Fig 2: Transcriptional analysis highlights THBS1 as central regulator of GBM invasion. A network of proteins encoded by genes that are overexpressed in the invasive area of P3 tumours when compared to the core area, or are expressed by the host. Relationships in the network represent known protein–protein interactions. Human genes are represented in purple and mouse genes are colour coded according to their expression (green (2) – red (10); normalised counts in log2 scale)

Fig 3: Thrombospondin-1 is overexpressed in senescent cells and blocks cell proliferation.a Quantitative RT-PCR analysis of SASP components in emergent cells (n = 3). Emergent cells were generated as described above and mRNA expression was analyzed as compared with parental cells. b Analysis of TSP1 expression, by RT-QPCR or by western blot in LS174T and MCF7 cells treated or not and after emergence. The presence of TSP1 in the secretome of emergent cells was assessed by western blot assay following serum starvation for 24 h. Intracellular hsc70 expression was evaluated in parallel on the corresponding adherent cells (n = 4, Mann–Whitney test, ** = p < 0.01, *** = p < 0.001). c Cell sorting of dividing PLD and senescent PLS clones according to FSC/SSC and Ki67 parameters (n = 4 +/− sd). The image illustrates the gates that have been used during cell sorting. THBS1 mRNA expression was analyzed by quantitative RT-PCR (n = 5 +/− sd, Mann–Whitney test, * = p < 0.05). d Analysis of LS174T (n = 3 +/− sd) and MCF7 (n = 5) proliferation and survival using clonogenic tests. Cells have been treated with the indicated concentrations of TSP1 or PBS for 7 days. e Following TSP1 stimulation of LS174T cells (10 µg/ml), PML staining and p21 and p15 expressions were evaluated by immunofluorescence and western blot (n = 3)

Fig 4: Senescence escape in primary fibroblasts generates CD47low cells with a reduced expression of p21waf1.a Analysis of WI38 proliferation and survival using clonogenic tests (n = 3 +/− sd). Cells have been treated with TSP1 (10 µg/ml) or PBS for 7 days. b WI38 cells have been infected with a lentivirus expressing a K-RasG12V-estrogen receptor chimera. After KRasG12V induction, senescence was evaluated at day 20 by crystal violet staining (left), SA-β-galactosidase activity (middle), and mRNA expression of p15INK4b, p16INK4a, p21waf1, IL1-beta, IL-6, and IL-8 (n = 5 +/− sd). c Senescence was induced in WI38 cells as described above and cells were then infected with a lentivirus expressing the SV40 large T antigen or GFP as a control. Emergence was observed after 1 month. d One week after emergence detection, dividing and senescent WI38 clones were recovered and analyzed by intracellular flow cytometry. p21waf1 and KI-67 expressions were quantified by flow cytometry in CD47low cells or CD47high cells and normalized to control IgG staining (n = 6, n = 4 for KI-67 staining, Kolmogorov–Smirnov test, ** = p < 0.01). One illustrative image of p21waf1 staining is presented

Fig 5: THBS1/CD47 interaction in P3 tumour invasion and growth. a P3 cells were included into collagen I gels and then incubated in normoxia (21 % O2) or hypoxia (1 % O2). P3 spheroid invasion was measured in collagen I gels after 24 h. Scale: 50 µm. The graph represents the results as means ± s.d. of three independent experiments, each done in 6–8 replicates for each condition. *P < 0.05; ns, non-significant (ANOVA). b P3 tumours were treated with 10 mg/kg (BW) of bevacizumab (Bev), control peptide (control), TAX2 alone (TAX2) or in combination (Bev + TAX2). Tumours sections were stained with anti-CD31 antibody. Scale: 20 µm. The graphs below represent the vascular density (left panel) and the quantification of vessel types according to size (right panel: small vessels < 10 μm; medium size vessels between 10 and 20 μm; large vessels > 20 μm) (average of 5 tumours with 8 sections/tumour analysed). Results are represented as means ± s.d. *P < 0.05; ***P < 0.001; ****P < 0.0001 for small vessel comparison; ##P < 0.01 for medium vessel comparison; ns, non-significant (ANOVA). c P3 tumours were stained for Nestin (grey) to evaluate contra-lateral or single-cell invasions (black dashed lines around tumour edges and red dashed lines around invasive areas). Scale: 100 µm. The graphs below represent core (left panel), contra-lateral invasion (medium panel) and single-cell invasion (right panel) areas of tumours from treated and untreated mice (average of 5 tumours with 8 sections/tumour analysed). Results are represented as means ± s.d. *P < 0.05; **P < 0.01; ****P < 0.0001; ns, non-significant (ANOVA). d Kaplan–Meier survival curves of P3 xenotransplanted mice treated with control peptide, TAX2 and bevacizumab alone or in combination (n = 5 mice per group); P-values were calculated with log-rank test; *P-value < 0.05; ***P-value < 0.001