High-resolution Magnetic Resonance Spectroscopy Research Articles

Nuclear magnetic resonance-biochemical correlation toward deep learning of theranosis and precision medicine

Efforts have been made to employ the nuclear magnetic resonance (NMR)-biochemical correlation concept or a combination of MR imaging (MRI) and MR spectroscopy (MRS) as an established diagnostic tool for medical practice in clinical settings. Recent reviews and meta-analyses indicate the great possibility of using integrated multimodal multiparametric MRI and MRS for deep learning (DL) of soft-tissue pathophysiology, enabling improved decision-making and disease progression monitoring in precision medicine. Recent guidelines and clinical trials suggest the need for DL of the biophysical and biochemical nature of the brain, breast, prostate, liver, and heart tissue from digital spectromics analysis, along with other molecular imaging modalities. The current opinions, based on recent recommendations, available literature on evidence-based MR spectromics, clinical trials, and meta-analyses on high-resolution MRI and MRS suggest that utilizing MRI and MRS signals as theranostic biomarkers for various soft tissues can demonstrate NMR-biochemical correlation and employ MRI with MRS as adjunct real-time tools, generating robust, and fast tissue digital images with metabolic screening. The integration of DL features can aid in evaluating patient disease diagnosis and therapy within a clinical setting, considering the available medical practices and their limitations.

Metabolic profiling of colorectal cancer organoids: A comparison between high-resolution magic angle spinning magnetic resonance spectroscopy and solution nuclear magnetic resonance spectroscopy of polar extracts.

Patient-derived cancer cells cultured in vitro are a cornerstone of cancer metabolism research. More recently, the introduction of organoids has provided the research community with a more versatile model system. Physiological structure and organization of the cell source tissue are maintained in organoids, representing a closer link to in vivo tumor models. High-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) is a commonly applied analytical approach for metabolic profiling of intact tissue, but its use has not been reported for organoids. The aim of the current work was to compare the performance of HR MAS MRS and extraction-based nuclear magnetic resonance (NMR) in metabolic profiling of wild-type and tumor progression organoids (TPOs) from human colon cancer, and further to investigate how the sequentially increased genetic alterations of the TPOs affect the metabolic profile. Sixteen metabolites were reliably identified and quantified both in spectra based on NMR of extracts and HR MAS MRS of intact organoids. The metabolite concentrations from the two approaches were highly correlated (r = 0.94), and both approaches were able to capture the systematic changes in metabolic features introduced by the genetic alterations characteristic of colorectal cancer progression (e.g., increased levels of lactate and decreased levels of myo-inositol and phosphocholine with an increasing number of mutations). The current work highlights that HR MAS MRS is a well-suited method for metabolic profiling of intact organoids, with the additional benefit that the nondestructive nature of HR MAS enables subsequent recovery of the organoids for further analyses based on nucleic acids or proteins.

Maternal exposure to polystyrene microplastics alters placental metabolism in mice.

The rapid growth in the worldwide use of plastics has resulted in a vast accumulation of microplastics in the air, soil and water. The impact of these microplastics on pregnancy and fetal development remains largely unknown. In pregnant mice, we recently demonstrated that exposure to micro- and nanoplastics throughout gestation resulted in significant fetal growth restriction. One possible explanation for reduced fetal growth is abnormal placental metabolism. To evaluate the effect of maternal exposure to microplastics on placental metabolism. In the present study, CD-1 pregnant mice were exposed to 5 μm polystyrene microplastics in filtered drinking water at one of four concentrations (0 ng/L (controls), 102 ng/L, 104 ng/L, 106 ng/L) throughout gestation (n = 7-11/group). At embryonic day 17.5, placental tissue samples were collected (n = 28-44/group). Metabolite profiles were determined using 1H high-resolution magic angle spinning magnetic resonance spectroscopy. The relative concentration of lysine (p = 0.003) and glucose (p < 0.0001) in the placenta were found to decrease with increasing microplastic concentrations, with a significant reduction at the highest exposure concentration. Multivariate analysis identified shifts in the metabolic profile with MP exposure and pathway analysis identified perturbations in the biotin metabolism, lysine degradation, and glycolysis/gluconeogenesis pathways. Maternal exposure to microplastics resulted in significant alterations in placental metabolism. This study highlights the potential impact of microplastic exposure on pregnancy outcomes and that efforts should be made to minimize exposure to plastics, particularly during pregnancy.

