CCR1-Mediated Formation of Liver Microenvironment for Cancer Metastasis

Accumulating evidence suggests that bone marrow-derived myeloid cells constitute the major components of cancer microenvironment and play significant roles in the progression of cancer. We have previously shown that myeloid cells expressing CCR1, a chemokine receptor, are recruited to the microenvironment of disseminated colon cancer cells in the liver (PNAS 2010). These CCR1⁺ cells promote metastatic colonization of cancer cells in the liver through producing metalloproteinases MMP9 and MMP2. However, the cell lineage and precise characteristics of these myeloid cells were largely unknown. The aim of this study is to characterize the cell types among these myeloid cells expressing CCR1 and/or MMPs.

In order to characterize the CCR1-expressing myeloid cells accumulating at liver metastatic foci, 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 Ccr1cis-regulatory elements. A flow cytometric analysis of adult transgenic mouse bone marrow cells showed that expression of the reporter mVenus fluorescence was restricted to the CD11b⁺ Gr-1⁺ cells and the level of endogenous Ccr1 mRNA correlated well with that of mVenus fluorescence, suggesting that expression of the reporter precisely reflected that of the endogenous CCR1.

Inoculation of CCL9-expressing mouse colon cancer cell line CMT93 into the spleen of syngeneic hosts causes efficient metastatic colonization of the cancer cells in the liver. Employing this model, we monitored the behavior of the Ccr1-mVenus⁺ myeloid cells in the cancer metastasis microenvironment, and found that CD45⁺ CD11b⁺Ccr1-mVenus⁺ cells purified from liver metastatic foci were mostly neutrophils. In contrast, within CD45⁺ CD11b⁺Ccr1-mVenus^– fraction, we identified eosinophils, monocytes and fibrocytes. Notably, the major cell types in the metastatic foci were monocytes and fibrocytes, with the latter producing abundant collagen.

Chronologically, accumulation of neutrophils and monocytes preceded that of eosinophils and fibrocytes. In Ccr1^−/− mice, early accumulation of Ccr1-mVenus⁺ Gr-1⁺ cells was significantly compromised and later recruitment of fibrocytes to the metastatic foci was reduced. Antibody-mediated depletion of neutrophils caused a significant reduction in the fibrocyte number at the metastatic foci. These results suggest that CCR1 plays critical roles in early accumulation of neutrophils and subsequent recruitment of fibrocytes.

After having identified four types of myeloid cells in the metastatic foci, we determined the expression levels of CCR1, MMP9 and MMP2 mRNAs in each purified population, and found that CCR1 and MMP9 are expressed preferentially by neutrophils, whereas MMP2 is expressed exclusively by monocytes/fibrocytes. Considering the chronological difference in the appearance of myeloid cells in liver metastatic foci, these results suggest that production of MMP9 and MMP2 in succession by different myeloid cells is necessary for disseminated colon cancer cells to colonize in the liver.

In summary, these results suggests that CCR1 is required for recruitment of neutrophils in the early phase of colon cancer dissemination and the subsequent accumulation of fibrocytes, and that MMP9 and MMP2 are produced by neutrophils and fibrocytes, respectively, in a successive manner during the formation of liver metastasis. Further investigation of these cancer-driven host reactions in human colon cancer dissemination may lead to effective preventive measures against cancer metastasis.