Interplay between V-ATPase G1 and small EV-miRNAs modulates ERK1/2 activation in GBM stem cells and non-neoplastic milieu

Abstract

The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and in vivo tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EVs). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures.

To this end, small EVs were isolated from patients-derived GSC grown as neurospheres (NS) with high (V1G1^HIGH-NS) or low (V1G1^LOW-NS) V1G1 expression and analyzed for V-ATPase subunits presence, microRNA contents and cellular responses in recipient cultures.

Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1^HIGH-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The microRNA (miRNA) profile of V1G1^HIGH-NS-derived small EV differed significantly from that of V1G1^LOW-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the pro-oncogenic effects of V1G1^HIGH small EV.

These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration.

Introduction

Despite a better understanding of the molecular pathogenesis of glioblastoma (GBM), this remains an aggressive and invariably fatal disease. Local tumor recurrence is the main cause of GBM-related death and surgery remains the only potentially curative option. We previously showed that the vacuolar proton pump V-ATPase is key for GBM stem cell (GSC) survival ¹ and that GSC with higher V-ATPase G1 expression exhibit heightened tumorigenicity in vivo ². This is achieved, at least in part, through GSC secretion of large extracellular vescicles (EVs) called large oncosomes in the tumor milieu. This is in line with other studies ^3–5 that identified V-ATPase subunits in smaller EVs such as exosomes. Small EVs, including exosomes, are nano-sized (40-150 nm) particles that originate from the late endosomal trafficking machinery ^{6, 7}. These small EVs can exert pleiotropic biological functions both in physiologic and pathologic conditions and can influence the proximal or distant microenvironment via the horizontal transfer of bioactive molecules. Because of these characteristics, small EVs or exosomes have been implicated in every stage of tumorigenesis, from tumor onset ⁸, maintenance and propagation of the cancer stem cell niche ^{9, 10}, to metastatic dissemination ^{11, 12}. Specifically, both tumor- and stroma-derived exosomes have been also involved in metastatic dissemination ^13–15 affecting vascular permeability or by conditioning pre-metastatic sites in distant organs ^{11, 16–18}. The biological functions exerted by exosomes in recipient cells involve microRNAs (miRNAs) ⁸, a class of the epigenetic regulators qualitatively and quantitatively altered in transformed cells.

In this study we investigated the interplay between V-ATPase and small EV-miRNAs in glioblastoma taking advantage of primary, patients-derive GSC and non-neoplastic brain cultures.

Materials and methods

Generation of primary, patients derived, GBM cultures.

Primary cells were isolated directly from patient’s sample after surgery at Fondazione IRCCS Ca’ Granda-Ospedale Maggiore Policlinico, Milan, Italy. Written informed consent was obtained from all subjects involved in the study and the protocol was approved by a local Ethic Committee (IRB#275/2013).

Samples obtained from tumor cores (n=51) and/or non-neoplastic tumor margins (MG, n=20) were disaggregated enzymatically and mechanically using a tumor dissociation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Then, tumor cells were washed twice with HBSS (Thermo Fisher Scientific, Waltham, MA USA) and seeded in Neurocult media supplemented with NS-A Proliferation Kit (Human) and growth factors (bFGF and EGF; all from Stem Cell, Vancouver, BC, Canada) to maintain undifferentiated state, or RPMI supplemented with 10% FBS (both from Gibco-Thermo Fisher Scientific) for differentiated condition. MG cultures were seeded in RPMI supplemented with 10% FBS and maintained for up to one month in culture.

Small EVs isolation and characterization.

Small EVs (sEV) were isolated from supernatant of neurospheres (NS) cultures (n=12) left untreated (controls), or after 24 h of the indicated treatment. In all cases, cells were seeded at a concentration of 15 NS/ml and media was recovered after 48 h (controls), or cultures were left untreated for 24 h followed by incubation with V-ATPase or ERK1/2 targeting drugs for 24 h and then media were recovered. Cells were pelleted at 250 rcf for 5 min and supernatants were collected and concentrated using Amicon Ultra centrifugal filter tubes (MilliporeSigma, part of Merck KGaA, Darmstadt, Germany). Then, the collected supernatants were centrifuged at 1000 rcf for 10 min at 4°C to remove debris and/or apoptotic bodies (pellet), followed by a second centrifugation at 10.000 rcf for 30 min at 4°C to remove large EVs.

Finally, the sEV fraction was isolated from supernatants by qEV size exclusion column (SEC – iZON, Oxford, UK) as described by Lobb et al. ¹⁹. Briefly, 500ul of concentrated supernatant, obtained from 50ml of starting supernatant, were overlaid on qEV column, eluted with PBS and collected in 1.5ml eppendorf. sEVs are present in phases 7-8-9 ^{20, 21}. The concentration and size distribution of particles was assessed by Nanoparticle Tracking Analysis (NTA) using the Nanosight NS300 instrument (Malvern, Instruments Ltd Worcestershire, UK).

