PROJECT 3 SUMMARY Cisplatin (CDDP) remains the gold-standard for chemotherapeutic treatment for multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). High rates of treatment failure result from the development of acquired resistance following this relatively toxic chemotherapy. Despite the frequent use of CDDP, no robust predictors of tumor response or development of acquired resistance exist. Treatment failure is uniformly fatal. Given the critical unmet need for predictors of tumor response and acquired resistance, we have focused our efforts on the assessment of tumor response with minimally invasive imaging (hyperpolarized magnetic resonance imaging; HP-MRI) and detection of biological shifts in circulating tumor cells (CTCs) while patients are undergoing cisplatin-based therapy. We have shown that CDDP and other genotoxic agents trigger measurable fluctuations in tumor cell metabolism detectable by both conventional biochemical assays and HP- MRI with [1-13C]-pyruvate. The recycling of key coenzymes in several reductive metabolic pathways links the effects of genotoxic stress to carbon flux from pyruvate into lactate via lactate dehydrogenase (LDH). We previously showed that genotoxic agents alter the intracellular redox state, shift pyruvate/lactate metabolism, and suppress the apparent rate of pyruvate conversion to lactate (kPL) in a manner that correlates with anti-tumor effectiveness. Genomic and transcriptomic analysis of regulatory shifts associated with the acquisition of cisplatin resistance in CTCs will re-enforce the imaging-based quantification of cisplatin sensitivity and resistance. Based on these preliminary data, we hypothesize that metabolic reprogramming driven by Nrf-2 in cisplatin- resistant HNSCC is detectable using a combination of non-invasive imaging of carbon flux (kPL- via HP- MRI) and scCTC analysis. To assess this premise, we will use well-characterized preclinical models of HNSCC that are sensitive and resistant to cisplatin. Alterations in glycolytic metabolism will be measured at baseline and following cisplatin administration through hyperpolarized imaging and biochemical assays. These measurements will be validated with biochemical assays in vitro and in vivo (Aim 1). We will also measure treatment response in HNSCC patients relative to alterations in tumor kPL by acquiring HP-MRI data prior to and following induction chemotherapy (Aim 2). Biological confirmation will be performed in CTCs to identify biomarkers associated with cisplatin resistance through genomic and transcriptomic analysis. Successful completion of this study will establish HP-MRI as a minimally invasive imaging approach to characterize relative tumor resistance to cisplatin and provide real-time feedback to optimize treatment. CTC biomarker analysis will provide critical biological support for the imaging findings. This work has the potential to change the basis for clinical decision-making regarding the use of cis...