Global extracellular vesicle proteomic signature defines U87-MG glioma cell hypoxic status with potential implications for non-invasive diagnostics

Abstract

PurposeGlioblastoma multiforme (GBM) is the most common and lethal of primary malignant brain tumors. Hypoxia constitutes a major determining factor for the poor prognosis of high-grade glioma patients, and is known to contribute to the development of treatment resistance. Therefore, new strategies to comprehensively profile and monitor the hypoxic status of gliomas are of high clinical relevance. Here, we have explored how the proteome of secreted extracellular vesicles (EVs) at the global level may reflect hypoxic glioma cells.MethodsWe have employed shotgun proteomics and label free quantification to profile EVs isolated from human high-grade glioma U87-MG cells cultured at normoxia or hypoxia. Parallel reaction monitoring was used to quantify the identified, hypoxia-associated EV proteins. To determine the potential biological significance of hypoxia-associated proteins, the cumulative Z score of identified EV proteins was compared with GBM subtypes from HGCC and TCGA databases.ResultsIn total, 2928 proteins were identified in EVs, out of which 1654 proteins overlapped with the ExoCarta EV-specific database. We found 1034 proteins in EVs that were unique to the hypoxic status of U87-MG cells. We subsequently identified an EV protein signature, “HYPSIGNATURE”, encompassing nine proteins that strongly represented the hypoxic situation and exhibited close proximity to the mesenchymal GBM subtype.ConclusionsWe propose, for the first time, an EV protein signature that could comprehensively reflect the hypoxic status of high-grade glioma cells. The presented data provide proof-of-concept for targeted proteomic profiling of glioma derived EVs, which should motivate future studies exploring its utility in non-invasive diagnosis and monitoring of brain tumor patients.Electronic supplementary materialThe online version of this article (10.1007/s11060-019-03262-4) contains supplementary material, which is available to authorized users.

Proteome of Glioblastoma-Derived Exosomes as a Source of Biomarkers

“…A more detailed analysis of the involvement of these proteins in the cellular processes is presented in Supplementary Tables S2 and S3 . Similar results for protein functions in glioblastoma exosomes have been described in the literature [ 20 , 21 ]…”

“…A more detailed analysis of the involvement of these proteins in the cellular processes is presented in Supplementary Tables S2 and S3. Similar results for protein functions in glioblastoma exosomes have been described in the literature [20,21] (1433E). Two more proteins, nucleophosmin (NPM) and cofilin (COF1), are present in four exosome samples ( Supplementary Table S1).…”

Hypoxia and extracellular vesicles: A review on methods, vesicular cargo and functions

“…Indira Chandran et al. (2019)) utilized proteomics and analysed hypo‐ and normo‐EVs from glioblastoma cells and confirmed the increased release of IGFBP3 and CA9 in hypoxia. They showed enrichment of HIF signalling pathway proteins in hypo‐EVs and also validated this altered release at the transcriptional level.…”

Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration

“…It is now clear that GBMs are able to release EVs [ 14 ], thanks to studies that have identified many of the intracellular molecules secreted by EVs. Investigation has been mostly performed on EVs deriving from different human glioma cell lines [ 15 , 16 ] and, also, in GSCs [ 17 , 18 , 19 ] or plasma from patients with primary GBM [ 20 ]. However, it has been limited to isolation and characterization of Exos.…”