Fig. 3: Tumor cell secreted CSF-1 increases CSF-1R-dependent macrophage VEGF-A secretion. | Oncogene

Fig. 3: Tumor cell secreted CSF-1 increases CSF-1R-dependent macrophage VEGF-A secretion.

From: Targeting CSF-1 signaling between tumor cells and macrophages at TMEM doorways inhibits breast cancer dissemination

Fig. 3

A VEGF-A ELISA of conditioned media obtained from BAC1.2F5 macrophages co-cultured with MDA-MB-231 tumor cells treated with control antibody (Control Ab), CSF-1R blocking Ab or CSF-1R inhibitor (GW2580), denoted as concentration (pg/mL) of secreted protein. n = 3 individual experiments performed in duplicate, *p < 0.05, ***p < 0.001, ****p < 0.0001 analyzed by two-way ANOVA. B ELISA determination of VEGF-A (pg/mL) in medium obtained from BAC1.2F5 macrophages cultured in medium conditioned by MDA-MB-231 tumor cells (tumor cell CM) and treated with control Ab, CSF-1R blocking Ab (CSF-1R Ab) or CSF-1R inhibitor (GW2580). n = 3 individual experiments performed in duplicate, ***p < 0.001, ****p < 0.0001 analyzed by two-way ANOVA. C ELISA determination of VEGF-A (pg/mL) in medium conditioned by bone marrow macrophages (BMMs) either expressing (Csf1r+/+) or lacking (Csf1r−/−) CSF-1R co-cultured with MDA-MB-231 tumor cells for the times indicated. n = 3 individual experiments done in duplicate, ns= not significant, ****p < 0.0001 analyzed by two-way ANOVA. D ELISA determination of VEGF-A (pg/mL) in medium from Csf1r+/+ or Csf1r−/− BMMs cultured in MDA-MB-231 tumor cell conditioned medium (tumor cell CM) for the times indicated. n = 3 individual experiments performed in duplicate, ns= not significant, ****p < 0.0001 analyzed by two-way ANOVA. E BAC1.2F5 macrophages (Mφ), labelled with CellTrackerTM Green, were co-cultured with or without MDA-MB-231 tumor cells (TC), labelled with CellTrackerTM Red, and treated with either control or CSF-1R blocking antibodies for 48 h. Cells were fixed, permeabilized and stained for VEGF-A. Macrophages were identified by the CellTracker Green label and only images of macrophages are shown. The heat map scale to right shows intensity of VEGF-A staining. Scale bar = 5 µm. F Quantitation of the VEGF-A fluorescence intensity in macrophages in (E). The amount of VEGF-A in the macrophage was quantified using ImageJ, as described in the methods section. ns not significant, *p < 0.05, **p < 0.01, ****p < 0.0001, analyzed by one-way ANOVA. Experiment performed in triplicate, one representative experiment shown with at least 10 cells analyzed per group, each dot represents a cell.

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