New Horizons in Hyperpolarized 13C MRI.

Hyperpolarization techniques significantly enhance the sensitivity of magnetic resonance (MR) and thus present fascinating new directions for research and applications with in vivo MR imaging and spectroscopy (MRI/S). Hyperpolarized 13C MRI/S, in particular, enables real-time non-invasive assessment of metabolic processes and holds great promise for a diverse range of clinical applications spanning fields like oncology, neurology, and cardiology, with a potential for improving early diagnosis of disease, patient stratification, and therapy response assessment. Despite its potential, technical challenges remain for achieving clinical translation. This paper provides an overview of the discussions that took place at the international workshop "New Horizons in Hyperpolarized 13C MRI," in March 2023 at the Bavarian Academy of Sciences and Humanities, Munich, Germany. The workshop covered new developments, as well as future directions, in topics including polarization techniques (particularly focusing on parahydrogen-based methods), novel probes, considerations related to data acquisition and analysis, and emerging clinical applications in oncology and other fields.

Quo Vadis Hyperpolarized 13C MRI?

Over the last two decades, hyperpolarized 13C MRI has gained significance in both preclinical and clinical studies, hereby relying on technologies like PHIP-SAH (ParaHydrogen-Induced Polarization-Side Arm Hydrogenation), SABRE (Signal Amplification by Reversible Exchange), and dDNP (dissolution Dynamic Nuclear Polarization), with dDNP being applied in humans. A clinical dDNP polarizer has enabled studies across 24 sites, despite challenges like high cost and slow polarization. Parahydrogen-based techniques like SABRE and PHIP offer faster, more cost-efficient alternatives but require molecule-specific optimization. The focus has been on imaging metabolism of hyperpolarized probes, which requires long T1, high polarization and rapid contrast generation. Efforts to establish novel probes, improve acquisition techniques and enhance data analysis methods including artificial intelligence are ongoing. Potential clinical value of hyperpolarized 13C MRI was demonstrated primarily for treatment response assessment in oncology, but also in cardiology, nephrology, hepatology and CNS characterization. In this review on biomedical hyperpolarized 13C MRI, we summarize important and recent advances in polarization techniques, probe development, acquisition and analysis methods as well as clinical trials. Starting from those we try to sketch a trajectory where the field of biomedical hyperpolarized 13C MRI might go.

Lithium isotopes differentially modify mitochondrial amorphous calcium phosphate cluster size distribution and calcium capacity.

Lithium is commonly prescribed as a mood stabilizer in a variety of mental health conditions, yet its molecular mode of action is incompletely understood. Many cellular events associated with lithium appear tied to mitochondrial function. Further, recent evidence suggests that lithium bioactivities are isotope specific. Here we focus on lithium effects related to mitochondrial calcium handling. Lithium protected against calcium-induced permeability transition and decreased the calcium capacity of liver mitochondria at a clinically relevant concentration. In contrast, brain mitochondrial calcium capacity was increased by lithium. Surprisingly, 7Li acted more potently than 6Li on calcium capacity, yet 6Li was more effective at delaying permeability transition. The size distribution of amorphous calcium phosphate colloids formed in vitro was differentially affected by lithium isotopes, providing a mechanistic basis for the observed isotope specific effects on mitochondrial calcium handling. This work highlights a need to better understand how mitochondrial calcium stores are structurally regulated and provides key considerations for future formulations of lithium-based therapeutics.

Parahydrogen-Polarized [1-13 C]Pyruvate for Reliable and Fast Preclinical Metabolic Magnetic Resonance Imaging.

Hyperpolarization techniques increase nuclear spin polarization by more than four orders of magnitude, enabling metabolic MRI. Even though hyperpolarization has shown clear value in clinical studies, the complexity, cost and slowness of current equipment limits its widespread use. Here, a polarization procedure of [1-13 C]pyruvate based on parahydrogen-induced polarization by side-arm hydrogenation (PHIP-SAH) in an automated polarizer is demonstrated. It is benchmarked in a study with 48 animals against a commercial dissolution dynamic nuclear polarization (d-DNP) device. Purified, concentrated (≈70-160 mM) and highly hyperpolarized (≈18%) solutions of pyruvate are obtained at physiological pH for volumes up to 2 mL within 85 s in an automated process. The safety profile, image quality, as well as the quantitative perfusion and lactate-to-pyruvate ratios, are equivalent for PHIP and d-DNP, rendering PHIP a viable alternative to established hyperpolarization techniques.

