Hyperpolarized Lactate Research Articles

Hyperpolarized [1-13 C] pyruvate MRSI to detect metabolic changes in liver in a methionine and choline-deficient diet rat model of fatty liver disease.

Nonalcoholic fatty liver disease is an important cause of chronic liver disease. There are limited methods for monitoring metabolic changes during progression to steatohepatitis. Hyperpolarized 13 C MRSI (HP 13 C MRSI) was used to measure metabolic changes in a rodent model of fatty liver disease. Fifteen Wistar rats were placed on a methionine- and choline-deficient (MCD) diet for 1-18 weeks. HP 13 C MRSI, T2 -weighted imaging, and fat-fraction measurements were obtained at 3 T. Serum aspartate aminotransaminase, alanine aminotransaminase, and triglycerides were measured. Animals were sacrificed for histology and measurement of tissue lactate dehydrogenase (LDH) activity. Animals lost significant weight (13.6% ± 2.34%), an expected characteristic of the MCD diet. Steatosis, inflammation, and mild fibrosis were observed. Liver fat fraction was 31.7% ± 4.5% after 4 weeks and 22.2% ± 4.3% after 9 weeks. Lactate-to-pyruvate and alanine-to-pyruvate ratios decreased significantly over the study course; were negatively correlated with aspartate aminotransaminase and alanine aminotransaminase (r = -[0.39-0.61]); and were positively correlated with triglycerides (r = 0.59-0.60). Despite observed decreases in hyperpolarized lactate signal, LDH activity increased by a factor of 3 in MCD diet-fed animals. Observed decreases in lactate and alanine hyperpolarized signals on the MCD diet stand in contrast to other studies of liver injury, where lactate and alanine increased. Observed hyperpolarized metabolite changes were not explained by alterations in LDH activity, suggesting that changes may reflect co-factor depletion known to occur as a result of oxidative stress in the MCD diet. HP 13 C MRSI can noninvasively measure metabolic changes in the MCD model of chronic liver disease.

Lactate and Lactylation: Clinical Applications of Routine Carbon Source and Novel Modification in Human Diseases

Cell metabolism generates numerous intermediate metabolites that could serve as feedback and feed-forward regulation substances for posttranslational modification. Lactate, a metabolic product of glycolysis, has recently been conceptualized to play a pleiotropic role in shaping cell identities through metabolic rewiring and epigenetic modifications. Lactate-derived carbons, sourced from glucose, mediate the crosstalk among glycolysis, lactate, and lactylation. Furthermore, the multiple metabolic fates of lactate make it an ideal substrate for metabolic imaging in clinical application. Several studies have identified the crucial role of protein lactylation in human diseases associated with cell fate determination, embryonic development, inflammation, neoplasm, and neuropsychiatric disorders. Herein, this review will focus on the metabolic fate of lactate-derived carbon to provide useful information for further research and therapeutic approaches in human diseases. We comprehensively discuss its role in reprogramming and modification during the regulation of glycolysis, the clinical translation prospects of the hyperpolarized lactate signal, lactyl modification in human diseases, and its application with other techniques and omics.

Toward Optimizing and Understanding Reversible Hyperpolarization of Lactate Esters Relayed from para-Hydrogen.

The SABRE-Relay hyperpolarization method is used to enhance the 1H and 13C NMR signals of lactate esters, which find use in a wide range of medical, pharmaceutical, and food science applications. This is achieved by the indirect relay of magnetization from para-hydrogen, a spin isomer of dihydrogen, to OH-containing lactate esters via a SABRE-hyperpolarized NH intermediary. This delivers 1H and 13C NMR signal enhancements as high as 245- and 985-fold, respectively, which makes the lactate esters far more detectable using NMR. DFT-calculated J-couplings and spin dynamics simulations indicate that, while polarization can be transferred from the lactate OH to other 1H nuclei via the J-coupling network, incoherent mechanisms are needed to polarize the 13C nuclei at the 6.5 mT transfer field used. The resulting sensitivity boost is predicted to be of great benefit for the NMR detection and quantification of low concentrations (<mM) of lactate esters and could provide a useful precursor for the production of hyperpolarized lactate, a key metabolite.

