Hyperpolarized 13C NMR Research Articles

Differentiating leukemia subtypes based on metabolic signatures using hyperpolarized 13C NMR.

Leukemia is a group of blood cancers that are classified in four major classes. Within these four classes, many different subtypes exists with similar origin, genetic mutations, and level of maturity, which can make them difficult to distinguish. Despite their similarities, they might respond differently to treatment, and therefore distinguishing between them is of crucial importance. A deranged metabolic phenotype (Warburg effect) is often seen in cancer cells, leukemia cells included, and is increasingly a target for improved diagnosis and treatment. In this study, hyperpolarized 13C NMR spectroscopy was used to characterize the metabolic signatures of the six leukemia cell lines ML-1, CCRF-CEM, THP-1, MOLT-4, HL-60, and K562. This was done using [1-13C]pyruvate and [1-13C]alanine as bioprobes for downstream metabolite quantification and kinetic analysis on cultured cells with and without 2-deoxy-D-glucose treatment. The metabolic signatures of similar leukemia subtypes could be readily distinguished. This includes ML-1 and THP-1, which are of the similar M4 and M5 AML subtypes, CCRF-CEM and MOLT-4, which are of the similar T-ALL lineage at different maturation states, and HL-60 and K562, which are of the closely related M1 and M2 AML subtypes. The data presented here demonstrate the potential of hyperpolarized 13C NMR spectroscopy as a method to differentiate between leukemia subtypes of similar origin. Combining this method with bioreactor setups could potentially allow for better leukemia disease management as metabolic signatures could be acquired from a single biopsy through repeated experimentation and intervention.

Hyperpolarized 13C NMR Reveals Pathway Regulation in Lactococcus lactis and Metabolic Similarities and Differences Across the Tree of Life.

The control of metabolic networks is incompletely understood, even for glycolysis in highly studied model organisms. Direct real-time observations of metabolic pathways can be achieved in cellular systems with 13C NMR using dissolution Dynamic Nuclear Polarization (dDNP NMR). The method relies on a short-lived boost of NMR sensitivity using a redistribution of nuclear spin states to increase the alignment of the magnetic moments by more than four orders of magnitude. This temporary boost in sensitivity allows detection of metabolism with sub-second time resolution. Here, we hypothesized that dDNP NMR would be able to investigate molecular phenotypes that are not easily accessible with more conventional methods. The use of dDNP NMR allows real-time insight into carbohydrate metabolism in a Gram-positive bacterium (Lactoccocus lactis), and comparison to other bacterial, yeast and mammalian cells shows differences in the kinetic barriers of glycolysis across the kingdoms of life. Nevertheless, the accumulation of non-toxic precursors for biomass at kinetic barriers is found to be shared across the kingdoms of life. We further find that the visualization of glycolysis using dDNP NMR reveals kinetic characteristics in transgenic strains that are not evident when monitoring the overall glycolytic rate only. Finally, dDNP NMR reveals that resting Lactococcus lactis cells use the influx of carbohydrate substrate to produce acetoin rather than lactate during the start of glycolysis. This metabolic regime can be emulated using suitably designed substrate mixtures to enhance the formation of the C4 product acetoin more than 400-fold. Overall, we find that dDNP NMR provides analytical capabilities that may help to clarify the intertwined mechanistic determinants of metabolism and the optimal usage of biotechnologically important bacteria.

Hyperpolarized 1H and 13C NMR Spectroscopy in a Single Experiment for Metabolomics.

The application of NMR spectroscopy to complex mixture analysis and, in particular, to metabolomics is limited by the low sensitivity of NMR. We recently showed that dissolution dynamic nuclear polarization (d-DNP) could enhance the sensitivity of 13C NMR for complex metabolite mixtures, leading to the detection of highly sensitive 13C NMR fingerprints of complex samples such as plant extracts or urine. While such experiments provide heteronuclear spectra, which are complementary to conventional NMR, hyperpolarized 1H NMR spectra would also be highly useful, with improved limits of detection and compatibility with the existing metabolomics workflow and databases. In this technical note, we introduce an approach capable of recording both 1H and 13C hyperpolarized spectra of metabolite mixtures in a single experiment and on the same hyperpolarized sample. We investigate the analytical performance of this method in terms of sensitivity and repeatability, and then we show that it can be efficiently applied to a plant extract. Significant sensitivity enhancements in 1H NMR are reported with a repeatability suitable for metabolomics studies without compromising on the performance of hyperpolarized 13C NMR. This approach provides a way to perform both 1H and 13C hyperpolarized NMR metabolomics with unprecedented sensitivity and throughput.

