Magnetic Resonance in Medicine 72:1375–1380 (2014)

Functional Molecular Imaging of Tumors by Chemical Exchange Saturation Transfer MRI of 3-O-Methyl-DGlucose Michal Rivlin,1 Ilan Tsarfaty,2 and Gil Navon1* enables obtaining images of endogenous cellular components or exogenous agents by MRI (7–9). The imaging of glucose, 2DG and FDG using CEST MRI was recently demonstrated and suggested to be useful for cancer diagnosis (10–13). The CEST signal obtained from glucose is lower than that obtained from 2DG and FDG due to its rapid conversion to lactic acid by glycolysis. However, the toxicity of these analogues at high concentrations (14–17) limits their use to laboratory animals. 3-O-Methyl-D-glucose (3OMG), a nonmetabolizable derivative of glucose, is taken up rapidly and preferentially by tumors (18), but, unlike glucose, 2DG or FDG, 3OMG does not serve as substrate for hexokinase and therefore does not undergo phosphorylation (18,19) and is entirely excreted by the kidneys (20,21). It is generally considered to be nontoxic, but detailed studies of its toxicity are lacking. In the present work, we demonstrate an enhanced CEST MRI image in mice carrying xenograph breast tumors following the injection of 3OMG. Our results suggest that 3OMG-CEST may serve as an alternative to PET/CT in cases in which the radiation associated with the latter modality needs to be avoided. Like PET, it has the potential to detect tumors and metastases, distinguish between malignant and benign tumors and monitor tumor response to therapy.

Purpose: To evaluate the feasibility to detect tumors and metastases by the chemical exchange saturation transfer (CEST) MRI technique using 3-O-Methyl-D-glucose (3OMG), a nonmetabolizable derivative of glucose that is taken up rapidly and preferentially by tumors and is entirely excreted by the kidneys. Methods: In vivo CEST MRI experiments were performed on a Bruker 7 Tesla Biospec on implanted orthotopic mammary tumors of mice before and following i.p. injection of 3OMG. The CEST images were generated by a series of gradientecho images collected from a single 1 mm coronal slice after a 1.2 s presaturation pulse, applied at offsets of 61.2 ppm from the water and at B1 power of 2.5 mT. Results: Following 3OMG (1.5 g/kg) i.p. injection, an enhanced CEST effect of approximately 20% was visualized at the tumor within a few minutes. The signal slowly declined reaching half of its maximum at approximately 80 min. Conclusion: Due to the large CEST effect of 3OMG and its low toxicity 3OMG-CEST may serve for the detection of tumors and metastases in the clinic. Magn Reson Med C 2014 Wiley Periodicals, Inc. 72:1375–1380, 2014. V Key words: MRI; breast cancer; chemical exchange saturation transfer; CEST; 3-O-Methyl-D-glucose

INTRODUCTION Glucose and its analogues 2-deoxy-D-glucose (2DG) and 2fluoro-deoxy-D-glucose (FDG) are known to be taken up preferentially by cancer cells, a phenomenon known as the “Warburg effect” (1). Like glucose, these analogues are subject to phosphorylation catalyzed by the hexokinase, but they do not undergo further metabolism by means of the glycolysis pathway and accumulate in the cells. The positron emission tomography (PET) method makes use of this phenomenon by imaging the accumulation of the radioactive fluorine atom 18F of the FDG (2–5). The chemical exchange saturation transfer (CEST) MRI method takes advantage of magnetization transfer between residues with exchangeable protons, such as amine, amide or hydroxyl and water, enabling their detection at low concentrations (6). The enhanced sensitivity of the method


School of Chemistry, Tel Aviv University, Tel Aviv, Israel. Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel Aviv University, Israel. Grant sponsor: Israel Science Foundation; Grant sponsor: Breast Cancer Research Foundation; Grant sponsor: the United States-Israel Binational Science Foundation. *Correspondence to: Gil Navon, School of Chemistry, Tel Aviv University, Tel Aviv, 69978, Israel. E-mail: [email protected] 2

Received 2 May 2014; revised 28 August 2014; accepted 28 August 2014 DOI 10.1002/mrm.25467 Published online 18 September 2014 in Wiley Online Library ( C 2014 Wiley Periodicals, Inc. V

