PROJECT SUMMARY/ABSTRACT “The role of MIF in mitochondrial metabolism and M2-TAM polarization.” Tumor-associated macrophages (TAMs) promote intratumoral microvessel density, increase tumor stromal matrix remodeling and potently suppress anti-tumor immunity in late-stage cancer patients. Although substantial research has identified how “M2” TAM polarization promotes tumor progression, much less is known about how TAMs obtain this M2 phenotype within tumor microenvironments. Metabolic reprogramming is a critical determinant of macrophage polarization as recent studies demonstrate that M2 TAMs adopt an oxidative phosphorylation/mitochondrial metabolic phenotype and uniquely respond to metabolites within the tumor microenvironment. This proposal’s long-term objective is to investigate the mechanisms by which macrophage migration inhibitory factor (MIF) dictates lactate-enhanced M2 polarization. Our preliminary studies demonstrate that M2 macrophages metabolize extracellular lactate to pyruvate which, in turn, is oxidized in the mitochondria and is both necessary and sufficient to significantly enhancing Th2 cytokine-dependent M2 TAM polarization. Our data further indicates that MIF promotes mitochondrial metabolism which is required for maximal lactate/pyruvate-enhanced M2 polarization and acquisition of pro-tumorigenic phenotypes. Two aims are proposed to test our hypotheses: Specific Aim 1: Delineate the mechanistic effectors of lactate/pyruvate to HIF1α-dependent M2-macrophage polarization. Objective: To use a combination of metabolic flux analyses, metabolomics, gene expression assays, transgenic mice and mechanistic studies to answer three primary questions: 1) Whether M2 macrophages actively metabolize lactate; 2) Which metabolic pathways are utilized to accomplish this, and; 3) How is lactate metabolism mechanistically linked to M2 polarization Specific Aim 2: Determine the mechanistic contributions of MIF in lactate/pyruvate-mediated M2 TAM polarization. Objective: Use mutant MIF protein re-expression, small-molecule rescue studies, flow cytometry and gene expression assays to make three determinations: 1) Whether MIF dictates mitochondrial biogenesis and metabolism in M2 macrophages; 2) Whether and how MIF influence mitochondrial metabolism by binding to CSN5 and regulating NRF2 stability, and; 3) If MIF is functionally linked to lactate-amplified HIF1α stability and ensuing M2 polarization.