Lactate Signal Research Articles

Asiatic acid inhibits lactate-induced cardiomyocyte apoptosis through the regulation of the lactate signaling cascade.

The lactate signaling cascade has recently been linked to mitochondrial energy metabolism and cardiomyocyte apoptosis in several cardiovascular diseases. Asiatic acid(AA) exhibits a variety of pharmacological effects, including antioxidant and anti-apoptotic effects. In this study, we investigated the protective effects of AA against the lactate-induced apoptosis of cardiomyocytes, as well as its mechanisms of action. Neonatal rat cardiomyocytes were pre-treated with 20µMAA for 24h, followed by exposure to 20mM lactate for a further 24h. Cell viability was determined by a cell counting kit-8(CCK-8) assay. Cell apoptosis, reactive oxygen species(ROS) levels and mitochondrial membrane potential(Δψm) were evaluated by flow cytometry or fluorescence microscopy. The expression levels of mitochondrial monocarboxylate transporter1(MCT1) and cytoplasmic cytochromec, cleaved caspase-9 and caspase-3 were assayed by western blot analysis. Our results revealed that AA significantly inhibited lactate-induced apoptosis, intracellular ROS generation and the loss of Δψm. AA also increased the expression of mitochondrial MCT1 and reduced the expression of cytoplasmic cytochromec, cleaved caspase-9 and caspase-3 in the lactate-stimulated cardiomyocytes. To the best of our knowledge, our data demonstrate for the first time that AA plays a cytoprotective role in lactate-induced apoptosis by regulating the lactate signaling cascade, involving the inhibition of oxidative stress and mitochondria-dependent caspase activation, as well as the upregulation of mitochondrial MCT1 expression.

In Vivo Monitoring of Rat Spinal Cord Metabolism Using Hyperpolarized Carbon-13 MR Spectroscopic Imaging.

This study demonstrated the feasibility of using hyperpolarized 13C-MR spectroscopic imaging with [1-13C]-pyruvate to evaluate in vivo spinal cord metabolism. High pyruvate and relatively small lactate signal were observed in the cervical spinal cords of naive rats. Lactate and pyruvate measures were similar for spinal cord and supratentorial brain. The results from this study establish baseline measures for spinal cord hyperpolarized MRS imaging with 13C pyruvate. This technique holds promise as a valuable molecular imaging tool for monitoring biochemical processes in the normal and diseased spinal cord.

Abstract LB-312: The regulation of lactate metabolism in the context of EMT of breast cancer cell lines