Imaging biomarkers of TERT or GABPB1 silencing in TERT-positive glioblastoma.

TERT promoter mutations are observed in 80% of wild-type IDH glioblastoma (GBM). Moreover, the upstream TERT transcription factor GABPB1 was recently identified as a cancer-specific therapeutic target for tumors harboring a TERT promoter mutation. In that context, noninvasive imaging biomarkers are needed for the detection of TERT modulation. Multiple GBM models were investigated as cells and in vivo tumors and the impact of TERT silencing, either directly or by targeting GABPB1, was determined using 1H and hyperpolarized 13C magnetic resonance spectroscopy (MRS). Changes in associated metabolic enzymes were also investigated. 1H-MRS revealed that lactate and glutathione (GSH) were the most significantly altered metabolites when either TERT or GABPB1 was silenced, and lactate and GSH levels were correlated with cellular TERT expression. Consistent with the drop in lactate, 13C-MRS showed that hyperpolarized [1-13C]lactate production from [1-13C]pyruvate was also reduced when TERT was silenced. Mechanistically, the reduction in GSH was associated with a reduction in pentose phosphate pathway flux, reduced activity of glucose-6-phosphate dehydrogenase, and reduced NADPH. The drop in lactate and hyperpolarized lactate were associated with reductions in glycolytic flux, NADH, and expression/activity of GLUT1, monocarboxylate transporters, and lactate dehydrogenase A. Our study indicates that MRS-detectable GSH, lactate, and lactate production could serve as metabolic biomarkers of response to emerging TERT-targeted therapies for GBM with activating TERT promoter mutations. Importantly these biomarkers are readily translatable to the clinic, and thus could ultimately improve GBM patient management.

Co-Polarized [1-13C]Pyruvate and [1,3-13C2]Acetoacetate Provide a Simultaneous View of Cytosolic and Mitochondrial Redox in a Single Experiment.

Cellular redox is intricately linked to energy production and normal cell function. Although the redox states of mitochondria and cytosol are connected by shuttle mechanisms, the redox state of mitochondria may differ from redox in the cytosol in response to stress. However, detecting these differences in functioning tissues is difficult. Here, we employed 13C magnetic resonance spectroscopy (MRS) and co-polarized [1-13C]pyruvate and [1,3-13C2]acetoacetate ([1,3-13C2]AcAc) to monitor production of hyperpolarized (HP) lactate and β-hydroxybutyrate as indicators of cytosolic and mitochondrial redox, respectively. Isolated rat hearts were examined under normoxic conditions, during low-flow ischemia, and after pretreatment with either aminooxyacetate (AOA) or rotenone. All interventions were associated with an increase in [Pi]/[ATP] measured by 31P NMR. In well-oxygenated untreated hearts, rapid conversion of HP [1-13C]pyruvate to [1-13C]lactate and [1,3-13C2]AcAc to [1,3-13C2]β-hydroxybutyrate ([1,3-13C2]β-HB) was readily detected. A significant increase in HP [1,3-13C2]β-HB but not [1-13C]lactate was observed in rotenone-treated and ischemic hearts, consistent with an increase in mitochondrial NADH but not cytosolic NADH. AOA treatments did not alter the productions of HP [1-13C]lactate or [1,3-13C2]β-HB. This study demonstrates that biomarkers of mitochondrial and cytosolic redox may be detected simultaneously in functioning tissues using co-polarized [1-13C]pyruvate and [1,3-13C2]AcAc and 13C MRS and that changes in mitochondrial redox may precede changes in cytosolic redox.

Cardiac metabolic imaging using hyperpolarized [1-13 C]lactate as a substrate.

