Researcher Profile and Settings

Affiliation

• Faculty of Information Science and Technology　Bioengineering and Bioinformatics　Bioengineering

Job Title

• Associate Professor

URL

http://www.ist.hokudai.ac.jp/labo/mre/index-j.html

J-Global ID

• 200901060827679837

Research Interests

• Molecular Imaging   Metabolism   pathophysiology   Neuroscience   Hyperpolarized 13C MRI   Physical Chemistry   Radiology   Quantum Chemistry   Radiation Biology   Nuclear Hyperpolarization   Electron Spin Resonance   

Research Areas

• Life sciences / Pharmaceuticals - analytical and physicochemistry
• Life sciences / Physiology
• Life sciences / Cognitive neuroscience
• Informatics / Biological, health, and medical informatics
• Life sciences / Tumor diagnostics and therapeutics
• Manufacturing technology (mechanical, electrical/electronic, chemical engineering) / Measurement engineering
• Life sciences / Medical systems

Educational Organization

•  Bachelor's degree program　School of Engineering
•  Master's degree program　Graduate School of Information Science and Technology
•  Doctoral (PhD) degree program　Graduate School of Information Science and Technology

Academic & Professional Experience

• 2015/02 - Today Hokkaido University Information Science and Technology Associate Professor
• 2014/07 - 2015/01 National Cancer Institute, NIH (Radiation Biology Branch) Staff Scientitst
• 2009/12 - 2014/07 National Cancer Institute, NIH (Radiation Biology Branch) Research Fellow
• 2005/12 - 2009/12 National Cancer Institute, NIH
• 2005/04 - 2006/11 Kyushu University
• 2004/04 - 2005/03 Kyushu University

Education

• 2002/04 - 2005/03  Kyushu University
• 2000/04 - 2002/04  Kyushu University
• 1996/04 - 2000/03  Kyushu University

Association Memberships

• 日本医学物理学会   The Parahydrogen Enhanced Resonance Meeting PERM   International Society of Magnetic Resonance   日本分子イメージング学会   

Research Activities

Published Papers

• Parahydrogen-induced ¹³C hyperpolarizer using a flow guide for magnetic field cycling to evoke ¹H-¹³C spin order transfer toward metabolic MRI

  Koudai Sawami, Tatsuya Naganuma, Hana Yabe, Toshihiko Taki, Neil J. Stewart, Yoshiki Uchio, Norihiko Takeda, Noriyuki Hatae, Takuya Hashimoto, Hiroshi Hirata, Shingo Matsumoto

  IEEE Transactions on Biomedical Engineering 1 - 8 0018-9294 2024 [Refereed][Not invited]
  
• Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo

  Ririko Nakaoka, Kazuhiro Kato, Kumiko Yamamoto, Hironobu Yasui, Shingo Matsumoto, Igor A. Kirilyuk, Valery V. Khramtsov, Osamu Inanami, Hiroshi Hirata

  Analytical Chemistry 0003-2700 2023/02/01
• Continuous monitoring of post-irradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

  Tatsuya Kawai, Masayuki Matsuo, Yoichi Takakusagi, Keita Saito, Fuminori Hyodo, Nallathamby Devasahayam, Shingo Matsumoto, Shun Kishimoto, Hironobu Yasui, Kazutoshi Yamamoto, Murali C Krishna

  NMR in biomedicine 35 (10) e4783  2022/06/03 

  BACKGROUND: Reoxygenation has a significant impact on the tumor response to radiotherapy. With the developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. PURPOSE: In the present study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2 ) in tumor tissues. MATERIALS AND METHODS: Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an X-ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2-weighted anatomical images for reference. The differences in the mean pO2 were determined through two-tailed Student's t-test and one-way analysis of variance (ANOVA). RESULTS: The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, P = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose-dependent increase in the hypoxic areas (pO2 < 10 mmHg) 2 h and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2 > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. CONCLUSION: We demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings might lead to new knowledge of the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation-based radiation therapy.
• Hyperpolarized 13C Magnetic Resonance Imaging as a Tool for Imaging Tissue Redox State, Oxidative Stress, Inflammation, and Cellular Metabolism

  Stewart NJ, Sato T, Takeda N, Hirata H, Matsumoto S

  Antioxid Redox Signal 36 (1-3) 81 - 94 2022/01 [Refereed][Invited]
  
• Redox-sensitive mapping of a mouse tumor model using sparse projection sampling of electron paramagnetic resonance

  Kimura K, Iguchi N, Nakano H, Yasui H, Matsumoto S, Inanami O, Hirata H

  Antioxid Redox Signal 36 (1-3) 57 - 69 1523-0864 2022/01 [Refereed][Not invited]
  
• Synthesis of [1-13C-5-12C]-alpha-ketoglutarate enables noninvasive detection of 2-hydroxyglutarate.

  Miura N, Mushti C, Sail D, AbuSalim JE, Yamamoto K, Brender JR, Seki T, AbuSalim DI, Matsumoto S, Camphausen KA, Krishna MC, Swenson RE, Kesarwala AH

  NMR Biomed 34 (11) e4588  2021/11 [Refereed]
  
• Biomedical Applications of the Dynamic Nuclear Polarization and Parahydrogen Induced Polarization Techniques for Hyperpolarized ¹³C MR Imaging

  Stewart NJ, Matsumoto S

  Magn Reson Med Sci 20 (1) 1 - 17 1347-3182 2021/05 [Refereed][Invited]
  
• Hyperpolarized 13 C Magnetic Resonance Imaging of Fumarate Metabolism by Parahydrogen-induced Polarization: A Proof-of-Concept in vivo Study

  Stewart NJ, Nakano H, Sugai S, Tomohiro M, Kase Y, Uchio Y, Yamaguchi T, Matsuo Y, Naganuma T, Takeda N, Nishimura I, Hirata H, Hashimoto T, Matsumoto S

  ChemPhysChem 22 (10) 915 - 923 1439-4235 2021/02 [Refereed][Invited]
  
• Macrophage derived TNFα promotes hepatic reprogramming to Warburg-like metabolism.

  Tarasenko TN, Jestin M, Matsumoto S, Saito K, Hwang S, Gavrilova O, Trivedi N, Zerfas PM, Barca E, DiMauro S, Senac J, Venditti CP, Cherukuri M, McGuire PJ

  J Mol Med 97 (9) 1231 - 1243 2019/09 [Refereed][Not invited]
  
• Imaging of glucose metabolism by 13C-MRI distinguishes pancreatic cancer subtypes in mice.

  Kishimoto S, Brender JR, Crooks DR, Matsumoto S, Seki T, Oshima N, Merkle H, Lin P, Reed G, Chen AP, Ardenkjaer-Larsen JH, Munasinghe J, Saito K, Yamamoto K, Choyke PL, Mitchell J, Lane AN, Fan TW, Linehan WM, Krishna MC

  eLIFE 8 e46312  2019/08 [Refereed][Not invited]
  
• Synthesis and evaluation of 13C-labeled 5-5-dimethyl-1-pyrroline-N-oxide aimed at in vivo detection of reactive oxygen species using hyperpolarized 13C-MRI.

  Saito K, Sail D, Yamamoto K, Matsumoto S, Blackman B, Kishimoto S, Brender JR, Swenson RE, Mitchell JB, Krishna MC

  Free Radic Biol Med 1 (131) 18 - 26 2019/02 [Refereed][Not invited]
  
• Simultaneous T2^* mapping of ¹⁴N- and ¹⁵N-labeled dicarboxy-PROXYLs using CW-EPR-based single-point imaging

  Yokoyama, T., Taguchi, A., Kubota, H., Stewart, N.J., Matsumoto, S., Kirilyuk, I.A., Hirata, H.

  Journal of Magnetic Resonance 305 2019 [Refereed][Not invited]
  
• Proteasome inhibition disrupts the metabolism of fumarate hydratase- deficient tumors by downregulating p62 and c-Myc

  Sourbier, C., Ricketts, C.J., Liao, P.-J., Matsumoto, S., Wei, D., Lang, M., Railkar, R., Yang, Y., Wei, M.-H., Agarwal, P., Krishna, M., Mitchell, J.B., Trepel, J.B., Neckers, L., Linehan, W.M.

  Scientific Reports 9 (1) 2019 [Refereed][Not invited]
  
• Long-range heteronuclear J-coupling constants in esters: Implications for ¹³C metabolic MRI by side-arm parahydrogen-induced polarization

  Neil J. Stewart, Hiroyuki Kumeta, Mitsushi Tomohiro, Takuya Hashimoto, Noriyuki Hatae, Shingo Matsumoto

  Journal of Magnetic Resonance 296 85 - 92 1090-7807 2018/11 [Refereed][Not invited]
   

  © 2018 Elsevier Inc. Side-arm parahydrogen induced polarization (PHIP-SAH) presents a cost-effective method for hyperpolarization of 13C metabolites (e.g. acetate, pyruvate) for metabolic MRI. The timing and efficiency of typical spin order transfer methods including magnetic field cycling and tailored RF pulse sequences crucially depends on the heteronuclear J coupling network between nascent parahydrogen protons and 13C, post-parahydrogenation of the target compound. In this work, heteronuclear nJHC (1 < n ≤ 5) couplings of acetate and pyruvate esters pertinent for PHIP-SAH were investigated experimentally using selective HSQMBC-based pulse sequences and numerically using DFT simulations. The CLIP-HSQMBC technique was used to quantify 2/3-bond JHC couplings, and 4/5-bond JHC ≲ 0.5 Hz were estimated by the sel-HSQMBC-TOCSY approach. Experimental and numerical (DFT-simulated) nJHC couplings were strongly correlated (P < 0.001). Implications for 13C hyperpolarization by magnetic field cycling, and PH-INEPT and ESOTHERIC type spin order transfer methods for PHIP-SAH were assessed, and the influence of direct nascent parahydrogen proton to 13C coupling when compared with indirect homonuclear TOCSY-type transfer through intermediate (non-nascent parahydrogen) protons was studied by the density matrix approach.
• In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

  Komarov DA, Ichikawa Y, Yamamoto K, Stewart NJ, Matsumoto S, Yasui H, Kirilyuk IA, Khramtsov VV, Inanami O, Hirata H

  Anal Chem 2018/11 [Refereed][Not invited]
  
• Pulsed Electron Paramagnetic Resonance Imaging: Applications in the Studies of Tumor Physiology.

  Kishimoto S, Matsumoto KI, Saito K, Enomoto A, Matsumoto S, Mitchell JB, Devasahayam N, Krishna MC

  Antioxid Redox Signal 28 (15) 1378 - 1393 2018/11 [Refereed][Not invited]
  
• EPR-based oximetric imaging: a combination of single point-based spatial encoding and T1 weighting.

  Ken-Ichiro Matsumoto, Shun Kishimoto, Nallathamby Devasahayam, Gadisetti V R Chandramouli, Yukihiro Ogawa, Shingo Matsumoto, Murali C Krishna, Sankaran Subramanian

  Magnetic resonance in medicine 80 (5) 2275 - 2287 2018/11 [Refereed][Not invited]
   

  PURPOSE: Spin-lattice relaxation rate (R1 )-based time-domain EPR oximetry is reported for in vivo applications using a paramagnetic probe, a trityl-based Oxo71. METHODS: The R1 dependence of the trityl probe Oxo71 on partial oxygen pressure (pO2 ) was assessed using single-point imaging mode of spatial encoding combined with rapid repetition, similar to T1 -weighted MRI, for which R1 was determined from 22 repetition times ranging from 2.1 to 40.0 μs at 300 MHz. The pO2 maps of a phantom with 3 tubes containing 2 mM Oxo71 solutions equilibrated at 0%, 2%, and 5% oxygen were determined by R1 and apparent spin-spin relaxation rate ( R2*) simultaneously. RESULTS: The pO2 maps derived from R1 and R2* agreed with the known pO2 levels in the tubes of Oxo71. However, the histograms of pO2 revealed that R1 offers better pO2 resolution than R2* in low pO2 regions. The SDs of pixels at 2% pO2 (15.2 mmHg) were about 5 times lower in R1 -based estimation than R2*-based estimation (mean ± SD: 13.9 ± 1.77 mmHg and 18.3 ± 8.70 mmHg, respectively). The in vivo pO2 map obtained from R1 -based assessment displayed a homogeneous profile in low pO2 regions in tumor xenografts, consistent with previous reports on R2*-based oximetric imaging. The scan time to obtain the R1 map can be significantly reduced using 3 repetition times ranging from 4.0 to 12.0 μs. CONCLUSION: Using the single-point imaging modality, R1 -based oximetry imaging with useful spatial and oxygen resolutions for small animals was demonstrated.
• Impact of the characteristic impedance of coaxial lines on the sensitivity of a 750-MHz electronically tunable EPR resonator

  R. Nakaoka, D. A. Komarov, S. Matsumoto, H. Hirata

  Applied Magnetic Resonance 49 (8) 853 - 867 0937-9347 2018/08 [Refereed][Not invited]
  
• Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

  Matsumoto S, Kishimoto S, Saito K, Takakusagi Y, Munasinghe JP, Devasahayam N, Hart CP, Gillies RJ, Mitchell JB, Krishna MC

  Cancer Res. 78 (14) 3783 - 3792 2018/07 [Refereed][Not invited]
  
• Pulsed Electron Paramagnetic Resonance Imaging: Applications in the Studies of Tumor Physiology.

