Heat shock proteins (HSPs) are recognized as significant participants in immune reactions. We previously reported that expression of HSP70 in response to hyperthermia, produced using our original magnetite cationic liposomes (MCLs), induces antitumor immunity. In the present study, we examine whether the antitumor immunity induced by hyperthermia is enhanced by hsp70 gene transfer. A human hsp70 gene mediated by cationic liposomes was injected into a B16 melanoma nodule in C57BL/6 mice in situ. At 24 hours after the injection of the hsp70 gene, MCLs were injected into melanoma nodules in C57BL/6 mice, which were subjected to an alternating magnetic field for 30 minutes. The temperature at the tumor reached 431C and was maintained by controlling the magnetic field intensity. The combined treatment strongly arrested tumor growth over a 30-day period, and complete regression of tumors was observed in 30% (3/10) of mice. Systemic antitumor immunity was induced in the cured mice. This study demonstrates that this novel therapeutic strategy combining the use of hsp70 gene therapy and hyperthermia using MCLs may be applicable to patients with advanced malignancies. Cancer Gene Therapy (2003) 10, 918-925. doi:10.1038/sj.cgt.7700648Keywords: heat shock proteins; hyperthermia; magnetite; cancer immunotherapy H yperthermia is a promising approach for cancer therapy. 1,2 However, the inevitable technical problem with hyperthermia is the difficulty in heating only the local tumor region to the intended temperature without damaging the surrounding normal tissue. Magnetic nanoparticles have been used for hyperthermia treatment in an attempt to overcome these disadvantages. 3,4 Magnetic nanoparticles generate heat in an alternating magnetic field (AMF) due to hysteresis loss. 5 We have developed magnetite cationic liposomes (MCLs) for intracellular hyperthermia. 6,7 The hyperthermic effect of MCLs was examined in an in vivo study, and complete tumor regression was observed. 8 Since some researchers have reported that heat treatment itself can enhance the immunogenicity of cancer cells, 9-12 we previously investigated hyperthermia-induced antitumor immunity in T-9 rat glioma cells in vivo. 13 This induced immunity continued for an extended period of time, and the rats treated with hyperthermia completely rejected T-9 cells as a metastasis model. Moreover, we investigated the mechanisms of antitumor immunity induced by intracellular hyperthermia with regard to the role of heat shock proteins (HSPs). 14,15 HSPs are a highly conserved group of intracellular proteins whose synthesis is increased by a large variety of stressors including heat shock. 16 As expression of HSP70 protects cells from heat-induced apoptosis, 17 HSP70 expression is considered a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90, and glucose-regulated protein 96 (gp96), in immune reactions. 18,19 A possible mechanism by which HSPs influence tumor cell immunogenicity is associated with...