Purified sEV were stored in aliquots (20μl each) at −80°C before they were used for functional experiments or RNA/protein extraction. The sEV dose was selected based on initial results from dose-response analysis (Supplementary Fig. S1a). Specifically, each sEV aliquot contained 4×10⁷ sEV. For controls (mock samples), fresh NC medium was processed as before and stored at −80°C in aliquots of 20 μl each.

sEV ultrastructure and V1G1 protein presence was evaluated by electron microscopy (EM) and immunogold staining following the Lobb protocol ¹⁹ and as previously described ².

We have submitted all relevant data of our experiments to the EV-TRACK knowledgebase (EV-TRACK ID: EV200059) ²².

Functional characterization of recipient cells after co-cultures with NS-small EV.

To investigate sEV biological effects on non-neoplastic brain (MG), or on tumor cell maintained in differentiated condition (Diff-TC) cell growth, cell cycle progression, Ki67 proliferation index and cell motility assays were evaluated. Instead, to evaluate their effect on non-sphere forming tumor recipient cultures (NSF-TC, maintained in undifferentiated growth condition) a sphere formation assay was used. Recipient cells were seeded in multi-well plates and let reach 60% confluence; specifically, 2×10⁴ or 2×10⁵ cells were plated in 24-wells or 6-wells plates respectively. Then, recipient cultures were supplemented with NS-derived sEV at the concentration of 20ul per ml of culture media, with a ratio of 4×10⁷ sEV / 2×10⁴ cells.

For the analysis of cell growth, cells were stained with CellTrace dye (5μM for 20 min at 37°C) and the number of cell divisions was evaluated using flow cytometry by scoring the reduction of the dye intensity caused by redistribution of probe in daughter cells as described ². Specifically, Diff-TC or MG cells were followed for 24 h or 17 days, respectively. Cell cycle progression was investigated by propidium iodide (PI) staining and flow-cytometry. Briefly, cells were fixed with cold absolute EtOH in ice overnight, then washed two times with PBS-1% FBS and stained with 500μl of PI/RNase Staining Buffer (BD Biosciences). The fluorescence activated cell sorting (FACS) Canto I instrument and FlowJo V.10.1 software were used for flow-cytometry experiments (Becton-Dickinson). Cell proliferation was also evaluated by Ki67 nuclear staining after 24 h or 72 h of sEV co-culture with glioma or MG recipient cells, respectively. Images were acquired using a confocal microscope (Leica TCS SP5; Leica Microsystems, Milan, Italy) at 40X magnification.

To evaluate cell motility, MG cells were co-cultured with the indicated sEV preparations. After 1h, cell movement was followed using Nikon time-lapse microscope (Eclipse Ti-E, Nikon Instruments; Florence, Italy) for 12 h capturing images every hour. Conversely, glioma cell migration was analyzed with a wound-healing assay. Briefly, after 24h of sEV co-culture a wound was created in the monolayer using a 200μl tip. Gap closure was monitored over a 64 h and images were captured every 4 h using the time-lapse microscope. For both experiments, cells were maintained in controlled gas-humidified chamber at 37°C and images were captured in bright field with 10X objective. Gap closure was measured using ImageJ manual tracking plugin and then normalized on initial gap dimension. Spheres formation was evaluated in non-sphere forming glioma culture after supplementation with the indicated sEVs preparation. Then, the number of generated tumor spheres was scored after 3 and 6 days.

Protein synthesis

For the detection of nascent protein synthesis the Click-iT™ HPG Alexa Fluor™ 488 Protein Synthesis Assay Kit was used following manufacturer’s instruction (Thermo Fisher Scientific). Specifically, MG monolayer cultures were grown on cover glasses and let reach 60% confluence. Then recipient cultures were supplemented with NS sEV at the concentration of 4×10⁷ sEV / 2×10⁴ cells and, after 2, 4 and 6 h, 50 μM Click-iT were added to the cells and incubated for 30 min. Then, cells were fixed and permeabilized in 3.7% paraformaldehyde (PFA) and 0.5% Triton respectively for 20 min at room temperature. Click-iT was detected with Click-iT reaction cocktail while nuclei were stained with Hoechst 33342. Images were acquired using a confocal microscope (Leica TCS SP5) at 40X magnification. Nascent protein synthesis was assessed by determining signal intensity (MFI) in the area proximal to the nucleus.

Cancer pathway reporter array

To measures the activity of signaling pathways in NS, the Cignal Finder Cancer 10-Pathway Reporter Array (Qiagen, Hilden, Germany) was used. NS at basal condition were transfected with the indicated gene reporter (pathway specific responsive element fused with firefly luciferase gene) and a constitutive Renilla luciferase construct. Dual-luciferase emissions were then measured using the Tecan Infinite F200 luminometer (Tecan Trading AG, Switzerland) and Luciferase to Renilla signal were computed for each pathway. The experiment was performed four times for either V1G1^LOW or V1G1^HIGH-NS.

