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Imaging the metabolism of [2,3-2 H2 ]fumarate to produce malate can be used to detect tumor cell death post-treatment. Here we assess the sensitivity of the technique for detecting cell death by lowering the concentration of injected [2,3-2 H2 ]fumarate and by varying the extent of tumor cell death through changes in drug concentration. Mice were implanted subcutaneously with human triple negative breast cancer cells (MDA-MB-231) and injected with 0.1, 0.3 and 0.5 g/kg [2,3-2 H2 ]fumarate before and after treatment with a multivalent TRAlL-R2 agonist (MEDI3039) at 0.1, 0.4 and 0.8 mg/kg. Tumor conversion of [2,3-2 H2 ]fumarate to [2,3-2 H2 ]malate was assessed from a series of 13 spatially localized 2 H MR spectra acquired over 65 min using a pulse-acquire sequence with a 2 ms BIR4 adiabatic excitation pulse. Tumors were then excised and stained for histopathological markers of cell death; cleaved caspase 3 (CC3) and DNA damage (terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)). The rate of malate production and the malate/fumarate ratio plateaued at tumor fumarate concentrations of 2 mM, which were obtained with injected [2,3-2 H2 ]fumarate concentrations of 0.3 g/kg and above. Tumor malate concentration and the malate/fumarate ratio increased linearly with the extent of cell death determined histologically, which was varied by changing drug concentration. At an injected [2,3-2 H2 ]fumarate concentration of 0.3 g/kg, 20% CC3 staining corresponded to a malate concentration of 0.62 mM and a malate/fumarate ratio of 0.21. Extrapolation indicated that there would be no detectable malate at 0% CC3 staining. The use of low and non-toxic fumarate concentrations and the production of [2,3-2 H2 ]malate at concentrations that are within the range that can be detected clinically suggest this technique could translate to the clinic.

PURPOSE: To compare carbon-13 (13 C) MRSI of hyperpolarized [1-13 C]pyruvate metabolism in a murine tumor model with mass spectrometric (MS) imaging of the corresponding tumor sections in order to cross validate these metabolic imaging techniques and to investigate the effects of pyruvate delivery and tumor lactate concentration on lactate labeling. METHODS: [1-13 C]lactate images were obtained from tumor-bearing mice, following injection of hyperpolarized [1-13 C]pyruvate, using a single-shot 3D 13 C spectroscopic imaging sequence in vivo and using desorption electrospray ionization MS imaging of the corresponding rapidly frozen tumor sections ex vivo. The images were coregistered, and levels of association were determined by means of Spearman rank correlation and Cohen kappa coefficients as well as linear mixed models. The correlation between [1-13 C]pyruvate and [1-13 C]lactate in the MRS images and between [12 C] and [1-13 C]lactate in the MS images were determined by means of Pearson correlation coefficients. RESULTS: [1-13 C]lactate images generated by MS imaging were significantly correlated with the corresponding MRS images. The correlation coefficient between [1-13 C]lactate and [1-13 C]pyruvate in the MRS images was higher than between [1-13 C]lactate and [12 C]lactate in the MS images. CONCLUSION: The inhomogeneous distribution of labeled lactate observed in the MRS images was confirmed by MS imaging of the corresponding tumor sections. The images acquired using both techniques show that the rate of 13 C label exchange between the injected pyruvate and endogenous tumor lactate pool is more correlated with the rate of pyruvate delivery to the tumor cells and is less affected by the endogenous lactate concentration.

