Supplementary Figure 2 from Hyperpolarized 13C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

Supplementary Methods, Figure Legends 1-4 from Hyperpolarized ¹³C Spectroscopic Imaging Informs on Hypoxia-Inducible Factor-1 and Myc Activity Downstream of Platelet-Derived Growth Factor Receptor

PurposeMagnetic resonance spectroscopy of hyperpolarized substrates allows for the observation of label exchange catalyzed by enzymes providing a powerful tool to investigate tissue metabolism and potentially kinetics in vivo. However, the accuracy of current methods to calculate kinetic parameters has been limited by T1 relaxation effects, extracellular signal contributions, and reduced precision at lower signal‐to‐noise ratio.Theory and MethodsTo address these challenges, we investigated a new modeling technique using metabolic activity decomposition‐stimulated echo acquisition mode. The metabolic activity decomposition‐stimulated echo acquisition mode technique separates exchanging from nonexchanging metabolites providing twice the information as conventional techniques.ResultsThis allowed for accurate measurements of rates of conversion and of multiple T1 values simultaneously using a single acquisition.ConclusionThe additional measurement of T1 values for the reaction metabolites provides further biological information about the cellular environment of the metabolites. The new technique was investigated through simulations and in vivo studies of transgenic mouse models of cancer demonstrating improved assessments of kinetic rate constants and new T1 relaxation value measurements for hyperpolarized 13C‐pyruvate, 13C‐lactate, and 13C‐alanine. Magn Reson Med 71:1–11, 2014. © 2013 Wiley Periodicals, Inc.

Purpose:To implement and evaluate combined parallel magnetic resonance imaging (MRI) and partial Fourier acquisition and reconstruction for rapid hyperpolarized carbon‐13 (13C) spectroscopic imaging. Short acquisition times mitigate hyperpolarized signal losses that occur due to T1 decay, metabolism, and radiofrequency (RF) saturation. Human applications additionally require rapid imaging to permit breath‐holding and to minimize the effects of physiologic motion.Materials and Methods:Numerical simulations were employed to validate and characterize the reconstruction. In vivo MR spectroscopic images were obtained from a rat following injection of hyperpolarized 13C pyruvate using an 8‐channel array of carbon‐tuned receive elements.Results:For small spectroscopic matrix sizes, combined parallel imaging and partial Fourier undersampling resulted primarily in decreased spatial resolution, with relatively less visible spatial aliasing. Parallel reconstruction qualitatively restored lost image detail, although some pixel spectra had persistent numerical error. With this technique, a 30 × 10 × 16 matrix of 4800 3D MR spectroscopy imaging voxels from a whole rat with isotropic 8 mm3 resolution was acquired within 11 seconds.Conclusion:Parallel MRI and partial Fourier acquisitions can provide the shorter imaging times and wider spatial coverage that will be necessary as hyperpolarized 13C techniques move toward human clinical applications. J. Magn. Reson. Imaging 2013;38:701–713. © 2013 Wiley Periodicals, Inc.

The development of hyperpolarized technology utilizing dynamic nuclear polarization (DNP) has enabled the rapid measurement of 13C metabolism in vivo with very high SNR. However, with traditional DNP equipment, consecutive injections of a hyperpolarized compound in an animal have been subject to a practical minimum time between injections governed by the polarization build-up time, which is on the order of an hour for [1-13C]pyruvate. This has precluded the monitoring of metabolic changes occurring on a faster time scale. In this study, we demonstrated the ability to acquire in vivo dynamic magnetic resonance spectroscopy (MRS) and 3D magnetic resonance spectroscopic imaging (MRSI) data in normal rats with a 5 min interval between injections of hyperpolarized [1-13C]pyruvate using a prototype, sub-Kelvin dynamic nuclear polarizer with the capability to simultaneously polarize up to 4 samples and dissolve them in rapid succession. There were minimal perturbations in the hyperpolarized spectra as a result of the multiple injections, suggesting that such an approach would not confound the investigation of metabolism occurring on this time scale. As an initial demonstration of the application of this technology and approach for monitoring rapid changes in metabolism as a result of a physiological intervention, we investigated the pharmacodynamics of the anti-cancer agent dichloroacetate (DCA), collecting hyperpolarized data before administration of DCA, 1 min after administration, and 6 min after administration. Dramatic increases in 13C-bicarbonate were detected just 1 min (as well as 6 min) after DCA administration.

