Hyperpolarized 13C Magnetic Resonance Spectroscopy Research Articles

Hyperpolarized [1-13C]-pyruvate MRS evaluates immune potential and predicts response to radiotherapy in cervical cancer

BackgroundMonitoring pyruvate metabolism in the spleen is important for assessing immune activity and achieving successful radiotherapy for cervical cancer due to the significance of the abscopal effect. We aimed to explore the feasibility of utilizing hyperpolarized (HP) [1-13C]-pyruvate magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) to evaluate pyruvate metabolism in the human spleen, with the aim of identifying potential candidates for radiotherapy in cervical cancer.MethodsThis prospective study recruited six female patients with cervical cancer (median age 55 years; range 39–60) evaluated using HP [1-13C]-pyruvate MRI/MRS at baseline and 2 weeks after radiotherapy. Proton (1H) diffusion-weighted MRI was performed in parallel to estimate splenic cellularity. The primary outcome was defined as tumor response to radiotherapy. The Student t-test was used for comparing 13C data between the groups.ResultsThe splenic HP [1-13C]-lactate-to-total carbon (tC) ratio was 5.6-fold lower in the responders than in the non-responders at baseline (p = 0.009). The splenic [1-13C]-lactate-to-tC ratio revealed a 1.7-fold increase (p = 0.415) and the splenic [1-13C]-alanine-to-tC ratio revealed a 1.8-fold increase after radiotherapy (p = 0.482). The blood leukocyte differential count revealed an increased proportion of neutrophils two weeks following treatment, indicating enhanced immune activity (p = 0.013). The splenic apparent diffusion coefficient values between the groups were not significantly different.ConclusionsThis exploratory study revealed the feasibility of HP [1-13C]-pyruvate MRS of the spleen for evaluating baseline immune potential, which was associated with clinical outcomes of cervical cancer after radiotherapy.Trial registrationClinicalTrials.gov NCT04951921, registered 7 July 2021.Relevance statementThis prospective study revealed the feasibility of using HP 13C MRI/MRS for assessing pyruvate metabolism of the spleen to evaluate the patients’ immune potential that is associated with radiotherapeutic clinical outcomes in cervical cancer.Key points• Effective radiotherapy induces abscopal effect via altering immune metabolism.• Hyperpolarized 13C MRS evaluates patients’ immune potential non-invasively.• Pyruvate-to-lactate conversion in the spleen is elevated following radiotherapy.Graphical

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.

Magnetic Resonance Imaging (MRI) and MR Spectroscopic Methods in Understanding Breast Cancer Biology and Metabolism.

A common malignancy that affects women is breast cancer. It is the second leading cause of cancer-related death among women. Metabolic reprogramming occurs during cancer growth, invasion, and metastases. Functional magnetic resonance (MR) methods comprising an array of techniques have shown potential for illustrating physiological and molecular processes changes before anatomical manifestations on conventional MR imaging. Among these, in vivo proton (1H) MR spectroscopy (MRS) is widely used for differentiating breast malignancy from benign diseases by measuring elevated choline-containing compounds. Further, the use of hyperpolarized 13C and 31P MRS enhanced the understanding of glucose and phospholipid metabolism. The metabolic profiling of an array of biological specimens (intact tissues, tissue extracts, and various biofluids such as blood, urine, nipple aspirates, and fine needle aspirates) can also be investigated through in vitro high-resolution NMR spectroscopy and high-resolution magic angle spectroscopy (HRMAS). Such studies can provide information on more metabolites than what is seen by in vivo MRS, thus providing a deeper insight into cancer biology and metabolism. The analysis of a large number of NMR spectral data sets through multivariate statistical methods classified the tumor sub-types. It showed enormous potential in the development of new therapeutic approaches. Recently, multiparametric MRI approaches were found to be helpful in elucidating the pathophysiology of cancer by quantifying structural, vasculature, diffusion, perfusion, and metabolic abnormalities in vivo. This review focuses on the applications of NMR, MRS, and MRI methods in understanding breast cancer biology and in the diagnosis and therapeutic monitoring of breast cancer.

