This laboratory is investigating the potential use of recombinant TNF as an antineoplastic agent in combination with chemotherapeutics using surgical isolation perfusion techniques to obtain high intravascular levels in clinical trials of limb and liver perfusion. Laboratory studies are being conducting to assess the mechanism of this highly successful antitumor treatment. Tissue specimens from the clinical trials taken before and after perfusion treatment, murine tumor models including xenografts in nude mice as well as a variety of murine tumors, tumor cell lines in vitro, endothelial cells in vitro, and an in vivo model of a neovascularized sponge are all being used as reagents or models to investigate the antitumor effect seen in the clinical trials of regional perfusion. Sequential biopsies from the clinical trials of melanoma have shown there is a very rapid and specific necrosis in the tumor which spares adjacent normal tissue. This necrosis occurs without any evidence of fibrin or thrombus within the tumor microvasculature but with specific damage to tumor endothelial cells in TNF treated specimens. Human melanoma cell lines obtained from patients with variable response to TNF in melphalan limb perfusion that grow in nude mice have been developed and initial analysis shows that the response in the murine model correlates with the human clinical response. Responder lines have greater IL-8 production and fas antigen expression. Murine cell lines have been characterized in terms of their response to TNF in vitro and in vivo and their production of angiogenic growth factors in vitro and in vivo. Only production of VEGF in vivo correlates with TNF antitumor effect in vivo. In vitro studies using endothelial cells(HUVEC) as well as a murine endothelial cell line have evaluated the effects of TNF on endothelial cell viability as well as ability to form a permeability barrier. An in vivo model of neovascularized tissue using a subcutaneous sponge has demonstrated that TNF has specific effects against proliferating endothelium outside of a tumor environment in terms of flow through newly formed vessels as well as permeability of a neovascularized bed. These models are being further explored to define the molecular and cellular responses of growing endothelium to intravascular TNF.
