13C magnetic resonance spectroscopy measurements with hyperpolarized [1‐13C] pyruvate can be used to detect the expression of transgenic pyruvate decarboxylase activity in vivo

Purpose Dissolution dynamic nuclear polarization can increase the sensitivity of the 13C magnetic resonance spectroscopy experiment by at least four orders of magnitude and offers a novel approach to the development of MRI gene reporters based on enzymes that metabolize 13C‐labeled tracers. We describe here a gene reporter based on the enzyme pyruvate decarboxylase (EC 4.1.1.1), which catalyzes the decarboxylation of pyruvate to produce acetaldehyde and carbon dioxide. Methods Pyruvate decarboxylase from Zymomonas mobilis (zmPDC) and a mutant that lacked enzyme activity were expressed using an inducible promoter in human embryonic kidney (HEK293T) cells. Enzyme activity was measured in the cells and in xenografts derived from the cells using 13C MRS measurements of the conversion of hyperpolarized [1‐13C] pyruvate to H13 CO3–. Results Induction of zmPDC expression in the cells and in the xenografts derived from them resulted in an approximately two‐fold increase in the H13 CO3–/[1‐13C] pyruvate signal ratio following intravenous injection of hyperpolarized [1‐13C] pyruvate. Conclusion We have demonstrated the feasibility of using zmPDC as an in vivo reporter gene for use with hyperpolarized 13C MRS. Magn Reson Med 76:391–401, 2016. © 2015 The Authors. Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.


INTRODUCTION
Reporter genes provide a means of visualizing gene expression in living organisms. The introduction of fluorescence and bioluminescence reporter genes has revolutionized molecular cell biology, to a large extent due to the relative simplicity and speed of these imaging modalities. However, despite good sensitivity, the use of these techniques is restricted in vivo due to poor depth penetration, which limits their use in superficial tissues. Reporter genes developed to work with positron emission tomography have very good depth penetration and sensitivity but have limited spatial resolution and may require an on-site cyclotron for probe synthesis (1), whereas MR reporters can have good spatiotemporal resolution and depth penetration but may be limited by a lack of sensitivity. Several systems have been developed that use T 1 and T 2 (*) MRI contrast agents [reviewed in Lyons et al. (1) and Patrick et al. (2)]. However, these can suffer from background signal, as water proton relaxation rates may change independently of reporter gene expression, for example due to hemorrhage, changes in vascularity, or the development of air cavities and calcifications (3). Furthermore, phagocytosis by infiltrating macrophages of dead cells that contain contrast agents can give rise to persistent nonspecific contrast (3). In addition, high magnetic fields may be required to achieve sufficient contrast with some agents that modulate T 2 /T 2 * relaxation rates (4).
The first enzyme-based magnetic resonance spectroscopy (MRS) gene reporter was creatine kinase, which was expressed in Escherichia coli (5), yeast (6), and murine liver (7), where the activity of the enzyme was detected by 31 P MRS. This approach exploited the fact that creatine kinase is not expressed endogenously in these systems; however, the low sensitivity of 31 P MRS limited the temporal, and in the liver, spatial resolution.
There are several examples of 19 F-based gene reporters, where the reporter is an enzyme that catalyzes transformation of a fluorinated substrate. 19 F MR-based reporters have the advantage that there is effectively no background signal in biological systems, the nucleus has a broad chemical shift range and it is relatively sensitive to MRS detection. The possibility of noninvasive monitoring of therapeutic gene expression in vivo using 19 F MRS was demonstrated in xenografts stably expressing carboxypeptidase G2, a bacterial enzyme that mediates the release of DNA alkylating agents by enzymatic cleavage of labile pro-drugs (8). The conversion of 3,5-difluorobenzoyl-L-glutamic acid to 3,5-difluorobenzoic acid was measured using 19 F MRS and thus provided a readout of the activity of this therapeutic transgene within the target tissue (8). Another 19 F MRS-based reporter exploited the enzyme b-galactosidase, encoded by the lacZ gene, which has been widely used as an optical reporter. b-Galactosidase catalyzes the cleavage of 2-fluoro-4-nitrophenyl b-D-galactopyranoside (OFPNPG) to aglycone 2-fluoro-4-nitrophenol, which results in the appearance of a new resonance in the 19 F spectrum (9). The expression of lacZ in human PC-3 prostate carcinoma cells grown as xenografts in mice was detected by 19 F MRS after intratumoral injection of OFPNPG (9).
The introduction of dissolution dynamic nuclear polarization, a method that can increase the sensitivity of 13 C MRS by at least four orders of magnitude, has allowed imaging of some 13 C labeled substrates and the products of their metabolism in real time in vivo (10). Reporter genes encoding enzymes that process hyperpolarized 13 C-labeled tracers would give rise to signal, which like 19 F has no background and could be imaged within a few minutes. The short half-life of the hyperpolarization and rapid clearance of small molecule metabolites means that reporter gene expression could be reinterrogated very rapidly. This is in contrast to reporters that use T 1 or T 2 /T 2 * contrast agents, which may require several hours or days to clear (2,11), which limits the temporal resolution of longitudinal assessment of gene expression using T 1 or T 2 /T 2 *-weighted imaging.
The enzyme-based reporter genes that have been developed for use with hyperpolarized 13 C-labeled cell substrates are summarized in Table 1. However, a number of factors have prevented their successful implementation in vivo, including insufficient levels of polarization, a short T 1 relaxation time of the hyperpolarized 13 C label (12), low specific activity of the enzyme with the labeled substrate (13), and insufficient levels of enzyme activity in the expressing cells (12,14).
We demonstrate here the use of PDC, from the bacterium Zymomonas mobilis, as an MR gene reporter in HEK293T human embryonic kidney cells (15), and in implanted xenografts that were derived from these cells. The PDC-catalyzed decarboxylation of pyruvate to give acetaldehyde and carbon dioxide is a reaction that is not present in mammalian tissues and can be monitored using 13 C MRS measurements of 13 C bicarbonate production following injection of hyperpolarized [1-13 C] pyruvate (Fig. 1a). Labeled bicarbonate is produced by spontaneous and carbonic anhydrase-catalyzed exchange of the hyperpolarized 13 C label between 13 CO 2 and the intracellular bicarbonate pool (16).

