Diffusion weighted magnetic resonance spectroscopy revealed neuronal specific microstructural alterations in Alzheimer's disease.

In Alzheimer's disease, reconfiguration and deterioration of tissue microstructure occur before substantial degeneration become evident. We explored the diffusion properties of both water, a ubiquitous marker measured by diffusion MRI, and N-acetyl-aspartate, a neuronal metabolite probed by diffusion-weighted magnetic resonance spectroscopy, for investigating cortical microstructural changes downstream of Alzheimer's disease pathology. To this aim, 50 participants from the Swedish BioFINDER-2 study were scanned on both 7 and 3 T MRI systems. We found that in cognitively impaired participants with evidence of both abnormal amyloid-beta (CSF amyloid-beta42/40) and tau accumulation (tau-PET), the N-acetyl-aspartate diffusion rate was significantly lower than in cognitively unimpaired participants (P < 0.05). This supports the hypothesis that intraneuronal tau accumulation hinders diffusion in the neuronal cytosol. Conversely, water diffusivity was higher in cognitively impaired participants (P < 0.001) and was positively associated with the concentration of myo-inositol, a preferentially astrocytic metabolite (P < 0.001), suggesting that water diffusion is sensitive to alterations in the extracellular space and in glia. In conclusion, measuring the diffusion properties of both water and N-acetyl-aspartate provides rich information on the cortical microstructure in Alzheimer's disease, and can be used to develop new sensitive and specific markers to microstructural changes occurring during the disease course.

Investigation of metabolite correlates of CEST in the human brain at 7 T.

Metabolite-weighted chemical exchange saturation transfer MRI can be used to indirectly image metabolites such as creatine and glutamate. This study aims to further explore the contrast of CEST at 2 ppm in the human brain at 7T and investigate the metabolite correlates of CEST at 2 ppm via correlations with magnetic resonance spectroscopy (MRS). Simulations were performed to establish the optimal acquisition parameters, such as total saturation time (tsat) and B1 root mean squared (B1rms) for CEST at 2 ppm in the human brain. Parameters were validated via in vitro phantom studies at 7T using concentrations, pH and temperature comparable to what is found in the human brain. Finally, 10 healthy volunteers were scanned at 7T for comparison with MRS. Our results show that the optimal parameters to acquire CEST at 2 ppm images are: B1rms = 2.14 µT & tsat = 1500 ms, respectively. Comparison with MRS showed no significant correlation between CEST at 2 ppm and total Creatine measured by MRS (R = 0.19; p-value = 0.273). However, a significant correlation was found between CEST at 2 ppm and Glu (R = 0.39; p-value = 0.033), indicating the broad Glutamate-weighted CEST as the main measurable contributor to CEST at 2 ppm. We identified and confirmed optimal CEST at 2 ppm sequence parameters and validated CEST at 2 ppm measurements in a controlled in vitro environment. Our findings suggest that glutamate is a substantial contributor to the CEST at 2 ppm contrast observed in the human brain, whereas the creatine contribution to CEST at 2 ppm in the brain did not show a measurable contribution.

Diffusion-weighted MR spectroscopy: Consensus, recommendations, and resources from acquisition to modeling.

Brain cell structure and function reflect neurodevelopment, plasticity, and aging; and changes can help flag pathological processes such as neurodegeneration and neuroinflammation. Accurate and quantitative methods to noninvasively disentangle cellular structural features are needed and are a substantial focus of brain research. Diffusion-weighted MRS (dMRS) gives access to diffusion properties of endogenous intracellular brain metabolites that are preferentially located inside specific brain cell populations. Despite its great potential, dMRS remains a challenging technique on all levels: from the data acquisition to the analysis, quantification, modeling, and interpretation of results. These challenges were the motivation behind the organization of the Lorentz Center workshop on "Best Practices & Tools for Diffusion MR Spectroscopy" held in Leiden, the Netherlands, in September 2021. During the workshop, the dMRS community established a set of recommendations to execute robust dMRS studies. This paper provides a description of the steps needed for acquiring, processing, fitting, and modeling dMRS data, and provides links to useful resources.

Study protocol of IMAGINE-HD: Imaging iron accumulation and neuroinflammation with 7T-MRI + CSF in Huntington's disease.