Hyperpolarized (HP) [1-13 C]lactate is an attractive alternative to [1-13 C]pyruvate as a substrate to investigate cardiac metabolism in vivo: it can be administered safely at a higher dose and can be polarized to a degree similar to pyruvate via dynamic nuclear polarization. While 13 C cardiac experiments using HP lactate have been performed in small animal models, they have not been demonstrated in large animal models or humans. Utilizing the same hardware and data acquisition methods as the first human HP 13 C cardiac study, 13 C metabolic images were acquired following injections of HP [1-13 C]lactate in porcine hearts. Data were also acquired using HP [1-13 C]pyruvate for comparison. The 13 C bicarbonate signal was localized to the myocardium and had a similar appearance with both substrates for all animals. No 13 C pyruvate signal was detected in the experiments following injection of HP 13 C lactate. The signal-to-noise ratio (SNR) of injected lactate was 88 ± 4% of the SNR of injected pyruvate, and the SNR of bicarbonate in the experiments using lactate as the substrate was 52 ± 19% of the SNR in the experiments using pyruvate as the substrate. The lower SNR was likely due to the shorter T1 of [1-13 C]lactate as compared with [1-13 C]pyruvate and the additional enzyme-catalyzed metabolic conversion step before the 13 C nuclei from [1-13 C]lactate were detected as 13 C bicarbonate. While challenges remain, the potential of HP lactate as a substrate for clinical metabolic imaging of human heart has been demonstrated.

Modeling hyperpolarized lactate signal dynamics in cells, patient-derived tissue slice cultures and murine models.

Determining the aggressiveness of renal cell carcinoma (RCC) noninvasively is a critical part of the diagnostic workup for treating this disease that kills more than 15,000 people annually in the USA. Recently, we have shown that not only the amount of lactate produced, as a consequence of the Warburg effect, but also its efflux out of the cell, is a critical marker of RCC aggressiveness and differentiating RCCs from benign renal tumors. Enzymatic conversions can now be measured in situ with hyperpolarized (HP) 13 C magnetic resonance (MR) on a sub-minute time scale. Using RCC models, we have shown that this technology can interrogate in real time both lactate production and compartmentalization, which are associated with tumor aggressiveness. The dynamic HP MR data have enabled us to robustly characterize parameters that have been elusive to measure directly in intact living cells and murine tumors thus far. Specifically, we were able to measure the same intracellular lactate longitudinal relaxation time in three RCC cell lines of 16.42 s, and lactate efflux rate ranging from 0.14 to 0.8 s-1 in the least to the most aggressive RCC cell lines and correlate it to monocarboxylate transporter isoform 4 expression. We also analyzed dynamic HP lactate and pyruvate data from orthotopic murine RCC tumors using a simplified one-compartment model, and showed comparable apparent pyruvate to lactate conversion rate (kPL ) values with those measured in vitro. This kinetic modeling was then extended to characterize the lactate dynamics in patient-derived living RCC tissue slices; and even without direct measurement of the extracellular lactate signal the efflux parameter was still assessed and was distinct between the benign renal tumors and RCCs. Across all these preclinical models, the rate parameters of kPL and lactate efflux correlated to cancer aggressiveness, demonstrating the validity of our modeling approach for noninvasive assessment of RCC aggressiveness.

Simultaneous Metabolic and Perfusion Imaging Using Hyperpolarized 13C MRI Can Evaluate Early and Dose-Dependent Response to Radiation Therapy in a Prostate Cancer Mouse Model