METHODS
All chemicals were purchased from Sigma Aldrich (Gillingham, Dorset, UK), unless stated otherwise.

Analysis of Cell Growth
Twenty-five thousand cells were seeded per well in 48-well plates (NUNC; Thermo Fisher Scientific, Waltham, Massachusetts, USA), with six replicates per experimental condition. Eight hours after seeding, an appropriate volume of a 1 mg/mL solution of doxycycline in sterile water, or water alone, was added to the media. Confluence of the monolayer was measured using an IncuCyte instrument (Essen Instruments, Essen, Michigan, USA), which was then used to calculate the cell doubling time using Prism 6.0 (GraphPad Software, Inc., La Jolla, California, USA) .

Time Course of Doxycycline-Induced PDC Expression
The growth medium was refreshed 24 h after cell seeding in T25 tissue culture flasks. For dose-response experiments, doxycycline was added to the media at the indicated concentrations, and the cells were harvested 48 h later. For time course experiments, the medium was supplemented with 2 mg/mL of doxycycline at 24, 48, or 96 h before cell harvesting.

Cell Extraction and Western Blotting
Cells were lysed in passive lysis 5Â buffer (Promega, Madison, Wisconsin, USA), diluted in water, and contained complete ethylenediaminetetraacetic acid-free protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland) as recommended by the manufacturer. Protein concentration was measured using a Bradford protein assay and a Sunrise 96-well plate reader (Tecan Group Ltd, M€ annedorf, Switzerland). The protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto a polyvinylidene difluoride membrane, and probed for expression of the V5 epitope and a-actin (see Supporting Information Section S3).

C T 1 Measurements In Vitro
Measurements were made using a 11.7 T Bruker Avance II þ 500 MHz spectrometer and an inversion recovery pulse sequence in samples containing the molecule of interest, 10% D 2 O and 4% bovine serum albumin with a pH between 7.4 and 7.6. Table 2 shows the sample composition and the delays in the inversion recovery sequence.