  Shun Kishimoto, Ken-Ichiro Matsumoto, Keita Saito, Ayano Enomoto, Shingo Matsumoto, James B Mitchell, Nallathamby Devasahayam, Murali C Krishna

  Antioxidants & redox signaling 28 (15) 1378 - 1393 2018/05/20 [Refereed][Not invited]
   

  SIGNIFICANCE: Electron paramagnetic resonance imaging (EPRI) is capable of generating images of tissue oxygenation using exogenous paramagnetic probes such as trityl radicals or nitroxyl radicals. The spatial distribution of the paramagnetic probe can be generated using magnetic field gradients as in magnetic resonance imaging and, from its spectral features, spatial maps of oxygen can be obtained from live objects. In this review, two methods of signal acquisition and image formation/reconstruction are described. The probes used and its application to study tumor physiology and monitor treatment response with chemotherapy drugs in mouse models of human cancer are summarized. Recent Advances: By implementing phase encoding/Fourier reconstruction in EPRI in time domain mode, the frequency contribution to the spatial resolution was avoided and images with improved spatial resolution were obtained. The EPRI-generated pO2 maps in tumor were useful to detect and evaluate the effects of various antitumor therapies on tumor physiology. Coregistration with other imaging modalities provided a better understanding of hypoxia-related alteration in physiology. CRITICAL ISSUES: The high radiofrequency (RF) power of EPR irradiation and toxicity profile of radical probes are the main obstacles for clinical application. The improvement of RF low power pulse sequences may allow for clinical translation. FUTURE DIRECTIONS: Pulsed EPR oximetry can be a powerful tool to research various diseases involving hypoxia such as cancer, ischemic heart diseases, stroke, and diabetes. With appropriate paramagnetic probes, it can also be applied for various other purposes such as detecting local acid-base balance or oxidative stress. Antioxid. Redox Signal. 28, 1378-1393.
• A 750-MHz Electronically Tunable Resonator Using Microstrip Line Couplers for Electron Paramagnetic Resonance Imaging of a Mouse Tumor-Bearing Leg.

  Tatsuya Amida, Ririko Nakaoka, Denis A Komarov, Kumiko Yamamoto, Osamu Inanami, Shingo Matsumoto, Hiroshi Hirata

  IEEE transactions on bio-medical engineering 65 (5) 1124 - 1132 2018/05 [Refereed][Not invited]
   

  OBJECTIVE: The purpose of this work was to develop an electronically tunable resonator operating at 750 MHz for continuous-wave electron paramagnetic resonance (CW-EPR) imaging of a mouse tumor-bearing leg. METHODS: The resonator had a multi-coil parallel-gap structure with a sample space of 16 mm in diameter and 20 mm in length. Microstrip line couplers were used in conjunction with varactor diodes to enable resonance frequency adjustment and to reduce the nonlinear effects of the varactor diodes. The resonator was modeled by the finite-element method and a microwave circuit simulation was performed to clarify its radiofrequency characteristics. RESULTS: A tunable resonator was evaluated in terms of its resonance frequency, tunable frequency band, and conversion efficiency of the RF magnetic field. The developed resonator provided a tunable frequency band of 4 MHz at a central frequency of 747 MHz and a conversion efficiency of 34 μT/W1/2. To demonstrate the application of this tunable resonator to EPR imaging, three-dimensional EPR images of a sample solution and a mouse tumor-bearing leg were obtained. CONCLUSION: The developed tunable resonator satisfied our initial requirements for in vivo EPR imaging and may be able to be further improved using the present finite-element and circuit models if any problems arise during future practical applications. SIGNIFICANCE: This work may help to promote EPR imaging of tumor-bearing mice in cancer-related studies.
• Radiotherapy Synergizes with the Hypoxia-Activated Prodrug Evofosfamide: In Vitro and In Vivo Studies

  Yoichi Takakusagi, Shun Kishimoto, Sarwat Naz, Shingo Matsumoto, Keita Saito, Charles P. Hart, James B. Mitchell, Murali C. Krishna

  ANTIOXIDANTS & REDOX SIGNALING 28 (2) 131 - 140 1523-0864 2018/01 [Refereed][Not invited]
   

  Aims: Evofosfamide (TH-302) is a hypoxia-activated prodrug (HAP) that releases the DNA-damaging bromo-isophosphoramide mustard (Br-IPM) moiety selectively under hypoxic conditions. Since solid tumors are known to have hypoxic regions, HAPs in combination with chemotherapy or radiotherapy (XRT) will be beneficial. We tested the oxygen dependence of release kinetics of Br-IPM using electron paramagnetic resonance (EPR) with spin trapping by monitoring redox cycling of the nitroimidazole moiety of TH-302, and oxygen dependence of TH-302 on in vitro cytotoxicity at different levels of hypoxia was also examined. Two tumor implants (SCCVII and HT29) in mice were studied. Results: TH-302 fragmentation to release Br-IPM was noticed at oxygen levels <76 mmHg, which increased with higher levels of hypoxia. Enhanced cellular cytotoxicity was also observed at oxygen levels <76 mmHg. In vivo pO(2) imaging in the two tumor implants showed that the SCCVII tumor implant had higher level of hypoxia compared with the HT29 xenograft. TH-302 as a monotherapy in vivo showed modest effects in SCCVII implants and minimal effects in HT29 xenografts, whereas TH-302 in combination with ionizing radiation showed significant benefit in both tumor models. Innovation: We examined the kinetics of redox cycling versus fragmentation of TH-302. The combination of oxygen-dependent XRT with TH-302 is effective even in tumors with significant hypoxia. Conclusions: Imaging studies identifying the magnitude of hypoxia in tumors indicated that the responsiveness to TH-302 and the antitumor effect of TH-302 were enhanced by combining with XRT in both the TH-302-sensitive SCCVII tumor and -resistant HT29 tumor. Antioxid. Redox Signal. 28, 131-140.
• Feasibility of in vivo three-dimensional T-2(*) mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging

  Harue Kubota, Denis A. Komarov, Hironobu Yasui, Shingo Matsumoto, Osamu Inanami, Igor A. Kirilyuk, Valery V. Khramtsov, Hiroshi Hirata

  MAGNETIC RESONANCE MATERIALS IN PHYSICS BIOLOGY AND MEDICINE 30 (3) 291 - 298 0968-5243 2017/06 [Refereed][Not invited]
   

  Objectives The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T-2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. Materials and methods Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-( 3,4-H-2(2))-(1-N-15)-1-oxyl (H-2, N-15-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(H-2(3)) methylpyrrolidin-(3,4-H-2(2))1- oxyl (H-2-DCP), were used in the study. A clonogenic cell survival assay was performed with the H-2-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected H-2, N-15-DCP and H-2-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T-2* mapping. Results H-2-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of H-2, N-15-DCP in tumor tissues was 24.7 +/- 2.9 min (mean +/- standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the H-2, N-15-DCP radical showed a plateau of 10.2 +/- 1.2 min (mean +/- SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T-2* maps with H-2, N-15-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. Conclusion EPR signals of H-2, N-15-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T-2* mapping with CW-EPR-based SPI.
• Effect of amifostine, a radiation-protecting drug, on oxygen concentration in tissue measured by EPR oximetry and imaging

  Megumi Ueno, Shingo Matsumoto, Atsuko Matsumoto, Sushma Manda, Ikuo Nakanishi, Ken-ichiro Matsumoto, James B. Mitchell, Murali C. Krishna, Kazunori Anzai

  JOURNAL OF CLINICAL BIOCHEMISTRY AND NUTRITION 60 (3) 151 - 155 0912-0009 2017/05 [Refereed][Not invited]
   

  Effect of amifostine, a radiation-protecting drug, on muscle tissue partial pressure of oxygen was investigated by electron paramagnetic resonance spectroscopy and imaging. When amifostine was administered intraperitoneally or intravenously to mice, the linewidth of the electron paramagnetic resonance spectra of the lithium octa-n-butoxy-substituted naphthalocyanine implanted in the mouse leg muscle decreased. Electron paramagnetic resonance oximetry using a lithium octa-n-butoxy-substituted naphthalocyanine probe and electron paramagnetic resonance oxygen mapping using a triarylmethyl radical paramagnetic probe was useful to quantify pressure of oxygen in the tissues of living mice. The result of electron paramagnetic resonance oximetric imaging showed that administration of amifostine could decrease pressure of oxygen in the muscle and also tumor tissues. This finding suggests that lowering pressure of oxygen in tissues might contribute in part to the radioprotection of amifostine.
• Quantitative imaging of pO(2) in orthotopic murine gliomas: hypoxia correlates with resistance to radiation

  Hironobu Yasui, Tatsuya Kawai, Shingo Matsumoto, Keita Saito, Nallathamby Devasahayam, James B. Mitchell, Kevin Camphausen, Osamu Inanami, Murali C. Krishna

  FREE RADICAL RESEARCH 51 (9-10) 861 - 871 1071-5762 2017 [Refereed][Not invited]
   

  Hypoxia is considered one of the microenvironmental factors associated with the malignant nature of glioblastoma. Thus, evaluating intratumoural distribution of hypoxia would be useful for therapeutic planning as well as assessment of its effectiveness during the therapy. Electron paramagnetic resonance imaging (EPRI) is an imaging technique which can generate quantitative maps of oxygen in vivo using the exogenous paramagnetic compound, triarylmethyl and monitoring its line broadening caused by oxygen. In this study, the feasibility of EPRI for assessment of oxygen distribution in the glioblastoma using orthotopic U87 and U251 xenograft model is examined. Heterogeneous distribution of pO(2) between 0 and 50mmHg was observed throughout the tumours except for the normal brain tissue. U251 glioblastoma was more likely to exhibit hypoxia than U87 for comparable tumour size (median pO(2); 29.7 and 18.2 mmHg, p = .028, in U87 and U251, respectively). The area with pO(2) under 10mmHg on the EPR oximetry (HF10) showed a good correlation with pimonidazole staining among tumours with evaluated size. In subcutaneous xenograft model, irradiation was relatively less effective for U251 compared with U87. In conclusion, EPRI is a feasible method to evaluate oxygen distribution in the brain tumour.
• C-13-MR Spectroscopic Imaging with Hyperpolarized [1-C-13]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors

  Keita Saito, Shingo Matsumoto, Yoichi Takakusagi, Masayuki Matsuo, H. Douglas Morris, Martin J. Lizak, Jeeva P. Munasinghe, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  CLINICAL CANCER RESEARCH 21 (22) 5073 - 5081 1078-0432 2015/11 [Refereed][Not invited]
   

  Purpose: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized C-13-MRI enabled detecting metabolic changes in tumors using a tracer [1-C-13] pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized C-13-MRI. Experimental Design: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-C-13] pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and C-13-MR signals were acquired using a 4.7 T scanner. Results: [1-C-13] pyruvate and [1-C-13] lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-C-13] pyruvate administration. The [1-C-13] lactate to [1-C-13] pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy x 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on C-13-MRI. Conclusions: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized C-13-MRI could be useful for the evaluation of early response to radiotherapy. (C) 2015 AACR.
• Accelerated 4D Quantitative Single Point EPR Imaging Using Model-Based Reconstruction

  Hyungseok Jang, Shingo Matsumoto, Nallathamby Devasahayam, Sankaran Subramanian, Jiachen Zhuo, Murali C. Krishna, Alan B. McMillan

  MAGNETIC RESONANCE IN MEDICINE 73 (4) 1692 - 1701 0740-3194 2015/04 [Refereed][Not invited]
   

  PurposeElectron paramagnetic resonance imaging has surfaced as a promising noninvasive imaging modality that is capable of imaging tissue oxygenation. Due to extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from single-point imaging and inherently suffers from limited spatial and temporal resolution, preventing localization of small hypoxic tissues and differentiation of hypoxia dynamics, making accelerated imaging a crucial issue. MethodsIn this study, methods for accelerated single-point imaging were developed by combining a bilateral k-space extrapolation technique with model-based reconstruction that benefits from dense sampling in the parameter domain (measurement of the T-2(*) decay of a free induction delay). In bilateral kspace extrapolation, more k-space samples are obtained in a sparsely sampled region by bilaterally extrapolating data from temporally neighboring k-spaces. To improve the accuracy of T-2(*) estimation, a principal component analysis-based method was implemented. ResultsIn a computer simulation and a phantom experiment, the proposed methods showed its capability for reliable T-2(*) estimation with high acceleration (8-fold, 15-fold, and 30-fold accelerations for 61x61x61, 95x95x95, and 127x127x127 matrix, respectively). ConclusionBy applying bilateral k-space extrapolation and model-based reconstruction, improved scan times with higher spatial resolution can be achieved in the current single-point electron paramagnetic resonance imaging modality. Magn Reson Med 73:1692-1701, 2015. (c) 2014 Wiley Periodicals, Inc.
• Pyruvate sensitizes pancreatic tumors to hypoxia-activated prodrug TH-302

  Wojtkowiak JW, Cornnell HC, Matsumoto S, Saito K, Takakusagi Y, Dutta P, Kim M, Zhang X, Leos R, Bailey KM, Martinez G, Lloyd MC, Weber C, Mitchell JB, Lynch RM, Baker AF, Gatenby RA, Rejniak KA, Hart C, Krishna MC, Gillies RJ

  Cancer Metab 3 (1) 2  2015/01 [Refereed][Not invited]
  
• Targeting ABL1-Mediated Oxidative Stress Adaptation in Fumarate Hydratase-Deficient Cancer

