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CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages.

Kitamura T, Qian BZ, Soong D, Cassetta L, Noy R, Sugano G, Kato Y, Li J, Pollard JW - J. Exp. Med. (2015)

Bottom Line: Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice.Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM-cancer cell interactions.These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets.

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Affiliation: MRC Centre for Reproductive Health, Queen's Medical Research Institute, the University of Edinburgh, Edinburgh EH16 4TJ, Scotland, UK.

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CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2−/− mice were assessed by RT-PCR (n = 6 per genotype, 6 independent experiments) and ELISA (n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P < 0.01 versus WT. (B) Ccl3 mRNA (left) and CCL3 protein (right) were assessed by RT-PCR (n = 3, 2 independent experiments) and ELISA (n = 3 per genotype, 2 independent experiments) in macrophages cultured with PBS or recombinant CCL2 (100 ng/ml). The BMDMs were isolated from WT or Ccr2−/− mice. Data are means ± SEM. *, P < 0.01. (C) Expression of Ccl3 mRNA was assessed by RT-PCR (n = 4, four independent experiments) in monocytes and macrophages obtained from E0771-LG tumor-injected mice treated with neutralizing anti-CCL2 or control IgG antibodies. IM, inflammatory monocytes (CD115+CD11b+Ly6C+); RM, resident monocytes (CD115+CD11b+Ly6C–); MAMs (F4/80+CD11b+CD11c–Ly6C–); Rmac, resident macrophages (F4/80+CD11b–CD11c+Ly6C–). Data are means ± SEM. *, P < 0.01 versus IgG-IM; †, P < 0.05 versus IgG-MAM. Gating strategies are shown Fig. S1. (D) Expression of Ccl3 mRNA was assessed by RT-PCR in Rmac, MAM (n = 7, 7 independent experiments) or neutrophil (Neu), CD4+ T cell, CD8+ T cell, B cell, and NK cell (n = 3, 3 independent experiments). The leukocytes were isolated from tumor-bearing mouse lung. Data are means ± SEM. *, P < 0.01 versus Rmac. (E) Relative CCL3 mRNA expression was assessed by RT-PCR (n = 4, 4 independent experiments) in human monocytes (Mo) and those differentiated into macrophages by CSF-1 (Mac). Data are means ± SEM. *, P < 0.05. (F) Relative CCL3 mRNA level was assessed by RT-PCR (n = 4, 4 independent experiments) in human macrophages cultured with recombinant human CCL2 (100 ng/ml). Data are means ± SEM. *, P < 0.05. (G) Expression of CCL3 mRNA was assessed by RT-PCR in human MDMs (hMDM) cultured with conditioned medium (C.M.) from human cancer cell lines as indicated. As a control (–), the cells were cultured in nonconditioned medium (αMEM including 10% FBS and 103 U/ml CSF-1). n = 5, 5 independent experiments. Data are means ± SEM. *, P < 0.01. (H) Expression of CCL3 mRNA was assessed by RT-PCR in human macrophages cultured in MDA231 conditioned medium with neutralizing anti-CCL2 or control IgG antibodies. n = 3. Data are means ± SEM. *, P < 0.05.
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fig2: CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2−/− mice were assessed by RT-PCR (n = 6 per genotype, 6 independent experiments) and ELISA (n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P < 0.01 versus WT. (B) Ccl3 mRNA (left) and CCL3 protein (right) were assessed by RT-PCR (n = 3, 2 independent experiments) and ELISA (n = 3 per genotype, 2 independent experiments) in macrophages cultured with PBS or recombinant CCL2 (100 ng/ml). The BMDMs were isolated from WT or Ccr2−/− mice. Data are means ± SEM. *, P < 0.01. (C) Expression of Ccl3 mRNA was assessed by RT-PCR (n = 4, four independent experiments) in monocytes and macrophages obtained from E0771-LG tumor-injected mice treated with neutralizing anti-CCL2 or control IgG antibodies. IM, inflammatory monocytes (CD115+CD11b+Ly6C+); RM, resident monocytes (CD115+CD11b+Ly6C–); MAMs (F4/80+CD11b+CD11c–Ly6C–); Rmac, resident macrophages (F4/80+CD11b–CD11c+Ly6C–). Data are means ± SEM. *, P < 0.01 versus IgG-IM; †, P < 0.05 versus IgG-MAM. Gating strategies are shown Fig. S1. (D) Expression of Ccl3 mRNA was assessed by RT-PCR in Rmac, MAM (n = 7, 7 independent experiments) or neutrophil (Neu), CD4+ T cell, CD8+ T cell, B cell, and NK cell (n = 3, 3 independent experiments). The leukocytes were isolated from tumor-bearing mouse lung. Data are means ± SEM. *, P < 0.01 versus Rmac. (E) Relative CCL3 mRNA expression was assessed by RT-PCR (n = 4, 4 independent experiments) in human monocytes (Mo) and those differentiated into macrophages by CSF-1 (Mac). Data are means ± SEM. *, P < 0.05. (F) Relative CCL3 mRNA level was assessed by RT-PCR (n = 4, 4 independent experiments) in human macrophages cultured with recombinant human CCL2 (100 ng/ml). Data are means ± SEM. *, P < 0.05. (G) Expression of CCL3 mRNA was assessed by RT-PCR in human MDMs (hMDM) cultured with conditioned medium (C.M.) from human cancer cell lines as indicated. As a control (–), the cells were cultured in nonconditioned medium (αMEM including 10% FBS and 103 U/ml CSF-1). n = 5, 5 independent experiments. Data are means ± SEM. *, P < 0.01. (H) Expression of CCL3 mRNA was assessed by RT-PCR in human macrophages cultured in MDA231 conditioned medium with neutralizing anti-CCL2 or control IgG antibodies. n = 3. Data are means ± SEM. *, P < 0.05.