C MRS Measurements with Hyperpolarized C-Labeled Pyruvate In Vitro
Cells (5 Â 10 7 ) in 2 mL of pyruvate-, and FBS-free DMEM in a 10 mm NMR tube were placed in a vertical bore 9.4T NMR spectrometer with a broadband probe tuned to 13 C (Varian NMR Instruments, Palo Alto, California, USA). Typically 9-12 s after dissolution, 2 mL of the dissolved polarized sample containing 75 mM 13 Clabeled pyruvate were injected into the tube, and 180 spectra with a flip angle of 12 were acquired (data points ¼ 5440, acquisition time ¼ 0.170 s, repetition time [TR] ¼ 0.25 s, number of transients ¼ 4, sweep width ¼ 16 kHz) at 37 C.

H MRS Measurements with [U-2 H 3 ] Pyruvate In Vitro
Sodium pyruvate was deuterated as described previously (19). Approximately 5.0 Â 10 7 cells were suspended in pyruvate-free DMEM. Each cell suspension was split into two equal volumes, one of which was pelleted and frozen in liquid nitrogen. The remaining sample was resuspended in 0.54 mL of pyruvate-free DMEM (containing 5 mM 2,2,3,3-D4 sodium-3-trimethylsilylpropionate and 12.5% D 2 O) in a 5-mm-diameter NMR tube and placed in a vertical bore 11.7 T Bruker Avance II þ 500 MHz spectrometer. Two or three control spectra were acquired before 60 mL of a 0.25 M solution of sodium [U-2 H 3 ] pyruvate in H 2 O was added to the cell suspension and mixed. After the addition of 100 mL of mineral oil, the sample was returned to the spectrometer and, after thermal equilibration, a series of 16 1 H spectra were collected using a pulse and acquire sequence with 90 flip angle pulses (data points ¼ 16384, The cell pellets were lysed in 0.45 mL of ice-cold lysis buffer, essentially as described above, except that the lysis buffer was diluted in 200 mM citrate buffer (pH 6.0). A 480-mL sample of the lysate was then mixed with 60-mL of a solution of 10 mM thiamine pyrophosphate and 10 mM MgCl 2 in 2 H 2 0 and used for 1 H MRS measurements.

Animal Experiments
All animal experiments were performed in compliance with a project license issued under the Animals (Scientific Procedures) Act of 1986 and were designed with reference to the UK Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia (20). Protocols were approved by the Cancer Research UK, Cambridge Institute Animal Welfare and Ethical Review Body. Six to eight week-old female severe combined immune-deficient (SCID) mice (Charles River Ltd, Margate, UK) were used in each experimental group.

Xenograft Implantation and Induction of PDC Expression In Vivo
Each animal was injected subcutaneously in the left flank with 0.1 mL of a cell suspension containing 2.5 Â 10 7 cells stably transfected with the tet-on PDC-GFP-V5/ His plasmid expressing the wild-type or mutant enzymes. Xenograft diameters were measured using calipers, and the length (L) and width (W) were used to calculate their volumes (V) according to the formula The xenografts typically reached a volume of approximately 0.5-0.7 cm 3 within 21-25 days from implantation. To induce PDC expression, a solution of 10 mg mL À1 doxycycline with 5% w/v sucrose was given to the mice in their drinking water ad libitum for 96 h prior to imaging experiments.

C MRS Measurements In Vivo
Animals were anesthetized and monitored as described previously (22). A 20-mm-diameter surface coil (Rapid Biomedical GmbH, Rimpar, Germany) was placed over the xenograft, and the entire assembly was placed in a 13 C/ 1 H volume coil (Rapid Biomedical) in a 7T horizontal bore magnet (Varian). Xenografts were localized in transverse 1 H images acquired using a spin-echo pulse sequence (TR ¼ 1.5 s; echo time ¼ 10 ms; field of view ¼ 40 Â 40 mm; data matrix ¼ 128 Â 128; slice thickness ¼ 2 mm; number of slices ¼ 15). Immediately after dissolution, 10 mL/g body weight of the hyperpolarized pyruvate solution (typically 240 mL in total) was injected via the tail vein over a period of 2 s. Spectroscopic acquisitions were started 30 s from the injection. Single transient spectra (128 spectra with TR ¼ 500 ms, nominal flip angle ¼ 20 , sweep width ¼ 6 kHz; the delay from the middle of the excitation pulse to the beginning of data acquisition was 900 ms) from an 8-mm slice through the tumor were acquired. The center frequencies for the spectra were selected to be 175 ppm and 200 ppm for experiments with [1-13 C] pyruvate and [2-13 C] pyruvate, respectively. For experiments with [2-13 C] pyruvate, the protons were decoupled using a WALTZ decoupling sequence during acquisition (B1 $1.65 kHz), centered at 9.7 ppm.