Local Structure and Protons in Non-Stoichiometric Pseudo-Cubic Pollucite Mineral by Multinuclear NMR

The pollucite structure is considered as a candidate ceramic crystalline matrix for the ceramic immobilization and long-term storage of 135Cs and 137Cs fission products, and thus, their structural characteristics have particular importance. However, its local structure has not been fully resolved from reciprocal-space techniques and infrared spectroscopy, and important discrepancies exist in the available literature. Two birefringent and non-stoichiometric pollucite specimens from Tanco pegmatite (Cs0.83Na0.20Al1.13Si2.56O6) and from Mt. Mica pegmatite (Cs0.94Na018Al1.23Si2.78O6), with powder X-ray diffraction patterns fully consistent with the cubic Ia-3d space-group symmetry, and with a very different degree of hydrothermal alteration, were used in this work. High-resolution magic-angle spinning multinuclear magnetic resonance (MAS NMR) spectroscopy, including 29Si, 27Al, 23Na, 133Cs, and 1H spectra at 9.4 T, as well as 1H, 27Al, 27Al{1H} dipolar evolutions and 27Al{29Si} Heteronuclear Multiple Quantum Coherence (HMCQ) spectra at 17.6 T, has been used to investigate the local structure of pollucite and the role of protons. The 29Si spectra suggest a local structure with a disordered Si/Al distribution in only one tetrahedral T site, but with a preference of Si atoms for Q41 (3Si,1Al) and Q42 (2Si,2Al) environments, in comparison with random and Loewenstein distributions, due to charge dispersion effects. However, the 27Al{1H} dipolar evolutions suggest two spectroscopically distinct T sites for Al atoms. The 23Na and 133Cs spectra indicate broad site distributions for these cavity cations. The anisotropic character of the long-range disordered pollucite structure, with a pseudo-cubic symmetry and lack of strict periodicity, can be explained from an incipient displacive transition to lower symmetry. These pollucite specimens are essentially anhydrous minerals despite the 1H and the cross-polarization experiments suggesting that some protons exist in the structure as -OH groups, whereas water molecules were only found in relation to the phyllosilicate impurities from alteration in specimen Tanco and perhaps also as liquid water in fluid inclusions.

Characterization of lipomatous tumors with high-resolution 1H MRS at 17.6T: Do benign lipomas, atypical lipomatous tumors and liposarcomas have a distinct metabolic signature?

BackgroundDistinguishing between some benign lipomas (BLs), atypical lipomatous tumors (ALTs), and dedifferentiated liposarcomas (DDLs) can be challenging due to overlapping magnetic resonance imaging characteristics, and poorly understood molecular mechanisms underlying the malignant transformation of liposarcomas.PurposeTo identify metabolic biomarkers of the lipomatous tumor spectrum by examining human tissue specimens using high-resolution 1H magnetic resonance spectroscopy (MRS).Materials and methodsIn this prospective study, human tissue specimens were obtained from participants who underwent surgical resection for radiologically-indeterminate lipomatous tumors between November 2016 and May 2019. Tissue specimens were obtained from normal subcutaneous fat (n=9), BLs (n=10), ALTs (n=7) and DDLs (n=8). Extracts from specimens were examined with high-resolution MRS at 17.6T. Computational modeling of pattern recognition-based cluster analysis was utilized to identify significant differences in metabolic signatures between the lipomatous tumor types.ResultsSignificant differences between BLs and ALTs were observed for multiple metabolites, including leucine, valine, branched chain amino acids, alanine, acetate, glutamine, and formate. DDLs were distinguished from ALTs by increased glucose and lactate, and increased phosphatidylcholine. Multivariate principal component analysis showed clear clustering identifying distinct metabolic signatures of the tissue types.ConclusionMetabolic signatures identified in 1H MR spectra of lipomatous tumors provide new insights into malignant progression and metabolic targeting. The metabolic patterns identified provide the foundation of developing noninvasive MRS or PET imaging biomarkers to distinguish between BLs, ALTs, and DDLs.

Endothelial nitric oxide deficiency results in abnormal placental metabolism

Placental metabolism determines the amount of nutrients available to the fetus and may be altered in pregnancies complicated by fetal growth restriction (FGR). To study which metabolites are associated with FGR, we performed 1H high-resolution magic angle spinning magnetic resonance spectroscopy of placental tissue from endothelial nitric oxide synthase knockout (eNOS KO) mice, a model of FGR, and C57BL/6J controls at embryonic day 17.5 (n = 24/genotype). The relative concentration of glucose was increased in the placentas of eNOS KO mice compared to controls (p = 0.006). This study highlights the potential for glucose as a biomarker of abnormal placental metabolism that leads to FGR.

