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. 2013 Jun;15(6):641-8.
doi: 10.1593/neo.121866.

Control of both myeloid cell infiltration and angiogenesis by CCR1 promotes liver cancer metastasis development in mice

Mathieu Paul Rodero  1 , Constance Auvynet, Lucie Poupel, Behazine Combadière, Christophe Combadière
Affiliations

Control of both myeloid cell infiltration and angiogenesis by CCR1 promotes liver cancer metastasis development in mice

Mathieu Paul Rodero et al. Neoplasia. 2013 Jun.

Abstract

Expression of the CC chemokine receptor 1 (CCR1) by tumor cells has been associated with protumoral activity; however, its role in nontumoral cells during tumor development remains elusive. Here, we investigated the role of CCR1 deletion on stromal and hematopoietic cells in a liver metastasis tumor model. Metastasis development was strongly impaired in CCR1-deficient mice compared to control mice and was associated with reduced liver monocyte infiltration. To decipher the role of myeloid cells, sublethally irradiated mice were reconstituted with CCR1-deficient bone marrow (BM) and showed better survival rates than the control reconstituted mice. These results point toward the involvement of CCR1 myeloid cell infiltration in the promotion of tumor burden. In addition, survival rates were extended in CCR1-deficient mice receiving either control or CCR1-deficient BM, indicating that host CCR1 expression on nonhematopoietic cells also supports tumor growth. Finally, we found defective tumor-induced neoangiogenesis (in vitro and in vivo) in CCR1-deficient mice. Overall, our results indicate that CCR1 expression by both hematopoietic and nonhematopoietic cells favors tumor aggressiveness. We propose CCR1 as a potential therapeutical target for liver metastasis therapy.

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Figures

Figure 1
Figure 1
Lack of CCR1 reduces tumor development. (A) Survival curve of C57BL/6 control mice (solid line) or C57BL/6 CCR1-/- mice (dotted line) injected IV with 5 x 105 EL-4. Mean survival time of CCR1-/- mice (n = 28) was 20% longer than in control mice (n = 19). (B) Twenty-one days after tumor inoculation, the increase in liver weight is smaller in CCR1-/- mice compared to WT mice. Each data point represents the mean tumoral liver weight ± SEM of 10 mice. (C) CCR1-/- mice (dotted line, n = 11) showed delay tumor apparition compared to control mice (solid line, n = 11), log-rank test, P = .005. (D) Tumor size growth in control mice (solid line, n = 11) or CCR1-/- mice (dotted line, n = 11) injected s.c. with 1 x 105 EL-4. Tumor development is delayed in CCR1-/- mice. *P < .05; **P < .01 (mice with tumor size of >3 cm3 were killed).
Figure 2
Figure 2
CCR1 deficiency alters leukocyte recruitment at the tumor site. (A) Dot plot analysis identified tumoral leukocyte subpopulation infiltrates by flow cytometry analysis: 1, myeloid cells; 2, inflammatory monocytes; 3, resident monocytes; 4, neutrophils. (B) CCR1-/- mice show defect in the myeloid cell mobilization, mainly due to a defect recruitment of the 7/4 + monocyte population. Percentages ± SEM were indicated for each group (control in black, n = 10; CCR1-/- in white, n = 10) and were obtained from two independent experiments. Np, neutrophils; significant value of *P < .05, **P < .001, and ***P < .001.
Figure 3
Figure 3
CCR1 expression by both BM and non-BM-derived cells promotes tumor metastasis. Survival curve of CCR1-/- and control chimeric mice injected IV with 5 x 105 EL-4. Chimeric mice resulted from control and CCR1-/- irradiated hosts reconstituted with CCR1-/- GFP or control GFP BM. CCR1-/- hosts with control BM (open squares; n = 7) and WT host with control BM (filled squares; n = 9) had significantly decreased mean survival than mice of the same genotype with CCR1 BM, respectively, WT host with CCR1 BM (open circles; n = 10) and CCR1 host with CCR1 BM (filled circles; n = 11 mice).
Figure 4
Figure 4
Reduced numbers of microvessels in tumoral liver of CCR1-/- mice. (A) Frozen liver sections from CCR1-/- and control mice injected with tumor cells were stained with anti-CD31 antibody. Control staining was performed by only incubating with the secondary antibody. (B) Number of microvessels were counted in six randomly chosen fields. Each value represents mean ± SEM (n = 6). *P < .05, **P < .001 compared to D0, #P < .05 compared to control mice.
Figure 5
Figure 5
Lack of CCR1 reduces neoangiogenesis in a vessel sprouting assay. (A) Representative photomicrography of aortic ring with vessel sprouting. The outgrowth area was measured between the vessel growth front and the base of the aortic ring. (B) Vessel sprouting in control and CCR1-deficient aortic ring. Outgrowth area was measured in the absence or presence of EL-4-conditioned media. Each value represents mean area ± SEM (n = 12). (C) Level of CCR1 transcripts in hepatic ECs and PBMCs from WT and CCR1-/- mice. ECs were isolated by flow cytometry and defined as CD45- F4/80- CD31+ cells. (D) Level of CCR1 ligand transcripts (CCL3 and CCL5) in EL-4. ***P < .001 compared to WT and ##P < .01 compared to control media.
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