Fig. 6

Reprogramming of glutamate metabolism drives invasive phenotypes in GBM. A Lower methylation signals by MeDIP-qPCR on the GRIA1 promoter and higher expression of GRIA1 transcripts were observed in U87 cells with lentivirus-mediated overexpression of human Rictor (Rictor OE) in comparison with control (GFP). Met, methylation (5-mC). B GBM tumors from rats infected with PDGFB-HA-IRES-EGFP retroviral vectors (n = 4) were probed by immunohistochemistry against GRIA1. Note intratumoral heterogeneity of GRIA1 immunoreactivity in accordance with the status of mTORC2 activation (pAkt) and 5-mC expression. Peri-necrotic area indicates pAkt_low/5-mC_high region, and non-necrotic to pAkt_high/5-mC_low region. Refer to Fig. 1B. Nec, necrosis. Scale bar, 20 µm. C Scratch assays using the co-culture of U87-EGFRvIII (GBM) cells with siRNA-mediated knockdown of GRIA1 and SH-SY5Y (neuroblastoma) cells. The area of gap was calculated 24 h after scratch. Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. D Knockdown of GRIA1 significantly (p < 0.01) affected GBM cell migration in the co-culture of U87-EGFRvIII and SH-SY5Y cells. E Measurement of glutamate (Glu) indicated that GRIA1 knockdown increased extracellular Glu (reduced intracellular Glu) in U87-EGFRvIII GBM cells. Conc, concentration. F Wound healing/migration assay on the co-culture of SH-SY5Y neuroblastoma cells with U87-EGFRvIII GBM cells treated by PhTx-74 (GRIA1/GRIA2 inhibitor: 20 μM) or a combination of PhTx-74 (20 μM) with GSK2256098 (FAK inhibitor: 100 nM). Cells were colored in red with the binary mode (red) of ImageJ software. Scale bar, 100 µm. G PhTx-74 treatment increased extracellular Glu (reduced intracellular Glu), and FAK inhibitor decreased phosphorylation of FAK (Tyr397) in U87-EGFRvIII GBM cells. Conc, concentration. H TCGA datasets on overall survival and progression free survival of GBM cases stratified by the expression level of GRIA1 transcripts