Abstract 6353: Metabolic changes in the spleen and pancreas induced by PDAC xenografts with or without glutamine transporter downregulation

Abstract Introduction: Our ongoing studies are focused on characterizing metabolic changes induced in the organs of mice with cachexia-inducing Pa04C human pancreatic cancer xenografts. Because pancreatic cancer cells are glutamine dependent [1], we downregulated the glutamine transporter SLC1A5 in Pa04C cells to determine if metabolic changes induced in the spleen and pancreas by Pa04C tumors were normalized when SLC1A5 was downregulated in these tumors. Metabolic patterns were characterized using high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen and pancreas tissue obtained from normal mice and mice with Pa04C tumors and mice with Pa04C tumors with SLC1A5 downregulated. Method: Patient derived cachexia-inducing Pa04C pancreatic cancer cells were lentivirally transduced to express shRNA to stably downregulate SLC1A5. Mice were euthanized once tumors were ~500 mm3, the spleen and pancreas were excised and snap frozen. Snap frozen spleen (normal n= 5, Pa04C n= 11, Pa04C_SLC1A5 n= 10) and pancreas (normal n= 4, Pa04C n= 16, Pa04C_SLC1A5 n= 10) tissue samples were pulverized for dual phase extraction. The aqueous phase was used for 1H MRS analysis. Topspin 3.5 software was used for data processing and analyses. Results and Discussion: Significant downregulation of SLC1A5 mRNA and protein was confirmed in Pa04C_SLC1A5 cells and tumors. SLC1A5 downregulation resulted in significant growth delay and attenuation of weight loss. A comparison of normal mice vs empty vector/wild type tumor (EV/WT) bearing mice identified significant changes in succinate, aspartate and fumarate in the spleen, lactate, acetate, pyruvate, methionine, asparagine, creatine, choline phosphocholine, uracil, histidine and phenylalanine in the pancreas, with leucine, isoleucine, valine, alanine, glutamate, glutamine, glutathione, glycerophosphocholine, glycine, glucose and tyrosine commonly altered in the spleen and pancreas. A comparison of normal vs Pa04C_ SLC1A5 tumor bearing mice identified similar metabolic changes in the spleen and pancreas but these were reduced. Fumarate did not change in the spleen, and of the metabolic changes common to spleen and pancreas, glutamine did not change when tumor SLC1A5 was downregulated. Metabolite changes induced only in the pancreas were also similar to normal vs EV/WT with the exception of a change in glutamine with SLC1A5 downregulation and no change in lactate. Our data highlight the profound metabolic changes in spleen and pancreas metabolism that occur with growth of a cachexia-inducing pancreatic cancer xenograft, and the impact on these metabolic patterns as a result of downregulating the glutamine transporter in these cancer cells. The metabolic patterns identified in the spleen and pancreas may provide novel targets to reduce the morbidity from cachexia. Reference: 1. Son J et al, Nature. 2013;496(7443):101-5. Citation Format: Raj Kumar Sharma, Santosh Kumar Bharti, Balaji Krishnamachary, Yelena Mironchik, Paul Winnard Jr., Marie-France Penet, Zaver M. Bhujwalla. Metabolic changes in the spleen and pancreas induced by PDAC xenografts with or without glutamine transporter downregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6353.

Atomic-Level Structure of Mesoporous Hexagonal Boron Nitride Determined by High-Resolution Solid-State Multinuclear Magnetic Resonance Spectroscopy and Density Functional Theory Calculations

Mesoporous hexagonal boron nitride (p-BN) has received significant attention over the last decade as a promising candidate for water cleaning/pollutant removal and hydrogen storage applications. Here, high-resolution solid-state NMR spectroscopy and plane-wave density-functional theory (DFT) calculations are used to obtain an atomic-level description of p-BN. 1H–15N or 1H–14N heteronuclear (HETCOR) correlation experiments recorded with either conventional NMR at room temperature or dynamic nuclear polarization surface-enhanced spectroscopy (DNP-SENS) at ca. 100 K reveal NB2H, NBH2, NBH3+ species residing on the edges of BN sheets. Ultra-high field 35.2 T 11B NMR spectroscopy was used to resolve 11B NMR signals from BN3, BN2Ox(OH)1–x (x = 0–1), BNOx(OH)2–x (x = 0–2), BOx(OH)3–x (x = 0–3), and BOx(OH)4–x– (x = 0–4). Importantly, 2D 11B dipolar double-quantum–single-quantum homonuclear correlation spectra reveal that many pore/defect sites are composed of boron oxide/hydroxide clusters connected to the BN framework through BN2O units. 1D and 2D 11B{15N} HETCOR NMR experiments, in addition to plane-wave DFT calculations of nine different structural models, further confirm the assignment of all NMR signals. The detailed structure determination of the pore and edge/defect sites within p-BN should further enable the rational design and development of next-generation p-BN-based materials. In addition, the techniques outlined here should be applicable to determine structure within other porous and/or boron-based materials.

Impact of intratumoral heterogeneity on the metabolic profiling of breast cancer tissue using high-resolution magic angle spinning magnetic resonance spectroscopy.