INTRODUCTION: Strong evidence suggests a significant role for iron accumulation in the brain in addition to the well-documented neurodegenerative aspects of Huntington's disease (HD). The putative mechanisms by which iron is linked to the HD pathogenesis are multiple, including oxidative stress, ferroptosis and neuroinflammation. However, no previous study in a neurodegenerative disease has linked the observed increase of brain iron accumulation as measured by MRI with well-established cerebrospinal fluid (CSF) and blood biomarkers for iron accumulation, or with associated processes such as neuroinflammation. This study is designed to link quantitative data from iron levels and neuroinflammation metabolites obtained from 7T MRI of HD patients, with specific and well-known clinical biofluid markers for iron accumulation, neurodegeneration and neuroinflammation. Biofluid markers will provide quantitative measures of overall iron accumulation, neurodegeneration and neuroinflammation, while MRI measurements on the other hand will provide quantitative spatial information on brain pathology, neuroinflammation and brain iron accumulation, which will be linked to clinical outcome measures. METHODS: This is an observational cross-sectional study, IMAGINE-HD, in HD gene expansion carriers and healthy controls. We include premanifest HD gene expansion carriers and patients with manifest HD in an early or moderate stage. The study includes a 7T MRI scan of the brain, clinical evaluation, motor, functional, and neuropsychological assessments, and sampling of CSF and blood for the detection of iron, neurodegenerative and inflammatory markers. Quantitative Susceptibility Maps will be reconstructed using T2* weighted images to quantify brain iron levels and Magnetic Resonance Spectroscopy will be used to obtain information about neuroinflammation by measuring cell-specific intracellular metabolites' level and diffusion. Age and sex matched healthy subjects are included as a control group. DISCUSSION: Results from this study will provide an important basis for the evaluation of brain iron levels and neuroinflammation metabolites as an imaging biomarker for disease stage in HD and their relationship with the salient pathomechanisms of the disease on the one hand, and with clinical outcome on the other.

Imaging immunomodulatory treatment responses in a multiple sclerosis mouse model using hyperpolarized 13C metabolic MRI.

BACKGROUND: In recent years, the ability of conventional magnetic resonance imaging (MRI), including T1 contrast-enhanced (CE) MRI, to monitor high-efficacy therapies and predict long-term disability in multiple sclerosis (MS) has been challenged. Therefore, non-invasive methods to improve MS lesions detection and monitor therapy response are needed. METHODS: We studied the combined cuprizone and experimental autoimmune encephalomyelitis (CPZ-EAE) mouse model of MS, which presents inflammatory-mediated demyelinated lesions in the central nervous system as commonly seen in MS patients. Using hyperpolarized 13C MR spectroscopy (MRS) metabolic imaging, we measured cerebral metabolic fluxes in control, CPZ-EAE and CPZ-EAE mice treated with two clinically-relevant therapies, namely fingolimod and dimethyl fumarate. We also acquired conventional T1 CE MRI to detect active lesions, and performed ex vivo measurements of enzyme activities and immunofluorescence analyses of brain tissue. Last, we evaluated associations between imaging and ex vivo parameters. RESULTS: We show that hyperpolarized [1-13C]pyruvate conversion to lactate is increased in the brain of untreated CPZ-EAE mice when compared to the control, reflecting immune cell activation. We further demonstrate that this metabolic conversion is significantly decreased in response to the two treatments. This reduction can be explained by increased pyruvate dehydrogenase activity and a decrease in immune cells. Importantly, we show that hyperpolarized 13C MRS detects dimethyl fumarate therapy, whereas conventional T1 CE MRI cannot. CONCLUSIONS: In conclusion, hyperpolarized MRS metabolic imaging of [1-13C]pyruvate detects immunological responses to disease-modifying therapies in MS. This technique is complementary to conventional MRI and provides unique information on neuroinflammation and its modulation.