V-ATPase activity impairment.

V-ATPase block was achieved either by V1G1 siRNA or by incubation of NS with Bafilomycin A1 (BafA1) or Concanamycin A. A V1G1 targeting siRNA or mock control molecules were previously described ^{1, 2}. BafA1 was used at the non-toxic dose of 10nM for 24 h ¹. Similarly, we identified the adequate Concanamycin A concentration, corresponding to 10nM, as the dose able to decrease V-ATPase activity in NS without inducing extensive cell death after 24 h of treatment (Supplementary Fig. S1b,c).

Statistical analyses.

Differences among two or more samples’ groups were evaluated using non-parametric Mann-Whitney U or Kruskal-Wallis (with post-test) test, respectively (GraphPad Prism software; La Jolla, CA, USA). Data are expressed as mean±SD. All biological experiments were performed at least four times and experiments have a technical duplicate unless otherwise indicated in the legend.

For miRNA analysis, miRNA raw data were normalized using the global mean method and relative quantities were obtained applying the 2^-DCt formula. Then miRNA relative quantities were median-normalized, log2-transformed and imported in R environment for statistical analysis. The R packages ComplexHeatmap from Bioconductor was used for heatmap visualization. The samR package from CRAN was used to identify miRNAs differentially expressed according to V1G1 levels in NS (q.value <0.05, FC> 1 abs). Targets identification of significant microRNAs was achieved using the online tool miRTargetLink Human (https://ccb-web.cs.uni-saarland.de/mirtargetlink/) and transcripts with “strong” and experimentally validated interaction with at least two miRNAs were then imported in STRING database of protein-protein interactions ²³ and in WebGestalt (WEB-based Gene SeT AnaLysis Toolkit) functional enrichment analysis web tool ²⁴.

Results

Small EVs from V1G1^HIGH NS-drive tumorigenic reprogramming in non-neoplastic recipient cells.

We started this study characterizing sEV preparations obtained from GBM NS with high (V1G1^HIGH-NS) or low (V1G1^LOW-NS) V-ATPase G1 expression (Supplementary Fig. S2a, b) and, therefore, different tumorigenic potential in vivo ^{2, 25}. We verified by western blot, electron microscopy, NTA and multiparametric flow-cytometry assays that NS-isolated sEV had the expected size, morphology and phenotype (Supplementary Fig. S2c–f). In agreement with MISEV guidelines ^{26, 27}, sEV purified from either NS types had a diameter of 50-200 nm (Supplementary Fig. S2d, f), had an integral membrane (as evidenced by cell trace staining) and contained ssRNA (as evidenced by sytoRNA staining; Supplementary Fig. S2g). As for immunophenotypic characterization, sEV expressed the exosomal and extracellular vesicles markers CD9, CD63, Tsg101, Clathrin, whereas the plasma membrane protein Calnexin was not present (Supplementary Fig. S2c). Moreover, sEV displayed CD9 and CD81 antigens on their surface (Supplementary Fig. S2e). Finally, NTA showed that V1G1^LOW and V1G1^HIGH-NS produced similar amounts of sEV (Supplementary Fig. S2f).

To gain functional clues into signals induced by sEV from GBM NS in the surrounding environment, we co-cultured sEV from V1G1^HIGH and V1G1^LOW-NS with three types of recipient cells: non-neoplastic brain cultures obtained from tumor margins (MG), non-sphere forming primary low grade glioma cells maintained under differentiating (Diff-TC) or undifferentiating (NSF-TC) conditions ². sEV from either NS types were comparably internalized by all recipient cultures tested within 6 h from addition. However, at later time points, sEV derived from V1G1^HIGH-NS (sEV^HIGH) were more efficiently uptaken by recipient cells compared to sEV^LOW (Supplementary Fig. S2h, i). Under these conditions, co-culture with sEV^HIGH increased the survival time of MG cultures from 9 to 17 days, whereas sEV^LOW-supplemented cultures showed a survival time comparable to control (Mock; Fig. 1a, b and Supplementary Fig. S3a). Moreover, sEV^HIGH stimulated MG cell-cycle progression and proliferation, as showed by an increase in the fraction of cell with S phase DNA content (Fig. 1c and Supplementary Fig. S3b) and greater percentage of Ki67-positive nuclei (Fig. 1d). Similar effects were detected when sEV were provided to Diff-TC cells (Supplementary Fig. S3c, d), with sEV^HIGH inducing the strongest effects on cell proliferation. Consistent with these results, sEV^HIGH stimulated protein synthesis in MG cultures (Fig. 1e).

Fig. 1.