High-resolution magic angle spinning (HR-MAS) magnetic resonance spectroscopy (MRS) is a useful metabolic profiling technique for human tissue. However, the impact of intratumoral heterogeneity on the metabolite levels of breast cancers is not yet established. The purpose of this prospective study was to investigate whether the tumor cell fraction of core needle biopsy (CNB) specimens of breast cancers affect metabolic profiles assessed with HR-MAS MRS. From June 2015 to December 2016, 46 patients with 47 breast cancers were enrolled. HR-MAS MRS was used for the metabolic profiling of 285 CNB specimens from the 47 cancers. Multiple CNB samples (range 2-8) for the HR-MAS MRS experiment were obtained from surgical specimens under ultrasound guidance following surgical removal of the tumor. Tumor cell fraction was expressed as a percentage of the tumor cell volume relative to the total tumor volume contained in each CNB sample. Metabolite quantification levels were compared according to primary tumor characteristics using the t-test. Multivariate analyses were performed including primary tumor characteristics and tumor cell percentages as variables. Correlations between tumor cell percentage and metabolite levels in the CNB specimens were assessed according to the immunohistochemical status of the primary tumor. In univariate analysis, levels of choline-containing compounds, glutamate, glutamine, glycine, serine, and taurine were correlated with primary tumor characteristics. In multivariate analysis, most metabolite levels were not affected by tumor cell percentage. Tumor cell percentage showed poor correlation with metabolite levels in hormone receptor-positive cancer and triple-negative cancer, and poor to fair correlation with metabolite levels in HER2-positive cancer. This study showed that differences in the tumor cell fraction of CNB samples do not affect predictions on the primary cancer from which the samples are obtained.

Abstract 2353: Metabolic reprogramming by SLC1A5 downregulation in pancreatic cancer cells

Abstract Introduction: Pancreatic cancers exhibit limited response to chemo- and radiation therapy. Identifying novel targets for pancreatic cancer treatment is important for new treatment strategies. Because pancreatic cancer cells are ‘glutamine avid' [1], targeting glutamine metabolic pathways can provide novel options for treatment. The glutamine transporter, SLC1A5, is being actively investigated as a pharmacological target in cancer [2]. Here we have engineered human pancreatic cells expressing shRNA to downregulate the glutamine transporter SLC1A5. We performed high-resolution 1H magnetic resonance spectroscopy (MRS) to understand the metabolic reprograming that occurs with SLC1A5 downregulation in these two cell lines. Method: Panc1 and Pa04C pancreatic cancer cell lines were genetically engineered to express shRNA against SLC1A5 using lentiviral transduction. Downregulation of SLC1A5 was verified with qRTPCR and immunoblotting. Control cells expressing an empty vector (EV) were also engineered. Dual phase extraction was performed as previously described [3]. High-resolution 1H MRS was performed on the aqueous phase extracts and recorded on a 750 MHz spectrometer. All data acquisition, processing and quantification was performed with TOPSPIN 3.5 software. Spectra were obtained from Panc1EV (n=4), Pa04CEV (n=4), Panc1_SLC1A5_shRNA (n=4) and Pa04C_SLC1A5_shRNA (n=4) cells. Results and Discussion: SLC1A5 downregulation significantly decreased the glutamine/glutamate ratio in Pa04C_SLC1A5_shRNA cells but not in Panc1_SLC1A5_shRNA cells. Instead, in Panc1_SLC1A5_shRNA cells, a significant decrease of the amino acids leucine, isoleucine, valine, tyrosine, histidine, and phenylalanine was observed. Lactate and fumarate also significantly decreased in Panc1_SLC1A5_shRNA cells. Other than the significant decrease of glutamine/glutamate in the Pa04C_SLC1A5_shRNA cells, no other significant metabolic differences were observed in these cells. Our data expand the understanding of the diverse metabolic reprogramming that occurs following SLC1A5 downregulation in two human pancreatic cancer cell lines that may lead to the development of additional metabolic targets. Supported by NIH R01CA193365 and R35CA209960.1. Son J et al., Nature. 2013; 2. Bhutia YD et al., Biochim Biophys Acta. 2016; 3. Winnard PT Jr. et al., J Cachexia Sarcopenia Muscle. 2020. Citation Format: Raj Kumar Sharma, Balalji Krishnamachary, Ishwarya Sivakumar, Yelena Mironchik, Santosh Kumar Bharti, Zaver M. Bhujwalla. Metabolic reprogramming by SLC1A5 downregulation in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2353.