PURPOSE: Imaging tumor metabolism in vivo using hyperpolarized [1-13 C]pyruvate is a promising technique for detecting disease, monitoring disease progression, and assessing treatment response. However, the transient nature of the hyperpolarization and its depletion following excitation limits the available time for imaging. We describe here a single-shot multi spin echo sequence, which improves on previously reported sequences, with a shorter readout time, isotropic point spread function (PSF), and better signal-to-noise ratio. METHODS: The sequence uses numerically optimized spectrally selective excitation pulses set to the resonant frequencies of pyruvate and lactate and a hyperbolic secant adiabatic refocusing pulse, all applied in the absence of slice selection gradients. The excitation pulses were designed to be resistant to the effects of B0 and B1 field inhomogeneity. The gradient readout uses a 3D cone trajectory composed of 13 cones, all fully refocused and distributed among 7 spin echoes. The maximal gradient amplitude and slew rate were set to 4 G/cm and 20 G/cm/ms, respectively, to demonstrate the feasibility of clinical translation. RESULTS: The pulse sequence gave an isotropic PSF of 2.8 mm. The excitation profiles of the optimized pulses closely matched simulations and a 46.10 ± 0.04% gain in image SNR was observed compared to a conventional Shinnar-Le Roux excitation pulse. The sequence was demonstrated with dynamic imaging of hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate in vivo. CONCLUSION: The pulse sequence was capable of dynamic imaging of hyperpolarized 13 C labeled metabolites in vivo with relatively high spatial and temporal resolution and immunity to system imperfections.

PURPOSE: Hyperpolarized 15 N-labeled molecules have been proposed as imaging agents for investigating tissue perfusion and pH. However, the sensitivity of direct 15 N detection is limited by the isotope's low gyromagnetic ratio. Sensitivity can be increased by transferring 15 N hyperpolarization to spin-coupled protons provided that there is not significant polarization loss during transfer. However, complete polarization transfer would limit the temporal window for imaging to the order of the proton T1 (2-3 s). To exploit the long T1 offered by storing polarization in 15 N and the higher sensitivity of 1 H detection, we have developed a pulse sequence for partial polarization transfer. METHODS: A polarization transfer pulse sequence was modified to allow partial polarization transfer, as is required for dynamic measurements, and that can be implemented with inhomogeneous B1 fields, as is often the case in vivo. The sequence was demonstrated with dynamic spectroscopy and imaging measurements with [15 N2 ]urea. RESULTS: When compared to direct 15 N detection, the sequence increased the signal-to-noise ratio (SNR) by a factor of 1.72 ± 0.25, where both experiments depleted ~20% of the hyperpolarization (>10-fold when 100% of the hyperpolarization is used). Simulations with measured cross relaxation rates showed that this sequence gave up to a 50-fold increase in urea proton polarization when compared to spontaneous polarization transfer via cross relaxation. CONCLUSION: The sequence gave an SNR increase that was close to the theoretical limit and can give a significant SNR benefit when compared to direct 13 C detection of hyperpolarized [13 C]urea.

Glioma is difficult to detect or characterize using current liquid biopsy approaches. Detection of cell‐free tumor DNA (cftDNA) in cerebrospinal fluid (CSF) has been proposed as an alternative to detection in plasma. We used shallow whole‐genome sequencing (sWGS, at a coverage of < 0.4×) of cell‐free DNA from the CSF of 13 patients with primary glioma to determine somatic copy number alterations and DNA fragmentation patterns. This allowed us to determine the presence of cftDNA in CSF without any prior knowledge of point mutations present in the tumor. We also showed that the fragmentation pattern of cell‐free DNA in CSF is different from that in plasma. This low‐cost screening method provides information on the tumor genome and can be used to target those patients with high levels of cftDNA for further larger‐scale sequencing, such as by whole‐exome and whole‐genome sequencing., EMBO Molecular Medicine, 10 (12), ISSN:1757-4676, ISSN:1757-4684

