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PURPOSE The combined hyperpolarized (HP) 13 C pyruvate and urea MRI has provided a simultaneous assessment of glycolytic metabolism and tissue perfusion for improved cancer diagnosis and therapeutic evaluation in preclinical studies. This work aims to translate this dual-probe HP imaging technique to clinical research. METHODS A co-polarization system was developed where [1-13 C]pyruvic acid (PA) and [13 C, 15 N2 ]urea in water solution were homogeneously mixed and polarized on a 5T SPINlab system. Physical and chemical characterizations and toxicology studies of the combined probe were performed. Simultaneous metabolic and perfusion imaging was performed on a 3T clinical MR scanner by alternatively applying a multi-slice 2D spiral sequence for [1-13 C]pyruvate and its downstream metabolites and a 3D balanced steady-state free precession (bSSFP) sequence for [13 C, 15 N2 ]urea. RESULTS The combined PA/urea probe has a glass-formation ability similar to neat PA and can generate nearly 40% liquid-state 13 C polarization for both pyruvate and urea in 3-4 h. A standard operating procedure for routine on-site production was developed and validated to produce 40 mL injection product of approximately 150 mM pyruvate and 35 mM urea. The toxicology study demonstrated the safety profile of the combined probe. Dynamic metabolite-specific imaging of [1-13 C]pyruvate, [1-13 C]lactate, [1-13 C]alanine, and [13 C, 15 N2 ]urea was achieved with adequate spatial (2.6 mm × 2.6 mm) and temporal resolution (4.2 s), and urea images showed reduced off-resonance artifacts due to the JCN coupling. CONCLUSION The reported technical development and translational studies will lead to the first-in-human dual-agent HP MRI study and mark the clinical translation of the first HP 13 C MRI probe after pyruvate.

Purpose To investigate use of a novel imaging approach, hyperpolarized (HP) 13C magnetic resonance imaging (MRI) for simultaneous metabolism and perfusion assessment, to evaluate early and dose-dependent response to radiation therapy (RT) in a prostate cancer mouse model. Methods and Materials Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (n = 18) underwent single-fraction RT (4-14 Gy steep dose across the tumor) and were imaged serially at pre-RT baseline and 1, 4, and 7 days after RT using HP 13C MRI with combined [1-13C]pyruvate (metabolic active agent) and [13C]urea (perfusion agent), coupled with conventional multiparametric 1H MRI including T2-weighted, dynamic contrast-enhanced, and diffusion-weighted imaging. Tumor tissues were collected 4 and 7 days after RT for biological correlative studies. Results We found a significant decrease in HP pyruvate-to-lactate conversion in tumors responding to RT, with concomitant significant increases in HP pyruvate-to-alanine conversion and HP urea signal; the opposite changes were observed in tumors resistant to RT. Moreover, HP lactate change was dependent on radiation dose; tumor regions treated with higher radiation doses (10-14 Gy) exhibited a greater decrease in HP lactate signal than low-dose regions (4-7 Gy) as early as 1 day post-RT, consistent with lactate dehydrogenase enzyme activity and expression data. We also found that HP [13C]urea MRI provided assessments of tumor perfusion similar to those provided by 1H dynamic contrast-enhanced MRI in this animal model. However, apparent diffusion coefficien , a conventional 1H MRI functional biomarker, did not exhibit statistically significant changes within 7 days after RT. Conclusion These results demonstrate the ability of HP 13C MRI to monitor radiation-induced physiologic changes in a timely and dose-dependent manner, providing the basic science premise for further clinical investigation and translation.

There is an unmet clinical need for new and robust imaging biomarkers to distinguish indolent from aggressive prostate cancer. Hallmarks of aggressive tumors such as a decrease in extracellular pH (pHe) can potentially be used to identify aggressive phenotypes. In this study, we employ an optimized, high signal-to-noise ratio hyperpolarized (HP) 13C pHe imaging method to discriminate between indolent and aggressive disease in a murine model of prostate cancer. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice underwent a multiparametric MR imaging exam, including HP [13C] bicarbonate MRI for pHe, with 1H apparent diffusion coefficient (ADC) mapping and HP [1-13C] pyruvate MRI to study lactate metabolism. Tumor tissue was excised for histological staining and qRT-PCR to quantify mRNA expression for relevant glycolytic enzymes and transporters. We observed good separation in pHe between low- and high-grade tumor regions, with high-grade tumors demonstrating a lower pHe. The pHe also correlated strongly with monocarboxylate transporter Mct4 gene expression across all tumors, suggesting that lactate export via MCT4 is associated with acidification in this model. Our results implicate extracellular acidification as an indicator of indolent-to-aggressive transition in prostate cancer and suggest feasibility of HP pHe imaging to detect high-grade, clinically significant disease in men as part of a multiparametric MRI examination.

