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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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Fibrocyte characteristics exhibited by the macrophage-like cells recovered from the liver metastatic foci (a) Quantification of mRNAs for vimentin and collagens by RT-PCR. Myeloid cell subpopulations; neutrophils, eosinophils, monocytes and fibrocytes were collected by cell sorting, and analyzed by RT-PCR. Gapdh mRNA is shown as the loading control. b Photomicrographs of the sorted subpopulations that were immunostained for collagen, type I α1 (Col1a1). Nuclei were stained with DAPI. Scale bars, 10 μm. c Immunohistochemical staining of a metastatic liver focus for collagen, type I α1 (Col1a1; right) together with CD45 and DAPI (left). Note the strong expression of collagen, type I that formed similar structures to the ‘cap’ formation surrounding the metastatic foci [15]. Scale bar, 100 μm. Col1a1: collagen type I α1, Col1a2: collagen type I α2
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Fig4: Fibrocyte characteristics exhibited by the macrophage-like cells recovered from the liver metastatic foci (a) Quantification of mRNAs for vimentin and collagens by RT-PCR. Myeloid cell subpopulations; neutrophils, eosinophils, monocytes and fibrocytes were collected by cell sorting, and analyzed by RT-PCR. Gapdh mRNA is shown as the loading control. b Photomicrographs of the sorted subpopulations that were immunostained for collagen, type I α1 (Col1a1). Nuclei were stained with DAPI. Scale bars, 10 μm. c Immunohistochemical staining of a metastatic liver focus for collagen, type I α1 (Col1a1; right) together with CD45 and DAPI (left). Note the strong expression of collagen, type I that formed similar structures to the ‘cap’ formation surrounding the metastatic foci [15]. Scale bar, 100 μm. Col1a1: collagen type I α1, Col1a2: collagen type I α2

Mentions: It is known that a monocyte-derived lineage identified as ‘fibrocytes’ expresses a series of mesenchymal molecules as vimentin and collagens together with markers of hematopoietic cells [22]. We found that these monocytes and macrophage-like cells obtained from the metastatic foci expressed such non-hematopoietic marker molecules at substantial levels whereas neutrophils or eosinophils did not (Fig. 4a). Namely, immunofluorescence microscopy of the sorted cells confirmed expression of collagen type I by monocytes and macrophage-like cells derived from the metastatic foci (Fig. 4b). Moreover, most CD45+ hematopoietic cells that formed the ‘cap’ [15, 16] around the liver metastatic foci expressed abundant collagen type I (Fig. 4c). Therefore, we identify these macrophage-like cells as fibrocytes. As we reported earlier, these ‘cap’ cells were stained with a particular anti-CD34 antibody (clone: RAM34, Supplementary Fig. 3). However, we recently found that they were not stained with either of two other anti-CD34 antibodies from different sources (MEC14.7 or 3H1240), suggesting that the staining with RAM34 was caused by a non-specific binding of the antibody, conceivably to collagens. In fact, a qRT-PCR analysis of each sorted myeloid population showed that they contained little CD34 mRNA (data not shown). As mentioned above, it was difficult to dissociate and collect fibrocytes from the liver foci due to abundant collagen fibers in the ECM (Supplementary Fig. 1d). Accordingly, fibrocytes did not constitute the major population when isolated cells were analyzed by flow cytometry (see also below).Fig. 4


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)

Fibrocyte characteristics exhibited by the macrophage-like cells recovered from the liver metastatic foci (a) Quantification of mRNAs for vimentin and collagens by RT-PCR. Myeloid cell subpopulations; neutrophils, eosinophils, monocytes and fibrocytes were collected by cell sorting, and analyzed by RT-PCR. Gapdh mRNA is shown as the loading control. b Photomicrographs of the sorted subpopulations that were immunostained for collagen, type I α1 (Col1a1). Nuclei were stained with DAPI. Scale bars, 10 μm. c Immunohistochemical staining of a metastatic liver focus for collagen, type I α1 (Col1a1; right) together with CD45 and DAPI (left). Note the strong expression of collagen, type I that formed similar structures to the ‘cap’ formation surrounding the metastatic foci [15]. Scale bar, 100 μm. Col1a1: collagen type I α1, Col1a2: collagen type I α2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Fig4: Fibrocyte characteristics exhibited by the macrophage-like cells recovered from the liver metastatic foci (a) Quantification of mRNAs for vimentin and collagens by RT-PCR. Myeloid cell subpopulations; neutrophils, eosinophils, monocytes and fibrocytes were collected by cell sorting, and analyzed by RT-PCR. Gapdh mRNA is shown as the loading control. b Photomicrographs of the sorted subpopulations that were immunostained for collagen, type I α1 (Col1a1). Nuclei were stained with DAPI. Scale bars, 10 μm. c Immunohistochemical staining of a metastatic liver focus for collagen, type I α1 (Col1a1; right) together with CD45 and DAPI (left). Note the strong expression of collagen, type I that formed similar structures to the ‘cap’ formation surrounding the metastatic foci [15]. Scale bar, 100 μm. Col1a1: collagen type I α1, Col1a2: collagen type I α2
Mentions: It is known that a monocyte-derived lineage identified as ‘fibrocytes’ expresses a series of mesenchymal molecules as vimentin and collagens together with markers of hematopoietic cells [22]. We found that these monocytes and macrophage-like cells obtained from the metastatic foci expressed such non-hematopoietic marker molecules at substantial levels whereas neutrophils or eosinophils did not (Fig. 4a). Namely, immunofluorescence microscopy of the sorted cells confirmed expression of collagen type I by monocytes and macrophage-like cells derived from the metastatic foci (Fig. 4b). Moreover, most CD45+ hematopoietic cells that formed the ‘cap’ [15, 16] around the liver metastatic foci expressed abundant collagen type I (Fig. 4c). Therefore, we identify these macrophage-like cells as fibrocytes. As we reported earlier, these ‘cap’ cells were stained with a particular anti-CD34 antibody (clone: RAM34, Supplementary Fig. 3). However, we recently found that they were not stained with either of two other anti-CD34 antibodies from different sources (MEC14.7 or 3H1240), suggesting that the staining with RAM34 was caused by a non-specific binding of the antibody, conceivably to collagens. In fact, a qRT-PCR analysis of each sorted myeloid population showed that they contained little CD34 mRNA (data not shown). As mentioned above, it was difficult to dissociate and collect fibrocytes from the liver foci due to abundant collagen fibers in the ECM (Supplementary Fig. 1d). Accordingly, fibrocytes did not constitute the major population when isolated cells were analyzed by flow cytometry (see also below).Fig. 4

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