Nuclear spin hyperpolarization can dramatically increase the sensitivity of the 13C magnetic resonance experiment, allowing dynamic measurements of the metabolism of hyperpolarized 13C-labeled substrates in vivo. Here, we report a preclinical study of the response of lymphoma tumors to the vascular disrupting agent (VDA), combretastatin-A4-phosphate (CA4P), as detected by measuring changes in tumor metabolism of hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate. These measurements were compared with dynamic contrast agent–enhanced magnetic resonance imaging (DCE-MRI) measurements of tumor vascular function and diffusion-weighted MRI (DW-MRI) measurements of the tumor cell necrosis that resulted from subsequent loss of tumor perfusion. The rate constant describing flux of hyperpolarized 13C label between [1-13C]pyruvate and lactate was decreased by 34% within 6 hours of CA4P treatment and remained at this lower level at 24 hours. The rate constant describing production of labeled malate from hyperpolarized [1,4-13C2]fumarate increased 1.6-fold and 2.5-fold at 6 and 24 hours after treatment, respectively, and correlated with the degree of necrosis detected in histologic sections. Although DCE-MRI measurements showed a substantial reduction in perfusion at 6 hours after treatment, which had recovered by 24 hours, DW-MRI showed no change in the apparent diffusion coefficient of tumor water at 6 hours after treatment, although there was a 32% increase at 24 hours (P < 0.02) when regions of extensive necrosis were observed by histology. Measurements of hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate metabolism may provide, therefore, a more sustained and sensitive indicator of response to a VDA than DCE-MRI or DW-MRI, respectively. Mol Cancer Ther; 9(12); 3278–88. ©2010 AACR.
Targeting the tumor vasculature is an attractive treatment option due to the ease of drug delivery and the distinctive phenotype shown by tumor blood vessels, which are frequently angiogenic, lacking in smooth muscle, and with a rapidly proliferating endothelial cell population (1, 2). Vascular disrupting agents (VDA) selectively target these endothelial cells, causing the vessels to collapse, which, in turn, eliminates the supply of oxygen and nutrients and leads ultimately to tumor cell necrosis (2).
Combretastatin-A4-phosphate (CA4P), the water-soluble prodrug of CA4, is a tubulin-binding agent that induces a rapid shape change in proliferating endothelial cells by disrupting microtubule assembly, leading eventually to apoptotic endothelial cell death (3). CA4 at 100 mg/kg has been shown to yield rapid and prolonged interruption of blood flow in both human xenograft (MDA-MB-231 breast carcinoma) and murine (CaNT adenocarcinoma) tumor models, resulting in extensive tumor cell necrosis within 24 hours of drug treatment (4). Following successful phase I clinical trials (5), phase II/III trials have been initiated with CA4P as both a single agent in anaplastic thyroid carcinoma (6) and a combination of carboplatin and paclitaxel for ovarian cancer (7) and chemotherapy-naive non–small cell lung carcinoma (8).
Because VDAs rarely cause tumor shrinkage in the early stages of treatment, clinical trials of these agents have used functional biomarkers of response, including reductions in tumor perfusion, changes in tumor cell metabolism, and ultimately cell death. Measurements of vascular function using dynamic contrast agent–enhanced magnetic resonance imaging (DCE-MRI) and H2 15O positron emission tomography (PET) have been most commonly used in CA4P clinical trials (5, 9, 10). Although DCE-MRI provides high sensitivity, repeatability between centers is limited by the wide variety of acquisition parameters and kinetic models (11). The need for an on-site cyclotron to produce the short half-life isotope 15O more significantly limits PET studies of the vasculature (12).
Diffusion-weighted MRI (DW-MRI) has been used to image the tumor cell necrosis that results from a positive response to CA4P treatment, with a range of preclinical studies done in a rat rhabdomyosarcoma (13, 14). In these studies, changes in apparent diffusion coefficient (ADC) were highly dependent on the timing of the DW-MRI measurements relative to the administration of the treatment. At present, the use of DW-MRI as a clinical biomarker of VDAs has been limited by a lack of uniform standards for data acquisition and processing (15). A recent phase I trial combining CA4P with bevacizumab found good reproducibility between 2 centers, although no significant change in ADC was observed until after 2 doses of CA4P, again highlighting the importance of experiment timing (16).