  Carole Sourbier, Christopher J. Ricketts, Shingo Matsumoto, Daniel R. Crooks, Pei-Jyun Liao, Philip Z. Mannes, Youfeng Yang, Ming-Hui Wei, Gaurav Srivastava, Sanchari Ghosh, Viola Chen, Cathy D. Vocke, Maria Merino, Ramaprasad Srinivasan, Murali C. Krishna, James B. Mitchell, Ann Marie Pendergast, Tracey A. Rouault, Len Neckers, W. Marston Linehan

  CANCER CELL 26 (6) 840 - 850 1535-6108 2014/12 [Refereed][Not invited]
   

  Patients with germline fumarate hydratase (FH) mutation are predisposed to develop aggressive kidney cancer with few treatment options and poor therapeutic outcomes. Activity of the proto-oncogene ABL1 is upregulated in FH-deficient kidney tumors and drives a metabolic and survival signaling network necessary to cope with impaired mitochondrial function and abnormal accumulation of intracellular fumarate. Excess fumarate indirectly stimulates ABL1 activity, while restoration of wild-type FH abrogates both ABL1 activation and the cytotoxicity caused by ABL1 inhibition or knockdown. ABL1 upregulates aerobic glycolysis via the mTOR/HIF1 alpha pathway and neutralizes fumarate-induced proteotoxic stress by promoting nuclear localization of the antioxidant response transcription factor NRF2. Our findings identify ABL1 as a pharmacologically tractable therapeutic target in glycolytically dependent, oxidatively stressed tumors.
• Pyruvate Induces Transient Tumor Hypoxia by Enhancing Mitochondrial Oxygen Consumption and Potentiates the Anti-Tumor Effect of a Hypoxia-Activated Prodrug TH-302

  Yoichi Takakusagi, Shingo Matsumoto, Keita Saito, Masayuki Matsuo, Shun Kishimoto, Jonathan W. Wojtkowiak, William DeGraff, Aparna H. Kesarwala, Rajani Choudhuri, Nallathamby Devasahayam, Sankaran Subramanian, Jeeva P. Munasinghe, Robert J. Gillies, James B. Mitchell, Charles P. Hart, Murali C. Krishna

  PLOS ONE 9 (9) e107995  1932-6203 2014/09 [Refereed][Not invited]
   

  Background: TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. Methodology/Results: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O-2), with minimal effect under aerobic conditions (21% O-2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (similar to 550 mm(3)), significantly delayed tumor growth. Conclusions/Significance: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.
• In Vivo Imaging of Tumor Physiological, Metabolic, and Redox Changes in Response to the Anti-Angiogenic Agent Sunitinib: Longitudinal Assessment to Identify Transient Vascular Renormalization

  Shingo Matsumoto, Keita Saito, Yoichi Takakusagi, Masayuki Matsuo, Jeeva P. Munasinghe, Herman D. Morris, Martin J. Lizak, Hellmut Merkle, Keiji Yasukawa, Nallathamby Devasahayam, Sankaran Suburamanian, James B. Mitchell, Murali C. Krishna

  ANTIOXIDANTS & REDOX SIGNALING 21 (8) 1145 - 1155 1523-0864 2014/09 [Refereed][Not invited]
   

  Aims: The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. Results: Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO(2) imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2-4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized C-13 MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. Innovation: Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. Conclusion: A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment.
• Magnetic Resonance Imaging of the Tumor Microenvironment in Radiotherapy: Perfusion, Hypoxia, and Metabolism

  Masayuki Matsuo, Shingo Matsumoto, James B. Mitchell, Murali C. Krishna, Kevin Camphausen

  SEMINARS IN RADIATION ONCOLOGY 24 (3) 210 - 217 1053-4296 2014/07 [Refereed][Not invited]
   

  The tumor microenvironment is characterized by hypoxia, low pH, and high interstitial fluid pressure. Hypoxic regions in tumors with low partial pressure of oxygen (pO(2)) levels can result in resistance to radiotherapy, thus causing local failure. Therefore, it would be desirable to noninvasively measure pO(2) levels in the tumor before, during, and after treatment to better customize therapy and follow treatment response. Several techniques used in preclinical and clinical studies to obtain the pO(2) status of tissue, such as dynamic contrast-enhanced magnetic resonance imaging, blood oxygen level dependent imaging, and electron paramagnetic resonance imaging, are reviewed. Furthermore, the ability to hyperpolarize specific metabolic substrates that are isotopically labeled with C-13 coupled with magnetic resonance spectroscopy enables noninvasive imaging of tissue metabolism, such as glycolysis. Published by Elsevier Inc.
• Four-Channel Surface Coil Array for 300-MHz Pulsed EPR Imaging: Proof-of-Concept Experiments

  Ayano Enomoto, Hiroshi Hirata, Shingo Matsumoto, Keita Saito, Sankaran Subramanian, Murali C. Krishna, Nallathamby Devasahayam

  MAGNETIC RESONANCE IN MEDICINE 71 (2) 853 - 858 0740-3194 2014/02 [Refereed][Not invited]
   

  Time-domain electron paramagnetic resonance imaging is currently a useful preclinical molecular imaging modality in experimental animals such as mice and is capable of quantitatively mapping hypoxia in tumor implants. The microseconds range relaxation times (T-1 and T-2) of paramagnetic tracers and the large bandwidths (tens of MHz) to be excited by electron paramagnetic resonance pulses for spatial encoding makes imaging of large objects a challenging task. The possibility of using multiple array coils to permit studies on large sized object is the purpose of the present work. Toward this end, the use of planar array coils in different configurations to image larger objects than cannot be fully covered by a single resonator element is explored. Multiple circular surface coils, which are arranged in a plane or at suitable angles mimicking a volume resonator, are used in imaging a phantom and a tumor-bearing mouse leg. The image was formed by combining the images collected from the individual coils with suitable scaling. The results support such a possibility. By multiplexing or interleaving the measurements from each element of such array resonators, one can scale up the size of the subject and at the same time reduce the radiofrequency power requirements and increase the sensitivity. Magn Reson Med 71:853-858, 2014. (c) 2013 Wiley Periodicals, Inc.
• Single Acquisition Quantitative Single-Point Electron Paramagnetic Resonance Imaging

  Hyungseok Jang, Sankaran Subramanian, Nallathamby Devasahayam, Keita Saito, Shingo Matsumoto, Murali C. Krishna, Alan B. McMillan

  MAGNETIC RESONANCE IN MEDICINE 70 (4) 1173 - 1181 0740-3194 2013/10 [Refereed][Not invited]
   

  PurposeElectron paramagnetic resonance imaging has emerged as a promising noninvasive technology to dynamically image tissue oxygenation. Owing to its extremely short spin-spin relaxation times, electron paramagnetic resonance imaging benefits from a single-point imaging scheme where the entire free induction decay signal is captured using pure phase encoding. However, direct T-2(*)/pO(2) quantification is inhibited owing to constant magnitude gradients which result in time-decreasing field of view. Therefore, conventional acquisition techniques require repeated imaging experiments with differing gradient amplitudes (typically 3), which results in long acquisition time. MethodsIn this study, gridding was evaluated as a method to reconstruct images with equal field of view to enable direct T-2(*)/pO(2) quantification within a single imaging experiment. Additionally, an enhanced reconstruction technique that shares high spatial k-space regions throughout different phase-encoding time delays was investigated (k-space extrapolation). ResultsThe combined application of gridding and k-space extrapolation enables pixelwise quantification of T-2(*) from a single acquisition with improved image quality across a wide range of phase-encoding time delays. The calculated T-2(*)/pO(2) does not vary across this time range. ConclusionsBy utilizing gridding and k-space extrapolation, accurate T-2(*)/pO(2) quantification can be achieved within a single data set to allow enhanced temporal resolution (by a factor of 3). Magn Reson Med, 70:1173-1181, 2013. (c) 2013 Wiley Periodicals, Inc.
• Magnetic resonance imaging of tumor oxygenation and metabolic profile

  Murali C. Krishna, Shingo Matsumoto, Keita Saito, Masayuki Matsuo, James B. Mitchell, Jan H. Ardenkjaer-Larsen

  ACTA ONCOLOGICA 52 (7) 1248 - 1256 0284-186X 2013/10 [Refereed][Not invited]
   

  The tumor microenvironment is distinct from normal tissue as a result of abnormal vascular network characterized by hypoxia, low pH, high interstitial fluid pressure and elevated glycolytic activity. This poses a barrier to treatments including radiation therapy and chemotherapy. Imaging methods which can characterize such features non-invasively and repeatedly will be of significant value in planning treatment as well as monitoring response to treatment. The three techniques based on magnetic resonance imaging (MRI) are reviewed here. Tumor pO(2) can be measured by two MRI methods requiring an exogenous contrast agent: electron paramagnetic resonance imaging (EPRI) and Overhauser magnetic resonance imaging (OMRI). Tumor metabolic profi le can be assessed by a third method, hyperpolarized metabolic MR, based on injection of hyperpolarized biological molecules labeled with C-13 or N-15 and MR spectroscopic imaging. Imaging pO(2) in tumors is now a robust pre-clinical imaging modality with potential for implementation clinically. Pre-clinical studies and an initial clinical study with hyperpolarized metabolic MR have been successful and suggest that the method may be part of image-guided radiotherapy to select patients for tailored individual treatment regimens.
• Evaluation of partial k-space strategies to speed up time-domain EPR imaging

  Sankaran Subramanian, Gadisetti V. R. Chandramouli, Alan McMillan, Rao P. Gullapalli, Nallathamby Devasahayam, James B. Mitchell, Shingo Matsumoto, Murali C. Krishna

  MAGNETIC RESONANCE IN MEDICINE 70 (3) 745 - 753 0740-3194 2013/09 [Refereed][Not invited]
   

  Narrow-line spin probes derived from the trityl radical have led to the development of fast in vivo time-domain EPR imaging. Pure phase-encoding imaging modalities based on the single-point imaging scheme have demonstrated the feasibility of three-dimensional oximetric images with functional information in minutes. In this article, we explore techniques to improve the temporal resolution and circumvent the relatively short biological half-lives of trityl probes using partial k-space strategies. There are two main approaches: one involves the use of the Hermitian character of the k-space by which only part of the k-space is measured and the unmeasured part is generated using the Hermitian symmetry. This approach is limited in success by the accuracy of numerical estimate of the phase roll in the k-space that corrupts the Hermiticy. The other approach is to measure only a judicially chosen reduced region of k-space (a centrosymmetric ellipsoid region) that more or less accounts for >70% of the k-space energy. Both of these aspects were explored in Fourier transform-EPR imaging with a doubling of scan speed demonstrated by considering ellipsoid geometry of the k-space. Partial k-space strategies help improve the temporal resolution in studying fast dynamics of functional aspects in vivo with infused spin probes. Magn Reson Med 70:745-753, 2013. (c) 2012 Wiley Periodicals, Inc.
• EPR Oxygen Imaging and Hyperpolarized C-13 MRI of Pyruvate Metabolism as Noninvasive Biomarkers of Tumor Treatment Response to a Glycolysis Inhibitor 3-Bromopyruvate

  Shingo Matsumoto, Keita Saito, Hironobu Yasui, H. Douglas Morris, Jeeva P. Munasinghe, Martin Lizak, Hellmut Merkle, Jan Henrik Ardenkjaer-Larsen, Rajani Choudhuri, Nallathamby Devasahayam, Sankaran Subramanian, Alan P. Koretsky, James B. Mitchell, Murali C. Krishna

  MAGNETIC RESONANCE IN MEDICINE 69 (5) 1443 - 1450 0740-3194 2013/05 [Refereed][Not invited]
   

  The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO(2) < 10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized C-13-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs. (C) 2012 Wiley Periodicals, Inc.
• The relationship between tissue oxygenation and redox status using magnetic resonance imaging

  Fuminori Hyodo, Ryan M. Davis, Emi Hyodo, Shingo Matsumoto, Murali C. Krishna, James B. Mitchell

  INTERNATIONAL JOURNAL OF ONCOLOGY 41 (6) 2103 - 2108 1019-6439 2012/12 [Refereed][Not invited]
   

  The recent development of a bi-modality magnetic resonance imaging/electron paramagnetic resonance imaging (MRI/EPRI) platform has enabled longitudinal monitoring of both tumor oxygenation and redox status in murine cancer models. The current study used this imaging platform to test the hypothesis that a more reducing tumor microenvironment accompanies the development of tumor hypoxia. To test this, the redox status of the tumor was measured using Tempo! as a redox-sensitive MRI contrast agent, and tumor hypoxia was measured with wOxo63, which is an oxygen-sensitive EPRI spin probe. Images were acquired every 1-2 days in mice bearing SCCVII tumors. The median PO2 decreased from 14 mmHg at 7 days after tumor implantation to 7 mmHg at 15 days after implantation. Additionally, the hypoxic fraction, defined as the percentage of the tumor that exhibited a pO(2)<10mmHg, increased with tumor size (from 10% at 500 m(3) to 60% at 3,500 mm(3)). The rate of Tempol reduction increased as a function of tumor volume (0.4 min(-1) at 500 mm(3) to 1.7 min(-1) at 3,500 mm(3)), suggesting that the tumor microenvironment became more reduced as the tumor grew. The results show that rapid Tempol reduction correlates with decreased tumor oxygenation, and that the Tempol decay rate constant may be a surrogate marker for tumor hypoxia.
• Longitudinal Imaging Studies of Tumor Microenvironment in Mice Treated with the mTOR Inhibitor Rapamycin

  Keita Saito, Shingo Matsumoto, Hironobu Yasui, Nallathamby Devasahayam, Sankaran Subramanian, Jeeva P. Munasinghe, Vyomesh Patel, J. Silvio Gutkind, James B. Mitchell, Murali C. Krishna