Mentions: Consistent with the results from the microarray and real-time PCR, we found 40–50% reduction in CCL3 protein secretion from Ccr2-deficient macrophages compared with that from WT macrophages (Fig. 2, A and B). In the WT macrophages, stimulation with recombinant CCL2 significantly increased CCL3 mRNA and protein expression (Fig. 2 B). Because such an increase in CCL3 secretion was not found in Ccr2-deficient macrophages (Fig. 2 B), these results indicate that CCL2 signaling via CCR2 can increase CCL3 secretion from macrophages. Recombinant CCL2 failed to increase CCL3 secretion in Ccr2−/− BMDMs, and thus it is unlikely that any increase in CCL3 secretion is induced by contaminating endotoxin. Indeed, endotoxin contamination is reported as <0.01 ng/ml in our culture conditions and this is much less than is needed to increase CCL3 secretion from alveolar and peritoneal macrophages (Wang et al., 2000). To investigate whether CCL2 promotes Ccl3 expression in MAMs in vivo, we injected anti-CCL2 neutralizing antibody into WT mice having a similar load of lung metastases. After 2 d of antibody treatment, we isolated MAMs (F4/80+CD11b+CD11c–Ly6C–) and resident pulmonary macrophages (F4/80+CD11b–CD11c+Ly6C–) from tumor-bearing lung and inflammatory monocytes (IM; CD115+CD11b+Ly6C+) and resident monocytes (RM; CD115+CD11b+Ly6C–) from peripheral blood (Fig. S1). The treatment with anti-CCL2 antibody significantly suppressed Ccl3 transcript levels in MAMs (Fig. 2 C), indicating that CCL2 can increase CCL3 expression in macrophages at the metastasis sites as well. Interestingly, MAMs expressed 10-fold higher Ccl3 mRNA compared with either circulating IMs or RMs, or resident macrophages in the lung. It is notable that other major leukocyte populations in the tumor-bearing lung expressed low levels of Ccl3 mRNA comparable with resident macrophages, suggesting MAMs are the major source of CCL3 in the metastasis site (Fig. 2 D). Consistent with these data, CCL3 mRNA level in human monocyte-derived macrophages (hMDMs) was significantly higher than freshly isolated monocytes, and this level was increased by recombinant human CCL2 (Fig. 2, E and F). We have reported that CCL3 secretion from cultured macrophages is increased by conditioned medium from mouse mammary tumor cells (Ojalvo et al., 2009). Likewise, conditioned medium from various subtypes of human breast cancer cells increased CCL3 levels in hMDMs (Fig. 2 G). The conditioned medium contained CCL2 (MDA435, 56.5 ± 3.7; MDA231, 37.8 ± 5.2; T47D, 13.0 ± 0.6; MCF7, 59.4 ± 3.5 pg/ml), and the increase in CCL3 by the conditioned medium from MDA231 cells that induced highest amount of CCL3 was suppressed by anti-human CCL2 antibody treatment (67% compared with control IgG treatment; Fig. 2 H). The anti-CCL2 antibody treatment also suppressed CCL3 expression induced by conditioned medium from MDA435 (78% compared with control IgG treatment) but not from T47D and MCF7 cells (unpublished data). Collectively, these results indicate that MAMs secrete high level of CCL3 once they differentiate from IMs, and it is regulated, at least in part, by the CCL2–CCR2 signaling pathway. Our results, however, do not exclude the contribution of other pathways in the regulation of CCL3 synthesis.


CCL2-induced chemokine cascade promotes breast cancer metastasis by enhancing retention of metastasis-associated macrophages.

