Exhaustion of Protective Heat Shock Response Induces Significant Tumor Damage by Apoptosis after Modulated Electro-Hyperthermia Treatment of Triple Negative Breast Cancer Isografts in Mice

Abstract

Modulated electro-hyperthermia (mEHT) is a complementary antitumor therapy applying capacitive radiofrequency at 13.56 MHz. Here we tested the efficiency of mEHT treatment in a BALB/c mouse isograft model using the firefly luciferase-transfected triple-negative breast cancer cell line, 4T1. Tumors inoculated orthotopically were treated twice using a novel ergonomic pole electrode and an improved mEHT device (LabEHY 200) at 0.7 ± 0.3 W for 30 min. Tumors were treated one, two, or three times every 48 h. Tumor growth was followed by IVIS, caliper, and ultrasound. Tumor destruction histology and molecular changes using immunohistochemistry and RT-qPCR were also revealed. In vivo, mEHT treatment transitionally elevated Hsp70 expression in surviving cells indicating heat shock-related cell stress, while IVIS fluorescence showed a significant reduction of viable tumor cell numbers. Treated tumor centers displayed significant microscopic tumor damage with prominent signs of apoptosis, and major upregulation of cleaved/activated caspase-3-positive tumor cells. Serial sampling demonstrated substantial elevation of heat shock (Hsp70) response twelve hours after the treatment which was exhausted by twenty-four hours after treatment. Heat shock inhibitors Quercetin or KRIBB11 could synergistically amplify mEHT-induced tumor apoptosis in vitro. In conclusion, modulated electro-hyperthermia exerted a protective heat shock response as a clear sign of tumor cell stress. Exhaustion of the HSR manifested in caspase-dependent apoptotic tumor cell death and tissue damage of triple-negative breast cancer after mEHT monotherapy. Inhibiting the HSR synergistically increased the effect of mEHT. This finding has great translational potential.

Modulated Electro-Hyperthermia Induces a Prominent Local Stress Response and Growth Inhibition in Mouse Breast Cancer Isografts

“…In our previous study we demonstrated that modulated electro-hyperthermia (mEHT) induced Hsp70 upregulation, and exhaustion of this defense mechanism resulted in apoptotic cell death of the mouse 4T1 triple negative breast cancer (TNBC) model. Hsp70 inhibition synergistically enhanced the tumor-killing effect of mEHT [ 1 ]. In the present study, our aim was to investigate the molecular effects of mEHT using multiplex methods.…”

“…Hyperthermia decreases the activity of hypoxia-inducible factor 1 (HIF-1) and contributes to the vascular endothelial growth factor (VEGF)-mediated angiogenesis inhibition of Bevacizumab [ 5 ]. mEHT’s potential as a monotherapy is currently under investigation [ 1 , 6 ]. mEHT uses a 13.56 MHz frequency capacitively coupled electromagnetic field that transmits energy to the tumor.…”

“…mEHT can effectively induce caspase-dependent apoptosis as demonstrated by elevated cleaved caspase 3 (cC3) expression in mEHT-treated tumors [ 1 , 9 ]. mEHT induces a heat shock response and subsequent heat shock protein (Hsp70) upregulation in treated tumors [ 1 , 10 ]. In addition, mEHT potently inhibits tumor cell proliferation indicated by the attenuation of Ki67-positive cell nuclei [ 11 , 12 ], a widely used proliferation marker.…”

Suppression of Metastatic Melanoma Growth in Lung by Modulated Electro-Hyperthermia Monitored by a Minimally Invasive Heat Stress Testing Approach in Mice

“…In colorectal adenocarcinoma models, mEHT treatment has been demonstrated to induce both caspase-dependent and caspase-independent AIF-mediated apoptosis [ 17 , 18 , 19 ]. In triple-negative breast cancer isografts, exhaustion of mEHT-induced heat shock response resulted in significant tumor damage via a caspase-dependent mechanism in mice [ 20 ].…”

Modulated Electro-Hyperthermia Facilitates NK-Cell Infiltration and Growth Arrest of Human A2058 Melanoma in a Xenograft Model

“…The benefits of mEHT as a complementary therapy relies in its property to induce tumor-damage by direct cell killing, mediated by irreversible heat and cell stress and its tolerability by patients as it has almost no side-effects. In the recent years numerous papers have described the growth inhibiting effect of mEHT in various tumor types, mostly in preclinical mouse models ( 2 – 6 ). These results provide evidence that besides the direct cell killing, mEHT has an immunomodulatory effect supporting the anti-tumor immune response ( 3 , 4 , 7 ).…”