Non-invasive assessment of the biological aggressiveness of prostate cancer (PCa) is needed for men with localized disease. Hyperpolarized (HP) 13C magnetic resonance (MR) spectroscopy is a powerful approach to image metabolism, specifically the conversion of HP [1-13C]pyruvate to [1-13C]lactate, catalyzed by lactate dehydrogenase (LDH). Significant increase in tumor lactate was measured in high-grade PCa relative to benign and low-grade cancer, suggesting that HP 13C MR could distinguish low-risk (Gleason score &le, 3 + 4) from high-risk (Gleason score &ge, 4 + 3) PCa. To test this and the ability of HP 13C MR to detect these metabolic changes, we cultured prostate tissues in an MR-compatible bioreactor under continuous perfusion. 31P spectra demonstrated good viability and dynamic HP 13C-pyruvate MR demonstrated that high-grade PCa had significantly increased lactate efflux compared to low-grade PCa and benign prostate tissue. These metabolic differences are attributed to significantly increased LDHA expression and LDH activity, as well as significantly increased monocarboxylate transporter 4 (MCT4) expression in high- versus low- grade PCa. Moreover, lactate efflux, LDH activity, and MCT4 expression were not different between low-grade PCa and benign prostate tissues, indicating that these metabolic alterations are specific for high-grade disease. These distinctive metabolic alterations can be used to differentiate high-grade PCa from low-grade PCa and benign prostate tissues using clinically translatable HP [1-13C]pyruvate MR.

Author(s): Tang, Shuyu; Bok, Robert; Qin, Hecong; Reed, Galen; VanCriekinge, Mark; Delos Santos, Romelyn; Overall, William; Santos, Juan; Gordon, Jeremy; Wang, Zhen Jane; Vigneron, Daniel B; Larson, Peder EZ | Abstract: PurposeThe balanced steady-state free precession sequence has been previously explored to improve the efficient use of nonrecoverable hyperpolarized 13C magnetization, but suffers from poor spectral selectivity and long acquisition time. The purpose of this study was to develop a novel metabolite-specific 3D bSSFP ("MS-3DSSFP") sequence with stack-of-spiral readouts for improved lactate imaging in hyperpolarized [1-13 C]pyruvate studies on a clinical 3T scanner.MethodsSimulations were performed to evaluate the spectral response of the MS-3DSSFP sequence. Thermal 13C phantom experiments were performed to validate the MS-3DSSFP sequence. In vivo hyperpolarized [1-13 C], pyruvate studies were performed to compare the MS-3DSSFP sequence with metabolite-specific gradient echo ("MS-GRE") sequences for lactate imaging.ResultsSimulations, phantom, and in vivo studies demonstrate that the MS-3DSSFP sequence achieved spectrally selective excitation on lactate while minimally perturbing other metabolites. Compared with MS-GRE sequences, the MS-3DSSFP sequence showed approximately a 2.5-fold SNR improvement for lactate imaging in rat kidneys, prostate tumors in a mouse model, and human kidneys.ConclusionsImproved lactate imaging using the MS-3DSSFP sequence in hyperpolarized [1-13 C]pyruvate studies was demonstrated in animals and humans. The MS-3DSSFP sequence could be applied for other clinical applications such as in the brain or adapted for imaging other metabolites such as pyruvate and bicarbonate.

