Extracellular Vesicles Released by Glioblastoma Cells Stimulate Normal Astrocytes to Acquire a Tumor-Supportive Phenotype Via p53 and MYC Signaling Pathways

Abstract

The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES⁺/CD133⁺) and their differentiated (diff) progeny cells (NES^-/CD133^-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFβ1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53β in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.

Keywords: Astrocytes; Extracellular vesicles; Glioblastoma stem-like cells; MYC; Podosome formation; Senescence-associated secretory phenotype; TP53.

Fig. 1

a, b Nanoparticle tracking analysis measured particle size distributions of EVs purified from multiple sources. Mean and modal sizes of EVs isolated from GBM-stem cells (CD133⁺/NES⁺) were larger than EVs isolated from differentiated progeny cells (CD133⁻/NES⁻; averages of four experiments). Transmission electron microscopy confirmed the presence of 30–150-nm-sized particles with vesicular morphologies in JK2 c -stem and d -diff EV preparations. e Mass spectrometry analysis of EV proteomes prepared from GBM stem/diff cells and primary astrocytes identified all top 10 exosome proteins in every preparation and f showed significant enrichment of protein characteristic of exosomes, the plasma membrane and extracellular regions, greater than that identified in proteomes prepared from the originating cells

Fig. 2

Uptake of GBM-derived EVs by primary human astrocytes induces podosome formation. Primary human astrocytes (14,000/cm²) cultured for 24 h in the presence of a unlabeled JK2-stem EV and DiI-labeled, b JK2-stem, and c U87MG EVs. Images are ×10, bright field (gray) images merged with red (DiI-labeled EV) images. d Primary fetal astrocytes (14,000 cells/cm²) were incubated with and without GBM-EVs (1 μg EVs per 1120 cells) in triplicate, then fixed, and stained with FITC-phalloidin (green) and DAPI (blue). Astrocytes treated with WK1-stem and -diff EVs are visualized here on an Olympus BX51 fluorescence microscope. Cy3-gelatin (red) images corresponding to the same fields of view show dark patches void of fluorescence indicating areas of gelatin degradation (white arrows). e Graphed fold changes of percent gelatin degradation relative to untreated astrocytes ± SEM (error bars). Measurements are averaged across quadruplicate assays, each with four randomly sampled fields of view (×10 magnification). Asterisks represent a significant induction of podosome formation relative to untreated astrocytes (p < 0.05), where pound sign denotes significant changes relative to corresponding stem cells (p < 0.05)

Fig. 3

Ingenuity pathway analysis predicted significantly associated a canonical pathways and b upstream regulators in astrocytes treated with EVs from all GBM cells. Non-significant findings (p > 0.05) indicated with a dot

Fig. 4

Interaction mapping of overlapping proteome changes in astrocytes treated with GBM a -stem and b -diff EVs. Genes corresponding to differentially abundant proteins were mapped using Ingenuity Pathway Analysis. Proteins with significantly higher or lower levels in astrocytes have red and green symbols, respectively. Networks are annotated with significantly associated biological and canonical pathways (see legends for symbols and p values). IPA generated networks converged on significant upstream regulator molecules with predicted activations (orange) or inhibition (blue)