EXOSOMES SIGNALLING IN HUMAN GLIOMA STEM CELLS: THE CENTRAL ROLE OF V-ATPASE PROTON PUMP ACTIVITY

Recent evidences highlighted that glioblastomas (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. 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. The aim of this project was investigate production, biological effect and content of extracellular vesicles according to proton pump activity in glioma stem cells. Our data show that GSC are able to produce different types of EVs, which are internalized by recipient cells of different histology, such as non-neoplastic brain tumor margins, primary GBM monolayers (both differentiated and undifferentiated), and commercial glioma cultures. Exo and LO from GSC induces in recipient cells distinct effects. In particular, Exo significantly increased cell growth and cell motility, and these effects were stronger with Exo produced by NS with higher V-ATPase expression (V1G1HIGH NS). On the other hand, LO were able to strongly induce the sphere formation ability of primary GBM cultures. This effect lasted up 90 days after co-culture. In both situations, the block of V-ATPase activity by Bafilomycin A1 in NS-producing EVs completely reverted the effects. Interestingly, exosomes are able to vehiculate on their surface the V-ATPase G1 subunit, and its protein level increased in recipient cells after co-culture with EVs. At the molecular level, profiling of Exo-derived miRNAs distinguishes V1G1HIGH NS from V1G1LOW cultures. In silico analysis and annotation of miRNA target genes showed an enrichment of cancer, cell cycle and MAPK/Erk pathways. Regarding signaling pathway modulation by Exo in recipient cells, exosomes from V1G1HIGH NS activated the MAPK/Erk pathway. 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 exosomes contents.