Kitamura T, Qian BZ, Soong D, Cassetta L, Noy R, Sugano G, Kato Y, Li J, Pollard JW - J. Exp. Med. (2015)

CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2−/− mice were assessed by RT-PCR (n = 6 per genotype, 6 independent experiments) and ELISA (n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P < 0.01 versus WT. (B) Ccl3 mRNA (left) and CCL3 protein (right) were assessed by RT-PCR (n = 3, 2 independent experiments) and ELISA (n = 3 per genotype, 2 independent experiments) in macrophages cultured with PBS or recombinant CCL2 (100 ng/ml). The BMDMs were isolated from WT or Ccr2−/− mice. Data are means ± SEM. *, P < 0.01. (C) Expression of Ccl3 mRNA was assessed by RT-PCR (n = 4, four independent experiments) in monocytes and macrophages obtained from E0771-LG tumor-injected mice treated with neutralizing anti-CCL2 or control IgG antibodies. IM, inflammatory monocytes (CD115+CD11b+Ly6C+); RM, resident monocytes (CD115+CD11b+Ly6C–); MAMs (F4/80+CD11b+CD11c–Ly6C–); Rmac, resident macrophages (F4/80+CD11b–CD11c+Ly6C–). Data are means ± SEM. *, P < 0.01 versus IgG-IM; †, P < 0.05 versus IgG-MAM. Gating strategies are shown Fig. S1. (D) Expression of Ccl3 mRNA was assessed by RT-PCR in Rmac, MAM (n = 7, 7 independent experiments) or neutrophil (Neu), CD4+ T cell, CD8+ T cell, B cell, and NK cell (n = 3, 3 independent experiments). The leukocytes were isolated from tumor-bearing mouse lung. Data are means ± SEM. *, P < 0.01 versus Rmac. (E) Relative CCL3 mRNA expression was assessed by RT-PCR (n = 4, 4 independent experiments) in human monocytes (Mo) and those differentiated into macrophages by CSF-1 (Mac). Data are means ± SEM. *, P < 0.05. (F) Relative CCL3 mRNA level was assessed by RT-PCR (n = 4, 4 independent experiments) in human macrophages cultured with recombinant human CCL2 (100 ng/ml). Data are means ± SEM. *, P < 0.05. (G) Expression of CCL3 mRNA was assessed by RT-PCR in human MDMs (hMDM) cultured with conditioned medium (C.M.) from human cancer cell lines as indicated. As a control (–), the cells were cultured in nonconditioned medium (αMEM including 10% FBS and 103 U/ml CSF-1). n = 5, 5 independent experiments. Data are means ± SEM. *, P < 0.01. (H) Expression of CCL3 mRNA was assessed by RT-PCR in human macrophages cultured in MDA231 conditioned medium with neutralizing anti-CCL2 or control IgG antibodies. n = 3. Data are means ± SEM. *, P < 0.05.
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fig2: CCR2 signaling regulates CCL3 expression in MAMs. (A) Levels of Ccl3 mRNA (left) and CCL3 protein (right) in BMDMs isolated from WT or Ccr2−/− mice were assessed by RT-PCR (n = 6 per genotype, 6 independent experiments) and ELISA (n = 3 per genotype, two independent experiments). Data are means ± SEM. *, P < 0.01 versus WT. (B) Ccl3 mRNA (left) and CCL3 protein (right) were assessed by RT-PCR (n = 3, 2 independent experiments) and ELISA (n = 3 per genotype, 2 independent experiments) in macrophages cultured with PBS or recombinant CCL2 (100 ng/ml). The BMDMs were isolated from WT or Ccr2−/− mice. Data are means ± SEM. *, P < 0.01. (C) Expression of Ccl3 mRNA was assessed by RT-PCR (n = 4, four independent experiments) in monocytes and macrophages obtained from E0771-LG tumor-injected mice treated with neutralizing anti-CCL2 or control IgG antibodies. IM, inflammatory monocytes (CD115+CD11b+Ly6C+); RM, resident monocytes (CD115+CD11b+Ly6C–); MAMs (F4/80+CD11b+CD11c–Ly6C–); Rmac, resident macrophages (F4/80+CD11b–CD11c+Ly6C–). Data are means ± SEM. *, P < 0.01 versus IgG-IM; †, P < 0.05 versus IgG-MAM. Gating strategies are shown Fig. S1. (D) Expression of Ccl3 mRNA was assessed by RT-PCR in Rmac, MAM (n = 7, 7 independent experiments) or neutrophil (Neu), CD4+ T cell, CD8+ T cell, B cell, and NK cell (n = 3, 3 independent experiments). The leukocytes were isolated from tumor-bearing mouse lung. Data are means ± SEM. *, P < 0.01 versus Rmac. (E) Relative CCL3 mRNA expression was assessed by RT-PCR (n = 4, 4 independent experiments) in human monocytes (Mo) and those differentiated into macrophages by CSF-1 (Mac). Data are means ± SEM. *, P < 0.05. (F) Relative CCL3 mRNA level was assessed by RT-PCR (n = 4, 4 independent experiments) in human macrophages cultured with recombinant human CCL2 (100 ng/ml). Data are means ± SEM. *, P < 0.05. (G) Expression of CCL3 mRNA was assessed by RT-PCR in human MDMs (hMDM) cultured with conditioned medium (C.M.) from human cancer cell lines as indicated. As a control (–), the cells were cultured in nonconditioned medium (αMEM including 10% FBS and 103 U/ml CSF-1). n = 5, 5 independent experiments. Data are means ± SEM. *, P < 0.01. (H) Expression of CCL3 mRNA was assessed by RT-PCR in human macrophages cultured in MDA231 conditioned medium with neutralizing anti-CCL2 or control IgG antibodies. n = 3. Data are means ± SEM. *, P < 0.05.
