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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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Accumulation of four distinct types of myeloid cells in the metastatic liver foci a Cells were isolated from the liver, on or off the foci, on day 14 post-inoculation, and were analyzed by flow cytometry. CD45+ hematopoietic cells circled in the upper panels were further analyzed for expression of CD11b and Gr-1 in the lower panels. Numbers in panels are the percentage of CD45+ cells (top) or those of the gated subpopulations within CD45+ cells (bottom). b Identification of four distinct types of myeloid cells from the metastatic liver foci. The CD45+ cells collected from the metastatic foci on day 14 were analyzed for CD11b and Gr-1 (top left). CD11b+ Gr-1high cells (top center) and CD11b+ Gr-1low cells (lower center panel) were further analyzed for the forward and side scatters (FSC and SSC, respectively). Lower left panel shows the distribution of cell populations ‘(ii)–(iv)’ re-analyzed for expression of CD11b versus. Gr-1. Cytospin specimens of four distinct populations (i)–(iv) were subjected to Wright-Giemsa staining (right). Scale bars, 10 μm
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Fig3: Accumulation of four distinct types of myeloid cells in the metastatic liver foci a Cells were isolated from the liver, on or off the foci, on day 14 post-inoculation, and were analyzed by flow cytometry. CD45+ hematopoietic cells circled in the upper panels were further analyzed for expression of CD11b and Gr-1 in the lower panels. Numbers in panels are the percentage of CD45+ cells (top) or those of the gated subpopulations within CD45+ cells (bottom). b Identification of four distinct types of myeloid cells from the metastatic liver foci. The CD45+ cells collected from the metastatic foci on day 14 were analyzed for CD11b and Gr-1 (top left). CD11b+ Gr-1high cells (top center) and CD11b+ Gr-1low cells (lower center panel) were further analyzed for the forward and side scatters (FSC and SSC, respectively). Lower left panel shows the distribution of cell populations ‘(ii)–(iv)’ re-analyzed for expression of CD11b versus. Gr-1. Cytospin specimens of four distinct populations (i)–(iv) were subjected to Wright-Giemsa staining (right). Scale bars, 10 μm

Mentions: We then analyzed the myeloid cells in the liver metastatic foci in more detail. In large metastatic foci (Fig. 2a), most CD45+ hematopoietic cells lacked expression of Ccr1-mVenus, although a few expressing cells were also found. To further characterize the hematopoietic cells accumulated at the site of metastasis, we recovered cells from livers of control mice, and from livers with or without metastatic foci, and analyzed them by flow cytometry (Fig. 3a). The live hematopoietic (PI− CD45+) fraction was significantly larger in the metastatic foci (~11 vs. ~1 % in non-transplanted control, Fig. 3a top), and contained more CD11b+ myeloid cells (~70 vs. ~15 % in the control, Fig. 3a bottom). Then, CD11b+ myeloid cells were divided into two subpopulations in terms of Gr-1 expression (Gr-1high and Gr-1low; Fig. 3b, top left). The Gr-1high cells clustered as a distinct population in the scatter analysis (FSC vs. SSC; Fig. 3b, top right), and showed the neutrophil morphology in the sorted specimens [Fig. 3b, Photo (i)]. On the other hand, the Gr-1low cells were constituted of three subpopulations in the scatter analysis and re-analysis of the data sets for CD11b vs. Gr-1 (Fig. 3b, bottom). When sorted, two subpopulations with lower scatters (FSClow SSClow) showed morphologies of eosinophils and monocytes (Fig. 3b, right photos (ii) and (iii), respectively). In addition, we found cells with a profile of high FSC and SSC with the expression level of CD11b higher than that of eosinophils or monocytes (Fig. 3b, bottom left). The Giemsa staining of the sorted specimens revealed large cells with expanded cytoplasm containing vacuoles, suggesting that they are of the monocyte-macrophage lineage (Fig. 3b, right photo (iv)). We found that all these four subsets of CD11b+ myeloid cells were of the BM origin because liver metastasis experiments following BM transplantation using congenic donor cells (CD45.1 as the marker [21]) proved their donor origin (Supplementary Fig. 2).Fig. 3


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)