The NS-derived sEV ability to reprogram the brain microenvironment is correlated to V1G1 expression and activity in NS. (a) MG cells growth was evaluated over 17 days using Cell Trace dye after co-culture with the indicated sEV preparation. n=6 independent experiments; ***, p<0.001 by Kruskal-Wallis with post-test. (b) Representative images of MG cells after 9 days of co-culture with the indicated sEV preparation or vehicle (Mock). Scale bar=500μm or 100μm for enlargements. (c) Cell cycle progression was evaluated by flow cytometry after 3 days of co-culture with the indicated sEV. n=6 independent experiments; *, p= 0.01 (S phase of mock vs sEV^HIGH) by Mann-Whitney U test. (d) Nuclear Ki67 staining was performed in MG cells after co-culture with the different sEV for 3 days. Left, representative images, scale bar=50μm; right, quantification of 5 independent experiments. **, p=0.007 and §, p=0.009 by Mann-Whitney U test. (e) Protein synthesis was analyzed at different times in MG cells cultured with sEV or vehicle using Click-iT assay. Left, Representative images of MG cells co-cultured with sEV for 6 h; scale bar, 50μm. Right, Quantification of median fluorescence intensity (MFI). *, p=0.02 by Kruskal-Wallis with post-test (n=3). (f) MG cells movement after sEV or vehicle supplementation was investigated over a 12 hours interval. n=4 independent experiments; ***, p<0.0001 by Kruskal-Wallis with post-test. (g) The number of MG cell division in 24 h was investigated using Cell Trace dye and flow cytometry. n=4 independent experiments; **, p= 0.007 by Mann-Whitney U test. (h) Nuclear Ki67 staining was performed in MG cells after co-culture for 3 days with sEV^HIGH, or sEV derived from NS treated with BafA1 (sEV^BafA1) or vehicle (Mock). Right, quantification of Ki67-positive nuclei. **, p=0.02; ***, p=0.0009, by Mann-Whitney U test - each dot represents an independent experiment. (i) Diff-TC cells migration was measured as wound closure in the indicated culture conditions. n=3; *, p=0.01; **, p=0.003 comparing sEV^CTRL to sEV ^BafA1 by 2-way Anova with Turkey’s multiple comparison test. (j) NSF-TC cultivated with sEV^CTRL, sEV^BafA1 or with vehicle (Mock) were investigated for sphere-formation ability up to 6 days from co-culture. n=4; ***, p=0.008; §, p=0.004, $, p=0.01 by 2 way Anova with Bonferroni’s multiple comparisons test. Data are presented as mean±SD.

We next examined the effect of sEV on the motility of recipient cells. In these experiments, sEV^HIGH stimulated the motility of MG recipient cells, compared to control conditions or sEV^LOW addition (Fig. 1f). Similar effects were observed also when sEV^HIGH were co-cultured with Diff-TC cells (Supplementary Fig. S3e). Finally, only sEV^HIGH induced NSF-TC to form NS (Supplementary Fig. S3f), a tumor cell population known to be enriched in cancer stem cells ²⁸.

These set of data suggest that sEV secreted by V1G1^HIGH-NS induce a pro-tumorigenic response in the surrounding environment by promoting greater cell survival, proliferation, motility and clonogenicity, all events crucial for glioma progression ²⁹.

Bafilomycin A1 treatment of NS abolishes sEV^HIGH effects on recipient cultures.

To verify that the V-ATPase was responsible for sEV^HIGH effects on recipient cells, we impaired the pump activity in NS with BafA1. BafA1 treatment did not alter vesicles secretion or their immunophenotype (Supplementary Fig. S4a–c), and significantly increased sEV production by NS (Supplementary Fig. S4b). Also, sEV produced by V1G1^HIGH treated with BafA1 (sEV^BafA1) were internalized by recipient cells comparably to sEV purified from V1G1^HIGH-NS treated with vehicle (sEV^CTRL) (Supplementary Fig. S4d, e). Functionally, BafA1 treatment abolished the biological effects of sEV^HIGH in either type of recipient cells, shutting down proliferation of MG and Diff-TC (Fig. 1g, h and Supplementary Fig. S4f), Diff-TC motility (Fig. 1i), and NSF-TC sphere formation (Fig. 1j). Therefore, V-ATPase activity is required for the observed pro-tumorigenic functions of NS with elevated expression of V1G1.

Small EVs produced by GBM NS vehiculate V1G1 protein on their surface.

