Abstract
Background
A generation of therapies targeting tumor metabolism is becoming available for treating glioma. Hyperpolarized MRI is uniquely suited to directly measure the metabolic effects of these emerging treatments.
Purpose
To explore the feasibility of the use of hyperpolarized [1–carbon 13 {13C}]-pyruvate for real-time measurement of metabolism and response to treatment with a glycolytic inhibitor in an orthotopic mouse model of glioma.
Materials and Methods
In this animal study, anatomic MRI and dynamic 13C MR spectroscopy were performed at 7 T during intravenous injection of hyperpolarized [1-13C]-pyruvate on mice with orthotopic U87MG glioma and healthy control mice. Anatomic MRI and dynamic 13C MR spectroscopy were repeated after administration of the glycolytic inhibitor WP1122, a prodrug of 2-deoxy-d-glucose. All experiments were conducted in athymic nude mice between October 2016 and March 2017. Hyperpolarized lactate production was quantified as an apparent reaction rate, or kPL, and normalized lactate ratio (nLac). The Wilcoxon signed-rank test was used to assess changes in paired measures of lactate production before and after treatment.
Results
Thirteen 12–16-week-old female mice and five healthy female mice underwent anatomic MRI and hyperpolarized [1-13C]-pyruvate spectroscopy. Large contrast agent–enhanced tumors were shown in mice with glioma at T2-weighted and T1-weighted postcontrast MRI by postimplantation day 40. After treatment with WP1122, a decrease in lactate was observed in mice with glioma (baseline and treatment mean kPL, 0.027 and 0.018 sec−1, respectively, P = .01; baseline and posttreatment mean nLac, 0.28 and 0.22, respectively, P = .01) whereas no significant decrease was observed in healthy control mice (baseline and posttreatment mean kPL, 0.011 and 0.017 sec−1, respectively, P = .91; baseline and posttreatment mean nLac, 0.16 and 0.21, respectively, P = .84).
Conclusion
Hyperpolarized carbon 13 measurements of pyruvate metabolism can provide rapid feedback for monitoring treatment response in glioma.
© RSNA, 2019
Summary
Carbon 13 MR spectroscopy of hyperpolarized pyruvate provides measurements of baseline metabolism and immediate treatment response to glycolytic inhibition in an orthotopic mouse model of glioma.
Key Results
■ After intravenous administration of pyruvate, quantitative measurements of hyperpolarized lactate were higher in mice with orthotopic gliomas relative to healthy control animals (mean apparent reaction rate, or kPL, 0.027 vs 0.011 sec−1, respectively, P = .001; mean normalized lactate ratio [nLac], 0.28 vs 0.16, respectively, P = .001).
■ Thirty minutes after administration of the glycolytic inhibitor WP1122 by oral gavage, hyperpolarized lactate production decreased in mice with orthotopic gliomas (baseline and posttreatment mean kPL, 0.027 and 0.018 sec−1, respectively, P = .01; baseline and posttreatment mean nLac, 0.28 and 0.22, respectively, P = .01) whereas no significant decrease was observed in healthy control mice (baseline and posttreatment mean kPL, 0.011 sec−1 and 0.017 sec−1, respectively, P = .91; baseline and posttreatment mean nLac, 0.16 and 0.21, respectively, P = .84).
■ Hyperpolarized pyruvate spectroscopy has the capability to help measure metabolic state in brain tissue and to delineate the efficacy of metabolic therapies for glioma in a rapid, noninvasive, and direct way.
Introduction
Glioblastoma is the most commonly diagnosed primary brain tumor in adults and has a poor prognosis, with deterioration of cognitive function and quality of life (1). Despite considerable research investment in improved therapies, the median survival is 12–14 months (2). Treatment for glioblastoma and most other gliomas typically consists of surgical resection with subsequent chemotherapy and radiation therapy.
During and after therapy, most patients undergo serial MRI to delineate treatment response and disease recurrence. Enhancement on T1-weighted postcontrast images remains the reference standard for identification of high-grade malignant tissue in the brain, but these images often depict misleading changes in patients undergoing chemotherapy, radiation therapy, or symptomatic therapies (1). There is therefore a need for imaging methods capable of accurately and quantitatively measuring treatment response in glioma. As new therapies for glioma are developed to exploit the propensity of tumors to utilize glycolysis (3), new imaging approaches for monitoring the efficacy of these treatments will also be needed.
