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. 2011 Jun 8;475(7355):222-5.
doi: 10.1038/nature10138.

CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis

Bin-Zhi Qian  1 , Jiufeng Li, Hui Zhang, Takanori Kitamura, Jinghang Zhang, Liam R Campion, Elizabeth A Kaiser, Linda A Snyder, Jeffrey W Pollard
Affiliations

CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis

Bin-Zhi Qian et al. Nature. .

Abstract

Macrophages, which are abundant in the tumour microenvironment, enhance malignancy. At metastatic sites, a distinct population of metastasis-associated macrophages promotes the extravasation, seeding and persistent growth of tumour cells. Here we define the origin of these macrophages by showing that Gr1-positive inflammatory monocytes are preferentially recruited to pulmonary metastases but not to primary mammary tumours in mice. This process also occurs for human inflammatory monocytes in pulmonary metastases of human breast cancer cells. The recruitment of these inflammatory monocytes, which express CCR2 (the receptor for chemokine CCL2), as well as the subsequent recruitment of metastasis-associated macrophages and their interaction with metastasizing tumour cells, is dependent on CCL2 synthesized by both the tumour and the stroma. Inhibition of CCL2-CCR2 signalling blocks the recruitment of inflammatory monocytes, inhibits metastasis in vivo and prolongs the survival of tumour-bearing mice. Depletion of tumour-cell-derived CCL2 also inhibits metastatic seeding. Inflammatory monocytes promote the extravasation of tumour cells in a process that requires monocyte-derived vascular endothelial growth factor. CCL2 expression and macrophage infiltration are correlated with poor prognosis and metastatic disease in human breast cancer. Our data provide the mechanistic link between these two clinical associations and indicate new therapeutic targets for treating metastatic breast cancer.

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Figures

Figure 1
Figure 1
Pulmonary metastases preferentially recruit inflammatory monocytes through CCL2. a, Schematic for monocyte adoptive transfer into PyMT tumor mice with pulmonary metastases. b, Ratio of IM versus RM in different tissues of recipient mice bearing PyMT tumors and metastases. n=6, p<0.0001. c, Ratio of IM versus RM in control lung and lungs with i.v. injected Met-1 cells 7 hours before. n=4, p=0.0039. d, Relative donor IMs recruited in lungs challenged with Met-1 cells for 7 hours with control or anti-mouse CCL2 antibody treatment. n=3, p=0.045. e, Ratio of adoptively transferred CD14+CD16− and CD14loCD16+ human monocytes recruited into normal mice (open bars) and mice challenged with LM2 cells (solid bars) for 7 hours, n=5, p=0.0163. b-e, All bar = mean + s.e.m f, Number of adoptively transferred human CD14+CD16− monocytes that migrated into different tissues of mice challenged with LM2 cells via i.v. injection with control or anti-mouse CCL2 Ab treatment n=5, p=0.016, each line connects data from same donor.
Figure 2
Figure 2
CCL2-recruited monocytes promote metastatic seeding. a, Representative H&E stained sections showing Met-1 metastasis with Ab treatment. Bar=1 mm. b, Met-1 metastasis (Mets) burden with different Ab treatment. n=6, p=0.006. c and d, Representative snapshots of 3D reconstructed confocal images of tumor cells (blue) and macrophages (green) in lung vasculature (red) 24 hours after tumor cell tail vein injection in mice treated with control (c) or anti-mouse CCL2 Ab (d). Bar = 20 um. e, Number of macrophage-tumor cell interactions in mice with Ab treatment. f, Tumor cell extravasation in mice with different Ab treatment. (e, p= 0.0066 and f, p=0.00163 are based upon 3D images of 15-20 tumor clusters per mouse, n=3 mice/group.) g, Number of transmigrated Met-1 cells in presence of RMs or IMs. n=5, p<0.0001. h, Number of transmigrated Met-1 cells in presence of IMs with different Ab. n=3, p=0.0204. All bars shown are mean + s.e.m.
Figure 3
Figure 3
Tumor cell and host CCL2 promote metastatic seeding. a, Number of spontaneous pulmonary metastases from orthotopic MDA-MB-231 tumors with total CCL2 blockade or control treatment. Bar equals mean, n= 8/group, p<0.001. b, Mets burden of i.v. injected 4173 cells with different Ab treatment. n=6, p=2.14E-05. Bar equals mean + s.e.m. c, Representative fluorescent micrograph of transmigrated human 4173 cells pre-stained with cell tracker dye in the presence of different monocyte populations. Bar equals 20 um. d, Number of transmigrated 4173 cells in presence of IMs or RMs. Bar equals mean + s.e.m. of 3 experiments with duplicates. e, Relative number of transmigrated 4173 cells in presence of IMs with control, anti-human or anti-mouse CCL2 Ab normalized to average of control Ab which is arbitrarily set to 100. Bar represents mean + s.e.m. of 5 experiments with duplicates. One way anova with Bonferroni’s multiple comparison test, **p<0.01, ***p<0.001. f and g, CCL2 blockade starting from 2 days after MDA-MB-231 cells i.v. injection significantly reduces Mets burden as measured by realtime PCR of human Alu repeats normalized to mouse β actin on day 22 (f, n=10, ***p<0.001) and prolongs survival (g, n=10, p<0.001).
Figure 4
Figure 4
Monocyte-specific ablation of VEGF blocks pulmonary seeding. a, PCR of VEGF exon3 of BMMs of VEGFflox/flox mice with or without Csf1r-Mer-iCre-Mer transgene treated with 4-hydroxytamoxifen. Wild type (WT) and knockout (KO) bands are indicated. b and c, Number of trans-endothelial migrated Met-1 cells without BMM (b) and albumin permeability of endothelial monolayer (c), with WT or VEGF knockout BMMs. n=3 with duplicates. **p<0.01 with ANOVA. d, Relative VEGF exon 3 copy number in leukocytes from the peripheral blood of tamoxifen treated VEGFflox/flox;Csf1r-Mer-iCre-Mer and VEGFflox/flox mice. e, Met-1 Mets burden in VEGFflox/flox mice with or without Cre with same tamoxifen treatment. n=6, p=0.0004. f, Met-1 Mets burden in VEGFflox/flox;Csf1r-Mer-iCre-Mer mice with tamoxifen treatment with or without IM co-injection. n=6, p<0.0001. All data are mean+s.e.m.
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