Fig. 8

Validation of putative tumor suppressors using an ex vivo glioma model derived from EGFRvIII-mouse GBMs. a Cells from mouse GBMs were propagated as gliomaspheres in culture and subcutaneously transplanted into NOD-SCID-γ mice. b Histology of allografted cells confirmed tumor formation with diffuse expression of EGFRvIII in tumor cells (left panel), and a high level of Ki67 expression (right panel). Scale bar = 100 μm c Kaplan-Meier curve of survival of mice with allografts of EGFRvIII-tumor cells; 6/6 transplanted mice developed tumors with a median survival of 17 days. d Bar plot showing cell growth 4 weeks after CRISPR-cas9 induced mutations in Tead2, Nav3, and Spred1 in EGFRvIII-glioma cells, demonstrating significantly increased viable cells relative to wild-type cells (with non-targeting, NT, sgRNA). Measurements were performed in triplicate with three independent experiments. Data are represented as mean values ± SEM. Significance was determined using the one-way ANOVA test. e TIDE confirms a high level of gene editing of Spred1 using CRISPR-cas9 in glioma cells. f Treatment of EGFRvIII-glioma cells carrying CRISPR-induced Nav3 mutations and Spred1 mutations (g) with trametinib shows significantly reduced cell viability compared with WT cells (carrying NT sgRNA) at 0.1 μM. Significance was determined using the two-sided t test. ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05. Data are represented as mean values ± SEM