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Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

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Table S1. Proteins investigated by the PathScan Intracellular Signaling Membrane Array Kit in MG cells after coculture with small EVs. Table S2. Significant analysis of microarray (SAM) of small EVs miRNAs. Table S3. KEGG or Reactome pathways enrichment of miRNA's predicted targets. Table S4. Analysis of possible targeted pathways of the predicted miRNA targets with WebGestalt. Table S5. List of genes investigated with the Human Molecular Mechanisms of Cancer 96-well plate. Table S6. Targets of the differentially expressed miRNAs searched using miRTargetLink Human web tool. Table S7. List of TaqMan Assay used to evaluate gene expression. Table S8. List of investigated genes in sEV derived from V1G1LOW or -HIGH NS.

Figure S1. Selection of optimal sEV concentration and Concanamycin A dose. Figure S2. Small EVs characterization. Figure S3. Functional effects of NS-derived small EVs. Figure S4. Bafilomycin A1 treatment in NS does not interfere with their ability to secrete small EVs. Figure S5. sEV-mediated modulation of cancer pathway in recipient cells. Figure S6. miRNA content in NS small EV.

Pathologically activated neutrophils (PMN) with immunosuppressive activity, which are termed myeloid-derived suppressor cells (PMN-MDSC), play a critical role in regulating tumor progression. These cells have been implicated in promoting tumor metastases by contributing to premetastatic niche formation. This effect was facilitated by enhanced spontaneous migration of PMN from bone marrow to the premetastatic niches during the early-stage of cancer development. The molecular mechanisms underpinning this phenomenon remained unclear. In this study, we found that syntaphilin (SNPH), a cytoskeletal protein previously known for anchoring mitochondria to the microtubule in neurons and tumor cells, could regulate migration of PMN. Expression of SNPH was decreased in PMN from tumor-bearing mice and patients with cancer as compared with PMN from tumor-free mice and healthy donors, respectively. In Snph-knockout (SNPH-KO) mice, spontaneous migration of PMN was increased and the mice showed increased metastasis. Mechanistically, in SNPH-KO mice, the speed and distance travelled by mitochondria in PMN was increased, rates of oxidative phosphorylation and glycolysis were elevated, and generation of adenosine was increased. Thus, our study reveals a molecular mechanism regulating increased migratory activity of PMN during cancer progression and suggests a novel therapeutic targeting opportunity.

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 (EV). 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 GSCs grown as neurospheres (NS) with high (V1G1HIGH-NS) or low (V1G1LOW-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA 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 V1G1HIGH-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 miRNA profile of V1G1HIGH-NS–derived small EVs differed significantly from that of V1G1LOW-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 prooncogenic effects of V1G1HIGH 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. 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 EVs.

Small extracellular vesicles (sEV) contribute to the crosstalk between tumor cells and stroma, but the underlying signals are elusive. Here, we show that sEV generated by breast cancer cells in hypoxic (sEV(HYP)), but not normoxic (sEV(NORM)) conditions activate NFκB in recipient normal mammary epithelial cells. This increases the production and release of inflammatory cytokines, promotes mitochondrial dynamics leading to heightened cell motility and disrupts 3D mammary acini architecture with aberrant cell proliferation, reduced apoptosis and EMT. Mechanistically, Integrin-Linked Kinase packaged in sEV(HYP) via HIF1α is sufficient to activate NFκB in the normal mammary epithelium, in vivo. Therefore, sEV(HYP) activation of NFκB drives multiple oncogenic steps of inflammation, mitochondrial dynamics, and mammary gland morphogenesis in a breast cancer microenvironment.

Cancer metabolism, including in mitochondria, is a disease hallmark and therapeutic target, but its regulation is poorly understood. Here, we show that many human tumors have heterogeneous and often reduced levels of Mic60, or Mitofilin, an essential scaffold of mitochondrial structure. Despite a catastrophic collapse of mitochondrial integrity, loss of bioenergetics, and oxidative damage, tumors with Mic60 depletion slow down cell proliferation, evade cell death, and activate a nuclear gene expression program of innate immunity and cytokine/chemokine signaling. In turn, this induces epithelial-mesenchymal transition (EMT), activates tumor cell movements through exaggerated mitochondrial dynamics, and promotes metastatic dissemination in vivo. In a small-molecule drug screen, compensatory activation of stress response (GCN2) and survival (Akt) signaling maintains the viability of Mic60-low tumors and provides a selective therapeutic vulnerability. These data demonstrate that acutely damaged, "ghost" mitochondria drive tumor progression and expose an actionable therapeutic target in metastasis-prone cancers.