To investigate use of a novel imaging approach, hyperpolarized (HP) 13C magnetic resonance imaging (MRI) for simultaneous metabolism and perfusion assessment, to evaluate early and dose-dependent response to radiation therapy (RT) in a prostate cancer mouse model. Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (n = 18) underwent single-fraction RT (4-14 Gy steep dose across the tumor) and were imaged serially at pre-RT baseline and 1, 4, and 7 days after RT using HP 13C MRI with combined [1-13C]pyruvate (metabolic active agent) and [13C]urea (perfusion agent), coupled with conventional multiparametric 1H MRI including T2-weighted, dynamic contrast-enhanced, and diffusion-weighted imaging. Tumor tissues were collected 4 and 7 days after RT for biological correlative studies. We found a significant decrease in HP pyruvate-to-lactate conversion in tumors responding to RT, with concomitant significant increases in HP pyruvate-to-alanine conversion and HP urea signal; the opposite changes were observed in tumors resistant to RT. Moreover, HP lactate change was dependent on radiation dose; tumor regions treated with higher radiation doses (10-14 Gy) exhibited a greater decrease in HP lactate signal than low-dose regions (4-7 Gy) as early as 1 day post-RT, consistent with lactate dehydrogenase enzyme activity and expression data. We also found that HP [13C]urea MRI provided assessments of tumor perfusion similar to those provided by 1H dynamic contrast-enhanced MRI in this animal model. However, apparent diffusion coefficien , a conventional 1H MRI functional biomarker, did not exhibit statistically significant changes within 7 days after RT. These results demonstrate the ability of HP 13C MRI to monitor radiation-induced physiologic changes in a timely and dose-dependent manner, providing the basic science premise for further clinical investigation and translation.

HDAC inhibition in glioblastoma monitored by hyperpolarized 13C MRSI

Vorinostat is a histone deacetylase (HDAC) inhibitor that inhibits cell proliferation and induces apoptosis in solid tumors, and is in clinical trials for the treatment of glioblastoma (GBM). The goal of this study was to assess whether hyperpolarized 13 C MRS and magnetic resonance spectroscopic imaging (MRSI) can detect HDAC inhibition in GBM models. First, we confirmed HDAC inhibition in U87 GBM cells and evaluated real-time dynamic metabolic changes using a bioreactor system with live vorinostat-treated or control cells. We found a significant 40% decrease in the 13 C MRS-detectable ratio of hyperpolarized [1-13 C]lactate to hyperpolarized [1-13 C]pyruvate, [1-13 C]Lac/Pyr, and a 37% decrease in the pseudo-rate constant, kPL , for hyperpolarized [1-13 C]lactate production, in vorinostat-treated cells compared with controls. To understand the underlying mechanism for this finding, we assessed the expression and activity of lactate dehydrogenase (LDH) (which catalyzes the pyruvate to lactate conversion), its associated cofactor nicotinamide adenine dinucleotide, the expression of monocarboxylate transporters (MCTs) MCT1 and MCT4 (which shuttle pyruvate and lactate in and out of the cell) and intracellular lactate levels. We found that the most likely explanation for our finding that hyperpolarized lactate is reduced in treated cells is a 30% reduction in intracellular lactate levels that occurs as a result of increased expression of both MCT1 and MCT4 in vorinostat-treated cells. In vivo 13 C MRSI studies of orthotopic tumors in mice also showed a significant 52% decrease in hyperpolarized [1-13 C]Lac/Pyr when comparing vorinostat-treated U87 GBM tumors with controls, and, as in the cell studies, this metabolic finding was associated with increased MCT1 and MCT4 expression in HDAC-inhibited tumors. Thus, the 13 C MRSI-detectable decrease in hyperpolarized [1-13 C]lactate production could serve as a biomarker of response to HDAC inhibitors.

