Towards hyperpolarized ¹³C-succinate imaging of brain cancer

Abstract

We describe a novel ¹³C enriched precursor molecule, sodium 1-¹³C acetylenedicarboxylate, which after hydrogenation by PASADE-NA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized ¹³C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized ¹³C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized ¹³C sodium succinate, contained significant concentrations of the injected substrate, ¹³C sodium succinate, together with ¹³C maleate and succinate metabolites 1-¹³C-glutamate, 5-¹³C-glutamate, 1-¹³C-glutamine and 5-¹³C-glutamine. The ¹³C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood–brain barrier. These ex vivo results indicate that hyperpolarized ¹³C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in ¹³C MR spectral-spatial images.

1. Introduction

The low signal to noise ratio (SNR) in ¹³C NMR spectroscopy is due to the fact that only a small percentage of the available ¹³C nuclei become polarized by the externally applied magnetic field B₀ and contribute to the NMR signal in addition to low gyromagnetic ratio. Analytical chemistry applications of NMR spectroscopy overcome this low SNR problem by using concentrated samples and signal averaging. The application of NMR spectroscopy and imaging to biological systems, however, has yet to reach its full potential because of the extremely long imaging and spectroscopy acquisition times that would be required to obtain high SNR under the biological constraints of low concentration, physiological temperature, and high dielectric losses [1,2]. Nowhere is this more relevant than in the brain, where neurochemical events occur on the spatial (nm–cm) and temporal (ms–s) scales of electrical neurotransmission [3].

It is well known that metabolic substrates are transported across the blood brain barrier before undergoing neuronal and glial metabolism (Fig. 1) [4,5]. Currently in vivo ¹³C MRS of human brain measures concentrations of important fuels and neurotransmitters between 1 and 10 mM and reaction rates of 1–5 μmol/min/g [4,5]. Two novel methods of hyperpolarization of the ¹³C nucleus, dynamic nuclear polarization (DNP) [6,7] and parahydrogen and synthesis allow dramatically enhanced nuclear alignment (PASADENA) [8–10] provide a ¹³C NMR signal enhancement in excess of 10,000-fold compared to Boltzmann polarization in strong magnetic field, and offer the potential for in vivo measurement of nanomolar quantities of metabolites and metabolic reaction rates in seconds. Several investigators have successfully hyperpolarized test reagents and imaged the resulting ¹³C signal in vivo [11–16]. Subsecond Magnetic Resonance Angiography (MRA) has been demonstrated using ¹³C reagents hyperpolarized using either PASADENA or DNP that remain in the vasculature [7,11–13,15,16]. When a hyperpolarized ¹³C reagent exits the vasculature and enters the cells it may be metabolized, while conserving hyperpolarization of the ¹³C nucleus, allowing the acquisition of ¹³C images and spectra of intra-cellular metabolites. This application has been demonstrated in vivo by NMR detection of the conversion of ¹³C-pyruvate to ¹³C-lactate, ¹³C-alanine, and ¹³C-bicarbonate within seconds following injection of hyperpolarized ¹³C-pyruvate at high concentration directly into mouse and dog tumors [7,13,16–18] and for skeletal and cardiac muscle of larger animals [13,16].

Fig. 1.

A representation of intra-cerebral metabolite cycling between neurons and glia believed to underlie glutamate neurotransmission. The diagram of experimental design is shown above. Twenty-five millimolar aqueous solution of ADC precursor is hydrogenated in the polarizer to yield 3 mL of maleate and succinate products, which is then injected in the carotid artery of 9L tumor bearing rat. We propose that the hydrogenated products reach the brain through an altered blood–brain barrier in tumor tissue and enter glial and neuronal TCA cycle to yield glutamine and glutamate as final product in vivo.

Although DNP ¹³C hyperpolarization techniques have shown utility for fast in vivo ¹³C NMR imaging and spectroscopy, these earlier experiments were performed at ¹³C reagent concentrations of 300 mM, considerably higher than physiological, presenting possibly problematic biochemical and osmotic stress [10–12,19]. The purpose of this work is to demonstrate the feasibility of using physiologically relevant concentrations of PASADENA hyperpolarized molecules [19] (¹³C-labelled succinate and maleate (Fig. 1), injected in vivo, as biomarkers of tumor metabolism. We observed hyperpolarized 1-¹³C-succinate in vivo and its subsequent conversion to ¹³C-glutamine and ¹³C-glutamate in 9L brain tumor examined ex vivo.