Optimization of heteronuclear ultrafast 2D NMR for the study of complex mixtures hyperpolarized by dynamic nuclear polarization.

Hyperpolarized 13C NMR at natural abundance, based on dissolution dynamic nuclear polarization (d-DNP), provides rich, sensitive and repeatable 13C NMR fingerprints of complex mixtures. However, the sensitivity enhancement is associated with challenges such as peak overlap and the difficulty to assign hyperpolarized 13C signals. Ultrafast (UF) 2D NMR spectroscopy makes it possible to record heteronuclear 2D maps of d-DNP hyperpolarized samples. Heteronuclear UF 2D NMR can provide correlation peaks that link quaternary carbons and protons through long-range scalar couplings. Here, we report the analytical assessment of an optimized UF long-range HETCOR pulse sequence, applied to the detection of metabolic mixtures at natural abundance and hyperpolarized by d-DNP, based on repeatability and sensitivity considerations. We show that metabolite-dependent limits of quantification in the range of 1-50 mM (in the sample before dissolution) can be achieved, with a repeatability close to 10% and a very good linearity. We provide a detailed comparison of such analytical performance in two different dissolution solvents, D2O and MeOD. The reported pulse sequence appears as an useful analytical tool to facilitate the assignment and integration of metabolite signals in hyperpolarized complex mixtures.

Fine optimization of a dissolution dynamic nuclear polarization experimental setting for 13C NMR of metabolic samples.

NMR-based analysis of metabolite mixtures provides crucial information on biological systems but mostly relies on 1D H experiments for maximizing sensitivity. However, strong peak overlap of H spectra often is a limitation for the analysis of inherently complex biological mixtures. Dissolution dynamic nuclear polarization (d-DNP) improves NMR sensitivity by several orders of magnitude, which enables C NMR-based analysis of metabolites at natural abundance. We have recently demonstrated the successful introduction of d-DNP into a full untargeted metabolomics workflow applied to the study of plant metabolism. Here we describe the systematic optimization of d-DNP experimental settings for experiments at natural C abundance and show how the resolution, sensitivity, and ultimately the number of detectable signals improve as a result. We have systematically optimized the parameters involved (in a semi-automated prototype d-DNP system, from sample preparation to signal detection, aiming at providing an optimization guide for potential users of such a system, who may not be experts in instrumental development). The optimization procedure makes it possible to detect previously inaccessible protonated C signals of metabolites at natural abundance with at least 4times improved line shape and a high repeatability compared to a previously reported d-DNP-enhanced untargeted metabolomic study. This extends the application scope of hyperpolarized C NMR at natural abundance and paves the way to a more general use of DNP-hyperpolarized NMR in metabolomics studies.

Real-Time Non-Invasive and Direct Determination of Lactate Dehydrogenase Activity in Cerebral Organoids-A New Method to Characterize the Metabolism of Brain Organoids?

Organoids are a powerful tool in the quest to understand human diseases. As the developing brain is extremely inaccessible in mammals, cerebral organoids (COs) provide a unique way to investigate neural development and related disorders. The aim of this study was to utilize hyperpolarized 13C NMR to investigate the metabolism of COs in real-time, in a non-destructive manner. The enzymatic activity of lactate dehydrogenase (LDH) was determined by quantifying the rate of [1-13C]lactate production from hyperpolarized [1-13C]pyruvate. Organoid development was assessed by immunofluorescence imaging. Organoid viability was confirmed using 31P NMR spectroscopy. A total of 15 organoids collated into 3 groups with a group total weight of 20–77 mg were used in this study. Two groups were at the age of 10 weeks and one was at the age of 33 weeks. The feasibility of this approach was demonstrated in both age groups, and the LDH activity rate was found to be 1.32 ± 0.75 nmol/s (n = 3 organoid batches). These results suggest that hyperpolarized NMR can be used to characterize the metabolism of brain organoids with a total tissue wet weight of as low as 20 mg (<3 mm3) and a diameter ranging from 3 to 6 mm.

