Microenvironment and Immunology Primary Tumor Hypoxia Recruits CD11bþ/Ly6Cmed/Ly6Gþ Immune Suppressor Cells and Compromises NK Cell Cytotoxicity in the Premetastatic Niche

Hypoxia within a tumor acts as a strong selective pressure that promotes angiogenesis, invasion, and metastatic spread. In this study, we used immune competent bone marrow chimeric mice and syngeneic orthotopic mammary cancer models to show that hypoxia in the primary tumor promotes premetastatic niche formation in secondary organs. Injection of mice with cell-free conditioned medium derived from hypoxic mammary tumor cells resulted in increased bone marrow–derived cell infiltration into the lung in the absence of a primary tumor and led to increased metastatic burden in mammary and melanoma experimental metastasis models. By characterizing the composition of infiltrating bone marrow–derived cells, we identified CD11bþ/Ly6Cmed/Ly6Gþ myeloid and CD3 /NK1.1þ immune cell lineages as key constituents of the premetastatic niche. Furthermore, the cytotoxicity of natural killer (NK) cells was significantly decreased, resulting in a reduced antitumor response that allowedmetastasis formation in secondary organs to a similar extent as ablation of NK cells. In contrast, metastatic burden was decreased when active NK cells were present in premetastatic lungs. Together, our findings suggest that primary tumor hypoxia provides cytokines and growth factors capable of creating a premetastatic niche through recruitment of CD11bþ/Ly6Cmed/Ly6Gþmyeloid cells and a reduction in the cytotoxic effector functions of NK cell populations. Cancer Res; 1–6. 2012 AACR. Introduction Hypoxia is a common feature and poor prognostic marker in several solid cancers (1). Cells respond to hypoxia through stabilization of the Hypoxia-inducible factor (Hif) transcription factor, resulting in the expression of genes involved in angiogenesis, invasion, and metastasis (1). Hypoxia controls the composition of the tumor microenvironment and the invasive and metastatic capacity of various cancers (2). Factors capable of inducing bonemarrow–derived cell (BMDC) mobilization and recruitment to the tumor microenvironment include Hif targets VEGF, angiopoietin-1, PIGF, PDGF-B, and SDF1a (3). Emerging evidence suggests primary tumor hypoxia and BMDCs drive premetastatic niche development in distant tissues, making them permissive for metastatic spread (4, 5). Premetastatic niche formation was first attributed to tumor-derived VEGFand PIGF-induced secretion of fibronectin from fibroblasts, promoting adhesion of VEGFR1þ hematopoietic progenitor cells (6). Additional studies reported alternate mechanisms for premetastatic niche formation involving TNFa, TGFb, lysyl-oxidase (LOX), MMP2, MMP9, CXCR4, and SDF1a among others (7). Most of these are direct or indirect Hif targets and generally important in the metastatic process (1). Although secretion of these factors by the primary tumor increases BMDCs in premetastatic organs, in-depth characterization of the responsive BMDCs lineages is lacking. Here we report that conditioned medium containing monocyte chemotactic protein-1 (MCP-1/CCL2), derived from hypoxic mammary tumor cells, can induce premetastatic niche formation in the lungs of immune-competent animals. Furthermore, we define lung infiltrating, bone marrow–derived CD11bþ/Ly6Cmed/Ly6Gþ myeloid cells and natural killer (NK) cells with reduced antitumor responses, which form a premetastatic niche conducive to increased metastatic burden independent of tumor type. Authors' Affiliations: Cancer Genomics and Genetics Laboratory, Cancer Immunology Program, and Metastasis Research Group, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne; Department of Pathology, Sir Peter MacCallum Department of Oncology, Department of Biochemistry, The University of Melbourne; and Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). M.J. Smyth and A. M€ oller contributed equally to this work. Corresponding Authors: Andreas M€ oller and Mark Smyth, Peter MacCallum Cancer Centre, St. Andrews Place, East Melbourne, Victoria 3002, Australia. Phone: 613-9656-1423; Fax: 613-9656-1411; E-mail: [email protected] and [email protected]. doi: 10.1158/0008-5472.CAN-11-3873 2012 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 16, 2017. © 2012 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst July 2, 2012; DOI: 10.1158/0008-5472.CAN-11-3873