Figure 1.
FGFR activation in mammary epithelial cells and breast cancer cells promotes macrophage recruitment.
A) Conditioned media from HC-11/R1 cells containing soluble factors induced following treatment of cells with either B/B to activate iFGFR1 or ethanol as a solvent control for 24 were used to assess migration of RAW 264.7 cells. A significant induction in macrophage recruitment was observed in response to media containing iFGFR1-induced soluble factors. ***p<0.001. B) Conditioned media from HS578T cells treated with either PD173074, which inhibits FGFR activity, or DMSO as a solvent control were used to assess recruitment of THP-1 cells. A significant decrease in macrophage recruitment was observed in response to media from cells treated with an FGFR1 inhibitor. *p<0.05. Error bars represent SEM. C) Expression of CX3CR1 in macrophages isolated from mammary glands of mice treated with either B/B or solvent control for either 48 hours or 4 weeks. Expression levels were assessed using microarray analysis and expression levels are normalized to levels of GAPDH.
Table 1.
Proteomic analysis of soluble proteins after iFGFR1 activation.
Figure 2.
iFGFR1 activation induces secretion of CX3CL1 via the NFκB pathway.
Treatment of HC-11/R1 cells with B/B induces production and secretion of CX3CL1. A) CX3CL1 gene expression was induced after 4 hours of B/B treatment as determined by qRT-PCR analysis. CX3CL1 levels were normalized to cyclophilin. B) ELISA analysis demonstrated that CX3CL1 is secreted by HC-11/R1 cells after 24 hours of B/B treatment. C) Treatment of HC-11/R1 cells with B/B for 6 hours increased the transcriptional activity of NFκB as measured by luciferase assay. D) Treatment of HC-11/R1 cells with the NFκB inhibitor peptide SN50 in conjunction with B/B significantly reduced CX3CL1 transcript levels as measured by qRT-PCR in comparison to HC-11/R1 cells treated with inactive SN50 in the presence of B/B demonstrating that iFGFR1-induced CX3CL1 is mediated by NFκB signaling. *p<0.05, **p<0.001, ***p<0.0001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.
Figure 3.
iFGFR1-induced CX3CL1 secretion by epithelial cells mediates macrophage migration.
siRNA techniques were used to directly target CX3CL1 in order to determine the role of CX3CL1 in iFGFR1-mediated macrophage recruitment. A) CX3CL1 gene expression levels were significantly elevated in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, CX3CL1 gene expression levels were significantly reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. B) ELISA was performed to verify reduction in CX3CL1 protein secretion in HC-11/R1 cells transfected with CX3CL1siRNA. Results indicated that soluble CX3CL1 protein concentrations were significantly induced in HC-11/R1 cells transfected with non-targeting (NT) siRNA. In comparison, soluble protein concentrations were reduced in CX3CL1siRNA HC-11/R1 cells treated with B/B after 24 hours. C) Conditioned medium from non-targeting HC-11/R1 cells treated with B/B significantly increased migration of RAW 264.7 cells relative to conditioned medium from cells treated with solvent alone. Exposure to conditioned medium from CX3CL1 siRNA HC-11/R1 cells significantly decreased RAW 264.7 cell migration. Furthermore, addition of 50ng/mL rmCX3CL1 to CX3CL1siRNA HC-11/R1 cells at the time of B/B treatment rescued the loss of RAW 264.7 cell migration in cells exposed to conditioned medium from CX3CL1siRNA HC-11/R1 cells. These results indicate that CX3CL1 is a key mediator of macrophage recruitment. *p<0.05, **p<0.001, ***p<0.0001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.
Figure 4.
Human breast cancer cells secrete CX3CL1 in an FGF-dependent manner to promote macrophage cell migration.
FGF-dependent production and secretion of CX3CL1 by HS578T cells mediates recruitment of human macrophages. A) HS578T cells treated with 50ng/mL bFGF demonstrated significant induction of CX3CL1 gene expression after 4 hours relative to PBS solvent control-treated cells. B) Soluble CX3CL1 protein concentrations were significantly upregulated in HS578T cells treated with bFGF for 8 hours in comparison to control-treated cells. C) HS578T cells, which produce high levels of endogenous bFGF, were treated with the PD173074 for 8 hours to inhibit autocrine FGFR activation. A significant reduction in CX3CL1 gene expression was observed relative to DMSO-treated cells as measured by qRT-PCR. D) THP1 cells that had been differentiated into macrophages using PMA demonstrated increased migratory potential when exposed to conditioned medium from CX3CL1-expressing HS578T cells in comparison to serum free medium. Furthermore, this enhanced cell migration was significantly reduced when THP1 cells were treated with a CX3CL1 blocking antibody (aCX3CL1) relative to IgG-treated THP1 cells. *p<0.05, **p<0.001. Results in each figure panel are representative of a minimum of three different experiments. Error bars represent SEM.
Figure 5.
iFGFR1 activation in vivo promotes recruitment of CX3CR1-positive macrophages.
MMTV-iFGFR1 transgenic mice were treated with B/B in order to analyze the population of macrophages that are recruited to the mammary epithelium during early stages of iFGFR1-induced mammary tumorigenesis. A) MMTV-iFGFR1 mice treated with B/B demonstrated an increase in macrophage recruitment after 10 days as indicated by an increased in the number of F4/80 positive cells. MMTV-iFGFR1 mice treated with anti-CX3CR1 in conjunction with B/B demonstrated a reduction in macrophage recruitment at 10 days indicating that iFGFR1 activation is responsible for recruiting a subset of macrophages that express CX3CR1. ***p<0.0001. Error bars represent SEM. B) Representative image of macrophages associated with budding epithelial structures in mammary glands from mice treated with control IgG antibody. C) Representative image of macrophages associated with budding epithelial structures in mammary glands from mice treated with anti-CX3CR1. Red = F4/80 staining, blue = DAPI. Scale bars represent 50 µM. Results in each figure panel are representative of a minimum of three different mice for each treatment group and genotype.
Figure 6.
Decreased macrophage recruitment correlates with decreased angiogenesis.
A) Quantification of BrdU incorporation demonstrates that decreased macrophage infiltration does not significantly correlate with a change in epithelial cell proliferation (N.S. = not significant). B) Quantification of VWF staining reveals decreased blood vessels associated with epithelial structures in mammary glands from mice treated with B/B and anti-CX3CR1 blocking antibody compared with mice treated with B/B and IgG control antibody. *p<0.05. C, D) Representative images of VWF stained mammary glands from mice treated with B/B and either control IgG antibody (C) or anti-CX3CR1 (D). Green = VWF staining, blue = DAPI. Arrows indicate VWF-positive structures. Scale bars represent 50 µM. Results in each figure panel are representative of a minimum of three different mice for each treatment group and genotype.