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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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Expression of Ccr1-mVenus reporter is restricted to Gr-1+ mouse neutrophils. a Schematic illustration of the bacterial artificial chromosome (BAC) carrying mouse Ccr1 gene. A gene encoding Venus targeted to plasma membrane (mVenus) was recombined in frame with the first ATG in exon 2 of Ccr1 followed by a polyadenylation sequences, so that no functional CCR1 is expressed from the BAC transgene. It carried ~8 kb of upstream and ~34 kb downstream genomic sequences from the Ccr1 gene locus. PDGFR TM domain: platelet-derived growth-factor receptor transmembrane domain. pA: polyadenylation signal. Ex: exon. b Expression of Ccr1-mVenus reporter gene specifically in the Gr-1+ neutrophils. Presented are flow cytometric profiles of BM cells from one of the four Ccr1-mVenus reporter transgenic lines (line #15; see also Supplementary Fig. 1). Most Gr-1+ cells are CD11b+ (left) and a subpopulation of Gr-1+ cells express Ccr1-mVenus (center). In the right panel, cells within the Ccr1-mVenus+ gate are shown. c Expression of Ccr1-mVenus reporter correlated well with the abundance of endogenous CCR1 transcripts. CD11b+ cells were divided into four populations based on the expression level of Ccr1-mVenus reporter (1–4 in the left panel), and RNA extracted from each population was subjected to real-time RT-PCR for quantification of endogenous Ccr1 transcripts (right). Results are shown as the mean ± S.D. (n = 3). dCcr1-mVenus reporter is expressed by mature Gr-1+ neutrophils in the BM. Ccr1-mVenus– and CCR1-mVenus+ BM cells were sorted (left) and subjected to modified Wright Giemsa staining of the cytospin specimens (center and right). Scale bars, 10 μm
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Fig1: Expression of Ccr1-mVenus reporter is restricted to Gr-1+ mouse neutrophils. a Schematic illustration of the bacterial artificial chromosome (BAC) carrying mouse Ccr1 gene. A gene encoding Venus targeted to plasma membrane (mVenus) was recombined in frame with the first ATG in exon 2 of Ccr1 followed by a polyadenylation sequences, so that no functional CCR1 is expressed from the BAC transgene. It carried ~8 kb of upstream and ~34 kb downstream genomic sequences from the Ccr1 gene locus. PDGFR TM domain: platelet-derived growth-factor receptor transmembrane domain. pA: polyadenylation signal. Ex: exon. b Expression of Ccr1-mVenus reporter gene specifically in the Gr-1+ neutrophils. Presented are flow cytometric profiles of BM cells from one of the four Ccr1-mVenus reporter transgenic lines (line #15; see also Supplementary Fig. 1). Most Gr-1+ cells are CD11b+ (left) and a subpopulation of Gr-1+ cells express Ccr1-mVenus (center). In the right panel, cells within the Ccr1-mVenus+ gate are shown. c Expression of Ccr1-mVenus reporter correlated well with the abundance of endogenous CCR1 transcripts. CD11b+ cells were divided into four populations based on the expression level of Ccr1-mVenus reporter (1–4 in the left panel), and RNA extracted from each population was subjected to real-time RT-PCR for quantification of endogenous Ccr1 transcripts (right). Results are shown as the mean ± S.D. (n = 3). dCcr1-mVenus reporter is expressed by mature Gr-1+ neutrophils in the BM. Ccr1-mVenus– and CCR1-mVenus+ BM cells were sorted (left) and subjected to modified Wright Giemsa staining of the cytospin specimens (center and right). Scale bars, 10 μm

Mentions: We demonstrated recently that CCR1 plays critical roles in liver metastasis of Smad4-deficient colon cancer in a mouse model [16]. In the BM, CD11b+ Gr-1+ myeloid cells expressed 10 times higher level of Ccr1 mRNA than CD11b– Gr-1– non-myeloid cells (Supplementary Fig. 1a), suggesting that CCR1 expressing cells were highly enriched in the myeloid cells. To isolate and characterize the CCR1-expressing cells by cell sorting, we tested antibodies from various sources, but were unable to find one that bound to mouse CCR1 specifically and reliably. Accordingly, we resorted to the construction of a reporter transgenic mouse model whose marker gene (membrane-targeted Venus; mVenus) was placed under the control of the Ccr1 promoter. As the source of regulatory elements to reconstitute the endogenous CCR1 expression, we used a BAC clone spanning 8 kb upstream and 34 kb downstream of the mouse Ccr1 gene (Fig. 1a). Thus, we established four independent transgenic lines (Fig. 1b, and Supplementary Fig. 1b, c; see "Materials and methods" section).Fig. 1


