Fig. 4: Inhibition of CSF-1R signaling reduces TAVO and trans-endothelial migration.

A IF staining of tumor tissue from mice treated with control or CSF-1R blocking antibodies (CSF-1R Ab) were stained for CD31 (green), TMR-dextran (red), and ZO-1 (magenta). The right panel shows increased magnification of yellow box from middle panel to demonstrate overlap between CD31 and ZO-1 stains (merge/overlap indicated with white signal and indicated with white arrows). Scale bar left and middle panels= 50 µm; right panel= 25 µm, B Quantification of extravascular 155 kDa dextran-TMR. *p < 0.05. C Vascular ZO-1 *p < 0.05, and D circulating tumor cells for mice treated with control Ab or CSF-1R blocking Ab in A. *p < 0.05, analyzed by Student’s t-test. (B–D, n = 9 mice treated with control antibody, 7 mice treated with CSF-1R antibody). E Quantitation of subluminal to luminal trans-endothelial migration (iTEM activity, which is a measure of number of intravasating tumor cells in the intravasation direction) of MDA-MB-231 cells plated on the confluent and sealed endothelium either alone or with BAC1.2F5 macrophages. Tumor cells were treated with control antibody (Control Ab and DMSO), CSF-1R blocking Ab (CSF-1R Ab) or CSF-1R inhibitor (GW2580). Tumor cells were labelled with CellTracker^TM green, which allowed us to identify and quantify the cells crossing the endothelial monolayer. The endothelium is stained with ZO-1 antibodies to ensure endothelial confluence and tight junctions at locations of TC crossing. The number of MDA-MB-231 cells that crossed the endothelium were imaged using a confocal microscope and quantified using ImageJ software. n = 3 experiments, each point represents the average fold change for that treatment group for each experiment, *p < 0.05 analyzed by one-way ANOVA. F Quantitation of iTEM activity of MDA-MB-231 cells plated on the endothelium alone or with BMMs expressing or lacking CSF-1R expression. The assay was imaged and analyzed as in (E). n = 3 experiments, each point represents the average fold change for that treatment group for each experiment, ***p < 0.001 analyzed by one-way ANOVA. G Quantitation of iTEM activity of MDA-MB-231 cells transfected with either control siRNA or two different siRNAs targeting CSF-1 (#1, #2). Transfected cells were plated on the endothelium either alone or with BAC1.2F5 macrophages. The assay was imaged and analyzed as in (E). n = 3 experiments, each point represents the average fold change for that treatment group for each experiment, ns not significant, **p < 0.01, ***p < 0.001 analyzed by one-way ANOVA.