METHODS Chemicals and Media 3OMG and D-glucose were obtained from Sigma-Aldrich, Israel. Animals The animal model was essentially the same as that reported in our previous publication (13). BALB/C female mice were purchased and kept in the breeding facility of the Sackler School of Medicine, Tel Aviv University. To induce orthotropic xenograph tumors in the mice, DA3-D1-DMBA-3 cells were injected into the lower left mammary gland of 8-week-old (17–22 gram) animals (5  105 cells in 100 mL saline). The tumors were allowed to grow for 10–14 days, reaching an average tumor volume of 5 mm3. All experiments with animal models were carried out in compliance with the principles of the National Research Council (NRC) and were approved by the institutional animal care and use committee (IACUC) (#M-12–035). Preparation of tumor extracts The tumors were surgically excised and immediately weighed and immersed in liquid nitrogen. The frozen



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FIG. 1. Spectra of the hydroxyl protons of 100 mM 3-OMG in H2O at pH 5.77, temperature ¼ 4 C. Spectrum taken a short time after dissolution (a) and 24 h after dissolution (b). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

tumors were homogenized with a tissue homogenizer, using the methanol/CHCl3/H2O extraction method (22) with a volume ratio of 2/2/1.8, respectively. After centrifugation (4 C, 4000  g, 12 min), only the upper aqueous phase was kept for analysis. The samples were dried gently by evaporator, frozen at 80 C, and lyophilized to dryness for 24 h. Each sample was dissolved in 0.5 mL D2O (99.98%, Biolab, Israel), adjusted to pH of 6.5–7 and inserted into a 5-mm tube for NMR. NMR Spectroscopy 1

H NMR spectra were recorded at B0 ¼ 11.7T in 5-mm tubes on a Bruker 500 MHz DRX with the following parameters: spectral width 7500 Hz, pulse width 5.5 us (corresponding to a 45 flip angle); data size 16 K; relaxation delay 10 s; number of scans ¼ 16. The spectrometer was de-tuned to avoid radiation damping effects. CEST NMR experiments were performed by applying a long off-resonance presaturation pulse before acquisition. A series of frequencies (V) were used in the range of 3.5 to þ3.5 ppm relative to the water signal. Several rf saturation fields (B1) in the range of 1–6 mT (50–250 Hz) and durations of 2 s were used. The chemical exchange contrast was measured by magnetization transfer asymmetry,MTRasym: MTRasym(V) ¼ [MCEST(-V)-MCEST(V)]/Mo. CEST MRI A Bruker 7 Tesla (T) Biospec scanner with 30 cm bore size was used to scan implanted xenograph mammary tumors of mice before and after i.p. injection of the glucose analog 3OMG in saline, pH 7.4. In our previous work on 2DG and FDG (13), we found no major difference between i.p or i.v. injections. DA3 tumor-bearing mice, with an average tumor volume of 5 mm3, were anesthetized with isoflurane (1–2%) and scanned with surface coil. Their temperature was maintained at 37 C.

In Vivo CEST Images were generated as follows: a series of gradientecho images were collected from a single 1 mm coronal slice of the abdominal area (acquisition matrix 128  64, field of view of 40  40 mm2) after a 1.2 s presaturation pulse of 2.5 mT (106 Hz) at 61.2 ppm from the water signal. The mean intensities in the selected region of interest (ROI) in the tumor were used for the MTRasym plot. No corrections for the B0 inhomogeneity were used in the present work because no significant change was observed after such correction in our previous work (13) using the same mouse model. The linewidth of the water peak was approximately 60 Hz. RESULTS In Vitro Studies To isolate the peaks of the hydroxyl protons of 3OMG, the potential source of the CEST effect, the conditions of pH ¼ 5.77 and a temperature of 4 C were chosen. Figure 1 depicts the spectra of 3OMG a short time after dissolution and after 24 h. Apparently 3OMG, like glucose, exists in the solid state as the a-anomer, and the rate of mutarotation is slow. As a point of reference, the rates of the mutarotation of glucose are 1.0  103 min1 and 1.0  102 min1 at temperature ¼ 3 C and 25 C, respectively (23). This slow rate should be taken into consideration when using freshly prepared solutions. The assignment of the hydroxyl protons peaks in Figure 1 was based on the assignment of a-3OMG in DMSO-d6 (24) and the correlation between the peaks of the hydroxyl protons of glucose in water and DMSO (25). The peaks of the hydroxyl protons of 3OMG at pH ¼ 5.77 are broadened considerably with rising temperature or increasing pH, and at pH 7.4 and temperature ¼ 37 C they are completely invisible. However, under these conditions they give rise to a considerable

