Simple SummaryModerate loco-regional hyperthermia (40–45 °C) is a therapeutic modality that can improve the effects of chemotherapy and radiotherapy and, in addition, improve our immune response against cancer. However, the effects of these combinations on many different cancers are modest. The use of much higher temperatures (>60 °C, thermal ablation) can cause severe damage to healthy tissues. In fact, our results show that cancer cells show extraordinary resistance to moderate hyperthermia. However, we found that the combination of hyperthermia (not higher than 52 °C) with low-strength electromagnetic fields acts synergistically, causing irreversible damage to different cancer cells. Moreover, these externally applied energies can be combined with chemotherapy and/or targeted therapies to achieve complete cancer eradication. In vivo, the energy causing focal hyperthermia can be distributed in multiple beams that can be concentrated in the tumor, thus avoiding damaging the healthy tissues that it passes through.AbstractAt present, the applications and efficacy of non-ionizing radiations (NIR) in oncotherapy are limited. In terms of potential combinations, the use of biocompatible magnetic nanoparticles as heat mediators has been extensively investigated. Nevertheless, developing more efficient heat nanomediators that may exhibit high specific absorption rates is still an unsolved problem. Our aim was to investigate if externally applied magnetic fields and a heat-inducing NIR affect tumor cell viability. To this end, under in vitro conditions, different human cancer cells (A2058 melanoma, AsPC1 pancreas carcinoma, MDA-MB-231 breast carcinoma) were treated with the combination of electromagnetic fields (EMFs, using solenoids) and hyperthermia (HT, using a thermostated bath). The effect of NIR was also studied in combination with standard chemotherapy and targeted therapy. An experimental device combining EMFs and high-intensity focused ultrasounds (HIFU)-induced HT was tested in vivo. EMFs (25 µT, 4 h) or HT (52 °C, 40 min) showed a limited effect on cancer cell viability in vitro. However, their combination decreased viability to approximately 16%, 50%, and 21% of control values in A2058, AsPC1, and MDA-MB-231 cells, respectively. Increased lysosomal permeability, release of cathepsins into the cytosol, and mitochondria-dependent activation of cell death are the underlying mechanisms. Cancer cells could be completely eliminated by combining EMFs, HT, and standard chemotherapy or EMFs, HT, and anti-Hsp70-targeted therapy. As a proof of concept, in vivo experiments performed in AsPC1 xenografts showed that a combination of EMFs, HIFU-induced HT, standard chemotherapy, and a lysosomal permeabilizer induces a complete cancer regression.