Abstract Lactate is both a metabolite of glycolysis, and a component of several signaling pathways. In recent years, there has been an increased interest in tumor lactate metabolism and signaling, with several studies indicating that lactate is a critical regulator of cancer development. Indeed, tumor lactate levels correlate with increased metastasis, tumor recurrence, and poor overall survival of cancer patients. This recent interest in lactate metabolism has been exclusively studied in the context of neoplastic transformation per se. However, little is known about shifts in lactate metabolism in the context of metastatic transformation that confers increased motility and invasiveness on cancer cells. Epithelial-Mesenchymal transition (EMT) has been proposed as a mechanism involved in the early stage of metastasis. EMT is a physiological process during embryogenesis, but is also activated in some epithelial-derived cancers. EMT reduces cell-cell contacts and incrases the motility and invasiveness of cancer cells. Previously our lab reported that prolonged mammosphere culture induced EMT in two epithelial breast cancer cell lines, MCF-7 and BT-474 cells. This generated the respective mesenchymal types, MCF-7EMT and BT-474EMT. This study has examined whether shifts in lactate metabolism occur during EMT, using these four cell lines. All cells were cultured in a 3-dimensional Matrigel matrix with modified medium that contains glucose, glutamine, pyruvate and lactate at physiological concentrations in order to more accurately mimic the tumor microenvironment than standarad 2-dimensional cell culture conditions. We observed that EMT-induced MCF-7EMT and BT-474EMT exhibit higher glycolytic rates and export more lactate compared to their respective parental epithelial cell lines. Moreover, EMT-induced MCF-7EMT and BT-474EMT cells display altered expression of lactate-related genes. In particular, both the endogenous lactate receptor, GPR81, and the lactate importer, MCT1, are highly expressed in epithelial MCF-7 and BT-474 cells, but are suppressed in post-EMT mesenchymal MCF7EMT and BT474EMT cells. Additionally, the knockdown of GPR81 in epithelial MCF-7 and BT-474 cell lines significantly reduced the expression of MCT1. NMR analysis of cytoplasmic extracts revealed that intracellular lactate levels are significantly higher in the epithelial MCF7 cells vs. the corresponding mesenchymal MCF7EMT cells. The role of GPR81 in cellular lactate balance and cell proliferation was examined by siRNA approach. GPR81 knockdown significantly decreased MCT1 and intracellular lactate, as well as cell proliferation in both epithelial MCF7 and BT474 cells. Interestingly, we found that β-estradiol (E2) treatment of MCF7 cells up-regulates MCT1 expression, whereas Tamoxifen treatment suppresses MCT1 expression. These hormonal effects link the ability of E2 and Tamoxifen to stimulate or inhibit, respectively, cell proliferation through their actions on MCT1-mediated lactate import. Further study will examine whether changes in intracellular lactate levels feedback on either GPR81 and/or MCT1. Citation Format: Denisse G. Tafur, Bruce A. White. The regulation of lactate metabolism in the context of EMT of breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-312.

Abstract SY19-03: Translation of hyperpolarized 13C imaging to the clinic

Abstract A decade after the first demonstration of dissolution-DNP by Ardenkjaer-Larsen et al.(1), metabolic imaging using HP [1-13C]pyruvate was accomplished in prostate cancer patients(2). Translation of this technology into patients required the development and optimization of a clinical DNP polarizer, the implementation of multinuclear MR capabilities on clinical MRI scanners, the construction of specialized radio frequency (RF) coils to detect 13C nuclei, and developing new MR pulse sequences to efficiently capture the signal (2). These steps towards translation will be presented with emphasis on where the translation process stands currently. Additionally, studies involving preclinical prostate cancer models played an important role in establishing the potential clinical value of HP [1-13C]pyruvate in prostate cancer. Preclinical studies in prostate cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment(3). This preclinical data was critical to obtaining the US Food and Drug Administration (FDA) Investigational New Drug (IND) approval for the first human trial and setting the stage for ongoing clinical trials of HP [1-13C]pyruvate. The Phase 1 clinical trial evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Increasing evidence points to prostate cancer being a disease strongly linked to abnormal metabolism, and several important metabolic shifts have been associated with the presence and progression of prostate cancer (4). The median time to delivery of hyperpolarized 13C pyruvate was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1- 13C]pyruvate in regions of biopsy-proven cancer. There was increased conversion of [1-13C]pyruvate to [1-13C]lactate in the tumor, with the [1-13C]pyruvate reaching a maximum by 18 ± 4 s and the [1-13C]lactate reached a maximum at 27 ± 2 s after the start of acquisition, which was 5 s after the end of injection. The timings of maximal signal were similar to those observed in preclinical murine and human tissue slice culture hyperpolarized [1-13C]pyruvate studies (4). Overall, the pyruvate to lactate flux (mean ± SD) was 0.045 ± 0.025 s−1 for [1-13C]pyruvate to [1-13C]lactate in tumor voxels and 0.009 ± 0.003 s−1 for voxels coming from regions that included blood vessels. Although there was sufficient lactate signal to determine a pyruvate to lactate flux for the contralateral normal prostate in this example (0.016 s−1), a majority of normal prostate demonstrated hyperpolarized lactate levels that were to low to accurately calculate a pyruvate to lactate flux. Ongoing hyperpolarized [1-13C]pyruvate studies in prostate cancer patients are investigating the test-retest reproducibility of [1-13C]pyruvate delivery and metabolism, response to androgen deprivation therapy, the development of androgen-independent disease and its response to new 2nd line therapeutic approaches. The current results of these studies will be presented. Sixteen sites from around the world currently have DNP Polarizers capable of performing patient studies, with a number of new installations planned for the coming year. Additionally, eighteen hyperpolarized patient studies are currently funded involving a total of 864 patients with prostate, brain and breast cancer prior to and after therapy. Most of these trials are focused on the use of HP [1-13C]pyruvate, but there are a number of new hyperpolarized 13C labeled probes that are in the clinical pipeline and these will be summarized in this lecture.

