Abstract Metastasis is the main cause of cancer-associated deaths, but the mechanisms underlying this complex phe- nomenon are incompletely understood. Macrophages are multifunctional immune cells found in all tissues, in- cluding in tumor microenvironments (TME). It has been known for over three decades that high densities of TME macrophages correlate with cancer progression, metastatic spread and shorter patient survival, but TME mac- rophages have not yet been specifically targeted in a successful therapy. The objective of this project is to elu- cidate the cellular and molecular mechanisms by which macrophages promote metastatic cancer spread, and thereby provide the scientific basis for the development of effective therapies. Our work relies on an innovative “humanized mouse” model that carries a transplanted human immune system, including functional human mac- rophages, and in which co-implanted solid tumors grow and metastasize. Using this experimental system to model human melanoma, we have shown that human macrophages play an essential role in promoting metas- tasis, recapitulating clinical observations. By single cell RNA sequencing, we have identified a unique, exclusively TME-localized human macrophage population, as well as candidate mechanisms underlying the differentiation and survival of these cells. Our central hypothesis is that pharmacologically targeting these TME macrophages could be used to prevent and/or manage metastatic spread in cancer patients. Guided by our strong preliminary data, we will test our hypothesis, using state-of-the-art technologies and following three specific aims. We will: (1) determine whether the same metastasis-supporting TME macrophages are present across biopsy samples representing individual patients and different cancer types, and functionally test the in vivo role of these macro- phages in humanized mouse patient-derived xenografts (“immuno-PDX”); (2) define the mechanisms by which these macrophages differentiate and survive in the TME; and (3) determine the mechanisms by which TME macrophages support metastasis. The predicted outcome of the proposed research is that we will rigorously characterize metastasis-supporting macrophages in human cancer, and advance candidate targets for novel cancer therapies. Specifically targeting these macrophages, as a monotherapy or in combination with other available therapies, could have a positive impact on patients for whom current treatments are ineffective. Finally, positive findings in the proposed experiments would further advance our humanized mouse model as a highly valuable research tool for translational research in immuno-oncology, including evaluations of novel therapies in relevant pre-clinical conditions.