Mark Van Criekinge

Metabolic imaging of patients with prostate cancer using hyperpolarized [1-¹³C]pyruvate.

Metabolic imaging with hyperpolarized pyruvate was used to safely and noninvasively visualize prostate tumors in patients. The Hyperpolarized Prostate Cancer cells have a different metabolism than healthy cells. Specifically, they consume more pyruvate—a key component in glycolysis—than their normal counterparts. Nelson and colleagues therefore used a hyperpolarized form of pyruvate ([1-13C]pyruvate) to sensitively image increased levels of its product, [1-13C]lactate, as well as the flux of pyruvate to lactate. The [1-13C]pyruvate agent was used here in a first-in-human study in men with prostate cancer. Patients received varying doses of [1-13C]pyruvate that were found to be safe. These patients were then rapidly imaged with hyperpolarized 13C magnetic resonance (MR), which was able to provide dynamic (time course) information as well as three-dimensional (3D) (spatial) data at a single time point. Tumors were detected in all patients with biopsy-proven cancer. And, importantly, with 13C MR imaging (MRI), Nelson et al. were able to see cancer in regions of the prostate that were previously considered to be tumor-free upon inspection with other conventional anatomic imaging methods. With the ability to safely image tumor location and also follow tumor metabolism over time, hyperpolarized 13C MRI may be useful both for initial diagnosis and for monitoring therapy. Although the patients in this study had early-stage disease, the authors believe that [1-13C]lactate/[1-13C]pyruvate flux will only increase with tumor grade, making this imaging technology amenable to more advanced and aggressive cancers. Future studies will focus on optimizing agent preparation and delivery to ensure that this imaging technology can benefit patients in all clinical settings. This first-in-man imaging study evaluated the safety and feasibility of hyperpolarized [1-13C]pyruvate as an agent for noninvasively characterizing alterations in tumor metabolism for patients with prostate cancer. Imaging living systems with hyperpolarized agents can result in more than 10,000-fold enhancement in signal relative to conventional magnetic resonance (MR) imaging. When combined with the rapid acquisition of in vivo 13C MR data, it is possible to evaluate the distribution of agents such as [1-13C]pyruvate and its metabolic products lactate, alanine, and bicarbonate in a matter of seconds. Preclinical studies in cancer models have detected elevated levels of hyperpolarized [1-13C]lactate in tumor, with the ratio of [1-13C]lactate/[1-13C]pyruvate being increased in high-grade tumors and decreased after successful treatment. Translation of this technology into humans was achieved by modifying the instrument that generates the hyperpolarized agent, constructing specialized radio frequency coils to detect 13C nuclei, and developing new pulse sequences to efficiently capture the signal. The study population comprised patients with biopsy-proven prostate cancer, with 31 subjects being injected with hyperpolarized [1-13C]pyruvate. The median time to deliver the agent was 66 s, and uptake was observed about 20 s after injection. No dose-limiting toxicities were observed, and the highest dose (0.43 ml/kg of 230 mM agent) gave the best signal-to-noise ratio for hyperpolarized [1-13C]pyruvate. The results were extremely promising in not only confirming the safety of the agent but also showing elevated [1-13C]lactate/[1-13C]pyruvate in regions of biopsy-proven cancer. These findings will be valuable for noninvasive cancer diagnosis and treatment monitoring in future clinical trials.

Multi-compound Polarization by DNP Allows Simultaneous Assessment of Multiple Enzymatic Activities In Vivo

Methods for the simultaneous polarization of multiple 13C-enriched metabolites were developed to probe several enzymatic pathways and other physiologic properties in vivo, using a single intravenous bolus. A new method for polarization of 13C sodium bicarbonate suitable for use in patients was developed, and the co-polarization of 13C sodium bicarbonate and [1-(13)C] pyruvate in the same sample was achieved, resulting in high solution-state polarizations (15.7% and 17.6%, respectively) and long spin-lattice relaxation times (T1) (46.7 s and 47.7 s respectively at 3 T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-(13)C] pyruvate were even longer at 3 T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-(13)C] pyruvate were injected into normal mice and a murine prostate tumor model at 3T. Rapid equilibration of injected hyperpolarized 13C sodium bicarbonate with 13C CO2 allowed calculation of pH on a voxel by voxel basis, and simultaneous assessment of pyruvate metabolism with cellular uptake and conversion of [1-(13)C] pyruvate to its metabolic products. Initial studies in a Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model demonstrated higher levels of hyperpolarized lactate and lower pH within tumor, relative to surrounding benign tissues and to the abdominal viscera of normal controls. There was no significant difference observed in the tumor lactate/pyruvate ratio obtained after the injection of co-polarized 13C bicarbonate and [1-(13)C] pyruvate or polarized [1-(13)C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-(13)C]pyruvic acid, 13C sodium bicarbonate, [1,4-(13)C]fumaric acid, and 13C urea with high levels of solution polarization (17.5%, 10.3%, 15.6% and 11.6%, respectively) and spin-lattice relaxation values similar to those recorded for the individual metabolites. These studies demonstrated the feasibility of simultaneously measuring in vivo pH and tumor metabolism using nontoxic, endogenous species, and the potential to extend the multi-polarization approach to include up to four hyperpolarized probes providing multiple metabolic and physiologic measures in a single MR acquisition.

