High-resolution MRS Research Articles

Integration of MRI and MRS approaches to monitor molecular imaging and metabolomic effects of trabectedin on a preclinical ovarian cancer model.

Although several drugs are available to treat recurrences of human epithelial ovarian cancer (EOC), clinical responses often remain short lived and lead to only marginal improvements in patients' survival. One of the new drugs proposed for recurrent platinum-resistant EOC patients is trabectedin (Trab), a marine-derived antitumor agent initially isolated from the tunicate Ecteinascidia turbinata and currently produced synthetically. Predictive biomarkers of therapy response to this drug and the potential use of non-invasive functional MRI and MRS approaches for an early assessment of Trab efficacy have not yet been evaluated, although they might be relevant for improving the clinical management of EOC patients. In the present work we combined functional and spectroscopic magnetic resonance technologies, such as in vivo diffusion-weighted MRI and 1 H MRS, with ex vivo high resolution MRS (HR-MRS) metabolomic analyses, with the aim of identifying new pharmacodynamic markers of Trab effectiveness on well characterized, highly aggressive human SKOV3.ip (a HER2-enriched cell variant derived from SKOV3 cells) EOC xenografts. In vivo treatment with Trab (three consecutive weekly 0.2mg/kg i.v. injections) resulted in the following: (1) a significant reduction of in vivo tumor growth, along with the formation in cancer lesions of diffuse hyper-intense areas detected by T2 -weighted MRI and attributed to necrosis, in agreement with histopathology findings; (2) significant increases in the apparent diffusion coefficient mean and median values versus saline-treated control tumors; and (3) a significant reduction in the choline-containing metabolites' signal detected by quantitative in vivo MRS. Multivariate and quantitative HR-MRS analyses on ex vivo tissue samples revealed Trab-induced alterations in phospholipid and glucose metabolism identified as a decrease in phosphocholine and an increase in lactate. Collectively, these data identify Trab-induced functional MRI and MRS alterations in EOC models as a possible basis for further developments of these non-invasive imaging methods to improve the clinical management of EOC patients.

Abstract 2505: High-resolution MRS characterization of malignant ascites in two models of ovarian cancer

Abstract Build-up of malignant ascites occurs in more than one third of ovarian cancer patients and significantly contributes to poor quality of life and mortality. Advances in understanding malignant ascites formation and finding new therapeutic options are urgently needed. High-resolution 1H MRS has been described as extremely useful in detecting endogenous metabolites to diagnose cancer, by providing a detailed overview of metabolic pathways in a single measurement. In the present study, we are using two ovarian cancer cell lines, the murine ID8-VEGF-Defb29 cell line and the human OVCAR3 cell line. Implanted orthotopically, these two ovarian cancer models are characterized by different profile of ascites formation. The mouse cell line ID8-VEGF-Defb29 induces large volumes of ascites, often more than 10 mL, while the human OVCAR3 cell line induces ascites less frequently and at smaller volumes, usually less than 0.2 mL. We applied high-resolution 1H MRS to compare the metabolic composition of both ascitic fluids. To better understand the differences observed, we characterized the metabolism of these ovarian cancer cells in culture by analyzing cell lysates and conditioned culture media with 1H MRS to advance our understanding of cancer cell metabolic reprogramming in malignant ascites formation and the role of the tumor microenvironment in ascites formation and composition. The two tumor models used in this study induced different ascitic profiles. While OVCAR3 tumor bearing mice developed small viscous volume of ascites, ID8-VEGF-Defb29 induced higher volumes. ID8-VEGF-Defb29 ascitic fluids were characterized by higher levels of glutamine, glucose, poly-unsaturated fatty acids and pyruvate compared to the OVCAR3 fluids, while all the other metabolites, including glutamate, lactate, myo-inositol, choline and acetate, were lower. To determine if the differences observed in the ascitic fluids were only due to a different metabolism of the cancer cells, we investigated their metabolism in vitro. We analyzed the metabolites present in the conditioned cell culture media, and in the cells, and observed differences in OVCAR3 and ID8-VEGF-Defb29 cells metabolism in vitro, without replicating the differences observed in vitro. A Venn diagram of the different metabolites present in the cells, media and ascites showed differences in the metabolites present in those 3 compartments, and highlighted the ones in common. Beta-hydroxybutyrate, lipids, maleic and citrate were found in both ascites, and not in the cells or media. Ascites MRS derived biomarkers could help in ovarian cancer diagnosis, and enhance our understanding of the biochemical and metabolic changes associated with ovarian cancer, and with ascites formation. Supported by Tina’s Wish Foundation, NIH P50CA013175 and P30CA06973. Citation Format: Santosh Bharti, Flonne Wildes, Chien-Fu Hung, TC Wu, Zaver Bhujwalla, Marie-France Penet. High-resolution MRS characterization of malignant ascites in two models of ovarian cancer [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 2505. doi:10.1158/1538-7445.AM2017-2505

