Figure 6 from Inhibition of Extracellular Matrix Protein Fibulin-3 Reduces Immunosuppressive Signaling and Increases Macrophage Activation in Glioblastoma

Anti–fibulin-3 induces macrophage attack of GBM cells. A, Quantification of cytotoxicity in fLuc-expressing GBM cells co-cultured with U937 macrophages (effector:target = 5:1) in the presence of anti–fibulin-3 mAb428.2, control IgG, or vehicle. Control conditions included GBM cells incubated alone in the presence or absence of the same antibodies. The results were analyzed by one-way ANOVA for each cell line. B, Cytotoxicity of GBM cells cocultured with U937 macrophages in the presence of anti–fibulin-3 antibodies mAb428.2 or mAb3-5. C, Effect of anti–fibulin-3 antibodies on the expression of immunosuppressive signals by GBM cells. D, mAb428.2-induced cytotoxicity of GBM cells (GBM09) cocultured with U937 macrophages that were not pretreated (vehicle) or pretreated with excess of IgG-Fc fragment to block immunoglobulin receptors (FcR block). The results in (B–D) were analyzed by two-way ANOVA. E, Representative images of DiI-stained macrophages (red) cocultured with eGFP-expressing GBM9 cells (green) in the presence of mAb428.2 or control IgG. Tumor cell fragments were rarely observed inside macrophages in the control condition, even in direct contact with tumor cells. Confocal sectioning confirmed that eGFP cell fragments were inside the macrophages in presence of mAb428.2; scale bar, 25 μm. F, Flow cytometric analysis of phagocytic activity of U937 macrophages cocultured with GBM9:eGFP cells in the presence of control IgG, mAb428.2 (50 μg/mL), or an antibody against the cell surface phagocytosis inhibitor CD47 (10 μg/mL, positive control). The results were analyzed by one-way ANOVA. MFI, mean fluorescence intensity; ns, not significant.