Dynamic diffusion‐weighted hyperpolarized 13C imaging based on a slice‐selective double spin echo sequence for measurements of cellular transport

To develop a pulse sequence to dynamically measure the ADC of hyperpolarized substrates during their perfusion, metabolic conversion, and transport. We proposed a slice-selective double spin echo sequence for dynamic hyperpolarized 13 C diffusion-weighted imaging. The proposed pulse sequence was optimized for a high field preclinical scanner through theoretical analysis and simulation. The performance of the method was compared to non-slice-selective double spin echo via in vivo studies. We also validated the sequence for dynamic ADC measurement in both phantom studies and transgenic mouse model of prostate cancer studies. The optimized pulse sequence outperforms the traditional sequence with smaller saturation effects on the magnetization of hyperpolarized compounds that allowed more dynamic imaging frames covering a longer imaging time window. In pre-clinical studies (N = 8), the dynamic hyperpolarized lactate ADC maps of 6 studies in the prostate tumors showed an increase measured ADC over time, which might be related to lactate efflux from the tumor cells. The proposed sequence was validated and shown to improve dynamic diffusion weighted imaging compared to the traditional double spin echo sequence, providing ADC maps of lactate through time.

Effects of Deuteration of 13C-Enriched Phospholactate on Efficiency of Parahydrogen-Induced Polarization by Magnetic Field Cycling.

We report herein a large-scale (>10 g) synthesis of isotopically enriched 1-13C-phosphoenolpyruvate and 1-13C-phosphoenolpyruvate-d2 for application in hyperpolarized imaging technology. The 1-13C-phosphoenolpyruvate-d2 was synthesized with 57% overall yield (over two steps), and >98% 2H isotopic purity, representing an improvement over the previous report. The same outcome was achieved for 1-13C-phosphoenolpyruvate. These two unsaturated compounds with C=C bonds were employed for parahydrogen-induced polarization via pairwise parahydrogen addition in aqueous medium. We find that deuteration of 1-13C-phosphoenolpyruvate resulted in overall increase of 1H T1 of nascent hyperpolarized protons (4.30 ± 0.04 s versus 2.06 ± 0.01 s) and 1H polarization (~2.5% versus ~0.7%) of the resulting hyperpolarized 1-13C-phospholactate. The nuclear spin polarization of nascent parahydrogen-derived protons was transferred to 1-13C nucleus via magnetic field cycling procedure. The proton T1 increase in hyperpolarized deuterated 1-13C-phospholactate yielded approximately 30% better 13C polarization compared to non-deuterated hyperpolarized 1-13C-phospholactate. Analysis of T1 relaxation revealed that deuteration of 1-13C-phospholactate may have resulted in approximately 3-fold worse H→13C polarization transfer efficiency via magnetic field cycling. Since magnetic field cycling is a key polarization transfer step in the Side-Arm Hydrogenation approach, the presented findings may guide more rationale design of contrast agents using parahydrogen polarization of a broad range of 13C hyperpolarized contrast agents for molecular imaging employing 13C MRI. The hyperpolarized 1-13C-phospholactate-d2 is of biomedical imaging relevance because it undergoes in vivo dephosphorylation and becomes 13C hyperpolarized lactate, which as we show can be detected in the brain using 13C hyperpolarized MRI; an implication for future imaging of neurodegenerative diseases and dementia.

Metabolic Imaging of the Human Brain with Hyperpolarized 13C Pyruvate Demonstrates 13C Lactate Production in Brain Tumor Patients.

Hyperpolarized (HP) MRI using [1-13C] pyruvate is a novel method that can characterize energy metabolism in the human brain and brain tumors. Here, we present the first dynamically acquired human brain HP 13C metabolic spectra and spatial metabolite maps in cases of both untreated and recurrent tumors. In vivo production of HP lactate from HP pyruvate by tumors was indicative of altered cancer metabolism, whereas production of HP lactate in the entire brain was likely due to baseline metabolism. We correlated our results with standard clinical brain MRI, MRI DCE perfusion, and in one case FDG PET/CT. Our results suggest that HP 13C pyruvate-to-lactate conversion may be a viable metabolic biomarker for assessing tumor response.Significance: Hyperpolarized pyruvate MRI enables metabolic imaging in the brain and can be a quantitative biomarker for active tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3755/F1.large.jpg Cancer Res; 78(14); 3755-60. ©2018 AACR.

Noninvasive Immunometabolic Cardiac Inflammation Imaging Using Hyperpolarized Magnetic Resonance.

