Metabolically Active Macrophages: A novel mechanistic link between obesity and TNBC Triple negative breast cancer (TNBC) patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Previous study showed that obesity promote tumor burden in C3Tag mouse model. However, mechanisms by which obesity potentiates TNBC progression are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and adipose macrophages (ATMs) accumulation, which are a key source of this inflammation. While it is well established that tumor-associated macrophages (TAMs) are key effector cells that promote breast cancer, role of ATMs on tumorigenesis has largely been ignored. ATMs are an attractive mechanistic link between obesity and TNBC as ATMs are the predominant type of macrophage in the breast during early tumorigenesis. This raises the possibility that ATMs play an important role in tumor progression. Here we show that metabolic dysfunction promotes a mechanistically distinct pro-inflammatory phenotype (metabolic activation; MMe) in breast adipose tissue macrophages isolated from obese women and express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naïve macrophages with media conditioned by human breast adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. We further demonstrated that pre-treating human TNBC cells (MDA-MB-231) with conditioned media derived from MMe macrophages promotes colony formation and invasion in vitro, and intravasation of cancer cells into the blood in vivo. Moreover, pre-treated cancer cells showed a two fold increase in the expression of stem-like cell markers (CD90, OCT4, SOX2 and NANOG). Remarkably, we saw the same effect on ‘stemness’ of tumor cells in obese mice compared to lean mice. Based on these findings we hypothesize that obesity-induced changes to mammary adipose tissue reprogram macrophages to a MMe phenotype that potentiates TNBC initiation and metastasis. In the proposed work, we would determine the mechanism by which MMe macrophages promote TNBC progression (aim1) and their contribution in promoting obesity-associated TNBC (aim 2). In aim 1, we will test the hypothesis that inflammatory cytokines secreted by MMe macrophages are responsible for their pro- tumorigenic effect. Two test this we will deplete inflammatory cytokines in MMe macrophage conditioned media using two approaches: i). Genetic knockout (TLR2-/- and NOX2-/-) macrophages that inhibit inflammatory cytokine expression in MMe macrophages. ii). Neutralizing antibodies against inflammatory c...