Magnetic resonance imaging (MRI) is widely used in the clinic to diagnose multiple sclerosis (MS), which affects the central nervous system and leads to a range of disabling symptoms. However, MRI is often not capable of detecting how well a patient responds to therapies, in particular those targeting the immune system. We questioned whether an advanced MRI method called hyperpolarized ¹³C MRS could help. Using a mouse model for MS, we showed that hyperpolarized ¹³C MRS can detect response to two therapies used in the clinic, namely fingolimod and dimethyl fumarate when conventional MRI could not. We also showed that this method is sensitive to the immune response. As hyperpolarized ¹³C MRS is becoming available in many centers worldwide, it could be used to evaluate existing and new treatments for people living with MS, improving care and quality of life.

Improved detection limits of J-coupled neurometabolites in the human brain at 7 T with a J-refocused sLASER sequence.

In a standard spin echo, the time evolution due to homonuclear couplings is not reversed, leading to echo time (TE)-dependent modulation of the signal amplitude and signal loss in the case of overlapping multiplet resonances. This has an adverse effect on quantification of several important metabolites such as glutamate and glutamine. Here, we propose a J-refocused variant of the sLASER sequence (J-sLASER) to improve quantification of J-coupled metabolites at ultrahigh field (UHF). The use of the sLASER sequence is particularly advantageous at UHF as it minimizes chemical shift displacement error and results in relatively homogenous refocusing. We simulated the MRS signal from brain metabolites over a broad range of TE values with sLASER and J-sLASER, and showed that the signal of J-coupled metabolites was increased with J-sLASER with TE values up to ~80 ms. We further simulated "brain-like" spectra with both sequences at the shortest TE available on our scanner. We showed that, despite the slightly longer TE, the J-sLASER sequence results in significantly lower Cramer-Rao lower bounds (CRLBs) for J-coupled metabolites compared with those obtained with sLASER. Following phantom validation, we acquired spectra from two brain regions in 10 healthy volunteers (age 38 ± 15 years) using both sequences. We showed that using J-sLASER results in a decrease of CRLBs for J-coupled metabolites. In particular, we measured a robust ~38% decrease in the mean CRLB (glutamine) in parietal white matter and posterior cingulate cortex (PCC). We further showed, in 10 additional healthy volunteers (age 34 ± 15 years), that metabolite quantification following two separate acquisitions with J-sLASER in the PCC was repeatable. The improvement in quantification of glutamine may in turn improve the independent quantification of glutamate, the main excitatory neurotransmitter in the brain, and will simultaneously help to track possible modulations of glutamine, which is a key player in the glutamatergic cycle in astrocytes.

Astrocytic function is associated with both amyloid-ß and tau pathology in non-demented APOE ϵ4 carriers.

A growing body of evidence suggests that astrocytes play a major role in the pathophysiology of Alzheimer's disease. Given that APOE is primarily expressed in astrocytes, these cells might be an important link between the APOE ε4 allele and the development of Alzheimer's disease pathology. Here, we investigate this hypothesis in vivo by measuring myo-inositol, a metabolite involved in astrocytic functions, with magnetic resonance spectroscopy. Currently, there is conflicting evidence regarding the relationship between APOE ε4 and myo-inositol concentration. Furthermore, data supporting a relationship between APOE ε4, myo-inositol and Alzheimer's disease pathology (amyloid-beta and tau proteins) in the preclinical stage of Alzheimer's disease are limited. A previous study revealed differences in myo-inositol levels between APOE ε4 carriers and non-carriers already in preclinical Alzheimer's disease participants. However, other reports showed no impact of APOE genotype on the association between myo-inositol and the rate of amyloid-beta accumulation. In the present study, we determined the effect of APOE genotype on the association between myo-inositol and both amyloid-ß and tau deposition quantified by PET in 428 cognitively unimpaired elderly and patients with mild cognitive impairment from the Swedish BioFINDER-2 cohort. APOE genotype impacted the associations between myo-inositol and amyloid-ß pathology as revealed by an interaction effect between APOE genotype and levels of myo-inositol (P < 0.001) such that higher myo-inositol concentration was related to more amyloid-beta pathology in APOE ε4 carriers only. A similar interaction effect was also found when investigating the effect of APOE on the association between myo-inositol and tau pathology (P < 0.01). Focusing on the APOE ε4 subsample, myo-inositol partially (17%) mediated the association between amyloid-beta and tau pathology (P < 0.05). Furthermore, in a subgroup of participants with available plasma levels of glial fibrillary acidic protein, a marker of astroglial activation and astrocytosis, we found that glial fibrillary acidic protein correlated with myo-inositol only in APOE e4 carriers (APOE ε4 carriers: P < 0.01; APOE ε4 non-carriers: P > 0.8), suggesting that myo-inositol might reflect an aspect of the astrocytic involvement in Alzheimer's pathology which is specific to the impact of APOE ε4. Therefore, we suggest that myo-inositol is a candidate in vivo marker to study the impact of APOE ε4 on the interplay between astrocytes and the pathophysiology of Alzheimer's disease.