Tissue Extraction
Freeze-clamped xenograft tissues were homogenized using a manual Potter S homogenizer (Sartorius, Epsom, UK) in 4 volumes (milliliters per gram wet weight of tissue) of ice-cold lysis buffer. Homogenates were centrifuged at 16,200 g and 4 C in an Eppendorf 5415D bench top centrifuge (Eppendorf, Hamburg, Germany) to remove debris.

Immunohistochemistry
Immunohistochemical protocols and image analysis were performed using instruments, reagents, and software from Leica (Leica Microsystems, Milton Keynes, UK) unless stated otherwise. Xenografts were excised and fixed in 4% neutral buffered formalin solution for 24 h and then transferred to 70% ethanol. After paraffin embedding, tissue sections were taken from the center of the xenograft and stained for cleaved caspase 3 (CC3), V5 epitope, and GFP expression. Hematoxylin and eosin (H&E) staining was performed to assess tissue viability (see Supporting Information Section S5).

Image Analysis
Slides were digitized using a Leica Aperio XT120 and analyzed using Aperio ImageScope software. For analysis of H&E staining, the area covered by nonviable xenograft tissue (as judged by the loss of structural coherence and visible chromatin condensation) was identified manually and compared with the total xenograft area, and the result was converted to percentage cell death. The Aperio ImageScope Positive PixelCount v9 macro, tuned to identify negatively and positively 3,3'-diaminobenzidine-stained tissue, was used for quantification of CC3 staining, which is reported as the number of positive pixels as a percentage of the total number of pixels.

Statistical Analysis
The results are presented as the mean 6 standard deviation unless stated otherwise. Statistical significance was assessed using Microsoft Excel with a two-tailed t test at the 95% confidence level. 394 Dzien et al.  (Table 2). Although these T 1 values were measured at 11.7T, we expect them to be shorter (because of the effect of chemical shift anisotropy) than those measured at 9.4 T and 7 T, which were the magnetic field strengths used for the cell and xenograft experiments, respectively. Expression of PDC in HEK293T cells transfected with the zmPDC-GFP-V5/His transgene was detectable on western blots by 24 h after addition of 2 mg/mL of doxycycline to the growth medium and showed further increases at 48 and 96 h. Expression was dose dependent, showing an increase between 0.5 and 2.0 mg/mL doxycycline and was undetectable in the absence of the drug (Fig. 2a). The H113Q mutant, which had no detectable PDC activity [data not shown and Huang et al. (17)], showed greater doxycycline-induced expression than the wild-type enzyme (Fig. 2b).

