Signaling pathways in tumoricidal macrophages in pancreatic cancer

Project Summary Macrophages are active phagocytes and play important roles in engulfment of pathogens, clearance of dead cells and immune complexes, and tissue repair, as well as clearance of live cells including aged red blood cells and neutrophils. Most of the known pro-phagocytic interactions rely on plasma membrane lipid changes in target cells as they start to become apoptotic; how neoplastic or damaged cells are distinguished from healthy cells is not fully understood. We discovered that mouse and human macrophages treated with cIAP1/2 antagonism and the cytokine lymphotoxin (LTα1β2) or IFNγ become strongly phagocytic and can engulf live tumor cells. This live cell phagocytosis occurs in the absence of antibodies and is independent of CD47 and calreticulin, demonstrating that unidentified pro-phagocytic pathways must exist. Moreover, chemogenomic screening of deubiquitinase inhibitors identified a chemical series that also induces live cell phagocytosis, indicating the robustness of this macrophage cell state. Our long-term goal is to identify the receptor-ligand interactions governing live cell phagocytosis. Our central hypothesis is that pro-phagocytic receptors on macrophages are dynamically regulated by the ubiquitin/proteasome system and recognize constitutively expressed ligands on malignant or damaged target cells. In Aim 1, we will define the role of deubiquitinases in regulation of macrophage surface receptors governing recognition and phagocytosis of live cells. We identified the ubiquitin-editing enzyme TNFAIP3 as a promising regulator of phagocytosis. We will validate the role of TNFAIP3 in induction of phagocidal macrophages and identify the downstream signaling and surface proteins on macrophages regulated by TNFAIP3 that mediate recognition and/or phagocytosis of live cancer cells. Unbiased proteomics analysis of phagocidal macrophages induced by deubiquitinase inhibitors revealed the upregulation of fatty acid desaturases, which we will study in aim 1.3. In Aim 2, we will determine the extent to which live cell phagocytosis by macrophages is a means of tumor destruction in vivo. Here we will develop fluorescence-based reporter constructs that can be inserted into any cancer model to distinguish between tumor cells that died and were taken up by efferocytosis versus true phagocytosis of live cells in vivo. Using this novel PhagoGLO in vivo reporter system, we will be able to quantify the ratio of live cell versus dead cell phagocytosis in vivo, both in terms of endogenous anti-tumor immunity and in the context of macrophage-targeted therapies or innate immune agonists. We will also test candidate genes from Aim 1 to determine their impact on live cell phagocytosis in vivo. Collectively this proposal will define novel regulators of live cell phagocytosis and expand our ability to therapeutically impinge or enhance macrophage phagocytic activity, as well as generate a technologically innovative in vivo platform for studying macrophage phagocytosis in tumors that will both meet the needs of this current project and launch my successful independent career studying macrophage cell biology in the context of cancer therapies.