Fig. 4

TAb2 tumors promote drastic expansion of F4/80⁺ TAMs. An in vitro co-culture assay was set up using BM cells and TAb2 or TCh3 tumor cells, for evaluating the effects of tumors on myeloid cells. Total numbers of TAMs (CD11b⁺Ly6C⁻Ly6G⁻F4/80⁺) were counted by flow cytometry at different time points (day 2, 3 and 4). A TAb2 tumors drive the expansion of TAMs. BM cells were either cultured alone (BM) or co-cultured with TAb2 (TAb2-BM) or TCh3 (TCh3-BM) tumor cells, respectively. Left panel: Growth curves of F4/80⁺ TAMs. Right panel: Representative flow plots of CD11b⁺F4/80⁺ population. B TAb2 tumors drive the expansion of TAMs independent of cell-cell contact. BM cells were either cultured alone or cultured with TAb2 or TCh3 tumor cells, respectively, in transwell plates. Left panel: Growth curve of F4/80⁺ TAMs. Right panel: Representative flow plots of CD11b⁺F4/80⁺ population. P values are shown for multiple comparisons to TAb2-BM by two-way ANOVA in (A) and (B). C-D TAb2 tumor-mediated TAM expansion requires CSF1 and VEGF. C Representative flow plots of CD11b vs F4/80 (top) and CD86 vs. CD206 (bottom) in the co-culture of TAb2 tumor cells and BM cells in the absence or presence of CSF1R mAb or VEGFR inhibitor. D Growth curves of F4/80⁺ TAMs (left) and CD206⁺CD86⁻ M2 TAMs (right). BM cells were co-cultured with TAb2 tumor cells in the absence (black) or presence of CSF1R mAb (red) or VEGFR inhibitor (blue). Results are representative of more than three independent experiments done in triplicates. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test