RESULTS
The cell doubling times of wild-type HEK293T cells, cells transfected with zmPDC-GFP-V5/His, and cells transfected with zmPDC/H113Q-GFP-V5/His increased after the addition of doxycycline to the growth medium in a dose-dependent manner. At 4 mg/mL doxycycline, the doubling times of cells expressing the wild-type and mutant enzymes were 129.4 6 7.6% and 146.8 6 35.6%, respectively, of their doubling times in the absence of doxycycline (Fig. 2c).
PDC activity in cells that had been transfected with zmPDC-GFP-V5/His and grown in the presence of 2 mg/ mL doxycycline for 48 h was first assessed using 1 H MRS measurements of [2,2,2-2 H 3 ] acetaldehyde production following the addition of [U-2 H 3 ] pyruvate (Fig. 3a). Lysed cells showed a 5.4-fold higher acetaldehyde production rate compared with intact cells (5.6 Â 10 À4 6 0.8 Â 10 À4 U/10 7 cells and 1.1 Â 10 À4 6 0.2 Â 10 À4 U/10 7 cells [SD, n ¼ 3], respectively), demonstrating that the activity of the enzyme in the cells was limited by the rate of pyruvate transport. PDC activity was not detectable in cells that did not express zmPDC-GFP-V5/His protein or that expressed zmPDC H113Q-GFP-V5/His protein at 48 h after addition of 2 mg/mL doxycycline to the growth medium [data not shown and Huang et al. (17)]. We were unable to detect PDC activity in vivo in xenografts derived from cells expressing zmPDC-GFP-V5/His, using similar 1 H MRS measurements, following intraperitoneal injection of [U-2 H 3 ] pyruvate (see Supporting Fig. S1). Addition of hyperpolarized [1-13 C] pyruvate to cells resulted in a time-dependent increase in the bicarbonate signal at 162.8 ppm, which could be followed using dynamic 13 C MRS measurements (Fig.  3b). Doxycycline-induced expression of the wild-type enzyme resulted in an approximately 2.5-fold higher 13 C bicarbonate/pyruvate signal ratio calculated from the sum of first 80 spectra acquired after the addition of 75 mM hyperpolarized [1-13 C] pyruvate (Fig. 3c, 3d), when compared with uninduced cells or wild-type cells incubated in the presence or absence of doxycycline.
Subcutaneous implantation of cells in SCID mice resulted in xenografts that consisted predominantly of HEK-293T cells (Fig. 4a). Xenografts derived from cells transfected with the zmPDC-GFP-V5/His vector expressed zmPDC-GFP-V5/His protein at 96 h after doxycycline administration, as demonstrated by western blots of tissue extracts. The protein was not detectable in the absence of doxycycline administration or in xenografts derived from wild-type HEK293T cells (Fig. 4b). zmPDC-GFP-V5/His protein expression was confirmed by staining of tissue sections with antibodies against GFP and the V5 epitope (Fig. 4c). Expression was also confirmed by imaging GFP fluorescence at 48 and 96 h after doxycycline administration (Fig. 4d). There was some evidence of a decrease in the growth rate of xenografts expressing the wild-type (zmPDC-GFP-V5/His) and mutant enzymes (zmPDC/H113Q-GFP-V5/ His) following doxycycline administration; however, this decrease was not statistically significant ( Fig. 5a and 5b, respectively). The area of the tissue sections that comprised nonviable (necrotic and apoptotic) cells, as assessed by H&E staining, did not differ significantly between doxycycline-treated and untreated xenografts (23.0 6 6.6% and 21.5 6 4.6%, respectively; SD, n ¼ 4; P ¼ 0.79), nor did the levels of apoptosis, as assessed by the levels of cleaved caspase 3 staining (14.3 6 5.6% and 15.5 6 8.4%, respectively; SD, n ¼ 3; P ¼ 0.85) (Fig. 4c).