Development of hyperpolarized technology utilizing dynamic nuclear polarization has enabled the measurement of 13C metabolism in vivo at very high signal-to-noise ratio (SNR). In vivo mitochondrial metabolism can, in principle, be monitored with pyruvate, which is catalyzed to acetyl-CoA via pyruvate dehydrogenase (PDH). The purpose of this work was to determine whether the compound sodium dichloroacetate (DCA) could aid the study of mitochondrial metabolism with hyperpolarized pyruvate. DCA stimulates PDH by inhibiting its inhibitor, pyruvate dehydrogenase kinase. In this work, hyperpolarized [1-13C]pyruvate and [2-13C]pyruvate were used to probe mitochondrial metabolism in normal rats. Increased conversion to bicarbonate (+181±69%, P=.025) was measured when [1-13C]pyruvate was injected after DCA administration, and increased glutamate (+74±23%, P=.004), acetoacetate (+504±281%, P=.009) and acetylcarnitine (+377±157%, P=.003) were detected when [2-13C]pyruvate was used.

Christine Leon, Peder Larson, Adam Kerr, Klaus Kruttwig, Cornelius Von Morze, Simon Hu, Robert Bok, Andrei Goga, John Pauly, Sarah Nelson, John Kurhanewicz, Daniel Vigneron Department of Radiology and Biomedical Imaging, University of California, San Francisco, UC Berkeley-UCSF Graduate Program in Bioengineering, Department of Electrical Engineering, Stanford University, and Department of Hematology and Oncology, University of California, San Francisco

High resolution compressed sensing hyperpolarized 13C magnetic resonance spectroscopic imaging was applied in orthotopic human glioblastoma xenografts for quantitative assessment of spatial variations in 13C metabolic profiles and comparison with histopathology. A new compressed sensing sampling design with a factor of 3.72 acceleration was implemented to enable a factor of 4 increase in spatial resolution. Compressed sensing 3D 13C magnetic resonance spectroscopic imaging data were acquired from a phantom and 10 tumor‐bearing rats following injection of hyperpolarized [1‐13C]‐pyruvate using a 3T scanner. The 13C metabolic profiles were compared with hematoxylin and eosin staining and carbonic anhydrase 9 staining. The high‐resolution compressed sensing 13C magnetic resonance spectroscopic imaging data enabled the differentiation of distinct 13C metabolite patterns within abnormal tissues with high specificity in similar scan times compared to the fully sampled method. The results from pathology confirmed the different characteristics of 13C metabolic profiles between viable, non‐necrotic, nonhypoxic tumor, and necrotic, hypoxic tissue. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.

Hyperpolarized 13 C MR spectroscopic imaging can detect not only the uptake of the pre‐polarized molecule but also its metabolic products in vivo, thus providing a powerful new method to study cellular metabolism. Imaging the dynamic perfusion and conversion of these metabolites provides additional tissue information but requires methods for efficient hyperpolarization usage and rapid acquisitions. In this work, we have developed a time‐resolved 3D MR spectroscopic imaging method for acquiring hyperpolarized 13 C data by combining compressed sensing methods for acceleration and multiband excitation pulses to efficiently use the magnetization. This method achieved a 2 sec temporal resolution with full volumetric coverage of a mouse, and metabolites were observed for up to 60 sec following injection of hyperpolarized [1‐ 13 C]‐pyruvate. The compressed sensing acquisition used random phase encode gradient blips to create a novel random undersampling pattern tailored to dynamic MR spectroscopic imaging with sampling incoherency in four (time, frequency, and two spatial) dimensions. The reconstruction was also tailored to dynamic MR spectroscopic imaging by applying a temporal wavelet sparsifying transform to exploit the inherent temporal sparsity. Customized multiband excitation pulses were designed with a lower flip angle for the [1‐ 13 C]‐pyruvate substrate given its higher concentration than its metabolic products ([1‐ 13 C]‐lactate and [1‐ 13 C]‐alanine), thus using less hyperpolarization per excitation. This approach has enabled the monitoring of perfusion and uptake of the pyruvate, and the conversion dynamics to lactate and alanine throughout a volume with high spatial and temporal resolution. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.

