This laboratory is investigating the potential use of recombinant TNF as an antineoplastic agent using surgical isolation perfusion techniques to obtain high intravascular levels. A porcine model of isolated hepatic perfusion to treat the liver for a 30 - 90 minute time period has been developed in which the animals are survived postoperatively. Using this model, a 60 minute hyperthermic TNF perfusion can be performed in which perfusate levels of TNF are obtained in the active range of 2 -5 mcg/ml. These TNF concentrations are effective to treat murine tumors as well as extremity melanoma and sarcoma in limb perfusion trials. The direct hepatotoxic effects appear to be limited measured by liver function tests and hepatic histology. A sensitive assay for nitric oxide has been developed to determine the role of this mediator in the toxicity of pig and human liver perfusions and in clinical TNF limb perfusions. In in vitro model using cultured endothelial cells has been developed to study interactions between TNF and endothelial cells under the influence of the tumor microenvironment. Studies of secondary mediators and endothelial cell interaction with TNF may lead to decreased toxicity and improve understanding of the mechanism of the TNF antitumor effect. Murine sarcoma cell lines have been transfected with an adenovirus E1A expression vector to obtain several stable clones that are highly sensitive to TNF in vitro. These E1A clones grow and then regress in vivo in immunocompetent mice based on a CD8/TNF dependent mechanism. However, sensitivity to TNF in vitro more than 5 log orders compared to parent line leads to absolutely no improved response to bolus intravenous TNF treatment in vivo in irradiated mice. These transfected cell lines are useful tools to study the mechanism of direct TNF cytotoxicity in vitro, and to correlate TNF sensitivity with response in vivo.
