Fig. 6: NOTCH1 inhibition leads to depletion of the perivascular niche but promotes TM-network formation.

a Left: Exemplary in vivo 2-PM images of S24 shControl (top, D40) and shCX43 tumors (bottom, D43). Right: Corresponding ratio of perivascular and parenchymal cells in S24 shControl and shCX43 knockdown tumors (D40± 4, n = 5 regions (S24 shControl)/6 regions (S24 shCX43) in 3 mice per group, two-tailed t-test). b Immunofluorescence staining of activated Notch1 in a S24 tumor section. Arrow: multi-TM parenchymal cell (low Notch1 activation); arrowhead: Notch1-low TM; asterisk: perivascular cell (high Notch1 activation). c Exemplary 2-PM images of S24 shControl (top) and shNOTCH1 (bottom) glioblastoma stem-like cells (GBMSCs) (green) with segmentation of co-opted blood vessels (white) demonstrating a reduced population of the perivascular compartment after NOTCH1 downregulation (D24 ± 1). Blood vessels (blue). d Ratio of the perivascular and parenchymal cell count 20 and 40 days after S24 shControl and shNOTCH1 tumor cell implantation (n = 5 regions in 3 mice (S24 shControl)/4 mice (S24 shNOTCH1), 38–445 cells, two-tailed t-tests). e Histogram and quantification of TM length in S24 shControl and shNOTCH1 GBSMSCs (D20, n = 3 mice (S24 shControl), n = 4 mice (S24 shNOTCH1). f Quantification of TM-devoid (0 TMs) vs. TM-rich (>4 TMs) S24 shControl and shNOTCH1 GBMSCs (n = 5 regions in 3 mice (S24 shControl)/4 mice (S24 shNOTCH1), 18–286 cells, one-way ANOVA on the ranks (p = 2.90 × 10⁻¹²), Student–Newman–Keuls post hoc test). g Ratio of connected and non-connected S24 shControl and shNOTCH1 GBMSCs 20 and 40 days after tumor cell implantation (n = 5 regions in 3 mice (S24 shControl)/4 mice (S24 shNOTCH1), 11–245 cells, two-tailed Mann–Whitney tests). h Corresponding 2-PM images demonstrating the TM richness of S24 shNOTCH1 tumors (D40). Data a, d, f, g are represented as mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.