Results: Figure 1 shows the pre and post DCA spectra when using hyperpolarized [1-C]pyruvate as the substrate. The injected pyruvate (and pyruvate-hydrate) levels did not change. Bicarbonate increased dramatically, and lactate and alanine decreased, suggesting that DCA was effective in stimulating PDH. Figure 2 shows the pre and post DCA spectra when using hyperpolarized [2-C]pyruvate as the substrate. The peak assignments and their ppm values are given in the Figure 2 legend. Peaks 1, 3, and 4 were assigned by comparing the ppm values with peaks assigned from a prior [2-C]pyruvate study [5], and peak 2 was assigned based on a standard metabolome database and consideration of plausible pathways. Once again, pyruvate did not increase, suggesting that injection volumes were consistent between pre and post acquisitions. Glutamate, increased perhaps slightly, and acetoacetate and acetylcarnitine increased dramatically.

Hyperpolarized technology utilizing dynamic nuclear polarization has enabled rapid and high-sensitivity measurements of 13C metabolism in vivo. The most commonly used in vivo agent for hyperpolarized 13C metabolic imaging thus far has been [1-13C]pyruvate. In preclinical studies, not only is its uptake detected, but also its intracellular enzymatic conversion to metabolic products including [1-13C]lactate and [1-13C]alanine. However, the ratio of 13C-lactate/13C-pyruvate measured in this data does not accurately reflect cellular values since much of the [1-13C]pyruvate is extracellular depending on timing, vascular properties, and extracellular space and monocarboxylate transporter activity. In order to measure the relative levels of intracellular pyruvate and lactate, in this project we hyperpolarized [1-13C]alanine and monitored the in vivo conversion to [1-13C]pyruvate and then the subsequent conversion to [1-13C]lactate. The intracellular lactate-to-pyruvate ratio of normal rat tissue measured with hyperpolarized [1-13C]alanine was 4.89±0.61 (mean±S.E.) as opposed to a ratio of 0.41±0.03 when hyperpolarized [1-13C]pyruvate was injected.

Tumor cells have an altered metabolic phenotype characterized by increased glycolysis and diminished oxidative phosphorylation. Despite the suspected importance of glycolysis in tumorigenesis, the temporal relationship between oncogene signaling, in vivo tumor formation, and glycolytic pathway activity is poorly understood. Moreover, how glycolytic pathways are altered as tumors regress remains unknown. Here, we use a switchable model of Myc-driven liver cancer, along with hyperpolarized 13C-pyruvate magnetic resonance spectroscopic imaging (MRSI) to visualize glycolysis in de novo tumor formation and regression. LDHA abundance and activity in tumors is tightly correlated to in vivo pyruvate conversion to lactate and is rapidly inhibited as tumors begin to regress, as are numerous glycolysis pathway genes. Conversion of pyruvate to alanine predominates in precancerous tissues prior to observable morphologic or histological changes. These results demonstrate that metabolic changes precede tumor formation and regression and are directly linked to the activity of a single oncogene.