2. Methods

2.1. PASADENA hyperpolarization

The instrumentation and polarization transfer technique necessary for generating hyperpolarized ¹³C molecules is described in detail by Goldman et al. [20], Johannesson et al. [21], and Bhattacharya et al. [11]. Briefly, the parahydrogen gas is used in a chemical reaction (hydrogenation) to produce the PASADENA precursors. In order to preserve the spin correlation between the protons immediately after hydrogenation a rhodium catalyst [22,23] is used, which transfers the protons as a unit on to the precursor, without scrambling. The chemistry takes place during 4 s, at an elevated temperature (60 °C) in a reactor where a solution containing both precursor and catalyst (constituted in ultrapure water (Millipore Super-Q Plus System); final pH 7.8) is injected, into an atmosphere of 10 bar pressure of parahydrogen gas. The first step in PASADENA hyperpolarization is the molecular addition of dihydrogen, first to a catalyst which then passes the two correlated protons on to the unsaturated precursor of the target species, disodium acetylenedicarboxylate (ADC). The chemical goal is to achieve this reaction in a timescale which is small compared to spin lattice relaxation times. Relaxation losses in spin order can occur either as ¹H relaxation on the dihydride or relaxation of the protons or the target heteronucleus on the addition product. Longitudinal relaxation was minimized by proton irradiation in the reactor prior to polarization transfer, which traps the singlet state [24]. In order to break the symmetry to achieve PASADENA hyperpolarization on ADC, the ¹³C label is confined to only one carbon nucleus (C1) in this symmetric molecule. The carboxyl C1 label was chosen to maximize the T₁ relaxation times for the hyperpolarized species. Theoretical analysis [20] and experimental results in vitro obtained with a toxic molecule hydroxyethypropionate (HEP) show ¹³C polarization in the order of unity can be generated routinely [25]. The delays of the low field pulse sequence [20] were chosen to be optimal for the three-spin system of maleate.

2.2. ¹³C NMR spectroscopy of hyperpolarized samples

In vitro ¹³C MR spectroscopy studies to confirm the chemical identity of the hydrogenated product(s) of the 1-¹³C-ADC substrate as a result of the PASADENA hyperpolarization process were performed on a horizontal wide-bore 4.7 T MR system (Bruker Avance, Bruker, Germany). Data acquisition were started immediately after the hyperpolarized samples were placed at the center of a dual tuned ¹H/¹³C 40 mm ID solenoid coil centered in the magnet bore. Manual transfer of the hyperpolarized materials was employed from the polarizer to the spectrometer or animal. Approximately 10 s elapsed between the completion of PASADENA hyperpolarization and the start of data acquisition in the 4.7 T MR system and 10 s on a 1.5 T GE clinical MR scanner.

2.3. In vivo ¹³C-succinate imaging at 1.5 T

Normal rats (250–350 g wt.) were prepared for MR imaging by cannulating the right jugular vein with 120 cm long silastic tubing (0.08 mm diameter) while the rat was under anesthesia (intra-peritoneal Nembutal at 0.1 mL/100 g body weight). The procedures used on rats in this work were approved by the HMRI animal subjects committee.

MR imaging to determine the distribution of hyperpolarized ¹³C-succinate in these normal rats was performed on a 1.5 T MR scanner (General Electric Medical Systems, Signa EchoSpeed, ESE 9.1 software, Milwaukee, WI.) The rats were sedated with intraperitoneal Nembutal (or ketamine-xylazine) as described above, placed supine on a dual tuned 12.5 cm diameter ¹H-¹³C surface coil, and positioned at the center of the magnet. The manufacturer’s three-dimensional FIESTA pulse sequence was modified to allow multi-nuclear 3D ¹³C FIESTA imaging (MN-3DFIESTA) [11]. Fast MN-3DFIESTA imaging was performed before, during and after injection of ¹³C-succinate hyperpolarized using the PASADENA technique. The MN-3DFIESTA images were acquired with TR/TE of 6.3/3.1 ms, a bandwidth of ±62.5 kHz, sixteen 5 mm slices, 44 phase encodings over 220 mm field-of-view (FOV) and 64 readout points over 320 mm FOV to yield an isotropic spatial resolution of 5 × 5 × 5 mm³. A 4.4 M ¹³C-acetate sphere was placed near the brain of the rat as a reference for the determination of the degree of hyperpolarization in vivo.

2.4. In vivo ¹³C-succinate distribution in tumor and normal brain tissue

Brain tumors were induced in Wistar rats (approximately 250 mg) by the injection of 9L tumor cells directly into the frontal hemisphere of the brain. The rats were allowed to recover from the procedure and were imaged when tumors reached >5 mm diameter (between 10 and 15 days following tumor cell injection).