Mentions: Consistent with the results from the microarray and real-time PCR, we found 40–50% reduction in CCL3 protein secretion from Ccr2-deficient macrophages compared with that from WT macrophages (Fig. 2, A and B). In the WT macrophages, stimulation with recombinant CCL2 significantly increased CCL3 mRNA and protein expression (Fig. 2 B). Because such an increase in CCL3 secretion was not found in Ccr2-deficient macrophages (Fig. 2 B), these results indicate that CCL2 signaling via CCR2 can increase CCL3 secretion from macrophages. Recombinant CCL2 failed to increase CCL3 secretion in Ccr2−/− BMDMs, and thus it is unlikely that any increase in CCL3 secretion is induced by contaminating endotoxin. Indeed, endotoxin contamination is reported as <0.01 ng/ml in our culture conditions and this is much less than is needed to increase CCL3 secretion from alveolar and peritoneal macrophages (Wang et al., 2000). To investigate whether CCL2 promotes Ccl3 expression in MAMs in vivo, we injected anti-CCL2 neutralizing antibody into WT mice having a similar load of lung metastases. After 2 d of antibody treatment, we isolated MAMs (F4/80+CD11b+CD11c–Ly6C–) and resident pulmonary macrophages (F4/80+CD11b–CD11c+Ly6C–) from tumor-bearing lung and inflammatory monocytes (IM; CD115+CD11b+Ly6C+) and resident monocytes (RM; CD115+CD11b+Ly6C–) from peripheral blood (Fig. S1). The treatment with anti-CCL2 antibody significantly suppressed Ccl3 transcript levels in MAMs (Fig. 2 C), indicating that CCL2 can increase CCL3 expression in macrophages at the metastasis sites as well. Interestingly, MAMs expressed 10-fold higher Ccl3 mRNA compared with either circulating IMs or RMs, or resident macrophages in the lung. It is notable that other major leukocyte populations in the tumor-bearing lung expressed low levels of Ccl3 mRNA comparable with resident macrophages, suggesting MAMs are the major source of CCL3 in the metastasis site (Fig. 2 D). Consistent with these data, CCL3 mRNA level in human monocyte-derived macrophages (hMDMs) was significantly higher than freshly isolated monocytes, and this level was increased by recombinant human CCL2 (Fig. 2, E and F). We have reported that CCL3 secretion from cultured macrophages is increased by conditioned medium from mouse mammary tumor cells (Ojalvo et al., 2009). Likewise, conditioned medium from various subtypes of human breast cancer cells increased CCL3 levels in hMDMs (Fig. 2 G). The conditioned medium contained CCL2 (MDA435, 56.5 ± 3.7; MDA231, 37.8 ± 5.2; T47D, 13.0 ± 0.6; MCF7, 59.4 ± 3.5 pg/ml), and the increase in CCL3 by the conditioned medium from MDA231 cells that induced highest amount of CCL3 was suppressed by anti-human CCL2 antibody treatment (67% compared with control IgG treatment; Fig. 2 H). The anti-CCL2 antibody treatment also suppressed CCL3 expression induced by conditioned medium from MDA435 (78% compared with control IgG treatment) but not from T47D and MCF7 cells (unpublished data). Collectively, these results indicate that MAMs secrete high level of CCL3 once they differentiate from IMs, and it is regulated, at least in part, by the CCL2–CCR2 signaling pathway. Our results, however, do not exclude the contribution of other pathways in the regulation of CCL3 synthesis.

Bottom Line: Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice.Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM-cancer cell interactions.These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets.

View Article: PubMed Central - HTML - PubMed

Affiliation: MRC Centre for Reproductive Health, Queen's Medical Research Institute, the University of Edinburgh, Edinburgh EH16 4TJ, Scotland, UK.

Show MeSH
Related in: MedlinePlus