  PLOS ONE 7 (11) e49456  1932-6203 2012/11 [Refereed][Not invited]
   

  Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3)) and measurements of tumor pO(2) and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2) levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2)<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.
• Echo-based Single Point Imaging (ESPI): A novel pulsed EPR imaging modality for high spatial resolution and quantitative oximetry

  Sankaran Subramanian, Nallathamby Devasahayam, Shingo Matsumoto, Keita Saito, James B. Mitchell, Murali C. Krishna

  JOURNAL OF MAGNETIC RESONANCE 218 105 - 114 1090-7807 2012/05 [Refereed][Not invited]
   

  A novel time-domain spectroscopic EPR imaging approach, that is a unique combination of already known techniques, is described. The first one is multi-gradient Single Point Imaging involving pure phase-encoding where the oximetry is based on T. Line width derived from T-2(+); is subject to susceptibility effects and therefore needs system-dependent line width calibrations. The second approach utilizes the conventional 90 degrees-tau-180 degrees Spin-Echo pulse sequence where the images are obtained by the filtered backprojection after FT of the echoes collected under frequency-encoding gradients. The spatially resolved oximetry information is derived from a set of T-2-weighted images. The back-projection images suffer susceptibility artifacts with resolution determined by T, but the oximetry based on T-2 is quite reliable. The current approach combines Single Point Imaging and the Spin-Echo procedure to take advantage the enhanced spatial resolution associated with the former and the T-2 dependent contrast of the latter. Pairs of images are derived choosing two time points located at identical time intervals on either side of the 180 pulse. The refocusing pulse being exactly in the middle of the two points ensures that artifacts associated with susceptibility and field inhomogeneities are eliminated. In addition, the net phase accumulated by the two time points being identical results in identical field of views, thus avoiding the zoomin effect as a function delay in regular SPI and the associated interpolation requirements employed in T-weighted oximetry. The end result is superior image resolution and reliable oximetry. In spite of the fact that projection-reconstruction methods require less number of measurements compared to SPI, the enormous advantage in SNR of the SPI procedure makes the echo-based SPI equally efficient in terms of measurement time. The Fourier reconstruction, line width independent resolution and the true T-2-weighting make this novel procedure very attractive for in vivo EPR imaging of tissue oxygen quantitatively.
• Electron Paramagnetic Resonance Imaging of Tumor pO(2)

  Murali C. Krishna, Shingo Matsumoto, Hironobu Yasui, Keita Saito, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell

  RADIATION RESEARCH 177 (4) 376 - 386 0033-7587 2012/04 [Refereed][Not invited]
   

  Electron paramagnetic resonance imaging (EPRI) can be used to noninvasively and quantitatively obtain three-dimensional maps of tumor PO,. The paramagnetic tracer triarylmethyl (TAM), a substituted trityl radical moiety, is not toxic to animals and provides narrow isotropic spectra, which is ideal for in vivo EPR imaging experiments. From the oxygen-induced spectral broadening of TAM, pO(2) maps can be derived using EPRI. The instrumentation consists of an EPRI spectrometer and 7T magnetic resonance imaging (MRI) system both operating at a common radiofrequency of 300 MHz. Anatomic images obtained by MRI can be overlaid with pO(2) maps obtained from EPRI. With imaging times of less than 3 min, it was possible to monitor the dynamics of oxygen changes in tumor and distinguish chronically hypoxic regions from acutely hypoxic regions. In this article, the principles of pO(2) imaging with EPR and some relevant examples of tumor imaging are reviewed. (C) 2012 by Radiation Research Society
• Transient decrease in tumor oxygenation after intravenous administration of pyruvate

  Keita Saito, Shingo Matsumoto, Nallathamby Devasahayam, Sankaran Subramanian, Jeeva P. Munasinghe, H. Douglas Morris, Martin J. Lizak, Jan Henrik Ardenkjaer-Larsen, James B. Mitchell, Murali C. Krishna

  MAGNETIC RESONANCE IN MEDICINE 67 (3) 801 - 807 0740-3194 2012/03 [Refereed][Not invited]
   

  MRI using hyperpolarized 13C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO2) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-13C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO2 after pyruvate injection in hyperpolarized 13C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies. Magn Reson Med, 2012. (c) 2011 Wiley Periodicals, Inc.
• Reporting of quantitative oxygen mapping in EPR imaging

  Sankaran Subramanian, Nallathamby Devasahayam, Alan McMillan, Shingo Matsumoto, Jeeva P. Munasinghe, Keita Saito, James B. Mitchell, Gadisetti V. R. Chandramouli, Murali C. Krishna

  JOURNAL OF MAGNETIC RESONANCE 214 (1) 244 - 251 1090-7807 2012/01 [Refereed][Not invited]
   

  Oxygen maps derived from electron paramagnetic resonance spectral-spatial imaging (EPRI) are based upon the relaxivity of molecular oxygen with paramagnetic spin probes. This technique can be combined with MRI to facilitate mapping of pO(2) values in specific anatomic locations with high precision. The co-registration procedure, which matches the physical and digital dimensions of EPR and MR images, may present the pO(2) map at the higher MRI resolution, exaggerating the spatial resolution of oxygen, making it difficult to precisely distinguish hypoxic regions from normoxic regions. The latter distinction is critical in monitoring the treatment of cancer by radiation and chemotherapy, since it is well-established that hypoxic regions are three or four times more resistant to treatment compared to normoxic regions. The aim of this article is to describe pO(2) maps based on the intrinsic resolution of EPRI. A spectral parameter that affects the intrinsic spatial resolution of EPRI is the full width at half maximum (FWHM) height of the gradient-free EPR absorption line in frequency-encoded imaging. In single point imaging too, the transverse relaxation times (T-2*) limit the resolution since the signal decays by exp(-t(p)/T-2*) where the delay time after excitation pulse, t(p), is related to the resolution. Although the spin densities of two point objects may be resolved at this separation, it is inadequate to evaluate quantitative changes of pO(2) levels since the linewidths are proportionately affected by pO(2). A spatial separation of at least twice this resolution is necessary to correctly identify a change in pO(2) level. In addition, the pO(2) values are blurred by uncertainties arising from spectral dimensions. Blurring due to noise and low resolution modulates the pO(2) levels at the boundaries of hypoxic and normoxic regions resulting in higher apparent pO(2) levels in hypoxic regions. Therefore, specification of intrinsic resolution and pO(2) uncertainties are necessary to interpret digitally processed pO(2) illustrations. Published by Elsevier Inc.
• Antiangiogenic Agent Sunitinib Transiently Increases Tumor Oxygenation and Suppresses Cycling Hypoxia

  Shingo Matsumoto, Sonny Batra, Keita Saito, Hironobu Yasui, Rajani Choudhuri, Chandramouli Gadisetti, Sankaran Subramanian, Nallathamby Devasahayam, Jeeva P. Munasinghe, James B. Mitchell, Murali C. Krishna

  CANCER RESEARCH 71 (20) 6350 - 6359 0008-5472 2011/10 [Refereed][Not invited]
   

  Structural and functional abnormalities in tumor blood vessels impact the delivery of oxygen and nutrients to solid tumors, resulting in chronic and cycling hypoxia. Although chronically hypoxic regions exhibit treatment resistance, more recently it has been shown that cycling hypoxic regions acquire prosurvival pathways. Angiogenesis inhibitors have been shown to transiently normalize the tumor vasculatures and enhance tumor response to treatments. However, the effect of antiangiogenic therapy on cycling tumor hypoxia remains unknown. Using electron paramagnetic resonance imaging and MRI in tumor-bearing mice, we have examined the vascular renormalization process by longitudinally mapping tumor partial pressure of oxygen (pO(2)) and microvessel density during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation was visualized by electron paramagnetic resonance imaging 2 to 4 days following antiangiogenic treatments, accompanied by a 45% decrease in microvessel density. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth. In addition, dynamic oxygen imaging obtained every 3 minutes was conducted to distinguish tumor regions with chronic and cycling hypoxia. Sunitinib treatment suppressed the extent of temporal fluctuations in tumor pO(2) during the vascular normalization window, resulting in the decrease of cycling tumor hypoxia. Overall, the findings suggest that longitudinal and noninvasive monitoring of tumor pO(2) makes it possible to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs. Cancer Res; 71(20); 6350-9. (C) 2011 AACR.
• Detecting Response of Rat C6 Glioma Tumors to Radiotherapy Using Hyperpolarized [1-C-13]Pyruvate and C-13 Magnetic Resonance Spectroscopic Imaging

  Sam E. Day, Mikko I. Kettunen, Murali Krishna Cherukuri, James B. Mitchell, Martin J. Lizak, H. Douglas Morris, Shingo Matsumoto, Alan P. Koretsky, Kevin M. Brindle

  MAGNETIC RESONANCE IN MEDICINE 65 (2) 557 - 563 0740-3194 2011/02 [Refereed][Not invited]
   

  We show here that hyperpolarized [1-C-13]pyruvate can be used to detect treatment response in a glioma tumor model; a tumor type where detection of response with (18)fluoro-2-deoxyglucose, using positron emission tomography, is limited by the high background signals from normal brain tissue. C-13 chemical shift images acquired following intravenous injection of hyperpolarized [1-C-13]pyruvate into rats with implanted C6 gliomas showed significant labeling of lactate within the tumors but comparatively low levels in surrounding brain.Labeled pyruvate was observed at high levels in blood vessels above the brain and from other major vessels elsewhere but was detected at only low levels in tumor and brain.The ratio of hyperpolarized C-13 label in tumor lactate compared to the maximum pyruvate signal in the blood vessels was decreased from 0.38 +/- 0.16 to 0.23 +/- 0.13, (a reduction of 34%) by 72 h following whole brain irradiation with 15 Gy. Magn Reson Med 65:557-563,2011. (C) 2010 Wiley-Liss, Inc.
• Magnetic resonance imaging of organic contrast agents in mice: capturing the whole-body redox landscape

  Ryan M. Davis, Shingo Matsumoto, Marcelino Bernardo, Anastasia Sowers, Ken-Ichiro Matsumoto, Murali C. Krishna, James B. Mitchell

  FREE RADICAL BIOLOGY AND MEDICINE 50 (3) 459 - 468 0891-5849 2011/02 [Refereed][Not invited]
   

  Nitroxides are a class of stable free radicals that have several biomedical applications including radioprotection and noninvasive assessment of tissue redox status. For both of these applications, it is necessary to understand the in vivo biodistribution and reduction of nitroxides. In this study, magnetic resonance imaging was used to compare tissue accumulation (concentration) and reduction of two commonly studied nitroxides: the piperidine nitroxide Tempol and the pyrrolidine nitroxide 3-CP. It was found that 3-CP was reduced 3 to 11 times slower (depending on the tissue) than Tempol in vivo and that maximum tissue concentration varies substantially between tissues (0.6-7.2 mM). For a given tissue, the maximum concentration usually did not vary between the two nitroxides. Furthermore, using electron paramagnetic resonance spectroscopy, we showed that the nitroxide reduction rate depends only weakly on cellular pO(2) in the oxygen range expected in vivo. These observations, taken with the marked variation in nitroxide reduction rates observed between tissues, suggest that tissue pO(2) is not a major determinant of the nitroxide reduction rate in vivo. For the purpose of redox imaging, 3-CP was shown to be an optimal choice based on the achievable concentrations and bioreduction observed in vivo. Published by Elsevier Inc.
• Fatty acid amide hydrolase is a key regulator of endocannabinoid-induced myocardial tissue injury

  Partha Mukhopadhyay, Bela Horvath, Mohanraj Rajesh, Shingo Matsumoto, Keita Saito, Sandor Batkai, Vivek Patel, Galin Tanchian, Rachel Y. Gao, Benjamin F. Cravatt, Gyoergy Hasko, Pal Pacher

  FREE RADICAL BIOLOGY AND MEDICINE 50 (1) 179 - 195 0891-5849 2011/01 [Refereed][Not invited]
   

  Previous studies have suggested that increased levels of endocannabinoids in various cardiovascular disorders (e.g., various forms of shock, cardiomyopathies, atherosclerosis) through the activation of CB(1) cannabinoid receptors may promote cardiovascular dysfunction and tissue injury. We have investigated the role of the main endocannabinoid anandamide-metabolizing enzyme (fatty acid amide hydrolase; FAAH) in myocardial injury induced by an important chemotherapeutic drug, doxorubicin (DOX; known for it cardiotoxicity mediated by increased reactive oxygen and nitrogen species generation), using well-established acute and chronic cardiomyopathy models in mice. The DOX-induced myocardial oxidative/nitrative stress (increased 4-hydroxynonenal, protein carbonyl, and nitrotyrosine levels and decreased glutathione content) correlated with multiple cell death markers, which were enhanced in FAAH knockout mice exhibiting significantly increased DOX-induced mortality and cardiac dysfunction compared to their wild type. The effects of DOX in FAAH knockouts were attenuated by CB(1) receptor antagonists. Furthermore, anandamide induced enhanced cell death in human cardiomyocytes pretreated with FAAH inhibitor and enhanced sensitivity to ROS generation in inflammatory cells of FAAH knockouts. These results suggest that in pathological conditions associated with acute oxidative/nitrative stress FAAH plays a key role in controlling the tissue injury that is, at least in part, mediated by the activation of CB(1) receptors by endocannabinoids. Published by Elsevier Inc.
• Intracellular Hypoxia of Tumor Tissue Estimated by Noninvasive Electron Paramagnetic Resonance Oximetry Technique Using Paramagnetic Probes

