in fact, able to modulate endothelial cells, contributing to their proliferation and migration [36]. A markedly decreased expression of inflammatory markers was found in Glioma patients' exosomes, compatible with the effects of tumor-mediated immunosuppression [37].…”