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Purpose: Targeting tumor vasculature using vascular disrupting agents (VDAs) is an attractive therapy. These agents induce rapid physiological changes long before any growth inhibitory effects become apparent. Detecting these early changes should allow us to predict anti-tumor effects. Hyperpolarized ¹³C magnetic resonance spectroscopy (HPMRS) allows dynamic measurements of the metabolism of 13C-labeled substrates and may be a new approach for reliably monitoring early effects of VDAs. The aim of this study was to investigate the potential of HPMRS to achieve this in a pre-clinical model. Methods: Mice bearing 200 cubic mm foot implanted C3H mammary carcinomas were intraperitoneally injected with combretastatin-A4-phosphate (CA4P) or the A1 analogue OXi4503. Tumor response was assessed by determining necrosis development, measured histologically 24-hours after treatment, and tumor growth time (TGT; time to reach 5 times treatment volume). Estimates of tumor perfusion and metabolism were performed 3h (CA4P) and 6h (OXi4503) after treatment. Perfusion was measured from Hoechst 33342 uptake in histological sections and by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) at 7-Tesla. Metabolic changes were assessed following intravenous injection of hyperpolarized [1-¹³C]pyruvate and observing the conversion to lactate with HPMRS in a 9.4-Tesla scanner. Additional mice were positron emission tomography (PET) scanned using a Mediso nanoScan PET/MRI scanner following administration of fluorodeoxyglucose (FDG). Statistical comparisons were made using a one-way analysis of variance (ANOVA), with significance level of p Results: A dose response relationship was found between injected drug doses and both tumor necrosis induction and TGT, with OXi4503 having a larger effect than CA4P. Two equally effective doses were selected for further analysis (250 mg/kg for CA4P and 50 mg/kg for OXi4503). Both drugs significantly decreased perfusion relative to controls; the respective mean (with 1 SE) values for CA4P and OXi4503 were decreased to 57% (50-64) and 38% (28-48) using Hoechst 33342, and 61% (57-65) and 61% (54-68) using DCE-MRI. FDG uptake (mean tumor-to-brain ratio) also significantly reduced to 43% (33-53) and 33% (25-41) for CA4P and OXi4503, respectively. However, an unaltered pyruvate to lactate ratio was found; the values being 98% (89-107) for CA4P and 113% (102-124) for OXi4503. Conclusion: No change in the HPMRS values was observed, despite changes in all other parameters, indicating that the metabolic fate of pyruvate remains unaltered despite reduced delivery of oxygen and nutrients. This could be because our data provides measures of whole-tumor mean responses that include a viable metabolically unaltered tumor rim and a tumor core with no blood flow (i.e., no pyruvate delivery) following VDA treatment. Citation Format: Ane B. Iversen, Morten Busk, Lotte B. Bertelsen, Christoffer Laustsen, Ole L. Munk, Thomas Nielsen, Thomas R. Wittenborn, Johan Bussink, Jasper Lok, Hans Stodkilde-Jorgensen, Michael R. Horsman. The potential of hyperpolarized 13-C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents [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 2854. doi:10.1158/1538-7445.AM2017-2854

Purpose: AMP-activated protein kinase (AMPK) responds to ATP depletion and orchestrates cell-adaptive responses to energy-restricted conditions like low glucose or hypoxia. AMPK activation stimulates energy producing pathways and inhibits ATP-consuming biosynthesis and controls post-irradiation metabolism. Pharmacological stimulation of AMPK to mimic energetic stress slows tumor growth, possible by inhibition of proliferation. However, since AMPK is critically involved in cell survival during energy restrictions (a tumor hallmark), inhibition of AMPK may effectively kill treatment resistant hypoxic tumor cells. In this study we investigate the antitumor effect of AMPK targeting using the AMPK inhibitor Compound C (CC). Methods: Since hypoxia-induced changes in energy-metabolism (e.g., Pasteur effect) is much more pronounced in cells inherently dependent on oxidative phosphorylation than in the typical glycolytic tumor energetic phenotype, cell lines representing both phenotypes were included. Cell viability, proliferation and energy metabolism (glucose consumption/lactate synthesis) was assessed during acute (3h) or chronic (48h) energy-replete (5 mM glucose and 21% O2) or energy-deplete (2 mM glucose and 0% O2) conditions in the absence or presence of CC. Furthermore, CC treated and untreated mice bearing SCCVII tumors were administered with pimonidazole (hypoxia marker) and FDG (glucose analogue) followed by tissue section analysis of the microregional distribution of hypoxia and glucose consumption. Results: Long-term CC treatment reduced cell viability/proliferation and suppressed glucose consumption and lactate synthesis both under energy replete and deplete conditions, but the relative effects were more pronounced during energy restriction, suggesting that treatment may show specificity towards cells in the hostile tumor microenvironment. Also acute changes in energy metabolism 3h following transition to energy-replete conditions were observed during CC treatment. In accordance, the hypoxia-elicited stimulation of glucose use (the Pasteur effect) was fully suppressed in CC-treated cells, resulting in much more dramatic effects on glycolytic flux in SiHa cells (atypical non-glycolytic phenotype) than in FaDuDD cells (typical glycolytic phenotype). Currently, we are investigating if CC treatment affects the tumor microenvironment in tumor-bearing mice and whether CC treatment sensitize tumors to irradiation. Conclusions: CC fully suppressed the hypoxia-elicited Pasteur effect which may be responsible for cell death. Metabolic effects were much more pronounced in inherently non-glycolytic than in glycolytic tumor cells. In vivo testing of the efficacy/specificity of CC treatment in tumor bearing mice is ongoing. Citation Format: Morten Busk, Brita S. Soerensen, Steen Jakobsen, Bent Honoré, Jens Overgaard, Michael R. Horsman. AMPK as a target to overcome hypoxia-induced treatment resistance in tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4958. doi:10.1158/1538-7445.AM2013-4958