Current cardiovascular clinical imaging techniques offer only limited assessment of innate immune cell-driven inflammation, which is a potential therapeutic target in myocardial infarction (MI) and other diseases. Hyperpolarized magnetic resonance (MR) is an emerging imaging technology that generates contrast agents with 10- to 20 000-fold improvements in MR signal, enabling cardiac metabolite mapping. To determine whether hyperpolarized MR using [1-13C]pyruvate can assess the local cardiac inflammatory response after MI. We performed hyperpolarized [1-13C]pyruvate MR studies in small and large animal models of MI and in macrophage-like cell lines and measured the resulting [1-13C]lactate signals. MI caused intense [1-13C]lactate signal in healing myocardial segments at both day 3 and 7 after rodent MI, which was normalized at both time points after monocyte/macrophage depletion. A near-identical [1-13C]lactate signature was also seen at day 7 after experimental MI in pigs. Hyperpolarized [1-13C]pyruvate MR spectroscopy in macrophage-like cell suspensions demonstrated that macrophage activation and polarization with lipopolysaccharide almost doubled hyperpolarized lactate label flux rates in vitro; blockade of glycolysis with 2-deoxyglucose in activated cells normalized lactate label flux rates and markedly inhibited the production of key proinflammatory cytokines. Systemic administration of 2-deoxyglucose after rodent MI normalized the hyperpolarized [1-13C]lactate signal in healing myocardial segments at day 3 and also caused dose-dependent improvement in IL (interleukin)-1β expression in infarct tissue without impairing the production of key reparative cytokines. Cine MRI demonstrated improvements in systolic function in 2-DG (2-deoxyglucose)-treated rats at 3 months. Hyperpolarized MR using [1-13C]pyruvate provides a novel method for the assessment of innate immune cell-driven inflammation in the heart after MI, with broad potential applicability across other cardiovascular disease states and suitability for early clinical translation.

Placental physiology monitored by hyperpolarized dynamic 13C magnetic resonance

Placental functions, including transport and metabolism, play essential roles in pregnancy. This study assesses such processes in vivo, from a hyperpolarized MRI perspective. Hyperpolarized urea, bicarbonate, and pyruvate were administered to near-term pregnant rats, and all metabolites displayed distinctive behaviors. Little evidence of placental barrier crossing was observed for bicarbonate, at least within the timescales allowed by 13C relaxation. By contrast, urea was observed to cross the placental barrier, with signatures visible from certain fetal organs including the liver. This was further evidenced by the slower decay times observed for urea in placentas vis-à-vis other maternal compartments and validated by mass spectrometric analyses. A clear placental localization, as well as concurrent generation of hyperpolarized lactate, could also be detected for [1-13C]pyruvate. These metabolites also exhibited longer lifetimes in the placentas than in maternal arteries, consistent with a metabolic activity occurring past the trophoblastic interface. When extended to a model involving the administration of a preeclampsia-causing chemical, hyperpolarized MR revealed changes in urea's transport, as well as decreases in placental glycolysis vs. the naïve animals. These distinct behaviors highlight the potential of hyperpolarized MR for the early, minimally invasive detection of aberrant placental metabolism.

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.

C Hyperpolarized magnetic resonance imaging of cardiac inflammation and repair

Myocardial infarction (MI) remains a major killer despite highly optimised systems for the delivery of primary percutaneous coronary intervention (PPCI), highlighting a need for novel therapeutics that could be administered...

