Identification of hypoxia in cells and tissues of epigastric 9L rat glioma using EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl) acetamide] (original) (raw)

Abstract

One of the most sensitive hypoxia detection methods is based on the observation that binding of nitroimidazoles to cellular macromolecules occurs as a result of hypoxia-dependent bioreduction by cellular nitroreductases. Nitroimidazole-binding techniques provide measurements of hypoxia to virtually any degree of spatial resolution and with a multiplicity of techniques. This paper demonstrates hypoxia imaging using in vivo EF5 binding with detection by a fluorochrome-conjugated monoclonal antibody. We investigated these techniques in the 9L glioma tumour, in part because the exact nature of the hypoxia in this tumour system is controversial. Our results demonstrate that following intravenous injection of EF5, binding and detection using a monoclonal antibody in 9L gliomas is specific and oxygen dependent. Detection of binding using fluorescence microscopy can be performed on frozen tissues; tissue sections can be counterstained with haematoxylin and eosin for light microscopic analysis. Alternatively, the distribution of hypoxia in a tumour can be inferred by examining individual tumour cells using flow cytometric techniques. Based upon the results presented herein, the radiation-resistant phenotype of 9L epigastric tumours grown in our laboratories can be associated with the presence of hypoxic cells.

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Selected References

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  1. Brown J. M., Giaccia A. J. Tumour hypoxia: the picture has changed in the 1990s. Int J Radiat Biol. 1994 Jan;65(1):95–102. doi: 10.1080/09553009414550131. [DOI] [PubMed] [Google Scholar]
  2. Chaplin D. J., Durand R. E., Olive P. L. Acute hypoxia in tumors: implications for modifiers of radiation effects. Int J Radiat Oncol Biol Phys. 1986 Aug;12(8):1279–1282. doi: 10.1016/0360-3016(86)90153-7. [DOI] [PubMed] [Google Scholar]
  3. Chapman J. D., Baer K., Lee J. Characteristics of the metabolism-induced binding of misonidazole to hypoxic mammalian cells. Cancer Res. 1983 Apr;43(4):1523–1528. [PubMed] [Google Scholar]
  4. Cline J. M., Thrall D. E., Rosner G. L., Raleigh J. A. Distribution of the hypoxia marker CCI-103F in canine tumors. Int J Radiat Oncol Biol Phys. 1994 Mar 1;28(4):921–933. doi: 10.1016/0360-3016(94)90113-9. [DOI] [PubMed] [Google Scholar]
  5. Franko A. J., Koch C. J., Garrecht B. M., Sharplin J., Hughes D. Oxygen dependence of binding of misonidazole to rodent and human tumors in vitro. Cancer Res. 1987 Oct 15;47(20):5367–5376. [PubMed] [Google Scholar]
  6. GRAY L. H., CONGER A. D., EBERT M., HORNSEY S., SCOTT O. C. The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol. 1953 Dec;26(312):638–648. doi: 10.1259/0007-1285-26-312-638. [DOI] [PubMed] [Google Scholar]
  7. Gutin P. H., Barcellos M. H., Shrieve D. C., Sano Y., Bernstein M., Deen D. F. Further evidence for the absence of a hypoxic fraction in the 9L rat tumour multicellular spheroid system. Br J Radiol. 1982 Sep;55(657):688–690. doi: 10.1259/0007-1285-55-657-688. [DOI] [PubMed] [Google Scholar]
  8. Hodgkiss R. J., Jones G., Long A., Parrick J., Smith K. A., Stratford M. R., Wilson G. D. Flow cytometric evaluation of hypoxic cells in solid experimental tumours using fluorescence immunodetection. Br J Cancer. 1991 Jan;63(1):119–125. doi: 10.1038/bjc.1991.24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hodgkiss R. J., Kelleher E., Parrick J. Hypoxia-specific inhibition of recovery from radiation damage by a novel 2-nitroimidazole with a theophylline side chain. Int J Radiat Biol. 1992 Jun;61(6):797–803. doi: 10.1080/09553009214551671. [DOI] [PubMed] [Google Scholar]
  10. Hodgkiss R. J., Middleton R. W., Parrick J., Rami H. K., Wardman P., Wilson G. D. Bioreductive fluorescent markers for hypoxic cells: a study of 2-nitroimidazoles with 1-substituents containing fluorescent, bridgehead-nitrogen, bicyclic systems. J Med Chem. 1992 May 15;35(10):1920–1926. doi: 10.1021/jm00088a030. [DOI] [PubMed] [Google Scholar]
  11. Howell R. L., Koch C. J. The disaggregation, separation and identification of cells from irradiated and unirradiated EMT6 mouse tumors. Int J Radiat Oncol Biol Phys. 1980 Mar;6(3):311–318. doi: 10.1016/0360-3016(80)90139-x. [DOI] [PubMed] [Google Scholar]
  12. Koch C. J., Giandomenico A. R., Iyengar C. W. Bioreductive metabolism of AF-2[2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide] combined with 2-nitroimidazoles. Implications for use as hypoxic cell markers. Biochem Pharmacol. 1993 Sep 14;46(6):1029–1036. doi: 10.1016/0006-2952(93)90667-l. [DOI] [PubMed] [Google Scholar]