Preliminary study for non-invasive magnetic resonance imaging and spectroscopy of the eye: A novel technique for monitoring pharmacokinetics of ocular drug delivery

Introduction Conventional pharmacokinetic methods for studying ocular drug delivery are invasive and cannot be conveniently applied to humans. The development of non-invasive techniques in monitoring pharmacokinetics of ocular drug delivery is of vital importance. Purpose The aim of this study was to develop a novel clinical technique that permits monitoring of in vivo intravitreal drug pharmacokinetics based on both Hydrogen Magnetic Resonance Spectroscopy (H-MRS) and/or quantitative Magnetic Resonance Imaging (qMRI) methodologies. Materials and methods Advanced qMRI and H-MRS were performed using a high resolution clinical MRI eye protocol based on T1w and T2w 3D-sequences as well as a standard single voxel. H-MRS protocol utilizing a clinical 1.5T whole-body scanner enforced with a specially designed small coil apparatus for signal detection. H-MRS single voxel dimensions were (1.5 × 1.5 × 1.5 cm 3 ). In vitro and ex vivo experiments were performed using pig eyes as reference standards and Flurbiprofen (a non-steroidal anti-inflammatory ocular drug) in concentrations 0.04 M–2 mM. All measurements performed at a fixed temperature of 37 °C utilizing an in-house developed temperature monitor/controller device. Results High resolution qMRI images and MRS spectra of the vitreous with and without the presence of ocular drugs at different concentrations were obtained. Characteristic H-MRS peaks on 1.5 and 7 ppm were observed on both Flurbiprofen concentrations. Differences on the relative NMR signal measurements were obtained using various Flurbiprofen concentrations. Characteristic water H-MRS peak on 4.7 ppm was observed in vitreous samples proved a valuable means for the assessment of pharmacokinetics on ocular drug delivery. Conclusion A clinical protocol of H-MRS and/or qMRI will become feasible with the development of a rapid, inexpensive, and automated technique that could be easily integrated on any conventional clinical MRI examination.

Investigation Of Muscle Metabolism Of The Quadriceps Via High-resolution Respirometry and 31P MRS In Connection With A 10 Week Endurance Training

Skeletal muscle is a highly adaptable tissue that can adjust to different stimuli. In the present study we investigated the impact of endurance training on muscle oxidative capacity with high resolution respirometry and 31P magnetic resonance spectroscopy (31P MRS). 40 healthy untrained subjects (UG) who performed an endurance training program 3 times a week lasting for 10 weeks were included in the study. 17 highly trained athletes (AG) were studied for comparison. Spatially-resolved dynamic 31P MRS measurements were obtained from the upper leg and biopsy samples were taken from the vastus lateralis to assess mitochondrial capacity with high-resolution respirometry. Subsequently, endurance and strength capacities of the subjects were determined via motor performance tests. After 10 weeks, the initial tests and muscle biopsies were repeated. We observed a significant increase in mass specific OXPHOS flux with training in the UG from 78.97 ± 16.05 to 101.39 ± 19.19 pmol.s-1.mg-1 (p<0.01). Flux between the UG and AG, both before and after training, respectively, was significantly different (AG: 120 ± 32.79 pmol.s-1.mg-1, UG see above, p<0.01). The capacity of the mitochondria to oxidize MCFA was significantly increased with training, observed as an increase in absolute flux (from 12.79 ± 4.67 pre-training to 29.58 ± 7.25 pmol.s-1.mg-1 post-training, p<0.01) and in the flux control ratio (FCR=fraction of a given flux relative to the maximal flux) of octanoyl-carnitine (0.14 ± 0.05 pre-trainig to 0.28 ± 0.04 post-training, p<0.01). However, no difference in the FCR of MCFA oxidation was found between UG after training and the AG (0.28 ± 0.04 post-training vs 0.26 ± 0.06). The FCR of Oxphos was increased after training (0.95 ± 0.09, p<0.01) but was not different between UG before training and AG (0.86 ± 0.09 pre-training vs. 0.86 ± 0.11). To date, analysis of the 31P MRS was still in progress. In conclusion, mitochondria seem to adapt to endurance training in a quantitative and qualitative way. The qualitative adaptations can most prominently be observed in the capacity of MCFA oxidation, which is increased due to training. The limitation of the OXPHOS system seems to be decreased temporarily in untrained subjects exposed to exercise training. Yet, this reversed decreased limitation in athletic subjects is unknown.