Tumor response to VDAs can also be detected through changes in tumor cell metabolism. Preclinical models have shown significant (up to 80%) and rapid (within 2 hours) reductions in uptake of the positron-emitting glucose analogue, [18F]fluorodeoxyglucose (FDG), after treatment with both CA4P (17) and AVE8062, a combretastatin derivative (18); however, measurements of [18F]fluorodeoxyglucose uptake can be confounded by the presence of inflammation and hypoxia, both of which may arise following CA4P treatment (19).
We have shown previously that a reduction in tumor cell energy status, measured using 31P magnetic resonance spectroscopy (MRS), is a sensitive indicator of tumor response to treatment with a CA4P (10). Clinically, MRS of nuclei other than protons has thus far been limited by an inherent lack of sensitivity. Dynamic nuclear polarization (DNP) of 13C-labeled cell metabolites in vitro and subsequent intravenous administration dramatically enhances their sensitivity to detection in vivo (>10,000-fold; ref. 20). Using this technique, both the location of the molecule and, more importantly, the dynamics of its subsequent conversion into other cell metabolites can be imaged in vivo. The rate of conversion of hyperpolarized [1-13C]pyruvate into [1-13C]lactate in a murine model of lymphoma was shown to decrease significantly as early as 24 hours after cytotoxic therapy (21, 22), whereas the conversion of hyperpolarized [1,4-13C2] fumarate, a tricarboxylic acid cycle intermediate, into [1,4-13C2]malate was increased and shown to correlate in vitro with levels of tumor cell necrosis (23).
We show here that the exchange of hyperpolarized 13C label between pyruvate and lactate is reduced in murine lymphoma tumors within 24 hours of treatment with CA4P and that the production of [1,4-13C2]malate from hyperpolarized [1,4-13C2]fumarate is increased over the same period. These measurements have been compared directly with DCE-MRI measurements of tumor perfusion and DW-MRI measurements of tumor cell necrosis. Despite the limited lifetime of the hyperpolarization and the need for specialist DNP equipment, the first clinical trials of hyperpolarized [1-13C]pyruvate are expected this year (24), so there is a reasonable expectation that these methods could translate to the clinic in the near future, where they could provide a sensitive adjunct to multiparametric MRI measurements of tumor responses to VDAs.
Materials and Methods
Tumor implantation, treatment, and histology
Tumors were established, as described previously, by subcutaneous inoculation of 5 × 106 EL4 murine lymphoma cells (no authentication was done by the authors) in female C57BL/6 mice and allowed to grow for 10 days. Drug-treated animals received an intraperitoneal administration of 100 mg/kg of CA4P (structure shown in Fig. 1; provided by Oxigene). Procedures were conducted in accordance with project and personal licenses issued under the United Kingdom Animals (Scientific Procedures) Act, 1986. Tumor cell death was assessed histologically by hematoxylin and eosin staining of tissue sections obtained at postmortem. Slides were scanned using an Aperio XT (Aperio Technologies), and the area fraction containing the fragmented nuclei of dead cells, relative to the total area of 2 fields of view, was evaluated in 2 sections per excised tumor by using the ImageJ software (National Institutes of Health).
Hyperpolarization of [1-13C]pyruvate and [1,4-13C2]fumarate
[1-13C]Pyruvic acid samples (44 mg, 14 mol/L; 91% 13C) contained 15 mmol/L of trityl radical, tris(8-carboxy- 2,2,6,6-tetra-(hydroxyethyl)-benzo-[1,2-4,5′]-bis-(1,3)- dithiole-4-yl)-methyl sodium salt (OX063; GE Healthcare, Amersham, UK) and 1.4 mmol/L of an aqueous solution of a gadolinium chelate (ProHance; Bracco International B.V.). [1,4-13C2]Fumaric acid (381.2 mg, 3.4 mol/L; 99% 13C; Cambridge Isotope Laboratories) was dissolved in 4.5 g of dimethyl sulfoxide (Sigma Aldrich), containing 18.5 mmol/L of trityl radical, (tris(8-carboxy- 2,2,6,6(tetra(methoxyethyl)-benzo-[1,2-4,5′]bis-(1,3)dithiole-4-yl)methyl sodium salt (AH111501; GE Healthcare) and 0.8 mmol/L of anhydrous gadolinium chelate, (1,3,5-tris-(N-(DO3A-acetamido)-N-methyl-4-amino-2-methylphenyl)-[1,3,5]tria-zinane-2,4,6-trione (Gd-3, GE Healthcare). A 40-mg aliquot of this fumarate solution was used for each experiment.