Radial Flow Perfusion Enables Real-Time Profiling of Cellular Metabolism at Low Oxygen Levels with Hyperpolarized 13C NMR Spectroscopy.

In this study, we describe new methods for studying cancer cell metabolism with hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS) that will enable quantitative studies at low oxygen concentrations. Cultured hepatocellular carcinoma cells were grown on the surfaces of non-porous microcarriers inside an NMR spectrometer. They were perfused radially from a central distributer in a modified NMR tube (bioreactor). The oxygen level of the perfusate was continuously monitored and controlled externally. Hyperpolarized substrates were injected continuously into the perfusate stream with a newly designed system that prevented oxygen and temperature perturbations in the bioreactor. Computational and experimental results demonstrated that cell mass oxygen profiles with radial flow were much more uniform than with conventional axial flow. Further, the metabolism of HP [1-13C]pyruvate was markedly different between the two flow configurations, demonstrating the importance of avoiding large oxygen gradients in cell perfusion experiments.

Using SABRE Hyperpolarized 13C NMR Spectroscopy to Interrogate Organic Transformations of Pyruvate.

Signal amplification by reversible exchange (SABRE) is a hyperpolarization technique that uses a metal complex to catalytically transfer magnetization from parahydrogen to molecules of interest. SABRE is used here to monitor the decarboxylation of sodium pyruvate-1,2-[13C2] at a 15 mM concentration to form ethanoic acid and CO2 upon reaction with hydrogen peroxide (150 mM). The rate constant of this reaction is determined by hyperpolarized 13C SABRE-NMR spectroscopy as 0.056 ± 0.003 dm3 mol-1 s-1 at 298 K and is comparable to that determined from thermal 1H NMR (k = 0.050 ± 0.003 dm3 mol-1 s-1) and UV measurements (k = 0.053 ± 0.001 dm3 mol-1 s-1). The hyperpolarized reaction intermediate 2-hydroperoxy-2-hydroxypropanoate is detected in a single scan hyperpolarized 13C NMR spectrum. This work highlights how SABRE hyperpolarization can be used as a tool for the precise monitoring of chemical transformations by hyperpolarized NMR spectroscopy.

Real-time hyperpolarized 13C magnetic resonance detects increased pyruvate oxidation in pyruvate dehydrogenase kinase 2/4\u2013double knockout mouse livers

The pyruvate dehydrogenase complex (PDH) critically regulates carbohydrate metabolism. Phosphorylation of PDH by one of the pyruvate dehydrogenase kinases 1–4 (PDK1–4) decreases the flux of carbohydrates into the TCA cycle. Inhibition of PDKs increases oxidative metabolism of carbohydrates, so targeting PDKs has emerged as an important therapeutic approach to manage various metabolic diseases. Therefore, it is highly desirable to begin to establish imaging tools for noninvasive measurements of PDH flux in rodent models. In this study, we used hyperpolarized (HP) 13C-magnetic resonance spectroscopy to study the impact of a PDK2/PDK4 double knockout (DKO) on pyruvate metabolism in perfused livers from lean and diet-induced obese (DIO) mice and validated the HP observations with high-resolution 13C-nuclear magnetic resonance (NMR) spectroscopy of tissue extracts and steady-state isotopomer analyses. We observed that PDK-deficient livers produce more HP-bicarbonate from HP-[1-13C]pyruvate than age-matched control livers. A steady-state 13C-NMR isotopomer analysis of tissue extracts confirmed that flux rates through PDH, as well as pyruvate carboxylase and pyruvate cycling activities, are significantly higher in PDK-deficient livers. Immunoblotting experiments confirmed that HP-bicarbonate production from HP-[1-13C]pyruvate parallels decreased phosphorylation of the PDH E1α subunit (pE1α) in liver tissue. Our findings indicate that combining real-time hyperpolarized 13C NMR spectroscopy and 13C isotopomer analysis provides quantitative insights into intermediary metabolism in PDK-knockout mice. We propose that this method will be useful in assessing metabolic disease states and developing therapies to improve PDH flux.

