Project Summary/Abstract Cancer is a metabolically heterogeneous disease. At the core of most metabolic pathways, mitochondria play central roles in cancer cell proliferation, migration, invasion, metastasis and therapeutic resistance. Due to significant amount of somatic and germline mutations, cancer-relevant modifications in mitochondria are found. While some mitochondrial modifications provide aggressive advantages to cancer cells, others are detrimental. More understandings of how cancer cells manipulate mitochondrial modifications, replenish mitochondrial repertoire, and regulate mitochondria related metabolisms would therefore hold great promises to tackle cancer down. Supported by our preliminary work, we hypothesize that a small population of cancer cells, the cancer stem cells (also known as cancer initiating cells), can horizontally transfer their mitochondria to macrophages. Due to the predisposed modifications in mitochondria of cancer stem cells, we further hypothesize that these cancer stem cells derived mitochondria could rewire metabolic pathways in macrophages thus leading to alternative activation of macrophages (M2). The hypotheses will be tested by two specific aims. We will initially test the reproducibility of the horizontal transfer of mitochondria initiated by different types of cancers (Aim 1). We will also determine if such mitochondrial transfer is actively conducted by cancer stem cells or passively achieved via phagocytosis by macrophages (Aim1). Once the mitochondria of cancer stem cells are present in macrophages, we will examine how these mitochondria mask arginine metabolism, oxidative phosphorylation and reactive oxygen species production in macrophages (Aim2). Additionally, we will determine the polarization spectrum of macrophages in response to the acquisition of exogenous mitochondria from cancer stem cells (Aim 2). With successful completion of this proposal, the information collected from this R03 will not only provide new insights of new immune evasion mediated through the horizontally mitochondrial transfer from cancer stem cells but also present an enticing target for more effective therapeutic interventions specifically targeting the cancer stem cells or the tumor associated macrophages. In addition, it may also serve as a proof-of-principle to apply to other types of stromal cells that may account for other cancer related diseases.