Figure 6 from A Compendium of Syngeneic, Transplantable Pediatric High-Grade Glioma Models Reveals Subtype-Specific Therapeutic Vulnerabilities

A, Left: acid-extracted histone and protein lysates prepared from ex vivo GS lines derived from the two-hit de novo models (KP, H3.1KP, KF, and KN) were probed for K27M, K27me3, HA, and total H3.3 levels. NSCs derived from the LRL (LRL NSC) were used as controls. Middle: validation of FGFR1 overexpression in ex vivo KF cells; GAPDH was used as a loading control. Right: validation of p53 and NF1 downregulation in ex vivo cells by qRT-PCR. Data are presented as means ± SD from at least n = 3 replicates. ****, P < 0.0001 and ***, P = 0.0003 were calculated using unpaired t test and one-way ANOVA. B, Left: acid-extracted histone and protein lysates prepared from ex vivo GS lines derived from the three- and four-hit de novo models (KPP, KPD, KPAP, KPAD, KPPMPIK, H3.1KACVPIK, KNF, KPF, and KPC) were probed for K27M, K27me3, HA, and total H3.3 levels. LRL NSCs were used as controls. Validation of PDGFRA, PPM1D, PIK3CA, ACVR1, FGFR1, and CCND2 overexpression in ex vivo GS cells; GAPDH and tubulin were used as loading controls. Right: validation of p53, ATRX, and NF1 downregulation in ex vivo GS cells by qRT-PCR. Data are presented as means ± SD from at least n = 3 replicates. ****, P < 0.0001 and ***, P = 0.0003 were calculated using unpaired t test and one-way ANOVA. C, Left: IHC detection of hematoxylin and eosin (H&E), GFP, K27M, and Ki-67 in coronal hindbrain sections from orthotopically engrafted, symptomatic mice. Mice were injected in either the pons (H3K27M) or striatum (H3.3G34R) with 150,000 to 300,000 cells from each cell GS cell line (n = 5 mice per condition). Tumor cells are GFP⁺. Scale bars, 2 mm. Right: BLI of brainstem tumors in orthotopically engrafted C57BL/6J mice without shaving/depilation using Akaluc imaging. D, Kaplan–Meier curves depicting survival of mice orthotopically injected with GS cells derived from H3.3K27M (left), H3.1K27M (middle), and H3.3G34R (right) models. Note that the following samples are shown twice in A and B: LRL NSC, KF, and KN; these repetitions are indicated with an asterisk. The sole purpose of this repetition is to make it easier for the reader, by collating all the Western blot (WB) data describing the “two-hit” models in A and all the WB data describing the “three-hit or more” models in B.