Abstract 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