Abstract 4215: Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts

Abstract Pancreatic cancer, the most lethal of solid tumors is an aggressive disease that develops relatively symptom-free. The absence of early symptoms has created a critical need for identifying and developing new noninvasive biomarkers and therapeutic intervention to improve the survival rate of pancreatic cancer patients. Our goal is to identify metabolic biomarkers for early detection with an understanding of pancreatic cancer progression from benign lesions to a malignant state. Hyperpolarized magnetic resonance imaging (HP-MRI) provides a &amp;gt;10,000-fold signal enhancement for detecting of endogenous metabolic substrates to monitor metabolic fluxes through the multiple key biochemical pathways including glycolysis and citric acid cycle. In particular, conversion between hyperpolarized 13C-labeled pyruvate and lactate, catalyzed by lactate dehydrogenase (LDH), has been shown to have a number of potential applications such as diagnosis, staging tumor grade and monitoring therapy response. Genetic mutation is evident in causing tumorigenesis and often time these mutations trigger the signaling pathways that are associated with “metabolic transformations. There is currently tremendous research interest in dissecting the mechanisms of metabolic transformation as the cancers progress. In this effort, we are applying high-resolution NMR (nuclear magnetic resonance) metabolomics and the real-time hyperpolarized metabolic imaging of patient-derived xenograft tumors for probing the underlying mechanism of altered-metabolism and correlate with pancreatic cancer progression and its aggressiveness. The protocol detailing heterotopic engraftment of pancreatic cancer patient tumors into immunodeficient mice and expansion of direct xenograft tumors was recently reported elsewhere. These patient-derived xenografts (PDX) were well annotated with original patient tumors. For the NMR metabolomics study, the fresh-frozen tumor samples were homogenized, lyophilized, and resuspended in D2O and 1-D proton NMR spectroscopy was employed to characterize the water-soluble portion of the metabolome. These data suggest that elevated glycolysis (production of high lactate) and alterations in choline metabolism may arise in pancreatic cancer development. Also, the metabolite concentrations were significantly higher in aggressive tumors compare to non-aggressive one. The real-time metabolic imaging shows the immediate conversion of lactate after injection (i.v.) of hyperpolarized 13C pyruvate and probe the upregulated LDH activity as the cancer progress. The basic genomic profiling of these tumors is in progress. This model may provide an excellent platform where mutational status of these patient tumors can be correlated with both high resolution and hyperpolarized dynamical metabolomics data for characterizing individual tumor phenotypes. Citation Format: Prasanta Dutta, Mayrim Rios Perez, Travis Cole Salzillo, Michael Pratt, Yaan Kang, Niki Zacharias, Anirban Maitra, Jason B. Fleming, Pratip Bhattacharya. Hyperpolarized magnetic resonance metabolic imaging and NMR metabolomics to assess the progression and aggressiveness of patient-derived pancreatic cancer xenografts. [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 4215.

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.

Mutant IDH1 expression is associated with down-regulation of monocarboxylate transporters.

Mutations in isocitrate dehydrogenase 1 (IDH1) are characteristic of low-grade gliomas. We recently showed that mutant IDH1 cells reprogram cellular metabolism by down-regulating pyruvate dehydrogenase (PDH) activity. Reduced pyruvate metabolism via PDH could lead to increased pyruvate conversion to lactate. The goal of this study was therefore to investigate the impact of the IDH1 mutation on the pyruvate-to-lactate flux. We used 13C magnetic resonance spectroscopy and compared the conversion of hyperpolarized [1-13C]-pyruvate to [1-13C]-lactate in immortalized normal human astrocytes expressing mutant or wild-type IDH1 (NHAIDHmut and NHAIDHwt). Our results indicate that hyperpolarized lactate production is reduced in NHAIDHmut cells compared to NHAIDHwt. This reduction was associated with lower expression of the monocarboxylate transporters MCT1 and MCT4 in NHAIDHmut cells. Furthermore, hyperpolarized lactate production was comparable in lysates of NHAIDHmut and NHAIDHwt cells, wherein MCTs do not impact hyperpolarized pyruvate delivery and lactate production. Collectively, our findings indicated that lower MCT expression was a key contributor to lower hyperpolarized lactate production in NHAIDHmut cells. The SLC16A3 (MCT4) promoter but not SLC16A1 (MCT1) promoter was hypermethylated in NHAIDHmut cells, pointing to possibly different mechanisms mediating reduced MCT expression. Finally analysis of low-grade glioma patient biopsy data from The Cancer Genome Atlas revealed that MCT1 and MCT4 expression was significantly reduced in mutant IDH1 tumors compared to wild-type. Taken together, our study shows that reduced MCT expression is part of the metabolic reprogramming of mutant IDH1 gliomas. This finding could impact treatment and has important implications for metabolic imaging of mutant IDH1 gliomas.

