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

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.

Figure 1

Immunohistochemical preparations of a D-12 tumour. An avidin-biotin peroxidase-based method was used for staining. Haematoxylin was used for counterstaining. Hypoxic focus visualized by anti-pimonidazole antibody staining (A) and section of a vascular hot spot visualized by anti-CD31 antibody staining (B).

Figure 2

Immunohistochemical preparations of adjacent sections of a D-12 tumour. An avidin-biotin peroxidase-based method was used for staining. Haematoxylin was used for counterstaining. IL-8 positive foci visualized by anti-IL-8 antibody staining (A) and hypoxic foci visualized by anti-pimonidazole antibody staining (B).

Figure 3

Density of hypoxic foci in the primary tumour (•), density of vascular hot spots in the primary tumour (○) and number of pulmonary metastases in the host (▪) vs wet weight of the primary tumour. The densities of hypoxic foci and vascular hot spots were determined in one experiment and the number of pulmonary metastases in another. Points represent single D-12 tumours (•, ○) or mean values of 10 D-12 tumours/mice (▪).

Figure 4

Hypoxia, IL-8 expression and neovascularization in metastatic and non-metastatic D-12 primary tumours. Points represent single tumours. Density of hypoxic foci (A), density of IL-8 positive foci (B) and density of vascular hot spots (C).

Figure 5

Density of vascular hot spots vs density of hypoxic foci in D-12 tumours. Points represent single tumours. Tumours with necrotic fractions below 25% (A) and tumours with necrotic fractions within the range of 25–50% (B).

Figure 6

Effects of anti-VEGF treatment, anti-IL-8 treatment, and combined anti-VEGF and anti-IL-8 treatment on development of hypoxia, neovascularization and spontaneous pulmonary metastasis in D-12 tumours. (A) Percentage of mice with metastasis. Points represent single experiments involving 10 mice each. (B) Densities of hypoxic foci and vascular hot spots. Columns represent mean values of 20 mice. Bars represent s.e.m.