The metabolically inactive hyperpolarized agents HP001 (bis-1,1-(hydroxymethyl)-[1-13C]cyclopropane-d8) and urea enable a new type of perfusion magnetic resonance imaging based on a direct signal source that is background-free. The addition of perfusion information to metabolic information obtained by spectroscopic imaging of hyperpolarized [1-13C]pyruvate would be of great value in exploring the relationship between perfusion and metabolism in cancer. In preclinical normal murine and cancer model studies, we performed both dynamic multislice imaging of the specialized hyperpolarized perfusion compound HP001 (T1=95 s ex vivo, 32 s in vivo at 3 T) using a pulse sequence with balanced steady-state free precession and ramped flip angle over time for efficient utilization of the hyperpolarized magnetization and three-dimensional echo-planar spectroscopic imaging of urea copolarized with [1-13C]pyruvate, with compressed sensing for resolution enhancement. For the dynamic data, peak signal maps and blood flow maps derived from perfusion modeling were generated. The spatial heterogeneity of perfusion was increased 2.9-fold in tumor tissues (P=.05), and slower washout was observed in the dynamic data. The results of separate dynamic HP001 imaging and copolarized pyruvate/urea imaging were compared. A strong and significant correlation (R=0.73, P=.02) detected between the urea and HP001 data confirmed the value of copolarizing urea with pyruvate for simultaneous assessment of perfusion and metabolism.

Purpose: To demonstrate dynamic imaging of a diffusible perfusion tracer, hyperpolarized [C-13]urea, for regional measurement of blood flow in preclinical cancer models.Materials and Methods: A pulse sequence using balanced steady state free precession (bSSFP) was developed, with progressively increasing flip angles for efficient sampling of the hyperpolarized magnetization. This allowed temporal and volumetric imaging of the [C-13]urea signal. Regional signal dynamics were quantified for kidneys and liver, and estimates of relative blood flows were derived from the data. Detailed perfusion simulations were performed to validate the methodology.Results: Significant differences were observed in the signal patterns between normal and cancerous murine hepatic tissues. In particular, a 19% reduction in mean blood flow was observed in tumors, with 26% elevation in the tumor rim. The blood flow maps were also compared with metabolic imaging results with hyperpolarized [1-C-13]pyruvate.Conclusion: Regional assessment of perfusion is possible by imaging of hyperpolarized [C-13]urea, which is significant for the imaging of cancer.

A new method was developed for simultaneous spatial localization and spectral separation of multiple compounds based on a single echo, by designing the acquisition to place individual compounds in separate frequency encoding bands. This method was specially designed for rapid and robust metabolic imaging of hyperpolarized 13C substrates and their metabolic products, and was investigated in phantom studies and studies in normal mice and transgenic models of prostate cancer to provide rapid metabolic imaging of hyperpolarized [1-13C]pyruvate and its metabolic products [1-13C]lactate and [1-13C]alanine at spatial resolutions up to 3mm in-plane. Elevated pyruvate and lactate signals in the vicinity of prostatic tissues were observed in transgenic tumor mice. The multi-band frequency encoding technique enabled rapid metabolic imaging of hyperpolarized 13C compounds with important advantages over prior approaches, including less complicated acquisition and reconstruction methods.

We report metabolic images of 13C, following injection of a bolus of hyperpolarized [1-13C] pyruvate in a live rat. The data were acquired on a clinical scanner, using custom coils for volume transmission and array reception. Proton blocking of all carbon resonators enabled proton anatomic imaging with the system body coil, to allow for registration of anatomic and metabolic images, for which good correlation was achieved, with some anatomic features (kidney and heart) clearly visible in a carbon image, without reference to the corresponding proton image. Parallel imaging with sensitivity encoding was used to increase the spatial resolution in the SI direction of the rat. The signal to noise ratio in was in some instances unexpectedly high in the parallel images; variability of the polarization among different trials, plus partial volume effects, are noted as a possible cause of this.

