CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis.

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. ©2011 Macmillan Publishers Limited. All rights reserved

To understand the origin of macrophages in primary tumors and their metastatic sites we measured monocyte trafficking. Mouse monocytes were identified by their expression of CD11b and CD115 (Fig. S3a) and sorted by FACS into sub-populatoins of Gr1+/Ly6C+ IMs and Gr1−/Ly6C- resident monocytes (RMs)[7,8] (Fig. S3b-d). 10^5 of each monocyte population which have similar Csf1r-GFP expression as a reporter (Fig. S3b) were adoptively transferred[9-11] into syngeneic FVB mice bearing autochthonous late stage PyMT mammary tumors with spontaneous pulmonary metastases (Fig. 1a). 18 hours after adoptive transfer the ratio of recovered GFP+ (Fig. S3e) IMs over RMs from the same donor were determined to measure their relative recruitment. This indicated similar numbers of donor cells in the blood (showing equivalent availability), but in the primary tumor RMs are preferentially recruited while in pulmonary metastases, IMs are preferentially recruited with greater than 3-fold enrichment (Fig. 1b). Consistent with this, a significant population of endogenous IMs is identified in metastasis-bearing but not normal lung (Fig. S4a). This preferential IM recruitment in lung is not observed in 7-week-old PyMT mice bearing pre-metastatic mammary tumors (Fig. S4b). In experimentally induced pulmonary foci of i.v. injected Met-1 cells[12], IMs are also preferentially recruited (Fig. S4c). GFP labeled cells are readily detectable in pulmonary metastases 5 days after transfer (data not shown) and within two days a significant portion of them have differentiated into F4/80+CD11b+Gr1−MAMs[2] that are not seen in normal lungs (Fig. S4d). To test if IMs were recruited early in the metastasis process, we transferred monocyte populations 7 hours after i.v. injection of Met-1 cells, a time point chosen before significant tumor-macrophage interaction and tumor cell extravasation[2]. Compared with control lungs, the recruitment of IMs to the tumor cell-challenged lung dramatically increased with the ratio of IMs over RMs increasing over 5-fold (Fig. 1c). However, this preferential recruitment of IMs was not observed following i.v. injection with PBS or latex beads as a control for injection or particle lodgment (data not shown and Fig. S4e). Consistent with this early recruitment of IMs, CCR2hi MAMs were preferentially recruited to lungs 36 hours after tumor cell inoculation[2]. However at this time B cells and T cells, including Foxp3+ Treg cells were not differentially recruited (Fig. S5a-c and data not shown). These data indicate that MAMs are derived from IMs that are specifically recruited early during pulmonary metastases before other immune cells.

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.

Distinct chemokine signals recruit monocytes[7] with IMs responding to CCL2[10,11]. Lung metastases of PyMT tumor homogeneously express CCL2, in contrast to heterogeneous expression in primary tumors (Fig. S6a-d). IMs but not RMs have high level of CCR2 expression (Fig. S6e). Neutralizing CCL2 using anti-mouse CCL2 Ab[13] significantly inhibited the recruitment of IMs to lungs challenged with metastatic tumor cells (Fig. 1d) and the increase in MAMs to the metastatic site (Fig. S4f). Other CCR2 expressing leukocytes (sub-population of T cell) and Treg cells in this model are unaffected by anti-CCL2 Ab treatment (Fig. S5d). Furthermore, preferential recruitment of IMs to tumor cell challenged lung was completely abrogated with adoptive transfer of monocytes sorted from CCR2 null mutant (KO) mice (Fig. S6f).