PURPOSE: To investigate using a novel imaging approach - hyperpolarized (HP) (13)C magnetic resonance imaging (MRI) for simultaneous metabolism and perfusion assessment - to evaluate early and does-dependent radiotherapy (RT) response in a prostate cancer mouse model. METHODS AND MATERIALS: Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mice (n = 18) underwent singlefraction RT (4 - 14 Gy steep dose across the tumor) and were imaged serially at pre-RT baseline and 1, 4, and 7 days post-RT, using HP (13)C MRI with combined [1-(13)C]pyruvate (metabolic active agent) and [(13)C]urea (perfusion agent), coupled with conventional multiparametric (1)H MRI including T(2)-weighted, dynamic contrast-enhanced (DCE), and diffusion-weighted imaging. Tumor tissues were collected at Day 4 and Day 7 for biological correlative studies. RESULTS: We found a significant decrease in HP pyruvate-to-lactate conversion in tumors responding to RT, with concomitant significant increases in HP pyruvate-to-alanine conversion and HP urea signal; whereas the opposite changes were observed in tumors resistant to RT. Moreover, HP lactate change was radiation-dose dependent: tumor regions treated with higher radiation doses (10-14 Gy) exhibited a greater decrease in HP lactate signal than low-dose regions (4-7 Gy) as early as 1 day post-RT, consistent with LDH enzyme activity and expression data. We also found that HP [(13)C]urea MRI provided similar assessments of tumor perfusion to those provided by (1)H DCE MRI in this animal model. However, apparent diffusion coefficient (ADC), a conventional (1)H MR functional biomarker, did not exhibit statistically significant changes within 7 days after RT. CONCLUSION: These results demonstrate the ability of HP (13)C MRI to monitor radiation-induced physiologic changes in a timely and dose-dependent manner, providing the basic science premise for further clinical investigations and translation.

Oxidative stress is a critical feature of several common neurologic disorders. The brain is well adapted to neutralize oxidative injury by maintaining a high steady-state concentration of small-molecule intracellular antioxidants including glutathione in astrocytes and ascorbic acid in neurons. Ascorbate-derived imaging probes for hyperpolarized 13C magnetic resonance spectroscopy and positron emission tomography have been used to study redox changes (antioxidant depletion and reactive oxygen species accumulation) in vivo. In this study, we applied these imaging probes to the normal rat brain and a rat model of glutathione depletion. We first studied hyperpolarized [1-13C]dehydroascorbate in the normal rat brain, demonstrating its robust conversion to [1-13C]vitamin C, consistent with rapid transport of the oxidized form across the blood-brain barrier. We next showed that the kinetic rate of this conversion decreased by nearly 50% after glutathione depletion by diethyl maleate treatment. Finally, we showed that dehydroascorbate labeled for positron emission tomography, namely [1-11C]dehydroascorbate, showed no change in brain signal accumulation after diethyl maleate treatment. These results suggest that hyperpolarized [1-13C]dehydroascorbate may be used to non-invasively detect oxidative stress in common disorders of the brain.

Imaging studies are frequently used to support the clinical diagnosis of infection. These techniques include computed tomography (CT) and magnetic resonance imaging (MRI) for structural information and single photon emission computed tomography (SPECT) or positron emission tomography (PET) for metabolic data. However, frequently, there is significant overlap in the imaging appearance of infectious and noninfectious entities using these tools. To address this concern, recent approaches have targeted bacteria-specific metabolic pathways. For example, radiolabeled sugars derived from sorbitol and maltose have been investigated as PET radiotracers, since these are efficiently incorporated into bacteria but are poor substrates for mammalian cells. We have previously shown that para-aminobenzoic acid (PABA) is an excellent candidate for development as a bacteria-specific imaging tracer as it is rapidly accumulated by a wide range of pathogenic bacteria, including metabolically quiescent bacteria and clinical st...

The growing demand for metabolism-specific imaging techniques has rekindled interest in Deuterium (2H) Metabolic Imaging (DMI), a robust method based on administration of a substrate (glucose, acetate, fumarate, etc.) labeled with the stable isotope of hydrogen and the observation of its metabolic fate in three-dimensions. This technique allows the investigation of multiple metabolic processes in both healthy and diseased states. Despite its low natural abundance, the short relaxation time of deuterium allows for rapid radiofrequency (RF) pulses without saturation and efficient image acquisition. In this review, we provide a comprehensive picture of the evolution of DMI over the course of recent decades, with a special focus on its potential clinical applications.