Abstract 2354: Downregulating the glutamine transporter, SLC1A5, significantly reduces cachexia in a PDAC xenograft

Abstract Cachexia occurs with high frequency and severity in pancreatic ductal adenocarcinoma (PDAC) patients [1]. Cachectic patients experience a wide range of symptoms affecting the function of organs such as muscle, liver, brain, and heart, causing significant morbidity [2]. In high-resolution 1H magnetic resonance spectroscopy (MRS) studies of extracts, we previously observed significant perturbation of glutamine in the brain and plasma of mice with a cachexia-inducing PDAC xenograft [3] that prompted us to evaluate the effects of modifying tumor glutamine metabolism on cachexia. We investigated tumors derived from cachexia-inducing Pa04C cells engineered to express shRNA against the glutamine transporter, SLC1A5, glutaminase (GLS) 1, and GLS 2. Patient-derived cachexia-inducing Pa04C cells were stably transduced with virions that expressed shRNA against GLS1 or GLS2 or SLC1A5. Pooled clones were obtained with puromycin selection. Empty vector (EV) cells were also established. Downregulation of target genes was confirmed with mRNA and protein expression characterization. The effects of gene knockdown on tumor growth and weight-loss were determined following subcutaneous inoculation of engineered cells or wild type cells in SCID mice. Longitudinal tumor growth, weights and percent weight changes were determined in 7 wild type (WT), 7 EV, 9 GLS1 downregulated, 9 GLS2 downregulated, and 9 SLC1A5 downregulated tumor bearing mice. Once tumors were ~500 mm3 in volume, tumors were harvested from euthanized mice, and snap frozen for molecular analysis. Protein and mRNA obtained from tumors was validated for downregulation of target genes. Efficient downregulation of SLC1A5, GLS1 and GLS2 mRNA and protein was confirmed in tumors. Downregulating SLC1A5 significantly reduced tumor growth. But, downregulating GLS1 or GLS2 did not reduce tumor growth and, in fact, GLS1 downregulated tumors grew significantly faster than WT or EV tumors. Importantly, for comparable tumor volumes, we found that body weight loss was markedly reduced in mice with SLC1A5 downregulated tumors. Although GLS1 downregulated tumors grew faster than WT tumors, weight loss was attenuated at comparable tumor volumes in these tumors although not to the same extent as in SLC1A5 downregulated tumors. These data highlight potential role of SLC1A5 in PDAC tumor treatment and in the treatment of PDAC-induced cachexia, and support targeting the glutamine/glutamate axis in PDAC to reduce or reverse cachexia.Supported by NIH R35 CA209960 and R01 CA193365. 1. Fearon KC, Baracos VE. Cachexia in pancreatic cancer: new treatment options and measures of success. 2010; 2. Inui A. Cancer anorexia-cachexia syndrome: current issues in research and management. CA Cancer J Clin. 2002; 3. Winnard PT, Jr., et al., Brain metabolites in cholinergic and glutamatergic pathways are altered by pancreatic cancer cachexia. J Cachexia Sarcopenia Muscle. 2020. Citation Format: Balaji Krishnamachary, Ishwarya Sivakumar, Yelena Mironchik, Raj Kumar Sharma, Santosh Kumar Bharti, Marie-France Penet, Paul Winnard, Flonne Wildes, Eibhlin Goggins, Anirban Maitra, Michael G. Goggins, Zaver M. Bhujwalla. Downregulating the glutamine transporter, SLC1A5, significantly reduces cachexia in a PDAC xenograft [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2354.

Biodistribution of Poly(alkyl cyanoacrylate) Nanoparticles in Mice and Effect on Tumor Infiltration of Macrophages into a Patient-Derived Breast Cancer Xenograft

We have investigated the biodistribution and tumor macrophage infiltration after intravenous injection of the poly(alkyl cyanoacrylate) nanoparticles (NPs): PEBCA (poly(2-ethyl-butyl cyanoacrylate), PBCA (poly(n-butyl cyanoacrylate), and POCA (poly(octyl cyanoacrylate), in mice. These NPs are structurally similar, have similar PEGylation, and have previously been shown to give large variations in cellular responses in vitro. The PEBCA NPs had the highest uptake both in the patient-derived breast cancer xenograft MAS98.12 and in lymph nodes, and therefore, they are the most promising of these NPs for delivery of cancer drugs. High-resolution magic angle spinning magnetic resonance (HR MAS MR) spectroscopy did not reveal any differences in the metabolic profiles of tumors following injection of the NPs, but the PEBCA NPs resulted in higher tumor infiltration of the anti-tumorigenic M1 macrophages than obtained with the two other NPs. The PEBCA NPs also increased the ratio of M1/M2 (anti-tumorigenic/pro-tumorigenic) macrophages in the tumors, suggesting that these NPs might be used both as a vehicle for drug delivery and to modulate the immune response in favor of enhanced therapeutic effects.

