Molecular Imaging and Biology

, Volume 21, Issue 1, pp 122–129 | Cite as

A Novel PET Probe “[18F]DiFA” Accumulates in Hypoxic Region via Glutathione Conjugation Following Reductive Metabolism

  • Yoichi ShimizuEmail author
  • Songji Zhao
  • Hironobu Yasui
  • Ken-ichi Nishijima
  • Hiroki Matsumoto
  • Tohru Shiga
  • Nagara Tamaki
  • Mikako Ogawa
  • Yuji Kuge
Research Article



Hypoxia in tumor has close relationship with angiogenesis and tumor progression. Previously, we developed 2,2-dihydroxymethyl-3-[18F]fluoropropyl-2-nitroimidazole ([18F]DiFA) as a novel positron emission tomography (PET) probe for diagnosis of hypoxia. In this study, we elucidated whether the accumulation of [18F]DiFA in cells is dependent on the hypoxic state and revealed how [18F]DiFA accumulates in hypoxic cells in combination with imaging mass spectrometry (IMS).


FaDu human head and neck cancer cells were treated with [18F]DiFA and then incubated under normoxia (21% O2) or hypoxia (1% O2) for 2 h. The cells were extracted using methanol, and the radioactivities of the precipitates (macromolecule fraction) and supernatants (low-molecular-weight fraction) were measured. FaDu-bearing mice were injected intravenously with [18F]DiFA and with pimonidazole 1 h later. The tumors were excised 2 h after the injection of [18F]DiFA. Autoradiography, IMS, and immunohistochemical (IHC) staining for pimonidazole were performed with serial tumor sections.


In the in vitro study, the radioactivity of FaDu cells was significantly higher under hypoxia than that under normoxia (0.53 ± 0.02 vs. 0.27 ± 0.02 %dose/mg protein, p < 0.05). The radioactivity of the low-molecular-weight fraction was 66.3 ± 0.6% in the hypoxic cell. In the in vivo study, [18F]DiFA accumulated in the tumor tissues existed mainly as low-molecular-weight compounds (90.4 ± 0.9%). In addition, the glutathione conjugate of reductive DiFA metabolite (amino-DiFA-GS) existed in tumor tissues revealed by the IMS study, and the distribution pattern of amino-DiFA-GS was very similar to that of the radioactivity and the positive staining area of pimonidazole.


Our results suggest that [18F]DiFA undergoes the glutathione conjugation reaction following reductive metabolism in hypoxic cells, which leads hypoxia-specific PET imaging with [18F]DiFA.

Key words

DiFA Hypoxia Imaging mass spectrometry Molecular imaging Glutathione 



We thank the staff of the Hokkaido University Hospital Cyclotron Facility for the synthesis of [18F]DiFA and Reimi Kishi for her skillful technical assistance.

Funding Information

This study was supported by the Acceleration Transformative Research for Medical Innovation program (ACT-M) from the Japan Agency for Medical Research and Development (AMED).

Compliance with Ethical Standards

The protocols for the experiments with tumor xenograft model mice were approved by the Laboratory Animal Care and Use Committee of Hokkaido University. The animal experiments were performed in accordance with the Guidelines for Animal Experiments of the Graduate School of Medicine, Hokkaido University.

Conflict of Interest

T. S., N. T., and Y. K. have grant support from Nihon Medi-Physics Co., Ltd. H. M is an employee of Nihon Medi-Physics Co., Ltd. The other authors declare that there is no conflict of interest associated with this manuscript.

Supplementary material

11307_2018_1214_MOESM1_ESM.pdf (407 kb)
ESM 1 (PDF 406 kb)


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Copyright information

© World Molecular Imaging Society 2018

Authors and Affiliations

  1. 1.Laboratory of Bioanalysis and Molecular Imaging, Graduate School of Pharmaceutical SciencesHokkaido UniversitySapporoJapan
  2. 2.Central Institute of Isotope ScienceHokkaido UniversitySapporoJapan
  3. 3.Kyoto University HospitalKyotoJapan
  4. 4.Graduate School of MedicineHokkaido UniversitySapporoJapan
  5. 5.Research CentreNihon Medi-Physics Co., Ltd.ChibaJapan

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