Hyperpolarized [2-13C]-fructose: a hemiketal DNP substrate for in vivo metabolic imaging.

Hyperpolarized (13)C labeled molecular probes have been used to investigate metabolic pathways of interest as well as facilitate in vivo spectroscopic imaging by taking advantage of the dramatic signal enhancement provided by DNP. Due to the limited lifetime of the hyperpolarized nucleus, with signal decay dependent on T(1) relaxation, carboxylate carbons have been the primary targets for development of hyperpolarized metabolic probes. The use of these carbon nuclei makes it difficult to investigate upstream glycolytic processes, which have been related to both cancer metabolism as well as other metabolic abnormalities, such as fatty liver disease and diabetes. Glucose carbons have very short T(1)s (<1 s) and therefore cannot be used as an in vivo hyperpolarized metabolic probe of glycolysis. However, the pentose analogue fructose can also enter glycolysis through its phosphorylation by hexokinase and yield complementary information. The C(2) of fructose is a hemiketal that has a relatively longer relaxation time (approximately 16 s at 37 degrees C) and high solution state polarization (approximately 12%). Hyperpolarized [2-(13)C]-fructose was also injected into a transgenic model of prostate cancer (TRAMP) and demonstrated difference in uptake and metabolism in regions of tumor relative to surrounding tissue. Thus, this study demonstrates the first hyperpolarization of a carbohydrate carbon with a sufficient T(1) and solution state polarization for ex vivo spectroscopy and in vivo spectroscopic imaging studies.

Hyperpolarized 13C-pyruvate magnetic resonance reveals rapid lactate export in metastatic renal cell carcinomas

Renal cell carcinomas (RCC) are a heterogeneous group of tumors with a wide range of aggressiveness. Noninvasive methods to confidently predict the tumor biologic behavior and select appropriate treatment are lacking. Here, we investigate the dynamic metabolic flux in living RCC cells using hyperpolarized (13)C-pyruvate magnetic resonance spectroscopy (MRS) combined with a bioreactor platform and interrogated the biochemical basis of the MRS data with respect to cancer aggressiveness. RCC cells have significantly higher pyruvate-to-lactate flux than the normal renal tubule cells. Furthermore, a key feature distinguishing the localized from the metastatic RCC cells is the lactate efflux rate, mediated by the monocarboxylate transporter 4 (MCT4). The metastatic RCC cells have significantly higher MCT4 expression and corresponding higher lactate efflux, which is essential for maintaining a high rate of glycolysis. We show that such differential cellular transporter expression and associated metabolic phenotype can be noninvasively assessed via real-time monitoring of hyperpolarized (13)C-pyruvate-to-lactate flux.

Hyperpolarized 13C spectroscopy and an NMR-compatible bioreactor system for the investigation of real-time cellular metabolism

The purpose of this study was to combine a three‐dimensional NMR‐compatible bioreactor with hyperpolarized 13C NMR spectroscopy in order to probe cellular metabolism in real time. JM1 (immortalized rat hepatoma) cells were cultured in a three‐dimensional NMR‐compatible fluidized bioreactor. 31P spectra were acquired before and after each injection of hyperpolarized [1‐13C] pyruvate and subsequent 13C spectroscopy at 11.7 T. 1H and two‐dimensional 1H‐1H‐total correlation spectroscopy spectra were acquired from extracts of cells grown in uniformly labeled 13C‐glucose, on a 16.4 T, to determine 13C fractional enrichment and distribution of 13C label. JM1 cells were found to have a high rate of aerobic glycolysis in both two‐dimensional culture and in the bioreactor, with 85% of the 13C label from uniformly labeled 13C‐glucose being present as either lactate or alanine after 23 h. Flux measurements of pyruvate through lactate dehydrogenase and alanine aminotransferase in the bioreactor system were 12.18 ± 0.49 nmols/sec/108 cells and 2.39 ± 0.30 nmols/sec/108 cells, respectively, were reproducible in the same bioreactor, and were not significantly different over the course of 2 days. Although this preliminary study involved immortalized cells, this combination of technologies can be extended to the real‐time metabolic exploration of primary benign and cancerous cells and tissues prior to and after therapy. Magn Reson Med, 2010.

Metabolic Reprogramming and Validation of Hyperpolarized 13C Lactate as a Prostate Cancer Biomarker Using a Human Prostate Tissue Slice Culture Bioreactor

The treatment of prostate cancer has been impeded by the lack of both clinically relevant disease models and metabolic markers that track tumor progression. Hyperpolarized (HP) 13C MR spectroscopy has emerged as a new technology to investigate the metabolic shifts in prostate cancer. In this study, we investigate the glucose reprogramming using HP 13C pyruvate MR in a patient‐derived prostate tissue slice culture (TSC) model.

