Abstract PR17: CCL2-induced chemokine cascade promotes breast cancer metastasis via retention of metastasis-associated macrophages
Authors: Kitamura Takanori , Pollard Jeffrey W. 
Published: May 2019

Abstract
Breast cancer is a leading cause of cancer death in women largely due to metastasis to the bone and lung. In an experimental model of breast cancer lung metastasis, macrophages characterized as F4/80+CD11b+Ly6Chigh accumulate in the lung and directly contact with disseminating cancer cells within 48 hours post-tumor injection, which promotes extravasation and metastatic outgrowth of cancer cells. We have reported that these metastasis-associated macrophages (MAMs) originate from circulating inflammatory monocytes (IMs) recruited by CC-chemokine ligand 2 (CCL2), and that anti-CCL2 antibody treatments reduce the numbers of MAMs and metastatic foci. These results indicate that the CCL2-CCR2 axis plays an important role in metastatic seeding of breast cancer cells via recruitment of IMs.
Although CCL2 is known as a monocyte chemoattractant, it also promotes phagocytosis, survival, and polarization of myeloid cells. Furthermore, the pro-metastatic CD11b+ MAMs express a much higher level of CCR2 compared with CD11c+ pulmonary resident macrophages that are not involved in breast cancer metastasis. We thus hypothesized that CCL2-CCR2 axis can also regulate pro-metastatic functions of MAMs by modulating their gene expression. To identify potential downstream targets of CCR2 signaling in MAMs, we compared their gene expression profile with that of resident macrophages in normal lung, and identified that CCL3 gene expression was increased in MAMs. By real-time PCR, we confirmed that MAMs expressed 10-fold higher Ccl3 mRNA compared with either circulating IMs or resident macrophages in the lung. Interestingly, the Ccl3 level in MAMs was significantly suppressed by in vivo treatment with anti-CCL2 antibody. Consistently, recombinant CCL2 can increase CCL3 secretion from wild type but not Ccr2 deficient macrophages in vitro, indicating that CCL2 can increase CCL3 expression in macrophages. To investigate the roles of CCL3 signaling in metastasis we intravenously injected mouse breast cancer cells into mice which blood cells lack CCL3 or its receptor CCR1, and found that loss of Ccl3 or Ccr1 significantly reduced the number of lung metastasis foci, as well as the number of MAMs accumulated in tumor-challenged lung within 24 hours. We also found that adoptive transfer of wild type IMs increased the reduced number of lung metastasis foci in Ccl3 deficient mice, suggesting that CCL3 secretion from IMs/MAMs can promote metastatic seeding of breast cancer cells by enhancing the early accumulation of MAMs via CCR1. To investigate how CCR1 promotes MAM accumulation, we injected fluorescently labeled wild type or Ccr1/ IMs into mice with lung metastasis. Although labeled monocytes from both genotypes infiltrated into the lung to a similar extent after 18 hours, there were significantly fewer Ccr1/ cells than wild type cells by 42 hours, indicating that activation of CCR1 signaling promotes retention of MAMs in the metastatic lung. Because most cancer cells extravasate within 48 hours post-injection and interact with MAMs in the lung, we hypothesized that the CCL3-CCR1 axis might enhance MAM retention through regulating the interaction between cancer cells and MAMs. We then cultured fluorescent macrophages on matrigel with cancer cells and tracked them for 24 hours by microscopy. Although majority of wild type macrophages were retained at the same position once they attached to cancer cells, Ccl3/ or Ccr1/ macrophages more frequently detached from cancer cells and moved around dishes, suggesting that the CCL3-CCR1 axis can enhance macrophage-cancer cell interaction which arrests the macrophages.
These findings collectively indicate that the CCL2-triggered chemokine cascade in macrophages promotes metastatic seeding of breast cancer cells and is a potential target to prevent metastatic disease.
Citation Format: Takanori Kitamura, Jeffrey W. Pollard. CCL2-induced chemokine cascade promotes breast cancer metastasis via retention of metastasis-associated macrophages. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr PR17.