PROJECT SUMMARY/ABSTRACT Mitochondria perform many crucial functions including energy production through oxidative phosphorylation (OxPhos) and their dysfunction has been implicated in pathologies including cancer. Evidence suggests metastasis is seeded by rare subpopulations of primary tumor cells with unique biological properties. Defining these unique properties is crucial for developing effective therapies. Using single-cell RNA sequencing (scRNAseq) on patient-derived xenograft (PDX) models of triple negative breast cancer, we generated the first single cell atlas of PDX micrometastatic breast cancer cells in lungs and their corresponding primary tumor cells. We found lung micrometastatic cells, and a small subpopulation of primary tumor cells, had elevated mitochondrial membrane potential and upregulated previously uncharacterized genes in context of breast cancer metastasis, including NME1 and RAN as well as genes associated with OxPhos. We also found breast cancer cells functionally require OxPhos for seeding in the lung. Increased OxPhos provides more ATP available for metastatic cells to use in energy consuming processes, including the production of GTP from ATP for activating G proteins and downstream pathways implicated with invasiveness in cancers. The NME1 gene encodes for NME1 nucleoside diphosphate kinase that catalyzes the production of GTP using ATP. One possible G protein that could be activated by NME1 through GTP production includes the Ran GTPase, encoded by the RAN gene, which was also highly expressed in lung metastatic cells in several PDX models. I hypothesize primary breast tumor cells will use prominent transcriptional and metabolic programs found in lung micrometastatic cells, including OxPhos, to promote metastasis. Furthermore, I hypothesize that OxPhos generates energy to fuel GTP production by NME1 to activate Ran and other G-proteins to facilitate metastatic progression of primary tumor cells. My preliminary work shows ectopic expression of NME1 and RAN promotes lung metastasis of primary tumor cells in breast cancer PDX mice. This work will define the role of OxPhos, transcriptional and metabolic pathways feeding into it, and potential OxPhos functions independent of ATP-generation in driving breast cancer metastasis using stable isotope tracing, metabolomics, and RNA sequencing. Additionally, I will define the role of NME1 and RAN and how they may work together or in parallel to promote breast cancer metastasis in PDX models. Findings of this project will reveal molecular and metabolic pathways preferentially used in metastatic cells to provide insight to metastasis etiology, present novel markers for improved detection and discover therapeutic opportunities to pursue to improve treatment for breast cancer metastasis.