Combined in vivo 13C-metabolomics and proteomics approach to optimise immunotherapy response in malignant melanoma Bernardus Evers1,2, Albert Gerding1,2, Jiske Tiersma3, Karen van Eunen1,2, Justina C. Wolters4, Dirk-Jan Reijngoud1,2, Mathilde Jalving3, and Barbara M. Bakker1,2 1) Laboratory of Pediatrics, Section Systems Medicine of Metabolism and Signalling, University of Groningen, University Medical Center Groningen, The Netherlands. 2) Systems Biology Centre for Energy Metabolism and Ageing, University of Groningen, University Medical Center Groningen, The Netherlands. 3) Department of Medical Oncology, University of Groningen, University Medical Center Groningen, The Netherlands 4) Department of Pharmacy, Analytical Biochemistry, University of Groningen, The Netherlands Metabolic reprogramming is a common feature during tumourigenesis that allows tumours to adapt to nutrient-poor microenvironments, thereby maintaining cell viability and produce biomass for cell proliferation. Increased aerobic lactate fermentation, known as the "Warburg effect" is a well-studied metabolic alteration in melanoma, as well as other cancer types, that renders the tumour microenvironment hypoglycaemic and acidic. Preclinical in vitro and in vivo data show that this phenomenon has immunosuppressive effects and may as well attenuate patient response to immunotherapy. Interestingly, this metabolic alteration distinguishes a tumour and its corresponding microenvironment from healthy tissue and make their metabolic processes susceptible to drug targeting. We hypothesise that drugs normalising tumour metabolism may revert metabolic-induced immunosuppression and increase patient response to immunotherapy. In this study, we use a combined proteomics and 13C-metabolomics approach to investigate the effect of dichloroacetate (DCA) on normalizing tumour metabolism in vivo. DCA reroutes the pyruvate produced in glycolysis to be oxidized in the mitochondria, thereby reducing the flux to lactic acid and neutralising the tumour microenvironment. A seven-day, phase 2 clinical trial of DCA has been planned in 36 patients with malignant melanoma prior to immunotherapy. Pre- and post-DCA treatment biopsies will be taken after intravenous [U-13C]glucose infusion in isotopic steady-state. We present the development of GC-EI-MS(MS) methods for quantitative analysis of 13C-label incorporation in glycolytic, TCA cycle and pentose phosphate pathway intermediates. In addition we have developed targeted proteomics for the absolute quantification of glycolytic and mitochondrial metabolic enzymes using in-house designed 13C-labelled peptide standards based on QConCat technology. These methods will be applied to the respective paired biopsies. Combing the patient-specific response to immunotherapy and DCA with subsequent analysis and computational modelling will enable detailed characterisation of metabolic activity and give insight into metabolic regulation upon treatment in vivoin melanoma patients. Thus, we expect to provide an unprecedented insight into melanoma tumour metabolism and proof-of-concept that targeting metabolism improves immunotherapy response in malignant melanoma patients.