  Atsuko Matsumoto, Ken-ichiro Matsumoto, Shingo Matsumoto, Fuminori Hyodo, Anastasia L. Sowers, Janusz W. Koscielniak, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 34 (1) 142 - 145 0918-6158 2011/01 [Refereed][Not invited]
   

  Electron paramagnetic resonance (EPR) oximetry at 700 MHz operating frequency employing a surface coil resonator is used to assess tissue partial pressure of oxygen (pO(2)) using paramagnetic media whose linewidth and decay constant are related to oxygen concentration. Differences in extracellular and intracellular pO(2) in squamous cell carcinoma (SCC) tumor tissue were tested using several types of water-soluble paramagnetic media, which localize extracellularly or permeate through the cell membrane. The nitroxide carboxy-PROXYL (CxP) can only be distributed in blood plasma and extracellular fluids whereas the nitroxides carbamoyl-PROXYL (CmP) and TEMPOL (TPL) can permeate cell membranes and localize intracellularly. EPR signal decay constant and the linewidth of the intravenously administered nitroxides in SCC tumor tissues implanted in mouse thigh and the contralateral normal muscle of healthy mice breathing gases with different pO(2) were compared. The pO(2) in the blood can depend on the oxygen content in the breathing gas while tissue PO2 was not directly influenced by pO(2) in the breathing gas. The decay constants of CmP and TPL in tumor tissue were significantly larger than in the normal muscles, and lower linewidths of CmP and TPL in tumor tissue was observed. The SCC tumor showed intracellular hypoxia even though the extracellular pO(2) is similar to normal tissue in the peripheral region.
• Imaging Cycling Tumor Hypoxia

  Shingo Matsumoto, Hironobu Yasui, James B. Mitchell, Murali C. Krishna

  CANCER RESEARCH 70 (24) 10019 - 10023 0008-5472 2010/12 [Refereed][Not invited]
   

  Cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. Cycling hypoxia can exist more than 100-mu m distances from a microvessel, and some of these regions have been shown to exist adjacent to normal tissue. Fluctuations in pO(2) of approximately 20 mm Hg can occur with periodicities of minutes to hours and even days. These fluctuations have been attributed to changes in erythrocyte flux, perfusion, and also development of newer vascular networks. Cycling hypoxia has been shown to induce the expression of hypoxia-inducible transcription factor-1 alpha (HIF-1 alpha) and also confer tumor cells and tumor vascular endothelial cells with enhanced prosurvival pathways, making tumors less responsive to radiation and chemotherapy. Imaging of cycling hypoxia in tumors can provide capabilities to help plan appropriate treatment, by taking into account the magnitude and frequency of fluctuations and also their locations adjacent to normal tissue. Electron paramagnetic resonance imaging (EPRI) provides the ability to distinguish chronic and cycling hypoxic regions and has the required spatial and temporal resolutions to provide quantitative maps of tumor pO(2). EPRI can serve as a valuable tool in examining tumor pO(2) longitudinally in response to treatment and in an experimentally chosen time window to spatially map fluctuations in pO(2) noninvasively in animal models of implanted or orthotopic tumors, with a potential for human applications. Cancer Res; 70(24); 10019-23. (C) 2010 AACR.
• Cannabidiol Attenuates Cardiac Dysfunction, Oxidative Stress, Fibrosis, and Inflammatory and Cell Death Signaling Pathways in Diabetic Cardiomyopathy

  Mohanraj Rajesh, Partha Mukhopadhyay, Sandor Batkai, Vivek Patel, Keita Saito, Shingo Matsumoto, Yoshihiro Kashiwaya, Bela Horvath, Bani Mukhopadhyay, Lauren Becker, Gyoergy Hasko, Lucas Liaudet, David A. Wink, Aristidis Veves, Raphael Mechoulam, Pal Pacher

  JOURNAL OF THE AMERICAN COLLEGE OF CARDIOLOGY 56 (25) 2115 - 2125 0735-1097 2010/12 [Refereed][Not invited]
   

  Objectives In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. Background Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. Methods Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. Results Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-kappa B and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38 alpha) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-alpha, markers of fibrosis (transforming growth factor-beta, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-kappa B activation, and cell death in primary human cardiomyocytes. Conclusions Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis. (J Am Coll Cardiol 2010;56:2115-25) (C) 2010 by the American College of Cardiology Foundation
• Low-Field Magnetic Resonance Imaging to Visualize Chronic and Cycling Hypoxia in Tumor-Bearing Mice

  Hironobu Yasui, Shingo Matsumoto, Nallathamby Devasahayam, Jeeva P. Munasinghe, Rajani Choudhuri, Keita Saito, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  CANCER RESEARCH 70 (16) 6427 - 6436 0008-5472 2010/08 [Refereed][Not invited]
   

  Tumors exhibit fluctuations in blood flow that influence oxygen concentrations and therapeutic resistance. To assist therapeutic planning and improve prognosis, noninvasive dynamic imaging of spatial and temporal variations in oxygen partial pressure (pO(2)) would be useful. Here, we illustrate the use of pulsed electron paramagnetic resonance imaging (EPRI) as a novel imaging method to directly monitor fluctuations in oxygen concentrations in mouse models. A common resonator platform for both EPRI and magnetic resonance imaging (MRI) provided pO(2) maps with anatomic guidance and microvessel density. Oxygen images acquired every 3 minutes for a total of 30 minutes in two different tumor types revealed that fluctuation patterns in pO(2) are dependent on tumor size and tumor type. The magnitude of fluctuations in pO(2) in SCCVII tumors ranged between 2- to 18-fold, whereas the fluctuations in HT29 xenografts were of lower magnitude. Alternating breathing cycles with air or carbogen (95% O(2) plus 5% CO(2)) distinguished higher and lower sensitivity regions, which responded to carbogen, corresponding to cycling hypoxia and chronic hypoxia, respectively. Immunohistochemical analysis suggests that the fluctuation in pO(2) correlated with pericyte density rather than vascular density in the tumor. This EPRI technique, combined with MRI, may offer a powerful clinical tool to noninvasively detect variable oxygenation in tumors. Cancer Res; 70(16); 6427-36. (C)2010 AACR.
• Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI

  Shingo Matsumoto, Hironobu Yasui, Sonny Batra, Yuichi Kinoshita, Marcelino Bernardo, Jeeva P. Munasinghe, Hideo Utsumi, Rajani Choudhuri, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 (42) 17898 - 17903 0027-8424 2009/10 [Refereed][Not invited]
   

  Architectural and functional abnormalities of blood vessels are a common feature in tumors. A consequence of increased vascular permeability and concomitant aberrant blood flow is poor delivery of oxygen and drugs, which is associated with treatment resistance. In the present study, we describe a strategy to simultaneously visualize tissue oxygen concentration and microvascular permeability by using a hyperpolarized (1)H-MRI, known as Over-hauser enhanced MRI (OMRI), and an oxygen-sensitive contrast agent OX63. Substantial MRI signal enhancement was induced by dynamic nuclear polarization (DNP). The DNP achieved up to a 7,000% increase in MRI signal at an OX63 concentration of 1.5 mM compared with that under thermal equilibrium state. The extent of hyperpolarization is influenced mainly by the local concentration of OX63 and inversely by the tissue oxygen level. By collecting dynamic OMRI images at different hyperpolarization levels, local oxygen concentration and microvascular permeability of OX63 can be simultaneously determined. Application of this modality to murine tumors revealed that tumor regions with high vascular permeability were spatio- temporally coincident with hypoxia. Quantitative analysis of image data from individual animals showed an inverse correlation between tumor vascular leakage and median oxygen concentration. Immunohistochemical analyses of tumor tissues obtained from the same animals after OMRI experiments demonstrated that lack of integrity in tumor blood vessels was associated with increased tumor microvascular permeability. This dual imaging technique may be useful for the longitudinal assessment of changes in tumor vascular function and oxygenation in response to chemotherapy, radiotherapy, or anti-angiogenic treatment.
• Estimation of tumor microvessel density by MRI using a blood pool contrast agent

  Fuminori Hyodo, Gadisetti V. R. Chandramouli, Shingo Matsumoto, Ken-Ichiro Matsumoto, James B. Mitchell, Murali C. Krishna, Jeeva P. Munasinghe

  INTERNATIONAL JOURNAL OF ONCOLOGY 35 (4) 797 - 804 1019-6439 2009/10 [Refereed][Not invited]
   

  Recognition of importance of angiogenesis to tumor growth, metastasis, and treatment outcome has led to efforts to develop non-invasive methods for longitudinal monitoring of tumor microvasculature. We describe a steady-state MRI technique to determine absolute blood volume (BV) as a marker of microvascular density with improved spatial and temporal resolution using an ultra small super paramagnetic iron oxide (USPIO). A noise reduction scheme for BV imaging was also proposed based on weighting factors derived by pre-contrast signal level as an adjustable additive constant. Gradient echo sequence was used for BV imaging with MRI at 7T. Optimal imaging conditions (USPIO dose and echo time) were determined by USPIO dose-dependent studies ex vivo and in vivo. Improved analysis strategies were at first applied for cerebral BV estimation in mice, which were found in good agreement with the literature values. These methods were then used to determine tumor BV in mice. The optimal concentration of USPIO for BV estimates was found to range from 3.6 to 4.48 mM (estimated as Fe concentration) in ex vivo experiments corresponding to an in vivo dosage of 215-287 mu mol/kg body weight, whereas a USPIO dose of 287 mu mol/kg leads to higher cerebral BV estimate in vivo than the reported values. Application of the BV imaging method to evaluation of anti-angiogenic effect of Sunitinib in squamous cell carcinoma (SCC) tumor bearing mice revealed similar to 46% reduction in tumor BV 4 days after start of Sunitinib treatment. The results show that the MRI. approach using USPIO yields high-resolution absolute BV images and the method can be conveniently applied to monitor longitudinal tumor microvessel density changes as a function of growth or in response to treatment.
• Pulsed EPR imaging of nitroxides in mice

  Fuminori Hyodo, Shingo Matsumoto, Nallathamby Devasahayam, Christopher Dharmaraj, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  JOURNAL OF MAGNETIC RESONANCE 197 (2) 181 - 185 1090-7807 2009/04 [Refereed][Not invited]
   

  Nitroxides, unlike trityl radicals, have shorter T(2)s which until now were not detectable in vivo by a time-domain Pulsed Electron Paramagnetic Resonance (EPR) spectrometer at 300 MHz since their phase memory times were shorter than the spectrometer recovery times. In the current version of the time-domain EPR spectrometer with improved spectrometer recovery times, the feasibility of detecting signals from nitroxide radicals was tested. Among the nitroxides evaluated, deuterated (15)N-Tempone ((15)N-PDT) was found to have the longest T(2). The signal intensity profile as a function of concentration of these agents was evaluated and a biphasic behavior was observed; beyond a nitroxide concentration of 1.5 mM, signal intensity was found to decrease as a result of self-broadening. Imaging experiments were carried out with (15)N-PDT in Solutions equilibrated with 0%, 5%, 10%, and 21% oxygen using the single point imaging (SPI) modality in EPR. The image intensity in these tubes was found to depend on the oxygen concentration which in turn influences the T(2) of (15)N-PDT. In vivo experiments were demonstrated with (15)N-PDT in anesthetized mice where the distribution and metabolism of (15)N-PDT could be monitored. This study, for the first time shows the capability to image a cell-permeable nitroxide in mice using pulsed EPR in the SPI modality. (C) 2009 Published by Elsevier Inc.
• Reconstruction for time-domain in vivo EPR 3D multigradient oximetric imaging--a parallel processing perspective

  Dharmaraj CD, Thadikonda K, Fletcher AR, Doan PN, Devasahayam N, Matsumoto S, Johnson CA, Cook JA, Mitchell JB, Subramanian S, Krishna MC

  Int J Biomed Imaging 2009 528639  2009 [Refereed][Not invited]
  
• Synthesis of nitroxyl radicals for overhauser-enhanced magnetic resonance imaging

  Ken-ichi Yamada, Yuichi Kinoshita, Toshihide Yamasaki, Hiromi Sadasue, Fumiya Mito, Mika Nagai, Shingo Matsumoto, Mariko Aso, Hiroshi Suemune, Kiyoshi Sakai, Hideo Utsumi

  ARCHIV DER PHARMAZIE 341 (9) 548 - 553 0365-6233 2008/09 [Refereed][Not invited]
   

  Non-invasive measurement and visualization of free radicals in vivo would be important to clarify their roles in the pathogenesis of free radical-associated diseases. Nitroxyl radicals can react with free radicals and be derivatized to achieve specific cellular / subcellular localizing capabilities while retaining the simple spectral features useful in imaging. Overhauser-enhanced magnetic resonance imaging (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity via the Overhauser Effect. In this study, we synthesized various nitroxyl probes having (15)N nuclei and deuterium, and measured the enhancement factor for Overhauser-enhanced magnetic resonance imaging experiments. (15)N-D-4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl ((15)N-D-oxo-TEMPO) has the highest enhancement factor compared with other nitroxyl probes. The proton signal enhancement was higher for (15)N-labeled nitroxyl probes when compared to the (14)N-labeled analogues because of the reduced spectral multiplicity of the I = 1/2 nucleus. Furthermore, this enhancement is proportional to the line width and number of electron spin resonance lines of nitroxyl radicals. Finally, we compared the Overhauser-enhanced magnetic resonance image of (15)N-labeled, deuterated 4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxyl with that of (14)N-H-TEMPOL. These results suggested that the selective deuteration of the nitroxyl probes enhanced the signal-to-noise ratio and thereby improved spatial and temporal resolutions.
• Assessment of tissue redox status using metabolic responsive contrast agents and magnetic resonance imaging

  Fuminori Hyodo, Benjamin P. Soule, Ken-Ichiro Matsumoto, Shingo Matusmoto, John A. Cook, Emi Hyodo, Anastasia L. Sowers, Murali C. Krishna, James B. Mitchell

  JOURNAL OF PHARMACY AND PHARMACOLOGY 60 (8) 1049 - 1060 0022-3573 2008/08 [Refereed][Not invited]
   

  Regulation of tissue redox status is important to maintain normal physiological conditions in the living body. Disruption of redox homoeostasis may lead to oxidative stress and can induce many pathological conditions such as cancer, neurological disorders and ageing. Therefore, imaging of tissue redox status could have clinical applications. Redox imaging employing magnetic resonance imaging (MRI) with nitroxides as cell-permeable redox-sensitive contrast agents has been used for non-invasive monitoring of tissue redox status in animal models. The redox imaging applications of nitroxide electron paramagnetic resonance imaging (EPRI) and MRI are reviewed here, with a focus on application of tumour redox status monitoring. While particular emphasis has been placed on differences in the redox status in turnours compared to selected normal tissues, the technique possesses the potential to have broad applications to the study of other disease states, inflammatory processes and other circumstances where oxidative stress is implicated.
• Low-field paramagnetic resonance imaging of tumor oxygenation and glycolytic activity in mice

  Matsumoto, Shingo, Hyodo, Fuminori, Subramanian, Sankaran, Devasahayam, Nallathamby, Munasinghe, Jeeva, Hyodo, Emi, Gadisetti, Chandramouli, Cook, John A., Mitchell, James B., Krishna, Murali C.

