Glioblastoma multiforme-derived extracellular vesicles drive normal astrocytes towards a tumour-enhancing phenotype

Abstract

Glioblastoma multiforme (GBM) is a devastating tumour with abysmal prognoses. We desperately need novel approaches to understand GBM biology and therapeutic vulnerabilities. Extracellular vesicles (EVs) are membrane-enclosed nanospheres released locally and systemically by all cells, including tumours, with tremendous potential for intercellular communication. Tumour EVs manipulate their local environments as well as distal targets; EVs may be a mechanism for tumourigenesis in the recurrent GBM setting. We hypothesized that GBM EVs drive molecular changes in normal human astrocytes (NHAs), yielding phenotypically tumour-promoting, or even tumourigenic, entities. We incubated NHAs with GBM EVs and examined the astrocytes for changes in cell migration, cytokine release and tumour cell growth promotion via the conditioned media. We measured alterations in intracellular signalling and transformation capacity (astrocyte growth in soft agar). GBM EV-treated NHAs displayed increased migratory capacity, along with enhanced cytokine production which promoted tumour cell growth. GBM EV-treated NHAs developed tumour-like signalling patterns and exhibited colony formation in soft agar, reminiscent of tumour cells themselves. GBM EVs modify the local environment to benefit the tumour itself, co-opting neighbouring astrocytes to promote tumour growth, and perhaps even driving astrocytes to a tumourigenic phenotype. Such biological activities could have profound impacts in the recurrent GBM setting.This article is part of the discussion meeting issue 'Extracellular vesicles and the tumour microenvironment'.

Keywords: astrocytes; exosomes; extracellular vesicles; glioblastoma multiforme; microenvironment; signalling.

Figure 1.

Extraction and purification of EVs from GBM cell lines. (a) Two GBM cell lines viewed under light microscopy. E8-5 and F3-8, were dissociated and grown under stem cell-like conditions in supplemented NBA medium (Invitrogen Technologies). Scale bar, 100 µm. (b) EV purification from cell culture media was conducted as described [16]. (c) NTA (Nanosight) was used to determine sizes and concentrations of vesicles; EVs were verified using ExoCheck arrays for eight purported EV markers.

Figure 2.

GBM EVs promote astrocyte migration. Astrocytes were obtained from a single individual. (a) Astrocytes were exposed to 50 µg ml⁻¹ of tumour EVs and observed over a 24 h period in three separate wound fill assays (three biological replicates with three technical replicates per experiment). Compared to controls (0 µg ml⁻¹ EVs), astrocytes displayed a significant increase in per cent wound closure in all three instances. Error bars, standard deviation. (b) Representative photos and ImageJ boundaries are shown.

Figure 3.

GBM EVs enhance astrocyte secretory outputs. Culture media from GBM EV-treated (500 µg ml⁻¹) astrocytes were analysed on antibody microarrays (R&D Systems) for relative cytokine/chemokine/growth factor concentrations. (a) Representative arrays shown (no EV treatment (i), EV treatment (ii), with potentially important molecules noted; C, positive control spot). (b) Heat map of secretome of untreated, short-term-treated (4 h), and 24 h-treated astrocytes is shown. Results are from two biological replicates, with two technical replicates for each. Astrocytes were obtained from a single individual.

Figure 4.

Results of core analysis/comparison analysis in IPA. Values from figure 3 heat map were entered into a comparison analysis in IPA. The top three networks (interactomes) are shown (named in table 1). Proteins from the array are in larger bold font, with elevated values from the array depicted in shades of red. The ‘scores’ listed are −log (p-values) and are based on the probabilities of random associations of these proteins. The significance threshold by default = 1.25. ‘Focus molecules’ refer to nodes that initiate networks. Solid blue lines show direct connections between proteins found in the array, derived from the IPA knowledgebase. Broken lines represent indirect connections arising from reasoned speculation, or via known intermediaries. Light blue/turquoise lines connect identified proteins within the network with proteins not part of the array. Line lengths (edges) between the protein nodes correlate to the degree of supportive knowledge documenting interactions. Note, we have shortened some edges to fit the interactomes into the figure.

Figure 5.

GBM EV-treated astrocytes generate a growth-stimulating medium. Astrocytes are from a single individual. Astrocytes were exposed to 0, 50 or 500 µg ml⁻¹ GBM EVs for 24 h. Conditioned astrocyte medium was collected, centrifuged to remove cells and debris, and then used as growth medium for GBM cells seeded at equal numbers. After 24 h, GBM cells were subjected to MTS assay (OD₄₉₀ as readout); fold increase over control (set = 1) is presented. Results are from three biological replicates, with six technical replicates per experiment. Error bars, standard deviation.

Figure 6.

GBM EV-treated astrocytes induce tumour-like signalling patterns that are concentration- and time-dependent. Astrocytes are from a single individual. Astrocytes were incubated with different concentrations of GBM EVs (0, 50 or 500 µg ml⁻¹) for 4, 8 and 24 h. Phospho-antibody arrays were incubated with lysates from EV-treated astrocytes; signal intensities were background-corrected and presented as fold change over control (untreated astrocytes at 4, 8 or 24 h = 0). Results are from two biological replicates with two technical replicates for each experiment.

Figure 7.

Results of core analysis/comparison analysis in IPA. Values from figure 6 were entered into a comparison analysis in IPA. The top three networks (interactomes) are shown (named in table 2). Protein/gene names, edges, scores and focus molecules are presented as in figure 4, except that proteins with reduced values are shown in shades of green.

Figure 8.

GBM EV treatment reduces sold-matrix growth dependence in astrocytes. Astrocytes are from a single individual. Astrocytes were incubated with 0, 50 or 500 µg ml⁻¹ of GBM EVs for 24 h. Astrocytes were washed, detached and plated in a soft agar assay for eight days, after which colony viability was measured by MTT assay (OD₅₇₀). Results are displayed as per cent growth over untreated astrocytes (control = 100%). Results are from two biological replicates with six technical replicates per experiment. Error bars, standard deviation.