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

ABSTRACT: 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.

SUBMITTER: Schvarcz CA

PROVIDER: S-EPMC8038813 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The benefits of high-fever range hyperthermia have been utilized in medicine from the Ancient Greek culture to the present day. Amplitude-modulated electro-hyperthermia, induced by a 13.56 MHz radiofrequency current (mEHT, or Oncothermia), has been an emerging means of delivering loco-regional clinical hyperthermia as a complementary of radiation-, chemo-, and molecular targeted oncotherapy. This unique treatment exploits the metabolic shift in cancer, resulting in elevated oxidative glycolysis (Warburg effect), ion concentration, and electric conductivity. These promote the enrichment of electric fields and induce heat (controlled at 42 °C), as well as ion fluxes and disequilibrium through tumor cell membrane channels. By now, accumulating preclinical studies using in vitro and in vivo models of different cancer types have revealed details of the mechanism and molecular background of the oncoreductive effects of mEHT monotherapy. These include the induction of DNA double-strand breaks, irreversible heath and cell stress, and programmed cells death; the upregulation of molecular chaperones and damage (DAMP) signaling, which may contribute to a secondary immunogenic tumor cell death. In combination therapies, mEHT proved to be a good chemosensitizer through increasing drug uptake and tumor reductive effects, as well as a good radiosensitizer by downregulating hypoxia-related target genes. Recently, immune stimulation or intratumoral antigen-presenting dendritic cell injection have been able to extend the impact of local mEHT into a systemic "abscopal" effect. The complex network of pathways emerging from the published mEHT experiments has not been overviewed and arranged yet into a framework to reveal links between the pieces of the "puzzle". In this paper, we review the mEHT-related damage mechanisms published in tumor models, which may allow some geno-/phenotype treatment efficiency correlations to be exploited both in further research and for more rational clinical treatment planning when mEHT is involved in combination therapies.

Project description:Modulated electro-hyperthermia (mEHT) is a form of mild hyperthermia (HT) used for cancer treatment. The principle utility of HT is the ability not only to increase cell temperature, but also to increase blood flow and associated pO2 to the microenvironment. While investigational evidence has shown the unique ability of mEHT to elicit apoptosis in cancer cells, in vivo and in vitro, the same trait has not been observed with conventional HT. There is dissension as to what allows mEHT to elicit apoptosis despite heating to only mild temperatures, with the predominant opinion in favor of increased temperature at a cellular level as the driving force. For this study, we hypothesized that in addition to temperature, the amount of electrical energy delivered is a major factor in induction of apoptosis by mEHT. To evaluate the impact of electrical energy on apoptosis, we divided generally practiced mEHT treatment into 3 phases: Phase I (treatment start to 10?min. mark): escalation from 25?°C to 37?°C Phase II (10?min. mark to 15?min. mark): escalation from 37?°C to 42?°C Phase III (15?min. mark to 45?min. mark): maintenance at 42?°C Combinations of mEHT at 18?W power, mEHT at 7.5?W power, water bath, and incubator were applied to each of the three phases. Power output was recorded per second and calculated as average power per second. Total number of corresponding Joules emitted per each experiment was also recorded. The biological effect of apoptotic cell death was assayed by annexin-V assay. In group where mEHT was applied for all three phases, apoptosis rate was measured at 31.18?±?1.47%. In group where mEHT was only applied in Phases II and III, apoptosis rate dropped to 20.2?±?2.1%. Where mEHT was only applied in Phase III, apoptosis was 6.4?±?1.7%. Interestingly, when mEHT was applied in Phases I and II, whether Phase III was conducted in either water bath at 42?°C or incubator at 37?°C, resulted in nearly identical apoptosis rates, 26?±?4.4% and 25.9?±?3.1%, respectively. These results showed that accumulation of mEHT at high-powered setting (18?W/sec) during temperature escalation (Phase I and Phase II), significantly increased apoptosis of tested cancer cells. The data also showed that whereas apoptosis rate was significantly increased during temperature escalation by higher power (18?W/sec), apoptosis was limited during temperature maintenance with lower power (7.5?W/sec). This presents that neither maintenance of 42?°C nor accumulation of Joules by mEHT has immediate correlating effect on apoptosis rate. These findings may offer a basis for direction of clinical application of mEHT treatment.

Project description:Background: Modulated electro-hyperthermia (mEHT), a non-invasive intervention using 13.56 MHz radiofrequency, can selectively target cancers due to their elevated glycolysis (Warburg-effect), extracellular ion concentration and conductivity compared to normal tissues. We showed earlier that mEHT alone can provoke apoptosis and damage associated molecular pattern (DAMP) signals in human HT29 colorectal cancer xenografts of immunocompromised mice. Materials: Here we tested the mEHT induced stress and immune responses in C26 colorectal cancer allografts of immunocompetent (BALB/c) mice between 12-72 h post-treatment. The right side of the symmetrical tumors grown in both femoral regions of mice were treated for 30 minutes, while the left side tumors served for untreated controls. Results: Loco-regional mEHT treatment induced an ongoing and significant tumor damage with the blockade of cell cycle progression indicated by the loss of nuclear Ki67 protein. Nuclear shrinkage, apoptotic bodies and DNA fragmentation detected using TUNEL assay confirmed apoptosis. Cleaved/activated-caspase-8 and -caspase-3 upregulation along with mitochondrial translocation of bax protein and release of cytochrome-c were consistent with the activation of both the extrinsic and intrinsic caspase-dependent programmed cell death pathways. The prominent release of stress-associated Hsp70, calreticulin and HMGB1 proteins, relevant to DAMP signaling, was accompanied by the significant tumor infiltration by S100 positive antigen presenting dendritic cells and CD3 positive T-cells with only scant FoxP3 positive regulatory T-cells. In addition, mEHT combined with a chlorogenic acid rich T-cell promoting agent induced significant cell death both in the treated and the untreated contralateral tumors indicating a systemic anti-tumor effect. Conclusions: mEHT induced caspase-dependent programmed cell death and the release of stress associated DAMP proteins in colorectal cancer allografts can provoke major immune cell infiltration. Accumulating antigen presenting dendritic cells and T-cells are likely to contribute to the ongoing tumor destruction by an immunogenic cell death mechanism both locally and through systemic effect at distant tumor sites.

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

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