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. 2000 Nov 22;83(11):1525–1531. doi: 10.1054/bjoc.2000.1489

Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia

P L Olive 1, R E Durand 1, J A Raleigh 2, C Luo 1, C Aquino-Parsons 1
PMCID: PMC2363410  PMID: 11076663

Abstract

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and whether the usual subjective criteria used to define ‘positive’ cells are optimal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and to validate this method by using a more direct indicator of radiobiologic hypoxia, the comet assay. SCCVII tumours in C3H mice were analysed for pimonidazole binding using flow cytometry and an iterative curve-fitting procedure, and the results were compared to the comet assay for the same cell suspensions. On average, cells defined as anoxic by flow analysis (n = 43 tumours) bound 10.8 ± 0.95 times more antibody than aerobic cells. In samples containing known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay under a wider range of tumour oxygen contents, mice were injected with hydralazine to reduce tumour blood flow, or allowed to breathe various gas mixtures during the 90 min exposure to pimonidazole. Hypoxic fraction estimated by the pimonidazole binding method agreed well with the hypoxic fraction measured using the comet assay in SCCVII tumours (r2 = 0.87, slope = 0.98), with similar results in human U87 glioma cells and SiHa cervical carcinoma xenografts. We therefore conclude that this objective analysis of pimonidazole labelling by flow cytometry gives a convenient and accurate estimate of radiobiological hypoxia. Preliminary analyses of biopsies from 3 patients given 0.5 g m–2 pimonidazole also suggest the suitability of this approach for human tumours. © 2000 Cancer Research Campaign http://www.bjcancer.com