Simultaneous magnetic resonance imaging of pH, perfusion and renal filtration using hyperpolarized 13C-labelled Z-OMPD.

pH alterations are a hallmark of many pathologies including cancer and kidney disease. Here, we introduce [1,5-13C2]Z-OMPD as a hyperpolarized extracellular pH and perfusion sensor for MRI which allows to generate a multiparametric fingerprint of renal disease status and to detect local tumor acidification. Exceptional long T1 of two minutes at 1 T, high pH sensitivity of up to 1.9 ppm per pH unit and suitability of using the C1-label as internal frequency reference enables pH imaging in vivo of three pH compartments in healthy rat kidneys. Spectrally selective targeting of both 13C-resonances enables simultaneous imaging of perfusion and filtration in 3D and pH in 2D within one minute to quantify renal blood flow, glomerular filtration rates and renal pH in healthy and hydronephrotic kidneys with superior sensitivity compared to clinical routine methods. Imaging multiple biomarkers within a single session renders [1,5-13C2]Z-OMPD a promising new hyperpolarized agent for oncology and nephrology.

Spectrally selective bSSFP using off-resonant RF excitations permits high spatiotemporal resolution 3D metabolic imaging of hyperpolarized [1-13 C]Pyruvate-to-[1-13 C]lactate conversion.

PURPOSE: To develop a high spatiotemporal resolution 3D dynamic pulse sequence for preclinical imaging of hyperpolarized [1-13 C]pyruvate-to-[1-13 C]lactate metabolism at 7T. METHODS: A standard 3D balanced SSFP (bSSFP) sequence was modified to enable alternating-frequency excitations. RF pulses with 2.33 ms duration and 900 Hz FWHM were placed off-resonance of the target metabolites, [1-13 C]pyruvate (by approximately -245 Hz) and [1-13 C]lactate (by approximately 735 Hz), to selectively excite those resonances. Relatively broad bandwidth (compared to those metabolites' chemical shift offset) permits a short TR of 6.29 ms, enabling higher spatiotemporal resolution. Bloch equation simulations of the bSSFP response profile guided the sequence parameter selection to minimize spectral contamination between metabolites and preserve magnetization over time. RESULTS: Bloch equation simulations, phantom studies, and in vivo studies demonstrated that the two target resonances could be cleanly imaged without substantial bSSFP banding artifacts and with little spectral contamination between lactate and pyruvate and from pyruvate hydrate. High spatiotemporal resolution 3D images were acquired of in vivo pyruvate-lactate metabolism in healthy wild-type and endogenous pancreatic tumor-bearing mice, with 1.212 s acquisition time per single-metabolite image and (1.75 mm)3 isotropic voxels with full mouse abdomen 56 × 28 × 21 mm3 FOV and fully-sampled k-space. Kidney and tumor lactate/pyruvate ratios of two consecutive measurements in one animal, 1 h apart, were consistent. CONCLUSION: Spectrally selective bSSFP using off-resonant RF excitations can provide high spatio-temporal resolution 3D dynamic images of pyruvate-lactate metabolic conversion.