DISCUSSION
We have demonstrated that the pyruvate decarboxylase activity expressed by the zmPDC-GFP-V5/His fusion protein can be detected in vivo using hyperpolarized [1-13 C] pyruvate and 13 C MRS and therefore that this system has the potential to be used as an MR gene reporter. The size  5. (a, b) Effect of induction of expression of wild-type (a) and mutant PDC (b) on xenograft growth. The size of the xenografts derived from cells transfected with the tet-on PDC or tet-on H113Q constructs are expressed as the percentage of the volume measured at the beginning of doxycycline treatment (0 h). Caliper measurements of two xenograft diameters were made every 48 h from 21 d after implantation, when doxycycline was added to the drinking water as indicated. Euhus' formula was used to calculate xenograft volume as described in the Methods section. Error bars represent one standard deviation of the mean. Only positive or negative error bars are shown for clarity. (c) Representative formalin-fixed sections taken from xenografts grown for $21-24 d following subcutaneous implantation of cells transfected with the tet-on PDC-GFP-V5/His construct. Where indicated, the mice were given 10 mg/mL doxycycline solution in their drinking water for 96 h prior to xenograft excision. The area of necrotic tissue was assessed via H&E staining, and apoptosis was investigated by probing with an anti-CC3 antibody. Scale bars ¼ 150 mm.
of the zmPDC coding sequence (1.60 kbp), although considerably larger than that of GFP (0.73 kbp) and similar fluorescent reporter genes, is comparable to that of the widely used bioluminescence reporter, Firefly luciferase (1.65 kbp). A compact reporter size is an advantage when the payload constraints of delivery vectors are considered. Because it is a spectroscopic method in principle, it could be used with multiple gene reporters; for example, we have shown recently that the urea transporter can be used as a gene reporter in conjunction with hyperpolarized [ 13 C]urea (24). The 13 C resonances of urea, [1-13 C] pyruvate, and bicarbonate are resolved, therefore both reporters could be used in the same system. This is in general not possible with MRI methods that employ contrast agents modulating T 1 or T 2 /T 2 * relaxation times, nor for positron emission tomographybased reporter genes (1), although there is this potential with chemical exchange saturation transfer (CEST)-based MRI gene reporters (25).
Doxycycline-induced expression of the zmPDC-GFP-V5/His fusion protein caused a decrease in cell growth rate in vitro (Fig. 2c). There was also some evidence for a decrease in growth rate in vivo of the xenografts derived from these cells following doxycycline administration in the drinking water (Fig. 5a, 5b). These effects on cell growth may be partially explained by the effects of doxycycline itself. Doxycycline, among other tetracyclines, is a potent inhibitor of mitochondrial translation, which inhibits formation of complexes I, III, IV, and V of the mitochondrial electron transfer chain (26)(27)(28)(29), leading to a decrease in citric acid cycle activity and, consequently, de novo pyrimidine synthesis (26). Consistent with this, we observed a decrease in the growth rate of untransfected wild-type HEK293T cells in vitro after the addition of doxycycline to the growth medium (Fig. 2c). This growth inhibition was more pronounced, however, in cells expressing the zmPDC-GFP-V5/His transgene when compared with wild-type cells (Fig. 2c), suggesting that growth could also be inhibited by expression of PDC activity. However, the observation that expression of the inactive (H113Q) mutant of the enzyme (17) also inhibited cell growth (Fig. 2c) suggests instead that this is a nonspecific effect of high-level protein expression rather than expression of PDC activity per se. Moreover, this may be due to the GFP component of the construct since this has, in some contexts, been shown to have a nonspecific toxic effect on expressing cells (30,31). The absence of a significant increase in apoptosis and necrosis in the cell line-derived xenografts in vivo, as assessed by anti-CC3 and H&E staining, respectively (Fig. 6c), indicates that zmPDC-GFP-V5/His expression has a cytostatic rather than a cytotoxic effect. Therefore, the reporter might be more appropriate for use in tissues with low cell turnover.
Cell lysis increased the rate of [2,2,2-2 H 3 ] acetaldehyde production from [U-2 H 3 ] pyruvate approximately fivefold in cells expressing zmPDC, implying that detection of PDC activity from measurements of labeled bicarbonate production in vivo is limited by the rate of pyruvate entry into the cell. This suggests that the reporter would work better in cells with higher rates of pyruvate transport and that there would be little to be gained from increasing the expression level or specific activity of zmPDC (eg, by removing the GFP tag), at least in HEK293T cells.  