Effects of hypotension and/or hypocapnia during sevoflurane anesthesia on perfusion and metabolites in the developing brain of piglets-a blinded randomized study.

Hypotension (HT) and/or hypocapnia (HC) are frequent complications occurring during pediatric anesthesia and may cause cerebral injury in the developing brain. The aim of this study is to investigate the effects of HT and/or HC on perfusion and metabolism in the developing brain. Twenty-eight piglets were randomly allocated to four groups: control (C), HT, HC, and hypotension and hyocapnia (HTC). Anesthesia was induced and maintained using sevoflurane. Fentanyl was added for instrumentation. Piglets were fully monitored and their lungs were artificially ventilated. Before treatment, conventional magnetic resonance imaging (MRI), dynamic susceptibility-contrast-enhanced T2*-weighted MRI (DSC-MRI), and single voxel proton MR spectroscopy ((1) H MRS) were performed. Hypotension (mean arterial blood pressure: 30 ± 3 mmHg) was induced by blood withdrawal and nitroprusside infusion, and hyperventilation was used to induce HC (PaCO2 : 2.7-3.3 kPa). (1) H MRS and DSC-MRI were repeated immediately once treatment goals were achieved and 120 min later. Radiologists were blinded to the groups. DSCI-MRI and (1) H MRS analyses were performed in the thalamus, occipital and parietal lobe, hippocampus, and watershed areas. In comparison to C, mean time to peak (TTP) increased with HTC in all brain areas as assessed with DSC-MRI (n = 26). Using (1) H MRS, a significant decrease in N-acetyl aspartate, choline, and myoinositol, as well as an increase in glutamine-glutamate complex (Glx) were detected independent of group. Compared to C, changes were more pronounced for Glx (due to an increase in glutamate) and myoinositol with HTC, for N-acetyl aspartate with HT, and for Glx with HC. No lactate signal was present. The combination of HT and HC during sevoflurane anesthesia resulted in alteration of cerebral perfusion with signs of neuronal dysfunction and early neuronal ischemia. HT and HC alone also resulted in signs of metabolic disturbances despite the absence of detectable cerebral perfusion alterations.

Voxel-by-voxel correlations of perfusion, substrate, and metabolite signals in dynamic hyperpolarized (13) C imaging.

In this study, a mixture of pyruvic acid and the perfusion agent HP001 was co-polarized for simultaneous assessment of perfusion and metabolism in vivo. The pre-polarized mixture was administered to rats with subcutaneous MDA-MB-231 breast cancer xenografts and imaged using an interleaved sequence with designed spectral-spatial pulses and flyback echo-planar readouts. Voxel-by-voxel signal correlations from 10 animals (15 data sets) were analyzed for tumour, kidney, and muscle regions of interest. The relationship between perfusion and hyperpolarized signal was explored on a voxel-by-voxel basis in various metabolically active tissues, including tumour, healthy kidneys, and skeletal muscle. Positive pairwise correlations between lactate, pyruvate, and HP001 observed in all 10 tumours suggested that substrate delivery was the dominant factor limiting the conversion of pyruvate to lactate in the tumour model used in this study. On the other hand, in cases where conversion is the limiting factor, such as in healthy kidneys, both pyruvate and lactate can act as excellent perfusion markers. In intermediate cases between the two limits, such as in skeletal muscle, some perfusion information may be inferred from the (pyruvate + lactate) signal distribution. Co-administration of pyruvate with a dynamic nuclear polarization (DNP) perfusion agent is an effective approach for distinguishing between slow metabolism and poor perfusion and a practical strategy for lactate signal normalization to account for substrate delivery, especially in cases of rapid pyruvate-to-lactate conversion and in poorly perfused regions with inadequate pyruvate signal-to-noise ratio for reliable determination of the lactate-to-pyruvate ratio. Copyright © 2016 John Wiley & Sons, Ltd.