  JOURNAL OF CLINICAL INVESTIGATION 118 (5) 1965 - 1973 0021-9738 2008/05 [Refereed][Not invited]
   

  A priori knowledge of spatial and temporal changes in partial pressure of oxygen (oxygenation; PO2) in solid tumors, a key prognostic factor in cancer treatment outcome, could greatly improve treatment planning in radiotherapy and chemotherapy. Pulsed electron paramagnetic resonance imaging (EPRI) provides quantitative 3D maps of tissue PO2 in living objects. In this study, we implemented an EPRI set-up that could acquire PO2 maps in almost real time for 2D and in minutes for 3D. We also designed a combined EPRI and MRI system that enabled generation of pO(2) maps with anatomic guidance. Using EPRI and an air/carbogen (95% O-2 plus 5% CO2) breathing cycle, we visualized perfusion-limited hypoxia in murine tumors. The relationship between tumor blood perfusion and PO2 status was examined, and it was found that significant hypoxia existed even in regions that exhibited blood flow. In addition, high levels of lactate were identified even in normoxic tumor regions, suggesting the predominance of aerobic glycolysis in murine tumors. This report presents a rapid, noninvasive method to obtain quantitative maps of PO2 in tumors, reported with anatomy, with precision. In addition, this method may also be useful for studying the relationship between PO2 status and tumor-specific phenotypes such as aerobic glycolysis.
• Dynamic monitoring of localized tumor oxygenation changes using RF pulsed electron paramagnetic resonance in conscious mice

  Shingo Matsumoto, Michael Graham Espey, Hideo Utsumi, Nallathamby Devasahayam, Ken-Ichiro Matsumoto, Atsuko Matsumoto, Hiroshi Hirata, David A. Wink, Periarman Kuppusamy, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna

  MAGNETIC RESONANCE IN MEDICINE 59 (3) 619 - 625 0740-3194 2008/03 [Refereed][Not invited]
   

  Oxygenation status is a key determinant in both tumor growth and responses to therapeutic interventions. The oxygen partial pressure (1302) was assessed using a novel pulsed electron paramagnetic resonance (EPR) spectroscopy at 750 MHz. Crystals of lithium phthalocyanine (LiPc) implanted into either squamous cell carcinoma (SCC) tumor or femoral muscle on opposing legs of mice were tested by pulsed EPR. The results showed pO(2) of SCC tumor was 2.7 +/- 0.4 mmHg, while in the femoral muscle it was 6.1 +/- 0.9 mmHg. A major advantage of pulsed EPR oximetry over conventional continuous-wave (CW) EPR oximetry is the lack of influence from subject motion, while avoiding artifacts associated with modulation or power saturation. Resonators in pulsed EPR are overcoupled to minimize recovery time. This makes changes in coupling associated with object motion minimal without influencing spectral quality. Consequently, pulsed EPR oximetry enables approximately a temporal resolution of similar to one second in pO(2) monitoring in conscious subjects, avoiding significant influence of anesthetics on the physiology being studied. The pO(2) in SCC tumor and muscle was found to be higher without anesthesia (3.9 +/- 0.5 mmHg for tumor, 8.8 +/- 1.2 mmHg for muscle). These results support the advantage of pulsed EPR in examining pO(2) in conscious animals with LiPc chronically implanted in predetermined regions.
• Advantageous application of a surface coil to EPR irradiation in Overhauser-enhanced MRI

  Shingo Matsumoto, Kenichi Yamada, Hiroshi Hirata, Keiji Yasukawa, Fuminori Hyodo, Kazuhiro Ichikawa, Hideo Utsumi

  MAGNETIC RESONANCE IN MEDICINE 57 (4) 806 - 811 0740-3194 2007/04 [Refereed][Not invited]
   

  The present study describes the advantageous application of a surface coil to electron paramagnetic resonance (EPR) irradiation in Overhauser-enhanced MRI (OMRI). OMRI is a double-resonance method for imaging free radicals based on the Over-hauser effect. Proton NMR images are recorded without and with EPR irradiation of the free radical resonance, which results in a difference proton image that shows signal enhancement in spatial regions that contain the free radical. To obtain good signal enhancement in OMRI, very high RF power and a long EPR irradiation time are required. To improve sensitivity and shorten the image acquisition time, especially for localized (and topical) applications, we developed and tested a surface-coil-type EPR irradiation coil. Theoretical calculations and experimental data showed that EPR irradiation through the surface coil could ameliorate the localized Overhauser enhancement, which was related to the ratio of 8, surface coil/B-1 volume coil in the region of interest (ROI), as expected. The increased sensitivity could also be converted into a shortened EPR irradiation time, resulting in fast data acquisition. For biomedical applications, the use of a surface coil (as opposed to a conventional volume coil) could decrease the total RF power deposition in the sample required to obtain the same Overhauser enhancement in the ROI.
• Are free radical reactions increased in the diabetic eye?

  Mayumi Yamato, Shingo Matsumoto, Katsuaki Ura, Ken-Ichi Yamada, Tatsuya Naganuma, Toyoshi Inoguchi, Toshiaki Watanabe, Hideo Utsumi

  ANTIOXIDANTS & REDOX SIGNALING 9 (3) 367 - 373 1523-0864 2007/03 [Refereed][Not invited]
   

  Reactive oxygen species (ROS) are thought to play a significant role in the development of diabetic retinopathy; however, no direct evidence supports ROS generation in vivo. This study used in vivo electron spin resonance (ESR) spectroscopy with a surface resonator to detect local free radical reactions. The ESR signal decay of carbamoyl-PROXYL was enhanced in the eyes of streptozotocin (STZ)-induced diabetic mice. This enhanced signal decay was suppressed by the administration of SOD or the pretreatment with aminoguanidine. We demonstrate, for the first time, specific free radical reactions in the eyes of mice with STZ-induced diabetes.
• Simultaneous molecular imaging of redox reactions monitored by Overhauser-enhanced MRI with 14N- and 15N-labeled nitroxyl radicals

  Utsumi H, Yamada K, Ichikawa K, Sakai K, Kinoshita Y, Matsumoto S, Nagai M

  Proc Natl Acad Sci USA 103 (5) 1463 - 1468 0027-8424 2006 [Refereed][Not invited]
   

  MRI has provided significant clinical utility in the diagnosis of diseases and will become a powerful tool to assess phenotypic changes in genetically engineered animals. Overhauser enhanced MRI (OMRI), which is a double resonance technique, creates images of free radical distributions in small animals by enhancing the water proton signal intensity by means of the Overhauser effect. Several studies have demonstrated noninvasive assessment of reactive oxygen species generation in small animals by using low frequency electron spin resonance (ESR) spectroscopy imaging and nitroxyl radicals. In vivo ESR signal intensities of nitroxyl radicals decrease with time after injection and the decreases are enhanced by reactive oxygen species, generated in oxidative disease models in a site-specific manner. In this study, we show images of nitroxyl radicals with different isotopes by changing the external magnetic field for ESR irradiation between 14N and 15N nuclei in field-cycled OMRI. OMRI simultaneously obtained dual images of two individual chemical processes. Oxidation and reduction were monitored in a rate-dependent manner at nanometer scale by labeling membrane-permeable and -impermeable nitroxyl radicals with 14N and 15N nuclei. Phantom objects containing ascorbic acid-encapsulated liposomes with membrane-permeable radicals but not membrane-impermeable ones show a time-dependent decrease of the OMRI image intensity. The pharmacokinetics in mice was assessed with OMRI after radical administration. This OMRI technique with dual probes should offer significant applicability to nanometer scale molecular imaging and simultaneous assessment of independent processes in gene-modified animals. Thus, it may become a powerful tool to clarify mechanisms of disease and to monitor pharmaceutical therapy. © 2006 by The National Academy of Sciences of the USA.
• Absolute oxygen tension (pO(2)) muscle tissue as determined by in murine fatty and EPR

  A Matsumoto, S Matsumoto, AL Sowers, JW Koscielniak, NJ Trigg, P Kuppusamy, JB Mitchell, S Subramanian, MC Krishna, K Matsumoto

  MAGNETIC RESONANCE IN MEDICINE 54 (6) 1530 - 1535 0740-3194 2005/12 [Refereed][Not invited]
   

  The absolute partial pressure of oxygen (pO(2)) in the mammary gland pad and femoral muscle of female mice was measured using EPR oximetry at 700 MHz. A small quantity of lithium phthalocyanine (LiPc) crystals was implanted in both mammary and femoral muscle tissue of female C3H mice. Subsequent EPR measurements were carried out 1-30 days after implantation with or without control of core body temperature. The pO(2) values in the tissue became stable 2 weeks after implantation of LiPc crystals. The pO(2) level was found to be higher in the femoral muscle than in the mammary tissue. However, the pO(2) values showed a strong dependence on the core body temperature of the mice. The pO(2) values were responsive to carbogen (95% O-2 5% CO2) breathing even 44-58 days after the implantation of LiPc. The LiPc linewidth was also sensitive to changes in the blood supply even 60 days after implantation of the crystals. This study further validates the use of LiPc crystals and EPR oximetry for long-term non-invasive assessment of pO(2) levels in tissues, underscores the importance of maintaining normal body core temperature during the measurements, and demonstrates that mammary tissue functions at a lower pO(2) level than muscle in female C3H mice.
• Influence of proton T-1 on oxymetry using Overhauser enhanced magnetic resonance imaging

  S Matsumoto, H Utsumi, T Aravalluvan, K Matsumoto, A Matsumoto, N Devasahayam, AL Sowers, JB Mitchell, S Subramanian, MC Krishna

  MAGNETIC RESONANCE IN MEDICINE 54 (1) 213 - 217 0740-3194 2005/07 [Refereed][Not invited]
   

  In Overhauser enhanced magnetic resonance imaging (OMRI) for in vivo measurement of oxygen partial pressure (pO(2)), a paramagnetic contrast agent is introduced to enhance the proton signal through dynamic nuclear polarization. A uniform proton T-1 is generally assumed for the entire region of interest for the computation of PO2 using OMRI. It is demonstrated here, by both phantom and in vivo (mice) imaging, that such an assumption may cause erroneous estimate of PO2. A direct estimate of pixel-wise T-1 is hampered by the poor native MR intensities, owing to the very low Zeeman field (15-20 mT) in OMRI. To circumvent this problem, a simple method for the pixel-wise mapping of proton T-1 using the OMRI scanner is described. A proton T-1 image of a slice through the center of an SCC tumor in a mouse clearly shows a range of T-1 distribution (0.2 similar to 1.6 s). Computation of PO2 images using pixel-wise T-1 values promises oximetry with minimal artifacts by OMRI. Published 2005 Wiley-Liss, Inc.
• In vivo imaging of oxidative stress in the kidney of diabetic mice and its normalization by angiotensin II type 1 receptor blocker

  T Sonta, T Inoguchi, S Matsumoto, K Yasukawa, M Inuo, H Tsubouchi, N Sonoda, K Kobayashi, H Utsumi, H Nawata

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 330 (2) 415 - 422 0006-291X 2005/05 [Refereed][Not invited]
   

  This study was undertaken to evaluate oxidative stress in the kidney of diabetic mice by electron spin resonance (ESR) imaging technique. Oxidative stress in the kidney was evaluated as organ-specific reducing activity with the signal decay rates of carbamoyl-PROXYL probe using ESR imaging. The signal decay rates were significantly faster in corresponding image pixels of the kidneys of streptozotocin-induced diabetic mice than in those of controls. This technique further demonstrated that administration of angiotensin II type I receptor blocker (ARB), olmesartan (5 mg/kg), completely restored the signal decay rates in the diabetic kidneys to control values. In conclusion, this study provided for the first time the in vivo evidence for increased oxidative stress in the kidneys of diabetic mice and its normalization by ARB as evaluated by ESR imaging. This technique would be useful as a means of further elucidating the role of oxidative stress in diabetic nephropathy. (c) 2005 Elsevier Inc. All rights reserved.
• A composite resonator assembly suitable for EPR/NMR coregistration imaging