There is an unmet clinical need for new and robust imaging biomarkers to distinguish indolent from aggressive prostate cancer. Hallmarks of aggressive tumors such as a decrease in extracellular pH (pHe) can potentially be used to identify aggressive phenotypes. In this study, we employ an optimized, high signal-to-noise ratio hyperpolarized (HP) 13C pHe imaging method to discriminate between indolent and aggressive disease in a murine model of prostate cancer. Transgenic adenocarcinoma of the mouse prostate (TRAMP) mice underwent a multiparametric MR imaging exam, including HP [13C] bicarbonate MRI for pHe, with 1H apparent diffusion coefficient (ADC) mapping and HP [1-13C] pyruvate MRI to study lactate metabolism. Tumor tissue was excised for histological staining and qRT-PCR to quantify mRNA expression for relevant glycolytic enzymes and transporters. We observed good separation in pHe between low- and high-grade tumor regions, with high-grade tumors demonstrating a lower pHe. The pHe also correlated strongly with monocarboxylate transporter Mct4 gene expression across all tumors, suggesting that lactate export via MCT4 is associated with acidification in this model. Our results implicate extracellular acidification as an indicator of indolent-to-aggressive transition in prostate cancer and suggest feasibility of HP pHe imaging to detect high-grade, clinically significant disease in men as part of a multiparametric MRI examination.

Optimal treatment selection for localized renal tumors is challenging due to their variable biological behavior and limited ability to pre-operatively assess their aggressiveness. We investigated hyperpolarized (HP) 13C pyruvate MRI to noninvasively assess tumor lactate production and compartmentalization, which are strongly associated with renal tumor aggressiveness. Orthotopic tumors were created in mice using human renal cell carcinoma (RCC) lines (A498, 786-O, UOK262) with varying expression of lactate dehydrogenase A (LDHA) which catalyzes the pyruvate-to-lactate conversion, and varying expression of monocarboxylate transporter 4 (MCT4) which mediates lactate export out of the cells. Dynamic HP 13C pyruvate MRI showed that the A498 tumors had significantly higher 13C pyruvate-to-lactate conversion than the UOK262 and 786-O tumors, corresponding to higher A498 tumor LDHA expression. Additionally, diffusion-weighted HP 13C pyruvate MRI showed that the A498 tumors had significantly higher 13C lactate apparent diffusion coefficients compared to 786-O tumors, with corresponding higher MCT4 expression, which likely reflects more rapid lactate export in the A498 tumors. Our data demonstrate the feasibility of HP 13C pyruvate MRI to inform on tumor lactate production and compartmentalization, and provide the scientific premise for future clinical investigation into the utility of this technique to noninvasively interrogate renal tumor aggressiveness and to guide treatment selection.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13C dehydroascorbate (DHA) and 13C pyruvate MR spectroscopy to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline, and 7 days after unilateral ischemia reperfusion injury (IRI). Compared to the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13C Vitamin C/(Vitamin C+DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13C lactate/pyruvate ratio at day 7 compared to baseline, although the 13C lactate/pyruvate ratio was not significantly different between the IRI and the contralateral sham-operated kidneys at day 7. Arterial spin labeling MRI demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS), and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13C MR for the noninvasive assessment of oxidative stress and mitochondrial PDH activity following renal ischemia reperfusion injury.

Acute kidney injury (AKI) is a major risk factor for the development of chronic kidney disease (CKD). Persistent oxidative stress and mitochondrial dysfunction are implicated across diverse forms of AKI and in the transition to CKD. In this study, we applied hyperpolarized (HP) 13 C dehydroascorbate (DHA) and 13 C pyruvate magnetic resonance spectroscopy (MRS) to investigate the renal redox capacity and mitochondrial pyruvate dehydrogenase (PDH) activity, respectively, in a murine model of AKI at baseline and 7 days after unilateral ischemia reperfusion injury (IRI). Compared with the contralateral sham-operated kidneys, the kidneys subjected to IRI showed a significant decrease in the HP 13 C vitamin C/(vitamin C + DHA) ratio, consistent with a decrease in redox capacity. The kidneys subjected to IRI also showed a significant decrease in the HP 13 C bicarbonate/pyruvate ratio, consistent with impaired PDH activity. The IRI kidneys showed a significantly higher HP 13 C lactate/pyruvate ratio at day 7 compared with baseline, although the 13 C lactate/pyruvate ratio was not significantly different between the IRI and contralateral sham-operated kidneys at day 7. Arterial spin labeling magnetic resonance imaging (MRI) demonstrated significantly reduced perfusion in the IRI kidneys. Renal tissue analysis showed corresponding increased reactive oxygen species (ROS) and reduced PDH activity in the IRI kidneys. Our results show the feasibility of HP 13 C MRS for the non-invasive assessment of oxidative stress and mitochondrial PDH activity following renal IRI.