1H-NMR-Based salivary metabolomics from females with temporomandibular disorders – A pilot study

Although temporomandibular disorder (TMD) is the second most common musculoskeletal disorder in the general population, the disease is multifactorial and presents symptoms common to other conditions which misdiagnosis can lead to treatment failure. In this case-control study, we performed, for the first time, a high-resolution 1H-nuclear magnetic resonance spectroscopy metabolomic analysis of the saliva of 26 women with TMD of muscular origin (experimental group [EG]) at the beginning (EG-pre) and at the end (EG-post) of a conservative treatment, and of 27 normal women (control group [CG]) to identify a metabolic signature for TMD. One-way analysis of variance showed changes in the concentration of phenylacetate, dimethylamine, maltose, acetoin, and isovalerate. Partial least-square discriminant analysis showed that metabolite signals did not overlap in CG X EG-pre and EG-pre X EG-post, but overlapped in CG X EG-post. The area under the receiver operating characteristic curve was 1 in CG X EG-pre (95% CI, 1.000–1.000; p < 0.002), 0.993 in EG-pre X EG-post (95% CI, 0.963–1.000), and 0.832 in CG X EG-post (95% CI, 0.699–0.961). These results suggest that the metabolomic profiles of women with and without TMD differ, while after treatment there is a lower distinction and slight tendency towards overlapping between CG and EG-post compared to pre treatment. We also found that phenylacetate, dimethylamine, maltose, acetoin, and isovalerate are potential biomarkers for TMD of muscular origin.

T160. HIGH-RESOLUTION WHOLE BRAIN MR SPECTROSCOPIC IMAGING IN YOUTHS AT CLINICAL HIGH RISK FOR PSYCHOSIS: A PILOT STUDY

BackgroundIn general, MR spectroscopy (MRS) studies report alterations of both glutamatergic indices and NAA not only in first episode psychosis and established schizophrenia but also in high risk populations, suggesting that altered excitatory neurotransmission and loss of neuronal integrity are early pathophysiological processes. However, interpretation of these findings is limited by the region-of-interest approach of current MRS techniques, limiting the measurement of metabolites to delimited cerebral volumes, selected by a priori hypotheses. In that context, we developed and implemented a new technique including specific MR sequence and data reconstruction that allows for whole brain high-resolution MRS imaging (MRSI) in two or three dimensions. The results enable the mapping of main metabolites in all brain regions (cortex, white matter, deep grey matter) of youths at clinical high risk for psychosis (CHR-P).MethodsAn FID-MRSI (Henning et al. NMR Biomed 2009) sequence with a 3D phase encoding accelerated by compressed-sensing was implemented on a 3T Prisma fit MRI (Siemens, Erlangen, Germany). The echo time (TE) was 0.65 ms, repetition time (TR) was 355 ms and the flip angle 35 degree. FID was acquired with 4 kHz bandwidth. The size of the excited Volume of Interest (VOI) was (A/P-R/L-H/F) 210 mm by 160 mm by 95 mm with a matrix of 42 x 32 x 20 resulting in 5 mm isotropic resolution. After reconstruction (Klauser A et al. Magn Reson Med. 2018), 3D MRSI data were quantified with LCModel to produce 3D metabolite maps. Concentration for total N-acetyl aspartate (tNAA), total creatinine (tCre), choline-containing compounds (Cho), myo-inositol (Ins), glutamate and glutamine (Glx) were calculated in every single voxel. A T1-weighted MPRAGE anatomical scan was acquired for positioning of the 3D MRSI and for the segmentation of the brain. For each participant, brain tissue was segmented into gray and white matter. Cerebral lobes and deep grey mater structures were also delineated using Freesurfer software package.CHR-P individuals were recruited in the service of child and adolescent psychiatry and in the service of general psychiatry, department of psychiatry at Lausanne university hospital. They were help-seeking adolescents and young adults aged between 14 and 35, who presented a psychosis-risk syndrome or basic symptoms as assessed by the Structured Interview for Psychosis-Risk Syndromes (SIPS) and the Schizophrenia Proneness Instrument, Adult (SPI-A) or Child & Youth version (SPI-CY). Healthy controls matched for age and sex were recruited in the general population.ResultsThree-dimension MRSI provides spatial specificity by allowing main metabolites (i.e., tNAA, tCre, Cho, Ins and Glx) to be reliably mapped in the volume of the entire brain. The resulting contrast allows the recognition of brain compartments and subcortical structures. Individual brain segments, cerebral lobes and subcortical structures were registered to 3D MRSI data and the mean concentration in each structure was computed to allow group comparisons between CHR-P and HC.DiscussionIn general, there is a strong need to develop new tools for the identification and stratification of CHR-P populations. Alterations of gross brain anatomy are relatively late events but early and subtle neurochemical changes and especially those reflecting oxidative stress and concomitant synaptic remodeling are promising candidates. This pilot study illustrates the potential of three-dimension MRSI to detect such alterations in the whole brain and with a good spatial resolution.

Using high-resolution magic angle spinning magnetic resonance spectroscopy to characterize the metabolomic profile of renal cell carcinoma.