In vitro proton magnetic resonance spectroscopy of liver tissue informs in vivo hepatic proton magnetic resonance spectroscopy studies

Proton magnetic resonance spectroscopy (1H MRS) has the potential to provide a noninvasive assessment of hepatic lipid composition. The spectral resolution of in vivo clinical MR data is currently limited by the magnetic field strengths available and by motion artifact. Therefore, interpretation of in vivo MR findings is aided by corroboration with in vitro MR studies. A recent study published in HEPATOLOGY used 1H MRS of oils to model lipid composition in human liver.1 Johnson and colleagues presented a polyunsaturation index (PUI), defined as the amplitude of the diallylic fatty acid functional group resonance divided by the sum of the allylic, diallylic, methylene, and methyl functional group resonances. The PUI decreased in obesity and hepatic steatosis.1 A number of resonances were identified in the proton MR spectra from oils and the human liver. On the basis of chemical shift and relative concentration, we suggest the resonance assigned to the diallylic lipid peak in liver should in fact be assigned to total choline-containing compounds (tCho). Scrutiny of the published MR spectra from the oils showed the chemical shift difference between the methylene resonance and the diallylic resonance (peak 2) was δ1.5 ppm,1 consistent with results from in vitro MR studies of intact liver of δ1.47 ppm2 and δ1.48 ppm.3 However, the relative chemical shift between the methylene resonance and the peak assigned as the “diallylic” resonance (peak 2) in humans was δ1.9 ppm,1 more consistent with the published chemical shift difference between methylene and tCho resonances of δ1.90 ppm2 and δ1.88 ppm.3 A reassignment of the resonances between δ2.8 and δ3.2 ppm has implications for the interpretation of results. Although significant differences were seen between groups, these may be interpreted as being a result of a reduced tCho:lipid ratio, rather than a reduction in PUI. The choice of an internal reference peak is not obvious when using 1H MRS, because each resonance may comprise a number of overlapping peaks. However, in this study, the use of a reference peak that is present in both the model oils and the human liver, such as the methylene resonance, would provide a suitable reference for chemical shift, even if not for the absolute concentration. Therefore, to validate the spectral assignment of lower resolution MRS in vivo, it should be borne in mind that oils, while providing a model of lipid composition, may not be fully representative of all the resonances observed from intact tissue. Comparison to high-resolution MRS of intact tissue or tissue extracts enables the identification of constituents such as choline-related compounds, amino acid, and carbohydrate resonances, in addition to lipids, at physiological concentrations. Jeremy F.L. Cobbold*, Simon D. Taylor-Robinson*, I. Jane Cox , * Department of Gastroenterology and Hepatology, Division of Medicine, Imperial College London, London, UK, Imaging Sciences Department, Division of Clinical Sciences, Imperial College London, London, UK.