Pyruvate was hyperpolarized as described previously (20). Briefly, the liquid sample was lowered into a GE Healthcare DNP prototype hyperpolarizer and cooled rapidly to ∼1.2 K by using liquid helium at a pressure of ∼1 mbar. Polarization of the electron spins on the trityl radical was transferred to the 13C label, using microwave irradiation at 93.975 GHz and 100 mW for 45 minutes. The sample was then rapidly dissolved in 6 mL of HEPES buffer (40 mmol/L of HEPES, 94 mmol/L of NaOH, 30 mmol/L of NaCl, and 100 mg/L of EDTA) pressurized to 10 bar and at 180°C; the eluted sample was cooled to ∼37°C before administration. The level of polarization ranged from 20% to 40% and the final concentration of [1-13C]pyruvate was 75 mmol/L.
For [1,4-13C2]fumarate, aliquots of 10 μL were dropped into liquid nitrogen to form pellets, which were transferred to a precooled dissolution cup (25) and lowered into the hyperpolarizer. The sample was dissolved using 6 mL of phosphate buffer (40 mmol/L of phosphate, 50 mmol/L of NaCl, 40 mmol/L of NaOH, and 100 mg/L of EDTA), yielding a final fumarate concentration of 20 mmol/L. The polarization ranged from 15% to 25%.
Magnetic resonance spectroscopy and imaging
Animals were anaesthetized by intraperitoneal administration of 10 mL/kg of a 5:4:31 mixture of Hypnorm (VetaPharma Ltd.), Hypnovel (Roche), and saline. A catheter was inserted into the tail vein and the animal was placed inside a cradle with a 24-mm diameter surface coil tuned to 13C (100 MHz) positioned over the tumor. The assembly was placed in a quadrature 1H-tuned volume coil (Varian), in a 9.4-T vertical wide-bore magnet (Oxford Instruments). Transverse 1H images were acquired using a gradient-echo pulse sequence [30° pulse; repetition time (TR), 300 milliseconds; echo time (TE), 2.2 milliseconds; field-of-view (FOV), 35 × 35 mm in a data matrix of 256 × 256, with 2 averages per increment; slice thickness, 2 mm, and 21 transverse slices]. The tumor slice for hyperpolarized experiments was selected from these images, and a slice-selective excitation pulse and acquire sequence was used for 13C spectroscopy.
After administration of 0.2 mL of hyperpolarized pyruvate, 160 13C spectra were acquired from a 6 mm thick tumor slice at 1-second intervals with a nominal flip angle of 10°. Approximately 45 minutes after the pyruvate experiment, which was the time taken to hyperpolarize the fumarate sample, 0.2 mL of hyperpolarized fumarate was injected into the same animal and 80 13C spectra were acquired at 2-second intervals with a nominal flip angle of 20°. In both experiments, every 16th spectrum was acquired from the entire sensitive volume of the surface coil. The body temperature of the animals was maintained using a flow of warm air. It was not possible to coinject pyruvate and fumarate, as the resonances of the resulting hyperpolarized lactate and malate were insufficiently resolved in the spectra obtained in vivo.
MRS data analysis
The integrated peak intensities of hyperpolarized [1-13C]pyruvate and [1-13C]lactate were fitted to the modified Bloch equations for 2-site exchange to obtain the rate constants k L and k P and the apparent spin lattice relaxation rates as described previously (21)(A) (B) (C)
where Lz and Pz are the z magnetizations of the 13C nucleus in the lactate and pyruvate carboxyl carbons, ρL and ρP are the spin lattice relaxation rates (1/T1L,P), and L∞ and P∞ are the equilibrium magnetizations (i.e., at t = ∞). Because of the large number of parameters to be fitted, it was assumed that the spin-lattice relaxation rates of the metabolites were equivalent (ρP = ρL). The integrated intensities of hyperpolarized [1,4-13C2]fumarate and [1,4-13C2]malate were fitted to the same equations, with M and F referring to malate and fumarate, respectively, the apparent rate constants being kF and kM and by setting ρF = ρM. We have shown previously, and again confirmed in these experiments (data not shown), that the fitted forward rate constants (kP or kF) were not affected by the assumption of equal T1s over the range of T1 values encountered for these metabolites (21, 23). Results are expressed as mean ± standard deviation, unless stated otherwise. Statistical significance was tested using Prism (Graphpad, San Diego, CA) with an unpaired 1-tailed t test at the 95% confidence level.
