Fig. 6
DGCs augment macrophage infiltration through secretion of CCN1 in GBM. a Bar graph showing the normalized enrichment score (NES) of GSEA analysis of hallmark gene sets upregulated in CCN1-high GBMs (n = 264) compared with CCN1-low GBMs (n = 264). Thirty gene sets were significantly enriched at a false discovery rate (FDR) of < 0.25 and nominal P value of < 0.05 in CCN1-high GBMs. TCGA GBM dataset (HG-UG133A, n = 528). Red bars indicate signatures relate to immune responses. b Immune score of CCN1-high (n = 264) and CCN1-low (n = 264) GBMs in TCGA GBM dataset (HG-UG133A, n = 528). Violin plots represent the median (thick dotted line) and quartiles (dotted line). ****P < 0.0001, Student’s t-test. c Stromal score of CCN1-high and CCN1-low GBMs in TCGA GBM dataset (HG-UG133A). Violin plots represent the median (thick dotted line) and quartiles (dotted line). ****P < 0.0001, Student’s t-test. d GSEA analysis of various types of immune cell signatures upregulated in CCN1-high GBMs (n = 264) compared with CCN1-low GBMs (n = 264) in TCGA GBM dataset (HG-UG133A, n = 528). Red bars indicate FDR < 0.25. e GSEA analysis of macrophage-related signatures upregulated in CCN1-high GBMs (n = 264) compared with CCN1-low GBMs (n = 264) in TCGA GBM dataset (HG-UG133A, n = 528). FDR < 0.25 was defined as significantly enriched. f Representative image (upper panel) and quantification (lower panel) of transwell analysis of U937 macrophages upon stimulation with or without recombinant CCN1 (10 ng/ml). Scale bar, 100 μm. n = 4 biological replicates, mean ± SEM, ****P < 0.0001, Student’s t-test. CONT: control. g Representative image (upper panel) and quantification (lower panel) of transwell analysis of U937 macrophages upon stimulation with conditioned medium (CM) from DGCs (MGG4 and MGG8 DGCs) transduced with shCONT or shCCN1. Scale bar, 100 μm. n = 4 biological replicates, mean ± SEM, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. h Representative confocal images of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs plus 1 × 104 matched DGCs transduced with shCONT or 1 × 103 GSCs plus 1 × 104 DGCs transduced with shCCN1 derived from MGG8. Scale bar, 100 µm. Iba1 (red), CD206 (green), and DAPI (blue). i Quantitation of pan-macrophages (Iba1+) and M2 macrophages (CD206+) densities in xenografts formed by 1 × 103 GSCs plus 1 × 104 matched DGCs transduced with shCONT or 1 × 103 GSCs plus 1 × 104 DGCs transduced with shCCN1 derived from MGG8 cells. The total number of macrophages was counted in five randomly selected fields per sample. n = 5 biological replicates, mean ± SEM, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test. j 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 × 103 GSCs plus 1 × 104 matched DGCs transduced with shCONT or 1 × 103 GSCs plus 1 × 104 DGCs transduced with shCCN1. 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 < 0.01, ***P < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. k Representative H&E stainings of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs plus 1 × 104 matched DGCs transduced with shCONT or 1 × 103 GSCs plus 1 × 104 DGCs transduced with shCCN1 derived from MGG8. Scale bars, 2000 µm. l Kaplan–Meier (left) and log-rank P value (right) analyses of mice bearing orthotopic xenografts of 1 × 103 GSCs plus 1 × 104 matched DGCs transduced with shCONT or 1 × 103 GSCs plus 1 × 104 DGCs transduced with shCCN1 derived from MGG8 cells