Diagnostic value of MRS-quantified brain tissue lactate level in identifying children with mitochondrial disorders

ObjectivesMagnetic resonance spectroscopy (MRS) of children with or without neurometabolic disease is used for the first time for quantitative assessment of brain tissue lactate signals, to elaborate on previous suggestions of MRS-detected lactate as a marker of mitochondrial disease.MethodsMultivoxel MRS of a transverse plane of brain tissue cranial to the ventricles was performed in 88 children suspected of having neurometabolic disease, divided into ‘definite’ (n = 17, ≥1 major criteria), ‘probable’ (n = 10, ≥2 minor criteria), ‘possible’ (n = 17, 1 minor criterion) and ‘unlikely’ mitochondrial disease (n = 44, none of the criteria). Lactate levels, expressed in standardized arbitrary units or relative to creatine, were derived from summed signals from all voxels. Ten ‘unlikely’ children with a normal neurological exam served as the MRS reference subgroup. For 61 of 88 children, CSF lactate values were obtained.ResultsMRS lactate level (>12 arbitrary units) and the lactate-to-creatine ratio (L/Cr >0.22) differed significantly between the definite and the unlikely group (p = 0.015 and p = 0.001, respectively). MRS L/Cr also differentiated between the probable and the MRS reference subgroup (p = 0.03). No significant group differences were found for CSF lactate.ConclusionMRS-quantified brain tissue lactate levels can serve as diagnostic marker for identifying mitochondrial disease in children.Key points• MRS-detected brain tissue lactate levels can be quantified.• MRS lactate and lactate/Cr are increased in children with mitochondrial disease.• CSF lactate is less suitable as marker of mitochondrial disease.

Assessing the transport rate of hyperpolarized pyruvate and lactate from the intra- to the extracellular space.

The use of [1-(13) C]pyruvate hyperpolarized by means of dynamic nuclear polarization provides a direct way to track the metabolic transformations of this metabolite in vivo and in cell cultures. The identification of the intra- and extracellular contributions to the (13) C NMR resonances is not straightforward. In order to obtain information about the rate of pyruvate and lactate transport through the cellular membrane, we set up a method that relies on the sudden 'quenching' of the extracellular metabolites' signal. The paramagnetic Gd-tetraazacyclododecane triacetic acid (Gd-DO3A) complex was used to dramatically decrease the longitudinal relaxation time constants of the (13) C-carboxylate resonances of both pyruvate and lactate. When Gd-DO3A was added to an MCF-7 cellular culture, which had previously received a dose of hyperpolarized [1-(13) C]pyruvate, the contributions of the extracellular pyruvate and lactate signals were deleted. From the analysis of the decay curves of the (13) C-carboxylate resonances of pyruvate and lactate it was possible to extract information about the exchange rate of the two metabolites across the cellular membrane. In particular, it was found that, in the reported experimental conditions, the lactate transport from the intra- to the extracellular space is not much lower than the rate of lactate formation. The method reported herein is non-destructive and it could be translated to in vivo studies. It opens a route for the use of hyperpolarized pyruvate to assess altered activity of carboxylate transporter proteins that may occur in pathological conditions. Copyright © 2016 John Wiley & Sons, Ltd.