Measurements of enzyme activities and metabolite concentrations
Tumors were excised and freeze-clamped 30 seconds after administration of a 0.2-mL bolus of 75 mmol/L of pyruvate and 20 mmol/L of fumarate (nonhyperpolarized). These freeze-clamped samples were used to determine lactate dehydrogenase (LDH) and fumarase activities, as well as metabolite concentrations, at different time points after CA4P treatment. A small amount of each freeze-clamped tumor was homogenized and enzyme activities were determined spectrophotometrically (26). The remaining frozen tumor was extracted using 7% perchloric acid and neutralized using 2 mol/L of K2CO3. The concentrations of pyruvate and lactate in these neutralized extracts were measured using NADH-linked enzymatic assays, assuming a millimolar extinction coefficient for NADH of 6.22 (27, 28). Fumarate and malate concentrations in the same extracts were measured fluorometrically by using the fluorescence of acetylpyridine-adenine dinucleotide (29) and concentrations were determined by comparison with known standards. Results are expressed as mean ± standard error and unpaired 2-tailed t tests at the 95% confidence level were done using Excel (Microsoft).
The remainder of each extract was then treated with 0.5 g of Chelex-100 resin (Sigma Aldrich) for 1 hour and then lyophilized for 1H NMR analysis. The dried sample was dissolved in a buffer containing 0.1 mol/L of Na2HPO4 and 0.1 mol/L of KH2PO4 (pH = 8.10) in and D2O and containing 4.2 mmol/L of sodium trimethylsilyl-2,2,3,3-tetradeuteroproprionate (TSP) as a chemical shift and intensity standard (Sigma Aldrich). 1H NMR spectra were acquired at 500 MHz as 32,768 data points, using a 90° pulse, a sweep width of 16 kHz, a TR of 6 seconds, and were the sum of 256 transients. Peaks from metabolites of interest were integrated with reference to TSP and compared with samples of known concentration.
Dynamic contrast-enhanced magnetic resonance imaging
As the gadolinium contrast agent can increase the rate of polarization loss from the hyperpolarized substrates, the DCE-MRI data were acquired from a separate cohort of animals, before and after CA4P treatment, using a T1-weighted spin-echo pulse sequence (coronal tumor slice; FOV, 60 mm × 30 mm; data matrix, 256 × 64; slice thickness, 1.5 mm, 5 slices with 0.25-mm gaps between slices; TR, 110 milliseconds; and TE, 9 milliseconds). In addition, spin-lattice relaxation rates [R 1(1/T 1)] were measured using an inversion recovery fast low angle shot (FLASH) pulse sequence (TR, 5.5 milliseconds; TE, 2.5 milliseconds; and 10 seconds delay between images, 11 inversion times between 50 milliseconds and 10 seconds, 2 averages per inversion time). Four control images and one R1 map were collected before gadolinium diethylenetriaminepentaacetate (Gd-DTPA; Magnevist, Schering), diluted in sterile saline (0.9% sodium chloride) was administered intravenously to give 200 μmole/kg of body weight; imaging was then continued for 10 minutes. Inversion recovery data were fitted, pixel-by-pixel, to the monoexponential function:(D)
where TI is the inversion time. This gave estimates for S0, spin density; R1,pre, the relaxation rate before contrast agent administration; and k, a factor accounting for imperfections in flip angles. Spin-echo images were converted to relaxation rate maps using:(E)
S0,pre was estimated from control images using the measured R1,pre values and was assumed not to change during the experiment. The effect of Gd-DTPA on transverse relaxation was assumed to be negligible at the short TE used. The area under ΔR1 curve (AUC) was calculated from a region of interest covering the whole tumor (30, 31). Statistical significance was tested using Prism (Graphpad), with 1-way analysis of variance (ANOVA) on the Gd-DTPA uptake curves and a paired 2-tailed t test at the 95% confidence interval for the AUC.
