Abstract. Emerging cell-based therapies may greatly improve therapeutic success of cancer treatment. NK cell immunotherapy is particularly important in treatment of pediatric cancers, where information on tumor immunogens is lacking. Recently, there has been increased interest in the use of adoptive transfer of ex-vivo expanded NK cells for the treatment of solid tumors due to their innate features, higher safety compared to T cells and lower cost. This emerging therapy is of particular importance in osteosarcoma where 30-35% of patients die of pulmonary metastases due to a treatment-failure rate of 85% in this patient group. However, an important problem with implementation of cell therapies in the clinic is our inability to readily assess the presence and distribution of therapeutic cells after administration. Visualization of immune cell distribution and migration with non-invasive imaging could offer significant new insight into the progression of immunotherapy between infusion and invasive biopsy or resection. Although there are a number of viable approaches for imaging of immunotherapy, all current strategies have limitations and no technology has been proven as a single solution for longitudinal imaging of cellular immunotherapies. Therefore, it is important to continue pushing the limits of available imaging tools in order to find solutions that would address the pressing clinical needs. Hyperpolarized (HP) 129Xe MRI has recently emerged as a promising modality for direct imaging of respiratory function and gas absorption. Spectral separation between HP 129Xe gas in the airspace, tissue/plasma, and red blood cells permits quantification of gas absorption and exchange. Further, a few exploratory studies have demonstrated that chemical exchange saturation transfer (CEST) of HP 129Xe in perfluorocarbon nanodroplets (PFC NDs) can significantly enhance sensitivity to the presence of PFC ND’s due to a strong chemical shift between 129Xe gas in PFC and water. We see these developments as a tremendous opportunity to develop clinically translatable strategy for imaging NK cell therapy in the lungs. Here we propose to develop foundation for HP 129Xe CEST (hyperCEST) MRI of NK cells in vivo. The secondary goal is identification of imaging biomarkers of functional HP 129Xe MRI that may provide additional insight into response to cell immunotherapy. We will rely on our extensive expertise in (i) synthesis and applications of PFC NDs in biomedical imaging including cell labeling, and (ii) quantitative MRI, multinuclear imaging (13C, 19F, 129Xe) and metabolic imaging. Our hypothesis is that labeling of NK cells with PFC NDs optimized for 129Xe hyperCEST MRI will not adversely affect their functionality and will allow non-invasive NK cell monitoring with high sensitivity and specificity in the lungs. At the completion of these studies we will have a combination of optimized and validated probes for NK cell 129Xe hyperCEST MRI with corresponding imaging p...