Hyperpolarized MRI with 13 C-labeled metabolites has enabled metabolic imaging of tumors in vivo. The heterogeneous nature of tumors and the limited lifetime of the hyperpolarization require high resolution, both temporally and spatially. We describe two sequences that make more efficient use of the 13 C polarization than previously described single-shot 3D sequences. With these sequences, the target metabolite resonances were excited using spectral-spatial pulses and the data acquired using spiral readouts from a series of echoes created using a fast-spin-echo sequence employing adiabatic 180° pulses. The third dimension was encoded with blipped gradients applied in an interleaved order to the echo train. Adiabatic inversion pulses applied in the absence of slice selection gradients allowed acquisition of signal from odd echoes, formed by unpaired adiabatic pulses, as well as from even echoes. The sequences were tested on tumor-bearing mice following intravenous injection of hyperpolarized [1-13 C]pyruvate. [1-13 C] pyruvate and [1-13 C] lactate images were acquired in vivo with a 4 × 4 × 2 cm3 field of view and a 32 × 32 × 16 matrix, leading to a nominal resolution of 1.25 × 1.25 × 1.25 mm3 and an effective resolution of 1.25 × 1.25 × 4.5 mm3 when the z-direction point spread function was taken into account. The acquisition of signal from more echoes also allowed for an improvement in the signal-to-noise ratio for resonances with longer T2 relaxation times. The pulse sequences described here produced hyperpolarized 13 C images with improved resolution and signal-to-noise ratio when compared with similar sequences described previously.

Single-shot echo planar imaging (EPI), which allows an image to be acquired using a single excitation pulse, is used widely for imaging the metabolism of hyperpolarized 13 C-labelled metabolites in vivo as the technique is rapid and minimizes the depletion of the hyperpolarized signal. However, EPI suffers from Nyquist ghosting, which normally is corrected for by acquiring a reference scan. In a dynamic acquisition of a series of images, this results in the sacrifice of a time point if the reference scan involves a full readout train with no phase encoding. This time penalty is negligible if an integrated navigator echo is used, but at the cost of a lower signal-to-noise ratio (SNR) as a result of prolonged T2 * decay. We describe here a workflow for hyperpolarized 13 C EPI that requires no reference scan. This involves the selection of a ghost-containing background from a 13 C image of a single metabolite at a single time point, the identification of phase correction coefficients that minimize signal in the selected area, and the application of these coefficients to images acquired at all time points and from all metabolites. The workflow was compared in phantom experiments with phase correction using a 13 C reference scan, and yielded similar results in situations with a regular field of view (FOV), a restricted FOV and where there were multiple signal sources. When compared with alternative phase correction methods, the workflow showed an SNR benefit relative to integrated 13 C reference echoes (>15%) or better ghost removal relative to a 1 H reference scan. The residual ghosting in a slightly de-shimmed B0 field was 1.6% using the proposed workflow and 3.8% using a 1 H reference scan. The workflow was implemented with a series of dynamically acquired hyperpolarized [1-13 C]pyruvate and [1-13 C]lactate images in vivo, resulting in images with no observable ghosting and which were quantitatively similar to images corrected using a 13 C reference scan.

Arthritic conditions are a major source of chronic pain. Furthering our understanding of disease mechanisms creates the opportunity to develop more targeted therapeutics. In rheumatoid arthritis (RA), measurements of pH in human synovial fluid suggest that acidosis occurs, but that this is highly variable between individuals. Here we sought to determine if tissue acidosis occurs in a widely used rodent arthritis model: complete Freund's adjuvant (CFA)-induced inflammation. CFA robustly evoked paw and ankle swelling, concomitant with worsening clinical scores over time. We used magnetic resonance spectroscopic imaging of hyperpolarized [1-13 C]pyruvate metabolism to demonstrate that CFA induces an increase in the lactate-to-pyruvate ratio. This increase is indicative of enhanced glycolysis and an increased lactate concentration, as has been observed in the synovial fluid from RA patients, and which was correlated with acidosis. We also measured the 13 CO2 /H13 CO3- ratio, in animals injected with hyperpolarized H13 CO3- , to estimate extracellular tissue pH and showed that despite the apparent increase in glycolytic activity in CFA-induced inflammation there was no accompanying decrease in extracellular pH. The pH was 7.23 ± 0.06 in control paws and 7.32 ± 0.09 in inflamed paws. These results could explain why mice lacking acid-sensing ion channel subunits 1, 2 and 3 do not display any changes in mechanical or thermal hyperalgesia in CFA-induced inflammation.