The rats were anaesthetized with ketamine-xylazine, and approximately 3 mL of hydrogenated ¹³C-labeled product (pH 7.8) in ultrapure water (Millipore Super-Q Plus System) was injected directly into the internal carotid artery. Following a 1 h delay to allow metabolism, the animal was sacrificed and the brain and tumor tissues were harvested and frozen in liquid nitrogen. Solid samples of brain and of brain tumor were prepared for subsequent NMR examination.

2.5. Ex vivo ¹³C NMR spectroscopy of brain tissues

¹³C spectroscopy of ex vivo samples was performed with Bruker Avance data acquisition system in 11.7 T wide bore magnet, equipped with Bruker H/X/Y VT triple resonance 4 mm magic angle spinning (MAS) probe tuned to ¹³C resonant frequency using the X channel. Normal brain and brain tumor tissues were thawed shortly before the experiment and packed in 4 mm zirconia Bruker MAS rotors. ¹³C MAS spectra were acquired under slow spinning conditions, low power ¹H decoupling and 4 °C utilizing spin echo pulse sequence and 1024 transients.

3. Results

3.1. Hydrogenation of acetylene dicarboxylate and hyperpolarization of 1-¹³C-succinate

We chose disodium 1-¹³C-acetylenedicarboxylate (1-¹³C-ADC) [26] as a starting reagent for creating hyperpolarized sodium succinate using the PASADENA technique since hydrogenation of the 1-¹³C-ADC triple bond should result in 1-¹³C-maleate and 1-¹³C-succinate. The ¹³C NMR spectrum of the PASADENA reaction product of 1-¹³C-ADC (Fig. 2a) clearly shows hyperpolarized 1-¹³C-succinate resonating at ~175 ppm. A sphere containing 2.8 mL of 4.4 M 1-¹³C-acetate solution with a resonance at ~182 ppm is included in the spectrum for reference. The NMR spectrum of the same PASADENA reaction solution acquired after decay of the hyperpolarized spins is shown in Fig. 2b. The expected 1-¹³C-ADC hydrogenation products of 1-¹³C-maleate and 1-¹³C-succinate are both visible in this non-hyperpolarized spectrum acquired with 128 transients Fig. 2b.

Fig. 2.

(a) ¹³C NMR spectrum of hyperpolarized 1-¹³C-succinate from ADC precursor acquired at 4.7 T Bruker horizontal wide-bore MR system. A sphere containing 2.8 mL of 4.4 M 1-¹³C-acetate solution is used as a reference at ~182 ppm. A single transient ¹³C NMR spectrum revealed ~4400-fold signal enhancement with respect to Boltzmann polarization. (b) ¹³C spectrum of the post-hyperpolarized reaction mixture at Boltzmann polarization, 128 transients.

3.2. Distribution of injected 1-¹³C-succinate in rat

In vitro studies confirm that 1-¹³C-ADC is first hydrogenated to 1-¹³C-maleate and then further converted to 1-¹³C-succinate in a second hydrogenation step (Fig. 1). When the resulting mixture was injected in carotid artery in normal rats, subsecond ¹³C images indicate its delivery to vasculature and the head, but with insufficient spatial resolution at 1.5 T to distinguish brain tumor (Fig. 3) [11,27].

Fig. 3.

In vivo¹³C 3D FIESTA imaging of rat brain with PASADENA-hyperpolarized succinate. Sub-second ¹³C images (0.3 s) were acquired using a multi-nuclear 3D fast imaging sequence employing balanced steady-state acquisition (3D FIESTA). To allow for the short lived polarization, the 3D FIESTA ¹³C data were acquired with a TR/TE of 6.3/3.1 ms, sixteen 5 mm slices, 44 phase encodings over 220 mm field-of-view (FOV) and 64 readout points over 320 mm FOV to yield an isotropic spatial resolution of 5 × 5 × 5 mm³. The image shown represents one slice of ¹³C data acquired 9 s after infusion. It is overlaid on a coronal 3D fast gradient echo proton image with matching FOV and slice location acquired prior to infusion to provide anatomical correlation. All data were acquired on a 1.5 T (GE LX 9.1, Waukesha, WI) MR scanner with a dual-tuned ¹H/¹³C custom saddle coil. The rat was anesthetized and cannulated in the carotid artery where upon 1.5 mL of 25 mM of hyperpolarized succinate was injected. For reference, a 4.4 M acetate phantom was placed next to the rat, demonstrating significant signal enhancement of hyperpolarized succinate in vivo given the difference in concentration (4.4 M vs. 0.025 M) and signal intensity.

