Molecular subtypes and differentiation programmes of glioma stem cells as determinants of extracellular vesicle profiles and endothelial cell‐stimulating activities

Abstract

We have previously uncovered the impact of oncogenic and differentiation processes on extracellular vesicles (EVs) in cancer. This is of interested in the context of glioma stem cells (GSC) that are responsible for recurrent nature of glioblastoma multiforme (GBM), while retaining the potential to undergo differentiation and self renewal. GSCs reside in vascular niches where they interact with endothelial cells through a number of mediators including bioactive cargo of EVs. GSCs can be classified as proneural (PN) or mesenchymal (MES) subtypes on the basis of their gene expression profiles and distinct biological characteristics. In the present study we investigated how GSC diversity and differentiation programmes influence their EV-mediated communication potentials. Indeed, molecular subtypes of GBMs and GSCs differ with respect to their expression of EV-related genes (vesiculome) and GSCs with PN or MES phenotypes produce EVs with markedly different characteristics, marker profiles, proteomes and endothelial stimulating activities. For example, while EVs of PN GSC are largely devoid of exosomal markers their counterparts from MES GSCs express ample CD9, CD63 and CD81 tetraspanins. In both GSC subtypes serum-induced differentiation results in profound, but distinct changes of cellular phenotypes including the enhanced EV production, reconfiguration of their proteomes and the related functional pathways. Notably, the EV uptake was a function of both subtype and differentiation state of donor cells. Thus, while, EVs produced by differentiated MES GSCs were internalized less efficiently than those from undifferentiated cells they exhibited an increased stimulatory potential for human brain endothelial cells. Such stimulating activity was also observed for EVs derived from differentiated PN GSCs, despite their even weaker uptake by endothelial cells. These findings suggest that the role of EVs as biological mediators and biomarkers in GBM may depend on the molecular subtype and functional state of donor cancer cells, including cancer stem cells.Abbreviations: CryoTEM: cryo-transmission electron microscopy; DIFF: differentiated GSCs; EGF: epidermal growth factor; DUC: differential ultracentrifugation; EV: extracellular vesicle; FGF: fibroblast growth factor; GBM: glioblastoma multiforme; GFAP: glial fibrillary acidic protein; GO: gene ontology; GSC: glioma stem cells; HBEC-5i: human brain endothelial cells; MES: mesenchymal cells; MTS - [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; PMT1: proneural-to-mesenchyman transition cell line 1; PN: proneural cells; TEM: transmission electron microscopy; WB: western blotting

Oncogenic Regulation of Extracellular Vesicle Proteome and Heterogeneity

“…Since cellular vesicles of near‐exosomal size could contain up to 200 proteins each, the predicted number of such non‐overlapping protein combinations (vesicle subsets) could be calculated to be on the order of 5–25 . Considering a marked overlap in the protein content between different known EV classes already characterized by differential centrifugation, nano‐flow cytometry, and other methods the possible number of EV subsets could be much greater than presently recognized. This combinatorial diversity could be further amplified by the heterogeneity of nucleic acids contained within known EV subpopulations and through superimposed distribution of other molecular species such as lipids and metabolites.…”

“…Importantly, interactions with target cells, uptake, and intracellular processing of EVs are regulated by both their properties (imposed by parental cells) and by the state of recipient cells. For example, different subsets of glioma stem cells produce EVs that are either avidly or poorly taken up by endothelial cells . In other cases, proteoglycan expression on the surface of recipient cells dictates the uptake of glioma exosomes by various cellular recipients …”

“…Notably, several elements of this circuitry are influenced by oncogenic pathways, as suggested by distinct patterns of corresponding mRNA in molecular subtypes of glioblastoma associated with specific driver mutations . Moreover, the expression levels of vesiculation‐related genes (vesiculome) are altered in cancer cell lines driven by specific oncogenes such as HRAS , EGFR , EGFRvIII , and other genes …”

“…While certain oncoproteins (e.g., EGFRvIII/EGFR) and oncogenic DNA (e.g., HRAS) appear to preferentially track with exosome‐like vesicles, others may accompany amoeboid ectosomes (e.g., AKT), microvesicles, or apoptotic bodies . Indeed, regulation of the vesiculation and packaging processes could be highly context‐dependent and vary between different tumor types, or molecular subtypes of cancer . It should also be noted that oncogenic drivers act in concert with epigenetic processes that modulate cellular phenotype, such as EMT, formation of cancer stem cell (CSC) populations, and their more differentiated or dormant progeny .…”

“…Indeed, regulation of the vesiculation and packaging processes could be highly context‐dependent and vary between different tumor types, or molecular subtypes of cancer . It should also be noted that oncogenic drivers act in concert with epigenetic processes that modulate cellular phenotype, such as EMT, formation of cancer stem cell (CSC) populations, and their more differentiated or dormant progeny . Cancer cells also respond to microenvironmental cues, such as hypoxia, in a manner that is modulated by their genetic and epigenetic programs .…”