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CCR1-mediated accumulation of myeloid cells in the liver microenvironment promoting mouse colon cancer metastasis.

Hirai H, Fujishita T, Kurimoto K, Miyachi H, Kitano S, Inamoto S, Itatani Y, Saitou M, Maekawa T, Taketo MM - Clin. Exp. Metastasis (2014)

Bottom Line: We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes.Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes.The results also suggest relevant mechanisms in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Transfusion Medicine and Cell Therapy, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
To understand colon cancer metastasis, we earlier analyzed a mouse model that developed liver metastasis of cancer cells disseminated from the spleen. We suggested that CCR1(+) bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells, and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization. In the present study, we have examined these myeloid cells expressing CCR1 and/or MMPs in detail. To this end, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1 gene promoter. Then, myeloid cells obtained from the BM and liver metastatic foci were analyzed by the combination of flow cytometry and cytology/immunohistochemistry, in situ RNA hybridization, or quantitative RT-PCR. We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes. These cell types exhibited distinct expression patterns for CCR1, MMP2 and MMP9. Namely, neutrophils found in the early phase of cancer cell dissemination expressed CCR1 exclusively and MMP9 preferentially, whereas fibrocytes accumulated in later phase expressed MMP2 exclusively. Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes. The recruitment of CCR1(+) neutrophils in early phase of colon cancer dissemination appears to cause that of fibrocytes in late phase. These results implicate the key role of CCR1 in colon cancer metastasis in this mouse model, and explain why both MMP9 and MMP2 are essential as genetically demonstrated previously. The results also suggest relevant mechanisms in humans.

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Neutrophils and fibrocytes facilitate liver metastasis by producing metalloproteinases, MMP9 and MMP2, respectively. a–c Myeloid cell subpopulations isolated from metastatic foci on day 14 post-transplantation were subjected to real-time RT-PCR quantification of mRNAs encoding Ccr1 (a), MMP9 (b), or MMP2 (c). Results are presented as relative ratios to the level of Gapdh mRNA, with the mean ± S.D. (n = 3) Neut., neutrophils; Eos., eosinophils; Mon., monocytes; Fib., fibrocytes. d Distinct localization of cells expressing MMP9 or MMP2 in the metastatic foci. Serial sections from the liver metastatic foci on day 14 were subjected to immunohistochemistry for MMP9 (left) or in situ hybridization for MMP2 mRNA (right). Lower left photo is a higher magnification of the boxed area in the upper left. Scale bar, 100 μm
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Fig7: Neutrophils and fibrocytes facilitate liver metastasis by producing metalloproteinases, MMP9 and MMP2, respectively. a–c Myeloid cell subpopulations isolated from metastatic foci on day 14 post-transplantation were subjected to real-time RT-PCR quantification of mRNAs encoding Ccr1 (a), MMP9 (b), or MMP2 (c). Results are presented as relative ratios to the level of Gapdh mRNA, with the mean ± S.D. (n = 3) Neut., neutrophils; Eos., eosinophils; Mon., monocytes; Fib., fibrocytes. d Distinct localization of cells expressing MMP9 or MMP2 in the metastatic foci. Serial sections from the liver metastatic foci on day 14 were subjected to immunohistochemistry for MMP9 (left) or in situ hybridization for MMP2 mRNA (right). Lower left photo is a higher magnification of the boxed area in the upper left. Scale bar, 100 μm

Mentions: Both MMP9 and MMP2 are required for promotion of liver metastasis of colon cancer in a mouse model [16] To identify the cell type(s) that produce these matrix metalloproteinases, we performed quantitative RT-PCR analyses on the sorted myeloid cells isolated from the liver metastatic foci (Fig. 7a). As anticipated from the results of the Ccr1-mVenus reporter transgenic mice, CCR1 was abundantly expressed by neutrophils, with much lower levels in other myeloid cells. On the other hand, metalloproteinases MMP9 and MMP2 were expressed in neutrophils and monocytes/fibrocytes, respectively, at significant levels. Notably, MMP9 and MMP2 were not expressed simultaneously in any of these myeloid cell types (Fig. 7b, c). To further characterize the cell types that express MMP9 or MMP2, we performed immunohistochemistry for MMP9, and in situ hybridization for MMP2 on serial sections of the metastatic mouse livers (Fig. 7d). As the result, MMP9 was expressed only by a limited number of cells with lobulated nuclei (polymorphonuclear cells). In contrast, MMP2 mRNA (that hybridized to the anti-sense oligonucleotide, but not to the sense oligonucleotide) was found in the majority of tumor-associated stromal cells. These results suggest that, in the liver metastatic lesions, MMP9 and MMP2 were produced by neutrophils and monocyte/fibrocytes, respectively; by distinctly different myeloid cell types.Fig. 7


CCR1-mediated accumulation of myeloid cells in the liver microenvironment promoting mouse colon cancer metastasis.