3.3. Metabolism of injected 1-¹³C-succinate in rat brain

When injected via internal carotid artery into 9L tumor bearing rat, followed 1 h later by harvesting of the brain and ex vivo ¹³C NMR spectroscopy, no measurable ¹³C enriched species were observed in normal brain tissues. In contrast, 9L brain tumor tissues collected from the same rat, showed high concentrations of glutamine and glutamate enriched in C1 and C5 positions, and 1-¹³C-maleate, in addition to the residual 1-¹³C-succinate (Fig. 4).

Fig. 4.

Ex vivo MAS ¹³C spectra of brain (lower) and brain tumor tissues (upper). 80 mg of tissue was used in each experiment. ¹³C MAS spectra were acquired under slow spinning, 1–2 kHz conditions, low power ¹H decoupling and 4 °C utilizing spin echo pulse sequence with t_90° = 4.0 μs, 1024 transients and recycling delay of 5 s. Representative in vivo RARE image of 9L tumor bearing brain is shown. ¹³C precursors, succinate and maleate, as well as putative products of tumor metabolism, glutamine (Gln) and glutamate (Glu) are assigned based on model solutions. Note the absence of ¹³C enrichment in normal brain tissue from the same animal.

4. Discussion and conclusion

The in vitro experiment shown in Fig. 2 demonstrates that ADC is a viable precursor molecule for the production of hyperpolarized 1-¹³C-succinate using the PASADENA hyperpolarization process. The absence of a hyperpolarized 1-¹³C-maleate peak in the spectrum shown in Fig. 2a indicates that the catalyzed hydrogenation process has proceeded past the initial hydrogenation of the triple bond which yields 1-¹³C-maleate, to the hydrogenation of the resulting double bond which yields 1-¹³C-succinate. Further kinetic studies and calculations are needed to establish the concentrations of maleate and succinate at the time of the polarization transfer and the optimal polarization sequence for this mixture.

The significant amount of ¹³C-labeled succinate and maleate seen in the ¹³C NMR spectrum of the tumor tissue in Fig. 4 1 h following injection of the hyperpolarized substrates indicates that 1-¹³C-succinate and 1-¹³C-maleate accumulate in the tumor tissue when they are not detectable in normal brain tissue. The increased concentration of 1-¹³C-maleate, which is not a metabolic substrate for glial or neuronal metabolism, in the brain tumor spectrum is most likely due to increased accumulation due to a compromised blood–brain barrier. On the other hand, the increased concentration of 1-¹³C-succinate in the brain tumor may be due to both increased metabolic demand and its accumulation due to the compromised blood–brain barrier. The presence of ¹³C-labeled glutamate and glutamine, metabolic products of 1-¹³C-succinate may act as specific tumor biomarkers. Recent studies link abnormalities in tumor TCA-cycle enzymes succinic dehydrogenase and fumarate hydratase to known oncogenes [28–30]. Sub-second ¹³C imaging and spectroscopy performed after administration of hyperpolarized 1-¹³C-succinate may be uniquely sensitive for the in vivo detection of tumors.

These experiments also indicate that it should be possible to perform in vivo dynamic MR imaging and spectroscopy of tumor metabolism following injection of millimolar concentrations of hyperpolarized 1-¹³C-succinate, correctly determining in vivo metabolic flux of the hyperpolarized tracer administered in low (physiologic) concentrations and pH buffered solutions. The PASADE-NA method can produce hyperpolarized product every 2 min [11] which allows repeated in vivo studies of metabolism in the same animal. Furthermore, the relatively long T₁ of the carbonyl carbons and the degradation of the blood–brain barrier in tumors suggest that these species may discriminate between normal brain and metabolically active brain tumor tissues on the time scale over which hyperpolarization is maintained. We are currently investigating the ability of fast spectral-spatial imaging of hyperpolarized 1-¹³C-succinate to capture this information from brain tumors in vivo. In the current chemical design of the hydrogenation experiment, T₁ of 1-¹³C-succinate is only 6 s. Moreover, the hydrogenation reaction of 1-¹³C-ADC is incompletely controlled, resulting in production of both maleate and succinate species in solution. Because maleate is toxic at concentrations above 10 mM [31], we would like to avoid this intermediate. Accordingly, alternative reagents with double bonds for controlled hydrogenation and proper isotope enrichment are being designed to alleviate the above limitations and permit routine production of hyperpolarized succinate.

The ability to increase the NMR signal of ¹³C-labeled biologically relevant metabolic substrates by several orders of magnitude in the PASADENA hyperpolarization process promises to provide new methods for measuring and understanding tumor metabolism in vivo.