Preferential topography of proteins regulating vascularization and apoptosis in a MX1 xenotransplant after treatment with hypoxia, hyperthermia, ifosfamide, and irradiation.

The MX1 xenotransplant growing in nude mice was used as a model for estrogen- and progesterone-receptor-negative breast cancer. The effects of different therapeutic regimens-combinations of hyperthermia, chemotherapy, and irradiation-on the expression of proteins playing a role in tumor vascularization and apoptosis were investigated. Additionally, MX-1 tumors were exposed to hypoxia to investigate changes in protein expression related to angiogenesis. This is of particular importance with respect to antiangiogenic therapies that may be combined with the treatments mentioned before. Endothelial and adhesion factors, extracellular matrix (ECM) factors, apoptosis-regulating factors, and neuronal factors were examined by immunohistochemical techniques. Concerning vascularization, the most prominent changes were seen in the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which increased strongly after hypoxia. The other cytokines, adhesion and ECM molecules, were either little affected or unaffected by the therapy. At the ultrastructural level, the walls of the tumor vessels are of the sinusoidal type, possessing many fenestrations. With regard to the second focus of this investigation, apoptosis, tumor cells again exerted the strongest differences after hypoxia where c-myc was clearly enhanced, whereas the effects on p53, bcl-2, and CD95 were extremely weak or not detectable. Furthermore, the neurotransmitter somatostatin, a possible "external" regulator of apoptosis, did not show treatment-related changes. In summary, it was shown that 1) within the group of apoptosis-regulating proteins c-myc was particularly affected by hypoxia, indicating a possible role for an activation-induced pathway of apoptosis in this context; 2) minor changes seen after treatment combined with hyperthermia point to a more acute vascular reaction (=dilatation), causing an increase of tissue pO2 rather than angiogenesis; and 3) the concentrations of the angiogenic factors VEGF and bFGF rose strongly under hypoxia, thereby possibly exerting counterproductive effects to antiangiogenic therapy but not to thermochemotherapy or irradiation. This supports the concept of a combined antiangiogenic, hyperthermia, chemo- and irradiation therapy.