Abstract B51: Metabolic imaging with hyperpolarized [1-13C]-pyruvate and DCE-MRI

Abstract Purpose: To improve the quality and specificity of metabolic imaging with hyperpolarized pyruvate using prior information from dynamic contrast-enhanced MRI (DCE-MRI). Background: Dynamic nuclear polarization (DNP) and MR spectroscopic imaging of [1-13C]-pyruvate shows tremendous promise for imaging aerobic glycolysis with sensitivity, specificity, and resolution that was previously impossible. Exogeneous, enriched hyperpolarized (HP) pyruvate is injected as a bolus and traverses multiple biological barriers before it is exposed to intracellular enzymes that mediate its fate. Preservation of the spin label through chemical reaction allows visualization of pyruvate and its chemical endpoints (lactate, alanine, and carbon dioxide/bicarbonate). We have previously observed a strong correlation between tumor perfusion and the normalized area under the curve for HP lactate, the chemical endpoint associated with aerobic glycolysis. A pharmacokinetic model of HP substrate evolution that balances physiological accuracy and computational burden includes two physical pools (intravascular/extravascular) and two chemical pools (pyruvate/lactate). In the analytical solution for this model, terms for substrate extravasation and the fractional intravascular volume can be recognized as analogues for KTRANS and Vb that are derived from analysis of DCE-MRI data using the extended Tofts model. Use of these DCE-MRI parameters as prior information could eliminate unknowns in the measurement of HP agents and improve the specificity of quantitative estimates for kPL, the apparent conversion rate for HP pyruvate into lactate. Experimental Procedures: Athymic nude mice bearing orthotopic anaplastic thyroid cancer tumors were anesthetized and scanned using a 7T Biospec small animal MR system (Bruker Biospin Corp.). A dual-tuned 1H/13C volume resonator was used for anatomic imaging and for 13C excitation, and a surface coil was used for 13C detection. HP pyruvate was prepared using a HyperSense DNP system (Oxford Instruments). Dynamic 13C spectroscopic imaging data was acquired using a radial multi-band frequency encoding sequence beginning prior to intravascular administration of HP pyruvate via tail-vein catheter. DCE-MRI data was acquired after HP measurements, using a fast spoiled gradient echo sequence. DCE-MRI data was fit to the extended Tofts model on a pixel-by-pixel basis using software developed in Matlab (The Mathworks). Parametric maps for perfusion and vascular blood volume fraction were resampled to the resolution of HP 13C scans and used as priors in a constrained reconstruction algorithm that enforces consistency between undersampled 13C projection data, the kinetic model, and prior information from 1H MRI. Results: With prior information about extravasation and fractional intravascular blood volume, the constrained reconstruction algorithm needs only solve for a global parameterized vascular input function and values for chemical conversion (kPL) and signal relaxation/loss for each voxel in the imaging volume. Elimination of nuisance parameters with strongly correlated effects reduces the computational burden of constrained reconstruction and clarifies interpretation of changes in biomarkers due to disease progression or response to therapy. Conclusions: The combination of DCE-MRI with constrained reconstruction reduces the number of unknowns in quantitative metabolic imaging using HP pyruvate, eases the computational burden, and decouples correlated effects of perfusion and chemical exchange. Citation Format: James A. Bankson, Christopher M. Walker, Yunyun Chen, Stephen Y. Lai, John D. Hazle. Metabolic imaging with hyperpolarized [1-13C]-pyruvate and DCE-MRI. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B51.