Accelerated high-bandwidth MR spectroscopic imaging using compressed sensing.

To develop a compressed sensing (CS) acceleration method with a high spectral bandwidth exploiting the spatial-spectral sparsity of MR spectroscopic imaging (MRSI). Accelerations were achieved using blip gradients during the readout to perform nonoverlapped and stochastically delayed random walks in kx -ky -t space, combined with block-Hankel matrix completion for efficient reconstruction. Both retrospective and prospective CS accelerations were applied to (13) C MRSI experiments, including in vivo rodent brain and liver studies with administrations of hyperpolarized [1-(13) C] pyruvate at 7.0 Tesla (T) and [2-(13) C] dihydroxyacetone at 3.0 T, respectively. In retrospective undersampling experiments using in vivo 7.0 T data, the proposed method preserved spectral, spatial, and dynamic fidelities with R(2) ≥ 0.96 and ≥ 0.87 for pyruvate and lactate signals, respectively, 750-Hz spectral separation, and up to 6.6-fold accelerations. In prospective in vivo experiments, with 3.8-fold acceleration, the proposed method exhibited excellent spatial localization of metabolites and peak recovery for pyruvate and lactate at 7.0 T as well as for dihydroxyacetone and its metabolic products with a 4.5-kHz spectral span (140 ppm at 3.0 T). This study demonstrated the feasibility of a new CS approach to accelerate high spectral bandwidth MRSI experiments. Magn Reson Med 76:369-379, 2016. © 2016 Wiley Periodicals, Inc.

Detection of localized changes in the metabolism of hyperpolarized gluconeogenic precursors 13 C-lactate and 13 C-pyruvate in kidney and liver.

The purpose of this study was to characterize tissue-specific alterations in metabolism of hyperpolarized (HP) gluconeogenic precursors 13 C-lactate and 13 C-pyruvate by rat liver and kidneys under conditions of fasting or insulin-deprived diabetes. Seven normal rats were studied by MR spectroscopic imaging of both HP 13 C-lactate and 13 C-pyruvate in both normal fed and 24 h fasting states, and seven additional rats were scanned after induction of diabetes by streptozotocin (STZ) with insulin withdrawal. Phosphoenolpyruvate carboxykinase (PEPCK) expression levels were also measured in liver and kidney tissues of the STZ-treated rats. Multiple sets of significant signal modulations were detected, with graded intensity in general between fasting and diabetic states. An approximate two-fold reduction in the ratio of 13 C-bicarbonate to total 13 C signal was observed in both organs in fasting. The ratio of HP lactate-to-alanine was markedly altered, ranging from a liver-specific 54% increase in fasting, to increases of 69% and 92% in liver and kidney, respectively, in diabetes. Diabetes resulted in a 40% increase in renal lactate signal. STZ resulted in 5.86-fold and 2.73-fold increases in PEPCK expression in liver and kidney, respectively. MRI of HP 13 C gluconeogenic precursors may advance diabetes research by clarifying organ-specific roles in abnormal diabetic metabolism. Magn Reson Med 77:1429-1437, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Hyperpolarized (13)C-lactate to (13)C-bicarbonate ratio as a biomarker for monitoring the acute response of anti-vascular endothelial growth factor (anti-VEGF) treatment.