Dissolution dynamic nuclear polarization can enhance the MRI signal of [1–carbon 13 {13C}]-pyruvate more than 10 000 fold compared with thermal equilibrium, enabling new insights into tumor metabolism in vivo (4). Previous work in rodent models of glioma has demonstrated that hyperpolarized [1-13C]-pyruvate MR spectroscopy depicts decreases in tumor lactate metabolism caused by radiation therapy (5) and chemotherapy (6–9) within a few days of treatment. Recent advances in hyperpolarized 13C MRI have enabled the real-time measurement of glycolytic metabolism in patients with brain cancer (10,11).
Our hypothesis was that dynamic hyperpolarized MR spectroscopy could be used in mouse models to depict an acute decrease in aerobic glycolysis following treatment with the glycolytic inhibitor WP1122. Our purpose was to investigate the feasibility of using hyperpolarized [1-13C]-pyruvate to probe the metabolic state of glioma as a direct readout of treatment effect with a glycolytic inhibitor.
Materials and Methods
Disclosure
W.P. is a founder, shareholder, and chairman of the scientific advisory board of Moleculin Biotech. A grant from Moleculin Biotech partially supported synthesis of WP1122 and salary of R.Z. K.A.M. had control of the data submitted for publication. The authors made the decision to publish and are solely responsible for this manuscript.
Animals
Thirteen 6–10-week-old female athymic nude mice were implanted with 5 × 105 U87MG cells (ATCC, Manassas, Va) expressing firefly luciferase. We administered cells by stereotactic injection into the right caudate nucleus through a transcranial plastic guide screw (12). Five female athymic nude mice were the experimental healthy control animals. We monitored initial tumor engraftment and early growth by bioluminescent imaging. Our Institutional Animal Care and Use Committee approved all animal experiment procedures, which were performed between October 2016 and March 2017.
Experimental Drug
2-Deoxy-d-glucose, or 2-DG, is a widely studied inhibitor of glycolysis; however, its therapeutic use in vivo is limited because of its rapid metabolism. WP1122 (3,6-di-O-acetyl-2-deoxy-d-glucose) is a prodrug of 2-DG that improves biodistribution and prolongs exposure. Increased levels of 2-DG were observed in the murine brain (10) when WP1122 was used instead of 2-DG. WP1122 was therefore expected to lead to enhanced metabolic effects in brain tumors.
MRI Protocol
Approximately 40 days after implantation, mice were imaged by using a 7-T small animal MRI system with ParaVision 6 and equipped with BGA-12SHP gradients and a 72-mm inner diameter hydrogen 1/13C volume resonator (Bruker Biospin MRI, Billerica, Mass). MRI was performed by K.A.M. and C.M.W., each with 5 years of experience. We anesthetized each mouse by using 2% isoflurane, inserted a catheter into the tail vein, and secured the animal on the MRI bed with a heating pad and respiratory monitoring pillow. A 20-mm mini-flex coil (Rapid MR International, Columbus, Ohio) placed on the cranium was used for hyperpolarized 13C signal reception. [1-13C]-pyruvate was hyperpolarized by using a dissolution dynamic nuclear polarization system (HyperSense; Oxford Instruments, Abingdon, England), as previously described (13,14). We injected 200 μL of 80-mmol/L hyperpolarized [1-13C]-pyruvate (approximately 60 mg/kg) into the tail vein at dynamic 13C MR spectroscopy. We performed baseline imaging around 40 days after implantation and repeated all MRI the following day, 30 minutes after administering 2.5 g/kg WP1122 by oral gavage.
At each MRI examination, the following were acquired: axial and coronal T2-weighted rapid acquisition with relaxation enhancement (echo time msec/repetition time msec, 57/3000), axial T1-weighted rapid acquisition with relaxation enhancement (7.25/1000), and axial section-selective dynamic 13C spectroscopy (90 repetitions; repetition time msec, 2000; 20° excitation angle; 10-mm section; 2048 complex points over 5000-Hz spectral width). The section position for 13C MR spectroscopy was centered on the tumor as viewed on T2-weighted coronal images. T1-weighted axial images were acquired before and after administration of 0.2 mmol/kg of gadopentetate dimeglumine (Magnevist, Bayer Healthcare, Berlin, Germany) via tail vein catheter.
Histologic Analysis
After the final imaging session, the brain of each mouse was immediately excised, frozen over liquid nitrogen, and stored at −80°C. Frozen sections were cut at a thickness of 10 μm, fixed with 4% paraformaldehyde, and stained with hematoxylin-eosin (L.L.R., with 20 years of experience) by using standard methods (15).
