Hypoxia-associated spontaneous pulmonary metastasis in human melanoma xenografts: involvement of microvascular hot spots induced in hypoxic foci by interleukin 8

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Abstract
The aim of this study was to investigate whether tumour hypoxia and/or vascular hot spots promote the development of metastatic disease. The D-12 human melanoma xenograft line was used as a tumour model. Hypoxia and vascular hot spots were detected by immunohistochemistry using pimonidazole as a hypoxia marker and anti-CD31 antibody to visualize endothelial cells. Vascular hot spots were found to be induced in hypoxic foci, owing to hypoxia-induced up-regulation of angiogenesis stimulatory factors. This effect was mediated by interleukin 8 and possibly also by vascular endothelial growth factor. Interleukin 8 positive foci showed a high degree of co-localization with hypoxic foci, as revealed by immunohistochemistry. The incidence of spontaneous pulmonary metastases was associated with the density of hypoxic foci, the density of interleukin 8 positive foci and the density of vascular hot spots in the primary tumour. Treatment with neutralizing antibody against interleukin 8 and/or vascular endothelial growth factor resulted in hypoxia-induced necrosis rather than hypoxia-induced vascular hot spots and inhibited metastasis. Our study suggests a cause-effect relationship between hypoxia and metastasis in cancer and hence an elevated probability of metastatic disease in patients having primary tumours characterized by high densities of hypoxic foci and vascular hot spots.