Hyperpolarized [1-(13)C]pyruvate MRS provides a unique imaging opportunity to study the reaction kinetics and enzyme activities of in vivo metabolism because of its favorable imaging characteristics and critical position in the cellular metabolic pathway, where it can either be reduced to lactate (reflecting glycolysis) or converted to acetyl-coenzyme A and bicarbonate (reflecting oxidative phosphorylation). Cancer tissue metabolism is altered in such a way as to result in a relative preponderance of glycolysis relative to oxidative phosphorylation (i.e. Warburg effect). Although there is a strong theoretical basis for presuming that readjustment of the metabolic balance towards normal could alter tumor growth, a robust noninvasive in vivo tool with which to measure the balance between these two metabolic processes has yet to be developed. Until recently, hyperpolarized (13)C-pyruvate imaging studies had focused solely on [1-(13)C]lactate production because of its strong signal. However, without a concomitant measure of pyruvate entry into the mitochondria, the lactate signal provides no information on the balance between the glycolytic and oxidative metabolic pathways. Consistent measurement of (13)C-bicarbonate in cancer tissue, which does provide such information, has proven difficult, however. In this study, we report the reliable measurement of (13)C-bicarbonate production in both the healthy brain and a highly glycolytic experimental glioblastoma model using an optimized (13)C MRS imaging protocol. With the capacity to obtain signal in all tumors, we also confirm for the first time that the ratio of (13)C-lactate to (13)C-bicarbonate provides a more robust metric relative to (13)C-lactate for the assessment of the metabolic effects of anti-angiogenic therapy. Our data suggest a potential application of this ratio as an early biomarker to assess therapeutic effectiveness. Furthermore, although further study is needed, the results suggest that anti-angiogenic treatment results in a rapid normalization in the relative tissue utilization of glycolytic and oxidative phosphorylation by tumor tissue.

Separation and quantification of lactate and lipid at 1.3 ppm by diffusion-weighted magnetic resonance spectroscopy.

To separate the spectrally overlapped lactate and lipid signals at 1.3 ppm using diffusion-weighted magnetic resonance spectroscopy (DW-MRS) based on their large diffusivity difference. DW-MRS was applied to the gel phantoms containing lactate and lipid droplets, and to the rat brain tumors. Lactate and lipid signals and their apparent diffusion coefficients were computed from the diffusion-weighted proton spectra. Biexponential fitting and direct spectral subtraction approaches were employed and compared. DW-MRS could effectively separate lactate and lipid signals both in phantoms and rat brain C6 glioma by biexponential fitting. In phantoms, lactate and lipid signals highly correlated with the known lactate concentration and lipid volume fractions. In C6 glioma, both lactate and lipid signals were detected, and the lipid signal was an order of magnitude higher than lactate signal. The spectral subtraction approach using three diffusion weightings also allowed the separation of lactate and lipid signals, yielding results comparable to those by the biexponential fitting approach. DW-MRS presents a new approach to separate and quantify spectrally overlapped molecules and/or macromolecules, such as lactate and lipid, by using the diffusivity difference associated with their different sizes or mobility within tissue microstructure. Magn Reson Med 77:480-489, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Astrocyte-Neuron Interactions during Learning May Occur by Lactate Signaling Rather than Metabolism.

Biology is full of instances of exaptation, the functional shift or co-optation of a trait during evolution (Gould and Vrba, 1982). Exaptation played a critical role in human brain evolution. For example, hominin brain expansion is thought to have happened opportunistically upon food resources rich in brain-selective nutrients (Tattersall, 2010). Prehensile hands and bipedalism were other enabling factors in this process, as both features preceded the expansion of the brain, and notably, the development and utilization of tools (Wood, 2010). Similarly, central and peripheral vocal structures, initially used for a variety of non-linguistic reasons (chewing, larynx protection, size exaggeration), were pre-existing conditions to, and provided the anatomical basis for, the evolution of language (e.g., MacNeilage, 2010). The very emergence of abstract cognitive abilities in humans are hypothesized to have evolved from faculties originally developed for other purposes (Pinker, 2010).