Data Analysis
Dynamic hyperpolarized 13C spectra were analyzed by using code written in-house in Matlab (Matlab R2016a; Mathworks, Natick, Mass). Spectral peaks corresponding to [1-13C]-pyruvate and [1-13C]-lactate were fit with Lorentzian line shapes to obtain relative metabolite amplitudes for each point. A simple precursor-product model of hyperpolarized lactate production was fit to these metabolite time curves to estimate the rate of pyruvate-to-lactate conversion in tissue, known as kPL. This model-based analysis assumes that all of the observed hyperpolarized pyruvate is in contact with the enzymes that mediate conversion to lactate. A longitudinal relaxation time constant of 30 seconds was assumed for [1-13C]-lactate in vivo (16). In addition, the normalized lactate ratio (nLac) was calculated as the sum of lactate amplitudes over time divided by the sum of pyruvate and lactate amplitudes over time.
Statistical Analysis
We made statistical comparisons of lactate metrics between mice with gliomas and healthy control mice by using the one-tailed Wilcoxon rank-sum test. By using the one-tailed Wilcoxon signed-rank test, we compared paired measurements obtained on subsequent days from the same mice before and after WP1122 treatment. We used built-in functions in Matlab R2016a to perform all statistical tests. P values less than .05 indicated statistically significant differences.
Results
Representative images from MRI and hematoxylin-eosin–stained images in a mouse with an orthotopic glioma are shown in Figure 1. Mean tumor volumes of 53.76 mm3 ± 36.01 (standard deviation) were observed at T2-weighted imaging at the second (final) imaging point. Hematoxylin-eosin staining confirmed the presence of tumors in the right deep gray matter of mice implanted with U87MG cells.
Figure 1a:
Representative images in a mouse with orthotopic glioma. Large well-vascularized tumors (arrows in a and b) were manifest 40 days after implantation of U87MG glioma cells, as shown by the hyperintense lesion on (a) T2-weighted and (b) gadolinium-enhanced T1-weighted axial images from MRI. The corresponding section from hematoxylin-eosin staining (c) shows a large infiltrative tumor (arrow) centered in the right-sided deep gray matter.
Figure 1b:
Representative images in a mouse with orthotopic glioma. Large well-vascularized tumors (arrows in a and b) were manifest 40 days after implantation of U87MG glioma cells, as shown by the hyperintense lesion on (a) T2-weighted and (b) gadolinium-enhanced T1-weighted axial images from MRI. The corresponding section from hematoxylin-eosin staining (c) shows a large infiltrative tumor (arrow) centered in the right-sided deep gray matter.
Figure 1c:
Representative images in a mouse with orthotopic glioma. Large well-vascularized tumors (arrows in a and b) were manifest 40 days after implantation of U87MG glioma cells, as shown by the hyperintense lesion on (a) T2-weighted and (b) gadolinium-enhanced T1-weighted axial images from MRI. The corresponding section from hematoxylin-eosin staining (c) shows a large infiltrative tumor (arrow) centered in the right-sided deep gray matter.
Figure 2a shows the typical location of the 1-cm section used for 13C MR spectroscopy. Representative 13C dynamic spectra from the animal depicted in Figure 2a are shown as waterfall plots in Figure 2b and 2c. Hyperpolarized pyruvate and lactate signals were observed with high signal-to-noise ratio after the pyruvate injection, and a reduction in the relative amount of lactate was usually observed after treatment with WP1122 for mice with orthotopic gliomas.
Figure 2a:
Representative hyperpolarized MR spectroscopic data from a mouse with orthotopic glioma. (a) Pulse-acquire spectroscopy was performed for a 1-cm axial section centered on the tumor viewed on coronal T2-weighted images during injection of hyperpolarized pyruvate through a tail-vein catheter. Waterfall plots show more lactate signal at (b) baseline than (c) 30 minutes after treatment with the glycolytic inhibitor WP1122.
Figure 2b:
Representative hyperpolarized MR spectroscopic data from a mouse with orthotopic glioma. (a) Pulse-acquire spectroscopy was performed for a 1-cm axial section centered on the tumor viewed on coronal T2-weighted images during injection of hyperpolarized pyruvate through a tail-vein catheter. Waterfall plots show more lactate signal at (b) baseline than (c) 30 minutes after treatment with the glycolytic inhibitor WP1122.