Metabolic progression markers of neurodegeneration in the transgenic G93A‐SOD1 mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. Visualizing corresponding metabolic changes in the brain of patients with ALS with proton magnetic resonance spectroscopy ((1)H-MRS) may provide surrogate markers for an early disease detection, for monitoring the progression and for evaluating a treatment response. The primary objective of our study was to evaluate whether modifications in MR metabolite levels occur before clinical disease onset, and whether these changes are directly linked to a distinct spatial progression pattern in the CNS. Therefore, age-dependent alterations in the cerebral and spinal metabolic profile in the mouse model of ALS overexpressing the mutated human G93A-superoxide dismutase 1 (G93A-SOD1) were determined by high-resolution MRS of tissue extracts at 14.1 Tesla. Both non-transgenic mice (control mice) and transgenic mice overexpressing the non-mutated human SOD1 (tg-SOD1) served as controls. In the spinal cord of G93A-SOD1 mice significantly decreased levels of N-acetyl aspartate were already detected 34 days postpartum, i.e. about 60 days before the average disease onset caused by motor neuron decline. In addition, glutamine and gamma-aminobutyric acid concentrations were significantly diminished at Day 75, which is still in the presymptomatic phase of the disease. These metabolic changes were further progressive in the course of the disease and started to involve the brainstem at Day 75. Overall, high-resolution (1)H-MRS allows a sensitive spatial and temporal metabolite profiling in the presymptomatic phase of ALS even before significant neuronal cell loss occurs.

Neurochemical effects of acute toluene exposure in juvenile and adolescent rats as determined by high-resolution MRS

Neurochemical effects of acute toluene exposure in juvenile and adolescent rats as determined by high-resolution MRS

Is Magnetic Resonance Image Guidance the Key to Opening Chronic Total Occlusions?

Through progressive plaque growth or fibrotic organization of occlusive thrombus, atherosclerosis may result in chronic total occlusion (CTO) of a major arterial conduit. If CTO develops slowly, collateral pathways may supply sufficient perfusion to retain tissue viability despite occlusion of the major inflow conduit. However, such collateral-mediated perfusion rarely matches that provided by an open main conduit, particularly during the peak demand of regional muscular activity. Although strategies to limit regional oxygen demand (eg, β-blockers for the treatment of angina pectoris) or to enhance collateral function (eg, a walking program for superficial femoral occlusion, experimental angiogenesis) may be of some benefit, these approaches generally offer less complete return of normal physiology and relief of ischemic symptoms than does direct revascularization of the occluded vessel. With surgical bypass, the nature of the occlusion (its length, duration, tortuosity, calcification) is less important than the presence of a suitable distal anastomotic target. On the other hand, with catheter-based techniques, success depends heavily on these lesion characteristics. In fact, crossing such occluded segments with a guidewire remains the dominant barrier to catheter-based treatments of occluded arterial segments. Once the guidewire has been passed successfully into the distal true lumen, the process of dilating and stenting the segment is usually straightforward and durable, at least with the use of drug-eluting stents in coronary CTOs.1,2 Article p 1101 Devices designed to cross CTOs should have 3 key features: an ability to distinguish a true luminal path from one created within (dissection) or through (perforation) the vessel wall of the occluded segment, an ability to change direction (steer) to correct deviations from the desired path through the occlusion, and an ability to penetrate the frequently fibrotic and focally calcific substance of the occlusion through the use of either mechanical stiffness or an alternative energy modality (laser, …

Evaluation of31P high-resolution magic angle spinning of intact tissue samples

The first detailed evaluation is presented of high-resolution (31)P MRS using magic angle spinning (MAS) of intact tissue samples and comparison with the conventional method of studying tissue extracts. The main motivation is that MAS leaves the sample intact at the end of the study for histopathological evaluation. While MAS of tissue samples has previously been demonstrated for (1)H MRS, (31)P MRS is better suited to study of the phospholipid metabolites of importance in cancer. Samples of rhabdomyosarcoma and RIF-1 experimental tumours were maintained at 4 degrees C, spun at 3 kHz and measured in 28-min acquisitions at 11.7 and 14 T. Metabolite stability was evaluated using four sequential 28-min acquisitions. High-resolution MRS was performed on extracts of the same tissue samples. (31)P HR-MAS yielded well-resolved high-resolution spectra, showing peaks from phosphoethanolamine (PE), phosphocholine (PC), inorganic phosphate, glycerophosphoethanolamine and glycerophosphocholine, with linewidths in the range 3-20 Hz. In tumour samples there was no significant change in peak areas over a 2-h period, while peaks sensitive to pH (inorganic phosphate, PE and PC) showed a small change in chemical shift, corresponding to a change of 0.13 +/- 0.06 pH units. Tissue metabolite concentrations showed good agreement with concentrations measured from extracts of the same pieces of tissue. For calculation of metabolite concentrations, the measurement of a reference compound in a separate measurement is more robust than using the signal from a reference compound in the rotor with the sample. Compared with performing tissue extracts, use of MAS of intact tissue samples requires less preparation, is quicker and permits the same sample to be used for subsequent histopathology. The methodology has particular application in studying phospholipid metabolism in cancer and in monitoring tumour response to treatment, where concentrations of phospholipid-related metabolites are found to alter following response to a wide range of anti-cancer therapies.