Multivariate metabotyping of plasma predicts survival in patients with decompensated cirrhosis

Predicting survival in decompensated cirrhosis (DC) is important in decision making for liver transplantation and resource allocation. We investigated whether high-resolution metabolic profiling can determine a metabolic phenotype associated with 90-day survival. Two hundred and forty-eight subjects underwent plasma metabotyping by (1)H nuclear magnetic resonance (NMR) spectroscopy and reversed-phase ultra-performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-TOF-MS; DC: 80-derivation set, 101-validation; stable cirrhosis (CLD) 20 and 47 healthy controls (HC)). (1)H NMR metabotyping accurately discriminated between surviving and non-surviving patients with DC. The NMR plasma profiles of non-survivors were attributed to reduced phosphatidylcholines and lipid resonances, with increased lactate, tyrosine, methionine and phenylalanine signal intensities. This was confirmed on external validation (area under the receiver operating curve [AUROC]=0.96 (95% CI 0.90-1.00, sensitivity 98%, specificity 89%). UPLC-TOF-MS confirmed that lysophosphatidylcholines and phosphatidylcholines [LPC/PC] were downregulated in non-survivors (UPLC-TOF-MS profiles AUROC of 0.94 (95% CI 0.89-0.98, sensitivity 100%, specificity 85% [positive ion detection])). LPC concentrations negatively correlated with circulating markers of cell death (M30 and M65) levels in DC. Histological examination of liver tissue from DC patients confirmed increased hepatocyte cell death compared to controls. Cross liver sampling at time of liver transplantation demonstrated that hepatic endothelial beds are a source of increased circulating total cytokeratin-18 in DC. Plasma metabotyping accurately predicts mortality in DC. LPC and amino acid dysregulation is associated with increased mortality and severity of disease reflecting hepatocyte cell death.

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<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.

Bladder cancer cells re-educate TAMs through lactate shuttling in the microfluidic cancer microenvironment.

In the present study, we aimed to investigate the influence of lactate shuttling on the functional polarization and spatial distribution of transitional cell carcinoma of the bladder (TCCB) cells and macrophages. We designed a microfluidic coculture chip for real-time integrative assays. The effect of lactate shuttling on the re-education of macrophages by TCCB cells was explored by measuring the levels of NO using a total NO assay kit and by evaluating the protein expression of iNOS, p-NFkB-p65, Arg-1 and HIF-1α via cell immunofluorescence and western blotting. Additionally, we examined TCCB cell viability using acridine orange/ethidium bromide (AO/EB) and MitoTracker staining. Moreover, the concentration distributions of lactate and large signaling proteins in the culture chambers were measured using 4',6-diamidino-2-phenylindole (DAPI) and fluorescein isothiocyanate-dextran (FITC-dextran). Furthermore, the recruitment of macrophages and the influence of macrophages on BC metastasis were observed via light microscopy. We confirmed that TCCB cells reprogrammed macrophages into an M2 phenotype. Moreover, lactate inhibited M1 polarization and induced M2 polarization of macrophages, but blockade of cancer cell-macrophage lactate flux significantly inhibited the re-education of macrophages by TCCB cells. In addition, lactate diffused faster and deeper than large signaling proteins in the microfluidic tumor microenvironment. Furthermore, lactate alone induced the migration of macrophages, and M1, but not M2, macrophages reduced the motility of TCCB cells. TCCB cells reprogrammed macrophages into an M2 phenotype in a manner that depended on cancer cell-TAM lactate flux. Furthermore, the lactate shuttle may be a determinant of the density of TAMs in tumor tissue.

Lactate does not activate NF-κB in oxidative tumor cells.