The role of mitochondria in cancer continues to be debated and paradoxically implicated in opposing functions in tumor growth and tumor suppression. To understand this dichotomy, we explored the fu...

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of phosphoinositide 3-kinase (PI3K). To understand the role of autophagy in the pathogenesis of glial tumors in vivo, we used an established Drosophila melanogaster model of glioma based on overexpression in larval glial cells of an active human EGFR and of the PI3K homolog Pi3K92E/Dp110. Interestingly, the resulting hyperplastic glia express high levels of key components of the lysosomal-autophagic compartment, including vacuolar-type H+-ATPase (V-ATPase) subunits and ref(2)P (refractory to Sigma P), the Drosophila homolog of SQSTM1/p62. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of V-ATPase, of Pdk1, or of the Tor (Target of rapamycin) complex 1 (TORC1) component raptor prevents overgrowth and normalize ref(2)P levels. In addition, downregulation of the V-ATPase subunit VhaPPA1-1 reduces Akt and Tor-dependent signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, our data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase and MTORC1 components acting at lysosomes could represent therapeutic targets against GBM.

Mitochondria are signaling hubs in eukaryotic cells. Here, we showed that the mitochondrial FUN14 domain-containing protein-1 (FUNDC1), an effector of Parkin-independent mitophagy, also participates in cellular plasticity by sustaining oxidative bioenergetics, buffering ROS production, and supporting cell proliferation. Targeting this pathway in cancer cells suppressed tumor growth but rendered transformed cells more motile and invasive in a manner dependent on ROS-mediated mitochondrial dynamics and mitochondrial repositioning to the cortical cytoskeleton. Global metabolomics and proteomics profiling identified a FUNDC1 interactome at the mitochondrial inner membrane, comprising the AAA+ protease, LonP1, and subunits of oxidative phosphorylation, complex V (ATP synthase). Independently of its previously identified role in mitophagy, FUNDC1 enabled LonP1 proteostasis, which in turn preserved complex V function and decreased ROS generation. Therefore, mitochondrial reprogramming by a FUNDC1-LonP1 axis controls tumor cell plasticity by switching between proliferative and invasive states in cancer.

Glioblastoma (GBM), a very aggressive and incurable tumor, often results from constitutive activation of EGFR (epidermal growth factor receptor) and of PI3K (phosphoinositide 3-kinase). To understand the role of autophagy in the pathogenesis of glial tumorsin vivo, we used an establishedDrosophila melanogastermodel of glioma based on overexpression in larval glial cells of an active humanEGFRand of the PI3K homologDp110. Interestingly, the resulting hyperplastic glia expresses high levels of ref(2)P (refractory to Sigma P), theDrosophilahomolog of p62/SQSTM1. However, cellular clearance of autophagic cargoes appears inhibited upstream of autophagosome formation. Remarkably, downregulation of subunits of the vacuolar-H+-ATPase (V-ATPase) prevents overgrowth, reduces PI3K signaling and restores clearance. Consistent with evidence in flies, neurospheres from patients with high V-ATPase subunit expression show inhibition of autophagy. Altogether, our data suggest that autophagy is repressed during glial tumorigenesis and that V-ATPase could represent a therapeutic target against GBM.

Summary Changes in mitochondrial size, shape, and subcellular position, a process collectively known as mitochondrial dynamics, are exploited for various cancer traits. Modulation of subcellular mitochondrial trafficking and accumulation at the cortical cytoskeleton has been linked to the machinery of cell movements, fueling cell invasion and metastatic spreading. Here, we detail a technique to track changes in mitochondrial volume using a commercial CellLight™ Mitochondria-RFP/GFP reporter and live confocal microscopy. This allows a real-time study of mitochondrial dynamics in live cells. For complete details on the use and execution of this protocol, please refer to Bertolini et al. (2020)., Graphical abstract, Highlights • Protocol to measure mitochondrial dynamics in live cells • Use of time-lapse confocal imaging to capture the dynamics of mitochondria • Deconvolution of 4D images • Steps for analysis of changes in mitochondrial volume, Changes in mitochondrial size, shape, and subcellular position, a process collectively known as mitochondrial dynamics, are exploited for various cancer traits. Modulation of subcellular mitochondrial trafficking and accumulation at the cortical cytoskeleton has been linked to the machinery of cell movements, fueling cell invasion and metastatic spreading. Here, we detail a technique to track changes in mitochondrial volume using a commercial CellLight™ Mitochondria-RFP/GFP reporter and live confocal microscopy. This allows a real-time study of mitochondrial dynamics in live cells.

