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Fluorescence imaging of tumour hypoxia in xenograft tumour models Sobhanifar, Solmaz


Hypoxic cells in solid tumours are known to resist radiotherapy as well as forms of chemotherapy. Hypoxia is also believed to promote tumour aggressiveness and metastasis. It is therefore important to develop a practical method to identify hypoxic tumour cells in order to assess which patients could benefit from hypoxic cell-targeted therapies. The goal of this study was to develop an immunohistochemical approach to the detection of hypoxia in xenograft models that may be applied to clinical samples. The hypotheses to be tested were that areas of chronic or transient hypoxia could be distinguished based on specific patterns of hypoxia markers, and that HIF-1α could be comparable as a hypoxia marker to pimonidazole. The objectives were: 1) to characterise the kinetics of development under anoxia and loss upon reoxygenation of the exogenous hypoxia marker pimonidazole and the endogenous hypoxia marker HIF-1α, 2) to use quantitative fluorescence image analysis to compare patterns of pimonidazole binding and HIF-1α expression in WiDr, SiHa and M006 human tumour xenografts, and to measure marker response under various oxygen- breathing conditions, and 3) to compare hypoxia marker patterns in xenograft tumours with patterns observed in cervical cancer biopsies. HIF-1α and pimonidazole colocalised in regions distant from blood vessels; perinecrotic regions however showed pimonidazole binding but no HIF-la expression. This lack was not a result of anoxia but likely a result of glucose and serum starvation. With time after pimonidazole administration, the extent of colocalisation with HIF-1α was reduced from 60% at 90min to 7% at 48hr, consistent with the movement of pimonidazole-labelled cells into necrosis. Patterns of HIF-1α and pimonidazole binding in clinical samples confirmed the dissociation between the markers when pimonidazole was administered 24hr before tumour excision. Overall our xenograft results indicated HIF-1α to be a reliable indicator of tumour hypoxia; however, it over-estimated radiobiological hypoxia (<1% O2) and was not expressed in the most hypoxic regions of tumours. The kinetics of marker development and loss, and occasional regions expressing one but not the other marker indicated that HIF-1α in combination with pimonidazole has the potential to measure the presence of transient hypoxia.

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