Cortical glutamate and gamma-aminobutyric acid over the course of a provoked migraine attack, a 7 Tesla magnetic resonance spectroscopy study.

Enhanced activity of the glutamatergic system has been linked to migraine pathophysiology. The present study aimed to assess the involvement of the glutamatergic system in the onset of attacks. We provoked attacks by infusion of glyceryl trinitrate (GTN; 0.5 µg/kg/min over 20 min) in 24 female episodic migraineurs without aura and 13 female age-matched healthy controls. Over the course of a single day participants were scanned three times at fixed time slots (baseline before GTN infusion, 90 min and 270 min after start of GTN infusion). Single-volume proton magnetic resonance spectra (1H-MRS) were acquired at 7 Tesla from a volume of interest (VOI, 2x2x3 cm) in the visual cortex. We assessed the concentrations of glutamate, its major precursor glutamine, and its product gamma-aminobutyric acid (GABA) over the course of a provoked attack. The preictal state was defined as the period after GTN infusion until the migraine-like headache started, independent of possible experienced premonitory symptoms, and the ictal state was defined as the period with provoked migraine-like headache. Data were analyzed using a linear mixed-effect model for repeated measures. Glutamate and glutamine levels did not change from interictal to the preictal and ictal state. GABA levels increased from interictal towards the preictal state for migraine patients compared with healthy controls. We conclude that high resolution 7T MRS is able to show changes in the glutamatergic system towards a triggered migraine attack, by revealing an increased GABA concentration associated with the onset of a migraine attack.

Imaging 6-Phosphogluconolactonase Activity in Brain Tumors In Vivo Using Hyperpolarized δ-[1-13C]gluconolactone.

INTRODUCTION: The pentose phosphate pathway (PPP) is essential for NADPH generation and redox homeostasis in cancer, including glioblastomas. However, the precise contribution to redox and tumor proliferation of the second PPP enzyme 6-phosphogluconolactonase (PGLS), which converts 6-phospho-δ-gluconolactone to 6-phosphogluconate (6PG), remains unclear. Furthermore, non-invasive methods of assessing PGLS activity are lacking. The goal of this study was to examine the role of PGLS in glioblastomas and assess the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive imaging. METHODS: To interrogate the function of PGLS in redox, PGLS expression was silenced in U87, U251 and GS2 glioblastoma cells by RNA interference and levels of NADPH and reduced glutathione (GSH) measured. Clonogenicity assays were used to assess the effect of PGLS silencing on glioblastoma proliferation. Hyperpolarized δ-[1-13C]gluconolactone metabolism to 6PG was assessed in live cells treated with the chemotherapeutic agent temozolomide (TMZ) or with vehicle control. 13C 2D echo-planar spectroscopic imaging (EPSI) studies of hyperpolarized δ-[1-13C]gluconolactone metabolism were performed on rats bearing orthotopic glioblastoma tumors or tumor-free controls on a 3T spectrometer. Longitudinal 2D EPSI studies of hyperpolarized δ-[1-13C]gluconolactone metabolism and T2-weighted magnetic resonance imaging (MRI) were performed in rats bearing orthotopic U251 tumors following treatment with TMZ to examine the ability of hyperpolarized δ-[1-13C]gluconolactone to report on treatment response. RESULTS: PGLS knockdown downregulated NADPH and GSH, elevated oxidative stress and inhibited clonogenicity in all models. Conversely, PGLS expression and activity and steady-state NADPH and GSH were higher in tumor tissues from rats bearing orthotopic glioblastoma xenografts relative to contralateral brain and tumor-free brain. Importantly, [1-13C]6PG production from hyperpolarized δ-[1-13C]gluconolactone was observed in live glioblastoma cells and was significantly reduced by treatment with TMZ. Furthermore, hyperpolarized δ-[1-13C]gluconolactone metabolism to [1-13C]6PG could differentiate tumor from contralateral normal brain in vivo. Notably, TMZ significantly reduced 6PG production from hyperpolarized δ-[1-13C]gluconolactone at an early timepoint prior to volumetric alterations as assessed by anatomical imaging. CONCLUSIONS: Collectively, we have, for the first time, identified a role for PGLS activity in glioblastoma proliferation and validated the utility of probing PGLS activity using hyperpolarized δ-[1-13C]gluconolactone for non-invasive in vivo imaging of glioblastomas and their response to therapy.

