Fig. 4

3D185 reversed M2-like macrophage-induced CD8⁺ T cell suppression as well as inhibited CSF-1-differentiated macrophages induced FGFR-aberrant cancer cell migration. a-c 3D185 reversed murine CSF1-differentiated macrophage-induced CD8⁺ T cell suppression. Murine bone marrow cells were induced to mature M2-like macrophages with CSF-1 for 7 days and then treated with 3D185 or PLX3397 for 48 h. Then, murine BMDMs were cocultured with CFSE-labeled spleen cells and stimulated with anti-CD3/CD28 beads and IL-2 for 72 h. CD8⁺ T cell proliferation was tested by flow cytometry (a). granzyme B⁺ CD8⁺ T cells (b) and IFN-γ⁺ CD8⁺ T cells (c) were tested by flow cytometry. Representative data from two independent experiments are shown. Data are shown as the mean ± SD. D-F, 3D185 reversed human CSF1-differentiated macrophage-induced CD8⁺ T cell suppression. Human monocytes cells were induced to M2-like macrophages with CSF-1 for 7 days and then treated with 3D185 or PLX3397 for 48 h. Then, such human macrophages were cocultured with CFSE-labeled human CD8⁺T cells and stimulated with anti-CD3/CD28 beads and IL-2 for 72 h. CD8⁺ T cell proliferation was tested by flow cytometry (d). granzyme B+ CD8⁺ T cells (e) and TNF-α⁺ CD8⁺ T cells (f) were tested by flow cytometry. Representative data from two independent experiments are shown. Data are shown as the mean ± SD. g, h CSF-1-differentiated macrophages induced FGFR3-amplified RT112 cancer cell migration. Representative images are shown (scale bars, 1 mm) in (g). The relative migration was plotted (h). The data shown are the mean ± SD from two independent experiments, assuming 100% migration or invasion of cells stimulated with macrophage. Significant differences were determined using one-way ANOVA with Tukey’s multiple-comparison test (*p < 0.05; **p < 0.01; ***p < 0.001)