Targeted Metabolomics with Quantitative Dissolution Dynamic Nuclear Polarization.

Metabolite profiles and their isotopomer distributions can be studied noninvasively in complex mixtures with NMR. The advent of hyperpolarized 13C-NMR using quantitative dissolution Dynamic Nuclear Polarization (qdDNP) and isotope enrichment add sensitivity to such metabolic studies, enabling mapping and quantification of metabolic pathways and networks. Here we describe a sample preparation method, including cell incubation, extraction, and signal enhancement, for reproducible and quantitative analysis of hyperpolarized 13C-NMR metabolite spectra. We further illustrate how qdDNP can be applied to gain metabolic insights into living cells.

13C NMR Signal Enhancement Using Parahydrogen-Induced Polarization Mediated by a Cobalt Hydrogenation Catalyst.

The use of a cobalt-based catalyst for the generation of hyperpolarized 13C NMR resonances by parahydrogenation of ethyl acrylate is presented herein. Comparisons of the carboxylate 13C NMR signal enhancement factor of ethyl propionate between using (MesCCC)Co-py and a commonly utilized cationic diphosphine rhodium complex demonstrates that the cobalt system is a viable PHIP catalyst alternative. Furthermore, the operative hydrogenation mechanism of the cobalt system was examined by using 1H, 13C, and parahydrogen-induced polarization NMR spectroscopies to elucidate reaction intermediates affiliated with the observed 1H and 13C NMR signal enhancements in ethyl propionate.

Molecular detection of inflammation in cell models using hyperpolarized 13C-pyruvate.

The detection and treatment monitoring of inflammatory states remain challenging in part due to the multifactorial mechanisms of immune activation and spectrum of clinical manifestations. Currently, diagnostic strategies tend to be subjective and limited quantitative tools exist to monitor optimal treatment strategies. Pro-inflammatory M1 polarized macrophages exhibit a distinct metabolic glycolytic phenotype compared to the continuum of M2 polarization states. In the present study, the distinct metabolic phenotypes of resting and activated macrophages were successfully characterized and quantified using hyperpolarized carbon-13 (13C) labeled pyruvate and its metabolic products, i.e. lactate, as a biomarker of resting, disease and treated states.Methods: Mouse macrophage J774A.1 cells were used as a model system in an NMR compatible bioreactor to facilitate dynamic hyperpolarized 13C measurements. The glycolytic metabolism of the cells in the quiescent or resting state were compared with macrophages stimulated by lipopolysaccharide, a classical M1 activator using hyperpolarized 13C labeled pyruvate. Additionally, the activated macrophages were also treated with a non-steroidal anti-inflammatory drug to assess the changes in hyperpolarized lactate signal. The hyperpolarized lactate signals were then correlated using biochemical and molecular assays.Results: We first validated our model system of inflammatory cells by the hallmarks of M1 polarization using steady state metabolic profiling with high resolution NMR in conjunction with nitric oxide Greiss assay, enzyme activity, and mRNA expression. Subsequently, we clearly showed that the cutting edge technology of hyperpolarized 13C NMR can be used to detect elevated lactate levels in M1 polarized macrophages in comparison to control and non-steroidal anti-inflammatory drug treated M2 states.Conclusion: Hyperpolarized 13C lactate has the potential to serve as a biomarker to non-invasively detect and quantify pro-inflammatory state of immune regulatory cells and its response to therapy.