Hirai H, Fujishita T, Kurimoto K, Miyachi H, Kitano S, Inamoto S, Itatani Y, Saitou M, Maekawa T, Taketo MM - Clin. Exp. Metastasis (2014)

Neutrophils and fibrocytes facilitate liver metastasis by producing metalloproteinases, MMP9 and MMP2, respectively. a–c Myeloid cell subpopulations isolated from metastatic foci on day 14 post-transplantation were subjected to real-time RT-PCR quantification of mRNAs encoding Ccr1 (a), MMP9 (b), or MMP2 (c). Results are presented as relative ratios to the level of Gapdh mRNA, with the mean ± S.D. (n = 3) Neut., neutrophils; Eos., eosinophils; Mon., monocytes; Fib., fibrocytes. d Distinct localization of cells expressing MMP9 or MMP2 in the metastatic foci. Serial sections from the liver metastatic foci on day 14 were subjected to immunohistochemistry for MMP9 (left) or in situ hybridization for MMP2 mRNA (right). Lower left photo is a higher magnification of the boxed area in the upper left. Scale bar, 100 μm
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig7: Neutrophils and fibrocytes facilitate liver metastasis by producing metalloproteinases, MMP9 and MMP2, respectively. a–c Myeloid cell subpopulations isolated from metastatic foci on day 14 post-transplantation were subjected to real-time RT-PCR quantification of mRNAs encoding Ccr1 (a), MMP9 (b), or MMP2 (c). Results are presented as relative ratios to the level of Gapdh mRNA, with the mean ± S.D. (n = 3) Neut., neutrophils; Eos., eosinophils; Mon., monocytes; Fib., fibrocytes. d Distinct localization of cells expressing MMP9 or MMP2 in the metastatic foci. Serial sections from the liver metastatic foci on day 14 were subjected to immunohistochemistry for MMP9 (left) or in situ hybridization for MMP2 mRNA (right). Lower left photo is a higher magnification of the boxed area in the upper left. Scale bar, 100 μm
Mentions: Both MMP9 and MMP2 are required for promotion of liver metastasis of colon cancer in a mouse model [16] To identify the cell type(s) that produce these matrix metalloproteinases, we performed quantitative RT-PCR analyses on the sorted myeloid cells isolated from the liver metastatic foci (Fig. 7a). As anticipated from the results of the Ccr1-mVenus reporter transgenic mice, CCR1 was abundantly expressed by neutrophils, with much lower levels in other myeloid cells. On the other hand, metalloproteinases MMP9 and MMP2 were expressed in neutrophils and monocytes/fibrocytes, respectively, at significant levels. Notably, MMP9 and MMP2 were not expressed simultaneously in any of these myeloid cell types (Fig. 7b, c). To further characterize the cell types that express MMP9 or MMP2, we performed immunohistochemistry for MMP9, and in situ hybridization for MMP2 on serial sections of the metastatic mouse livers (Fig. 7d). As the result, MMP9 was expressed only by a limited number of cells with lobulated nuclei (polymorphonuclear cells). In contrast, MMP2 mRNA (that hybridized to the anti-sense oligonucleotide, but not to the sense oligonucleotide) was found in the majority of tumor-associated stromal cells. These results suggest that, in the liver metastatic lesions, MMP9 and MMP2 were produced by neutrophils and monocyte/fibrocytes, respectively; by distinctly different myeloid cell types.Fig. 7

Bottom Line: We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes.Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes.The results also suggest relevant mechanisms in humans.

View Article: PubMed Central - PubMed

Affiliation: Department of Transfusion Medicine and Cell Therapy, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

ABSTRACT
To understand colon cancer metastasis, we earlier analyzed a mouse model that developed liver metastasis of cancer cells disseminated from the spleen. We suggested that CCR1(+) bone marrow (BM)-derived cells are recruited to the microenvironment of disseminated colon cancer cells, and produce metalloproteinases MMP9 and MMP2, helping metastatic colonization. In the present study, we have examined these myeloid cells expressing CCR1 and/or MMPs in detail. To this end, we have established bacterial artificial chromosome (BAC)-based transgenic mouse lines in which membrane-targeted Venus fluorescent protein (mVenus) was expressed under the control of Ccr1 gene promoter. Then, myeloid cells obtained from the BM and liver metastatic foci were analyzed by the combination of flow cytometry and cytology/immunohistochemistry, in situ RNA hybridization, or quantitative RT-PCR. We have found four distinct types of myeloid cells recruited to the metastatic foci; neutrophils, eosinophils, monocytes and fibrocytes. These cell types exhibited distinct expression patterns for CCR1, MMP2 and MMP9. Namely, neutrophils found in the early phase of cancer cell dissemination expressed CCR1 exclusively and MMP9 preferentially, whereas fibrocytes accumulated in later phase expressed MMP2 exclusively. Either genetic inactivation of Ccr1 or antibody-mediated neutrophil depletion reduced subsequent recruitment of fibrocytes. The recruitment of CCR1(+) neutrophils in early phase of colon cancer dissemination appears to cause that of fibrocytes in late phase. These results implicate the key role of CCR1 in colon cancer metastasis in this mouse model, and explain why both MMP9 and MMP2 are essential as genetically demonstrated previously. The results also suggest relevant mechanisms in humans.

Show MeSH
Related in: MedlinePlus