Modulated electro-hyperthermia (mEHT) is a selective cancer treatment used in human oncology complementing other therapies. During mEHT, a focused electromagnetic field (EMF) is generated within the tumor inducing cell death by thermal and nonthermal effects. Here we investigated molecular changes elicited by mEHT using multiplex methods in an aggressive, therapy-resistant triple negative breast cancer (TNBC) model. 4T1/4T07 isografts inoculated orthotopically into female BALB/c mice were treated with mEHT three to five times. mEHT induced the upregulation of the stress-related Hsp70 and cleaved caspase-3 proteins, resulting in effective inhibition of tumor growth and proliferation. Several acute stress response proteins, including protease inhibitors, coagulation and heat shock factors, and complement family members, were among the most upregulated treatment-related genes/proteins as revealed by next-generation sequencing (NGS), Nanostring and mass spectrometry (MS). pathway analysis demonstrated that several of these proteins belong to the response to stimulus pathway. Cell culture treatments confirmed that the source of these proteins was the tumor cells. The heat-shock factor inhibitor KRIBB11 reduced mEHT-induced complement factor 4 (C4) mRNA increase. In conclusion, mEHT monotherapy induced tumor growth inhibition and a complex stress response. Inhibition of this stress response is likely to enhance the effectiveness of mEHT and other cancer treatments.

The Heat Shock Response (HSR) is a cellular stress reaction crucial for cell survival against stressors, including heat, in both healthy and cancer cells. Modulated electro-hyperthermia (mEHT) is an emerging non-invasive cancer therapy utilizing electromagnetic fields to selectively target cancer cells via temperature-dependent and independent mechanisms. However, mEHT triggers HSR in treated cells. Despite demonstrated efficacy in cancer treatment, understanding the underlying molecular mechanisms for improved therapeutic outcomes remains a focus. This review examines the HSR induced by mEHT in cancer cells, discussing potential strategies to modulate it for enhanced tumor-killing effects. Approaches such as HSF1 gene-knockdown and small molecule inhibitors like KRIBB11 are explored to downregulate the HSR and augment tumor destruction. We emphasize the impact of HSR inhibition on cancer cell viability, mEHT sensitivity, and potential synergistic effects, addressing challenges and future directions. This understanding offers opportunities for optimizing treatment strategies and advancing precision medicine in cancer therapy.

Modulated electro-hyperthermia (mEHT) is an adjuvant cancer therapy that enables tumor-selective heating (+2.5 °C). In this study, we investigated whether mEHT accelerates the tumor-specific delivery of doxorubicin (DOX) from lyso-thermosensitive liposomal doxorubicin (LTLD) and improves its anticancer efficacy in mice bearing a triple-negative breast cancer cell line (4T1). The 4T1 cells were orthotopically injected into Balb/C mice, and mEHT was performed on days 9, 12, and 15 after the implantation. DOX, LTLD, or PEGylated liposomal DOX (PLD) were administered for comparison. The tumor size and DOX accumulation in the tumor were measured. The cleaved caspase-3 (cC3) and cell proliferation were evaluated by cC3 or Ki67 immunohistochemistry and Western blot. The LTLD+mEHT combination was more effective at inhibiting tumor growth than the free DOX and PLD, demonstrated by reductions in both the tumor volume and tumor weight. LTLD+mEHT resulted in the highest DOX accumulation in the tumor one hour after treatment. Tumor cell damage was associated with cC3 in the damaged area, and with a reduction in Ki67 in the living area. These changes were significantly the strongest in the LTLD+mEHT-treated tumors. The body weight loss was similar in all mice treated with any DOX formulation, suggesting no difference in toxicity. In conclusion, LTLD combined with mEHT represents a novel approach for DOX delivery into cancer tissue.