Diffusion-weighted magnetic resonance imaging
The ADC was measured before and after treatment in all animal cohorts; 8 to 10 slices covering the whole tumor were acquired using a navigated dual-echo spin-echo pulse sequence (FOV, 35 × 35 mm2; data matrix, 128 × 64; slice thickness 2 mm; TR, 1.0 seconds; TE, 35 milliseconds and 45 milliseconds, the second echo being used as the navigator echo; 2 signal averages per b-value; ref. 32), with diffusion-sensitizing gradients applied along the slice axis [gradient length (∂), 7.5 milliseconds; delay between gradients (Δ), 13 milliseconds; and 5 b-values (0, 68, 271, 609, and 1,082 s/mm2).
ADC maps were generated for each slice on a pixel-by-pixel basis by fitting the signal intensity of the 5 diffusion-weighted images and the corresponding b-values to the monoexponential function:(F)
where S0 is the signal amplitude without diffusion gradients; b is the b-value, given by the amplitude, length, and separation of the diffusion gradients; and D is the ADC in the direction of the diffusion gradients. A region of interest (ROI) just inside the boundary of the tumor was selected in each slice, using a custom routine written in Matlab (Mathworks, Natick, MA). ADC histograms were generated for each ROI, as well as for the whole tumor. No statistically significant differences were found between the ADC histogram distributions or median pixel values of each tumor slice or the whole tumor compared with the central tumor slice as assessed by a paired 2-tailed t test at the 95% confidence interval. The mean ADC obtained from the multislice data was (0.45 ± 0.04) × 10−3 mm2/s for untreated animals and 0.51 ± 0.03 at 24 hours after treatment, whereas for the single slice data the mean ADC was 0.44 ± 0.04 and 0.55 ± 0.09, respectively (mean ± SD, n = 6 animals). The ADC maps and median ADC values of the central tumor slice were therefore considered representative and used to assess treatment response, using a paired 2-tailed t test at the 95% confidence interval.
Intravenous administration of hyperpolarized [1-13C] pyruvate or [1,4-13C2]fumarate resulted in readily detectable signals from these metabolites and in the production of [1-13C]lactate and [1,4-13C2]malate, respectively, in the lymphoma tumors (Fig. 2). The flux of 13C label from hyperpolarized [1-13C]pyruvate at 173 ppm to [1-13C]lactate at 185 ppm was clearly decreased 24 hours after treatment with CA4P (Fig. 2A and B), whereas the fumarase-catalyzed hydration of hyperpolarized [1,4-13C2]fumarate (177 ppm) to [1,4-13C2]malate (182, 184 ppm), which was barely detectable before treatment (Fig. 2C), showed a large increase at 24 hours after treatment (Fig. 2D).
The time courses of the pyruvate, lactate, fumarate, and malate signal intensities are shown in Fig. 3. These data were fitted to the modified Bloch equations for 2-site exchange. The rate constant describing label flux between pyruvate and lactate (k P) was reduced by 34% within 6 hours of treatment, from 0.10 ± 0.03 to 0.07 ± 0.02 s−1 (n = 10 animals, P < 0.01) and was at the same level at 24 hours (0.07 ± 0.03 s−1; n = 7 animals, P < 0.05), whereas the rate constant describing the production of labeled malate (k F) increased 1.6-fold at 6 hours after treatment (0.010 ± 0.004 to 0.016 ± 0.012 s−1; n = 7 animals, P < 0.1) and 2.5-fold at 24 hours (0.025 ± 0.018 s−1; n = 7 animals, P < 0.05). The peak of the malate signal appeared later in untreated tumors (19.7 seconds) compared with both treatment time points (13 seconds at 6 and 24 hours, respectively), as was observed previously in etoposide-treated lymphoma tumors (23).
