Fig. 1: In vitro models of H3K27M DIPG molecularly mimic oligodendrocyte precursor (OPC)-like and differentiated astrocyte (AC)-like DIPG and exhibit distinct gene expression programs. | Nature Communications

Fig. 1: In vitro models of H3K27M DIPG molecularly mimic oligodendrocyte precursor (OPC)-like and differentiated astrocyte (AC)-like DIPG and exhibit distinct gene expression programs.

From: Therapeutic targeting of differentiation-state dependent metabolic vulnerabilities in diffuse midline glioma

Fig. 1

A Schematic depicting the generation of DIPG gliomaspheres (GS) and differentiated glioma cells (DGC): GS (3-D floating spheres) were cultured in serum-free tumor stem cell media containing growth factors and supplements. Monolayer (adherent) DGC were generated by dissociating GS to single cells and culturing for up to 14 days in media containing 10% fetal bovine serum (FBS). B Growth kinetics of DIPG-007, SF7761 and DIPG-XIII GS vs. DGC. Error bars are mean ± SD from three biological replicates; ns (not significant) p = 0.7589, ****p < 0.0001 by area under proliferation curve test followed by two-tailed Student’s t-test. C, D Venn diagrams derived using DESeq2 package in R with adjustment for multiple comparisons illustrating the overlap of upregulated (C) and downregulated (D) genes in GS vs. DGC across three cell lines. E Western blot for OLIG2 in GS and DGC pairs of DIPG cell lines. HSP90 was used as a loading control. The figure shows representative blots from two independent experiments. F Analysis of the gene expression signature of DIPG GS vs. DGC cross-referenced with patient gene signature capturing OPC-like, AC-like gene signatures. G Differential expression of DIPG genes in OPC-like and AC-like tumors from patient single-cell RNAseq. F, G) Data was analyzed using unpaired, two-sided, two-tailed, Students t-test with 95% confidence intervals *p < 0.05; **p < 0.01; ***p < 0.001. H Survival analysis of DIPG/DMG patients (n = 78; female = 41, male = 28, unknown = 9. Age range was 1.2–17.9 years (average 9.8 ± 12 years) based on “GS high” versus “GS low” gene signature. Median survival is 11.9 months versus 8 months; Log Rank p = 0.0176. I, J “Hallmark” gene set enrichment analysis (GSEA) indicating pathways that are (I) upregulated and (J) downregulated in DIPG GS vs. DGC. GSEA plots show enrichment scores and include values for normalized enrichment score (NES), nominal p-value (P), and false discovery rate (FDR) q-value. For all panels, red indicates GS; blue, DGC.

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