EXTH-46. MRS BASED BIOMARKERS OF IDH1 MUTANT GLIOMA RESPONSE TO THE IDH INHIBITOR BAY-1436032

Abstract Gliomas are the most prevalent type of brain tumor in the central nervous system. Mutations in the cytosolic enzyme isocitrate dehydrogenase 1 (IDH1) are a common feature of primary low-grade gliomas, catalyzing the conversion of α-ketoglutarate (αKG) to the oncometabolite 2-hydroxyglutarate (2HG), and mutant IDH1 is a therapeutic target for these tumors. Several mutant IDH inhibitors are currently in clinical trials, nonetheless, complementary non-invasive early biomarkers to assess drug delivery and potential therapeutic response are still needed. The goal of this study was therefore to determine the potential of 1H and hyperpolarized 13C magnetic resonance spectroscopy (MRS)-based biomarkers as indicators of mutant IDH1 low-grade glioma response to treatment with the clinically-relevant IDH1 inhibitor BAY-1436032 in cells and animal models. Immortalized human astrocytes engineered to express mutant IDH1 were treated with 500nM (IC50 value) of BAY-1436032 and BT257 tumors implanted in rats were treated with 150mg/kg of BAY-1436032. To assess steady-state metabolite levels, 1H MRS spectra were acquired on a 500 MHz MRS cancer for cells and a 3 T scanner for animal studies. To assess metabolic fluxes, we used hyperpolarized 13C MRS and probed the fate of hyperpolarized [1-13C]αKG. 1H MRS showed a significant decrease in 2HG as well as a significant increase in glutamate (Glu) and phosphocholine (PCh) following BAY-1436032 treatment in both cell and animal models compared to controls. Furthermore, hyperpolarized 13C MRS showed that hyperpolarized 2HG production from hyperpolarized [1-13C]αKG was decreased and hyperpolarized glutamate production from hyperpolarized [1-13C]αKG was increased in the BAY-1436032 treated groups compared to controls. These findings are consistent with our previous study, which investigated the MRS-detectable consequences of two other mutant IDH inhibitors: AG120 and AG881. Collectively, our work identifies translatable MRS-based metabolic biomarkers of mutant IDH1 inhibition.

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.

Non-Invasive Differentiation of M1 and M2 Activation in Macrophages Using Hyperpolarized 13C MRS of Pyruvate and DHA at 1.47 Tesla.

Macrophage activation, first generalized to the M1/M2 dichotomy, is a complex and central process of the innate immune response. Simply, M1 describes the classical proinflammatory activation, leading to tissue damage, and M2 the alternative activation promoting tissue repair. Given the central role of macrophages in multiple diseases, the ability to noninvasively differentiate between M1 and M2 activation states would be highly valuable for monitoring disease progression and therapeutic responses. Since M1/M2 activation patterns are associated with differential metabolic reprogramming, we hypothesized that hyperpolarized 13C magnetic resonance spectroscopy (HP 13C MRS), an innovative metabolic imaging approach, could distinguish between macrophage activation states noninvasively. The metabolic conversions of HP [1-13C]pyruvate to HP [1-13C]lactate, and HP [1-13C]dehydroascorbic acid to HP [1-13C]ascorbic acid were monitored in live M1 and M2 activated J774a.1 macrophages noninvasively by HP 13C MRS on a 1.47 Tesla NMR system. Our results show that both metabolic conversions were significantly increased in M1 macrophages compared to M2 and nonactivated cells. Biochemical assays and high resolution 1H MRS were also performed to investigate the underlying changes in enzymatic activities and metabolite levels linked to M1/M2 activation. Altogether, our results demonstrate the potential of HP 13C MRS for monitoring macrophage activation states noninvasively.

Iron-Deficiency Anemia Results in Transcriptional and Metabolic Remodeling in the Heart Toward a Glycolytic Phenotype.

Iron deficiency is the most prevalent micronutrient disorder globally. When severe, iron deficiency leads to anemia, which can be deleterious to cardiac function. Given the central role of iron and oxygen in cardiac biology, multiple pathways are expected to be altered in iron-deficiency anemia, and identifying these requires an unbiased approach. To investigate these changes, gene expression and metabolism were studied in mice weaned onto an iron-deficient diet for 6 weeks. Whole-exome transcriptomics (RNAseq) identified over 1,500 differentially expressed genes (DEGs), of which 22% were upregulated and 78% were downregulated in the iron-deficient group, relative to control animals on an iron-adjusted diet. The major biological pathways affected were oxidative phosphorylation and pyruvate metabolism, as well as cardiac contraction and responses related to environmental stress. Cardiac metabolism was studied functionally using in vitro and in vivo methodologies. Spectrometric measurement of the activity of the four electron transport chain complexes in total cardiac lysates showed that the activities of Complexes I and IV were reduced in the hearts of iron-deficient animals. Pyruvate metabolism was assessed in vivo using hyperpolarized 13C magnetic resonance spectroscopy (MRS) of hyperpolarized pyruvate. Hearts from iron-deficient and anemic animals showed significantly decreased flux through pyruvate dehydrogenase and increased lactic acid production, consistent with tissue hypoxia and induction of genes coding for glycolytic enzymes and H+-monocarboxylate transport-4. Our results show that iron-deficiency anemia results in a metabolic remodeling toward a glycolytic, lactic acid-producing phenotype, a hallmark of hypoxia.