The Myc gene is a universal oncogene that promotes aggressive cancer, but its role in metastasis has remained elusive. Here, we show that Myc transcriptionally controls a gene network of subcellular mitochondrial trafficking that includes the atypical mitochondrial GTPases RHOT1 and RHOT2, the adapter protein TRAK2, the anterograde motor Kif5B, and an effector of mitochondrial fission, Drp1. Interference with this pathway deregulates mitochondrial dynamics, shuts off subcellular organelle movements, and prevents the recruitment of mitochondria to the cortical cytoskeleton of tumor cells. In turn, this inhibits tumor chemotaxis, blocks cell invasion, and prevents metastatic spreading in preclinical models. Therefore, Myc regulation of mitochondrial trafficking enables tumor cell motility and metastasis.

Hypoxia is a universal driver of aggressive tumor behavior, but the underlying mechanisms are not completely understood. Using a phosphoproteomics screen, we now show that active Akt accumulates in the mitochondria during hypoxia and phosphorylates pyruvate dehydrogenase kinase 1 (PDK1) on Thr346 to inactivate the pyruvate dehydrogenase complex. In turn, this pathway switches tumor metabolism towards glycolysis, antagonizes apoptosis and autophagy, dampens oxidative stress, and maintains tumor cell proliferation in the face of severe hypoxia. Mitochondrial Akt-PDK1 signaling correlates with unfavorable prognostic markers and shorter survival in glioma patients and may provide an “actionable” therapeutic target in cancer.

The crosstalk between tumor cells and the adjacent normal epithelium contributes to cancer progression, but its regulators have remained elusive. Here, we show that breast cancer cells maintained in hypoxia release small extracellular vesicles (sEVs) that activate mitochondrial dynamics, stimulate mitochondrial movements, and promote organelle accumulation at the cortical cytoskeleton in normal mammary epithelial cells. This results in AKT serine/threonine kinase (Akt) activation, membrane focal adhesion turnover, and increased epithelial cell migration. RNA sequencing profiling identified integrin-linked kinase (ILK) as the most upregulated pathway in sEV-treated epithelial cells, and genetic or pharmacologic targeting of ILK reversed mitochondrial reprogramming and suppressed sEV-induced cell movements. In a three-dimensional (3D) model of mammary gland morphogenesis, sEV treatment induced hallmarks of malignant transformation, with deregulated cell death and/or cell proliferation, loss of apical-basal polarity, and appearance of epithelial-to-mesenchymal transition (EMT) markers. Therefore, sEVs released by hypoxic breast cancer cells reprogram mitochondrial dynamics and induce oncogenic changes in a normal mammary epithelium.

Background The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Fund This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF).

Background Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. Methods V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients' cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. Findings GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0·03, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. Interpretation Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. Fund This work was supported by Fondazione Cariplo (2014–1148 to VV), Fondazione IRCCS Ca' Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV).

Background: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. Methods: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. Findings: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. Interpretation: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. Funding Statement: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF). Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Primary culture were obtained from fresh glioma specimens using surgical surplus of neoplastic or non-neoplastic tissue. All patients received a gross total resection of the tumor at Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico (Milan, Italy) and no patient received neoadjuvant therapy. The study was approved by an Institutional Review Board (IRB#275/2013) and signed informed consent was obtained from all enrolled subjects.

Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. We investigated deregulation of V-ATPase and associated signaling in aggressive gliomas. In vivo, we found that V-ATPase subunit expression correlates with glioma growth. Using patients' series, we observed that glioblastoma (GBM) and relapsed gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis. Molecularly, GBM-like V-ATPase expression correlated with switch in epigenetic enzymes and upregulation of homeobox genes. GBM neurospheres influenced the non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via large oncosomes (LO). LO reprogrammed recipient cells to grow as spheres and to migrate. Moreover, LO were particularly abundant in the circulation of GBM and of patients who died during follow-up. Finally, impairment of V-ATPase reduced LO activity. These data identify a global cell fate change that underlies glioma aggressiveness and suggest new entry points for glioma stratification, follow-up, and therapy. Funding Statement: This work was supported by Fondazione Cariplo (2014-1148 to VV), Fondazione IRCCS Ca’ Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV) and by the Ricerca Corrente program 2017 (to SF). AMS and MF were supported by a Fellowship from the Doctorate School in Molecular and Translational Medicine of Milan University. Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: Animal experiments were carried out in compliance with the institutional guidelines for the care and use of experimental animals (European Directive 2010/63/UE and the Italian law 26/2014), authorized by the Italian Ministry of Health and approved by the Animal Use and Care Committee of the University of Milan. The patient series of the study: The study was approved by an Institutional Review Board (IRB#275/2013) and signed informed consent was obtained from all patients.