  S Matsumoto, M Nagai, K Yamada, F Hyodo, K Yasukawa, M Muraoka, H Hirata, M Ono, H Utsumi

  CONCEPTS IN MAGNETIC RESONANCE PART B-MAGNETIC RESONANCE ENGINEERING 25B (1) 1 - 11 1552-5031 2005/04 [Refereed][Not invited]
   

  The development of a composite resonator assembly for electron paramagnetic resonance (EPR)/nuclear magnetic resonance (NMR) imaging and its first application to EPR/NMR coregistration imaging are described. Anatomic registration would be useful for the meaningful interpretation of images by EPR of free radical distributions in vivo. The composite resonator assembly consists of a parallel coil resonator, a field modulation coil for EPR imaging, and a solenoidal detection coil for NMR imaging. Radio-frequency (RF) shielding was used in addition for the EPR imaging. The homogeneity of the RF magnetic field of the EPR resonator was measured experimentally using a small-loop antenna; it was reasonably homogeneous along the long axis and in the radial plane. EPR and NMR images were obtained with both phantom and animal tests. The presence of the parallel coil resonator did not significantly affect NMR images. The superposition of the distribution of free radicals and protons was computed using three positional markers. The assembly performs the registration of two multimodal images. It is compatible with an orthogonal crossed-coil configuration for open-type clinical NMR imagers. (c) 2005 Wiley Periodicals, Inc.
• Evalution of oxidative stress in diabetic animals by in vivo electron spin resonance measurement - role of protein kinase C

  T Sonta, T Inoguchi, H Tsubouchi, N Sekiguchi, K Kobayashi, S Matsumoto, H Utsumi, H Nawata

  DIABETES RESEARCH AND CLINICAL PRACTICE 66 (Suppl 1) S109 - S113 0168-8227 2004/12 [Refereed][Not invited]
   

  Enhanced oxidative stress may be an important contributor to the pathogenesis of diabetic vascular complication. Although hyperglycemia-induced oxidative stress in diabetes has been well documented, exact source in vivo remains to be elucidated. Here we report a role of protein kinase C (PKC) in oxidative stress in diabetic animals using a technique of in vivo electron spin resonance (ESR) measurement that has been developed for direct and non-invasive analysis of free radical generation in living animals. First, using this measurement, we confirmed that streptozotocin-induced diabetic rats which showed a significant increase in free radical generation, which was restored by alpha-tocopherol treatment. Treatment of PKC inhibitor CGP41251 (50 mg/kg) or NAD(P)H oxidase inhibitor apocynin (5 mg/kg) restored the increased free radical generation in those diabetic animals. In conclusion, the present study provided the evidence that PKC-dependent activation of vascular NAD(P)H oxidase may be a major source in enhanced oxidative stress in diabetes in vivo. This may contribute to the pathogenesis of diabetic vascular complications. (C) 2004 Elsevier Ireland Ltd. All rights reserved.
• Evidence for contribution of vascular Nad(P)H oxidase to increased oxidative stress in animal models of diabetes and obesity

  T Sonta, T Inoguchi, H Tsubouchi, N Sekiguchi, K Kobayashi, S Matsumoto, H Utsumi, H Nawata

  FREE RADICAL BIOLOGY AND MEDICINE 37 (1) 115 - 123 0891-5849 2004/07 [Refereed][Not invited]
   

  At is well established that oxidative stress is enhanced in diabetes. However, the major in vivo source of oxidative stress is not clear. Here we show that vascular NAD(P)H oxidase may be a major source of oxidative stress in diabetic and obese models. In vivo electron spin resonance (ESR)/spin probe was used to evaluate systemic oxidative stress in vivo. The signal decay rate of the spin probe (spin clearance rate; SpCR) significantly increased in streptozotocin-induced diabetic rats 2 weeks after the onset of diabetes. This increase was completely normalized by treatment with the antioxidants a-tocopherol (40 mg/kg) and superoxide dismutase (5000 units/kg), and was significantly inhibited by treatment with a PKC-specific inhibitor, CGP41251 (50 mg/kg), and a NAD(P)H oxidase inhibitor, apocynin (5 mg/kg). Both obese ob/ob mice (10 weeks old) with mild hyperglycemia and Zucker fatty rats (11 weeks old) with normoglycemia exhibited significantly increased SpCR as compared with controls. Again, this increase was inhibited by treatment with both CGP41251 and apocynin. Oral administration of insulin sensitizer, pioglitazone (10 mg/ kg), for 7 days also completely normalized SpCR values. These results suggest that vascular NAD(P)H oxidase may be a major source of increased oxidative stress in diabetes and obesity. (C) 2004 Elsevier Inc. All rights reserved.
• In vivo measurement of redox status in streptozotocin-induced diabetic rat using targeted nitroxyl probes

  KI Yamada, D Inoue, S Matsumoto, H Utsumi

  ANTIOXIDANTS & REDOX SIGNALING 6 (3) 605 - 611 1523-0864 2004/06 [Refereed][Not invited]
   

  In vivo electron paramagnetic resonance (EPR) with nitroxyl spin probes has been used for the evaluation of in vivo free radical reactions and redox status in living animals. The aim of this study was to clarify the location of free radical reactions induced by hyperglycemia in osteogenic disorder shionogi (ODS) rats using in vivo EPR spectroscopy. Diabetes was induced by intravenous injection of streptozotocin (STZ). The amount of ascorbic acid (AsA) in ODS rats was controlled by feeding AsA-containing water. Fourteen days after STZ injection, blood glucose and plasma malondialdehyde levels in STZ-treated rats significantly increased compared with untreated rats. Signal decay rates of intravenously injected 3-carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxy (carbamoyl-PROXYL) (less membrane permeable) and 3-carboxy-PROXYL (membrane impermeable) were enhanced in STZ-treated rats in agreement with the previous reports. The decay rate of 3-acetoxymethoxy-PROXYL (membrane permeable) was significantly enhanced by STZ treatment in AsA-depleted rats, and this enhancement was partially restored to the control value by xanthine oxidase inhibitor, although the rate in AsA-supplemented rats was not changed by STZ treatment. These results suggested that the enhancement of signal decay occurred mainly in the intravascular region in STZ-induced diabetic rats and that AsA depletion induced the enhancement of intracellular signal decay through xanthine oxidase, although it is not clear whether the enhancement of signal decay is the cause or the effect of STZ-induced diabetes.
• Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice

  S Matsumoto, Koshiishi, I, T Inoguchi, H Nawata, H Utsumi

  FREE RADICAL RESEARCH 37 (7) 767 - 772 1071-5762 2003/07 [Refereed][Not invited]
   

  Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50 +/- 33 muU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15 +/- 6 muU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r = 0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.

MISC

• Feasibility study of tooth irradiation dose mapping using EPR

  新井之紘, 山口一郎, 松元慎吾, 赤羽英夫, 三宅実, 平田拓  電子スピンサイエンス学会年会講演要旨集  62nd (CD-ROM)-  2023
• [Imaging of Tumor-Specific Hypoxia Dynamics and Its Significance in Radiation Biology].

  Hironobu Yasui, Shingo Matsumoto, Osamu Inanami, Murali Cherukuri Krishna  Igaku butsuri : Nihon Igaku Butsuri Gakkai kikanshi = Japanese journal of medical physics : an official journal of Japan Society of Medical Physics  40-  (1)  13  -18  2020  [Not refereed][Not invited]
   

  Hypoxia has been known to be a feature associated with tumor radioresistance. So far, clinical strategies to overcome chronic hypoxia due to the limitation of the oxygen diffusion have been designed. However, intermittent or acute/cycling hypoxia, whose frequency can range between a few cycles per minutes to hours, is receiving increased attention, because this type of hypoxia has been reported to have an influence on tumor malignancy as well as treatment resistance via increased expression of pro-survival pathways. Therefore, a priori information on fluctuating hypoxia can be important in clinical treatment planning, but complicated dynamics makes it difficult to elucidate biological significance of intermittent hypoxia.Here, we illustrate the use of pulsed electron spin resonance imaging (ESRI) as a novel imaging method to directly monitor fluctuating oxygenation i.e. cycling hypoxia in transplanted tumors. A common resonator platform for both ESRI and magnetic resonance imaging (MRI) provided pO2 maps with anatomical guidance without positional movement. Oxygen images every 3 min in pO2 could visualize the rapid oxygen fluctuation and distinguish the cycling hypoxia and chronic hypoxia. Furthermore, we have examined the vascular renormalization process by longitudinally pO2 mapping during treatments with a multi-tyrosine kinase inhibitor sunitinib. Transient improvement in tumor oxygenation and the decrease of cycling tumor hypoxia were visualized by ESRI 2 to 4 days following antiangiogenic treatments. Radiation treatment during this time period of improved oxygenation by antiangiogenic therapy resulted in a synergistic delay in tumor growth.In conclusion, this ESRI technique combined with MRI, may offer a powerful clinical tool to noninvasively detect variable hypoxic status in tumors and to identify a window of vascular renormalization to maximize the effects of combination therapy with antiangiogenic drugs.
• マルチモダル分子イメージングによる腫瘍内微小環境の多角的評価と天然由来含硫糖脂質の誘発する放射線増感作用メカニズムの解明

  高草木 洋一, 松元 慎吾, 松尾 政之, 高草木 香織, 齋藤 圭太, 岸本 俊, 三浦 雅彦, 宮崎 年恭, 菅原 二三男, 坂口 謙吾, James B Mitchell, Murali C Krishna  第 14 回 日本分子イメージング学会総会・学術集会  2019/05  [Refereed][Not invited]
  
• Application of ESR pO₂ imaging to evaluation of tumor radiosensitivity in intracranial glioma mouse model

  安井博宣, 河合辰哉, 松元慎吾, 齋藤圭太, CAMPHAUSEN Kevin, KRISHNA Murali, 稲波修  電子スピンサイエンス学会年会講演要旨集  57th-  106‐107  2018/11/01  [Not refereed][Not invited]
  
• Serial Imaging Analysis by both absolute partial pressure of oxygen (pO2) and hyperpolarized [1-13C] pyruvate metabolism Changes in Cancer Treatment

  Masayuki Matsuo, Shingo Matsumoto, Keita Saito, Yoichi Takakusagi, Hidekazu Tanaka, Fuminori Hyodo, James B. Mitchell, Murali C. Krishna  46th JSMRM Annual Meeting  2018/09  [Refereed][Not invited]
  
• Monitoring effect of rapamaycin on pyruvate metabolism in SCC tumor using hyperpolarized 13C-MRI

  Keita Saito, Shingo Matsumoto, Yoichi Takakusagi, Masayuki Matsuo, Hellmut Merkle, James B. Mitchell, Murali C. Krishna  International Society for Magnetic Resonance in Medicine (ISMRM) 2018  3069  2018/06  [Refereed][Not invited]
  
• 最先端量子イメージングによる膵臓がん個別化医療のモデル研究

  松元慎吾, 高草木洋一, 齋藤圭太, 岸本俊 Mitchell, James B. Hart, Charles P. Gillies, Robert J. Krishna, Murali C  第 2 回 量子生命科学研究会  2018/05  [Not refereed][Invited]
  
• 電子スピン共鳴酸素イメージングによる脳室内移植グリオーマの放射線感受性評価

  安井博宣, 安井博宣, 河合辰哉, 松元慎吾, 松元慎吾, 齋藤圭太, CAMPHAUSEN Kevin, 稲波修, KRISHNA Murali  日本放射線影響学会大会抄録(Web)  61st-  92 (WEB ONLY)  -92  2018  [Not refereed][Not invited]
  
• 放射線照射における超偏極MRIおよびEPRI画像解析

  松尾政之, 芝本雄太, 松元慎吾, 齋藤圭太, 高草木洋一  第 43 回 日本磁気共鳴医学会大会  2015  [Not refereed][Not invited]
  
• Noninvasive assessment of drug actions in tumor microenvironment by the EPR oxygen imaging

  Yoichi Takakusagi, Shingo Matsumoto, Keita Saito, Masayuki Matsuo Shun, Kishimoto Kaori, Takakusagi Masahiko Miura Fumio Sugawara Kengo, Sakaguchi Robert, J. Gillies Charles P. Hart James, B. Mitchell Murali C. Krishna  第 54 回 電子スピンサイエンス学会年会 (SEST2015)  2015  [Not refereed][Not invited]
  
• 13C-MRI with hyperpolarized [1-13C]pyruvate detects metabolic changes in SCC tumors and HT-29 tumors by radiotherapy

  Keita Saito, Shingo Matsumoto, Yoichi Takakusagi, Masayuki Matsuo, H. Douglas Morris, Martin J. Lizak, Jeeva, P. Munasinghe, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna  International Conference of Radiation Research (ICRR2015)  2015  [Not refereed][Not invited]
  
• Increase in tumor oxygenataion and radiosensitivity caused by SQAP (CG-0321): the mechanism of actions revealed by in vivo imaging studies

  Yoichi Takakusagi, Shingo Matsumoto, Keita Saito, Masayuki Matsuo, Kaori Takakusagi, Shun Kishimoto, Masahiro Ishima, Hiroshi Murata, Keisuke Ohta, Masahiko Miura, Fumio Sugawara, Kengo Sakaguchi, James B. Mitchell, Murali C. Krishna  International Conference of Radiation Research (ICRR2015)  2015  [Not refereed][Not invited]
  
