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 p53 β 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. Electronic supplementary material The online version of this article (10.1007/s12035-018-1385-1) contains supplementary material, which is available to authorized users.

Extracellular Vesicle-Mediated Communication between the Glioblastoma and Its Microenvironment

“…In a later study, Hallal et al [60] reported increased podosome formation and extracellular matrix degradation in astrocytes cultured with GBM-derived EVs. In addition, they showed that the proteome of astrocytes exposed to GBM-derived EVs presented significant modifications in signaling pathways involved in tumorigenesis, such as decreased TP53 and increased MYC.…”

“…This messenger activates the astrocytic production of inflammatory cytokines such as interferon-α and tumor necrosis factor, which in turn induce the activation of STAT-1 and NF-κβ pathways in the brain's metastatic cells, thereby supporting tumor cell invasion and resistance to chemotherapy [58]. More recently, two articles have reported tumor-supportive effects of astrocytes driven by EVs released by GBMs [59,60]. Oushy et al demonstrated that astrocytes cultured with EVs from patient-derived GBMs display profound modifications of the secretome that results in positive effects on tumor growth [59].…”

“…They all have advantages and disadvantages, and still, an ideal and universal method for EV isolation is lacking. The studies mentioned in this section have used ultracentrifugation-based methods such as differential ultracentrifugation [28,29,40,43,51,52,59,60,73] or density gradient ultracentrifugation [37][38][39]. These are the most used technical procedures (at least in 2018 [77]), and have been validated by the ISEV2018 position paper [25].…”

Splicing Dysregulation as Oncogenic Driver and Passenger Factor in Brain Tumors

“…EVs are key messengers of intercellular communications that control the establishment and maintenance of TME [99], are actively secreted by GBM cells, and promote their oncogenic features [100][101][102][103]. For instance, GBM-secreted EVs stimulate recipient astrocytes to acquire a pro-tumoral phenotype by delivering several factors, including MYC and MYCN [101]. Furthermore, EV-mediated transfer of splicing factors from apoptotic to healthy GBM cells has also been proposed as a mechanism to alter splicing patterns and to promote malignancy.…”

A comprehensive proteomic SWATH-MS workflow for profiling blood extracellular vesicles: a new avenue for glioma tumour surveillance

“…A range of GBM samples (EVs, cells and tumour tissue fractions) were used to generate a comprehensive, custom glioma IDA spectral library. Specimens were prepared as described before from neurosurgical aspirate EVs [24], patient-derived GBM-stem-like cells [50], and soluble and microsomal proteomes [51] prepared from GBM tumour tissue (Supplementary Table 2). The peptide samples were fractionated by HILIC, a reverse-phase chromatographic method, to reduce sample complexity and increase protein ID coverage by LC-MS/MS [52].…”