Abstract 833: Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response

Abstract The purpose of this study was to characterize a three-dimensional calcium alginate culture system for real-time monitoring of cancer cell metabolism and radiation response. HCT116 colorectal cancer cells were encapsulated in 2.5% calcium alginate using a custom electrostatic rig adapted from a previously published system. Bead diameter was a function of voltage and alginate concentration: 4 x 107 cells/mL encapsulated at 4 and 8 kV resulted in mean diameters of 880 and 584 µm, respectively. The distribution of bead radii for all voltages showed positive excess kurtosis (4 kV = 1.6, 8 kV = 3.0) and 72.9% of 4 kV and 77.2% of 8 kV bead diameters fell within one standard deviation of the mean. Encapsulated cell viability was assessed by staining with Trypan Blue and clonogenic survival assays (CSAs). The baseline viability of cells immediately after encapsulation was 93.2%, which decreased 2 days after a single 10 Gy dose (90.7%, p&amp;lt;0.05). After two weeks of continuous culturing, 84.1% of non-irradiated and 73.8% of irradiated encapsulated cells remained viable (p&amp;lt;0.003). Pimonidazole staining demonstrated the presence of hypoxic cores proportional to bead size. CSAs of 8 kV beads showed a dose modifying factor (DMF) of 1.06 relative to cells grown in a monolayer, while larger 4 kV beads showed a DMF of 1.58. Mean oxygen consumption rate rose with increasing number of 8 kV beads/well: 1 bead, 35.5; 2 beads, 97.4; 3 beads, 175.5; 4 beads, 209.9; 2.0 x 104 monolayer cells, 92.6 (all pmol/min, ~8.2 x103 cells/bead, p&amp;lt;0.005). Hyperpolarized 13C-NMR spectroscopy of 400 uL of 8 kV beads containing 1.2 x 108 cells/mL showed a detectable conversion of [1-13C]-pyruvate to [1-13C]-lactate; 1 hour after a single 10 Gy dose, the lactate:pyruvate ratio decreased by 25%. Unpaired 2-tailed Student’s t-tests were used to determine significance between and one-way ANOVA was used to determine significance among groups with α set at p≤0.05. These data demonstrate the versatility of alginate hydrogels for real-time metabolic and radiation response studies which are non-invasive, higher throughput, and lower cost compared to in vivo systems. Future directions include additional metabolic flux analysis and hyperpolarized 13C-NMR spectroscopy to further investigate the cancer cell metabolic response to drug and/or radiation therapy. Citation Format: Aparna H. Kesarwala, Jenna L. Carter, Graham H. Read, Natsuko Miura, Kazutoshi Yamamoto, James B. Mitchell, Murali C. Krishna. Alginate hydrogels for three-dimensional culture and real-time monitoring of cancer cell metabolism and radiation response [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 833. doi:10.1158/1538-7445.AM2017-833

High-resolution hyperpolarized in vivo metabolic 13C spectroscopy at low magnetic field (48.7 mT) following murine tail-vein injection

High-resolution 13C NMR spectroscopy of hyperpolarized succinate-1-13C-2,3-d2 is reported in vitro and in vivo using a clinical-scale, biplanar (80cm-gap) 48.7mT permanent magnet with a high homogeneity magnetic field. Non-localized 13C NMR spectra were recorded at 0.52MHz resonance frequency over the torso of a tumor-bearing mouse every 2s. Hyperpolarized 13C NMR signals with linewidths of ∼3Hz (corresponding to ∼6ppm) were recorded in vitro (2mL in a syringe) and in vivo (over a mouse torso). Comparison of the full width at half maximum (FWHM) for 13C NMR spectra acquired at 48.7mT and at 4.7T in a small-animal MRI scanner demonstrates a factor of ∼12 improvement for the 13C resonance linewidth attainable at 48.7mT compared to that at 4.7T in vitro. 13C hyperpolarized succinate-1-13C resonance linewidths in vivo are at least one order of magnitude narrower at 48.7mT compared to those observed in high-field (≥3T) studies employing HP contrast agents. The demonstrated high-resolution 13C in vivo spectroscopy could be useful for high-sensitivity spectroscopic studies involving monitoring HP agent uptake or detecting metabolism using HP contrast agents with sufficiently large 13C chemical shift differences.

Quantifying reaction kinetics of the non-enzymatic decarboxylation of pyruvate and production of peroxymonocarbonate with hyperpolarized 13C-NMR.

