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.
Funding
ALEMBIC-Advanced Light
Electron Microscopy BioImaging Center
Ricerca Corrente program
NIH
Doctorate School in Molecular and Translational Medicine of Milan University
ARTICLE 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 (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.
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.