Elevated Tumor Lactate and Efflux in High-grade Prostate Cancer demonstrated by Hyperpolarized 13C Magnetic Resonance Spectroscopy of Prostate Tissue Slice Cultures

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score ≤3 + 4) from high-risk (Gleason score ≥4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR.

Metabolic imaging using hyperpolarized 13 C-pyruvate to assess sensitivity to the B-Raf inhibitor vemurafenib in melanoma cells and xenografts.

Nearly all melanoma patients with a BRAF‐activating mutation will develop resistance after an initial clinical benefit from BRAF inhibition (BRAFi). The aim of this work is to evaluate whether metabolic imaging using hyperpolarized (HP) 13C pyruvate can serve as a metabolic marker of early response to BRAFi in melanoma, by exploiting the metabolic effects of BRAFi. Mice bearing human melanoma xenografts were treated with the BRAFi vemurafenib or vehicle. In vivo HP 13C magnetic resonance spectroscopy was performed at baseline and 24 hours after treatment to evaluate changes in pyruvate‐to‐lactate conversion. Oxygen partial pressure was measured via electron paramagnetic resonance oximetry. Ex vivo qRT‐PCR, immunohistochemistry and WB analysis were performed on tumour samples collected at the same time‐points selected for in vivo experiments. Similar approaches were applied to evaluate the effect of BRAFi on sensitive and resistant melanoma cells in vitro, excluding the role of tumour microenvironment. BRAF inhibition induced a significant increase in the HP pyruvate‐to‐lactate conversion in vivo, followed by a reduction of hypoxia. Conversely, the conversion was inhibited in vitro, which was consistent with BRAFi‐mediated impairment of glycolysis. The paradoxical increase of pyruvate‐to‐lactate conversion in vivo suggests that such conversion is highly influenced by the tumour microenvironment.

Metabolic alterations in acute myocardial ischemia-reperfusion injury and necrosis using in vivo hyperpolarized [1-13C] pyruvate MR spectroscopy

This study aimed to investigate real-time early detection of metabolic alteration in a rat model with acute myocardial ischemia-reperfusion (AMI/R) injury and myocardial necrosis, as well as its correlation with intracellular pH level using in vivo hyperpolarized [1-13C] pyruvate magnetic resonance spectroscopy (MRS). Hyperpolarized 13C MRS was performed on the myocardium of 8 sham-operated control rats and 8 rats with AMI/R injury, and 8 sham-operated control rats and 8 rats with AMI-induced necrosis. Also, the correlations of levels of [1-13C] metabolites with pH were analyzed by Spearman’s correlation test. The AMI/R and necrosis groups showed significantly higher ratios of [1-13C] lactate (Lac)/bicarbonate (Bicar) and [1-13C] Lac/total carbon (tC), and lower ratios of 13C Bicar/Lac + alanine (Ala), and 13C Bicar/tC than those of the sham-operated control group. Moreover, the necrosis group showed significantly higher ratios of [1-13C] Lac/Bicar and [1-13C] Lac/tC, and lower ratios of 13C Bicar/Lac + Ala and 13C Bicar/tC than those of the AMI/R group. These results were consistent with the pattern for in vivo the area under the curve (AUC) ratios. In addition, levels of [1-13C] Lac/Bicar and [1-13C] Lac/tC were negatively correlated with pH levels, whereas 13C Bicar/Lac + Ala and 13C Bicar/tC levels were positively correlated with pH levels. The levels of [1-13C] Lac and 13C Bicar will be helpful for non-invasively evaluating the early stage of AMI/R and necrosis in conjunction with reperfusion injury of the heart. These findings have potential application to real-time evaluation of cardiac malfunction accompanied by changes in intracellular pH level and enzymatic activity.

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.