Functional noninvasive detection of glycolytic pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) lacks effective treatment options beyond chemotherapy. Although molecular subtypes such as classical and QM (quasi-mesenchymal)/basal-like with transcriptome-based distinct signatures have been identified, deduced therapeutic strategies and targets remain elusive. Gene expression data show enrichment of glycolytic genes in the more aggressive and therapy-resistant QM subtype. However, whether the glycolytic transcripts are translated into functional glycolysis that could further be explored for metabolic targeting in QM subtype is still not known. METHODS: We used different patient-derived PDAC model systems (conventional and primary patient-derived cells, patient-derived xenografts (PDX), and patient samples) and performed transcriptional and functional metabolic analysis. These included RNAseq and Illumina HT12 bead array, in vitro Seahorse metabolic flux assays and metabolic drug targeting, and in vivo hyperpolarized [1-13C]pyruvate and [1-13C]lactate magnetic resonance spectroscopy (HP-MRS) in PDAC xenografts. RESULTS: We found that glycolytic metabolic dependencies are not unambiguously functionally exposed in all QM PDACs. Metabolic analysis demonstrated functional metabolic heterogeneity in patient-derived primary cells and less so in conventional cell lines independent of molecular subtype. Importantly, we observed that the glycolytic product lactate is actively imported into the PDAC cells and used in mitochondrial oxidation in both classical and QM PDAC cells, although more actively in the QM cell lines. By using HP-MRS, we were able to noninvasively identify highly glycolytic PDAC xenografts by detecting the last glycolytic enzymatic step and prominent intra-tumoral [1-13C]pyruvate and [1-13C]lactate interconversion in vivo. CONCLUSION: Our study adds functional metabolic phenotyping to transcriptome-based analysis and proposes a functional approach to identify highly glycolytic PDACs as candidates for antimetabolic therapeutic avenues.

Prediction of multiple pH compartments by deep learning in magnetic resonance spectroscopy with hyperpolarized 13C-labelled zymonic acid.

BACKGROUND: Hyperpolarization enhances the sensitivity of nuclear magnetic resonance experiments by between four and five orders of magnitude. Several hyperpolarized sensor molecules have been introduced that enable high sensitivity detection of metabolism and physiological parameters. However, hyperpolarized magnetic resonance spectroscopy imaging (MRSI) often suffers from poor signal-to-noise ratio and spectral analysis is complicated by peak overlap. Here, we study measurements of extracellular pH (pHe) by hyperpolarized zymonic acid, where multiple pHe compartments, such as those observed in healthy kidney or other heterogeneous tissue, result in a cluster of spectrally overlapping peaks, which is hard to resolve with conventional spectroscopy analysis routines. METHODS: We investigate whether deep learning methods can yield improved pHe prediction in hyperpolarized zymonic acid spectra of multiple pHe compartments compared to conventional line fitting. As hyperpolarized 13C-MRSI data sets are often small, a convolutional neural network (CNN) and a multilayer perceptron (MLP) were trained with either a synthetic or a mixed (synthetic and augmented) data set of acquisitions from the kidneys of healthy mice. RESULTS: Comparing the networks' performances compartment-wise on a synthetic test data set and eight real kidney data shows superior performance of CNN compared to MLP and equal or superior performance compared to conventional line fitting. For correct prediction of real kidney pHe values, training with a mixed data set containing only 0.5% real data shows a large improvement compared to training with synthetic data only. Using a manual segmentation approach, pH maps of kidney compartments can be improved by neural network predictions for voxels including three pH compartments. CONCLUSION: The results of this study indicate that CNNs offer a reliable, accurate, fast and non-interactive method for analysis of hyperpolarized 13C MRS and MRSI data, where low amounts of acquired data can be complemented to achieve suitable network training.

Conversion of Hyperpolarized [1-13C]Pyruvate in Breast Cancer Cells Depends on Their Malignancy, Metabolic Program and Nutrient Microenvironment.

Hyperpolarized magnetic resonance spectroscopy (MRS) is a technology for characterizing tumors in vivo based on their metabolic activities. The conversion rates (kpl) of hyperpolarized [1-13C]pyruvate to [1-13C]lactate depend on monocarboxylate transporters (MCT) and lactate dehydrogenase (LDH); these are also indicators of tumor malignancy. An unresolved issue is how glucose and glutamine availability in the tumor microenvironment affects metabolic characteristics of the cancer and how this relates to kpl-values. Two breast cancer cells of different malignancy (MCF-7, MDA-MB-231) were cultured in media containing defined combinations of low glucose (1 mM; 2.5 mM) and glutamine (0.1 mM; 1 mM) and analyzed for pyruvate uptake, intracellular metabolite levels, LDH and pyruvate kinase activities, and 13C6-glucose-derived metabolomics. The results show variability of kpl with the different glucose/glutamine conditions, congruent with glycolytic activity, but not with LDH activity or the Warburg effect; this suggests metabolic compartmentation. Remarkably, kpl-values were almost two-fold higher in MCF-7 than in the more malignant MDA-MB-231 cells, the latter showing a higher flux of 13C-glucose-derived pyruvate to the TCA-cycle metabolites 13C2-citrate and 13C3-malate, i.e., pyruvate decarboxylation and carboxylation, respectively. Thus, MRS with hyperpolarized [1-13C-pyruvate] is sensitive to both the metabolic program and the nutritional state of cancer cells.

pH Dependence of T2 for Hyperpolarizable 13C-Labelled Small Molecules Enables Spatially Resolved pH Measurement by Magnetic Resonance Imaging.

