Abstract Background: Although the introduction of the HER2-specific humanized monoclonal antibody Trastuzumab (T) in clinical practice revolutionized the treatment of HER2-positive breast cancer (BC), about half of the T-treated patients do not benefit or become refractory after its administration. Despite different molecular tumor profiles significantly associated with T benefit were reported, the identification of a non invasive clinical approach to select patients responsive or not to T is still a clinical unmet task. We recently reported that mammary cancer cell lines derived from spontaneous nodules arisen in mice transgenic for the human d16HER2 splice variant (HER2-addicted) were significantly enriched in genes involved in glycolysis pathway versus those developed in mice transgenic for the human full-length HER2-positive (WTHER2) (HER2-non addicted) (1,2). Accordingly, the aim of our study was to define whether tumor lactate levels could reflect HER2 addiction and, in turn, T susceptibility. Methods: The metabolite content in murine and human HER2-positive cell lines and specimens was determined by quantitative Nuclear Magnetic Resonance (NMR) spectroscopy. The optical metabolic imaging that quantifies the levels of the anaerobic glycolysis-related metabolite nicotinamide adenine dinucleotide (NADH) and the aerobic metabolite flavin adenine dinucleotide (FAD) was represented by Optical redox ratio (NADH/FAD). The extracellular levels of lactate were calculated by blood gas analyzer. Results: The metabolic profile of HER2-addicted (MI6, MI7) versus -non-addicted (WTHER2_1, WTHER2_2) murine tumor cells revealed higher intracellular levels of metabolites associated to glycolysis in MI6 and MI7 cells versus WTHER2-positive cells. In keeping, also the evaluation of the metabolic profile of human HER2-positive BC cell lines classified as addicted (SKBR3 and BT474) or not (MDAMB453 and MDAMB361) to HER2-signaling (3) confirmed significant higher intracellular and extracellular levels of lactate in HER2-addicted versus -non-addicted cells. The extracellular levels of NADH and NADH/FAD ratio further sustained a significantly higher activity of an anaerobic metabolism in HER2-addicted cells. Noteworthy, the metabolic profile of 27 HER2-positive BC cases treated with T, classified as HER2-addicted or not on the basis of the activation status of d16HER2 (1), provided further evidence of significantly higher levels of lactate in tumors HER2-addicted. Conclusions: Overall, even if a dysregulated glucose metabolism is a tumor hallmark, our results provide relevant in vitro and in vivo evidences of the potential future non-invasive lactate detection as a possible biomarker of HER2-addiction and T-responsiveness. Supported by the Italian Ministry of Health and AIRC. References: 1) Castagnoli L et Al, Cancer Res 2014 2) Castagnoli L et Al ,Oncogene 2016 3) Shiu KK et Al, Oncogene 2014 Citation Format: Lorenzo Castagnoli, Egidio Iorio, Ada Koschorke, Cristina Ghirelli, Gianmauro Palombelli, Debora Giani, Matteo Dugo, Daniele Morelli, Rossella Canese, Elda Tagliabue, Serenella M. Pupa. Lactate production as a potential marker of HER2-addiction and Trastuzumab susceptibility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5428. doi:10.1158/1538-7445.AM2017-5428