Fig. 7: Angiocrine factors induce VC-Resist state transition, cell survival and partial proneural-to-mesenchymal transition.

A Nestin expression in Nestin^P-dTomato MGG4 cells in presence of conditioned media from blood vessels (CM-BV) or conditioned media from brain endothelial cells (CM-bEnd) using time-lapse imaging. Data are means ± SEM (n = 4 independent experiments; p = 0.0003; Spearman correlation). B Enrichment of GL261 NesHI cell population when cultured with bEnd-CM or control (CT) media by FACS. Data are means ± SEM (n = 3 independent experiments; p = 0.0016; paired two-side t test). C Nestin mRNA per cell in Nestin^P-dTomato MGG4 cells in the presence of bEnd.3 conditioned media (CM) or control media (CT). Total number of cells=46, unpaired two-sided t test. D (Left) Real-time micrographs of Nestin^P-dTomato MGG4 cells embedded in agarose gel showing the induced NesLO-to-NesHI transitions. (Right) Quantification of the reprogramming rate in Nestin^P-dTomato MGG4 cells pre-conditioned in CT or CM for 3 days (n = 76; ****p < 0.0001; unpaired two-sided t test). E Pearson correlation analysis of basal Nestin expression and fold change (FC) of Nestin expression in presence of conditioned media (bEnd-CM) in 4 GBM cell lines. X-axis: 1/basal CT values for Nestin expression by RT-PCR, Y-axis: Nestin expression FC with bEnd-CM. Patient-derived (blue) mouse (green) cell lines (n = 3; R = 0,88; p = 0,001; Pearson test). F Enrichment of MGG4 NesHI cell population in the presence of CM-bEnd (CM) both with (5 Gy) and without IR analyzed at day5 by FACS. Data are means (n = 2 independent experiments; *p < 0.05; **p < 0.01; two-way ANOVA, Turkey’s multiple comparisons test). G (Top), GSEA plots of the VC-Resist signature and senescence geneset (FRIDMAN_SENESCENCE_UP) in MGG4 co-cultured with blood vessels (Bottom), or GSC2 co-cultured with endothelial cells. The normalized enrichment score (NES) and q.value are indicated. H β-Gal senescence in Nestin^P-dTomato MGG4 cells after treatments (5 Gy of IR, 50 μM TMZ) or stimulated with CM-BV. Data are means ± SEM (n = 3 independent experiments; *p < 0.05; unpaired two-sided t test). I FACS analysis of CellTrace^TM dye dilution during cell division in CT or CM-bEnd cells. Time to undergo cell division was calculated based on the mean fluorescent intensity values. Data are means ± SEM (n = 4, **p < 0.01). J Cell death (percentage of Sytox+ cells) in Nestin^P-dTomato MGG4 cells conditioned with bEnd.3 conditioned media (CM) or control (CT). Data are means ± SEM (n = 3 independent experiments; ***p < 0.001; unpaired two-sided t test).