Compartmental diffusion and microstructural properties of human brain gray and white matter studied with double diffusion encoding magnetic resonance spectroscopy of metabolites and water.

Double diffusion encoding (DDE) of the water signal offers a unique ability to separate the effect of microscopic anisotropic diffusion in structural units of tissue from the overall macroscopic orientational distribution of cells. However, the specificity in detected microscopic anisotropy is limited as the signal is averaged over different cell types and across tissue compartments. Performing side-by-side water and metabolite DDE spectroscopic (DDES) experiments provides complementary measures from which intracellular and extracellular microscopic fractional anisotropies (µFA) and diffusivities can be estimated. Metabolites are largely confined to the intracellular space and therefore provide a benchmark for intracellular µFA and diffusivities of specific cell types. By contrast, water DDES measurements allow examination of the separate contributions to water µFA and diffusivity from the intra- and extracellular spaces, by using a wide range of b values to gradually eliminate the extracellular contribution. Here, we aimed to estimate tissue and compartment specific human brain microstructure by combining water and metabolites DDES experiments. We performed our DDES measurements in two brain regions that contain widely different amounts of white matter (WM) and gray matter (GM): parietal white matter (PWM) and occipital gray matter (OGM) in a total of 20 healthy volunteers at 7 Tesla. Metabolite DDES measurements were performed at b = 7199 s/mm2, while water DDES measurements were performed with a range of b values from 918 to 7199 s/mm2. The experimental framework we employed here resulted in a set of insights pertaining to the morphology of the intracellular and extracellular spaces in both gray and white matter. Results of the metabolite DDES experiments in both PWM and OGM suggest a highly anisotropic intracellular space within neurons and glia, with the possible exception of gray matter glia. The water µFA obtained from the DDES results at high b values in both regions converged with that of the metabolite DDES, suggesting that the signal from the extracellular space is indeed effectively suppressed at the highest b value. The µFA measured in the OGM significantly decreased at lower b values, suggesting a considerably lower anisotropy of the extracellular space in GM compared to WM. In PWM, the water µFA remained high even at the lowest b value, indicating a high degree of organization in the interstitial space in WM. Tortuosity values in the cytoplasm for water and tNAA, obtained with correlation analysis of microscopic parallel diffusivity with respect to GM/WM tissue fraction in the volume of interest, are remarkably similar for both molecules, while exhibiting a clear difference between gray and white matter, suggesting a more crowded cytoplasm and more complex cytomorphology of neuronal cell bodies and dendrites in GM than those found in long-range axons in WM.

Early Noninvasive Metabolic Biomarkers of Mutant IDH Inhibition in Glioma.

Approximately 80% of low-grade glioma (LGGs) harbor mutant isocitrate dehydrogenase 1/2 (IDH1/2) driver mutations leading to accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). Thus, inhibition of mutant IDH is considered a potential therapeutic target. Several mutant IDH inhibitors are currently in clinical trials, including AG-881 and BAY-1436032. However, to date, early detection of response remains a challenge. In this study we used high resolution 1H magnetic resonance spectroscopy (1H-MRS) to identify early noninvasive MR (Magnetic Resonance)-detectable metabolic biomarkers of response to mutant IDH inhibition. In vivo 1H-MRS was performed on mice orthotopically-implanted with either genetically engineered (U87IDHmut) or patient-derived (BT257 and SF10417) mutant IDH1 cells. Treatment with either AG-881 or BAY-1436032 induced a significant reduction in 2-HG. Moreover, both inhibitors led to a significant early and sustained increase in glutamate and the sum of glutamate and glutamine (GLX) in all three models. A transient early increase in N-acetylaspartate (NAA) was also observed. Importantly, all models demonstrated enhanced animal survival following both treatments and the metabolic alterations were observed prior to any detectable differences in tumor volume between control and treated tumors. Our study therefore identifies potential translatable early metabolic biomarkers of drug delivery, mutant IDH inhibition and glioma response to treatment with emerging clinically relevant therapies.