Functional Molecular Imaging of Tumors


FIG. 2. Z spectra (a) and MTRasym (b) plot of 10 mM 3OMG solution (containing 10 mM phosphate buffer and 10% D2O) as a function of the rf saturation field at pH ¼ 7.5, temper(B1) ature ¼ 37 C. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]

CEST effect. The Z-spectra and the MTRasym plots for 10 mM 3OMG at pH ¼ 7.5 and temperature ¼ 37 C at different rf saturation fields (B1) are shown in Figure 2. The lack of resolution in these plots is a result of the extensive broadening due to enhanced chemical exchange at this pH and temperature. It is interesting to note that the MTRasym plots shift to a lower field when the rf saturation field is raised, most likely because of the different exchange rates of the different hydroxyl protons. Based on the assignment given in Figure 1 it can be concluded that the exchange rate is slowest for the hydroxyls at the 6th position and fastest for the anomeric hydroxyls at position 1. A simple calculation indicates that because the proton concentration in water is 111 M, 14% CEST for a 3OMG concentration of 10 mM corresponds to an enhancement factor of approximately 1500 relative to the direct detection of 3OMG. Figure 3 shows the CEST curves for 10mM 3OMG solutions at temperature ¼ 25 C and a frequency offset of 1.2 ppm for different pH and rf saturation field values. At this temperature and the pH range of 7.1–7.5, peaks at 0.3, 1, 2.1 and 2.7 ppm are clearly resolved for the different OH groups. The pH dependence is different at different frequency offsets. For instance, at a frequency

FIG. 3. MTRasym plot of a 10 mM 3OMG solution (containing 10 mM phosphate buffer and 10% D2O) with pH values from 6.3–8 measured at different frequencies offset from water (B1 ¼ 2.5 mT) (a) and at frequency offset of 1.2 ppm as a function of the rf saturation field (b). (temperature ¼ 25 C). (B1) [Color figure can be viewed in the online issue, which is available at]

offset of around 3 ppm, there is a sharp increase in the CEST effect when the pH decreases from 8.02 to 6.34. This is due to the pH dependence of the exchange rate of the anomeric hydroxyl proton of a-3OMG. To compare the extent of accumulation of D-glucose and 3OMG in the tumors, they were excised 25 min after i.p. injection of the agents (1.5 g/kg) and their extracts were prepared. The MTRasym plot of the combined extracts of three tumors for each agent is shown in Figure 4. As can be seen, the CEST effect for the peak at 1.2 ppm was significantly higher for the tumors treated with 3OMG compared with those treated with D-glucose. Because the CEST effects of 3OMG and glucose solutions at the same concentrations were approximately the same, the higher CEST effect of 3OMG in the extracts reflects its higher concentration in the tumors. In Vivo Studies Having demonstrated in vitro that 3OMG concentrations of only a few millimolar could be detected with CEST (Figs. 2–4), we set out to determine the ability of 3OMG CEST MRI to image mammary tumors, in vivo. Toward this end, we injected i.p. a 3OMG solution into mice


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As seen in Figure 5, a strong and sharp CEST effect was visualized at the tumor within a few min of the 3OMG injection (1.5 g/kg), and reached 20% above the control at 9 min. With the exception of the urinary bladder no other organs showed a significant CEST effect on the MRI scans session. The enhanced CEST declined slowly reaching half its maximum value after approximately 80 min. These results were replicated in five experiments with initial CEST enhancement of 20.7 6 1.6%. DISCUSSION

FIG. 4. MTRasym plots of combined extracts of 3 tumors treated with 3OMG or with D-glucose (B1 ¼ 2.5 mT, pH ¼ 7.0, temperature ¼ 25 C).

bearing DA3 tumors after imaging the tumor anatomy by T2-weighted spin echo sequence (RARE). The images obtained in one of the experiments are shown in Figure 5 together with the calculated CEST values for a total of five experiments. The uneven contrast observed in the images most likely results from B1 and not B0 inhomogeneities, because they are apparent in the T2-weighted images as well.