The maximum amplitude of the [1,4-13C2]malate signals relative to the signal from [1,4-13C2]fumarate increased from 8 ± 1% in untreated tumors to 13 ± 3% at 6 hours and 20 ± 2% at 24 hours after CA4P treatment (mean ± SE; n = 7 animals per group). Histologic assessment of tumor sections obtained post mortem showed the presence of necrosis in the drug-treated tumors. This increased from a background level of 6.4 ± 1.4% (n = 7 animals) in untreated tumors to 15.9 ± 4% (2.5-fold higher; n = 3 animals, P < 0.05) at 6 hours after treatment and 35.6 ± 5.2% (6-fold higher; n = 7 animals, P < 0.01) at 24 hours after treatment. Tumors at 6 hours after treatment (Fig. 4B) showed small and diffuse areas of necrosis compared with the untreated tumors (Fig. 4A), whereas widespread necrosis was observed at 24 hours (Fig. 4C). We have previously shown that malate production from fumarate is a measure of tumor cell necrosis in vitro. The data presented here suggest that this is also the case in vivo, as there was a good correlation between the rate of malate production (y; Fig. 4D) and the percentage necrosis (x) observed in histologic sections.
The hyperpolarized signals from all metabolites were observed to decay more rapidly following drug treatment (Fig. 3), and there was a significant decrease in the fitted relaxation time constants for pyruvate (25.4 and 28.1 seconds at 6 and 24 hours after treatment compared with 30.9 seconds before; n = 7 animals, P < 0.05) and fumarate (17.7 and 18.2 seconds compared with 26.4 seconds before; n = 7 animals, P < 0.05). The relaxation time for tissue water protons was also found to decrease from 2.25 ± 0.05 seconds before treatment (n = 8 animals) to 2.11 ± 0.06 seconds (n = 4 animals, P = 0.019) and 2.09 ± 0.04 seconds (n = 5 animals, P = 0.023) at 6 and 24 hours after treatment with CA4P, respectively. These decreases in T 1 relaxation times may be explained by dipole–dipole interactions between the 13C and 1H nuclei and paramagnetic deoxyhemoglobin and methemoglobin, which are known to accumulate in the regions of hemorrhage (33) induced by CA4P treatment (4).
Measurements on tumor extracts (Table 1) showed an increase in LDH activity at 6 hours after treatment but no significant change in fumarase activity over the treatment time course. The lactate concentration in tumor extracts appeared to decrease by ∼10% following treatment, confirmed also by 1H NMR, but because of the large spread in the untreated data, this was not significant. The pyruvate concentration decreased by 45% at 6 hours after treatment, but that by 24 hours was comparable with the pretreatment level; pyruvate could not be detected reliably in the 1H NMR spectra.
Fumarate could not be measured separately from malate in the fluorometric assay of tumor extracts, presumably because of contaminating fumarase activity that remained following the extraction process. The results from the assay, thus, provide a measure of the total concentrations of both fumarate and malate (Table 1). Instead, the ratio of the fumarate and malate signals observed in the 1H NMR measurements was used to probe the relative concentrations of these metabolites, increasing by 2.4-fold (P < 0.1, n = 3 animals) and 3.7-fold (n = 3 animals, P < 0.05) at 6 and 24 hours after treatment, respectively.
Treatment response was also assessed using DCE-MRI measurements of tumor perfusion and DW-MRI measurements of tumor cell necrosis. The pattern of changes in the DCE-MRI and DW-MRI parameters were similar to what has been observed previously in other tumor types with this drug (4, 34–36). One-way ANOVA between the curves of Gd-DTPA uptake measured in the DCE-MRI experiments (Fig. 5A) showed significant (1-way ANOVA; n = 3 animals, P < 0.001) suppression of uptake at 6 hours after treatment, returning to earlier mentioned pretreatment values (n = 5 animals, P < 0.05) at 24 hours. Because Gd-DTPA is neither freely diffusible nor a pure blood pool agent, uptake is affected by changes in both blood flow and vessel permeability; therefore, as CA4P is expected to increase the permeability of surviving vessels (37), a combination of restored perfusion and increased permeability may explain the increase in uptake beyond pretreatment levels at 24 hours. The corresponding AUC (Fig. 5B) up to 10 minutes after administration of the contrast agent decreased from 9.67 ± 0.43 to 6.85 ± 0.95 mmol/L (n = 3 animals, P = 0.030) at 6 hours after treatment and increased to 12.8 ± 0.9 mmol/L (n = 5 animals, P = 0.005) at 24 hours (mean ± SE).
