Extracellular pH (pH e ) is lower in many tumors than in the corresponding normal tissue. The significance of acidic pH e in the development of metastatic disease was investigated in the present work. Human melanoma cells (A-07, D-12, and T-22) were cultured in vitro at pH e 6.8 or 7.4 (control) before being inoculated into the tail vein of BALB/c nu/nu mice for formation of experimental pulmonary metastases. Cell invasiveness was studied in vitro by using Matrigel invasion chambers and angiogenesis was studied in vivo by using an intradermal assay. Protein secretion was measured by ELISA and immunocapture assays. Cells cultured at acidic pH e showed increased secretion of proteinases and proangiogenic factors, enhanced invasive and angiogenic potential, and enhanced potential to develop experimental metastases. Acidity-induced metastasis was inhibited by treatment with the general matrix metalloproteinase (MMP) inhibitor GM6001, the general cysteine proteinase inhibitor E-64, or blocking antibody against vascular endothelial growth factor-A (VEGF-A) or interleukin-8 (IL-8). Our study indicates that acidic pH e promotes experimental pulmonary metastasis in A-07, D-12, and T-22 human melanoma cells by a common mechanism involving acidity-induced up-regulation of the proteolytic enzymes MMP-2, MMP-9, cathepsin B, and cathepsin L and acidity-induced up-regulation of the proangiogenic factors VEGF-A and IL-8. One consequence of this observation is that treatment strategies involving deliberate tumor acidification to improve the efficacy of chemotherapy, photodynamic therapy, and hyperthermia should be avoided. Moreover, the possibility that the pH e of the primary tumor may be an important prognostic parameter for melanoma patients merits clinical investigation. (Cancer Res 2006; 66(13): 6699-707)

Cancer patients with recurrent local disease after radiation therapy have increased probability of developing regional and distant metastases. The mechanisms behind this observation were studied in the present work by using D-12 and R-18 human melanoma xenografts growing in preirradiated beds in BALB/c-nu/nu mice as preclinical models of recurrent primary tumors in humans. D-12 tumors metastasize to the lungs, whereas R-18 tumors develop lymph node metastases. Based on earlier studies, we hypothesized that metastasis was governed primarily by the proangiogenic factor interleukin-8 (IL-8) in D-12 tumors and by the invasive growth-promoting receptor urokinase-type plasminogen activator receptor (uPAR) in R-18 tumors. Pimonidazole was used as a hypoxia marker, and hypoxia, microvascular hotspots, and the expression of IL-8 and uPAR were studied by immunohistochemistry. The metastatic frequency was significantly higher in tumors in preirradiated beds than in control tumors in unirradiated beds, and it increased with the preirradiation dose. D-12 tumors showed increased fraction of hypoxic cells, increased fraction of IL-8-positive cells, and increased density of microvascular hotspots in preirradiated beds, and R-18 tumors showed increased fraction of hypoxic cells and increased fraction of uPAR-positive cells in preirradiated beds. Strong correlations were found between these parameters and metastatic frequency. IL-8 was up-regulated in hypoxic regions of D-12 tumors, and uPAR was up-regulated in hypoxic regions of R-18 tumors. Daily treatment with anti-IL-8 antibody (D-12) or anti-uPAR antibody (R-18) suppressed metastasis significantly. Our preclinical study suggests that primary tumors recurring after inadequate radiation therapy may show increased metastatic propensity because of increased fraction of hypoxic cells and hypoxia-induced up-regulation of metastasis-promoting gene products. Two possible mechanisms were identified: hypoxia may enhance metastasis by inducing neoangiogenesis facilitating hematogenous spread and by promoting invasive growth facilitating lymphogenous spread. The aggressive behavior of postirradiation local recurrences suggests that they should be subjected to curative treatment as early as possible to prevent further metastatic dissemination. Moreover, the possibility that patients with a high probability of developing local recurrences after radiation therapy may benefit from postirradiation treatment with antiangiogenic and/or anti-invasive agents merits clinical investigation. (Cancer Res 2005; 65(6): 2387-96)