IL-4/IL-13 Stimulated Macrophages Enhance Breast Cancer Invasion Via Rho-GTPase Regulation of Synergistic VEGF/CCL-18 Signaling

Abstract

Tumor associated macrophages (TAMs) are increasingly recognized as major contributors to the metastatic progression of breast cancer and enriched levels of TAMs often correlate with poor prognosis. Despite our current advances it remains unclear which subset of M2-like macrophages have the highest capacity to enhance the metastatic program and which mechanisms regulate this process. Effective targeting of macrophages that aid cancer progression requires knowledge of the specific mechanisms underlying their pro-metastatic actions, as to avoid the anticipated toxicities from generalized targeting of macrophages. To this end, we set out to understand the relationship between the regulation of tumor secretions by Rho-GTPases, which were previously demonstrated to affect them, macrophage differentiation, and the converse influence of macrophages on cancer cell phenotype. Our data show that IL-4/IL-13 in vitro differentiated M2a macrophages significantly increase migratory and invasive potential of breast cancer cells at a greater rate than M2b or M2c macrophages. Our previous work demonstrated that the Rho-GTPases are potent regulators of macrophage-induced migratory responses; therefore, we examined M2a-mediated responses in RhoA or RhoC knockout breast cancer cell models. We find that both RhoA and RhoC regulate migration and invasion in MDA-MB-231 and SUM-149 cells following stimulation with M2a conditioned media. Secretome analysis of M2a conditioned media reveals high levels of vascular endothelial growth factor (VEGF) and chemokine (C-C motif) ligand 18 (CCL-18). Results from our functional assays reveal that M2a TAMs synergistically utilize VEGF and CCL-18 to promote migratory and invasive responses. Lastly, we show that pretreatment with ROCK inhibitors Y-276332 or GSK42986A attenuated VEGF/CCL-18 and M2a-induced migration and invasion. These results support Rho-GTPase signaling regulates downstream responses induced by TAMs, offering a novel approach for the prevention of breast cancer metastasis by anti-RhoA/C therapies.

Keywords: Rho (Rho GTPase); breast cancer; invasion; metastasis; migration.

Figure 1

M2a macrophage conditioned media is a potent inducer of breast cancer cell 2D migration. Results from scratch wound migration assays display M2a conditioned media elicits a significantly greater migratory response in MDA-231 cells (A) and SUM-149 cells (B). Similar results observed in a modified donut cell migration assay in MDA-231 (C) and SUM-149 cells (D); 48 h post M2a C.M. addition in MDA-231 cells is statistically significant to all other 48 h time points, p < 0.001 (C). The three M2 macrophage conditioned medias do not alter cellular growth rates in MDA-231 cells (E). Results from YSI metabolite analysis display no changes in key metabolic analytes among the three M2 macrophages (F). All results are compiled from ≥2 independent experiments; error bars represent ± standard deviation; Statistical significance determined by one-way ANOVA; p-value is displayed as *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

3D cell invasion assays confirm M2a TAMs as the greatest enhancer of breast cancer cell invasion. (A) Results from spheroid invasion assays in MDA-231 cells display a strong response to M2a conditioned media (C.M.). (B) Spheroid invasion results from the inflammatory TNBC cell line, SUM-149. Statistical significance in (A,B) is displayed as the 144 h time point compared against all other 144 h time points. (C,D) Results from 3D transwell migration confirm M2a conditioned media (red) enhances MDA-MB-231 (C) and SUM-149 (D) cancer cell invasion. All results are compiled from ≥2 independent experiments; Statistical significance was determined by one-way ANOVA; error bars represent ± standard deviation; p-value is displayed as *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

The Rho-GTPases regulate migratory responses to M2a macrophage conditioned media. Scratch assays results display a reduction in M2a-induced migration in both MDA-MB-231 (A) and SUM-149 cells (B). Similar results obtained from 3D spheroid invasion assays (C: MDA-231; D: SUM-149). Pretreatment with 1μM ROCK inhibitor (either Y-27632 or GSK429286A e.g. “GSK”) reduces M2a macrophage-induced 2D migration or 3D spheroid invasion in both MDA-231 (E,G) and SUM-149 cells (F,H). All results are compiled from ≥2 independent experiments; error bars represent ± standard deviation; p-value is displayed as *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 4

Synergistic activity of CCL-18 and VEGF, secreted from M2a macrophages, enhance breast cancer cell migration. (A) ELISA results from concentrated cell supernatants indicate that M2a macrophages secrete higher levels of CCL-18, IL-4, and VEGF than their M2b or M2c counterparts. ELISA data is averaged across 4 independent experiments. Colors indicate row comparisons of a single analyte protein level [low levels (blue) vs. high levels (red)] compared among the macrophage populations. Square or box size is designed to provide quantitative global analysis of overall analyte protein levels e g., 1 pg/mL (small square) to 5,000 pg/mL (large square). Results from wound closure assay in MDA-MB-231 cells (B) and in SUM-149 cells (C) show removal of CCL-18 and VEGF from M2a C.M. significantly inhibits migration. (D) Representative images of MDA-MB-231 cells wound migration assays results. Similar results obtained from spheroid invasion assays in both MDA-231 (E) and SUM-149 cells (F). Representative images from MDA-231 invasion assays shown in (G). Data is reported as an average ±std. dev. of 3 independent experiments, analyzed by one-way ANOVA, **p > 0.01, ***p > 0.001.

Figure 5

ROCK inhibition significantly diminishes synergistic VEGF/CCL-18 included breast cancer cell migration and invasion. (A) Wound closure rates are dramatically slowed in MDA-231 cells pretreated with either 1 μM Y-27632 or 1 μM GSK429286A followed by VEGF and CCL-18 administration. (B) Wound closure rates in SUM-149 cells are slowed following pretreatment with the ROCK inhibitors, Y-27632 or GSK429286A followed by rVEGF/CCL-18 administration. (C) Results from spheroid invasion assays display VEGF/CCL-18-mediated invasion is reduced in MDA-231 cells pretreated with either ROCK inhibitor. (D) ROCK inhibition in SUM-149 cells does not significantly inhibit cellular invasion. Data is reported as an average ±std. dev. of at least 3 independent experiments, analyzed by one-way ANOVA, ***p > 0.001.

Figure 6

Human M2a macrophages enhance breast cancer cell migration and invasion through VEGF/CCL-18 signaling regulated by Rho-GTPases. 40X phase contrast images of fully differentiated human macrophages (A). ELISA results from concentrated conditioned media extracts from normal human macrophage growth media (serum containing; white), unstimulated M-CSF differentiated macrophages (orange), and IL-4/IL-13 polarized human M2a macrophages (purple/blue) (B). ELISA results from analyte removal assays (C). Results of wound closure 12 h post wound (left panel) or spheroid invasion 144 h post treatment/supplementation (right panel) experiments utilizing either human M2a conditioned media (M2a) analyte depleted hM2a media (e.g., hM2a ΔVEGF) or pretreatment with ROCK inhibitors with hM2a macrophage conditioned media in MDA-231 cells (D). Representative images of either wound closure (top) or spheroid invasion (bottom) experiments in MDA-231 cells are shown below quantified data. Similar results displayed for wound closure (left panel) or spheroid invasion (right panel) with representative images below quantified data for SUM-149 cells (E). All results are compiled from ≥2 independent experiments; Statistical significance was determined by one-way ANOVA; error bars represent ± standard deviation; p-value is displayed as *p < 0.05, **p < 0.01, ***p < 0.001.