Metastasis-associated macrophages constrain antitumor capability of natural killer cells in the metastatic site at least partially by membrane bound transforming growth factor β

BACKGROUND: Metastatic breast cancer is a leading cause of cancer-related death in women worldwide. Infusion of natural killer (NK) cells is an emerging immunotherapy for such malignant tumors, although elimination of the immunosuppressive tumor environment is required to improve its efficacy. The effects of this “metastatic” tumor environment on NK cells, however, remain largely unknown. Previous studies, including our own, have demonstrated that metastasis-associated macrophages (MAMs) are one of the most abundant immune cell types in the metastatic tumor niche in mouse models of metastatic breast cancer. We thus investigated the effects of MAMs on antitumor functions of NK cells in the metastatic tumor microenvironment. METHODS: MAMs were isolated from the tumor-bearing lung of C57BL/6 mice intravenously injected with E0771-LG mouse mammary tumor cells. The effects of MAMs on NK cell cytotoxicity towards E0771-LG cells were evaluated in vitro by real-time fluorescence microscopy. The effects of MAM depletion on NK cell activation, maturation, and accumulation in the metastatic lung were evaluated by flow cytometry (CD69, CD11b, CD27) and in situ hybridization (Ncr1) using colony-stimulating factor 1 (CSF-1) receptor conditional knockout (Csf1r-cKO) mice. Finally, metastatic tumor loads in the chest region of mice were determined by bioluminescence imaging in order to evaluate the effect of MAM depletion on therapeutic efficacy of endogenous and adoptively transferred NK cells in suppressing metastatic tumor growth. RESULTS: MAMs isolated from the metastatic lung suppressed NK cell-induced tumor cell apoptosis in vitro via membrane-bound transforming growth factor β (TGF-β) dependent mechanisms. In the tumor-challenged mice, depletion of MAMs increased the percentage of activated (CD69(+)) and mature (CD11b(+)CD27(–)) NK cells and the number of Ncr1(+) NK cells as well as NK cell-mediated tumor rejection in the metastatic site. Moreover, MAM depletion or TGF-β receptor antagonist treatment significantly enhanced the therapeutic efficacy of NK cell infusion in suppressing early metastatic tumor outgrowth. CONCLUSION: This study demonstrates that MAMs are a main negative regulator of NK cell function within the metastatic tumor niche, and MAM targeting is an attractive strategy to improve NK cell-based immunotherapy for metastatic breast cancer.