Our CEST NMR studies of 3OMG in aqueous solutions indicate that the method enables the detection of the compound with an enhanced sensitivity amounting to over 1000-fold compared with its direct detection. This, together with the selective uptake of the compound by tumors and its low toxicity, points to its potential usefulness in the detection and monitoring of tumors. 3OMG CEST MRI showed significant signal enhancement in the tumor regions of more than 20% that started a very short time after injection of the compound, slowly decayed and retained more than 70% of its maximum 1 h after injection. The initial enhancement with 3OMG in the present work is similar to levels reported in our previous studied with 2DG and FDG (13) indicates similar level of initial enhancement, but the enhancement with the latter agents persisted for more than 2 h. This is easily explained by fact that the CEST effect is a combined result of both 2DG and its phosphorylation product 2DG-6-P, both of which have similar CEST effects (12), and are eliminated

FIG. 5. CEST MRI kinetic measurements in the tumor at different times following injection of 3OMG, 1.5 g/kg. (B1 ¼ 2.5 mT, B0 ¼ 7 T). a: A T2-weighted image before the administration of the agent. b: A CEST image before the administration of the agent. c: A CEST image 9 min after the injection. The marked ROI was used for the CEST calculation. d: The time series of the %CEST for the five mice tested.

Functional Molecular Imaging of Tumors

over long periods of time. The phosphorylation process of 2DG was previously found in implanted MCF-7 tumors in mice to last for approximately 2 h (26). CEST MRI using 2DG and FDG as contrast agents to replace PET/CT or PET/MRI in cancer research has a place in laboratory animals, but the method is limited in human studies by the toxicity of the agents at high concentrations: a dose of 3 g/kg of 2DG was reported to be partially toxic in rats (14), and the dosage of 2DG for humans in the clinic was limited to 60–300 mg/kg (15–17). The lack of toxicity of glucose makes glucoCEST (10,11) an appealing modality, although glucose administration under the same experimental setups as we used here for 3OMG resulted in lower initial CEST enhancement, that declined sharply and reached only 3% above the control value at 25 min after the glucose administration (13). In agreement with our previous findings (13), Nasrallah et al (12) reported that the CEST effect of glucose in the brain was approximately half that of 2DG, and completely vanished 15 min after a bolus injection of glucose. These authors were, however able to maintain the CEST effect of glucose for a long time by continuous infusion of glucose. One problem with the CEST MRI of glucose and its derivatives is the relatively small frequency offset from the water peak that gives maximal CEST effect. Our results were obtained with a 7T MRI scanner, a field not routinely used clinically. CEST MRI with the same frequency offset of 1.2 ppm used here was successfully used in clinical 3T MRI scanners. One example is the gagCEST (27), based on sugar hydroxyl protons of the glycosaminoglycan, which are similar to those of the glucose-based CEST (glucoCEST). Clinical application of 3OMG CEST MRI may require a multi-slice or 3D imaging. The slow decay of the CEST allows enough time to perform such a procedure. The widely-used FDG-PET/CT has the advantage over other imaging modalities of sensitivity to small tumors and metastases and their levels of metabolic activity and malignancy. Because CEST MRI of 3OMG is based on the same physiological property of the glucose avidity of cancerous tumors, it may have similar advantages to those of PET/CT. However, the radiation hazard posed by PET/CT hampers its use for ongoing monitoring of the development of tumors or the effectiveness of therapeutic modalities such as chemotherapies. Thus, 3OMGCEST MRI may offer an alternative to PET/CT in cases where radiation must be avoided.

CONCLUSIONS The usefulness of 3OMG as the contrast agent with MRI CEST for cancer detection is demonstrated. The agent’s large CEST effect and low toxicity make it potentially useful for the detection and development of tumors and metastases and the monitoring of therapy in the clinic. ACKNOWLEDGMENTS The authors thank Sari Natan, for help with animal models, Dr. Yael Piontkewitz for assistance in handling the


