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

Abstract

Abstract Gliomas including glioblastoma (GBM) are the most common malignant brain tumors. Glioma extracellular vesicles (EVs), especially exosomes in plasma have biological effects such as mediating immunosuppression and contain signature tumor-specific cargo, therefore could serve as liquid biopsies and as a non-invasive biomarker. However, isolation of glioma plasma exosomes has not been optimized. The purpose of this study is to develop an optimized method for plasma-derived exosomes isolation and study the immunosuppressive capacity of these particles. Plasma exosomes from glioma patients and normal donors were isolated by brief centrifugation to remove cells/debris followed by one or two-step density gradient ultracentrifugation (DGU). EV size and concentration were determined by NanoSight analysis. Nanoscale Flow Cytometry analysis quantified and characterized the resulting exosome and microvesicle populations isolated by DGU. Protein cargo was screened by Western blot, protein array, and ELISA. One-step DGU efficiently isolated exosomes for NanoSight analysis. Isocitrate dehydrogenase (IDH) WT glioma patients’ plasma exosomes were smaller but higher concentration than normal donors. A second DGU efficiently concentrated exosomes for subsequent cargo analysis. Nanoscale Flow cytometry analysis confirmed that DGU allows isolation and purification of exosomes, not microvesicles. Exosome-specific markers and the immunosuppressive molecule programmed death-ligand 1 (PD-L1), were present in both glioma patient and normal donor exosomes. Screening cytokine and checkpoint molecule arrays suggested reduced pro-inflammatory molecules are found in GBM patients’ plasma exosomes compared to normal donors.