Modulated electro-hyperthermia induced loco-regional and systemic tumor destruction in colorectal cancer allografts

Abstract

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.

Consensus guidelines for the definition, detection and interpretation of immunogenic cell death

“…Open access the ubiquitin-specific peptidase inhibitor spautin-1, the antibiotic bleomycin, the protein phosphatase-2A inhibitor LB-100, the Chinese herbal medicine component shikonin and capsaicin [34][35][36][37][38] and (8) numerous physical interventions, encompassing various forms of ionizing radiation, extracorporeal photochemotherapy, hypericinbased photodynamic therapy (PDT), near-infrared photoimmunotherapy, high hydrostatic pressure, severe cytotoxic heat shock, nanopulse stimulation and electrohyperthermia. [39][40][41][42][43][44][45][46][47][48][49] Importantly, dose and administration schedules have a major impact on the ability of many of these agents to initiate productive ICD. [50][51][52] The aforementioned ICD inducers have been instrumental not only for identifying the molecular machinery that underlies the immunogenicity of some variants of RCD, 5 but also for elucidating the pathophysiological and therapeutic implications of the process.…”

Modulated Electro-Hyperthermia-Induced Tumor Damage Mechanisms Revealed in Cancer Models

“…The SAR distribution follows the thermal and electromagnetic heterogeneity of the malignant tissue, resulting in higher absorbance in the cancer than in the surrounding normal issue [ 27 , 28 ]. The general feature of tumor destruction using in vivo mEHT models was the extended morphological tumor damage, starting as early as after 24 h in the middle of the treated tumor lamps, which then almost evenly spread from inside to the periphery ( Figure 1 c) [ 11 , 12 ]. This suggested the instant penetration, concentration, and enhanced energy absorption in the middle of tumors.…”

“…SzIE, ÁOTK MÁB 26/2013 and the most recent one, no. PE/EA/633-5/2018), using mouse C26 colorectal cancer cell line allografted into BALB/c mice, are found in Vancsik et al, 2018 and 2019 [ 12 , 29 ]. In the studies, 10% neutral-buffered, formalin-fixed tumor tissues embedded in paraffin were cut for 2–3 µm thick serial sections and mounted on silanized adhesion glass slides.…”

“…The modulated electro-hyperthermia generated by 13.56 MHz amplitude-modulated radio-frequency (mEHT; trade name: Oncothermia) has lately been an emerging way of delivering loco-regional clinical hyperthermia with favorable safety and tolerance profiles [ 10 ]. Though mEHT has also been used in combination with chemo-, radiation-, or more recently immunotherapy, a growing number of preclinical studies have demonstrated that it can also induce significant tumor damage in monotherapy, primarily through provoking cell stress and regulated cell death response [ 11 , 12 ]. Tumor selectivity of mEHT treatment is based on the metabolic reprogramming in cancer, resulting in its elevated glycose uptake, oxidative glycolysis (Warburg effect), ion concentration, and electric permittivity (conductivity) compared to adjacent normal tissues [ 13 ].…”

Potentiation of the Abscopal Effect by Modulated Electro-Hyperthermia in Locally Advanced Cervical Cancer Patients

“…Preclinical research suggests that mEHT combined with immunotherapies is able to elicit an immune-mediated response (13) which may even extend to untreated tumors. Vancsik et al showed that mEHT induced DAMPs in murine models was followed by an invasion of antigen presenting cells (APC) and T-cells at the site of the treated tumor and that when mEHT was administered combined with a T-cell stimulating agent, APC and T-cell invasion was also seen in the untreated tumors of the same murine model (14). In an in vivo study, mEHT combined with dendritic cell therapy elicited a response to untreated tumors in murine squamous cell carcinoma (SCCVII) models (15).…”