MRSI using hyperpolarized (HP) C biomarkers can provide novel biochemical and physiological information and has been used to detect and characterize tumors in mouse models (1). For example, C1 pyruvate and its byproducts are commonly used biomarkers in murine studies. Due to the dynamic nature of the system, time course images need to be acquired with high temporal resolution to better understand the kinetics (2). The rapid loss of the HP signal due to spin relaxation (T1), RF pulse saturation, and tissue metabolism require special pulse sequences to acquire the magnetization rapidly and efficiently. In this project we developed single-shot imaging methods based on the echo-planar/fast-spin-echo hybrid (GRASE) imaging sequence (3) for acquiring 2D and 3D dynamic data at 14.1T. Using mouse models, images were acquired in about 154ms and provided excellent temporal and spatial resolution for our preliminary dynamic studies. Experimental Methods and Materials The experiments were done using a vertical, 14.1T Varian (Agilent) 600WB micro-imaging system equipped with 55mm 1000mT/m gradients and 40mm diameter proton and carbon RF coils. The mice were placed in a temperature controlled animal holder and anesthetized using a mixture of isoflurane/oxygen. An animal monitoring system (SA Instruments) was used to monitor respiration and trigger the scanner during all protocols. The proton coil was used for shimming and anatomical imaging and then the carbon coil was used for C imaging. C1 pyruvate was polarized using an Oxford HypersenseTM DNP instrument and 400ul of the resulting dissolution mixture containing 80mM pyruvate was administered via a jugular vein catheter. 3D images were acquired using the echo-planar based (GRASE) (3) pulse sequence shown in Figure 1, where the RF pulses are frequency selective for acquiring metabolite specific images during a time course study. For the 2D dynamic study, the 180 pulse was replaced with a slice selective pulse and the second phase encode gradients were omitted. The signals from the EPI trains were co-added to improve the SNR of the final image.

In order to compare in vivo metabolism between malignant gliomas and normal brain, 13C magnetic resonance (MR) spectroscopic imaging data were acquired from rats with human glioblastoma xenografts (U-251 MG and U-87 MG) and normal rats, following injection of hyperpolarized [1-13C]-pyruvate. The median signal-to-noise ratio (SNR) of lactate, pyruvate, and total observed carbon-13 resonances, as well as their relative ratios, were calculated from voxels containing Gadolinium-enhanced tissue in T1 postcontrast images for rats with tumors and from normal brain tissue for control rats. [1-13C]-labeled pyruvate and its metabolic product, [1-13C]-lactate, demonstrated significantly higher SNR in the tumor compared with normal brain tissue. Statistical tests showed significant differences in all parameters (P < .0004) between the malignant glioma tissue and normal brain. The SNR of lactate, pyruvate, and total carbon was observed to be different between the U-251 MG and U-87 MG models, which is consistent with inherent differences in the molecular characteristics of these tumors. These results suggest that hyperpolarized MR metabolic imaging may be valuable for assessing prognosis and monitoring response to therapy for patients with brain tumors.

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 [1-13C] 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) (46.7s and 47.7s respectively at 3T). Consistent with chemical shift anisotropy dominating the T1 relaxation of carbonyls, T1 values for 13C bicarbonate and [1-13C] pyruvate were even longer at 3T (49.7s and 67.3s, respectively). Co-polarized 13C bicarbonate and [1-13C] 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 [1-13C] 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 [1-13C] pyruvate or polarized [1-13C] pyruvate alone. The technique was extended to polarize four 13C labelled substrates potentially providing information on pH, metabolism, necrosis and perfusion, namely [1-13C]pyruvic acid, 13C sodium bicarbonate, [1,4-13C]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.

Abstract The recent development of hyperpolarized 13C magnetic resonance spectroscopic imaging provides a novel method for in vivo metabolic imaging with potential applications for detection of cancer and response to treatment. Chemotherapy-induced apoptosis was shown to decrease the flux of hyperpolarized 13C label from pyruvate to lactate due to depletion of NADH, the coenzyme of lactate dehydrogenase. In contrast, we show here that in PC-3MM2 tumors, inhibition of platelet-derived growth factor receptor with imatinib reduces the conversion of hyperpolarized pyruvate to lactate by lowering the expression of lactate dehydrogenase itself. This was accompanied by reduced expression of vascular endothelial growth factor and glutaminase, and is likely mediated by reduced expression of their transcriptional factors hypoxia-inducible factor-1 and c-Myc. Our results indicate that hyperpolarized 13C MRSI could potentially detect the molecular effect of various cell signaling inhibitors, thus providing a radiation-free method to predict tumor response. Cancer Res; 70(19); 7400–10. ©2010 AACR.