The transient nature of intermediate states in chemical reactions has made their detailed investigation difficult. In this study, we demonstrate the utility of hyperpolarized 13C-NMR to directly observe and quantify the kinetics of the intermediate compound in the non-enzymatic decarboxylation of pyruvate via H2O2 with time resolutions of <1 s. Reactants were sequentially added to a reaction vessel within a 9.4 T NMR magnet while continuously acquiring spectra with a low flip angle, producing the first direct observation at room temperature of the previously proposed reaction intermediate, 2-hydroperoxy-2-hydroxypropanoate. We also performed a series of NMR experiments to determine the identity of a previously unidentified peak, which was found to be peroxymonocarbonate, the product of the side reaction between HCO3-/CO2 and H2O2/OOH-. Using the information obtained from these experiments, we developed a kinetic model which fully describes the mechanism of reaction and can be fit to experimental data to simultaneously determine multiple kinetic rate constants over several orders of magnitude. We also discuss the application of this reaction to the production of hyperpolarized bicarbonate for pH imaging experiments. This study presents a template for the use of hyperpolarized 13C-NMR to study the kinetics and reaction mechanisms of innumerable organic reactions which involve polarizable substrates.

Abstract C113: The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells

Abstract Background: Monocarboxylate transporters (MCTs) are key modulators of lactate homeostasis and represent promising metabolic targets for molecular cancer therapeutics. The MCT1 inhibitor AZD3965 is now in clinical trial and understanding the impact of this drug on tumour cell metabolism may enable the discovery of pharmacodynamic (PD) biomarkers of target inhibition that will support the clinical development of such agents. Since MCT1 mediates the bidirectional transport of lactate and other monocarboxylates including pyruvate, here we use NMR spectroscopy to investigate the effect of AZD3965 on a) intracellular lactate levels and b) hyperpolarized 13C-pyruvate-lactate exchange, as biomarkers for MCT1 inhibition in human cancer cells with varying MCT4 expression (predictive of resistance to AZD3965). Materials and Methods: Human Raji (MCT4-) and Hut78 (MCT4 low (+)) lymphoma as well as HT29 (MCT4 high (+++)) colon carcinoma cells were treated with either 5nM or 500nM AZD3965 for 24h and levels of intracellular lactate determined by 1H NMR of cell extracts. Raji cells were also exposed to additional AZD3965 concentrations spanning 1nM-500nM. For 13C-pyruvate-lactate exchange studies, Raji cells were treated with either 5nM or 25nM AZD3965 for 24h. Cells were then incubated at 37°C in FBS-free medium and dynamic 13C NMR spectra acquired for 4 minutes with 2s intervals immediately after the addition of 10mM hyperpolarised [1-13C]pyruvic acid and 10mM unlabelled lactate. The ratio of the area under the curve for the summed lactate and pyruvate signals (LacAUC/PyrAUC) was determined to estimate pyruvate-lactate exchange. Data represent mean±SE. Results: 24h exposure to a low concentration of AZD3965 (5nM) led to increased intracellular lactate in MCT4- Raji and MCT4+ Hut78 human lymphoma cells to 2.65-fold and 10-fold respectively (p = ≤0.02) while the effect in MCT4+++ HT29 human colon carcinoma cells was insignificant (167±31% of controls, p = 0.1). Exposure to a high concentration of AZD3965 (500nM) increased intracellular lactate accumulation in HT29 cells albeit to a lesser degree than in Raji and Hut78 cells (4-fold (0.02) vs. 14 to 15-fold (p≤0.01) in the lymphoma lines). This effect is consistent with the expected blockade of lactate release in cells with low or no MCT4 expression following MCT1 inhibition. Lactate build-up in Raji cells was AZD3965 concentration-dependent being observed with as little as 1nM (up 1.7-fold), reaching a maximum at 25nM (12-fold) and plateauing thereafter. Analysis of the hyperpolarized 13C NMR data showed a significant decrease in LacAUC/PyrAUC to 31±6% in 5nM and 19±2% in 25nM AZD3965-treated Raji cells relative to controls (p&amp;lt;0.001). These results are consistent with MCT1-mediated transport of 13C-pyruvate being a rate limiting step in the 13C NMR-observed pyruvate-lactate exchange. Conclusions: Our data show that AZD3965 triggers intracellular lactate accumulation in a concentration- and MCT4 expression-dependent manner and inhibits 13C-pyruvate-lactate exchange (via blockade of 13C-pyruvate uptake). Intracellular lactate and hyperpolarized 13C-pyruvate-lactate exchange measurements are translatable to in vivo imaging studies and are therefore promising non-invasive metabolic biomarkers for AZD3965 and potentially other MCT1 inhibitors. Citation Format: Mounia Beloueche-Babari, Slawomir Wantuch, Markella Koniordou, Harry G. Parkes, Vaitha Arunan, Thomas R. Eykyn, Paul D. Smith, Martin O. Leach. The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C113.

