Fig. 6. YAP/TAZ are required for GBM initiation by preventing GSC differentiation.
(a-d) Immunocompromised mice were injected intracranially with shNF1/shp53-transformed cells derived from Yapfl/fl; Tazfl/fl newborn astroglial cells, also transduced with dual luciferase-GFP expression vectors. Control (YAP/TAZ wt) animals (n=5) were injected with cells transduced with Ad-GFP, whereas YAP/TAZ KO refers to animals (n=5) injected with cells transduced with Ad-Cre. (a) Representative images of brain bioluminescence. (b) Bioluminescence quantification shown as scatter dot plots and bar graphs showing mean with s.d.; p-value was calculated by unpaired two-tailed t-test. (c) Representative H&E staining; scale bar, 1 mm. (d) Magnification of the tumor generated by YAP/TAZ wt cells; scale bar, 250 μm. Arrowheads point to polynucleated giant cells, a characteristic trait of giant cells Glioblastoma.
(e-g) GL261 cells, an established mouse model of GBM, were injected intracranially in syngeneic (C57BL/6) mice. Control animals (n=5) were injected with cells transduced with lentiviral vectors coding for control shRNA, whereas YAP/TAZ-depleted refers to animals (n=5) injected with cells transduced with lentiviral vectors coding for doxycycline-inducible YAP and TAZ shRNAs and exposed to doxycycline prior to injection to induce YAP/TAZ depletion. Cells were also transduced with dual luciferase-GFP expression vectors. To sustain YAP/TAZ depletion after injection, doxycycline was added to the drinking water of all mice. (e) Representative images of brain bioluminescence at one day and 14 days after injection. (f) Bioluminescence quantification at three different time points shown as scatter dot plots and bar graphs showing mean with s.d.; unpaired two-tailed t-test p-values are shown. (g) Representative H&E stainings of brain sections from mice injected with control (upper panel and corresponding magnification) or with YAP/TAZ-depleted GL261 cells (middle and lower panels), the latters displaying either no remaining tumor cells (middle panel, representative of n=3 mice), or a residual amount of injected cells converging toward the right ventricle (lower panel and corresponding magnification, representative of n=2 mice). Scale bars, 2.5 mm in left panels and 250 μm in the magnifications shown on the right. ‘N’ indicates necrotic areas.
(h) Representative GFP and TUJ1 stainings (scale bars, 50 μm) in sections from the same mouse brains injected with control (upper panel) or YAP/TAZ depleted GL261 cells (lower panel) shown in the upper and lower panels of (g), respectively. The arrowhead point to a single TUJ1-positive cell in the control tumor.
(i) Gene Set Enrichment Analysis (GSEA) enrichment score curve of known markers of neuronal differentiation of NSC in YAP/TAZ KO vs. YAP/TAZ wt subcutaneous tumors from KRasG12V/shp53-transformed cells, following the experimental setup indicated in Extended Data Fig. 6. Signatures are available in Supplementary Table 7.
(j) GFP and TUJ1 stainings (scale bars, 50 μm) in sections from YAP/TAZ wt and YAP/TAZ KO tumors (representative of n=3 independent tumor samples each) derived from shNF1/shp53-transformed cells, following the experimental setup indicated in Extended Data Fig. 6.