Advertisement

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

Abstract

Purpose

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).

Procedures

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.

Results

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.

Conclusions

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 

Notes

Acknowledgments

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)

References

  1. 1.
    Wilson WR, Hay MP (2011) Targeting hypoxia in cancer therapy. Nat Rev Cancer 11:393–410CrossRefPubMedGoogle Scholar
  2. 2.
    Horsman MR, Mortensen LS, Petersen JB, Busk M, Overgaard J (2012) Imaging hypoxia to improve radiotherapy outcome. Nat Rev Clin Oncol 9:674–687CrossRefPubMedGoogle Scholar
  3. 3.
    Weissleder R, Pittet MJ (2008) Imaging in the era of molecular oncology. Nature 452:580–589CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Tamaki N, Hirata K (2016) Tumor hypoxia: a new PET imaging biomarker in clinical oncology. Int J Clin Oncol 21:619–625CrossRefPubMedGoogle Scholar
  5. 5.
    Lopci E, Grassi I, Chiti A, Nanni C, Cicoria G, Toschi L, Fonti C, Lodi F, Mattioli S, Fanti S (2014) PET radiopharmaceuticals for imaging of tumor hypoxia: a review of the evidence. Am J Nucl Med Mol Imaging 4:365–384PubMedPubMedCentralGoogle Scholar
  6. 6.
    Peeters SG, Zegers CM, Yaromina A, van Elmpt W, Dubois L, Lambin P (2015) Current preclinical and clinical applications of hypoxia PET imaging using 2-nitroimidazoles. Q J Nucl Med Mol Imaging 59:39–57PubMedGoogle Scholar
  7. 7.
    Troost EG, Laverman P, Philippens ME et al (2008) Correlation of [18F]FMISO autoradiography and pimonidazole [corrected] immunohistochemistry in human head and neck carcinoma xenografts. Eur J Nucl Med Mol Imaging 35:1803–1811CrossRefPubMedGoogle Scholar
  8. 8.
    Toyonaga T, Hirata K, Shiga T, Nagara T (2017) Players of ‘hypoxia orchestra’—what is the role of FMISO? Eur J Nucl Med Mol Imaging 44:1679–1681CrossRefPubMedGoogle Scholar
  9. 9.
    Biskupiak JE, Krohn KA (1993) Second generation hypoxia imaging agents. J Nucl Med 34:411–413PubMedGoogle Scholar
  10. 10.
    Okamoto S, Shiga T, Yasuda K, Ito YM, Magota K, Kasai K, Kuge Y, Shirato H, Tamaki N (2013) High reproducibility of tumor hypoxia evaluated by 18F-fluoromisonidazole PET for head and neck cancer. J Nucl Med 54:201–207CrossRefPubMedGoogle Scholar
  11. 11.
    Nakata N, Okumura Y, Nagata E et al (2012) Evaluation of a new PET hypoxia tracer, [18F] HIC101, in comparison with [18F] FMISO. J Nucl Med 53:1523Google Scholar
  12. 12.
    Masaki Y, Shimizu Y, Yoshioka T, Nishijima KI, Zhao S, Higashino K, Numata Y, Tamaki N, Kuge Y (2017) FMISO accumulation in tumor is dependent on glutathione conjugation capacity in addition to hypoxic state. Ann Nucl Med 31:596–604CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Masaki Y, Shimizu Y, Yoshioka T, Tanaka Y, Nishijima KI, Zhao S, Higashino K, Sakamoto S, Numata Y, Yamaguchi Y, Tamaki N, Kuge Y (2015) The accumulation mechanism of the hypoxia imaging probe “FMISO” by imaging mass spectrometry: possible involvement of low-molecular metabolites. Sci Rep 5:16802CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Masaki Y, Shimizu Y, Yoshioka T, Feng F, Zhao S, Higashino K, Numata Y, Kuge Y (2016) Imaging mass spectrometry revealed the accumulation characteristics of the 2-nitroimidazole-based agent “pimonidazole” in hypoxia. PLoS One 11:e0161639CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Raleigh JA, Chou SC, Arteel GE, Horsman MR (1999) Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors. Radiat Res 151:580–589CrossRefPubMedGoogle Scholar
  16. 16.
    Caprioli RM, Farmer TB, Gile J (1997) Molecular imaging of biological samples: localization of peptides and proteins using MALDI-TOF MS. Anal Chem 69:4751–4760CrossRefPubMedGoogle Scholar
  17. 17.
    Gobey J, Cole M, Janiszewski J, Covey T, Chau T, Kovarik P, Corr J (2005) Characterization and performance of MALDI on a triple quadrupole mass spectrometer for analysis and quantification of small molecules. Anal Chem 77:5643–5654CrossRefPubMedGoogle Scholar
  18. 18.
    Vaidyanathan S, Gaskell S, Goodacre R (2006) Matrix-suppressed laser desorption/ionisation mass spectrometry and its suitability for metabolome analyses. Rapid Commun Mass Spectrom 20:1192–1198CrossRefPubMedGoogle Scholar
  19. 19.
    Sugimoto M, Shimizu Y, Yoshioka T, Wakabayashi M, Tanaka Y, Higashino K, Numata Y, Sakai S, Kihara A, Igarashi Y, Kuge Y (2015) Histological analyses by matrix-assisted laser desorption/ionization-imaging mass spectrometry reveal differential localization of sphingomyelin molecular species regulated by particular ceramide synthase in mouse brains. Biochim Biophys Acta 1851:1554–1565CrossRefPubMedGoogle Scholar
  20. 20.
    Sugimoto M, Wakabayashi M, Shimizu Y, Yoshioka T, Higashino K, Numata Y, Okuda T, Zhao S, Sakai S, Igarashi Y, Kuge Y (2016) Imaging mass spectrometry reveals acyl-chain- and region-specific sphingolipid metabolism in the kidneys of sphingomyelin synthase 2-deficient mice. PLoS One 11:e0152191CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Prekeges JL, Rasey JS, Grunbaum Z, Krohn KH (1991) Reduction of fluoromisonidazole, a new imaging agent for hypoxia. Biochem Pharmacol 42:2387–2395CrossRefPubMedGoogle Scholar
  22. 22.
    Riddick DS, Lee C, Ramji S, Chinje EC, Cowen RL, Williams KJ, Patterson AV, Stratford IJ, Morrow CS, Townsend AJ, Jounaidi Y, Chen CS, Su T, Lu H, Schwartz PS, Waxman DJ (2005) Cancer chemotherapy and drug metabolism. Drug Metab Dispos 33:1083–1096CrossRefPubMedGoogle Scholar
  23. 23.
    Komiya S, Gebhardt MC, Mangham DC, Inoue A (1998) Role of glutathione in cisplatin resistance in osteosarcoma cell lines. J Orthop Res 16:15–22CrossRefPubMedGoogle Scholar

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

Personalised recommendations