Mechanism of action differences in the antitumor effects of transmembrane and secretory tumor necrosis factor-alpha in vitro and in vivo

The proinflammatory cytokine tumor necrosis factor-alpha (TNF alpha) exists naturally in two forms, a 26 kDa transmembrane form (TM-TNF alpha), and a 17 kDa secretory form (S-TNF alpha). The biological roles for each of these forms of TNF alpha in tumor killing have not been completely elucidated. Therefore, in this study, three different recombinant retroviral vectors, wild-type TNF alpha, solely secretable TNF alpha mutant, and uncleavable transmembrane TNF alpha mutant, were constructed by molecular techniques and stably transfected into a murine hepatic carcinoma cell line (H22). TNF alpha, either secreted in cell culture supernatants by secretable TNF alpha mutant- or wild-type TNF alpha-producing tumor cells, or as a treansmembrane form expressed on the cell surface of uncleavable TNF alpha mutant- or wild-type TNF alpha-synthesizing tumor cells, was demonstrated to be cytotoxic against the TNF sensitive L929 cell line. The H22 cells transfected with the three different forms of TNFa were shown to kill parental H22 cells in an in vitro cytotoxicity assay [effect/target (E/T) ratio-dependent manner], and their maximal killing rates were similar to 38-43% at E/T ratio of 5:1. The injection of total 2.5x10(5) mixed cells containing transfected and parental H22 tumor cells at different ratios into syngeneic mice resulted in the inhibition of tumor growth with a maximal inhibition rates of similar to 57 similar to 72% at E/T ratio of 5:1. A transient weight loss was found in mice bearing solely secretable TNF alpha mutant producing tumors, whereas no obvious side effects were seen in mice bearing uncleavable TNFa mutant or wild-type TNF alpha expressing tumors. Finally, we demonstrate that tumors secreting S-TNF alpha promoted the subsequent infiltration of CD4(+) T cells, and to a lesser extent CD8(+) T cells, to the tumor site. The TM-TNF alpha expressing tumors up-regulated Fas (CD95) expression and inhibited the expression of tumor metastasis associated molecule CD44v3. These results suggest that S-TNF alpha and TM-TNF alpha kill cancer cells in vivo through different mechanisms of action. We conclude that the non-secreted form of TNF alpha may be an ideal candidate for cancer gene therapy due to its therapeutic potential and lowered side effect profile.