13 C spectra acquired from 8-mm-thick slices of xenografts $21 d after subcutaneous implantation HEK293T cells transfected with the PDC-GFP-V5/His construct. Experiments were performed either without doxycycline treatment (no doxycycline) or after 96 h of 10 mg/mL doxycycline solution administration (96 hours post doxycycline). A total of 10 mL/g body weight of a 75-mM solution of hyperpolarized [1-13 C] pyruvate was injected over 2 s. A total of 128 single transient 13 C spectra were acquired, starting 30 s from the injection (TR ¼ 500 ms, nominal flip angle ¼ 20 , SW¼ 6 kHz; delay from the middle of the excitation pulse to beginning of data acquisition was 900 ms). The insets show a 10Â vertical scale expansion of the 162-168 ppm region of the resulting 13  The background hyperpolarized 13 C bicarbonate signal observed in the experiments described here can be explained by the activity of mitochondrial pyruvate dehydrogenase (PDH), which was shown previously in the rat heart to lead to the production of labeled bicarbonate from hyperpolarized [1-13 C] pyruvate (32). An alternative pathway involving pyruvate carboxylase has been reported in isolated perfused mouse liver (33); however, HEK293T cells do not express this enzyme activity (34). The background bicarbonate signal is expected to be much less in tumor cells; for example, EL4 lymphoma tumors show no detectable hyperpolarized bicarbonate signal following injection of [1-13 C] pyruvate (18).
The tet-on doxycycline-inducible gene expression system used here allowed rapid and reversible gene expression by oral administration of doxycycline (35,36), allowing each xenograft to be used as its own control, which was important in demonstrating a PDC-dependent increase in labeled bicarbonate production above the background level of labeled bicarbonate. In principle, the increased bicarbonate production in these experiments could also have been due to an effect of doxycycline on the production of CO 2 by mitochondrial PDH activity. However, this is unlikely, because doxycycline had no effect on CO 2 production in wild-type cells, where the bicarbonate/pyruvate signal ratio was unaffected by doxycycline addition and was similar to the ratio observed in untreated cells transfected with the inducible wildtype PDC vector (Fig. 3d). Moreover, doxycycline, by inhibiting the mitochondrial respiratory chain, would likely cause an increase in the mitochondrial NADH/ NAD þ ratio by decreasing the rate of NADH oxidation (37). An increase in mitochondrial NADH/NAD þ ratio would be expected to inhibit PDH (38,39), leading to a decrease in CO 2 and bicarbonate production rather than an increase. In a limited number of experiments with xenografts expressing the mutant enzyme, we were unable to demonstrate an increase in bicarbonate production following administration of doxycycline in the drinking water.
An alternative approach to circumvent the problem of background bicarbonate signal would be to use hyperpolarized [2-13 C] pyruvate and detect the signal from [1-13 C] acetaldehyde. However, we were unable to reproducibly detect [1-13 C] acetaldehyde, which may be explained by the short T 1 of [1-13 C] acetaldehyde (approximately 11.0 s compared with 40.7 s in bicarbonate) as well as its close proximity to the much larger pyruvate signal. In addition, rapid metabolic conversion of [1-13 C] acetaldehyde to [1-13 C] acetate, and further to [1-13 C] AcCoA and [1-13 C] acetyl-carnitine, has been observed previously in experiments with hyperpolarized [1-13 C] acetate in vivo (23,40), which will decrease the steady state pool size of labeled acetaldehyde. Measurements of 13 C label flux between hyperpolarized [1-13 C, U-2 H 5 ] ethanol and [1-13 C] acetate in mouse liver showed no evidence of the [1-13 C, U-2 H 4 ] acetaldehyde intermediate, despite the much longer T 1 of the deuterated acetaldehyde, which was approximately 19 s. Again this was attributed to a low steady-state concentration of the labeled intermediate (22). Because [1-13 C] acetaldehyde exists in equilibrium with the hydrated form, with the hydrate 13 C resonance at 91.4 ppm (data not shown), this will further decrease the sensitivity of its detection by 13 C MRS (41). The resonance detected in xenografts at 205.6 ppm with hyperpolarized [2-13 C] pyruvate is likely to be from [1-13 C] AcCoA produced by PDH activity, as has been observed previously in heart muscle following administration of hyperpolarized [2-13 C] pyruvate (42). However, this would not explain the increase in this signal observed following doxycycline administration in the experiments described here. A possible explanation for this is that there is an increase in [1-13 C] acetate formation resulting from aldehyde dehydrogenase 2-catalyzed oxidation of the [1-13 C] acetaldehyde produced by PDC and that this is then rapidly converted to AcCoA by thiokinase. Build-up of AcCoA has been observed in alcoholic fatty liver disease, where acetate produced by mitochondrial aldehyde dehydrogenase 2 from ethanolderived acetaldehyde is converted to AcCoA, but its further metabolism via the citric acid cycle is limited due to product inhibition of isocitrate dehydrogenase and aketoglutarate dehydrogenase by NADH produced during acetaldehyde oxidation (43).
In conclusion, we have demonstrated the feasibility of using zmPDC as an MR gene reporter when used with hyperpolarized [1-13 C] pyruvate. However, the levels of bicarbonate produced above background were low, which would make it challenging to use in conjunction with an imaging experiment. Increasing zmPDC expression, at least in the cells used here, is unlikely to improve the sensitivity of the reporter, because PDC activity appeared to be limited by pyruvate transport into the cell. However, the potential for increased temporal resolution resulting from rapid signal acquisition coupled with subsequent rapid decay of the hyperpolarized signals and the capability of this reporter to be combined with other spectroscopy-based gene reporters in the same experiment merits further development of this and other enzyme-based gene reporters for use with hyperpolarized 13 C MRS.