PROJECT SUMMARY In this proposal we want to understand the metabolic factors that are essential to tissue-resident macrophage (TRM) development and function. New advances in metabolomics technology have helped to decode how cellular metabolism helps to shape immune cell form and function, but these developments have so far generally stopped short of meaningfully probing the metabolism of immune cells within the tissues themselves. For cells like TRMs, that exclusively reside within the tissues, a full understanding the biology of these cells is missing until we can identify how they utilize cellular metabolism to support their tissue-specific identity and function. Given the important roles TRMs play in maintaining tissue homeostasis and shaping pathogenic environments, it is essential we begin to probe the biochemistry of these cells. We present an approach for exploring TRM metabolism in vivo. Through the use of metabolic tracers in vivo and rapid isolation of TRMs from the tissue, we detail how this approach will be used to identify metabolic programs essential to TRM development and function within the tissues. We show how proof of principle testing of this framework reveals the polyamine-hypusine axis as a novel metabolic node active in differentiating monocytes and TRMs. We implement in vitro and in vivo validation studies laid out in our framework, which include bone marrow chimera, parabiosis and novel mouse model creation, to show that this pathway is essential for the development and maintenance of TRMs across a multitude of organs. We also propose a highly novel approach to studying TRM metabolism in humans. Using normathemic perfusion machines, we will perfuse human organs with metabolic tracers to gain an understanding of human TRM biology in situ. Crucially, we will employ this approach to experimentally test the pathways identified as important in mouse TRMs in a human setting. Finally, a major focus of this proposal is to explore TRM metabolism within tumors. Using similar approaches outlined above for TRMs during homeostasis, we will evaluate the metabolic activity of TAMs using well defined murine tumor models. We will use our innovative human system that allows us to experimentally manipulate human organs to probe the metabolism of human hepatocellular carcinoma lesions and of the TRMs that reside inside them. Through these orthogonal approaches across species, we expect to build up a detailed picture of tumor macrophage metabolic activity that can be used to identify novel pathways that can be targeted to modulate these cells within tumors for therapeutic benefit.