Mammary tumor cells with high metastatic potential are hypersensitive to macrophage-derived hepatocyte growth factor that promotes metastatic tumor growth in mice Takanori Kitamura1,2, Yu Kato3, Demi Brownlie4, Daniel Soong2, Gaël Sugano2, Nicole Kippen2, Jiufeng Li3, Dahlia Doughty-Shenton4, Neil Carragher4,5, Jeffrey W. Pollard2,3 1 Royal (Dick) School of Veterinary Studies and Roslin Institute, The University of Edinburgh, Edinburgh, UK 2 MRC Centre for Reproductive Health, The University of Edinburgh, Edinburgh, UK 3 Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 4 Edinburgh Phenotypic Assay Centre, The University of Edinburgh, Edinburgh, UK 5 Cancer Research UK Edinburgh Centre, MRC Institute of Genetics & Molecular Medicine, The University of Edinburgh, Edinburgh, UK. Background: Metastasis-associated macrophages (MAMs) promote persistent growth of breast cancer cells in the metastatic site, and are thus an attractive therapeutic target to treat breast cancer metastasis, a leading cause of cancer related death in women. However, the precise mechanisms behind MAM-mediated metastatic tumor outgrowth have not been fully elucidated. Methods: E0771 mouse mammary tumor cells were injected into the mammary fat pad or the tail vein of C57BL/6 mice, and the cells from established lung metastatic foci were cultured as highly metastatic derivatives (E0771-HML2 or E0771-LG, respectively). Response to macrophage-derived factor and expression of hepatocyte growth factor (HGF) receptor, MET, was investigated by spheroid invasion assay and western blotting, respectively. E0771-LG cells were manipulated to express firefly luciferase gene and conditional shRNA (express by doxycycline treatment) against Met. These cells were intravenously injected into C57BL/6 mice and metastatic tumor growth was monitored by bioluminescent imaging. Results: The highly metastatic mammary tumor cells expressed higher levels of MET than low metastatic parental cells. This elevated expression resulted in the enhanced and prolonged MET activation in response to recombinant HGF. Moreover, highly metastatic tumor cells were more responsive to HGF released from macrophages compared to low metastatic parental cells. In mouse models of metastatic breast cancer, MAMs uniquely expressed HGF in the lung with metastatic tumors, and blockade of MET signaling in cancer cells suppressed metastatic tumor expansion. The metastasis suppression caused by MET inhibition was reversed by depletion of natural killer (NK) cells. Conclusion: Highly metastatic tumor cells express high level of MET and are responsive to macrophage-derived HGF, which promotes metastatic tumor outgrowth in part through activation of NK cells. Results from this study suggest a new approach to prevent life-threatening metastatic tumor formation, i.e., blockade of MAM-induced MET signal activation in metastatic cancer cells.