High Resolution

(13)C steady state free precession (SSFP) magnetic resonance imaging and effective spin-spin relaxation time (T2) mapping were performed using hyperpolarized [(13)C] urea and [(13) C,(15)N2] urea injected intravenously in rats. (15)N labeling gave large T2 increases both in solution and in vivo due to the elimination of a strong scalar relaxation pathway. The T2 increase was pronounced in the kidney, with [(13) C,(15) N2] urea giving T2 values of 6.3±1.3 s in the cortex and medulla, and 11±2 s in the renal pelvis. The measured T2 in the aorta was 1.3±0.3 s. [(13)C] urea showed shortened T2 values in the kidney of 0.23±0.03 s compared to 0.28±0.03 s measured in the aorta. The enhanced T2 of [(13)C,(15)N2] urea was utilized to generate large signal enhancement by SSFP acquisitions with flip angles approaching the fully refocused regime. Projection images at 0.94 mm in-plane resolution were acquired with both urea isotopes, with [(13)C,(15) N2] urea giving a greater than four-fold increase in signal-to-noise ratio over [(13)C] urea.

^{13}

In this manuscript, the remarkable NMR signal enhancement that can be provided by dynamic nuclear polarization (DNP) was combined with the reactivity of acetic anhydride with amines to perform rapid, high SNR analyses of amino acid mixtures and to hyperpolarize new biomolecules of interest. [1,1-13C] acetic anhydride is an excellent substrate for DNP hyperpolarization because it can be well polarized in only 30 min and has a relatively long T1 relaxation time (33.9 s at 11.7 T and 37 °C). The secondary hyperpolarization approach developed in this project takes advantage of the preferential reaction of acetic anhydride with amine nucleophiles, which occurs much more rapidly than hydrolysis to produce hyperpolarized N-acetyl adducts. This new approach was used to reproducibly and near-quantitatively (mean yield − 89.8%) resolve a mixture of amino acids Gly, Ser, Val, Leu, and Ala in a single acquisition (3 s) with a signal enhancement of up to 1,400-fold as compared with thermal equilibrium. Secondary hyperpolarization was performed for several small peptides and N-acetylcysteine, a drug administered intravenously to treat acetaminophen overdose. Although, in general the T1 of the N-acetyl adducts decreased with increasing molecular weight of the biomolecules, the T1 values were still on the order of 10 s, and the correlation of T1 with molecular weight was not exact suggesting the potential of secondarily polarizing relatively large biomolecules. This study demonstrates the feasibility of using prepolarized [1,1-13C] acetic anhydride and rapid chemical reactions to provide high SNR NMR spectra of amino acid derivatives and other biomolecules.

C MRI With Hyperpolarized Urea: In Vivo

Oxidative stress has been proposed to be a unifying cause for diabetic nephropathy and a target for novel therapies. Here we apply a new endogenous reduction-oxidation (redox) sensor, hyperpolarized (HP) 13C dehydroascorbate (DHA), in conjunction with MRI to noninvasively interrogate the renal redox capacity in a mouse diabetes model. The diabetic mice demonstrate an early decrease in renal redox capacity, as shown by the lower in vivo HP 13C DHA reduction to the antioxidant vitamin C (VitC), prior to histological evidence of nephropathy. This correlates with lower tissue reduced glutathione (GSH) concentration and higher NADPH oxidase 4 (Nox4) expression, consistent with increased superoxide generation and oxidative stress. ACE inhibition restores the HP 13C DHA reduction to VitC with concomitant normalization of GSH concentration and Nox4 expression in diabetic mice. HP 13C DHA enables rapid in vivo assessment of altered redox capacity in diabetic renal injury and after successful treatment.

T_{2}

Application of (13)C MRS in vivo on whole body MR system has been limited due to the low static field (and consequent low signal to noise ratio-SNR) of these scanners; thus there have been few reports of (1)H decoupled (13)C MRS in vivo using a clinical MR platform. The recent development of techniques to retain highly polarized spins in solution following DNP in a solid matrix has provided a mechanism to use endogenous pre-polarized (13)C labeled substrates to study real time cellular metabolism in vivo with high SNR. In a recent in vivo hyperpolarized metabolic imaging study using (13)C pyruvate, it has been demonstrated that the line shape (signal decay) of the resonances observed are greatly affected by J(CH) coupling in addition to inhomogeneous broadening. This study demonstrates the feasibility of improving hyperpolarized (13)C metabolic imaging in vivo by incorporating (1)H decoupling on a clinical whole body 3T MR scanner. No reduction of T1 of a pre-polarized (13)C substrate ([1-(13)C] lactate) in solution was observed when (1)H decoupling was applied with WALTZ16 sequence. Narrower linewidth for the [1-(13)C] lactate resonance was observed in hyperpolarized (13)C MRSI data in vivo with (1)H decoupling.