Background The vacuolar H+-ATPase (V-ATPase) is a multisubunit proton pump with a role in the acidification of the extra- and intra-cellular environments. Recent evidence connected V-ATPase deregulation and human diseases, including cancer. We previously demonstrated that the overexpression of the catalytic subunit V1G1 is correlated with glioma stem cells (GSC) maintenance. According to V1G1 expression, we sorted glioblastoma patients-derived neurospheres cultures (NS) into two categories: V1G1HIGH and V1G1LOW NS. These NS differ in terms of stem cell genes expression, sphere formation, invasion ability, lysosomal acidity and bioenergetics. Methods V1G1HIGH, V1G1LOW NS treated with 10nM of BafilomycinA1 (BafA1) or vehicle for 24h (n=3 per group) were used for the functional experiments. Lactate, glucose, ATP levels were measured by commercial kits. Metabolic proteins expression was assessed by western blot. ROS production and mitochondria function were measured by flow cytometry using MitoSox and TMRE assays, respectively. Results At basal conditions, V1G1LOW NS produce higher levels of lactate (p=0.0379) whereas V1G1HIGH NS show higher levels of extracellular glucose and intracellular ATP. Accordingly, the two NS cultures show an opposite trend in LDH subunits expression with LDHA, which pushes lactate production, being more expressed in V1G1LOW (p=0.0027). Conversely, LDHB that facilities the production of pyruvate from lactate, and the transporter MCT1 are more expressed in V1G1HIGH NS. Interestingly, using the V-ATPase specific inhibitor BafA1, metabolic characteristics of V1G1HIGH are redirected to the V1G1LOW phenotype. Moreover, in V1G1HIGH NS BafA1 induces ROS production and affects mitochondria activity, increasing their depolarization. Conclusions Taken together these data suggest two distinct metabolic behaviors of NS according to V-ATPase expression. Indeed for energy production V1G1LOW NS could rely on a glycolytic metabolism whereas V1G1HIGH NS on oxidative phosphorylation. Moreover, when overexpressed the proton pump could also have a role in preserving mitochondria polarity. Although preliminary, this is the first evidence reporting a role for V-ATPase in the regulation of cancer stem cell metabolism. Citation Format: Alessandra M. Storaci, Irene Bertolini, Manuela Caroli, Stefano Ferrero, Valentina Vaira. V-ATPase in glioma stem cells: V1G1 subunit expression correlates with metabolic behavior and mitochondria activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 791.

