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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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CCR1-dependent accumulation of neutrophils and fibrocytes (a) Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 1 post-transplantation of the tumor cells into Ccr1+/+ or Ccr1−/− host mice. Numbers indicate the percentages of the gated cells within the whole CD45+ cell population. The proportions of CD11b+ Gr-1+ cells and Ccr1-mVenus+ cells are summarized in (b), (top and bottom, respectively). Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with +/+. c Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 7 post-transplantation (top). The CD11b+ Gr-1low cells were analyzed further for scatter characteristics (FSC vs. SSC; bottom). Numbers indicate the percentages of the gated cells. d The frequencies for neutrophils (i), eosinophils (ii), monocytes (iii), and fibrocytes (iv) are summarized as the mean ± S.D. (n = 4). *P < .05 compared with +/+
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Fig6: CCR1-dependent accumulation of neutrophils and fibrocytes (a) Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 1 post-transplantation of the tumor cells into Ccr1+/+ or Ccr1−/− host mice. Numbers indicate the percentages of the gated cells within the whole CD45+ cell population. The proportions of CD11b+ Gr-1+ cells and Ccr1-mVenus+ cells are summarized in (b), (top and bottom, respectively). Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with +/+. c Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 7 post-transplantation (top). The CD11b+ Gr-1low cells were analyzed further for scatter characteristics (FSC vs. SSC; bottom). Numbers indicate the percentages of the gated cells. d The frequencies for neutrophils (i), eosinophils (ii), monocytes (iii), and fibrocytes (iv) are summarized as the mean ± S.D. (n = 4). *P < .05 compared with +/+

Mentions: To investigate the roles of CCR1 in the accumulation of these myeloid cell types, we introduced the Ccr1-mVenus reporter transgene into Ccr1−/− mice by successive crosses and subjected them to liver dissemination-metastasis experiments (Fig. 6). On day 1 post-splenic injection, we observed rapid accumulation of Ccr1-mVenus+ neutrophils (CD11b+ Gr-1high) in Ccr1+/+ control mice. In the Ccr1−/− mutant mice, however, accumulation of Ccr1-mVenus+ CD11b+ Gr-1high neutrophils was reduced significantly (Fig. 6a, b). On day 7, the relative abundance of neutrophils (Ccr1-mVenus+ CD11b+ Gr-1high cells) between wild type and knockout mice did not differ significantly (P = 0.32, Fig. 6c, d), whereas the proportion of eosinophils was increased in Ccr1−/− mice, with that of monocytes and fibrocytes rather decreased (Fig. 6c, d). These results suggest that CCR1 played key roles not only in the earlier mobilization and accumulation of neutrophils, but also in later accumulation of monocytes and fibrocytes in liver lesions of CRC metastasis, although the molecular mechanisms by which neutrophils recruit fibrocytes remain unclear.Fig. 6


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)

CCR1-dependent accumulation of neutrophils and fibrocytes (a) Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 1 post-transplantation of the tumor cells into Ccr1+/+ or Ccr1−/− host mice. Numbers indicate the percentages of the gated cells within the whole CD45+ cell population. The proportions of CD11b+ Gr-1+ cells and Ccr1-mVenus+ cells are summarized in (b), (top and bottom, respectively). Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with +/+. c Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 7 post-transplantation (top). The CD11b+ Gr-1low cells were analyzed further for scatter characteristics (FSC vs. SSC; bottom). Numbers indicate the percentages of the gated cells. d The frequencies for neutrophils (i), eosinophils (ii), monocytes (iii), and fibrocytes (iv) are summarized as the mean ± S.D. (n = 4). *P < .05 compared with +/+
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Fig6: CCR1-dependent accumulation of neutrophils and fibrocytes (a) Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 1 post-transplantation of the tumor cells into Ccr1+/+ or Ccr1−/− host mice. Numbers indicate the percentages of the gated cells within the whole CD45+ cell population. The proportions of CD11b+ Gr-1+ cells and Ccr1-mVenus+ cells are summarized in (b), (top and bottom, respectively). Results are presented as the mean ± S.D. (n = 3). *P < .05 compared with +/+. c Flow cytometric analysis of the CD45+ cells harvested from metastatic liver foci on day 7 post-transplantation (top). The CD11b+ Gr-1low cells were analyzed further for scatter characteristics (FSC vs. SSC; bottom). Numbers indicate the percentages of the gated cells. d The frequencies for neutrophils (i), eosinophils (ii), monocytes (iii), and fibrocytes (iv) are summarized as the mean ± S.D. (n = 4). *P < .05 compared with +/+
Mentions: To investigate the roles of CCR1 in the accumulation of these myeloid cell types, we introduced the Ccr1-mVenus reporter transgene into Ccr1−/− mice by successive crosses and subjected them to liver dissemination-metastasis experiments (Fig. 6). On day 1 post-splenic injection, we observed rapid accumulation of Ccr1-mVenus+ neutrophils (CD11b+ Gr-1high) in Ccr1+/+ control mice. In the Ccr1−/− mutant mice, however, accumulation of Ccr1-mVenus+ CD11b+ Gr-1high neutrophils was reduced significantly (Fig. 6a, b). On day 7, the relative abundance of neutrophils (Ccr1-mVenus+ CD11b+ Gr-1high cells) between wild type and knockout mice did not differ significantly (P = 0.32, Fig. 6c, d), whereas the proportion of eosinophils was increased in Ccr1−/− mice, with that of monocytes and fibrocytes rather decreased (Fig. 6c, d). These results suggest that CCR1 played key roles not only in the earlier mobilization and accumulation of neutrophils, but also in later accumulation of monocytes and fibrocytes in liver lesions of CRC metastasis, although the molecular mechanisms by which neutrophils recruit fibrocytes remain unclear.Fig. 6

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