710 Background: Renal cell carcinoma (RCC) is a metabolic disease, with the various subtypes exhibiting aberrations in different metabolic pathways. Metabolomics may offer greater sensitivity for revealing disease biology than evaluation of tissue morphology. In this study, we investigate the metabolomic profile of RCC using high resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS). Methods: Tissue samples were obtained from radical or partial nephrectomy specimens that were fresh frozen &amp; stored at -80ºC. Tissue HRMAS MRS was performed by a Bruker AVANCE spectrometer. Metabolomic profiles of RCC &amp; adjacent benign renal tissue were compared, and false discovery rates (FDR) were used to account for multiple testing. Regions of interest (ROI) with FDR &lt; 0.05 were selected as potential predictors of malignancy. The Wilcoxon rank sum test was used to compare median MRS relative intensities for the candidate predictors. Logistic regression was used to determine odds ratios for risk of malignancy based on abundance of each metabolite. Results: There were 38 RCC (16 clear cell, 11 papillary, 11 chromophobe) &amp; 13 adjacent normal tissue specimens (matched pairs). Metabolic predictors of malignancy based on FDR include histidine, phenylalanine, phosphocholine, serine, phosphocreatine, creatine, glycerophosphocholine, valine, glycine, myo-inositol, scylla-inositol, taurine, glutamine, spermine, acetoacetate &amp; lactate. Higher levels of spermine, histidine &amp; phenylalanine at 3.15-3.13 ppm were associated with a decreased risk of RCC (OR 4x10−5, 95% CI 7.42x10−8, 0.02), while 2.84-2.82 ppm increased the risk of malignant pathology (OR 7158.67, 95% CI 6.3, 8.3x106), and the specific metabolites characterizing this region remain to be identified. Tumor stage did not appear to affect the metabolomics of malignant tumors, suggesting that metabolites are more dependent on histologic subtype. Conclusions: HRMAS-MRS identified many metabolites that may predict RCC. We demonstrated that those in the 3.14-3.13 ppm ROI were present in lower levels in RCC, while higher levels of metabolites in the 2.84-2.82 ppm ROI substantially increased the risk of RCC.

Using high-resolution magic angle spinning magnetic resonance spectroscopy to characterize the metabolomic profile of fat-poor angiomyolipoma and renal cell carcinoma.

711 Background: Fat-poor angiomyolipoma (AML) can be difficult to differentiate from renal cell carcinoma (RCC) radiographically and may lead to biopsy or unnecessary intervention. In vivoplatforms with the ability to identify tumor histology based on metabolic profiles may avoid unnecessary procedures &amp; their complications. The metabolomics of AML have not been characterized, &amp; research into this area may provide targetable molecules for large AMLs. In this study, we investigate the metabolomic profile of AMLs compared to clear cell RCC (ccRCC) using high resolution magic angle spinning (HRMAS) magnetic resonance spectroscopy (MRS). Methods: Tissue samples were obtained from radical or partial nephrectomy specimens that were fresh frozen &amp; stored at -80ºC. Tissue HRMAS MRS was performed by a Bruker AVANCE spectrometer. Metabolomic profiles of RCC &amp; adjacent benign renal tissue were compared, and false discovery rates (FDR) accounted for multiple testing. Regions of interest (ROI) with FDR &lt;0.05 were considered potential predictors of ccRCC rather than AML. The Wilcoxon rank sum test was used to compare median MRS relative intensities for candidate predictors. Logistic regression was used to determine odds ratios for risk of malignancy based on abundance of each metabolite. Results: There were 16 ccRCC samples &amp; 7 AML specimens. Candidate predictors of malignancy rather than AML based on FDR p-values include histidine, phenylalanine, phosphocholine, serine, alanine, glutamate, glutathione, glycerophosphocholine, &amp; glutamine. While an abundance of these metabolites is associated with higher risk of malignancy, the odds ratio was particularly high in the 3.5-3.49 ppm spectral region (OR 2.99x106, 95% CI 3.27, 2.73x1012, p=0.033)in ccRCC samples. Conclusions: HRMAS MRS identified metabolites that may help differentiate fat-poor AML from ccRCC. In particular, metabolites in the 3.5-3.49 ppm spectral region increased the risk of harboring RCC. Our findings may contribute to future in vivostudies to help identify which patients require intervention for malignancy &amp; which may be observed for benign AML without requiring biopsy.

Historical Biobanks in Breast Cancer Metabolomics- Challenges and Opportunities.

Background: Metabolomic characterization of tumours can potentially improve prediction of cancer prognosis and treatment response. Here, we describe efforts to validate previous metabolomic findings using a historical cohort of breast cancer patients and discuss challenges with using older biobanks collected with non-standardized sampling procedures. Methods: In total, 100 primary breast cancer samples were analysed by high-resolution magic angle spinning magnetic resonance spectroscopy (HR MAS MRS) and subsequently examined by histology. Metabolomic profiles were related to the presence of cancer tissue, hormone receptor status, T-stage, N-stage, and survival. RNA integrity number (RIN) and metabolomic profiles were compared with an ongoing breast cancer biobank. Results: The 100 samples had a median RIN of 4.3, while the ongoing biobank had a significantly higher median RIN of 6.3 (p = 5.86 × 10−7). A low RIN was associated with changes in choline-containing metabolites and creatine, and the samples in the older biobank showed metabolic differences previously associated with tissue degradation. The association between metabolomic profile and oestrogen receptor status was in accordance with previous findings, however, with a lower classification accuracy. Conclusions: Our findings highlight the importance of standardized biobanking procedures in breast cancer metabolomics studies.