Next, we asked whether NS–derived sEVs carry the V1G1 subunit as a molecular cargo. Accordingly, we demonstrated by immunogold that all sEV types vehiculate the ATP6V1G1 subunit, (Fig. 2a), multiparametric flow-cytometry (Fig. 2b, c) and western blot (Fig. 2d) assays. Moreover, sEV^HIGH carried the highest amount of V1G1 compared with sEV^LOW and sEV^BafA1 (Fig. 2b, c). To assess if the EV-V1G1 was transferred to recipient cells, we examined its expression in MG cultures after co-culture with sEV^HIGH, sEV^LOW or control (Mock). In these experiments, V1G1 protein levels were increased in MG cells upon sEV^HIGH supplementation (Fig. 2f, g). Other pump subunits were equally modulated in recipient cultures, with V1F being more expressed in cells co-cultured with sEV^HIGH while V1D was more expressed after sEV^LOW supplementation (Fig. 2f). To exclude that this effect was related to de novo transcription, we next quantified the mRNA expression of V-ATPase subunits in recipient cultures after sEV supplementation. Here, no significant differences in V-ATPase subunit mRNA expression was observed between sEV^HIGH-supplemented and control cells (Fig. 2e). Finally, similar modulation of V-ATPase subunits was observed in Diff-TC after co-culture with different sEV (Supplementary Fig. S5a). Inhibition of V-ATPase activity in V1G1^HIGH-NS decreased the amount of V1G1 protein in secreted vesicles (Fig. 2b) and, accordingly, incubation of recipient cells with sEV^BafA1 did not result in increased V1G1 expression (Fig. 2f, g).

Fig. 2.

sEV vehiculate V1G1 protein to recipient cultures. (a) Representative images of immunogold staining for V1G1 protein on sEV isolated from NS with high (sEV^HIGH) or low (sEV^LOW) V1G1 expression or from V1G1^HIGH-NS treated with BafA1 (sEV^BafA1). Scale bars= 200nm. (b,c) Flow-cytometry analysis of V1G1 expression in the indicated sEV preparations bound to CD63-coated beads. Representative images and quantification (c) are shown. Bars, mean±SD (n=4); *, p=0.017; $, p=0.024 by Kruskal-Wallis with post-test. (d) The indicated proteins were analyzed by immunoblot in cell lysate (CL) or sEV^HIGH extracts (sEV). (e) The indicated transcripts were analyzed in MG cells after 48 h of co-culture with sEV^HIGH. Data are expressed relative to MG cells cultured with vehicle (Mock=1 for each target, n=4 different cultures). (f) The indicated proteins were analyzed by immunoblot in MG cells after 72 h of co-culture with the indicated sEV preparations. (g) Representative immunofluorescence images of MG co-cultured for 72 h with the indicated sEV. Scale bar=100μm. Right, quantification; MFI (mean fluorescent intensity). Symbols, mean±SD (n=3). ***, p= 0.001; ****, p<0.0001 by Mann-Whitney U test.

These data are in line with our previous description of a differential V-ATPase conformation according to glioma aggressiveness, with more aggressive tumors carrying higher levels of the V1G1 and V1F subunits ²⁵.

sEV^HIGH activates ERK1/2 signaling pathway in recipient cells

To better understand the mechanisms of sEV^HIGH pro-tumorigenic effects, we profiled cancer-related signaling pathways in MG recipient cells upon co-cultures with different sEV preparations for 72 h. Using an antibody array assay, we found that sEV^HIGH induced ERK1/2, Stat3, AMPKα, HSP27, PRAS40 and mTOR phosphorylation in recipient cultures (Fig. 3a and Supplementary Fig. S5b). Conversely, recipient cells treated with sEV^LOW showed reduced mTOR and Glycogen Synthase Kinase-3β (GSK3β) phosphorylation (Fig. 3a), suggesting that sEV^LOW blunt PI3K/ERK signaling in recipient cultures (Supplementary Table S1). In keeping with this finding, V1G1^HIGH treatment resulted in greater activation of ERK1/2 signaling compared to V1G1^LOW-NS at both transcriptional and protein level (Fig. 3b, c) and, accordingly, BafA1-mediated inhibition of V-ATPase activity decreased ERK1/2 phosphorylation particularly in V1G1^HIGH-NS (Fig. 3d). Other cancer related pathways, such as Wnt, Notch, NFkB and Myc, were not differentially induced in NS with different V1G1 level (Supplementary Fig. S5c) and were not modulated by sEV^HIGH (Supplementary Fig. S5d, e, f).

Fig. 3.

sEV^HIGH activate MAPK/ERK pathway in recipient cells. (a) Image of PathScan Intracellular Signaling Membrane Array Kit (upper) and quantification (n=1; bottom) performed with the indicated MG protein extracts. Data are expressed relative to control (Mock). Bars, mean±SD. (b) Status of MAPK signaling pathways in NS with high or low V1G1 expression was analyzed using a gene reporter assay. Luciferase to Renilla emissions were then measured and data are expressed using box and whiskers plot (n=3). *, p=0.01 by Unpaired t-test. (c,d) Total and phosphorylated ERK1/2 kinase levels were analyzed by immunoblot in V1G1^HIGH or V1G1^LOW-NS (c) or in NS treated with Bafilomycin A1 (BafA1; d) or vehicle. Bars, mean±SD (n=5). *, p=0.03; **, p=0.005 by Mann-Whitney U test. (e) Heatmap showing PI3K/Akt/MAPK genes expression modulation in MG cells after 48 h of co-culture with the indicated sEV preparations. (f) Validation by qPCR of the indicated transcripts expression in MG cells co-cultured for 48 h with the indicated sEV preparation. Bars, mean±SD (n=3). Dotted line indicates transcripts’ level in control samples (Mock used as calibrator). **, p=0.009 by Wilcoxon signed-rank test; ns= not significant.