Abstract 1501A: Detecting lung metastases by hyperpolarized NMR technique: A pilot study

Abstract Purpose: Early detection of lung-residing tumor is of high importance as 1) lung cancer is the number one leading cause of cancer death in the US and early-stage lung cancer was found to be associated with lower mortality than late-stage disease; 2) the lung is one of most common sites of metastasis for various cancers, and metastasis accounts for ∼90% of cancer death. Early confirmation of lung metastasis is expected to significantly change the therapeutic strategies. Currently, the major tools for screening lung-residing tumors in high risk populations include radiography and low dose CT. However, their false positive and over-diagnosis rates are very high (&amp;gt;90%). We aimed to explore the feasibility of employing hyperpolarized-13C NMR (HP-13C-NMR) technique for the early detection of primary lung cancer and lung metastases. HP-13C-NMR is a non-invasive spectroscopy/imaging technique that enhances regular NMR signal by ∼10000 times and has already been demonstrated in both preclinical and clinical cancer studies to quantify cancer enzymatic activities in vivo. Since cancer cells produce more lactate via the Warburg effect, by measuring the kinetics of lactate dehydrogenase, we expect to see the difference between the normal and cancerous lungs. Methods: We used the perfused mouse lungs infested with metastatic breast cancer cells to prove the concept. Nude mice of 7-9 weeks old were injected red fluorescent protein-transfected MDA-MB-231 cell line via tail vein. Tumors were allowed to grow for 4-39 weeks. Lung metastasis was confirmed by optical imaging in vivo. Perfused mouse lungs were placed in a NMR tube (9.4T vertical bore magnet). The lung viability was monitored using 31P spectroscopy before and after HP-13C-pyruvate injection. 8mM [1- 13C] pyruvate polarized via dynamic nuclear polarization (Hypersense, Oxford Instruments) was infused for HP-13C NMR. A series of 13C NMR spectra was acquired (α = 15°) every second for 5 min followed by data processing using custom MATLAB routines to obtain the time courses of pyruvate (Pyr) and lactate (Lac) signals and their ratio, Lac/Pyr. Results: Comparison between the normal lungs (N = 6) and the cancerous lungs (N = 8) shows 70% increase in maximum lactate (p = 0.041) and 94% increase in Lac/Pyr (p = 0.059) due to cancer formation. The standard deviations of the maximum lactate and the ratio in cancerous lung group are much larger than the control group, suggesting a large inter-lung heterogeneity in the cancerous lungs, presumably caused by different amount of metastases. Our next step is to evaluate the amount of metastases in these lungs by H&amp;E staining and quantitatively investigate the correlation between the hyperpolarized measurements and the amount of metastases. Conclusions: Our preliminary results warrant further investigation for the potential of developing a novel HP-NMR technique for the early detection of lung cancer/metastasis in vivo. Citation Format: He N. Xu, Mehrdad Pourfathi, Hoora Shaghaghi, Stephen Kadlececk, Harrilla Profka, Rahim Rizi, Lin Z. Li. Detecting lung metastases by hyperpolarized NMR technique: A pilot study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1501A. doi:10.1158/1538-7445.AM2015-1501A

Hyperpolarized 13C NMR observation of lactate kinetics in skeletal muscle.