• Serial Imaging of Physiological and Metabolic Changes in Response to Radiation Therapy With Tumor-Bearing Mice

  M. Matsuo, M. Shingo, S. Keita, T. Yoichi, K. Shun, D. Nallathamby, S. Sankaran, M. Douglas, M. Jeeva, M. James, K. Murali, S. Yuta  INTERNATIONAL JOURNAL OF RADIATION ONCOLOGY BIOLOGY PHYSICS  90-  S87  -S87  2014/09  [Not refereed][Not invited]
  
• Detection of metabolic changes in SCC tumor by mTOR inhibition using hyperpolarized 13C-pyruvate MRI

  Keita Saito, Shingo Matsumoto, Yoichi Takakusagi, Masayuki Matsuo, H. Douglas Morris, Hellmut Merkle, Martin J. Lizak, Jeeva, P. Munasinghe, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna  Joint Annual Meeting ISMRM-ESMRMB 2014  2014  [Not refereed][Not invited]
  
• 含硫糖脂質スルホキノボシルアシルグリセロール (SQAG) 誘導体による放射線増感作用: in vivo ライブイメージングによる増感機構の解明と治療後評価

  高草木洋一, 松元慎吾, 齋藤圭太, 松尾政之, 高草木香織, 岸本俊 Daryl Despres Jeeva, P. Munasinghe, Nallathamby Devasahayam, 石間正浩, 村田寛, 太田慶祐, 三浦雅彦, 坂口謙吾, 菅原二三男, James B. Mitchell Murali C. Krishna  文部科学省新学術領域研究 化学療法基盤支援活動第3回シンポジウム  2014  [Not refereed][Not invited]
  
• EPR spectroscopy and oxygen imaging for evaluation of the transient induction of tumor hypoxia and potentiation of bioreductively activated hypoxia-activated prodrug TH-302

  Yoichi Takakusagi, Shingo Matsumoto, Keita Saito, Masayuki Matsuo, Shun Kishimoto, Jonathan, W. Wojtkowiak, Robert J. Gillies, James B. Mitchell, Charles P. Hart, Murali C. Krishna  Joint Conference of APES2014・IES・SEST2014  2014  [Not refereed][Not invited]
  
• Effects of rapamycin on energy metabolism, oxygenation, and angiogenesis in SCC tumor

  Keita Saito, Shingo Matsumoto, Yoichi Takakusagi, Masayuki Matsuo, Jeeva, P. Munasinghe, H. Douglas Morris, Martin J. Lizak, Nallathamby Devasahayam, Sankaran Subramanian, Vyomesh Patel, Silvio J. Gutkind, James B. Mitchell, Murali C. Krishna  41st Annual ISOTT Meeting & EPR 2013 Tul4  2013  [Not refereed][Not invited]
  
• EPR oxygen imaging guided treatment in pancreatic cancer

  Shingo Matsumoto, Keita Saito, Yoichi Takakusagi, Masayuki Matsuo, Jeeva, P. Munasinghe, Nallathamby Devasahayam, Sankaran Subramanian, James B. Mitchell, Murali C. Krishna  World Molecular Imaging Congress  2013  [Not refereed][Not invited]
  
• Imaging-guided determination of the treatment regimens of the hypoxia-activated prodrug TH-302

  Yoichi Takakusagi, Shingo Matsumoto, Keita Saito, Masayuki Matsuo, William DeGraff, Rajani Choudhuri, Nallathamby Devasahayam, Sankaran Subramanian, Jeeva, P. Munasinghe, James B. Mitchell, Charles P. Hart, Murali C. Krishna  World Biotechnology Congress 2013  2013  [Not refereed][Not invited]
  
• Chronic and cyclic hypoxia in fractionated radiation therapy

  Masayuki Matsuo, Shingo Matsumoto, Keita Saito, Yoichi Takakusagi, H. Douglas, Morris, Jeeva, P. Munasinghe, Nallathamby Devasahayam, Sankallan Subramanian, James B. Mitchell, Murali C. Krishna  ASTRO Annual Meeting  2012  [Not refereed][Not invited]
  

Industrial Property Rights

• 特開US2012/034534:Transient hypoxia inducers and their use  2012/04/20

  Shingo Matsumoto, Keita Satito, Murali C. Krishna, James B. Mitchell
• 特表WO2010/062973:Quantitative oxygen imaging systems and methods using echo-based single point imaging  2010/06/03

  Sankaran Suramanian, Nallathamby Devasahayam, Shingo Matsumoto, James B. Mitchell, Cherukuri M. Krishna, John A. cook

Awards & Honors

• 2017 日本応用酵素協会 酵素研究奨励賞
  
• 2017 豊田理研 豊田理研スカラー
  
• 2015/05 International Society for Magnetic Resonance in Medicine (ISMRM) Distinguished Reviewer Award of Magnetic Resonance in Medicine
  
• 2013/09 World Molecular Imaging Congress (WMIC) Top Abstract
  
• 2012/09 World Molecular Imaging Congress (WMIC) Top Abstract
  
• 2008 World Molecular Imaging Congress Travel Stipend Award
   

  受賞者: MATSUMOTO Shingo;NCI;NIH
• 2008 EPR2008 Young Investigator Award
   

  受賞者: MATSUMOTO Shingo;NCI;NIH

Research Grants & Projects

• 超偏極13C脳代謝MRIによる末梢・全身性疾患に伴う認知機能障害の包括的理解

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2024/04 -2028/03 

  Author : 松元 慎吾
• 一重項水素を用いた脳代謝フラックスの超高感度センシングとストレス応答解析

  日本医療研究開発機構AMED：革新的先端研究開発支援事業PRIME

  Date (from‐to) : 2023/10 -2027/03 

  Author : 松元慎吾
• Development of parahydrogen-polarized 13C MRI of infectious sequelae.

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Grant-in-Aid for Challenging Research (Pioneering)

  Date (from‐to) : 2022/06 -2025/03 

  Author : Shingo Matsumoto
• 常温・低磁場核偏極による安定同位体PETイメージングの実現と実用展開

  日本学術振興機構：科学研究費補助金 基盤研究(A) 20H00654

  Date (from‐to) : 2020/04 -2023/03 

  Author : 松元慎吾, 武田憲彦

   

  超偏極タグは、安定同位体である13Cや15Nで標識した分子の核偏極率（= MRI感度に比例）を数万倍に励起することにより、放射性同位体標識に匹敵する高感度検出を実現する新しい分子センシング技術である。本研究では、１）水素ガスを用いた常温・低磁場核偏極により、現行の動的核偏極型の10分の1の低コストで導入可能な安定同位体標識によるPET様の分子イメージング技術（SI-PET）を実現する。このSI-PETを疾患モデルに適用し、２）心筋梗塞における心筋虚血の検出、代謝変容を指標とする認知症の早期検出、炎症性疾患における細胞死イメージング、の３つの診断法を確立することを目的とする。 ２年度目となる令和３年度は、初年度に構築した超偏極13C注射剤の全製造プロセスをマイコンPSoCにより自動制御するシステムを用いて励起装置を最適化することで、[1-13C]ピルビン酸の製造量30mL以上および13C偏極率7.5%を達成した。この30mLの反応系を４セット並列駆動することで、臨床応用に必要な100mL以上の超偏極13C注射剤の製造が期待される成果が得られた。励起装置の詳細とその応用研究として、常温低磁場核偏極により生成した超偏極13Cフマル酸を用いて、肝障害モデルにおける細胞死イメージングのin vivo撮像に成功した成果を合わせて学術雑誌への投稿準備を進めている。また、超偏極13C MRIの高解像度化を目指しDixon-IDEAL型の撮像シーケンスと再構成プログラムの開発を進め、数値ファントムを用いたシミュレーションでは、空間分解能1mmで４代謝物を区別した同時イメージングが可能であることを確認した。今後は上記の励起装置とMRI撮像技術を疾患モデル動物に適用し、改善した画質でのより実用的な代謝イメージング取得を目指す。
• Development of a method of 3D oxygen and pH mapping of tumors using electron spin resonance spectroscopy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2022/03 

  Author : Hirata Hiroshi

   

  We aimed to develop quantitative imaging techniques for the partial pressure of oxygen and extracellular pH for malignant tumors. In this study, electron spin resonance (ESR) spectroscopy was applied to the three-dimensional mapping of the partial pressure of oxygen and pH in solution samples. Using a molecular probe that can concurrently detect the partial pressure of oxygen and pH, we showed the feasibility of concurrent mapping the partial pressure of oxygen and pH in solution samples.
• On site metabolome analysis using ordinal temp and low-field nuclear polarization and deeplearning

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research Challenging Research (Exploratory)

  Date (from‐to) : 2020/07 -2022/03 

  Author : Matsumoto Shingo

   

  We have developed a high-speed NMR-based metabolome analysis system that collectively polarized metabolites obtained by biopsy and measured by increasing the NMR signals by tens of thousands of times using ordinal temperature and low magnetic field nuclear polarization technology. The 13C NMR signal in the 1.5T NMR apparatus was significantly increased up to 70,000 times, and metabolites with a concentration of several tens of μM could be detected by 13C NMR. On the other hand, due to the delay caused by equipment failure in the first year, the identification of disease-specific metabolic changes by LC/TOF-MS remained in the group of known disease marker metabolites. We constructed an automated system using an embedded microcomputer for all processes from sample injection, hyperpolarization induction, polarization transfer, and post-process to the metabolites containing derivatized carboxylic acid by the flow method.
• 低磁場核偏極による生体分子の超高感度センシング技術の開発

  Japan Agency for Medical Research and Development：AMED Sentankeisoku

  Date (from‐to) : 2018/08 -2021/03 

  Author : MATSUMOTO Shingo
• パラ水素誘起核偏極によるピルビン酸の超偏極１３Ｃ ＭＲＩ代謝イメージング法の発展

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2018/07 -2020/03 

  Author : 松元 慎吾, STEWART NEIL

   

  超偏極13C核磁気共鳴画像（Magnetic Resonance Imaging, MRI）は炭素の安定同位体である13Cで標識した化合物の13C NMR/MRI信号を一時的に数万倍に励起することで、その生体内における代謝反応をリアルタイムに可視化する最先端のMRI技術である。様々な臓器のがん診断や心機能評価において、欧米では既に数百人規模の臨床試験が行われ、その有用性が実証されている。その反面、現行の動的核偏極（Dynamic Nuclear Polarization, DNP）型の13C励起装置による臨床初期コストは3-4億円と非常に高額であり、本技術の国内普及を妨げる主因となっている。受入研究者・松元は、この超偏極13C MRIの臨床コストを10分の1に抑制すること目指し、水素ガスと量子技術を組み合わせたパラ水素誘起分極（PHIP）法による13C励起技術の研究開発を行っている。 特別研究員は昨年度までに、超偏極13C MRIの臨床応用において、最も重要な代謝プローブであるがPHIP法では通常励起できないピルビン酸を励起するためのサイドアーム型PHIP励起装置を構築した。NMR技術を駆使して決定したピルビン酸前駆体分子の全てのJ結合定数から、量子統計力学シミュレーションにより13C励起条件を最適化し、励起プロセスに実装することで、最終的に1.5Tの熱平衡状態と比較してピルビン酸の13C MRI信号の４万倍励起を達成した。2年度目は当研究室の他の大学院生と共に、スパースサンプリングされた部分的な13C MRI画像データから、人工知能技術の１つである深層学習とテンソル分解によるノイズ除去を組み合わせて画像を再構成する高速撮像法の研究開発も進め、代謝イメージングの画質を落とすことなく、時間分解能を約5倍向上することに成功した。
• パラ水素誘起分極13C MRIによる細胞死の非侵襲的イメージング技術の開発

  JSPS：JSPS Houga

  Date (from‐to) : 2018/07 -2020/03 

  Author : MATSUMOTO Shingo
• Imaging of acquired mutations using hydrogen energy

  JSPS：JSPS Young Investigator A

  Date (from‐to) : 2016/04 -2020/03 

  Author : MATSUMOTO Shingo
• Model experiments of partial pressure of oxygen-guided intensity modulated radiation therapy

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2016/04 -2018/03 

  Author : Hirata Hiroshi, MATSUMOTO Shingo

   

  The purpose of this study was to clarify the outcomes of the model experiments of oxygen partial pressure-guided radiation therapy. A miniature multi-leaf collimator (MLC) was developed for the model experiments using tumor-bearing mice. This miniature MLC was planned to control the intensity and the distribution of X-ray irradiation to tumor model mice. Also, the computation method for obtaining the collimator opening from the three-dimensional data of oxygen partial pressure in mouse tumors was developed and implemented. Moreover, tumor-growth curves were obtained for non-treated and uniform X-ray irradiated mice. These data will be the control data of tumor growth. For preparing tumor-bearing mice, murine squamous cell carcinoma (SCC VII) cells were subcutaneously implanted in mouse hind legs.
• Dynamic monitoring of homeostasis disruption using hyperpolarized 13C MRI

  Japan Science and Technology Agency：PRESTO

  Date (from‐to) : 2015/02 -2018/03 

  Author : MATSUMOTO Shingo
• Intensity Standard for L-band EPR Tooth Dosimetry

  Dart-CMCR：Pilot Project

  Date (from‐to) : 2013/12 -2014/12 

  Author : MATSUMOTO Shingo
• ESRI・MRI融合型画像解析システムの開発と臨床応用に関する研究

  Japan Society for The Promotion of Science：JSPS DC2

  Date (from‐to) : 2004/04 -2006/03 

  Author : MATSUMOTO Shingo

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