Bioenergetic Targeting during Organ Preservation: 31P Magnetic Resonance Spectroscopy Investigations into the Use of Fructose to Sustain Hepatic ATP Turnover during Cold Hypoxia in Porcine Livers

During liver preservation, ATP supplies become depleted, leading to loss of cellular homeostatic controls and a cascade of ensuing harmful changes. Anaerobic glycolysis is unable to prolong ATP production for a significant period because of metabolic blockade. Our aim was to promote glycolysis during liver cold hypoxia by supplying fructose as an additional substrate, compared to supplementation with an equivalent concentration of glucose. Porcine livers (two groups; n = 5 in each) were retrieved by clinical harvesting techniques and subjected to two cycles of cold hypoxia and oxygenated hypothermic reperfusion. In the second cycle of reperfusion, the perfusate was supplemented with either 10 mmol/L glucose (Group 1) or 10 mmol/L fructose (Group 2). During reperfusion in both groups, similar levels of ATP were detected by phosphorus magnetic resonance spectroscopy (31P MRS). However, during subsequent hypoxia, ATP was detected for much longer periods in the fructose-perfused group. The rate of ATP loss was sevenfold slower during hypoxia in the presence of fructose than in the presence of glucose (ATP consumption of −7.2 × 10−3% total 31P for Group 1 versus −1.0 × 10−3% total 31P for Group 2; P < 0.001). The changes in ATP were mirrored by differences in other MRS-detectable intermediates; e.g., inorganic phosphate was significantly higher during subsequent hypoxia in Group 1 (45.7 ± 2.7% total 31P) than in Group 2 (33.7 ± 1.1% total 31P; P < 0.01). High-resolution MRS of liver tissue extracts demonstrated that fructose was metabolized mainly via fructose 1-phosphate. We conclude that fructose supplied by brief hypothermic perfusion may improve the bioenergetic status of cold hypoxic livers by sustaining anaerobic glycolysis via a point of entry into the pathway that is different from that for glucose.

High resolution MRI and MRS: a feasibility study for the investigation of focal cerebral ischemia in mice.

Combined NMR imaging and spectroscopy have been applied to mouse brain during focal cerebral ischemia. The present study evaluated the feasibility of NMR measurements on mice in order to fine-tune the sequences and experimental setup for systematic investigations on stroke including future studies on transgenic animals. The acquisition of high quality diffusion-weighted, perfusion-weighted, and T2-weighted images (DWI, PWI, T2-WI, respectively) is demonstrated and complemented by measurements of 1H volume-selective spectroscopy and spectroscopic imaging (SI). Despite the small volume of the mouse brain, a satisfactory signal-to-noise ratio can be achieved with reasonably short measurement times. C57black/6J mice with an average body weight of 25 g were studied using state-of-the-art NMR sequences at 4.7 T. After induction of focal cerebral ischemia, the lesion was found clearly distinguishable in all imaging techniques. The apparent diffusion coefficient (ADC) was reduced in the ischemic region, and an expansion of the affected volume was observed with ongoing ischemia time. In the H spectra of ischemic animals a distinct change in the concentrations of NAA and lactate was visible. This is the first report on both SI data and perfusion-weighted imaging on mouse brain. It is demonstrated that the perfusion deficit during ischemia can be well demarcated. The spatial resolution of changes in metabolite concentrations allows the clear differentiation of elevated lactate levels in ischemic brain tissue.

High resolution intravascular MRI and MRS by using a catheter receiver coil.

