CommunicationHigh-throughput hyperpolarized 13C metabolic investigations using a multi-channel acquisition system
Graphical abstract
Introduction
The relatively high sensitivity and natural abundance of 1H nuclei in the body permit noninvasive morphological and functional investigation of disease with magnetic resonance (MR) imaging (MRI) and spectroscopy (MRS). The excellent chemical specificity of MR further enables assessment of certain biochemical characteristics of tissue. It is well-known, for example, that many malignancies undergo higher levels of glycolysis and lactic acid fermentation despite normal tissue oxygenation, a condition that is often referred to as aerobic glycolysis or the Warburg effect [1], [2]. Therefore, dynamic changes in pyruvate and lactate levels in vivo could serve as useful biomarkers of cancer metabolism. The unique 13C spectroscopic signatures of pyruvate and lactate could permit their non-invasive observation through MRS, MRI, and MR spectroscopic imaging (MRSI), though such measurements have low sensitivity due to low natural abundance (∼1%) and a gyromagnetic ratio that is one-quarter that of 1H. Low sensitivity necessitates prohibitively long scan times for signal averaging, rendering the use of 13C signal from endogenous metabolites impractical for real-time measurement of metabolism in vivo [3].
To overcome sensitivity limitations, methods that create a hyperpolarized (HP) nuclear spin population may be used to dramatically enhance the signal of exogenous substrates. One such method, known as dissolution dynamic nuclear polarization (DNP), involves combining an enriched substrate with a polarizing radical, and transferring polarization from unpaired electrons to nuclear spins through microwave radiation at very low temperatures [4], [5]. The HP sample is rapidly dissolved with a heated buffer solution and quickly transferred to the MR scanner, where it may be used to perform experiments with greater than 10,000-fold signal enhancement [5]. Preliminary studies involving HP [1-13C]-pyruvate include very promising investigations of real-time cancer metabolism in cells [6], [7], cancer diagnosis and staging [8], [9], therapeutic response [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], and detection of cardiac metabolism [20], [21].
Although HP 13C MR shows great potential for in vitro and both preclinical and clinical studies in vivo, it is not without drawbacks. In addition to the technical challenges for HP data acquisition, which are confounded by T1 relaxation and nonrenewable signal loss due to radiofrequency (RF) excitation, several practical limitations associated with cost, time, and efficiency may delay the widespread preclinical use of HP MRI. First, DNP requires significant build-up time to reach the desired polarization: ∼45 min to reach 90% of the maximum polarization level for [1-13C] pyruvate, limiting the number of observations that can be made in a given interval of time. Second, costs associated with the radical and the 13C-enriched substrate can be significant. Third, the DNP process proceeds most efficiently at very low temperature ∼1.4 K, requiring the use of cryogens that must often be replenished. System maintenance alone represents a large fraction of the overall cost. In addition, only on the order of 5% of the total dissolution volume may be used for in vivo studies in mice, and a smaller fraction may be used in vitro to maintain a physiologically relevant concentration in cell studies, resulting in substantial waste [22]. More efficient use of HP 13C agents would reduce cost and facilitate integration of this technology into routine biomedical research.
Although the vast majority of preclinical MR systems are equipped with no more than one channel which may be used for detection of 13C signals, a handful of clinical scanners support multi-channel HP 13C acquisition. These receivers, in combination with RF coil arrays, have permitted improved sensitivity [23], [24], increased spatial coverage [25], and a means to perform partially parallel imaging [26].
To improve the efficiency of ex vivo, in vitro, and in vivo preclinical experiments involving HP 13C, we have implemented a low-cost, multi-channel, broadband RF receiver with various array coils for performing multiple measurements in parallel. Signal-to-noise ratio (SNR) calculations, based on data acquired from an enriched 13C urea phantom at thermal equilibrium, were used to evaluate consistency among channels and to compare performance against standard scanner hardware. The capability to simultaneously capture spectral dynamics from four distinct volumes was established using a 4-channel RF coil array and phantoms that contained varying concentrations of enzyme infused with HP [1-13C] pyruvate. High-throughput in vitro experiments, involving human thyroid cancer cells, were then performed with a dual Helmholtz coil to demonstrate the feasibility for improving measurement efficiency while preserving experimental and environmental consistency. Finally, two surface coils were placed over distinct subcutaneous tumors on one mouse, to demonstrate simultaneous in vivo measurement of metabolism from separate and distinct anatomic sites. This establishes a platform where paired examination of experimental therapies may be readily and rapidly performed, with reduced deviation due to biological or experimental variations.
Section snippets
Multi-channel receiver
The home-built receiver system includes an RF module, a power supply module, an analog-to-digital conversion (ADC) acquisition board, and a Linux workstation. The RF module contains four RF channel cards, each supporting four independent receive channels, and a local oscillation (LO) signal distribution card. The LO signal is derived from the MRI console or alternatively provided by an external signal generator, and it provides a reference for down-converting the input RF signal to the baseband
SNR performance
Measurements from the 2 mL, 8 M 13C urea phantom at thermal equilibrium reveal that comparable SNR values are observed through the multi-channel receiver extension and the Biospec receiver. The paired SNR results are listed in Table 1, Table 2. The slightly higher average SNR in the multi-channel receiver (1.50 × 104 vs. 1.13 × 104) may be explained by imbalance in the splitter, differences in cable length and routing, and differences in gain and digital signal processing algorithms in the two
Discussion
In this work, we have demonstrated the versatility of simultaneous multi-sample HP 13C MR experiments made feasible by a broad-band multi-channel receiver extension and multinuclear array coils. Various responses to HP [1-13C] pyruvate were demonstrated in controlled enzyme phantoms ex vivo, cancer cells in vitro, and anaplastic thyroid tumors in vivo. The signal quality achieved with the multi-channel receiver closely matches that achieved with standard commercially available hardware. The
Acknowledgments
We thank Charles Kingsley, Jorge de la Cerda, Keith Michel, and Kiersten Maldonado for their support in preparing in vivo experiments at the Small Animal Imaging Facility. This work was supported in part by the National Institutes of Health (P30-CA016672, R21-CA178450) and the Cancer Prevention and Research Institute of Texas (RP101243-P5; RP140021-P5; RP101502; RP140106; RP140113). Funding for C.W. was provided by a Julia Jones Matthews Cancer Research Scholar training award; for M.R. as an
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