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.

Ultrafast acoustics for modulating matter / Bayer M.

Illumination of matter by intense ultrashort light pulses can lead to launching of coherent phonon pulses through the ultrafast thermal expansion. The duration of these pulses may be as short as a ps and their amplitude may reach one thousandth of a crystal lattice constant. This dynamic change of the lattice can vary the band gap of a semiconductor on corresponding time scales, which can be exploited to modulate light emitters on sub-THz frequencies. Further, in magnetic materials the acoustic pulses can be used to vary the magnetic anisotropy, by which a precession of the magnetization can be triggered. In suitably tailored structures, this precessing magnetization can serve as ultacompact source of microwave radiation with frequencies up to 100 GHz and more, by which high resolution magnetic resonance spectroscopy may be achieved. In this contribution we will discuss the basic principles of ultrafast acoustics of matter and gives examples of applications. The author acknowledges, among others, the long-lasting collaboration with Andrey Akimov (Nottingham), Alexey Scherbakov (St. Petersburg &amp; Dortmund), and Dmitri Yakovlev (Dortmund &amp; St. Pertersburg), without which the work would not have been possible

Abstract 5265: Spleen metabolism altered by human pancreatic cancer xenografts

Abstract Cancer-induced cachexia accounts for approximately 20% of all cancer deaths [1] and affects nearly 80% of pancreatic cancer patients [2, 3]. Cachectic patients experience a wide range of symptoms affecting several organ functions such as muscle, liver, brain, and heart that decrease quality of life and worsen prognosis. A major characteristic of cachexia is the accelerated skeletal muscle and fat storage wasting causing nutrient mobilization both directly as lipid and amino acids, and indirectly as glucose derived from the exploitation of liver gluconeogenesis that reaches the tumor through the bloodstream [4]. Here, for the first time, we have performed high-resolution quantitative 1H magnetic resonance spectroscopy (MRS) of spleen tissue obtained from normal mice and mice bearing PDAC that are cachectic (Pa04C) and non-cachectic (Panc1). The Panc1 (non-cachectic), and Pa04C (cachectic) PDAC cell lines were used [5]. Eight-week-old male severe combined immunodeficient mice were inoculated in the right flank with cancer cells (5×106). Mice were euthanized once tumors were ~ 300 mm3. Control, cachectic and non-cachectic groups consisted of 9, 10 and 9 mice per group respectively. Spleen water soluble metabolites were extracted, freeze dried, reconstituted in D2O PBS and transferred to an NMR tube for spectral acquisition with a 750 MHz (17.6T) MR spectrometer [6]. As anticipated, mice with cachexia-inducing Pa04C tumors showed significant weight loss with time. We observed, for the first time, that spleens from cachectic Pa04C tumor bearing mice showed a profound weight loss compared to normal mice and mice with Panc1 tumors. However, an increase of liver and spleen size has been previously reported in terminal cachectic human patients from colorectal cancer measured by CT scan 2-11 months prior to death [7]. A significant decrease in almost all amino acids was observed in cachectic (Pa04C) mouse spleens compared to normal and non-cachectic (Panc1) mouse spleens. Differences in choline metabolites, creatine, glutamine, glutamate, glutathione and aspartate were observed in cachectic mouse spleens compared to non-cachectic mouse spleens and spleens from healthy control mice. The significant decrease of amino acids in the cachectic spleens may reflect increased utilization of amino acids by the tumor or other organs during the cachexia muscle/protein wasting [4]. These results provide new insights into changes in spleen metabolism during cachexia, and support investigating metabolic targets to reduce cachexia associated morbidity. Supported by NIH R01CA193365 and R35CA209960. Reference: 1. Argiles et al. Nat. Rev. Cancer 2014, 14 (11), 754; 2. Fearon et al. HPB (Oxford) 2010, 12 (5), 323; 3. Ozola et al. Pancreatology 2015, 15 (1), 19; 4. Porporato et al. Oncogenesis 2016, 5, e200; 5. Winnard et al. Can. Res. 2016, 76(6): 1441; 6. Penet et al. Clin. Can. Res. 2015, 21 (2), 386; 7. Lieffers et al. Am J Clin Nutr. 2009, 89 (4), 1173 Citation Format: Santosh Kumar Bharti, Paul T. Winnard, Raj Kumar Sharma, Yelena Mironchik, Marie-France Penet, Zaver M. Bhujwalla. Spleen metabolism altered by human pancreatic cancer xenografts [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 5265.