Potentially important diagnostic information about atherosclerosis can be obtained by using magnetic resonance imaging and spectroscopy techniques. Because critical vessels such as the aorta, coronary arteries, and renal arteries are not near the surface of the body, surface coils are not adequate to increase the data quality to desired levels. A few catheter MR receiver coil designs have been proposed for imaging the walls of large blood vessels such as the aorta. These coils have limited longitudinal coverage and they are too thick to be placed into small vessels. A flexible, long and narrow receiver coil that can be placed on the tip of a catheter and will enable multi-slice high resolution imaging of small vessels has been developed. The authors describe the theory of the coil design technique, derive formulae for the signal-to-noise ratio characteristics of the coil, and show examples of high resolution cross-sectional images from isolated human aortas acquired by using this catheter coil. In addition, high resolution in vivo rabbit aorta images were obtained as well as a set of spatially resolved chemical shift spectra from a dog circumflex coronary artery.

Lactate production by human monocytes/macrophages determined by proton MR spectroscopy.

Elevated brain lactate has been observed by in vivo proton MRS in different pathological situations. The origin of this lactate remains controversial. The possibility that it was produced by the metabolism of phagocytic cells has been proposed. To investigate this hypothesis, the authors have employed high-resolution proton MRS to monitor changes in glucose, lactate, and other metabolites in the medium used to culture human monocyte-derived macrophages in vitro. Results show that the differentiation of human monocytes/macrophages in the presence of physiological stimulating factors (M-CSF or GM-CSF) was associated with an increase in lactate production and glucose utilization. The present results are consistent with the hypothesis that lactate detected by proton MRS in vivo may be produced by the metabolism of macrophages when infiltrates of these cells are present. The possible extrapolation of the authors' finding to the in vivo situation and its relevance are discussed.

Phosphorus metabolism during growth of lymphoma in mouse liver: a comparison of 31P magnetic resonance spectroscopy in vivo and in vitro

Large phosphomonoester (PME) signals are detected in the phosphorus magnetic resonance spectra (31P MRS) of many neoplastic and rapidly dividing tissues. In addition, alterations in phosphodiester (PDE) signals are sometimes seen. The present study of a murine lymphoma growing in liver showed a positive correlation between the hepatic PME/PDE ratio measured in vivo by 31P MRS at 4.7 T and the degree of lymphomatous infiltration in the liver, quantified by histology. High-resolution 31P MRS of liver extracts at 9.7 T showed that the PME peak consists largely of phosphoethanolamine (PE) and to a lesser extent of phosphocholine (PC). The concentration of both PE and PC increased positively with lymphomatous infiltration of the liver. In vivo, the PDE peak contains signals from phospholipids (mostly phosphatidylethanolamine and phosphatidylcholine) and the phospholipid breakdown products glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). Low levels of GPE and GPC were detected in the aqueous extracts of the control and infiltrated livers; their concentrations remained unchanged as the infiltration increased. The total concentration of phospholipids measured by 31P MRS of organic extracts decreased about 3-fold as the infiltration increased to 70%. Thus, our data showed that the increased PME/PDE ratio in vivo is due to both an increase in the PME metabolites and a decrease in the PDE metabolites. We propose that this ratio can be used as a non-invasive measure of the degree of lymphomatous infiltration in vivo.

Graphic-aided study of metabolic modifications of plasma in cancer using proton magnetic resonance spectroscopy.

Proton high-resolution MRS of human plasma allows the rapid detection, on the same spectrum, of many compounds originating from different metabolic pathways. In this paper, we illustrate the modifications of the plasma metabolic profiles recorded by proton NMR spectroscopy in different classes of cancers. These modifications can be easily monitored with graphic aids such as 'star plots' which define for each type of cancer a particular pattern describing the most altered metabolic pathways. By using 'star plots' three types of metabolic patterns have been distinguished: (i) the 'inflammatory' pattern characterized by an increase of glycosylated moieties of glycoproteins; (ii) a 'lipid modified' pattern, characterized by various modifications occurring mainly in the lipid moieties detected by MRS; and (iii) a pattern which is often observed in sarcomas and mainly characterized by an alteration in the N-acetyl glucosamine/N-acetyl neuraminic acid ratio. This study demonstrates the ability of proton MRS of plasma to rapidly detect the occurrence of metabolic modifications brought about by cancer evolution or therapy.