Glutamate Is a Noninvasive Metabolic Biomarker of IDH1-Mutant Glioma Response to Temozolomide Treatment.

Although lower grade gliomas are driven by mutations in the isocitrate dehydrogenase 1 (IDH1) gene and are less aggressive than primary glioblastoma, they nonetheless generally recur. IDH1-mutant patients are increasingly being treated with temozolomide, but early detection of response remains a challenge and there is a need for complementary imaging methods to assess response to therapy prior to tumor shrinkage. The goal of this study was to determine the value of magnetic resonance spectroscopy (MRS)-based metabolic changes for detection of response to temozolomide in both genetically engineered and patient-derived mutant IDH1 models. Using 1H MRS in combination with chemometrics identified several metabolic alterations in temozolomide-treated cells, including a significant increase in steady-state glutamate levels. This was confirmed in vivo, where the observed 1H MRS increase in glutamate/glutamine occurred prior to tumor shrinkage. Cells labeled with [1-13C]glucose and [3-13C]glutamine, the principal sources of cellular glutamate, showed that flux to glutamate both from glucose via the tricarboxylic acid cycle and from glutamine were increased following temozolomide treatment. In line with these results, hyperpolarized [5-13C]glutamate produced from [2-13C]pyruvate and hyperpolarized [1-13C]glutamate produced from [1-13C]α-ketoglutarate were significantly higher in temozolomide-treated cells compared with controls. Collectively, our findings identify 1H MRS-detectable elevation of glutamate and hyperpolarized 13C MRS-detectable glutamate production from either pyruvate or α-ketoglutarate as potential translatable metabolic biomarkers of response to temozolomide treatment in mutant IDH1 glioma. SIGNIFICANCE: These findings show that glutamate can be used as a noninvasive, imageable metabolic marker for early assessment of tumor response to temozolomide, with the potential to improve treatment strategies for mutant IDH1 patients.

Patient-derived cells from recurrent tumors that model the evolution of IDH-mutant glioma.

BACKGROUND: IDH-mutant lower-grade gliomas (LGGs) evolve under the selective pressure of therapy, but well-characterized patient-derived cells (PDCs) modeling evolutionary stages are lacking. IDH-mutant LGGs may develop therapeutic resistance associated with chemotherapy-driven hypermutation and malignant progression. The aim of this study was to establish and characterize PDCs, single-cell-derived PDCs (scPDCs), and xenografts (PDX) of IDH1-mutant recurrences representing distinct stages of tumor evolution. METHODS: We derived and validated cell cultures from IDH1-mutant recurrences of astrocytoma and oligodendroglioma. We used exome sequencing and phylogenetic reconstruction to examine the evolutionary stage represented by PDCs, scPDCs, and PDX relative to corresponding spatiotemporal tumor tissue and germline DNA. PDCs were also characterized for growth and tumor immortality phenotypes, and PDX were examined histologically. RESULTS: The integrated astrocytoma phylogeny revealed 2 independent founder clonal expansions of hypermutated (HM) cells in tumor tissue that are faithfully represented by independent PDCs. The oligodendroglioma phylogeny showed more than 4000 temozolomide-associated mutations shared among tumor samples, PDCs, scPDCs, and PDX, suggesting a shared monoclonal origin. The PDCs from both subtypes exhibited hallmarks of tumorigenesis, retention of subtype-defining genomic features, production of 2-hydroxyglutarate, and subtype-specific telomere maintenance mechanisms that confer tumor cell immortality. The oligodendroglioma PDCs formed infiltrative intracranial tumors with characteristic histology. CONCLUSIONS: These PDCs, scPDCs, and PDX are unique and versatile community resources that model the heterogeneous clonal origins and functions of recurrent IDH1-mutant LGGs. The integrated phylogenies advance our knowledge of the complex evolution and immense mutational load of IDH1-mutant HM glioma.

