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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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Chronological changes in the myeloid cell distribution in the liver metastatic foci. a Flow cytometric analysis of the CD45+ cells collected from the metastatic liver foci, performed on days 0–21 post-transplantation for expression of CD11b and Gr-1 (top). CD11b+ Gr-1low cells were further divided into three distinct populations; eosinophils (ii), monocytes (iii) and fibrocytes (iv) (bottom). b Results in (a) are redrawn as the subpopulation kinetics in the percentage of all CD45+ cells. Note that the accumulation of neutrophils and monocytes preceded that of fibrocytes and eosinophils in the metastatic liver foci in this mouse model. Results are shown as the mean ± S.D. (n = 3–7) (c and d) Fibrocyte accumulation was attenuated by neutrophil depletion. An anti–Ly-6G rat monoclonal antibody (1A8) was injected ip (500 μg/kg) on days −1 and +3. On day 0, CMT93 syngeneic colon cancer cells were transplanted to C57BL/6 hosts. On day 7, metastatic tissues in the liver were harvested and analyzed by flow cytometry. Ccr1-mVenus+ cells and fibrocytes among the CD45+ cell population were quantified in (c) and (d), respectively. Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with controls
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Fig5: Chronological changes in the myeloid cell distribution in the liver metastatic foci. a Flow cytometric analysis of the CD45+ cells collected from the metastatic liver foci, performed on days 0–21 post-transplantation for expression of CD11b and Gr-1 (top). CD11b+ Gr-1low cells were further divided into three distinct populations; eosinophils (ii), monocytes (iii) and fibrocytes (iv) (bottom). b Results in (a) are redrawn as the subpopulation kinetics in the percentage of all CD45+ cells. Note that the accumulation of neutrophils and monocytes preceded that of fibrocytes and eosinophils in the metastatic liver foci in this mouse model. Results are shown as the mean ± S.D. (n = 3–7) (c and d) Fibrocyte accumulation was attenuated by neutrophil depletion. An anti–Ly-6G rat monoclonal antibody (1A8) was injected ip (500 μg/kg) on days −1 and +3. On day 0, CMT93 syngeneic colon cancer cells were transplanted to C57BL/6 hosts. On day 7, metastatic tissues in the liver were harvested and analyzed by flow cytometry. Ccr1-mVenus+ cells and fibrocytes among the CD45+ cell population were quantified in (c) and (d), respectively. Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with controls

Mentions: Having identified four distinct myeloid cell types accumulating in the metastatic foci, we investigated the chronological changes in myeloid cells found in the liver metastasis lesions (Fig. 5a, b). The top panels in Fig. 5a show expression of CD11b and Gr-1 among the CD45+ hematopoietic cells whereas the bottom panels do FSC and SSC profiles of the CD11b+ Gr-1low population. After splenic injection of CMT93 cells, the number of CD45+ hematopoietic cells started to increase on day 1 and peaked on day 14, decreasing thereafter. Neutrophils and monocytes rapidly appeared on day 1 at high numbers before the appearance of eosinophils and fibrocytes. Accumulation of eosinophils and fibrocytes began on day 7 and peaked on day 14. These data suggest that the recruitment of neutrophils and monocytes may have caused that of eosinophils and fibrocytes.Fig. 5


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)

Chronological changes in the myeloid cell distribution in the liver metastatic foci. a Flow cytometric analysis of the CD45+ cells collected from the metastatic liver foci, performed on days 0–21 post-transplantation for expression of CD11b and Gr-1 (top). CD11b+ Gr-1low cells were further divided into three distinct populations; eosinophils (ii), monocytes (iii) and fibrocytes (iv) (bottom). b Results in (a) are redrawn as the subpopulation kinetics in the percentage of all CD45+ cells. Note that the accumulation of neutrophils and monocytes preceded that of fibrocytes and eosinophils in the metastatic liver foci in this mouse model. Results are shown as the mean ± S.D. (n = 3–7) (c and d) Fibrocyte accumulation was attenuated by neutrophil depletion. An anti–Ly-6G rat monoclonal antibody (1A8) was injected ip (500 μg/kg) on days −1 and +3. On day 0, CMT93 syngeneic colon cancer cells were transplanted to C57BL/6 hosts. On day 7, metastatic tissues in the liver were harvested and analyzed by flow cytometry. Ccr1-mVenus+ cells and fibrocytes among the CD45+ cell population were quantified in (c) and (d), respectively. Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with controls
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Related In: Results  -  Collection

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Fig5: Chronological changes in the myeloid cell distribution in the liver metastatic foci. a Flow cytometric analysis of the CD45+ cells collected from the metastatic liver foci, performed on days 0–21 post-transplantation for expression of CD11b and Gr-1 (top). CD11b+ Gr-1low cells were further divided into three distinct populations; eosinophils (ii), monocytes (iii) and fibrocytes (iv) (bottom). b Results in (a) are redrawn as the subpopulation kinetics in the percentage of all CD45+ cells. Note that the accumulation of neutrophils and monocytes preceded that of fibrocytes and eosinophils in the metastatic liver foci in this mouse model. Results are shown as the mean ± S.D. (n = 3–7) (c and d) Fibrocyte accumulation was attenuated by neutrophil depletion. An anti–Ly-6G rat monoclonal antibody (1A8) was injected ip (500 μg/kg) on days −1 and +3. On day 0, CMT93 syngeneic colon cancer cells were transplanted to C57BL/6 hosts. On day 7, metastatic tissues in the liver were harvested and analyzed by flow cytometry. Ccr1-mVenus+ cells and fibrocytes among the CD45+ cell population were quantified in (c) and (d), respectively. Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with controls
Mentions: Having identified four distinct myeloid cell types accumulating in the metastatic foci, we investigated the chronological changes in myeloid cells found in the liver metastasis lesions (Fig. 5a, b). The top panels in Fig. 5a show expression of CD11b and Gr-1 among the CD45+ hematopoietic cells whereas the bottom panels do FSC and SSC profiles of the CD11b+ Gr-1low population. After splenic injection of CMT93 cells, the number of CD45+ hematopoietic cells started to increase on day 1 and peaked on day 14, decreasing thereafter. Neutrophils and monocytes rapidly appeared on day 1 at high numbers before the appearance of eosinophils and fibrocytes. Accumulation of eosinophils and fibrocytes began on day 7 and peaked on day 14. These data suggest that the recruitment of neutrophils and monocytes may have caused that of eosinophils and fibrocytes.Fig. 5

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