The pattern of human monocytes recruitment in vivo to tumors is unknown. To test this, human CD14+CD16− IMs and CD14loCD16+ RMs[9] were sorted from enriched CD14+ cells from peripheral blood of healthy donors (Fig. S7a). 10^5 cells of each population were adoptively transferred into pairs of nude mice supplemented with recombinant human CSF-1 that is essential for monocyte/macrophage survival (Fig. S7e). Human monocytes were quantified using FACS analysis with anti-human CD45 antibody 18 hours after adoptive transfer (Fig. S7b). In normal mice transferred with monocytes from the same donor there are comparable numbers of human IMs and RMs in the circulation and recruited to the lung but about twice the numbers of IMs compared to RMs in the spleen (Fig. 1e, open bars). In mice given an i.v. injection of human MDA-MB-231-derived metastatic 4173 breast cancer cells[14] 7 hours before monocyte transfer, the ratio of the two monocyte populations in blood and spleen was similar to normal mice, but in the lungs IMs increased in ratio of >6-fold (Fig. 1e). In established pulmonary metastases derived from orthotopically injected 4173 cells, IMs were also preferentially recruited with a 5-fold increased ratio compared to normal lungs (Fig. S7d). Mouse IMs were also preferentially recruited to 4173 cells challenged lungs (data not shown). Human IMs express CCR2 with RMs expressing minimal levels of the receptor (Fig. S7c). Neutralizing host CCL2 using anti-mouse CCL2 Ab significantly reduced the recruitment of human IMs into lungs challenged with 4173 cells without change in the circulation or spleen (Fig. 1f). Treatment with an antibody specific to human CCL2[15] also inhibited IM recruitment (Fig. S7f) indicating the importance of CCL2 from both tumor and the target organ. This indicates that human IMs respond to the same CCL2/CCR2 signaling as the mouse cells for their specific recruitment during pulmonary metastasis

To test the effect of blocking IM recruitment on metastatic potential, we performed experimental metastasis assays with Met-1 cells mice treated with anti-mouse CCL2 or control Ab shortly before the tumor cell injection. This treatment reduced the total metastasis burden due to a significantly reduced number of metastasis nodules (Fig. 2a, b). Specific antibody to mouse CCL12, another ligand of mouse CCR2, had no effect on Met-1 cell metastasis (Fig. S8). This indicates that the specific CCL2-mediated IM recruitment is critical for tumor cell pulmonary seeding.

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.

Extravasation is a critical step for tumor cell metastatic seeding in lung[2]. We used an intact lung imaging system[16] to test the role of CCL2 recruited IMs in tumor cell extravasation. Csf1r-EGFP transgenic mice were injected i.v. with CFP-expressing Met-1 cells and harvested after 24 hours. Quantification of 3D reconstructed confocal images (Fig. 2c, d, Movie S1 and S2) shows that the number of macrophages directly interacting with tumor cells was significantly reduced by CCL2 neutralizing Ab compared with control Ab (Fig. 2e). Importantly, tumor cell extravasation was delayed and less efficient following IM blockade (Fig. 2f). Tumor cell extravasation involves cross talk between tumor cells, endothelial cells, basement membrane and macrophages. In an in vitro trans-endothelia migration assay (Fig. S9a)[17] tumor cell trans-endothelial migration is enhanced ~5-fold by mouse bone marrow derived macrophages (BMMs) located on the basal-lateral side of the endothelial monolayer. This effect is blocked by anti-mouse CCL2 neutralizing antibody but not control antibody (Fig. S9b). All three cell-types express CCL2, while only the macrophages express CCR2 (Fig. S9c) indicating that only macrophages respond to the CCL2 chemokine signaling. In confirmation of this macrophages from CCR2 KO mice are not capable of promoting tumor cell trans-endothelia migration (Fig. S9d). Importantly, FACS sorted IMs but not RMs significantly promoted tumor cell trans-endothelia migration that was also inhibited by anti-mouse CCL2 neutralizing antibody (Fig. 2g, h).

Total CCL2 blockade (both mouse and human) inhibited spontaneous lung metastasis of orthotopically injected MDA-MB-231 cells (Fig. 3a). Both tumor cell and host secreted ligands contribute to metastatic efficiency, since both anti-human and -mouse Ab alone significantly inhibit 4173 experimental metastasis (Fig. 3b) without affecting tumor cell proliferation in vitro (data not shown). This conclusion was also confirmed by knocking down CCL2 using siRNAs in 4173 cells which significantly reduced lung colonization in experimental metastasis assays (Fig. S10e, f). Consistent with this, a similar CCL2 knockdown in Met-1 cells did not affect tumor cell proliferation in vitro, but significantly inhibited their metastastic efficiency (Fig. S10a-c). In vitro 4173 trans-endothelia migration is also promoted by human IMs and inhibited by neutralizing either human or mouse CCL2 with specific Abs (Fig. 3c-e). These data indicate that tumor cell and target organ secreted CCL2 work together to promote tumor cell extravasation and metastatic seeding via action on IM recruitment. Consistent with the role of microenvironmental synthesized CCL2 in the lung, bone metastasis of MDA-MB-231 cells also recruit IMs and inhibition of CCL2 inhibits metastatic progression. In contrast liver metastasis of Met-1 cells did not recruit IMs and CCl2 inhibition did not reduce metastasis (data not shown). Furthermore, CCL2 blockade 2 days after i.v. injection of MDA-MB-231 cells reduces lung tumor burden and prolongs survival of mice indicating the importance of continuous recruitment of IM and their differentiation into MAMs for persistent metastatic growth (Fig. 3f, g).