We then looked at gene expression level of ERK1/2 pathway members in recipient MG cells upon co-culture with sEV^LOW or sEV^HIGH using a cancer-pathway array. The majority of these genes was upregulated after sEV^HIGH supplementation (Fig. 3e), with Bcl2 being the most upregulated target (p=0.009; Fig. 3f). To determine whether MAPK-related transcripts or Bcl2 upregulation in recipient cells was due to EV-mediated transfer or by de novo transcription, we profiled sEV-mRNA content. Bcl2 transcript was not present in sEV and, in general, MAPK-transcripts were not overrepresented in sEV^HIGH (Supplementary Fig. S5g). When MG cells were incubated with sEV^BafA1, protein phosphorylation, and Bcl2 or MAPK-transcripts were restored to basal levels (Fig. 4 a–c). Finally, we detected nuclear accumulation of phosphorylated ERK1/2 only in MG cells incubated with sEV^HIGH and not in control, sEV^LOW- or sEV^BafA1-treated cultures (Fig. 4d). These results suggest that the pro-tumorigenic reprogramming of V1G1^HIGH-NS-derived extracellular vesicles requires activation of ERK1/2 signaling and Bcl2 upregulation.

Fig. 4.

V-ATPase inhibition by Bafilomycin A1 blunts sEV^HIGH-mediated activation of MAPK/ERK pathway. (a) qRT-PCR of MAPK-related transcripts in MG cells after co-culture with sEV^HIGH, sEV^BafA1 or vehicle (Mock). Bars, mean±SD (n=3). ***, p<0.001 by Kruskal-Wallis with post-test. (b) A protein array was carried out with extracts from MG cells co-cultured with the indicated sEV preparations or vehicle (Mock). Upper panel, representative image; bottom panel, quantification of one experiment. The mock samples were used as calibrator. AU, arbitrary units. (c) Protein extracts from MG cells co-cultured for 72 h with the indicated sEV preparations were analyzed for total and phosphorylated (residues; pERK1/2) ERK by immunoblotting (n=4). (d) Nuclear presence of pERK1/2 in MG cells co-cultured with the indicated sEV was investigated by immunofluorescence and scored as the percentage of positive nuclei out the total number of cells. Right, quantification of 5 independent experiments. Bars, mean±SD. ****, p<0.0001 by Wilcoxon signed-rank test.

Regulation of MAPK signaling by a sEV^LOW-miRNA signature

Since MAPK-related genes were not vehiculated by sEV, we next asked whether small EVs miRNAs (sEV-miRNA) could contribute to ERK1/2 activation in recipient cells. In these experiments, we identified approximately 150 miRNAs in NS sEV and sEV-miRNome did not overlap with NS-miRNome, as outlined by PCA analysis (Supplementary Fig. S6a). Specifically, sEVs from V1G1^LOW-NS expressed a higher number of miRNAs that were expressed at higher levels, compared with sEV^HIGH (Supplementary Fig. S6a) and, globally, sEV-miRNAs distinguished between the two sEV types at unsupervised level (Fig. 5a). The diversity in miRNAs quantity observed between V1G1^LOW- and V1G1^HIGH-NS was not attributable to different expression of the miRNA processing enzymes Dicer and Drosha (Supplementary Fig. S6b). Next, we searched for miRNAs that differed between sEV^LOW and sEV^HIGH and we identified 45 miRNAs upregulated in sEV^LOW compared with sEV^HIGH samples (Supplementary Fig S6c and Supplementary Table S2). Among potentially repressed pathways targeted by sEV^LOW-miRNAs were MAPK signaling, PI3K/Akt signaling, and cell cycle (Fig. 5b, Supplementary Fig. S6d and Supplementary Tables S3 and S4). Therefore, we validated MAPK-associated miRNAs (n=32) in a second set of sEV and 10 miRNAs (31.3%) were confirmed to be significantly upregulated in sEV^LOW (Fig. 5c). These sEV-miRNAs were increased in recipient cells only after supplementation with sEV^LOW and not with sEV^HIGH, suggesting that the sEV-miRNAs were functional (Fig. 5d). Moreover, the level of these miRNAs was increased in EVs derived from V1G1^HIGH-NS treated with the V-ATPase inhibitors BafA1 (sEV^BafA1; Fig. 5e, Ctrl, sEV derived from V1G1^HIGH-NS) or Concanamycin A (Supplementary Fig. S6e), comparably to their expression detected in sEV^LOW. To test a potential role for the V1G1-overexpressing V-ATPase in regulating sEV-miRNA composition, we transiently silenced the V1G1 subunit in V1G1^HIGH-NS (Supplementary Fig. S6f) and analyzed these 10 miRNAs expression. The expression of five out of 10 sEV-miRNAs, namely miR-21-5p, −30b-5p, −30c-5p, −195-5p and miR-15b-5p, was increased after V1G1-siRNA (Supplementary Fig. S6g). Functionally, sEV^siV1G1 had no effect on the motility of recipient cells (Fig. 5f). Therefore, we ectopically overexpressed these pool of 5 miRNAs (miR-21-5p, −30b-5p, −30c-5p, −195-5p and miR-15b-5p; MAPK-miRNAs) or control molecules (Ctrl-miRNA) into MG recipient cells (Supplementary Fig. S6h) followed by co-culture with sEV^HIGH or control (Mock). After transfection, the expression of MAPK-miRNAs in MG cells was comparable to that achieved with sEV^LOW co-culture and it did not decrease after sEV^HIGH supplementation (Supplementary Fig. S6h). In this experimental setting, ectopic miRNAs expression abolished the functional effects of sEV^HIGH in recipient MG cells suppressing cell motility (Fig. 6a) and cell proliferation (Fig. 6b) to levels of control transfecting. In keeping with this, presence of the MAPK-miRNAs and sEV^HIGH prevented ERK signaling activity (Fig. 6c and Supplementary Fig. S6i, j), and Bcl2 or DUSP1 mRNA expression was not increased (Fig. 6d). The action of the MAPK-miRNAs was selective for ERK, since the expression of JNK transcripts (MAPK8, MAPK10) was unaffected by their presence as compared to cultures incubated with sEV^HIGH alone (Fig. 6d). Finally, treatment of recipient cells with the ERK inhibitor PD98059 after sEV supplementation prevented the pro-motile effect of sEV^HIGH (Supplementary Fig. S6k)

