The study of transient chemical phenomena by conventional NMR has proved elusive, particularly for non-(1)H nuclei. For (13)C, hyperpolarization using the dynamic nuclear polarization (DNP) technique has emerged as a powerful means to improve SNR. The recent development of rapid dissolution DNP methods has facilitated previously impossible in vitro and in vivo study of small molecules. This review presents the basics of the DNP technique, identification of appropriate DNP substrates, and approaches to increase hyperpolarized signal lifetimes. Also addressed are the biochemical events to which DNP-NMR has been applied, with descriptions of several probes that have met with in vivo success.

Metabolic imaging is a field of molecular imaging that focuses and targets changes in metabolic pathways for the evaluation of different clinical conditions. Targeting and quantifying metabolic changes noninvasively is a powerful approach to facilitate diagnosis and evaluate therapeutic response. This review addresses only techniques targeting metabolic pathways. Other molecular imaging strategies, such as affinity or receptor imaging or microenvironment-dependent methods are beyond the scope of this review. Here we describe the current state of the art in clinically translatable metabolic imaging modalities. Specifically, we focus on PET and MR spectroscopy, including conventional (1)H- and (13)C-MR spectroscopy at thermal equilibrium and hyperpolarized MRI. In this article, we first provide an overview of metabolic pathways that are altered in many pathologic conditions and the corresponding probes and techniques used to study those alterations. We then describe the application of metabolic imaging to several common diseases, including cancer, neurodegeneration, cardiac ischemia, and infection or inflammation.

Unlabelled

Reduction and oxidation (redox) chemistry is increasingly implicated in cancer pathogenesis. To interrogate the redox status of prostate tumors noninvasively, we developed hyperpolarized [1-(13)C]dehydroascorbate ((13)C-DHA), the oxidized form of vitamin C, as an MR probe. In a model of transgenic adenocarcinoma of the mouse prostate (TRAMP), increased reduction of hyperpolarized (13)C-DHA to vitamin C was observed in tumor, as compared with normal prostate and surrounding benign tissue. We hypothesized that this difference was due to higher concentrations of glutathione and increased transport of hyperpolarized (13)C-DHA via the glucose transporters (GLUT1, GLUT3, and GLUT4) in TRAMP tumor. To test these hypotheses, hyperpolarized (13)C-DHA MR spectroscopy (MRS) and (18)F-FDG PET were applied as complementary technologies in the TRAMP model.

Methods

Late-stage TRAMP tumors (>4 cm(3)) were studied at similar time points (MR studies conducted < 24 h after PET) in fasting mice by (18)F-FDG PET and hyperpolarized (13)C-DHA MR imaging on a small-animal PET/CT scanner and a (1)H/(3)C 3-T MR scanner. PET data were processed using open-source AMIDE software to compare the standardized uptake values of tumor with those of surrounding muscle, and (13)C-DHA MRS data were processed using custom software to compare the metabolite ratios (vitamin C/[vitamin C + (13)C-DHA]). After in vivo studies, the tumor glutathione concentrations were determined using a spectrophotometric assay, and thiol staining was performed using mercury orange. Real-time polymerase chain reaction was used to evaluate the relevant transporters GLUT1, GLUT3, and GLUT4 and vitamin C transporters SVCT1 and SVCT2. GLUT1 was also evaluated by immunohistochemistry.

Results

The average metabolite ratio was 0.28 ± 0.02 in TRAMP tumor, versus 0.11 ± 0.02 in surrounding benign tissue (n = 4), representing a 2.5-fold difference. The corresponding tumor-to-nontumor (18)F-FDG uptake ratio was 3.0. The total glutathione was 5.1 ± 0.4 mM in tumor and 1.0 ± 0.2 mM in normal prostate, whereas reduced glutathione was 2.0 ± 0.3 mM and 0.8 ± 0.3 mM, respectively, corresponding to a 2.5-fold difference. In TRAMP tumor, mercury orange staining demonstrated increased thiols. Real-time polymerase chain reaction showed no significant difference in GLUT1 messenger RNA between TRAMP tumor and normal prostate, with immunohistochemistry (anti-GLUT1) also showing comparable staining.

Conclusion

Both hyperpolarized (13)C-DHA and (18)F-FDG provide similar tumor contrast in the TRAMP model. Our findings suggest that the mechanism of in vivo hyperpolarized (13)C-DHA reduction and the resulting tumor contrast correlates most strongly with glutathione concentration. In the TRAMP model, GLUT1 is not significantly upregulated and is unlikely to account for the contrast obtained using hyperpolarized (13)C-DHA or (18)F-FDG.

We report the imaging of β-cyclodextrin-benzoic acid binding at 14T using hyperpolarized (13)C magnetic resonance (MR). Benzoic acid was polarized using a dynamic nuclear polarization (DNP) approach and combined with β-cyclodextrin in aqueous solution. As anticipated, decreases in the spin-lattice relaxation constant (T(1)) were observed with decreases in the ligand-receptor ratio. The calculated log K was approximately 1.7, similar to previously reported binding constants. Hyperpolarized [1-(13)C] benzoic acid was used to interrogate solutions of variable β-cyclodextrin concentrations, with the mixtures imaged at 14T using a 3D frequency-selective MR sequence. Differences in β-cyclodextrin concentration were easily visualized. These results suggest that hyperpolarized (13)C MR could be used in vivo to determine the presence and density of receptors for a given ligand-receptor pair.