MR-detectable metabolic biomarkers of response to mutant IDH inhibition in low-grade glioma.

Mutations in isocitrate dehydrogenase 1 (IDH1mut) are reported in 70-90% of low-grade gliomas and secondary glioblastomas. IDH1mut catalyzes the reduction of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG), an oncometabolite which drives tumorigenesis. Inhibition of IDH1mut is therefore an emerging therapeutic approach, and inhibitors such as AG-120 and AG-881 have shown promising results in phase 1 and 2 clinical studies. However, detection of response to these therapies prior to changes in tumor growth can be challenging. The goal of this study was to identify non-invasive clinically translatable metabolic imaging biomarkers of IDH1mut inhibition that can serve to assess response. Methods: IDH1mut inhibition was confirmed using an enzyme assay and 1H- and 13C- magnetic resonance spectroscopy (MRS) were used to investigate the metabolic effects of AG-120 and AG-881 on two genetically engineered IDH1mut-expressing cell lines, NHAIDH1mut and U87IDH1mut. Results:1H-MRS indicated a significant decrease in steady-state 2-HG following treatment, as expected. This was accompanied by a significant 1H-MRS-detectable increase in glutamate. However, other metabolites previously linked to 2-HG were not altered. 13C-MRS also showed that the steady-state changes in glutamate were associated with a modulation in the flux of glutamine to both glutamate and 2-HG. Finally, hyperpolarized 13C-MRS was used to show that the flux of α-KG to both glutamate and 2-HG was modulated by treatment. Conclusion: In this study, we identified potential 1H- and 13C-MRS-detectable biomarkers of response to IDH1mut inhibition in gliomas. Although further studies are needed to evaluate the utility of these biomarkers in vivo, we expect that in addition to a 1H-MRS-detectable drop in 2-HG, a 1H-MRS-detectable increase in glutamate, as well as a hyperpolarized 13C-MRS-detectable change in [1-13C] α-KG flux, could serve as metabolic imaging biomarkers of response to treatment.

In vivo detection of γ-glutamyl-transferase up-regulation in glioma using hyperpolarized γ-glutamyl-[1-13C]glycine.

Glutathione (GSH) is often upregulated in cancer, where it serves to mitigate oxidative stress. γ-glutamyl-transferase (GGT) is a key enzyme in GSH homeostasis, and compared to normal brain its expression is elevated in tumors, including in primary glioblastoma. GGT is therefore an attractive imaging target for detection of glioblastoma. The goal of our study was to assess the value of hyperpolarized (HP) γ-glutamyl-[1-13C]glycine for non-invasive imaging of glioblastoma. Nude rats bearing orthotopic U87 glioblastoma and healthy controls were investigated. Imaging was performed by injecting HP γ-glutamyl-[1-13C]glycine and acquiring dynamic 13C data on a preclinical 3T MR scanner. The signal-to-noise (SNR) ratios of γ-glutamyl-[1-13C]glycine and its product [1-13C]glycine were evaluated. Comparison of control and tumor-bearing rats showed no difference in γ-glutamyl-[1-13C]glycine SNR, pointing to similar delivery to tumor and normal brain. In contrast, [1-13C]glycine SNR was significantly higher in tumor-bearing rats compared to controls, and in tumor regions compared to normal-appearing brain. Importantly, higher [1-13C]glycine was associated with higher GGT expression and higher GSH levels in tumor tissue compared to normal brain. Collectively, this study demonstrates, to our knowledge for the first time, the feasibility of using HP γ-glutamyl-[1-13C]glycine to monitor GGT expression in the brain and thus to detect glioblastoma.

Hyperpolarized 13 C magnetic resonance spectroscopy detects toxin-induced neuroinflammation in mice.