Abstract 5263: 1H and 13C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy

Abstract Low-grade gliomas, driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene, are less aggressive than primary glioblastoma (GBM), but nonetheless always recur and ultimately lead to patient death. To improve patient survival, one therapeutic strategy is treatment with the alkylating chemotherapeutic agent Temozolomide (TMZ), previously reserved for the treatment of primary GBM. Though the treatment of IDH1 mutant patients with TMZ improves survival, early detection of treatment-associated effects remains challenging. The goal of this study was, therefore, to determine the value of magnetic resonance spectroscopy (MRS)-based biomarkers for detection of response to treatment. To this end, we examined the global metabolic alterations that occurred following TMZ treatment in a genetically engineered IDH1 mutant immortalized Normal Human Astrocyte (NHAIDHmut)-based cell model using 1H MRS combined with chemometrics and 13C MRS combined with labeling of cells with [1-13C]-glucose and [3-13C]-glutamine. Cells were treated either with the IC50 value of TMZ (100 μM; N=5), or with DMSO (0.2%; N=5) for 72 hours. Then, metabolites were extracted from cells using the dual-phase extraction method. 1H-MRS and proton-decoupled 13C-MRS spectra were acquired using a 500 MHz Bruker Avance spectrometer. 1H MRS data was subjected to multivariate analysis using SIMCA. Specific metabolites were also quantified using Mnova7, integrals normalized to TSP and to cell number and statistical significance of differences determined using unpaired Student’s t-test. Pathway enrichment analysis of dysregulated metabolites was performed using MetPA. principal component analysis score plot showed separation of TMZ-treated from DMSO-treated control cells. Further, improved separation between the groups was obtained by orthogonal-partial least squares discriminant analysis. Most significant metabolites contributing to class separation were identified using correlation ≥0.6 and variable importance in projection score ≥1. Glutamine, glutamate, 2-hydroxyglutarate (2-HG), pyruvate, succinate, glucose, phosphocholine, isoleucine, valine, lysine, phenylalanine, NAD(P)+ and AMP/ADP/ATP were observed to be significantly higher in TMZ-treated cells as compared to controls. Based on pathway impact score, tricarboxylic acid (TCA) cycle was identified to be the significantly altered pathway following treatment Further, there was a significant increases in glucose- and glutamine-derived glutamate and 2-HG, indicating an increase in flux to the TCA cycle following treatment. These findings demonstrate that IDH1 mutant glioma show a clear metabolomic fingerprint in response to TMZ therapy, which, combined with complementary hyperpolarized 13C MRS approaches, may help assess early response to TMZ therapy in mutant IDH1 glioma. Note: This abstract was not presented at the meeting. Citation Format: Elavarasan Subramani, Chloe Najac, Georgios Batsios, Pavithra Viswanath, Marina Radoul, Anne Marie Gillespie, Russell O. Pieper, Sabrina M. Ronen. 1H and 13C MRS-based metabolic markers of IDH1 mutant glioma response to temozolomide therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5263.

Hyperpolarized [1-13 C] pyruvate as a possible diagnostic tool in liver disease.

Introduction of hyperpolarized magnetic resonance in preclinical studies and lately translation to patients provides new detailed in vivo information of metabolic flux in organs. Hyperpolarized magnetic resonance based on 13C enriched pyruvate is performed without ionizing radiation and allows quantification of the pyruvate conversion products: alanine, lactate and bicarbonate in real time. Thus, this methodology has a promising potential for in vivo monitoring of energetic alterations in hepatic diseases. Using 13C pyruvate, we investigated the metabolism in the porcine liver before and after intravenous injection of glucose. The overall mean lactate to pyruvate ratio increased significantly after the injection of glucose whereas the bicarbonate to pyruvate ratio was unaffected, representative of the levels of pyruvate entering the tricarboxylic acid cycle. Similarly, alanine to pyruvate ratio did not change. The increased lactate to pyruvate ratio over time showed an exponential correlation with insulin, glucagon and free fatty acids. Together, these data, obtained by hyperpolarized 13C magnetic resonance spectroscopy and by blood sampling, indicate a hepatic metabolic shift in glucose utilization following a glucose challenge. Our findings demonstrate the capacity of hyperpolarized 13C magnetic resonance spectroscopy for quantifying hepatic substrate metabolism in accordance with well‐known physiological processes. When combined with concentration of blood insulin, glucagon and free fatty acids in the blood, the results indicate the potential of hyperpolarized magnetic resonance spectroscopy as a future clinical method for quantification of hepatic substrate metabolism.