Keywords: tumour hypoxia, pimonidazole, comet assay

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Selected References

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  1. Aquino-Parsons C., Luo C., Vikse C. M., Olive P. L. Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia. Radiother Oncol. 1999 May;51(2):179–185. doi: 10.1016/s0167-8140(99)00035-3. [DOI] [PubMed] [Google Scholar]
  2. Brown J. M., Giaccia A. J. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res. 1998 Apr 1;58(7):1408–1416. [PubMed] [Google Scholar]
  3. Bush R. S., Jenkin R. D., Allt W. E., Beale F. A., Bean H., Dembo A. J., Pringle J. F. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br J Cancer Suppl. 1978 Jun;3:302–306. [PMC free article] [PubMed] [Google Scholar]
  4. Chapman J. D., Dugle D. L., Reuvers A. P., Meeker B. E., Borsa J. Letter: Studies on the radiosensitizing effect of oxygen in Chinese hamster cells. Int J Radiat Biol Relat Stud Phys Chem Med. 1974 Oct;26(4):383–389. doi: 10.1080/09553007414551361. [DOI] [PubMed] [Google Scholar]
  5. Durand R. E., Raleigh J. A. Identification of nonproliferating but viable hypoxic tumor cells in vivo. Cancer Res. 1998 Aug 15;58(16):3547–3550. [PubMed] [Google Scholar]
  6. Falk S. J., Ward R., Bleehen N. M. The influence of carbogen breathing on tumour tissue oxygenation in man evaluated by computerised p02 histography. Br J Cancer. 1992 Nov;66(5):919–924. doi: 10.1038/bjc.1992.386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fyles A. W., Milosevic M., Wong R., Kavanagh M. C., Pintilie M., Sun A., Chapman W., Levin W., Manchul L., Keane T. J. Oxygenation predicts radiation response and survival in patients with cervix cancer. Radiother Oncol. 1998 Aug;48(2):149–156. doi: 10.1016/s0167-8140(98)00044-9. [DOI] [PubMed] [Google Scholar]
  8. Hill S. A., Collingridge D. R., Vojnovic B., Chaplin D. J. Tumour radiosensitization by high-oxygen-content gases: influence of the carbon dioxide content of the inspired gas on PO2, microcirculatory function and radiosensitivity. Int J Radiat Oncol Biol Phys. 1998 Mar 1;40(4):943–951. doi: 10.1016/s0360-3016(97)00892-4. [DOI] [PubMed] [Google Scholar]
  9. Hockel M., Schlenger K., Aral B., Mitze M., Schaffer U., Vaupel P. Association between tumor hypoxia and malignant progression in advanced cancer of the uterine cervix. Cancer Res. 1996 Oct 1;56(19):4509–4515. [PubMed] [Google Scholar]
  10. Horsman M. R., Khalil A. A., Siemann D. W., Grau C., Hill S. A., Lynch E. M., Chaplin D. J., Overgaard J. Relationship between radiobiological hypoxia in tumors and electrode measurements of tumor oxygenation. Int J Radiat Oncol Biol Phys. 1994 Jun 15;29(3):439–442. doi: 10.1016/0360-3016(94)90434-0. [DOI] [PubMed] [Google Scholar]
  11. Kavanagh M. C., Tsang V., Chow S., Koch C., Hedley D., Minkin S., Hill R. P. A comparison in individual murine tumors of techniques for measuring oxygen levels. Int J Radiat Oncol Biol Phys. 1999 Jul 15;44(5):1137–1146. doi: 10.1016/s0360-3016(99)00104-2. [DOI] [PubMed] [Google Scholar]
  12. Kennedy A. S., Raleigh J. A., Perez G. M., Calkins D. P., Thrall D. E., Novotny D. B., Varia M. A. Proliferation and hypoxia in human squamous cell carcinoma of the cervix: first report of combined immunohistochemical assays. Int J Radiat Oncol Biol Phys. 1997 Mar 1;37(4):897–905. doi: 10.1016/s0360-3016(96)00539-1. [DOI] [PubMed] [Google Scholar]
  13. Lee J., Siemann D. W., Koch C. J., Lord E. M. Direct relationship between radiobiological hypoxia in tumors and monoclonal antibody detection of EF5 cellular adducts. Int J Cancer. 1996 Jul 29;67(3):372–378. doi: 10.1002/(SICI)1097-0215(19960729)67:3<372::AID-IJC11>3.0.CO;2-A. [DOI] [PubMed] [Google Scholar]
  14. McLaren D. B., Pickles T., Thomson T., Olive P. L. Impact of nicotinamide on human tumour hypoxic fraction measured using the comet assay. Radiother Oncol. 1997 Nov;45(2):175–182. doi: 10.1016/s0167-8140(97)00110-2. [DOI] [PubMed] [Google Scholar]
  15. Olive P. L., Banáth J. P., Durand R. E. Heterogeneity in radiation-induced DNA damage and repair in tumor and normal cells measured using the "comet" assay. Radiat Res. 1990 Apr;122(1):86–94. [PubMed] [Google Scholar]
  16. Olive P. L. Detection of hypoxia by measurement of DNA damage in individual cells from spheroids and murine tumours exposed to bioreductive drugs. I. Tirapazamine. Br J Cancer. 1995 Mar;71(3):529–536. doi: 10.1038/bjc.1995.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Olive P. L. Distribution, oxygenation, and clonogenicity of macrophages in a murine tumor. Cancer Commun. 1989;1(2):93–100. doi: 10.3727/095535489820875273. [DOI] [PubMed] [Google Scholar]
  18. Olive P. L., Durand R. E. Detection of hypoxic cells in a murine tumor with the use of the comet assay. J Natl Cancer Inst. 1992 May 6;84(9):707–711. doi: 10.1093/jnci/84.9.707. [DOI] [PubMed] [Google Scholar]
  19. Olive P. L., Durand R. E., Jackson S. M., Le Riche J. C., Luo C., Ma R., McLaren D. B., Aquino-Parsons C., Thomson T. A., Trotter T. The comet assay in clinical practice. Acta Oncol. 1999;38(7):839–844. doi: 10.1080/028418699432527. [DOI] [PubMed] [Google Scholar]
  20. Olive P. L., Durand R. E., Le Riche J., Olivotto I. A., Jackson S. M. Gel electrophoresis of individual cells to quantify hypoxic fraction in human breast cancers. Cancer Res. 1993 Feb 15;53(4):733–736. [PubMed] [Google Scholar]
  21. Olive P. L., Horsman M. R., Grau C., Overgaard J. Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay. Br J Cancer. 1997;76(6):694–699. doi: 10.1038/bjc.1997.448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Overgaard J. Clinical evaluation of nitroimidazoles as modifiers of hypoxia in solid tumors. Oncol Res. 1994;6(10-11):509–518. [PubMed] [Google Scholar]
  23. Raleigh J. A., Calkins-Adams D. P., Rinker L. H., Ballenger C. A., Weissler M. C., Fowler W. C., Jr, Novotny D. B., Varia M. A. Hypoxia and vascular endothelial growth factor expression in human squamous cell carcinomas using pimonidazole as a hypoxia marker. Cancer Res. 1998 Sep 1;58(17):3765–3768. [PubMed] [Google Scholar]
  24. Raleigh J. A., Chou S. C., Arteel G. E., Horsman M. R. Comparisons among pimonidazole binding, oxygen electrode measurements, and radiation response in C3H mouse tumors. Radiat Res. 1999 May;151(5):580–589. [PubMed] [Google Scholar]
  25. Thrall D. E., Rosner G. L., Azuma C., McEntee M. C., Raleigh J. A. Hypoxia marker labeling in tumor biopsies: quantification of labeling variation and criteria for biopsy sectioning. Radiother Oncol. 1997 Aug;44(2):171–176. doi: 10.1016/s0167-8140(97)01931-2. [DOI] [PubMed] [Google Scholar]
  26. Varia M. A., Calkins-Adams D. P., Rinker L. H., Kennedy A. S., Novotny D. B., Fowler W. C., Jr, Raleigh J. A. Pimonidazole: a novel hypoxia marker for complementary study of tumor hypoxia and cell proliferation in cervical carcinoma. Gynecol Oncol. 1998 Nov;71(2):270–277. doi: 10.1006/gyno.1998.5163. [DOI] [PubMed] [Google Scholar]
  27. Walton M. I., Bleehen N. M., Workman P. Effects of localised tumour hyperthermia on pimonidazole (Ro 03-8799) pharmacokinetics in mice. Br J Cancer. 1989 May;59(5):667–673. doi: 10.1038/bjc.1989.138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang H., Koch C. J., Wallen C. A., Wheeler K. T. Radiation-induced DNA damage in tumors and normal tissues. III. Oxygen dependence of the formation of strand breaks and DNA-protein crosslinks. Radiat Res. 1995 May;142(2):163–168. [PubMed] [Google Scholar]

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