Skip to main content
PMC home page
British Journal of Cancer logoLink to British Journal of Cancer
. 1999 Feb;79(3-4):464–471. doi: 10.1038/sj.bjc.6690072

Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements

B M Fenton 1, S F Paoni 1, J Lee 2, C J Koch 3, E M Lord 2
PMCID: PMC2362405  PMID: 10027314

Abstract

Despite the possibility that tumour hypoxia may limit radiotherapeutic response, the underlying mechanisms remain poorly understood. A new methodology has been developed in which information from several sophisticated techniques is combined and analysed at a microregional level. First, tumour oxygen availability is spatially defined by measuring intravascular blood oxygen saturations (HbO2) cryospectrophotometrically in frozen tumour blocks. Second, hypoxic development is quantified in adjacent sections using immunohistochemical detection of a fluorescently conjugated monoclonal antibody (ELK3-51) to a nitroheterocyclic hypoxia marker (EF5), thereby providing information relating to both the oxygen consumption rates and the effective oxygen diffusion distances. Third, a combination of fluorescent (Hoechst 33342 or DiOC7(3)) and immunohistological (PECAM-1/CD31) stains is used to define the anatomical vascular densities and the fraction of blood vessels containing flow. Using a computer-interfaced microscope stage, image analysis software and a 3-CCD colour video camera, multiple images are digitized, combined to form a photo-montage and revisited after each of the three staining protocols. By applying image registration techniques, the spatial distribution of HbO2 saturations is matched to corresponding hypoxic marker intensities in adjacent sections. This permits vascular configuration to be related to oxygen availability and allows the hypoxic marker intensities to be quantitated in situ. © 1999 Cancer Research Campaign

Keywords: image analysis, angiogenesis, blood vessels, tumour oxygenation, oxyhaemoglobin, hypoxic marker

Full Text

The Full Text of this article is available as a PDF (3.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Fox S. B., Leek R. D., Weekes M. P., Whitehouse R. M., Gatter K. C., Harris A. L. Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol. 1995 Nov;177(3):275–283. doi: 10.1002/path.1711770310. [DOI] [PubMed] [Google Scholar]
  2. Horsman M. R., Nordsmark M., Khalil A. A., Hill S. A., Chaplin D. J., Siemann D. W., Overgaard J. Reducing acute and chronic hypoxia in tumours by combining nicotinamide with carbogen breathing. Acta Oncol. 1994;33(4):371–376. doi: 10.3109/02841869409098431. [DOI] [PubMed] [Google Scholar]
  3. Huang X., Molema G., King S., Watkins L., Edgington T. S., Thorpe P. E. Tumor infarction in mice by antibody-directed targeting of tissue factor to tumor vasculature. Science. 1997 Jan 24;275(5299):547–550. doi: 10.1126/science.275.5299.547. [DOI] [PubMed] [Google Scholar]
  4. Kayar S. R., Archer P. G., Lechner A. J., Banchero N. The closest-individual method in the analysis of the distribution of capillaries. Microvasc Res. 1982 Nov;24(3):326–341. doi: 10.1016/0026-2862(82)90020-6. [DOI] [PubMed] [Google Scholar]
  5. Lord E. M., Harwell L., Koch C. J. Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts. Cancer Res. 1993 Dec 1;53(23):5721–5726. [PubMed] [Google Scholar]
  6. O'Reilly M. S., Boehm T., Shing Y., Fukai N., Vasios G., Lane W. S., Flynn E., Birkhead J. R., Olsen B. R., Folkman J. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997 Jan 24;88(2):277–285. doi: 10.1016/s0092-8674(00)81848-6. [DOI] [PubMed] [Google Scholar]
  7. Olive P. L., Durand R. E. Characterization of a carbocyanine derivative as a fluorescent penetration probe. Cytometry. 1987 Nov;8(6):571–575. doi: 10.1002/cyto.990080607. [DOI] [PubMed] [Google Scholar]
  8. Rijken P. F., Bernsen H. J., van der Kogel A. J. Application of an image analysis system to the quantitation of tumor perfusion and vascularity in human glioma xenografts. Microvasc Res. 1995 Sep;50(2):141–153. doi: 10.1006/mvre.1995.1048. [DOI] [PubMed] [Google Scholar]
  9. Smith K. A., Hill S. A., Begg A. C., Denekamp J. Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours. Br J Cancer. 1988 Mar;57(3):247–253. doi: 10.1038/bjc.1988.54. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Thomson J. E., Rauth A. M. An in vitro assay to measure the viability of KHT tumor cells not previously exposed to culture conditions. Radiat Res. 1974 May;58(2):262–276. [PubMed] [Google Scholar]
  11. Trotter M. J., Chaplin D. J., Durand R. E., Olive P. L. The use of fluorescent probes to identify regions of transient perfusion in murine tumors. Int J Radiat Oncol Biol Phys. 1989 Apr;16(4):931–934. doi: 10.1016/0360-3016(89)90889-4. [DOI] [PubMed] [Google Scholar]
  12. Trotter M. J., Chaplin D. J., Olive P. L. Use of a carbocyanine dye as a marker of functional vasculature in murine tumours. Br J Cancer. 1989 May;59(5):706–709. doi: 10.1038/bjc.1989.148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Vecchi A., Garlanda C., Lampugnani M. G., Resnati M., Matteucci C., Stoppacciaro A., Schnurch H., Risau W., Ruco L., Mantovani A. Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur J Cell Biol. 1994 Apr;63(2):247–254. [PubMed] [Google Scholar]
  14. van Geel I. P., Oppelaar H., Rijken P. F., Bernsen H. J., Hagemeier N. E., van der Kogel A. J., Hodgkiss R. J., Stewart F. A. Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy. Br J Cancer. 1996 Feb;73(3):288–293. doi: 10.1038/bjc.1996.51. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from British Journal of Cancer are provided here courtesy of Cancer Research UK

RESOURCES