Summary
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.
Similar content being viewed by others
Article PDF
Change history
16 November 2011
This paper was modified 12 months after initial publication to switch to Creative Commons licence terms, as noted at publication
References
Brown, J. M. & Giaccia, A. J. (1994). Tumour hypoxia: the picture has changed in the 1990s. (Review). Int J Radiat Biol 65: 95–102.
Fenton, B. M. & Gayeski, T. E. J. (1990). Determination of microvascular oxyhemoglobin saturations using cryospectrophotometry. Am J Physiol 259: H1912–H1920.
Fenton, B. M. & Way, B. W. (1993). Vascular morphometry of KHT and RIF-1 murine sarcomas. Radiother Oncol 28: 57–62.
Fox, S. B., Leek, R. D., Weekes, M. P., Whitehouse, R. M., Gatter, K. C. & Harris, A. L. (1995). Quantitation and prognostic value of breast cancer angiogenesis: comparison of microvessel density, Chalkley count, and computer image analysis. J Pathol 177: 275–283.
Horsman, M. R., Nordsmark, M., Khalil, A. A., Hill, S. A., Chaplin, D. J., Siemann, D. W. & Overgaard, J. (1994). Reducing acute and chronic hypoxia in tumours by combining nicotinamide with carbogen breathing. Acta Oncologica 33: 371–376.
Huang, X., Molema, G., King, S., Watkins, L., Edgington, T. S. & Thorpe, P. E. (1997). Tumor infarction in mice by antibody-directed targeting of tissue factor to tumour vasculature. Science 275: 547–550.
Kayar, S. R., Archer, P. G., Lechner, A. J. & Banchero, N. (1982). The closest-individual method in the analysis of the distribution of capillaries. Microvasc Res 24: 326–341.
Klauber, N., Parangi, S., Flynn, E., Hamel, E. & Damato, R. J. (1997). Inhibition of angiogenesis and breast cancer in mice by the microtubule inhibitors 2-methoxyestradiol and taxol. Cancer Res 57: 81–86.
Loats, J. T., Sillau, A. H. & Banchero, N. (1978). How to quantify skeletal muscle capillarity. Adv Exp Med Biol 94: 41–48.
Lord, E. M., Harwell, L. & Koch, C. J. (1993). Detection of hypoxic cells by monoclonal antibody recognizing 2-nitroimidazole adducts. Cancer Res 53: 5721–5726.
Olive, P. L. & Durand, R. E. (1987). Characterization of a carbocyanine derivative as a fluorescent penetration probe. Cytometry 8: 571–575.
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. (1997). Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 88: 277–285.
Rijken, PFJW, Bernsen, HJJA & van der Kogel, A. J. (1995). Application of an image analysis system to the quantitation of tumor perfusion and vascularity in human glioma xenografts. Microvasc Res 50: 141–153.
Smith, K. A., Hill, S. A., Begg, A. C. & Denekamp, J. (1988). Validation of the fluorescent dye Hoechst 33342 as a vascular space marker in tumours. Br J Cancer 57: 247–253.
Thomson, J. E. & Rauth, A. M. (1974). An in vitro assay to measure the viability of KHT tumor cells not previously exposed to culture conditions. Radiat Res 58: 262–276.
Trotter, M. J., Chaplin, D. J., Durand, R. E. & Olive, P. L. (1989a). The use of fluorescent probes to identify regions of transient perfusion in murine tumors. Int J Radiat Oncol Biol Phys 16: 931–934.
Trotter, M. J., Chaplin, D. J. & Olive, P. L. (1989b). Use of a carbocyanine dye as a marker of functional vasculature in murine tumours. Br J Cancer 59: 706–709.
van Geel, I. P. J., Oppelaar, H., Rijken, PFJW, Bernsen, HJJA, Hagemeier, N. E. M., van der Kogel, A. J., Hodgkiss, R. J. & Stewart, F. A. (1996). Vascular perfusion and hypoxic areas in RIF-1 tumours after photodynamic therapy. Br J Cancer 73: 288–293.
Vaupel, P. W. (1993). Oxygenation of solid tumors. In Drug Resistance in Oncology, Teicher BA (ed), pp. 53–85, Marcel Dekker: New York
Vecchi, A., Garlanda, C., Lampugnani, M. G., Resnati, M., Matteucci, C., Stoppacciaro, A., Schnurch, H., Risau, W., Ruco, L., Mantovani, A. & Dejana, E. (1994). Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur J Cell Biol 63: 247–254.
Author information
Authors and Affiliations
Rights and permissions
From twelve months after its original publication, this work is licensed under the Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
About this article
Cite this article
Fenton, B., Paoni, S., Lee, J. et al. Quantification of tumour vasculature and hypoxia by immunohistochemical staining and HbO2 saturation measurements. Br J Cancer 79, 464–471 (1999). https://doi.org/10.1038/sj.bjc.6690072
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/sj.bjc.6690072
Keywords
This article is cited by
-
Radiation-Guided Targeting of Combretastatin Encapsulated Immunoliposomes to Mammary Tumors
Pharmaceutical Research (2009)
-
Antiangiogenic gene therapy with systemically administered sFlt-1 plasmid DNA in engineered gelatin-based nanovectors
Cancer Gene Therapy (2007)
-
Overexpression of VEGF121, but not VEGF165 or FGF-1, improves oxygenation in MCF-7 breast tumours
British Journal of Cancer (2004)
-
Inhibition of Rho pathways induces radiosensitization and oxygenation in human glioblastoma xenografts
Oncogene (2003)