Figure 2c:
Representative hyperpolarized MR spectroscopic data from a mouse with orthotopic glioma. (a) Pulse-acquire spectroscopy was performed for a 1-cm axial section centered on the tumor viewed on coronal T2-weighted images during injection of hyperpolarized pyruvate through a tail-vein catheter. Waterfall plots show more lactate signal at (b) baseline than (c) 30 minutes after treatment with the glycolytic inhibitor WP1122.
The Table and Figure 3 summarize the quantification of hyperpolarized lactate from dynamic 13C spectroscopy. At baseline, mice with orthotopic gliomas exhibited mean kPL of 0.027 sec−1 and mean nLac of 0.28, which were greater than those observed in healthy control mice (mean kPL, 0.011 sec−1; mean nLac, 0.16; P = .001 for both). After treatment with the glycolytic inhibitor WP1122, a decrease in kPL and nLac was observed for mice with glioma (baseline and posttreatment mean kPL, 0.027 and 0.018 sec−1, respectively, P = .01; baseline and posttreatment mean nLac, 0.28 and 0.22, respectively, P = .01). In healthy control mice, no decrease in hyperpolarized lactate was observed after WP1122 treatment (baseline and posttreatment mean kPL, 0.011 and 0.017 sec−1, respectively, P = .91; baseline and posttreatment mean nLac, 0.16 and 0.21, respectively, P = .84).
Hyperpolarized Lactate Quantifications in Diseased and Healthy Mouse Brains
Note.—Data are means ± standard deviation of the apparent rate constants of pyruvate-to-lactate conversion and normalized lactate, the time-integrated ratio of lactate to combined pyruvate and lactate. kPL = pyruvate-to-lactate constant, nLac = normalized lactate ratio.
Figure 3a:
Graphs show (a) apparent conversion rate of pyruvate to lactate (kPL) and (b) lactate signal normalized to sum of lactate and pyruvate signals (nLac) were greater in mice with orthotopic glioma than in healthy control mice (P = .001 for both kPL and nLac, one-tailed Wilcoxon rank-sum test). After treatment with the glycolytic inhibitor WP1122, a decrease in lactate was observed for mice with glioma (P = .01 for both kPL and nLac, one-tailed paired Wilcoxon signed-rank test), but no significant decrease was observed in healthy control mice (P = .91 for kPL and P = .84 for nLac, one-tailed paired Wilcoxon signed-rank test). * Statistical significance with P < .05, ** statistical significance with P < .01, n.s. = no statistically significant difference.
Figure 3b:
Graphs show (a) apparent conversion rate of pyruvate to lactate (kPL) and (b) lactate signal normalized to sum of lactate and pyruvate signals (nLac) were greater in mice with orthotopic glioma than in healthy control mice (P = .001 for both kPL and nLac, one-tailed Wilcoxon rank-sum test). After treatment with the glycolytic inhibitor WP1122, a decrease in lactate was observed for mice with glioma (P = .01 for both kPL and nLac, one-tailed paired Wilcoxon signed-rank test), but no significant decrease was observed in healthy control mice (P = .91 for kPL and P = .84 for nLac, one-tailed paired Wilcoxon signed-rank test). * Statistical significance with P < .05, ** statistical significance with P < .01, n.s. = no statistically significant difference.
Discussion
In this study, we investigated the use of dynamic hyperpolarized pyruvate MR spectroscopy to depict changes in tumor metabolism after administration of the glycolytic inhibitor WP1122. Hyperpolarized lactate production was quantified at baseline and 30 minutes after oral administration of WP1122 in mice with gliomas and healthy control mice, as both an apparent reaction rate, known as kPL, and normalized lactate ratio (nLac). Both metrics of hyperpolarized lactate decreased after WP1122 treatment in mice with gliomas, whereas no significant change was observed in healthy control mice.
Hyperpolarized [1-13C]-pyruvate MR spectroscopy delivers valuable insights into tumor metabolism that are not directly obtainable through other imaging modalities. Lactate dehydrogenase A expression is upregulated in glioma cells, resulting in higher lactate production and maintaining the cellular-reducing potential necessary to sustain glycolysis (17,18). Measurements of hyperpolarized pyruvate and lactate specifically probe the reaction catalyzed by lactate dehydrogenase A, delivering information on enzymatic flux that can assess treatment response and disease state in various models of malignant disease (13). Importantly, the changes in cancer metabolism depicted at hyperpolarized pyruvate MR spectroscopy reflect the real-time glycolytic state of tumors and precede morphologic changes observed at T2-weighted imaging (8,14). This technology therefore holds promise to provide early indications of response in glioma (19) after treatment with radiation therapy (5), chemotherapy (7,9), and immunotherapy. Hyperpolarized pyruvate MR spectroscopy measurements are particularly well suited for depicting the effects of emerging metabolic therapies for glioma, such as inhibitors of glycolytic and mitochondrial metabolism (20).
