Fig. 4: The CL3/NesHI state is already present in naïve GBM cells and is reversible, slow-cycling, senescent-like and resistant to therapy.
From: VC-resist glioblastoma cell state: vessel co-option as a key driver of chemoradiation resistance
A GSEA plot of the CL3 signature between NesLO and NesHI cells in NestinP-dTomato MGG4, MGG18 and GL261 sorted-cells. Normalized enrichment score (NES) and q.value are indicated. B Genes in common in CL3/NesHI genesets. C Feature plot and violin graph of NesHI common genes, i.e. the genes commonly upregulated in at least 3 genesets, among NesHI MGG4, MGG18, GL261 and CL3 (see Supplementary Fig. 5B) (2 independent experiments and 2 time-points per experiment). D Feature plot for CL3/NesHI signature (32 genes) in the database from Ruiz-Moreno, 2023. E Feature plot for CL3/NesHI signature (32 genes) in the Proneural-Mesenchymal axis RNA-velocity from Wang et al. 2023. F Bubble plot of GBM states genesets in NesHI MGG4, MGG18 and GL261 cells. G Biological functions by IPA in NesHI MGG4, MGG18 and GL261 cells. H Kinase enrichment analysis (KEA) on phosphoproteome of NesHI vs NesLO-sorted MGG4 GBM cells (n = 5; z-score is indicator of the kinase activity estimated; significant kinase groups are plotted). Volcano plot of the phosphoproteome in Supplementary Fig. 8F. I Cell death analysis in NesLO and NesHI cell populations in NestinP-dTomato MGG4 cells upon IR or TMZ. Data are means ± SEM. (n = 3 independent experiments, two-way ANOVA, p value between NesLO and NesHI). J Area under the curve analysis of Sytox+ cells in MGG4, MGG18, and GL261 cells treated with 2, 5, 8, 10, 12 Gy overtime (1–7 days) by FACS. Data are means ± SEM (n = 3 independent experiments, two-way ANOVA, Turkey’s multiple comparison test, p value between NesLO and NesHI are shown). Dose-effect plots in Supplementary Fig. 9. K FACS analysis of CellTraceTM dye dilution during cell division. Data are means ± SEM (n = 3 independent experiments; ns, non-significant; ***p < 0.001). L Bar plots showing decrease in SubG1 and increase in G2M cell cycle phases in NesHI cells compared to NesLO in NestinP-dTomato MGG4 cells. Data are means ± SEM (n = 3; *p < 0,05; **p < 0.01; paired two-sided t test). M Decrease of NesHI cell population overtime in FACS-sorted NesHI MGG4 and NesHI GL261 cells by FACS. Data are means ± SEM (n = 3). N β-Gal senescence staining in FACS-sorted NesLO and NesHI MGG4 or GL261 cells. Data are means ± SEM (n = 3 independent experiments; ***p < 0.001; unpaired two-sided t-test).