Lipopolysaccharide (LPS) is a commonly used agent for induction of neuroinflammation in preclinical studies. Upon injection, LPS causes activation of microglia and astrocytes, whose metabolism alters to favor glycolysis. Assessing in vivo neuroinflammation and its modulation following therapy remains challenging, and new noninvasive methods allowing for longitudinal monitoring would be highly valuable. Hyperpolarized (HP) 13 C magnetic resonance spectroscopy (MRS) is a promising technique for assessing in vivo metabolism. In addition to applications in oncology, the most commonly used probe of [1-13 C] pyruvate has shown potential in assessing neuroinflammation-linked metabolism in mouse models of multiple sclerosis and traumatic brain injury. Here, we aimed to investigate LPS-induced neuroinflammatory changes using HP [1-13 C] pyruvate and HP 13 C urea. 2D chemical shift imaging following simultaneous intravenous injection of HP [1-13 C] pyruvate and HP 13 C urea was performed at baseline (day 0) and at days 3 and 7 post-intracranial injection of LPS (n = 6) or saline (n = 5). Immunofluorescence (IF) analyses were performed for Iba1 (resting and activated microglia/macrophages), GFAP (resting and reactive astrocytes) and CD68 (activated microglia/macrophages). A significant increase in HP [1-13 C] lactate production was observed at days 3 and 7 following injection, in the injected (ipsilateral) side of the LPS-treated mouse brain, but not in either the contralateral side or saline-injected animals. HP 13 C lactate/pyruvate ratio, without and with normalization to urea, was also significantly increased in the ipsilateral LPS-injected brain at 7 days compared with baseline. IF analyses showed a significant increase in CD68 and GFAP staining at 3 days, followed by increased numbers of Iba1 and GFAP positive cells at 7 days post-LPS injection. In conclusion, we can detect LPS-induced changes in the mouse brain using HP 13 C MRS, in alignment with increased numbers of microglia/macrophages and astrocytes. This study demonstrates that HP 13 C spectroscopy has substantial potential for providing noninvasive information on neuroinflammation.

PI3K/mTOR inhibition of IDH1 mutant glioma leads to reduced 2HG production that is associated with increased survival.

70-90% of low-grade gliomas and secondary glioblastomas are characterized by mutations in isocitrate dehydrogenase 1 (IDHmut). IDHmut produces the oncometabolite 2-hydroxyglutarate (2HG), which drives tumorigenesis in these tumors. The phosphoinositide-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway represents an attractive therapeutic target for IDHmut gliomas, but noninvasive indicators of drug target modulation are lacking. The goal of this study was therefore to identify magnetic resonance spectroscopy (MRS)-detectable metabolic biomarkers associated with IDHmut glioma response to the dual PI3K/(mTOR) inhibitor XL765. 1H-MRS of two cell lines genetically modified to express IDHmut showed that XL765 induced a significant reduction in several intracellular metabolites including 2HG. Importantly, examination of an orthotopic IDHmut tumor model showed that enhanced animal survival following XL765 treatment was associated with a significant in vivo 1H-MRS detectable reduction in 2HG but not with significant inhibition in tumor growth. Further validation is required, but our results indicate that 2HG could serve as a potential noninvasive MRS-detectable metabolic biomarker of IDHmut glioma response to PI3K/mTOR inhibition.

In vivo investigation of hyperpolarized [1,3-13C2]acetoacetate as a metabolic probe in normal brain and in glioma.

Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to ß-hydroxybutyrate (ß-HB) by the mitochondrial enzyme ß-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The ß-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]ß-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]ß-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders.

HDAC inhibition in glioblastoma monitored by hyperpolarized 13 C MRSI.

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

Late-stage deuteration of 13C-enriched substrates for T1 prolongation in hyperpolarized 13C MRI.

A robust and selective late-stage deuteration methodology was applied to 13C-enriched amino and alpha hydroxy acids to increase spin-lattice relaxation constant T1 for hyperpolarized 13C magnetic resonance imaging. For the five substrates with 13C-labeling on the C1-position ([1-13C]alanine, [1-13C]serine, [1-13C]lactate, [1-13C]glycine, and [1-13C]valine), significant increase of their T1 was observed at 3 T with deuterium labeling (+26%, 22%, +16%, +25% and +29%, respectively). Remarkably, in the case of [2-13C]alanine, [2-13C]serine and [2-13C]lactate, deuterium labeling led to a greater than four fold increase in T1. [1-13C,2-2H]alanine, produced using this method, was applied to in vitro enzyme assays with alanine aminotransferase, demonstrating a kinetic isotope effect.