The median ADC of tumor water, measured using DW-MRI, was unchanged at 0.39 ± 0.05 × 10−3 mm2/s at 6 hours after treatment (n = 6 animals, P = 0.53) compared with untreated levels (0.42 ± 0.04 × 10−3 mm2/s; Fig. 6B), despite the onset of diffuse cellular necrosis observed in tumor sections obtained post mortem (Fig. 4). The tumor area in the ADC maps also remained largely homogeneous for both ADC histograms (Fig. 6A) and visual inspection (Fig. 6C and D), revealing no significant change from untreated tumors. At 24 hours after treatment, the median ADC showed a 32% increase to 0.55 ± 0.09 × 10−3 mm2/s (n = 7 animals, P = 0.018), much broader histograms were observed (Fig. 6A), and large necrotic regions with increased ADC could be distinguished within the tumor on the ADC maps (Fig. 6D).
Tumor responses to treatment are usually assessed from MRI and computed tomographic measurements of reductions in tumor size. The Response Evaluation Criteria in Solid Tumors (RECIST) define complete response as total tumor regression whereas partial response is defined as a 30% reduction in the sum of the longest tumor axes (38). However, because VDAs such as combretastatin often elicit a cytostatic response at early treatment time points, the RECIST criteria are of limited value (11, 38) and interest has focused instead on measuring functional biomarkers of response, including reductions in tumor perfusion, changes in tumor cell metabolism, and ultimately cell death.
We have shown here that hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate are sensitive markers of response to CA4P treatment. The apparent rate constant describing label exchange between [1-13C]pyruvate and [1-13C]lactate, k P, is influenced by a number of factors including tumor cell LDH activity, the concentration of the coenzyme NAD(H), tumor lactate concentration, membrane transport of pyruvate and lactate, and pyruvate delivery to the tumor (21). Treatment with CA4P resulted in a 34% decrease in k P at 6 hours after treatment and it remained at this lowered level at 24 hours after treatment. The decrease in k P at 6 hours is likely explained by a decrease in tumor perfusion. DCE-MRI measurements showed a decrease in the AUC of Gd-DTPA uptake of 29% at 6 hours, whereas independent ex vivo measurements of the tumor pyruvate concentration following administration of unlabeled pyruvate showed a decrease of 45%. The absence of a significant change in lactate concentration between 6 and 24 hours after CA4P treatment is consistent with the notion that the hyperpolarized [1-13C]pyruvate is labeling, by passive exchange, a preexisting lactate pool rather than there being significant net conversion of pyruvate to lactate. The evidence that we are measuring predominantly exchange, rather than net conversion of pyruvate into lactate, has been published previously (21, 39). The decrease in k P at 6 hours after CA4P treatment occurred despite a 50% increase in LDH activity, which by 24 hours had declined toward pretreatment levels. Such upregulation of LDH activity is likely to be a consequence of the disruption to blood flow resulting in increased tumor hypoxia, which can last for up to at least 6 hours after CA4P treatment (35); hypoxia induces the expression of HIF-1α, which mediates upregulation of the glycolytic enzymes, including LDH (40).
Restoration of tumor perfusion at 24 hours after CA4P treatment, which was evident from the DCE-MRI measurements, was not accompanied by an increase in k P, indicating that factors other than pyruvate delivery are determining the rate constant at this time point. Because there was no significant difference in LDH activity and only a small decline in lactate concentration relative to control, a loss of coenzyme NAD(H) or decreased membrane transport may be responsible. These factors could also have some influence in the decline at 6 hours. A further explanation may be that although there was restoration of perfusion, this was heterogeneous, with those regions of increased perfusion having lower levels of lactate and so showing lower levels of detectable label exchange.
From the perspective of response monitoring, however, the important point is that the hyperpolarized [1-13C]pyruvate experiment detects the initial and persistent damage to the tumor by probing first its perfusion and then metabolic state at a time when DCE-MRI measurements indicated recovery of perfusion. The hyperpolarized pyruvate experiment should, therefore, be less sensitive than DCE-MRI to the timing of the measurement after CA4P treatment, which could be advantageous in the clinic. The advantage of DCE-MRI, however, is that the technique has better spatial resolution; the projected resolution for hyperpolarized 13C imaging in the clinic is only of the order of 2.5 mm and therefore DCE-MRI may be better able to detect response if this is very heterogeneous.
