Recent reports have shown that tumor necrosis factor-alpha (TNF-alpha) can augment the effects of radiation against certain tumor types. However, the high concentrations of intravenous infusion of TNF-alpha needed to cause tumor regression can induce many systemic side effects. The aims of this study were to determine if TNF-alpha encapsulated in sterically stabilized (Stealth, ALZA Corporation, Mountain View, CA), PEGylated liposomes (SL) augments the antitumor effects of radiation and to compare its efficacy and possible toxicity with free TNF-alpha in the LS174T human colon tumor xenograft model. Nude mice were injected subcutaneously (s.c.) with LS174T cells and treated intravenously (i.v.) with Stealth-liposomal TNF-alpha (SL-TNF-alpha) with and without radiation or TNF-alpha with or without radiation when tumor size was approximately 200 mm(3). In phase 1, a significant decrease (p = 0.047) in tumor growth was observed with radiation at day 21 but not with SL-TNF-alpha or free TNF-alpha alone. By the end of phase 1 (day 27) with continued treatments, the SL-TNF-alpha plus radiation group had significantly smaller tumors (p = 0.044) than those in the free TNF-alpha plus radiation group. In phase 2, where a similar tumor growth reduction pattern was observed, the addition of TNF-alpha to radiation, either as free protein or within SL, increased lymphocyte activation and natural killer (NK) cell numbers in both blood and spleen. The effect was generally more pronounced with SL-TNF-alpha. Systemic toxicity, based on hematologic analyses and body weight, was absent or minimal. Collectively, the data show that pretreatment with SL-TNF-alpha can enhance more effectively, and possibly more safely, the effects of radiation against human colon tumor xenografts than can free TNF-alpha and that the increased antitumor action may involve upregulation of lymphocytes.