We combined the high MR signal enhancement achieved using dissolution dynamic nuclear polarization (DNP) with a pulsed gradient double spin echo diffusion MR sequence to rapidly and accurately measure the diffusion coefficients of various hyperpolarized (13)C molecules in solution. Furthermore, with a diffusion-weighted imaging sequence we generate diffusion coefficient maps of multiple hyperpolarized metabolites simultaneously. While hyperpolarized experiments can measure rapid, non-equilibrium processes by avoiding signal averaging, continuous signal loss due to longitudinal relaxation (T(1)) complicates quantitation. By correcting for this signal loss, we demonstrate the feasibility of using hyperpolarized (13)C diffusion-weighted MR to accurately measure real-time (seconds) molecular transport phenomena. Potential applications include rapidly measuring molecular binding, cellular membrane transport, in vivo metabolite distribution and establishing a magnetic field independent hyperpolarized parameter.

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.

Purpose

Hyperpolarized (13) C magnetic resonance allows for the study of real-time metabolism in vivo, including significant hyperpolarized (13) C lactate production in many tumors. Other studies have shown that aggressive and highly metastatic tumors rapidly transport lactate out of cells. Thus, the ability to not only measure the production of hyperpolarized (13) C lactate but also understand its compartmentalization using diffusion-weighted MR will provide unique information for improved tumor characterization.

Methods

We used a bipolar, pulsed-gradient, double spin echo imaging sequence to rapidly generate diffusion-weighted images of hyperpolarized (13) C metabolites. Our methodology included a simultaneously acquired B1 map to improve apparent diffusion coefficient (ADC) accuracy and a diffusion-compensated variable flip angle scheme to improve ADC precision.

Results

We validated this sequence and methodology in hyperpolarized (13) C phantoms. Next, we generated ADC maps of several hyperpolarized (13) C metabolites in a normal rat, rat brain tumor, and prostate cancer mouse model using both preclinical and clinical trial-ready hardware.

Conclusion

ADC maps of hyperpolarized (13) C metabolites provide information about the localization of these molecules in the tissue microenvironment. The methodology presented here allows for further studies to investigate ADC changes due to disease state that may provide unique information about cancer aggressiveness and metastatic potential.

Localized renal tumors are increasingly detected incidentally at imaging. Conventional imaging cannot reliably differentiate the 20% of these tumors that are benign from malignant renal cell carcinomas (RCCs), leading to unnecessary surgical resection and resulting morbidity associated with surgery. Here, we investigated hyperpolarized ¹³C pyruvate metabolism in live patient-derived renal tumor tissue slices using a novel magnetic resonance (MR) -compatible bioreactor platform. We demonstrated for the first time that clear cell RCCs (ccRCCs), which account for 70-80% of all RCCs, have increased lactate production as well as rapid lactate efflux compared to benign renal tumors. This difference is attributed to increased lactate dehydrogenase A and monocarboxylate transporter 4 expression in ccRCCs. This distinctive metabolic phenotype can be used to differentiate RCCs from benign renal tumors using clinically translatable hyperpolarized ¹³C pyruvate MR.

Background

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) (13) C 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 (13) C pyruvate MR in a patient-derived prostate tissue slice culture (TSC) model.

Methods

The steady-state metabolite concentrations in freshly excised human prostate TSCs were assessed and compared to those from snap-frozen biopsy samples. The TSCs were then applied to a perfused cell (bioreactor) platform, and the bioenergetics and the dynamic pyruvate flux of the TSCs were investigated by (31) P and HP (13) C MR, respectively.

Results

The prostate TSCs demonstrated steady-state glycolytic and phospholipid metabolism, and bioenergetics that recapitulate features of prostate cancer in vivo. (13) C spectra following injection of HP (13) C pyruvate showed significantly increased pyruvate to lactate flux in malignant as compared to the benign prostate TSCs. This increased flux in the malignant prostate TSCs correlated with both increased expression of monocarboxylate transporters (MCT) and activity of lactate dehydrogenase (LDH).

Conclusions

We provide the first mechanistic evidence for HP (13) C lactate as a prostate cancer biomarker in living human tissues, critical for the interpretation of in vivo studies. More broadly, the clinically relevant metabolic model system in combination with HP MR can facilitate the identification of clinically translatable biomarkers of prostate cancer presence, aggressiveness, and treatment response.

Nonalcoholic fatty liver disease (NAFLD) has become highly prevalent, now considered the most common liver disease in the western world. Approximately one-third of patients with NASH develop non-alchoholic steatohepatitis (NASH), histologically defined by lobular and portal inflammation, and accompanied by marked oxidative stress. Patients with NASH are at increased risk for cirrhosis and hepatocellular carcinoma, and diagnosis currently requires invasive biopsy. In animal models of NASH, particularly the methionine-choline deficient (MCD) model, profound changes are seen in redox enzymes and key intracellular antioxidants. To study antioxidant status in NASH non-invasively, we applied the redox probe hyperpolarized [1-¹³C] dehydroascorbic acid (HP DHA), which is reduced to Vitamin C (VitC) rapidly in the normal liver. In MCD mice, we observed a significant decrease in HP DHA to VitC conversion that accompanied hepatic fat deposition. When these animals were subsequently placed on a normal diet, resonance ratios reverted to those seen in control mice. These findings suggest that HP DHA, a potentially clinically translatable imaging agent, holds special promise in imaging NASH and other metabolic syndromes, to monitor disease progression and response to targeted therapies.