Cancer stem cells are the rare cells within a tumor that are capable of propagating the bulk tumor population indefinitelyanalogous to normal stem cells which propagate normal tissue regeneration throughout the lifetime of an organism. Cancer and leukemia stem cells (LSC) grow and spread throughout the body, often evading immunosurveillance mechanisms of the innate and adaptive immune system. Macrophages are effectors of the innate system, recognizing and phagocytosing altered cells eventually leading to their intracellular killing. Dendritic cells also phagocytose foreign cells, processing and presenting their peptide antigens to T cells of the adaptive immune system. We have found CD47, the cell surface ligand for macrophage and dendritic cell SIRPa receptors, to be significantly preferentially expressed on primary AML stem cells (LSC), and often their progeny, from both mice and humans. We hypothesize that upregulation of CD47 expression is a common mechanism for avoiding both macrophage phagocytosis and killing, and dendritic cell stimulation of immune responses. In preliminary data, we have demonstrated that blocking antibodies to human CD47 on primary LSC and leukemia cell lines lead to their phagocytosis and killing by mouse or human macrophages in vitro, and that CD47 antibody-coated LSC are unable to engraft in immunodeficient mice. This effect is specific for CD47 as LSC coated with isotype-matched anti-CD45 mAb or incubated with non-binding isotype control mAb are not killed in vitro and engraft robustly in vivo. In this grant, we propose to follow up these pilot studies with extensive investigation of in vivo preclinical therapies targeting LSC with anti-CD47 antibodies alone or in combination strategies, to optimize the elimination of human primary AML and AML LSC in immunodeficient mice. In gene expression studies carried out by the Clarke lab, they also found that CD47 was up regulated on breast CSCs (see Project 1). Thus between our labs, we will investigate the expression of CD47 on other cancer stem cells, especially epithelial cancers, investigate the regulation of CD47 and test the efficacy of therapeutic targeting of these cells in vivo with anti-CD47 antibodies. In collaboration with the Quake lab (Project 3) we will use microfluidic platforms to investigate the epigenetic and genetic regulators of CD47 gene expression and identify other differentially expressed signaling pathways in LSC and other CSC