The lactate anion is currently emerging as an oncometabolite. Lactate, produced and exported by glycolytic and glutaminolytic cells in tumors, can be recycled as an oxidative fuel by oxidative tumors cells. Independently of hypoxia, it can also activate transcription factor hypoxia-inducible factor-1 (HIF-1) in tumor and endothelial cells, promoting angiogenesis. These protumoral activities of lactate depend on lactate uptake, a process primarily facilitated by the inward, passive lactate-proton symporter monocarboxylate transporter 1 (MCT1); the conversion of lactate and NAD+ to pyruvate, NADH and H+ by lactate dehydrogenase-1 (LDH-1); and a competition between pyruvate and α-ketoglutarate that inhibits prolylhydroxylases (PHDs). Endothelial cells do not primarily use lactate as an oxidative fuel but, rather, as a signaling agent. In addition to HIF-1, lactate can indeed activate transcription factor nuclear factor-κB (NF-κB) in these cells, through a mechanism not only depending on PHD inhibition but also on NADH alimenting NAD(P)H oxidases to generate reactive oxygen species (ROS). While NF-κB activity in endothelial cells promotes angiogenesis, NF-κB activation in tumor cells is known to stimulate tumor progression by conferring resistance to apoptosis, stemness, pro-angiogenic and metastatic capabilities. In this study, we therefore tested whether exogenous lactate could activate NF-κB in oxidative tumor cells equipped for lactate signaling. We report that, precisely because they are oxidative, HeLa and SiHa human tumor cells do not activate NF-κB in response to lactate. Indeed, while lactate-derived pyruvate is well-known to inhibit PHDs in these cells, we found that NADH aliments oxidative phosphorylation (OXPHOS) in mitochondria rather than NAD(P)H oxidases in the cytosol. These data were confirmed using oxidative human Cal27 and MCF7 tumor cells. This new information positions the malate-aspartate shuttle as a key player in the oxidative metabolism of lactate: similar to glycolysis that aliments OXPHOS with pyruvate produced by pyruvate kinase and NADH produced by glyceraldehyde-3-phosphate dehydrogenase (GAPDH), oxidative lactate metabolism aliments OXPHOS in oxidative tumor cells with pyruvate and NADH produced by LDH1.

MRI with hyperpolarised [1-13C]pyruvate detects advanced pancreatic preneoplasia prior to invasive disease in a mouse model

ObjectivesPancreatic cancer (PCa) is treatable by surgery when detected at an early stage. Non-invasive imaging methods able to detect both established tumours and their precursor lesions are needed to select...

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 (>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&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 [1-(13) C]pyruvate MRI for noninvasive examination of placental metabolism and nutrient transport: A feasibility study in pregnant guinea pigs.

To test the feasibility of hyperpolarized [1-(13) C]pyruvate magnetic resonance imaging (MRI) for noninvasive examination of guinea pig fetoplacental metabolism and nutrient transport. Seven pregnant guinea pigs with a total of 30 placentae and fetuses were anesthetized and scanned at 3T. T1 -weighted (1) H images were obtained from the maternal abdomen. An 80 mM solution of hyperpolarized [1-(13) C]pyruvate (hereafter referred to as pyruvate) was injected into a vein in the maternal foot. Time-resolved 3D (13) C images were acquired starting 10 seconds after the beginning of bolus injection and every 10 seconds after to 50 seconds. The pregnant guinea pigs were recovered after imaging. Regions of interest (ROIs) were drawn around the maternal heart and each placenta and fetal liver in all slices in the (1) H images. These ROIs were copied to the (13) C images and were used to calculate the sum of the pyruvate and lactate signal intensities for each organ. The signal intensities were normalized by the volume of the organ and the maximum signal in the maternal heart. No adverse events were observed in the pregnant guinea pigs and natural pupping occurred at term (∼68 days). Pyruvate signal was observed in all 30 placentae, and lactate, a by-product of pyruvate metabolism, was also observed in all placentae. The maximum pyruvate and lactate signals in placentae occurred at 20 seconds. In addition to the observation of pyruvate and lactate signals in the placentae, both pyruvate and lactate signals were observed in all fetal livers. The maximum pyruvate and lactate signals in the fetal livers occurred at 10 seconds and 20 seconds, respectively. This work demonstrates the feasibility of using hyperpolarized [1-(13) C]pyruvate MRI to noninvasively examine fetoplacental metabolism and transport of pyruvate in guinea pigs. Hyperpolarized (13) C MRI may provide a novel method for longitudinal studies of fetoplacental abnormalities.