In a previous study, we presented evidence in vitro that the production of [1,4-13C2]malate from hyperpolarized [1,4-13C2]fumarate is related to tumor cell necrosis . Because fumarate shows only relatively slow transport across the plasma membrane, the onset of necrosis results in an increased rate of access of the labeled fumarate to intracellular fumarase and hence in the formation of labeled malate within the relatively short lifetime of the hyperpolarized 13C label. The results presented here, in which we have shown that the rate of production of [1,4-13C2]malate from [1,4-13C2]fumarate correlates with the levels of necrosis detected in histologic sections of tumors, show that this is also the case in vivo. This correlation between necrosis and the rate of malate production from fumarate has also been shown recently in a human breast cancer cell line (41).
The production of malate was detectable, although small, in untreated tumors and this increased within 6 hours of CA4P treatment, with a 1.6-fold increase in the rate constant describing malate production (k F), which occurred despite the decrease in tumor perfusion. The significant increase in the malate/fumarate 13C signal intensity ratio at this time point was paralleled by an increase in the malate/fumarate concentration ratio measured in tumor extracts by 1H spectroscopy. Thus, unlike the hyperpolarized [1-13C]pyruvate experiment in which we apparently measured primarily exchange of label between the injected pyruvate and a preexisting lactate pool, in the hyperpolarized [1,4-13C]fumarate experiment, we apparently measured principally the net production of malate from fumarate. The DW-MRI experiment, however, showed no change in the median ADC of tumor water over this period and the tumor area on diffusion images remained homogeneous. This is consistent with previous studies, which have shown that in xenograft tumors with small and diffuse regions of necrosis, there can be no change in ADC even with necrotic fractions of up to 40% (42). At 24 hours after CA4P treatment, the necrosis observed in tumor sections obtained post mortem had increased by a factor of 6 compared with untreated tumors, from 6.4% to 35.6%, and there was a 2.5-fold increase in the rate constant k F. The ADC maps at this time point showed regions of extensive necrosis, and there was a 32% increase in the median ADC. The hyperpolarized fumarate experiment, therefore, detects diffuse tumor necrosis and so may be a more sensitive and enduring measure of necrosis than the DW-MRI experiment. However, the hyperpolarized fumarate experiment requires intravenous administration and again does not have the spatial resolution that can be obtained with DW-MRI.
In conclusion, we have shown that the reduced rate of label exchange between [1-13C]pyruvate and [1-13C]lactate at 6 hours after treatment reflects reduced tumor perfusion, whereas it is sensitive to persistent damage to the tumor at 24 hours, at a time when DCE-MRI measurements showed that perfusion had recovered. Over the same time course, the increased hydration of [1,4-13C2]fumarate to [1,4-13C2]malate parallels the progressive increase in cellular necrosis observed in histologic sections and is sensitive to diffuse areas of necrosis that develop as early as 6 hours after drug administration. As the production of malate from fumarate is a positive contrast experiment, it is less susceptible to changes in substrate delivery than the pyruvate experiment. Diffusion-weighted imaging, in comparison, only detected response once the necrosis was widespread at 24 hours. The combination of hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate probes both the energy status of viable tumor cells and the onset of cell death following treatment. Hyperpolarized [1-13C]pyruvate and [1,4-13C2]fumarate, when combined with 13C spectroscopic imaging (21, 23), may be useful therefore in the clinic as sensitive imaging biomarkers of response to vascular targeted therapy.
Disclosure of Potential Conflicts of Interest
The polarizer and related materials in this work were provided by GE Healthcare. K.M. Brindle and M.I. Kettunen hold patents with GE Healthcare.
We thank Dai Chaplin (Oxigene) for the generous gift of CA4P. We also thank Wm. Brad O'Dell for assistance with metabolite concentration and enzyme activity assays.
The work was supported by a Cancer Research UK program grant to K.M. Brindle (C197/A3514) and by a Translational Research Program Award from The Leukemia & Lymphoma Society. F.A. Gallagher received a Cancer Research UK & Royal College of Radiologists (UK) clinical research training fellowship, T.H. Witney received a GE Healthcare-BBSRC CASE studentship, and B.W.C. Kennedy received a Cancer Research UK studentship.
- Received July 28, 2010.
- Revision received September 22, 2010.
- Accepted September 27, 2010.
- ©2010 American Association for Cancer Research.