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

Abstract

Purpose: Tumour hypoxia causes resistance to treatment and may promote the development of metastatic disease. The mean fraction of radiobiologically hypoxic cells has been determined for a large number of tumour cell lines, but 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 here was (1) to determine the fraction of radiobiologically hypoxic cells in individual tumours and (2) to differentiate quantitatively between chronic and acute hypoxia. Materials and methods: Four human melanoma xenograft lines (A-07, D-12, R-18, U-25) were included. A radiobiological assay based on the paired survival curve method was established to measure the fraction of radiobiologically hypoxic cells. An immunohistochemical assay using the hypoxia marker pimonidazole was developed to determine the fraction of chronically hypoxic cells. The fraction of acutely hypoxic cells was estimated from the fraction of radiobiologically hypoxic cells and the fraction of chronically hypoxic cells. Results: The fractions of radiobiologically hypoxic cells were in the ranges of 1-49% (A-07), 10-69% (D-12), 22-87% (R-18) and 23-85% (U-25); the fractions 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 cells were in the ranges of 1-47% (A-07), 1-57% (D-12), 9-80% (R-18) and 5-69% (U-25). The fraction of acutely hypoxic cells was higher than the fraction of chronically hypoxic cells in most A-07, R-18 and U-25 tumours. The fraction 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 more serious problem than chronic hypoxia and, consequently, it may be beneficial to focus on acute hypoxia rather than chronic hypoxia when searching for clinically useful predictive assays of hypoxia-induced radiation resistance and malignant progression and for methods to overcome treatment resistance caused by hypoxia.