The production of glycolytic end products, such as lactate, usually evokes a cellular shift from aerobic to anaerobic ATP generation and O2 insufficiency. In the classical view, muscle lactate must be exported to the liver for clearance. However, lactate also forms under well-oxygenated conditions, and this has led investigators to postulate lactate shuttling from non-oxidative to oxidative muscle fiber, where it can serve as a precursor. Indeed, the intracellular lactate shuttle and the glycogen shunt hypotheses expand the vision to include a dynamic mobilization and utilization of lactate during a muscle contraction cycle. Testing the tenability of these provocative ideas during a rapid contraction cycle has posed a technical challenge. The present study reports the use of hyperpolarized [1-(13)C]lactate and [2-(13)C]pyruvate in dynamic nuclear polarization (DNP) NMR experiments to measure the rapid pyruvate and lactate kinetics in rat muscle. With a 3s temporal resolution, (13)C DNP NMR detects both [1-(13)C]lactate and [2-(13)C]pyruvate kinetics in muscle. Infusion of dichloroacetate stimulates pyruvate dehydrogenase activity and shifts the kinetics toward oxidative metabolism. Bicarbonate formation from [1-(13)C]lactate increases sharply and acetyl-l-carnitine, acetoacetate and glutamate levels also rise. Such a quick mobilization of pyruvate and lactate toward oxidative metabolism supports the postulated role of lactate in the glycogen shunt and the intracellular lactate shuttle models. The study thus introduces an innovative DNP approach to measure metabolite transients, which will help delineate the cellular and physiological role of lactate and glycolytic end products.

Hyperpolarized 13C NMR detects rapid drug-induced changes in cardiac metabolism.

The diseased myocardium lacks metabolic flexibility and responds to stimuli differently compared with healthy hearts. Here, we report the use of hyperpolarized 13C NMR spectroscopy to detect sudden changes in cardiac metabolism in isolated, perfused rat hearts in response to adrenergic stimulation. Metabolism of hyperpolarized [1-(13)C]pyruvate was investigated in perfused rat hearts. The hearts were stimulated in situ by isoproterenol shortly after the administration of hyperpolarized [1-(13)C]pyruvate. The hyperpolarized 13C NMR results were corroborated with 1H NMR spectroscopy of tissue extracts. Addition of isoproterenol to hearts after equilibration of hyperpolarized [1-(13)C]pyruvate into the existing lactate pool resulted in a sudden, rapid increase in hyperpolarized [1-(13)C]lactate signal within seconds after exposure to drug. The hyperpolarized H(13)CO3 (-) and hyperpolarized [1-(13)C]alanine signals were not affected by the isoproterenol-induced elevated cardiac workload. Separate experiments confirmed that the new hyperpolarized [1-(13)C]lactate signal that arises after stimulation by isoproterenol reflects a sudden increase in total tissue lactate derived from glycogen. These results suggest that hyperpolarized pyruvate and 13C MRS may be useful for detecting abnormal glycogen metabolism in intact tissues.

Non-invasive In-cell Determination of Free Cytosolic [NAD+]/[NADH] Ratios Using Hyperpolarized Glucose Show Large Variations in Metabolic Phenotypes

Accumulating evidence suggest that the pyridine nucleotide NAD has far wider biological functions than its classical role in energy metabolism. NAD is used by hundreds of enzymes that catalyze substrate oxidation and, as such, it plays a key role in various biological processes such as aging, cell death, and oxidative stress. It has been suggested that changes in the ratio of free cytosolic [NAD(+)]/[NADH] reflects metabolic alterations leading to, or correlating with, pathological states. We have designed an isotopically labeled metabolic bioprobe of free cytosolic [NAD(+)]/[NADH] by combining a magnetic enhancement technique (hyperpolarization) with cellular glycolytic activity. The bioprobe reports free cytosolic [NAD(+)]/[NADH] ratios based on dynamically measured in-cell [pyruvate]/[lactate] ratios. We demonstrate its utility in breast and prostate cancer cells. The free cytosolic [NAD(+)]/[NADH] ratio determined in prostate cancer cells was 4 times higher than in breast cancer cells. This higher ratio reflects a distinct metabolic phenotype of prostate cancer cells consistent with previously reported alterations in the energy metabolism of these cells. As a reporter on free cytosolic [NAD(+)]/[NADH] ratio, the bioprobe will enable better understanding of the origin of diverse pathological states of the cell as well as monitor cellular consequences of diseases and/or treatments.