Recent studies (10,11) have established the safety and feasibility of hyperpolarized pyruvate MR in patients with brain tumors and demonstrated higher lactate production in some tumors relative to background brain metabolism. The unique information offered at hyperpolarized pyruvate imaging complements the advanced MRI methods often used in patients with brain tumors. Proton spectroscopy and recently introduced methods for deuterium spectroscopy (21) can interrogate the quantities of key metabolites including lactate in healthy and diseased tissues; however, these are steady-state measurements of metabolite pool size and not the direct delineations of enzymatic flux provided at hyperpolarized MR spectroscopy. The combination of more established advanced MRI methods and real-time metabolic imaging with hyperpolarized MR spectroscopy could offer improvements in evaluating glioma disease state and treatment response.
Although it provided detailed dynamic spectroscopic data, the acquisition method used in our study is limited by its lack of spatial resolution. Our data were acquired from the region of the brain containing the tumor, weighted by proximity to the 13C surface coil used for hyperpolarized signal reception. The tumors interrogated in our study were implanted at a relatively superficial depth. They were large and well-vascularized at the time of hyperpolarized pyruvate MR spectroscopy. Therefore, despite the contribution of signal from healthy tissue, the statistically significant decrease in lactate after administration of WP1122 in mice with tumors was greater than the modest increase in lactate observed in normal matched brain anatomy (Fig 3) and can be considered representative of tumor metabolism. Whereas there are imaging methods capable of discriminating hyperpolarized signals in tumors from other tissues, the application of these methods would incur a diminished signal-to-noise ratio relative to section-selective spectroscopy (22), and with relatively coarse spatial resolution would not eliminate partial volume averaging effects. Because of the heterogeneous distribution of tissues measured at hyperpolarized MR spectroscopy and the nonuniform signal reception profile, we chose to quantify our data with a precursor-product model and an area under the curve method. More complicated and physiologically accurate models account for the transport of hyperpolarized substrates between metabolically distinct physical compartments; however, they require estimation of a vascular input function and fitting of several additional unknown parameters (13). The quantification methods that we used are a compromise given the limited hyperpolarized signal available from the small quantity of pyruvate that can be administered in a mouse.
Further research is needed to establish the utility of hyperpolarized pyruvate measurements in clinical decision making for patients with glioma. Hyperpolarized pyruvate imaging in humans has resolved differences in metabolism between regions within healthy brains (23), between normal brain tissue and tumor, and between subregions of large tumors (10,11). More thorough clinical trials must be completed to quantify the variability in standardized acquisition and analysis methods before this technology can guide treatment decisions.
Our results show that hyperpolarized MR spectroscopy with [1–carbon 13]-pyruvate can depict both baseline metabolism and treatment effects of antiglycolytic therapy, and holds promise for rapidly and noninvasively quantifying the metabolic state of gliomas.
Acknowledgments
Acknowledgments
The authors gratefully acknowledge Jorge de la Cerda, BA, and Charles Kingsley, AAS, for their assistance with animal handling and preparation, as well as Verlene Henry, BS, and Caroline Carrillo, BS, for their help with tumor cell implantations.
Study supported by Marnie Rose Foundation, the National Cancer Institute (P30-CA016672), and Moleculin Biotech. The content is solely the responsibility of the authors and does not necessarily represent the official views of these agencies.
Disclosures of Conflicts of Interest: K.A.M. disclosed no relevant relationships. R.Z. disclosed no relevant relationships. C.M.W. disclosed no relevant relationships. L.L.R. disclosed no relevant relationships. W.P. Activities related to the present article: disclosed a grant from and shares in Moleculin Biotech; disclosed that author is the chair of the Scientific Advisory Board for Moleculin Biotech. Activities not related to the present article: disclosed royalties from patents issued and licensed related to WP1122 . Other relationships: disclosed no relevant relationships. J.A.B. disclosed no relevant relationships. D.S. disclosed no relevant relationships.
Abbreviation:
- nLac
- normalized lactate ratio
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