Fig. 5.

The sEV-miRNAs discriminates NS according V1G1 expression. (a) Unsupervised principal component analysis (PCA) of sEV derived from V1G1^LOW (n=6; orange dots) or V1G1^HIGH-NS (n=6; purple dots). The variance explained by each component is indicated in brackets. (b) Transcripts predicted to be targeted by sEV^LOW-enriched miRNAs (n=45; see also Supplementary Tables S2, S5, S6) were analyzed for pathways enrichment by STRING database or WebGestalt tool (see Supplementary Tables S3, S4) and visualized with Reactome. (c) Validation of differentially expressed miRNAs in sEV^LOW and sEV^HIGH samples was performed using qPCR. Bars, mean±SD (n=4). *, p=0.02 (miR-9-5p), p=0.015 (miR-30b-5p), p=0.02 (miR-195-5p). **, p=0.002 (miR-15b-5p), p=0.004 (miR-106b-5p) by Mann Whitney test. (d) Selected MAPK-related miRNAs were analyzed by qPCR in MG cells co-cultured for 48 h with the indicated sEV preparations. Bars, mean±SD (n=3). For each miRNA, the levels detected in mock sample were set equal to 1. $, p=0.0005; §, p=0.008; *, p=0.04 by 2way Anova with Turkey’s multiple comparison test. (e) The levels of the indicated miRNAs were analyzed by qPCR in sEV derived from untreated (Ctrl set equal to 1) or BafA1-treated V1G1^HIGH-NS. Bars, mean±SD (n=4). *, p=0.02 (miR-9-5p), p= 0.04 (miR-15b-5p), p=0.02 (miR-30c-5p) by Mann Whitney test. (f) The motility of MG cells co-cultured with the indicated sEV preparations or with vehicle (Mock) was monitored over 12 h and the distance covered by the cells was recorded. Symbols, mean±SD (n=3). **, p=0.004 (sEV^siCTRL vs sEV^siV1G1) by Kruskal-Wallis with post-test.

Fig. 6.

Up-regulation of MAPK-miRNAs prevents sEV^HIGH biological effect. (a) Cell motility of MG cells was investigated after transfection with a miRNA-Ctrl or miRNA-Pool while co-cultured with sEV^HIGH or vehicle. Symbols, mean±SD (n= 3). *, p=0.01; **, p=0.001; ***, p=0.0001 (miRNA-Pool/sEV^HIGH vs miRNA-Ctrl/sEV^HIGH) by Kruskal-Wallis with post-test. (b) Proliferation of MG cells transfected with the control or specific-miRNA Pool and co-cultured with vehicle or sEV^HIGH was investigated using a nuclear Ki67 staining assay. A representative image is shown for each condition. Scale bars, 50μm. Right, quantification of 3 independent experiments. Bars, mean±SD. ***, p=0.0009; ****, p<0.0001 by Kruskal-Wallis with post-test. ns, not significant. (c) ERK activation (pERK1/2) was analyzed by confocal immunofluorescence in MG cultures treated as indicated. Representative images are shown. Scale bar, 50 μm. Right, quantification of 3 independent experiments. Symbols, mean±SD. **, p=0.003 by Turkey’s multiple comparison test. (d) Genes expression in the indicated MG cells was analyzed by qPCR (n=3). (e) Schematic diagram of the proposed model.

