Hyperthermic treatments have been used for many years, particularly in anticancer therapy [6]. Typically, there are 2 ranges of targeting temperature used in such treatment. High temperatures (greater than 50°C) kill targeted tissue directly. Such direct heating methods using needle-type interstitial antenna materials are effective for inducing hyperthermia in local regions; however, these technologies are less effective for larger areas of tissue [7]. Lower temperatures of approximately 40°C to 43°C are also associated with an anticancer effect. There are several heating techniques employed in hyperthermic treatments using such a temperature range. For example, radiofrequency (RF) electric field application is one of the approved methods for cancer treatment. However, the effectiveness of this technique varies according to tumor size and the depth of the tumor region from the body surface. Furthermore, it is sometimes difficult to focus on the exact location of the tumor [8]. Microwaves, ultrasound applied as a heating method, are also used for inducing hyperthermia. For such hyperthermic therapy, magnetic nanoparticles have also used been used as a heating mediator. The use of these particles is based on fact that such multidomain ferro- or ferri-magnetic materials are heated by an alternating magnetic field due to hysteresis losses [9]. Single domain particles of magnetite can also generate heat by relaxation loss, not hysteresis loss, under irradiation of alternating magnetic field. Ten nanometer of diameter is boundary size to allocate particles between single-domain and multi-domain. Optimal diameter of magnetic nanoparticle for magnetic nanoparticle-mediated hyperthermia should be investigated in detail.

Our group has pioneered one of these technologies in order to selectively heat tumor regions [10]. We have introduced cationic liposome technology in order to enhance the surface interaction between cells and heating mediator, and thereby improve its localization. In previous animal studies, we have demonstrated the efficacy of hyperthermia induce using magnetite cationic liposomes (MCLs) [10-12] in several types of tumor model; for instance, B16 melanoma in mice [13,14], T9 glioma in rats [12,15], osteosarcoma in hamsters [16], MM46 mouse mammary carcinoma [17], PLS 10 rat prostate cancer [18], and VX-7 squamous cell carcinoma in rabbit tongue [19]. Furthermore, we have described the capability of immunologic reactivity in cancer therapy, which was enhanced by increasing the amount of heat-shock protein 70 (HSP70), following hyperthermic treatment with MCLs [20-22].

In several transplantable tumor models mentioned above, the magnetite nanoparticle-mediated hyperthermia proposed by us was found to be very effective for inducing complete tumor regression. In such models, however, transplantable cell is an explanted cell. To use the hyperthermia for human patients, the effect of the hyperthermia should be demonstrated by the use of more practical tumor model. We focused on oncogene transgenic mice as spontaneous tumor model and complete tumor regression of malignant melanoma induced by the oncogene ret was achieved by the hyperthermia. As a next step, we investigated here the application to carcinogen-induced spontaneous tumor models as a practical tumor model which can simulate practical tumor in the human patient.

Figure ​Figure11 shows that the temperature increases in the tumor region were slightly slower than those previously reported by our group [16]. We believe that this difference is due to the cooling effect of the vascular systems that developed in the spontaneous tumor tissue. However, it was possible to raise the temperature of the tumor region during the treatment periods, and we therefore followed former protocols in this study.

After the course of treatment, all 4 rats that developed multiple mammary tumors exhibited tumor regression in the immediate region of the treated site. In spite of this being our first attempt to apply our protocol to a hyperthermia effect similar to that obtained for a transplanted tumor model that we reported previously.

As shown in Fig. ​Fig.2,2, 1 of the 4 rats exhibited rapid regrowth the tumor within 2 weeks. Another rat exhibited slight regrowth. These results appear to indicate certain inadequacies of the present treatment protocol as applied to the spontaneous tumor model, despite it being applicable for the previously investigated transplanted tumor model. One of the possible reasons for this discrepancy is considered to be the highly developed vasculature and/or lymphoid system in the spontaneous tumors that were not present in transplanted tumors. A highly developed vasculature might create localized regions of tissue that do not reach sufficiently high temperatures. The developed nutrition/elimination systems also appear to promote the clearance of the administered MCLs from the injected region. Therefore, it is suggested that the treatment protocol should be optimized, particularly with respect to the dose of MCL, the temperature during treatment, and the number of repeat treatments. Given such optimization, it is expected that such regrowths would be repressed. However, tumor growth was remarkably controlled in the early going compared with untreated group of rats (Fig. ​(Fig.4).4). Therefore, we believe our hyperthermia system will be capable in the future.

In the case of the carcinogen-induced spontaneous tumor models, it is considered that the sites of origin in the animal body are heterogeneous. Indeed, in our experiments, tumor growth at several untreated sites progressed regardless of the treatment. Meanwhile, we also observed some tumor shrinkage at sites where no MCLs had been administered, and which had therefore not been exposed to the hyperthermic conditions during the treatment. These shrinkages were maintained stably for more than 50 days without additional treatment. These results suggest the presence of an immunological antitumor reaction induced by the MCL-mediated hyperthermia, as observed previously with a transplanted tumor model [20]. These responses are believed to be linked to a specific antigen, such as HSP70, that is generated by necrosis of the directly heated tumor cells [21,22]. We also reported the mechanisms of immune reactions which are induced by our hypertharmia system in the previous study [26,27]. We demonstrated the mechanism of immunological reaction which is enhanced by HSP70 dumped from dead tissue with our hyperthermia system. It was also revealed that cancer specific DC maturation was induced by our treatment system.