Background: Recent evidences highlighted that GBM secreted microvesicles (EVs), particularly exosomes (Exo) and large oncosomes (LO), play a major role in the cross-talk between tumor cell and non-neoplastic parenchyma. How GBMs manage to thrive in a highly unfavorable, acidic microenvironment is still unclear, but recent work from our group has identified the vacuolar pump H+-ATPase (V-ATPase) as an important effector of GBM growth and glioma stem cells (GSC) maintenance. Additionally, in ExoCarta database V-ATPase subunits have been described in Exo from different cancer cell types. Taken together, these data identify V-ATPase as an important driver of gliomagenesis, and a novel, actionable therapeutic target for disease intervention. However, the role of V-ATPase in reprogramming the GBM microenvironment has not been previously investigated. Methods: Exo and LO were isolated by an Invitrogen kit and serial centrifugation, respectively, from media of patients’ derived GBM neurospheres, enriched in GSC (NS, n=12) or differentiated cultures (n=8). For EVs internalization studies, Exo or LO were stained using FM 1-43 FX dye and the process was followed live for 30’ and at selected time points (30’-4h-24h), using a confocal microscopy or flow cytometry (FACS). Electron microscopy, FACS (of Exo stained with CellTrace and SytoRNA in combination with CD63 coated beads), Nanosight and immunoblotting (for CD63, CD9 and Clathrin) analyses were used to confirm EVs subtypes. Cultures from patients’ derived brain tumor margins or primary GBM (differentiated and not) were used as EVs-recipient cells. miRNA profiling was performed using Taqman Low density arrays and analyzed by R packages. Gene Ontology analysis was performed by DAVID. The study was approved by the Institutional Ethical Committee. Results: NS are able to produce different EVs, which are internalized by recipient cells after 4 and up to 24 hours of co-culture. Both Exo than LO from NS are able to significantly increase cell growth in recipient cells (brain tumor margins and primary GMB differentiated monolayers), and this effect is stronger with EVs produced by NS with higher V-ATPase expression (V-ATPaseHIGH NS). Primary GBM cells after co-culture with EVs are able to produce a higher number of NS and V-ATPase activity block by BafilomycinA1 in NS-producing EVs completely revert this effect. Finally, the co-culture of V-ATPaseLOW NS with EVs from V-ATPaseHIGH NS increases their motility in collagen matrixes. At molecular level, profiling of Exo-derived miRNAs distinguishes differentiated cultures from NS and, among NS, V-ATPaseHIGH cultures. In silico analysis and annotation of miRNA target genes from V-ATPaseHIGH–derived Exo showed an enrichment of cancer, cell cycle and PI3K/Akt pathways. Conclusions: Altogether, these data point toward the central role of different EV types in GBM communication and suggest a role of the V-ATPase proton pump in regulating EV’s contents. Citation Format: Irene Bertolini, Andrea Terrasi, Andrea Di Cristofori, Silvano Bosari, Valentina Vaira. V-ATPase control of EV signaling in glioma stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2889. doi:10.1158/1538-7445.AM2017-2889

// Andrea Di Cristofori 1,2,* , Stefano Ferrero 3,4,* , Irene Bertolini 1,3 , Gabriella Gaudioso 1,3 , Maria Veronica Russo 1,3 , Valeria Berno 5 , Marco Vanini 6 , Marco Locatelli 2 , Mario Zavanone 1,2 , Paolo Rampini 2 , Thomas Vaccari 7 , Manuela Caroli 2 and Valentina Vaira 3,5 1 Department of Pathophysiology and Organ Transplantation, University of Milan, Milan, Italy 2 Division of Neurosurgery, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy 3 Division of Pathology, Fondazione IRCCS Ca’ Granda, Ospedale Maggiore Policlinico, Milan, Italy 4 Department of Biomedical, Surgical and Dental Sciences, University of Milan, Milan, Italy 5 Istituto Nazionale Genetica Molecolare “Romeo ed Enrica Invernizzi”, Milan, Italy 6 Surgical Pathology Unit, St. Anna Hospital, Como, Italy 7 IFOM - The FIRC Institute of Molecular Oncology, Milan, Italy * These authors have equally contributed to this work Correspondence to: Manuela Caroli, email: // Valentina Vaira, email: // Keywords : glioblastoma, vacuolar H+-ATPase, bafilomycin A1, cancer stem cells, organotypic tissue cultures Received : January 16, 2015 Accepted : May 02, 2015 Published : May 22, 2015 Abstract The vacuolar H + ATPase (V-ATPase) is a proton pump responsible for acidification of cellular microenvironments, an activity exploited by tumors to survive, proliferate and resist to therapy. Despite few observations, the role of V-ATPase in human tumorigenesis remains unclear. We investigated the expression of ATP6V0C, ATP6V0A2, encoding two subunits belonging to the V-ATPase V0 sector and ATP6V1C, ATP6V1G1, ATPT6V1G2, ATP6V1G3, which are part of the V1 sector, in series of adult gliomas and in cancer stem cell-enriched neurospheres isolated from glioblastoma (GBM) patients. ATP6V1G1 expression resulted significantly upregulated in tissues of patients with GBM and correlated with shorter patients’ overall survival independent of clinical variables. ATP6V1G1 knockdown in GBM neurospheres hampered sphere-forming ability, induced cell death, and decreased matrix invasion, a phenotype not observed in GBM monolayer cultures. Treating GBM organotypic cultures or neurospheres with the selective V-ATPase inhibitor bafilomycin A1 reproduced the effects of ATP6V1G1 siRNA and strongly suppressed expression of the stem cell markers Nestin, CD133 and transcription factors SALL2 and POU3F2 in neurospheres. These data point to ATP6V1G1 as a novel marker of poor prognosis in GBM patients and identify V-ATPase inhibition as an innovative therapeutic strategy for GBM.