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)

Expression of Ccr1-mVenus reporter is restricted to Gr-1+ mouse neutrophils. a Schematic illustration of the bacterial artificial chromosome (BAC) carrying mouse Ccr1 gene. A gene encoding Venus targeted to plasma membrane (mVenus) was recombined in frame with the first ATG in exon 2 of Ccr1 followed by a polyadenylation sequences, so that no functional CCR1 is expressed from the BAC transgene. It carried ~8 kb of upstream and ~34 kb downstream genomic sequences from the Ccr1 gene locus. PDGFR TM domain: platelet-derived growth-factor receptor transmembrane domain. pA: polyadenylation signal. Ex: exon. b Expression of Ccr1-mVenus reporter gene specifically in the Gr-1+ neutrophils. Presented are flow cytometric profiles of BM cells from one of the four Ccr1-mVenus reporter transgenic lines (line #15; see also Supplementary Fig. 1). Most Gr-1+ cells are CD11b+ (left) and a subpopulation of Gr-1+ cells express Ccr1-mVenus (center). In the right panel, cells within the Ccr1-mVenus+ gate are shown. c Expression of Ccr1-mVenus reporter correlated well with the abundance of endogenous CCR1 transcripts. CD11b+ cells were divided into four populations based on the expression level of Ccr1-mVenus reporter (1–4 in the left panel), and RNA extracted from each population was subjected to real-time RT-PCR for quantification of endogenous Ccr1 transcripts (right). Results are shown as the mean ± S.D. (n = 3). dCcr1-mVenus reporter is expressed by mature Gr-1+ neutrophils in the BM. Ccr1-mVenus– and CCR1-mVenus+ BM cells were sorted (left) and subjected to modified Wright Giemsa staining of the cytospin specimens (center and right). Scale bars, 10 μm
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Fig1: Expression of Ccr1-mVenus reporter is restricted to Gr-1+ mouse neutrophils. a Schematic illustration of the bacterial artificial chromosome (BAC) carrying mouse Ccr1 gene. A gene encoding Venus targeted to plasma membrane (mVenus) was recombined in frame with the first ATG in exon 2 of Ccr1 followed by a polyadenylation sequences, so that no functional CCR1 is expressed from the BAC transgene. It carried ~8 kb of upstream and ~34 kb downstream genomic sequences from the Ccr1 gene locus. PDGFR TM domain: platelet-derived growth-factor receptor transmembrane domain. pA: polyadenylation signal. Ex: exon. b Expression of Ccr1-mVenus reporter gene specifically in the Gr-1+ neutrophils. Presented are flow cytometric profiles of BM cells from one of the four Ccr1-mVenus reporter transgenic lines (line #15; see also Supplementary Fig. 1). Most Gr-1+ cells are CD11b+ (left) and a subpopulation of Gr-1+ cells express Ccr1-mVenus (center). In the right panel, cells within the Ccr1-mVenus+ gate are shown. c Expression of Ccr1-mVenus reporter correlated well with the abundance of endogenous CCR1 transcripts. CD11b+ cells were divided into four populations based on the expression level of Ccr1-mVenus reporter (1–4 in the left panel), and RNA extracted from each population was subjected to real-time RT-PCR for quantification of endogenous Ccr1 transcripts (right). Results are shown as the mean ± S.D. (n = 3). dCcr1-mVenus reporter is expressed by mature Gr-1+ neutrophils in the BM. Ccr1-mVenus– and CCR1-mVenus+ BM cells were sorted (left) and subjected to modified Wright Giemsa staining of the cytospin specimens (center and right). Scale bars, 10 μm
Mentions: We demonstrated recently that CCR1 plays critical roles in liver metastasis of Smad4-deficient colon cancer in a mouse model [16]. In the BM, CD11b+ Gr-1+ myeloid cells expressed 10 times higher level of Ccr1 mRNA than CD11b– Gr-1– non-myeloid cells (Supplementary Fig. 1a), suggesting that CCR1 expressing cells were highly enriched in the myeloid cells. To isolate and characterize the CCR1-expressing cells by cell sorting, we tested antibodies from various sources, but were unable to find one that bound to mouse CCR1 specifically and reliably. Accordingly, we resorted to the construction of a reporter transgenic mouse model whose marker gene (membrane-targeted Venus; mVenus) was placed under the control of the Ccr1 promoter. As the source of regulatory elements to reconstitute the endogenous CCR1 expression, we used a BAC clone spanning 8 kb upstream and 34 kb downstream of the mouse Ccr1 gene (Fig. 1a). Thus, we established four independent transgenic lines (Fig. 1b, and Supplementary Fig. 1b, c; see "Materials and methods" section).Fig. 1

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