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).

To determine mechanism of the effects of IMs on tumor cell extravasation we analyzed the transcriptomes of RMs and IMs[18]. Among the differentially regulated genes VEGF is highly expressed by IMs, a fact we verified (Fig. S11a). To conditionally ablate VEGF in myeloid cells to test its role in the metastatic process, we generated a transgenic mouse expressing tamoxifen inducible Mer-iCre fusion protein driven by the Csf1r promoter crossed with VEGF mice[19]. Inducible ablation of Vegf was achieved in cultured BMMs treated with 4-hydroxytamoxifen (Fig. 4a) and these Vegf KO BMMs compared to control BMM are unable to promote tumor cell trans-endothelial migration and do not enhance permeability of the endothelial monolayer (Fig. 4b, c), a process important for metastasis[20]. In vivo injection of tamoxifen specifically ablates Vegf in monocytes without ablation in other circulating immune cells (Fig. 4d). This monocyte–specific depletion of VEGF significantly inhibited Met-1 cell experimental metastasis potential and seeding efficiency (Fig. 4e and S11b). Adoptive transfer experiments indicated that Vegf KO IMs infiltrate Met-1 lung metastasis at a comparable level as Vegf IMs, showing that this molecule is not required for IM recruitment (Fig. S11c). Importantly, co-injection of Met-1 cells and WT IMs into inducible macrophage VEGF knockout mice restored the tumor cell metastatic potential (Fig. 4f).

Figure 4

Monocyte-specific ablation of VEGF blocks pulmonary seeding.

a, PCR of VEGF exon3 of BMMs of VEGF 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 VEGF and VEGF mice. e, Met-1 Mets burden in VEGF mice with or without Cre with same tamoxifen treatment. n=6, p=0.0004. f, Met-1 Mets burden in VEGF mice with tamoxifen treatment with or without IM co-injection. n=6, p<0.0001. All data are mean+s.e.m.

Thus, these experiments have indicated that CCL2 synthesized by metastatic tumor cells and the target site tissue stroma is critical for recruitment of a sub-population of CCR2 expressing monocytes that enhance the subsequent extravasation of the tumor cells. Mechanistically this is at least in part through targeted delivery of molecules such as VEGF that promote extravasation. IMs are continually recruited by a CCL2 mechanism and differentiate into macrophages that promote the subsequent growth of metastatic cells. These data together with the clinical associations of CCL2 over-expression in human cancer noted above strongly argue for therapeutic approaches targeted against monocyte recruitment and function.

Methods Summary

Monocytes trafficking into primary tumors and their metastases were studied by adoptive transfer of mouse (Ly6C/Gr1+ or Ly6C/Gr1−) or human (CD14+CD16+ and CD16−) monocytes using MMTV-PyMT autochthonous, human and mouse experimental metastasis and human orthotopic tumor models. Monocytes and macrophages were recovered by enzymatic disaggregation of the tumors followed by FACS analysis. To test mechanisms behind monocyte recruitment and the effect of inhibition of this trafficking on metastasis anti-mouse or human neutralizing CCL2 antibodies or Ccr2 null mutant mice were used. In order to ablate VEGF expression in monocytes a myeloid specific (Csf1r promoter) tamoxifen inducible Cre expressing strain was crossed with VEGF mice and gene ablation induced by tamoxifen. Effects of monocyte depletion on tumor cell extravasation using Met-1, a FVB PyMT tumor derived metastatic cell line, was determined using an ex vivo intact lung imaging system and an in vitro extravasation assay.