This set of data proposes that there is a signature of sEV-miRNA associated with the V1G1^LOW phenotype that prevents activation of the ERK1/2 pathway in recipient cultures. On the contrary, V1G1^HIGH-NS do not vehiculate these miRNAs in their sEV and consequently reprogram the surrounding non-neoplastic microenvironment toward a pro-tumorigenic state through ERK1/2 activation (Fig. 6e).

Discussion

In this study we identified a novel mechanism exploited by the more tumorigenic V1G1^HIGH-NS to alter the non-neoplastic brain microenvironment by promoting pro-tumorigenic changes such as cell motility, proliferation and survival. Mechanistically, this involves activation and nuclear translocation of ERK1/2 and upregulation of its downstream effector Bcl2. Interestingly, Bcl2 or MAPK genes are not transferred to recipient cells via sEVs. Instead, we demonstrated that sEVs derived from V1G1^LOW-NS or from NS treated with the V-ATPase inhibitors carry a unique profile of inhibitory miRNAs, which are responsible for blunting the pro-tumorigenic response mediated by V1G1^HIGH-NS.

Taken together, these results are in line with the recent evidence showing that decreased level of tumor suppressor miRNAs in sEVs is a pro-metastatic mechanism ⁸. In our studies, target prediction analysis identified a panel of miRNAs, including miR-21-5p, −30b-5p, −30c-5p, −195-5p and miR-15b-5p, which were over-represented in sEV^LOW-miRNAs and played a key role in antagonizing MAPK signaling. Accordingly, ectopic expression of these miRNAs in MG recipient cells, or use of sEV derived from NS in which V-ATPase activity was abrogated, was sufficient to abolish ERK1/2 activation and the induction of pro-tumorigenic responses in recipient cells. Therefore, it is possible that the absence of MAPK-targeting miRNAs in secreted sEV from V1G1^HIGH-NS mediates unregulated ERK1/2 activation in recipient cells and downstream mechanisms of heightened cell proliferation and cell motility.

How the V1G1 subunit of the V-ATPase pump regulates the packaging of selective miRNAs in small EV remains to be elucidated. It is intriguing that this subunit is present on the NS plasma membrane, in sEV and that its inhibition decreases the proportion of sEVs vehiculating V1G1 while increasing sEV-miRNAs targeting MAPKs. We previously described that V-ATPase composition stratifies glioma according to aggressiveness and independently from clinical or molecular variables ²⁵. Now we show that one of the V-ATPase subunit associated to tumor aggressiveness plays a dominant role in sEV-mediated rewiring of the glioma surrounding microenvironment via ERK1/2 signaling.

The data presented here fit well with an emerging role of V-ATPase pump in the modulation of an intra- and extra-cellular milieu via acidification and modulation of protein trafficking in organelles such as endosomes and lysosomes ^{5, 15, 30, 31}. Moreover, a feedback loop exists between V-ATPase and ERK, since it has been described that V-ATPase inhibition blocks ERK/MEK signaling in regenerating epithelia ³² and in cisplatin-resistant ovarian cancer cells ³³. In addition, PI3K and ERK signaling are required for V-ATPase assembly upon viral infection ³². In this context, V-ATPase subunits are also involved in exosome biogenesis in physiologic and pathologic conditions ^{34, 35} and are found in EVs released from different cell types ^{2, 15}.

With respect to gliomagenesis, we know that GBM secreted EVs play a major role in the cross-talk between tumour cells and non-neoplastic parenchyma ^{16, 31, 36, 37} through the horizontal transfer of bioactive molecules ^{5, 16, 18, 38}. Initial studies on GBM use of EVs to reprogram the surrounding microenvironment showed that the oncogenic truncated EGFRVIII protein was transferred through large microvesicles to surrounding cancer cell that lacked the receptor ³⁹. This caused activation in recipient cells of oncogenic signaling that eventually led to phenotypical transformation and increased anchorage-independent cell growth. Moreover, GBM secreted EVs loaded with oncogenic cargoes have been described in glioma patients’ cerebrospinal fluid ⁴⁰ and in the circulation ². Therefore, it is becoming evident that aggressive glioma use EVs to favor tumor progression ^{2, 41}.

Altogether, our data identify novel molecular mechanisms of gliomagenesis, specifically affecting the GBM stem cell niche, via differential reprogramming of selected neighboring cell populations. As a key requirement in this pathway, the V-ATPase V1G1 subunit could provide an actionable therapeutic target for disease intervention both to prevent establishment of a stem cell niche, as well to limit local infiltration of non-neoplastic brain parenchyma.

Implications:

Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase dependent reprogramming of the brain microenvironment through the release of specialized extracellular vesicles.