Hyperpolarized 13C magnetic resonance imaging often uses spin-echo-based pulse sequences that are sensitive to the transverse relaxation time T2. In this context, local T2-changes might introduce a quantification bias to imaging biomarkers. Here, we investigated the pH dependence of the apparent transverse relaxation time constant (denoted here as T2) of six 13C-labelled molecules. We obtained minimum and maximum T2 values within pH 1-13 at 14.1 T: [1-13C]acetate (T2,min = 2.1 s; T2,max = 27.7 s), [1-13C]alanine (T2,min = 0.6 s; T2,max = 10.6 s), [1,4-13C2]fumarate (T2,min = 3.0 s; T2,max = 18.9 s), [1-13C]lactate (T2,min = 0.7 s; T2,max = 12.6 s), [1-13C]pyruvate (T2,min = 0.1 s; T2,max = 18.7 s) and 13C-urea (T2,min = 0.1 s; T2,max = 0.1 s). At 7 T, T2-variation in the physiological pH range (pH 6.8-7.8) was highest for [1-13C]pyruvate (ΔT2 = 0.95 s/0.1pH) and [1-13C]acetate (ΔT2 = 0.44 s/0.1pH). Concentration, salt concentration, and temperature alterations caused T2 variations of up to 45.4% for [1-13C]acetate and 23.6% for [1-13C]pyruvate. For [1-13C]acetate, spatially resolved pH measurements using T2-mapping were demonstrated with 1.6 pH units accuracy in vitro. A strong proton exchange-based pH dependence of T2 suggests that pH alterations potentially influence signal strength for hyperpolarized 13C-acquisitions.

Adenylate kinase derived ATP shapes respiration and calcium storage of isolated mitochondria.

The ratio of ADP and ATP is a natural indicator of cellular bioenergetic state and thus a prominent analyte in metabolism research. Beyond adenylate interconversion via oxidative phosphorylation and ATPase activities, ADP and ATP act as steric regulators of enzymes, e.g. cytochrome C oxidase, and are major factors in mitochondrial calcium storage potential. Consideration of all routes of adenylate conversion is critical to successfully predict their abundance in an experimental system and to correctly interpret many aspects of mitochondrial function. We showcase here how adenylate kinases elicit considerable impact on the outcome of a variety of mitochondrial assays through their drastic manipulation of the adenylate profile. Parameters affected include cytochrome c oxidase activity, P/O ratio, and mitochondrial calcium dynamics. Study of the latter revealed that the presence of ATP is required for mitochondrial calcium to be shaped into a particularly dense form of mitochondrial amorphous calcium phosphate.

Hyperpolarized 13C pyruvate magnetic resonance spectroscopy for in vivo metabolic phenotyping of rat HCC.

The in vivo assessment of tissue metabolism represents a novel strategy for the evaluation of oncologic disease. Hepatocellular carcinoma (HCC) is a high-prevalence, high-mortality tumor entity often discovered at a late stage. Recent evidence indicates that survival differences depend on metabolic alterations in tumor tissue, with particular focus on glucose metabolism and lactate production. Here, we present an in vivo imaging technique for metabolic tumor phenotyping in rat models of HCC. Endogenous HCC was induced in Wistar rats by oral diethyl-nitrosamine administration. Peak lactate-to-alanine signal ratios (L/A) were assessed with hyperpolarized magnetic resonance spectroscopic imaging (HPMRSI) after [1-13C]pyruvate injection. Cell lines were derived from a subset of primary tumors, re-implanted in nude rats, and assessed in vivo with dynamic hyperpolarized magnetic resonance spectroscopy (HPMRS) after [1-13C]pyruvate injection and kinetic modelling of pyruvate metabolism, taking into account systemic lactate production and recirculation. For ex vivo validation, enzyme activity and metabolite concentrations were spectroscopically quantified in cell and tumor tissue extracts. Mean peak L/A was higher in endogenous HCC compared to non-tumorous tissue. Dynamic HPMRS revealed higher pyruvate-to-lactate conversion rates (kpl) and lactate signal in subcutaneous tumors derived from high L/A tumor cells, consistent with ex vivo measurements of higher lactate dehydrogenase (LDH) levels in these cells. In conclusion, HPMRS and HPMRSI reveal distinct tumor phenotypes corresponding to differences in glycolytic metabolism in HCC tumor tissue.