Application of Hyperpolarized 13C Magnetic Resonance Imaging to Detect Target Inhibition of NFkB Activation in Preclinical Patient-Derived Models of CNS Lymphoma

To gain insights into the tumor microenvironment in primary and secondary CNS lymphomas, we applied LC/MS and GC/MS for differential metabolomic profiling of the cerebrospinal fluid (CSF) of CNS lymphoma patients compared to control subjects. Among 145 analytes identified, the majority were involved in energy metabolism; one of the most significantly upregulated metabolites in CNS lymphoma was lactate (1.8 fold, p<0.001). Subsequently we determined that baseline elevated CSF lactate, quantified by a Beckman Coulter Unicell Clinical Chemistry analyzer, correlated with short survival in three phase I investigations involving immunotherapy in relapsed CNS lymphoma (p<0.0001). (Blood Advances 2018). Given this, we hypothesize that tumor-associated lactate significantly contributes to the pathogenesis of CNS lymphoma as a potential mediator of tumor invasion as well as immunosuppression, and can serve as a novel biomarker in CNS lymphoma. To pursue this, we are testing the hypothesis that metabolic imaging techniques including proton magnetic resonance spectroscopy (MRS) and hyperpolarized (HP) 13C MRS can facilitate prognosis and response assessment to targeted therapies.Thus far we have applied these metabolic imaging approaches to preclinical murine models involving diffusely invasive, intracranial, patient-derived xenografts of CNS DLBCL in RAG-/- mice, to detect tumor-associated lactate production generated by infiltrating lymphoma. We demonstrated that each of these MRS approaches detect highly invasive lymphoma that is undetectable by conventional gadolinium-enhanced T1, T2 sequences, or diffusion-weighted imaging. Because of its ability to detect real-time changes in metabolic pathways, we focused on the application of HP13C pyruvate metabolic imaging as a non-invasive imaging tool for NF-kB pathway inhibition in CNS lymphoma using these models. We evaluated the metabolic response to AZ1495, a novel, CNS penetrant, orally-bioavailable inhibitor of IRAK4 kinase, comparing MYD88 wild type vs. MYD88 L265P mutant tumor models. Using a 14.1T imaging system for MR acquisition, we demonstrated similar tumor-associated production of HP 13C lactate in both MYD88wt and MY88 mutant tumors at 3 weeks post-implantation. We determined that while AZ1495 did not significantly impact lactate production in MYD88wt lymphoma, this agent significantly down-regulated tumor-associated HP pyruvate to lactate conversion (>47%) within 2 days in MYD88 mutant CNS lymphoma (p<0.02). (Figure 1). In parallel, we determined that AZ1495 potently antagonized phosphorylation of p65 REL-A selectively in intracranial xenografts harboring L265P MYD88 mutation. Transcriptional profiling by RNA-Seq demonstrated > 2-fold down-modulation of NF-kB gene expression at 4h of AZ1495 therapy, including transcripts encoding LDH-A as well as the catalytic subunit of PI3K, suggesting interaction with the B cell receptor pathway. Combination AZ1495 plus ibrutinib starting d+5 was synergistic in survival prolongation compared to AZ1495 monotherapy (p<0.003), ibrutinib monotherapy (p<0.005), or control gavage (p<0.002) in a MYD88 L265P mutant, clinically refractory patient-derived intracranial DLBCL xenograft model. Taken together, these data demonstrate the ability of HP13C MRI metabolic imaging to identify highly infiltrative CNS lymphoma, not detectable by conventional MR sequences, as well as its potential to provide an early, non-invasive pharmacodynamic biomarker of response in an NFkB pathway-specific manner, and to facilitate precision medicine in CNS lymphoma. Supported by the National Cancer Institute, the Leukemia and Lymphoma Society and by the Sandler Program for Breakthrough Biomedical Research. [Display omitted] DisclosuresGao:Glaxo Smith Kline: Employment. Drew:AstraZeneca: Employment. Degorce:Astra Zeneca: Employment. Mayo:Astra Zeneca: Employment. Dillman:Astra Zeneca: Employment. Anjum:Astra Zeneca: Employment. Bloecher:Astra Zeneca: Employment. Rubenstein:Celgene: Research Funding; Genentech: Research Funding.

