Fig. 4
DGCs promote macrophage infiltration and tumor progression in cooperation with GSCs. a Flow cytometric analysis of CD133 in MGG8 cells. Sorted CD133 + cells were cultured as GSCs and CD133-cells were cultured as DGCs. b Representative images of paired GSCs and DGCs derived from two primary human GBM specimens (MGG4 and MGG8). Scale bar, 300 µm. c Representative image (left panel) and quantification (right panel) of transwell analysis of U937 macrophages upon stimulation with control medium or conditioned medium (CM) from DGCs (MGG4 and MGG8). Scale bar, 100 μm. n = 4 biological replicates, mean ± SEM, ***P < 0.001, Student’s t-test. d Kaplan–Meier (upper) and log-rank P value (bottom) analyses of mice bearing orthotopic xenografts of 1 × 103 glioma stem-like cells (GSCs) alone, 1 × 105 differentiated glioblastoma cells (DGCs) alone, or cotransplanted 1 × 104 DGCs with 1 × 103 GSCs derived from MGG8 cells. e Representative H&E stainings of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs alone, or 1 × 103 GSCs plus 1 × 104 matched DGCs derived from MGG8. Scale bars indicate 2000, 100 and 50 µm in gross and detail views, respectively. Middle and right panels are high magnifications of the areas marked by rectangles in left panels. f Representative confocal images of tumor-bearing brains harvested at 30 days after implantation of 1 × 103 GSCs alone, or 1 × 103 GSCs plus 1 × 104 matched DGCs derived from MGG8. Scale bar, 100 µm. Iba1 (red), CD206 (green), and DAPI (blue). g Quantitation of pan-macrophage (Iba1+) and M2 macrophage (CD206+) densities in xenografts formed by 1 × 103 GSCs alone or 1 × 103 GSCs plus 1 × 104 matched DGCs 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 < 0.001, ****P < 0.0001, Student’s t-test. h Quantitation of the fraction of M2 macrophages (CD206+). The fraction was determined by M2 macrophages (CD206+) among pan-macrophages (Iba1+) in 1 × 103 GSCs alone or 1 × 103 GSCs plus 1 × 103 matched DGCs xenografts. 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, Student’s t-test