Hyperpolarized 13C Spectroscopy with Simple Slice-and-Frequency-Selective Excitation.

Hyperpolarized 13C nuclear magnetic resonance spectroscopy can characterize in vivo tissue metabolism, including preclinical models of cancer and inflammatory disease. Broad bandwidth radiofrequency excitation is often paired with free induction decay readout for spectral separation, but quantification of low-signal downstream metabolites using this method can be impeded by spectral peak overlap or when frequency separation of the detected peaks exceeds the excitation bandwidth. In this work, alternating frequency narrow bandwidth (250 Hz) slice-selective excitation was used for 13C spectroscopy at 7 T in a subcutaneous xenograft rat model of human pancreatic cancer (PSN1) to improve quantification while measuring the dynamics of injected hyperpolarized [1-13C]lactate and its metabolite [1-13C]pyruvate. This method does not require sophisticated pulse sequences or specialized radiofrequency and gradient pulses, but rather uses nominally spatially offset slices to produce alternating frequency excitation with simpler slice-selective radiofrequency pulses. Additionally, point-resolved spectroscopy was used to calibrate the 13C frequency from the thermal proton signal in the target region. This excitation scheme isolates the small [1-13C]pyruvate peak from the similar-magnitude tail of the much larger injected [1-13C]lactate peak, facilitates quantification of the [1-13C]pyruvate signal, simplifies data processing, and could be employed for other substrates and preclinical models.

Acquisition strategies for spatially resolved magnetic resonance detection of hyperpolarized nuclei.

Hyperpolarization is an emerging method in magnetic resonance imaging that allows nuclear spin polarization of gases or liquids to be temporarily enhanced by up to five or six orders of magnitude at clinically relevant field strengths and administered at high concentration to a subject at the time of measurement. This transient gain in signal has enabled the non-invasive detection and imaging of gas ventilation and diffusion in the lungs, perfusion in blood vessels and tissues, and metabolic conversion in cells, animals, and patients. The rapid development of this method is based on advances in polarizer technology, the availability of suitable probe isotopes and molecules, improved MRI hardware and pulse sequence development. Acquisition strategies for hyperpolarized nuclei are not yet standardized and are set up individually at most sites depending on the specific requirements of the probe, the object of interest, and the MRI hardware. This review provides a detailed introduction to spatially resolved detection of hyperpolarized nuclei and summarizes novel and previously established acquisition strategies for different key areas of application.

pH Dependence of T1 for 13 C-Labelled Small Molecules Commonly Used for Hyperpolarized Magnetic Resonance Imaging.

Hyperpolarization is a method to enhance the nuclear magnetic resonance signal by up to five orders of magnitude. However, the hyperpolarized (HP) state is transient and decays with the spin-lattice relaxation time (T1 ), which is on the order of a few tens of seconds. Here, we analyzed the pH-dependence of T1 for commonly used HP 13 C-labelled small molecules such as acetate, alanine, fumarate, lactate, pyruvate, urea and zymonic acid. For instance, the T1 of HP pyruvate is about 2.5 fold smaller at acidic pH (25 s, pH 1.7, B0 =1 T) compared to pH close to physiological conditions (66 s, pH 7.3, B0 =1 T). Our data shows that increasing hydronium ion concentrations shorten the T1 of protonated carboxylic acids of most of the analyzed molecules except lactate. Furthermore it suggests that intermolecular hydrogen bonding at low pH can contribute to this T1 shortening. In addition, enhanced proton exchange and chemical reactions at the pKa appear to be detrimental for the HP-state.