Accumulation of four distinct types of myeloid cells in the metastatic liver foci a Cells were isolated from the liver, on or off the foci, on day 14 post-inoculation, and were analyzed by flow cytometry. CD45+ hematopoietic cells circled in the upper panels were further analyzed for expression of CD11b and Gr-1 in the lower panels. Numbers in panels are the percentage of CD45+ cells (top) or those of the gated subpopulations within CD45+ cells (bottom). b Identification of four distinct types of myeloid cells from the metastatic liver foci. The CD45+ cells collected from the metastatic foci on day 14 were analyzed for CD11b and Gr-1 (top left). CD11b+ Gr-1high cells (top center) and CD11b+ Gr-1low cells (lower center panel) were further analyzed for the forward and side scatters (FSC and SSC, respectively). Lower left panel shows the distribution of cell populations ‘(ii)–(iv)’ re-analyzed for expression of CD11b versus. Gr-1. Cytospin specimens of four distinct populations (i)–(iv) were subjected to Wright-Giemsa staining (right). Scale bars, 10 μm
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Related In: Results  -  Collection

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Fig3: Accumulation of four distinct types of myeloid cells in the metastatic liver foci a Cells were isolated from the liver, on or off the foci, on day 14 post-inoculation, and were analyzed by flow cytometry. CD45+ hematopoietic cells circled in the upper panels were further analyzed for expression of CD11b and Gr-1 in the lower panels. Numbers in panels are the percentage of CD45+ cells (top) or those of the gated subpopulations within CD45+ cells (bottom). b Identification of four distinct types of myeloid cells from the metastatic liver foci. The CD45+ cells collected from the metastatic foci on day 14 were analyzed for CD11b and Gr-1 (top left). CD11b+ Gr-1high cells (top center) and CD11b+ Gr-1low cells (lower center panel) were further analyzed for the forward and side scatters (FSC and SSC, respectively). Lower left panel shows the distribution of cell populations ‘(ii)–(iv)’ re-analyzed for expression of CD11b versus. Gr-1. Cytospin specimens of four distinct populations (i)–(iv) were subjected to Wright-Giemsa staining (right). Scale bars, 10 μm
Mentions: We then analyzed the myeloid cells in the liver metastatic foci in more detail. In large metastatic foci (Fig. 2a), most CD45+ hematopoietic cells lacked expression of Ccr1-mVenus, although a few expressing cells were also found. To further characterize the hematopoietic cells accumulated at the site of metastasis, we recovered cells from livers of control mice, and from livers with or without metastatic foci, and analyzed them by flow cytometry (Fig. 3a). The live hematopoietic (PI− CD45+) fraction was significantly larger in the metastatic foci (~11 vs. ~1 % in non-transplanted control, Fig. 3a top), and contained more CD11b+ myeloid cells (~70 vs. ~15 % in the control, Fig. 3a bottom). Then, CD11b+ myeloid cells were divided into two subpopulations in terms of Gr-1 expression (Gr-1high and Gr-1low; Fig. 3b, top left). The Gr-1high cells clustered as a distinct population in the scatter analysis (FSC vs. SSC; Fig. 3b, top right), and showed the neutrophil morphology in the sorted specimens [Fig. 3b, Photo (i)]. On the other hand, the Gr-1low cells were constituted of three subpopulations in the scatter analysis and re-analysis of the data sets for CD11b vs. Gr-1 (Fig. 3b, bottom). When sorted, two subpopulations with lower scatters (FSClow SSClow) showed morphologies of eosinophils and monocytes (Fig. 3b, right photos (ii) and (iii), respectively). In addition, we found cells with a profile of high FSC and SSC with the expression level of CD11b higher than that of eosinophils or monocytes (Fig. 3b, bottom left). The Giemsa staining of the sorted specimens revealed large cells with expanded cytoplasm containing vacuoles, suggesting that they are of the monocyte-macrophage lineage (Fig. 3b, right photo (iv)). We found that all these four subsets of CD11b+ myeloid cells were of the BM origin because liver metastasis experiments following BM transplantation using congenic donor cells (CD45.1 as the marker [21]) proved their donor origin (Supplementary Fig. 2).Fig. 3

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