Isolation and characterization of exosomes from IDH mutant gliomas.

Abstract

152 Background: Mutations in the metabolic enzyme isocitrate dehydrogenase 1 (IDH1) are found in > 70% of intermediate grade gliomas. Gain-of-function mutations in IDH1 promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Tumor-derived exosomes (TEX) accumulate in the tumor microenvironment (TME) and in body fluids of patients. TEX serve as a communication system between tumor and normal cells. Circulating TEX carry a complex molecular and genetic cargo and interfere with functions of immune cells. This study characterizes TEX produced by IDH mutant glioma spheres and TEX from plasma of patients with IDH mutant gliomas. Methods: TEX produced by patient-derived IDH1(mut) or IDH1(wt) glioma spheres or TEX in patient’s plasma were isolated by mini size exclusion chromatography (mini-SEC). TEX morphology, size, numbers and molecular profile were characterized and the crossing of the blood-brain barrier (BBB) and biodistribution was investigated by in vivo imaging of TEX. Interactions of TEX with endothelial cells, astrocytes and immune cells were demonstrated by confocal microscopy. Results: TEX isolated from supernatants of IDH1(mut) or IDH1(wt) glioma spheres by mini-SEC carried TSG101 and showed the typical size and vesicular morphology of exosomes. BCA protein assays and qNano analysis revealed an elevated exosome production by IDH mutant cells ( p< 0.01). TEX carried immunosuppressive proteins (FasL, CTLA-4 and TRAIL) and IDH mutant exosomes carried higher levels of these proteins and of adenosine pathway components CD39 and CD73. Labeled TEX injected in the white matter of mice crossed the BBB and were detectable in distant organs 24h after injection. TEX were rapidly internalized by endothelial cells and astrocytes, but not by T cells, which interacted with TEX by surface signaling. These results were confirmed by investigating TEX isolated from glioma patient’s plasma. Conclusions: The data suggest that TEX play an important role in IDH mutant gliomas and drive tumor progression in part by inducing suppression of immune cells. Future efforts will focus on characterizing immunosuppressive effects of TEX derived from IDH mutant cells vs gliomas without IDH mutations.