animals during the MRI experiments, and Dr. Hadassah Shinar for obtaining the low temperature 3OMG spectra. We thank the Strauss Center for Computational Neuroimaging, the Sackler Institute for Biophysics at Tel Aviv University, and the Israel Science Foundation for the purchase of the MRI system. REFERENCES 1. Warburg O. On the origin of cancer cells. Science 1956;123:309– 314. 2. Foehrenbach H, Alberini JL, Maszelin P, Bonardel G, Tenenbaum F, de Dreuille O, Richard B, Gaillard JF, Devaux JY. [Positron emission tomography in clinical oncology]. Presse Med 2003;32:276–283. 3. Czernin J, Phelps ME. Positron emission tomography scanning: current and future applications. Annu Rev Med 2002;53:89–112. 4. Kubota K. From tumor biology to clinical Pet: a review of positron emission tomography (PET) in oncology. Ann Nucl Med 2001;15: 471–486. 5. Gambhir SS. Molecular imaging of cancer with positron emission tomography. Nat Rev Cancer 2002;2:683–693. 6. Ward KM, Aletras AH, Balaban RS. A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST). J Magn Reson 2000;143:79–87. 7. Vinogradov E, Sherry AD, Lenkinski RE. CEST: from basic principles to applications, challenges and opportunities. J Magn Reson 2013; 229:155–172. 8. Zaiss M, Bachert P. Chemical exchange saturation transfer (CEST) and MR Z-spectroscopy in vivo: a review of theoretical approaches and methods. Phys Med Biol 2013;58:R221–R269. 9. Liu G, Song X, Chan KW, McMahon MT. Nuts and bolts of chemical exchange saturation transfer MRI. NMR Biomed 2013;26:810– 828. 10. Chan KW, McMahon MT, Kato Y, Liu G, Bulte JW, Bhujwalla ZM, Artemov D, van Zijl PC. Natural D-glucose as a biodegradable MRI contrast agent for detecting cancer. Magn Reson Med 2012;68:1764–1773. 11. Walker-Samuel S, Ramasawmy R, Torrealdea F, et al. In vivo imaging of glucose uptake and metabolism in tumors. Nat Med 2013;19:1067– 1072. 12. Nasrallah FA, Pages G, Kuchel PW, Golay X, Chuang KH. Imaging brain deoxyglucose uptake and metabolism by glucoCEST MRI. J Cereb Blood Flow Metab 2013;33:1270–1278. 13. Rivlin M, Horev J, Tsarfaty I, Navon G. Molecular imaging of tumors and metastases using chemical exchange saturation transfer (CEST) MRI. Sci Rep 2013;3:1–7. 14. Miller LP, Villeneuve JB, Braun LD, Oldendorf WH. Effect of pharmacological doses of 3-0-methyl-D-glucose and 2-deoxy-D-glucose on rat brain glucose and lactate. Stroke 1986;17:957–961. 15. Stein M, Lin H, Jeyamohan C, Dvorzhinski D, Gounder M, Bray K, Eddy S, Goodin S, White E, Dipaola RS. Targeting tumor metabolism with 2-deoxyglucose in patients with castrate-resistant prostate cancer and advanced malignancies. Prostate 2010;70:1388–1394. 16. Raez LE, Papadopoulos K, Ricart AD, et al. A phase I dose-escalation trial of 2-deoxy-D-glucose alone or combined with docetaxel in patients with advanced solid tumors. Cancer Chemother Pharmacol 2013;71:523–530. 17. Dwarakanath BS, Singh D, Banerji AK, et al. Clinical studies for improving radiotherapy with 2-deoxy-D-glucose: present status and future prospects. J Cancer Res Ther 2009;5(Suppl. 1):S21–S26. 18. Hwang YY, Kim SG, Evelhoch JL, Ackerman JJ. Nonglycolytic acidification of murine radiation-induced fibrosarcoma 1 tumor via 3-O-methyl-D-glucose monitored by 1H, 2H, 13C, and 31P nuclear magnetic resonance spectroscopy. Cancer Res 1992;52: 1259–1266. 19. Xu YZ, Krnjevic K. Unlike 2-deoxy-D-glucose, 3-O-methyl-D-glucose does not induce long-term potentiation in rat hippocampal slices. Brain Res 2001;895:250–252. 20. Csaky TZ, Glenn JE. Urinary recovery of 3-methylglucose administered to rats. Am J Physiol 1957;188:159–162. 21. Jay TM, Dienel GA, Cruz NF, Mori K, Nelson T, Sokoloff L. Metabolic stability of 3-O-methyl-D-glucose in brain and other tissues. J Neurochem 1990;55:989–1000. 22. Martineau E, Tea I, Loaec G, Giraudeau P, Akoka S. Strategy for choosing extraction procedures for NMR-based metabolomic

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Functional molecular imaging of tumors by chemical exchange saturation transfer MRI of 3-O-Methyl-D-glucose.

To evaluate the feasibility to detect tumors and metastases by the chemical exchange saturation transfer (CEST) MRI technique using 3-O-Methyl-D-gluco...
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