Radiobiological and immunohistochemical assessment of hypoxia in human melanoma xenografts: acute and chronic hypoxia in individual tumours

Purpose: Tumour hypoxia causes resistance to treatment and may promote the development of metastatic disease. The mean fraction of radiobiologically h ypoxic cells has been determined for a large number of tumour cell lines, b ut quantitative information on intertumour heterogeneity in radiobiological hypoxia is sparse, and it is not known whether radiobiological hypoxia is mainly either chronic or acute in nature. The purpose of the work reported hens was (1) to determine the fraction of radiobiologically hypoxic cells i n individual rumours and (2) to differentiate quantitatively between chroni c and acute hypoxia. Materials and methods: Four human melanoma xenograft lines (A-07, D-12, R-1 8, U-25) were included. A radiobiological assay based on the paired surviva l curve method was established to measure the fraction of radiobiologically hypoxic cells. An immunohistochemical assay using the hypoxia marker pimon idazole was developed to determine the fraction of chronically hypoxic cell s. The fraction of acutely hypoxic cells was estimated from the fraction of radiobiologically hypoxic cells and the fraction of chronically hypoxic ce lls. Results: The fractions of radiobiologically hypoxic cells were in the range s of 1-49% (A-07), 10-69% (D-12), 22-87% (R-18) and 23-85% (U-25); the frac tions of chronically hypoxic cells were in the ranges of 0-15% (A-07), 5-25 % (D-12), 4-17% (R-18) and 9-25% (U-25); the fractions of acutely hypoxic c ells were in the ranges of 1-47% (A-07), 1-57% (D-12), 9-80% (R-18) and 5-6 9% (U-25). The fraction of acutely hypoxic cells was higher than the fracti on of chronically hypoxic cells in most A-07, R-18 and U-25 tumours. The fr action of chronically hypoxic cells was higher than the fraction of acutely hypoxic cells in 16 of 25 D-12 tumours. Conclusion: This study indicates that acute hypoxia in tumours is a far mor e serious problem than chronic hypoxia and, consequently, it may be benefic ial to focus on acute hypoxia rather than chronic hypoxia when searching fo r clinically useful predictive assays of hypoxia-induced radiation resistan ce and malignant progression and for methods to overcome treatment resistan ce caused by hypoxia.