Investigating real-time metabolic flux changes in a rat model of doxorubicin-induced cardiotoxicity using hyperpolarized 13C magnetic resonance spectroscopy

Investigating real-time metabolic flux changes in a rat model of doxorubicin-induced cardiotoxicity using hyperpolarized 13C magnetic resonance spectroscopy

Imaging glutathione depletion in the rat brain using ascorbate-derived hyperpolarized MR and PET probes

Oxidative stress is a critical feature of several common neurologic disorders. The brain is well adapted to neutralize oxidative injury by maintaining a high steady-state concentration of small-molecule intracellular antioxidants including glutathione in astrocytes and ascorbic acid in neurons. Ascorbate-derived imaging probes for hyperpolarized 13C magnetic resonance spectroscopy and positron emission tomography have been used to study redox changes (antioxidant depletion and reactive oxygen species accumulation) in vivo. In this study, we applied these imaging probes to the normal rat brain and a rat model of glutathione depletion. We first studied hyperpolarized [1-13C]dehydroascorbate in the normal rat brain, demonstrating its robust conversion to [1-13C]vitamin C, consistent with rapid transport of the oxidized form across the blood-brain barrier. We next showed that the kinetic rate of this conversion decreased by nearly 50% after glutathione depletion by diethyl maleate treatment. Finally, we showed that dehydroascorbate labeled for positron emission tomography, namely [1-11C]dehydroascorbate, showed no change in brain signal accumulation after diethyl maleate treatment. These results suggest that hyperpolarized [1-13C]dehydroascorbate may be used to non-invasively detect oxidative stress in common disorders of the brain.

Hyperpolarized 13C Magnetic Resonance Spectroscopy Reveals the Rate-Limiting Role of the Blood-Brain Barrier in the Cerebral Uptake and Metabolism of l-Lactate in Vivo.

The dynamics of l-lactate transport across the blood–brain barrier (BBB) and its cerebral metabolism are still subject to debate. We studied lactate uptake and intracellular metabolism in the mouse brain using hyperpolarized 13C magnetic resonance spectroscopy (MRS). Following the intravenous injection of hyperpolarized [1-13C]lactate, we observed that the distribution of the 13C label between lactate and pyruvate, which has been shown to be representative of their pool size ratio, is different in NMRI and C57BL/6 mice, the latter exhibiting a higher level of cerebral lactate dehydrogenase A (Ldha) expression. On the basis of this observation, and an additional set of experiments showing that the cerebral conversion of [1-13C]lactate to [1-13C]pyruvate increases after exposing the brain to ultrasound irradiation that reversibly opens the BBB, we concluded that lactate transport is rate-limited by the BBB, with a 30% increase in lactate uptake after its disruption. It was also deduced from these results that hyperpolarized 13C MRS can be used to detect a variation in cerebral lactate uptake of <40 nmol in a healthy brain during an in vivo experiment lasting only 75 s, opening new opportunities to study the role of lactate in brain metabolism.

Detection of Bacteria-Specific Metabolism Using Hyperpolarized [2-13C]Pyruvate.

The differentiation of bacterial infection from other causes of inflammation is difficult in clinical practice and is critical where patient outcomes rely heavily on early interventions. In addition to physical exam and laboratory markers, several imaging modalities are frequently employed, but these techniques generally target the host immune response, rather than the living microorganisms themselves. Here, we describe a method to detect bacteria-specific metabolism using hyperpolarized (HP) 13C magnetic resonance spectroscopy. This technology allows visualization of the real-time conversion of enriched 13C substrates to their metabolic products, identified by their distinct chemical shifts. We have identified the rapid metabolism of HP [2-13C]pyruvate to [1-13C]acetate as a metabolic signature of common bacterial pathogens. We demonstrate this conversion in representative Gram-negative and Gram-positive bacteria, namely, Escherichia coli and Staphylococcus aureus, and its absence in key mammalian cell types. Furthermore, this conversion was successfully modulated in three mutant strains, corresponding to deletions of relevant enzymes.

