Fig. 7
CCN1 secreted from DGCs augments macrophage infiltration through integrins. a Correlation analysis of mRNA expression of CCN1-binding integrins (ITGAV, ITGB3, ITGB5, ITGAM, ITGB2, ITGA6, and ITGB1) with DGC ssGSEA scores and mRNA expression of CCN1 in TCGA GBM (HG-U133A) dataset. Size and color indicate the correlation R-value. Pearson’s correlation test. b Correlation analysis of ITG (CCN1-binding integrin) ssGSEA scores with mRNA expression of CCN1 (left panel) and DGC ssGSEA scores (right panel) in TCGA GBM (HG-U133A) dataset. Red numbers indicate the correlation R-value and P value. Pearson’s correlation test. c Heat map shows expression of DGC, GSC, and ITG ssGSEA scores (top, second, and third from top) for each RNA sample in the anatomical structure study dataset (122 RNA sample data from 10 patients) from the IVY GAP database (columns). The corresponding GBM subtype and histology for each sample are labeled (bottom and second from bottom). d ssGSEA scores of ITG (CCN1-binding integrin) signature genes in multiple regions of the IVY GAP dataset (122 RNA sample data from 10 patients). Violin plots represents the median (thick dotted line) and quartiles (dotted line). **P < 0.01, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. e Schematic diagram showing the domain structure of wildtype CCN1 and D125A and DM mutants disrupted in binding sites for αvβ3/αvβ5 and αMβ2/α6β1 integrins, respectively. f Representative image (left panel) and quantification (right panel) of transwell analysis of U937 macrophages upon stimulation with conditioned medium (CM) from MGG8 DGCs with the indicated modification. Scale bar, 100 μm. n = 4 biological replicates, mean ± SEM, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. g Representative confocal images of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs plus 1 × 104 matched DGCs from MGG8 cells with the indicated modification by a lentivirus. Scale bar, 100 µm. Iba1 (red), CD206 (green), and DAPI (blue). h Quantitation of pan-macrophages (Iba1+) and M2 macrophages (CD206+) densities in xenografts formed by 1 × 103 GSCs plus 1 × 104 matched DGCs from MGG8 cells with the indicated modification by a lentivirus. The total number of macrophages was counted in five randomly selected fields per sample. n = 5, mean ± SEM, ****P < 0.0001, ns: not significant, one-way ANOVA with Tukey’s multiple comparisons test. i Quantitation of the fraction of M2 macrophages (CD206+). The fraction was determined by M2 macrophages (CD206+) among pan-macrophages (Iba1+) in xenografts formed by 1 × 102 GSCs plus 1 × 104 matched DGCs from MGG8 cells with the indicated modification by a lentivirus. The total number of macrophages was counted in five randomly selected fields per sample. n = 5 biological replicates. Data are represented as means ± SEM. *P < 0.05, **P < 0.01, ns: not significant, one-way ANOVA with Tukey’s multiple comparisons test. j Representative H&E stainings of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs plus 1 × 104 matched DGCs from MGG8 cells with the indicated modification by a lentivirus. Scale bars, 2000 µm. k Kaplan–Meier (upper) and log-rank P value (bottom) analyses of mice bearing orthotopic xenografts of 1 × 103 GSCs plus 1 × 104 matched DGCs from MGG8 cells with the indicated modification by a lentivirus. l Kaplan–Meier analyses between patients in CCN1-high and CCN1-low groups on the basis of median mRNA expression in TCGA GBM dataset (HG-UG133A). Log-rank P value analyses. m Kaplan–Meier analyses of patients in TCGA GBM dataset (HG-UG133A) on the basis of ssGSEA scores of ITG (CCN1-binding integrin) signature genes. Log-rank P value analyses. n Negative correlation between expression of CCN1 combined with ITG (CCN1-binding integrin) signature genes and overall patient survival in TCGA GBM (HG-U133A) dataset. Log-rank P value analyses. o Negative correlation between expression of DGC-signature genes combined with ITG (CCN1-binding integrin) signature genes and overall patient survival in TCGA GBM (HG-U133A) dataset. Log-rank P value analyses