  13. Leith J. T., Schilling W. A., Wheeler K. T. Cellular radiosensitivity of a rat brain tumor. Cancer. 1975 Jun;35(6):1545–1550. doi: 10.1002/1097-0142(197506)35:6<1545::aid-cncr2820350611>3.0.co;2-n. [DOI] [PubMed] [Google Scholar]
  14. Loeffler D. A., Keng P. C., Baggs R. B., Lord E. M. Lymphocytic infiltration and cytotoxicity under hypoxic conditions in the EMT6 mouse mammary tumor. Int J Cancer. 1990 Mar 15;45(3):462–467. doi: 10.1002/ijc.2910450315. [DOI] [PubMed] [Google Scholar]
  15. MacDonald H. R., Koch C. J. Energy metabolism and T-cell-mediated cytolysis. I. Synergism between inhibitors of respiration and glycolysis. J Exp Med. 1977 Sep 1;146(3):698–709. doi: 10.1084/jem.146.3.698. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Moulder J. E., Rockwell S. Hypoxic fractions of solid tumors: experimental techniques, methods of analysis, and a survey of existing data. Int J Radiat Oncol Biol Phys. 1984 May;10(5):695–712. doi: 10.1016/0360-3016(84)90301-8. [DOI] [PubMed] [Google Scholar]
  17. Muschel R. J., Bernhard E. J., Garza L., McKenna W. G., Koch C. J. Induction of apoptosis at different oxygen tensions: evidence that oxygen radicals do not mediate apoptotic signaling. Cancer Res. 1995 Mar 1;55(5):995–998. [PubMed] [Google Scholar]
  18. Nathan C. F., Mercer-Smith J. A., Desantis N. M., Palladino M. A. Role of oxygen in T cell-mediated cytolysis. J Immunol. 1982 Nov;129(5):2164–2171. [PubMed] [Google Scholar]
  19. Oleson J. R. Eugene Robertson Special Lecture. Hyperthermia from the clinic to the laboratory: a hypothesis. Int J Hyperthermia. 1995 May-Jun;11(3):315–322. doi: 10.3109/02656739509022467. [DOI] [PubMed] [Google Scholar]
  20. Olive P. L., Durand R. E. Fluorescent nitroheterocycles for identifying hypoxic cells. Cancer Res. 1983 Jul;43(7):3276–3280. [PubMed] [Google Scholar]
  21. Raleigh J. A., Miller G. G., Franko A. J., Koch C. J., Fuciarelli A. F., Kelly D. A. Fluorescence immunohistochemical detection of hypoxic cells in spheroids and tumours. Br J Cancer. 1987 Oct;56(4):395–400. doi: 10.1038/bjc.1987.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Secomb T. W., Hsu R., Dewhirst M. W., Klitzman B., Gross J. F. Analysis of oxygen transport to tumor tissue by microvascular networks. Int J Radiat Oncol Biol Phys. 1993 Feb 15;25(3):481–489. doi: 10.1016/0360-3016(93)90070-c. [DOI] [PubMed] [Google Scholar]
  23. Stone H. B., Brown J. M., Phillips T. L., Sutherland R. M. Oxygen in human tumors: correlations between methods of measurement and response to therapy. Summary of a workshop held November 19-20, 1992, at the National Cancer Institute, Bethesda, Maryland. Radiat Res. 1993 Dec;136(3):422–434. [PubMed] [Google Scholar]
  24. THOMLINSON R. H., GRAY L. H. The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer. 1955 Dec;9(4):539–549. doi: 10.1038/bjc.1955.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Teicher B. A., Herman T. S., Rose C. M. Effect of Fluosol-DA on the response of intracranial 9L tumors to X rays and BCNU. Int J Radiat Oncol Biol Phys. 1988 Nov;15(5):1187–1192. doi: 10.1016/0360-3016(88)90202-7. [DOI] [PubMed] [Google Scholar]
  26. Thompson J. M., Gralow J. R., Levy R., Miller R. A. The optimal application of forward and ninety-degree light scatter in flow cytometry for the gating of mononuclear cells. Cytometry. 1985 Sep;6(5):401–406. doi: 10.1002/cyto.990060503. [DOI] [PubMed] [Google Scholar]
  27. Urtasun R. C., Chapman J. D., Raleigh J. A., Franko A. J., Koch C. J. Binding of 3H-misonidazole to solid human tumors as a measure of tumor hypoxia. Int J Radiat Oncol Biol Phys. 1986 Jul;12(7):1263–1267. doi: 10.1016/0360-3016(86)90273-7. [DOI] [PubMed] [Google Scholar]
  28. Wallen C. A., Michaelson S. M., Wheeler K. T. Evidence for an unconventional radiosensitivity of rat 9L subcutaneous tumors. Radiat Res. 1980 Dec;84(3):529–541. [PubMed] [Google Scholar]
  29. Weber T., Seitz R. J., Liebert U. G., Gallasch E., Wechsler W. Affinity cytochemistry of vascular endothelia in brain tumors by biotinylated Ulex europaeus type I lectin (UEA I). Acta Neuropathol. 1985;67(1-2):128–135. doi: 10.1007/BF00688133. [DOI] [PubMed] [Google Scholar]
  30. Wong K. H., Wallen C. A., Wheeler K. T. Chemosensitization of the nitrosoureas by 2-nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structure-activity considerations. Int J Radiat Oncol Biol Phys. 1990 May;18(5):1043–1050. doi: 10.1016/0360-3016(90)90439-q. [DOI] [PubMed] [Google Scholar]
  31. Zeman E. M., Calkins D. P., Cline J. M., Thrall D. E., Raleigh J. A. The relationship between proliferative and oxygenation status in spontaneous canine tumors. Int J Radiat Oncol Biol Phys. 1993 Nov 15;27(4):891–898. doi: 10.1016/0360-3016(93)90465-8. [DOI] [PubMed] [Google Scholar]