The potential of hyperpolarized 13C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents

Background: Targeting tumor vasculature with vascular disrupting agents (VDAs) results in substantial cell death that precede tumor shrinkage. Here, we investigate the potential of hyperpolarized magnetic resonance spectroscopy (HPMRS) to monitor early metabolic changes associated with VDA treatment.Methods: Mice bearing C3H mammary carcinomas were treated with the VDAs combretastatin-A4-phosphate (CA4P) or the analog OXi4503, and HPMRS was performed following [1-13C]pyruvate administration. Similarly, treated mice were positron emission tomography (PET) scanned following administration of the glucose analog FDG. Finally, metabolic imaging parameters were compared to tumor regrowth delay and measures of vascular damage, derived from dynamic contrast-agent enhanced magnetic resonance imaging (DCE-MRI) and histology.Results: VDA-treatment impaired tumor perfusion (histology and DCE-MRI), reduced FDG uptake, increased necrosis, and slowed tumor growth. HPMRS, revealed that the [1-13C]pyruvate-to-[1-13C]lactate conversion remained unaltered, whereas [1-13C]lactate-to-[13C]bicarbonate (originating from respiratory CO2) ratios increased significantly following treatment.Conclusions: DCE-MRI and FDG-PET revealed loss of vessel functionality, impaired glucose delivery and reduced metabolic activity prior to cell death. [1-13C]lactate-to-[13C]bicarbonate ratios increased significantly during treatment, indicating a decline in respiratory activity driven by the onset of hypoxia. HPMRS is promising for early detection of metabolic stress inflicted by VDAs, which cannot easily be inferred based on blood flow measurements.

Abstract 2854: The potential of hyperpolarized 13-C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents

Abstract Purpose: Targeting tumor vasculature using vascular disrupting agents (VDAs) is an attractive therapy. These agents induce rapid physiological changes long before any growth inhibitory effects become apparent. Detecting these early changes should allow us to predict anti-tumor effects. Hyperpolarized ¹³C magnetic resonance spectroscopy (HPMRS) allows dynamic measurements of the metabolism of 13C-labeled substrates and may be a new approach for reliably monitoring early effects of VDAs. The aim of this study was to investigate the potential of HPMRS to achieve this in a pre-clinical model. Methods: Mice bearing 200 cubic mm foot implanted C3H mammary carcinomas were intraperitoneally injected with combretastatin-A4-phosphate (CA4P) or the A1 analogue OXi4503. Tumor response was assessed by determining necrosis development, measured histologically 24-hours after treatment, and tumor growth time (TGT; time to reach 5 times treatment volume). Estimates of tumor perfusion and metabolism were performed 3h (CA4P) and 6h (OXi4503) after treatment. Perfusion was measured from Hoechst 33342 uptake in histological sections and by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) at 7-Tesla. Metabolic changes were assessed following intravenous injection of hyperpolarized [1-¹³C]pyruvate and observing the conversion to lactate with HPMRS in a 9.4-Tesla scanner. Additional mice were positron emission tomography (PET) scanned using a Mediso nanoScan PET/MRI scanner following administration of fluorodeoxyglucose (FDG). Statistical comparisons were made using a one-way analysis of variance (ANOVA), with significance level of p&amp;lt;0.05. Results: A dose response relationship was found between injected drug doses and both tumor necrosis induction and TGT, with OXi4503 having a larger effect than CA4P. Two equally effective doses were selected for further analysis (250 mg/kg for CA4P and 50 mg/kg for OXi4503). Both drugs significantly decreased perfusion relative to controls; the respective mean (with 1 SE) values for CA4P and OXi4503 were decreased to 57% (50-64) and 38% (28-48) using Hoechst 33342, and 61% (57-65) and 61% (54-68) using DCE-MRI. FDG uptake (mean tumor-to-brain ratio) also significantly reduced to 43% (33-53) and 33% (25-41) for CA4P and OXi4503, respectively. However, an unaltered pyruvate to lactate ratio was found; the values being 98% (89-107) for CA4P and 113% (102-124) for OXi4503. Conclusion: No change in the HPMRS values was observed, despite changes in all other parameters, indicating that the metabolic fate of pyruvate remains unaltered despite reduced delivery of oxygen and nutrients. This could be because our data provides measures of whole-tumor mean responses that include a viable metabolically unaltered tumor rim and a tumor core with no blood flow (i.e., no pyruvate delivery) following VDA treatment. Citation Format: Ane B. Iversen, Morten Busk, Lotte B. Bertelsen, Christoffer Laustsen, Ole L. Munk, Thomas Nielsen, Thomas R. Wittenborn, Johan Bussink, Jasper Lok, Hans Stodkilde-Jorgensen, Michael R. Horsman. The potential of hyperpolarized 13-C magnetic resonance spectroscopy to monitor the effect of combretastatin based vascular disrupting agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2854. doi:10.1158/1538-7445.AM2017-2854