Enhancing therapeutic efficacy in triple-negative breast cancer and melanoma: synergistic effects of modulated electro-hyperthermia (mEHT) with NSAIDs especially COX-2 inhibition in in vivo models.

Triple-negative breast cancer (TNBC) is a leading cause of cancer mortality and lacks modern therapy options. Modulated electro-hyperthermia (mEHT) is an adjuvant therapy with demonstrated clinical efficacy for the treatment of various cancer types. In this study, we report that mEHT monotherapy stimulated interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) expression, and consequently cyclooxygenase 2 (COX-2), which may favor a cancer-promoting tumor microenvironment. Thus, we combined mEHT with nonsteroid anti-inflammatory drugs (NSAIDs): a nonselective aspirin, or the selective COX-2 inhibitor SC236, in vivo. We demonstrate that NSAIDs synergistically increased the effect of mEHT in the 4T1 TNBC model. Moreover, the strongest tumor destruction ratio was observed in the combination SC236 + mEHT groups. Tumor damage was accompanied by a significant increase in cleaved caspase-3, suggesting that apoptosis played an important role. IL-1ß and COX-2 expression were significantly reduced by the combination therapies. In addition, a custom-made nanostring panel demonstrated significant upregulation of genes participating in the formation of the extracellular matrix. Similarly, in the B16F10 melanoma model, mEHT and aspirin synergistically reduced the number of melanoma nodules in the lungs. In conclusion, mEHT combined with a selective COX-2 inhibitor may offer a new therapeutic option in TNBC.

Fine-scale spatial genetic structure across the species range reflects recent colonization of high elevation habitats in silver fir (Abies alba Mill.).

Variation in genetic diversity across species ranges has long been recognized as highly informative for assessing populations' resilience and adaptive potential. The spatial distribution of genetic diversity within populations, referred to as fine-scale spatial genetic structure (FSGS), also carries information about recent demographic changes, yet it has rarely been connected to range scale processes. We studied eight silver fir (Abies alba Mill.) population pairs (sites), growing at high and low elevations, representative of the main genetic lineages of the species. A total of 1,368 adult trees and 540 seedlings were genotyped using 137 and 116 single nucleotide polymorphisms (SNPs), respectively. Sites revealed a clear east-west isolation-by-distance pattern consistent with the post-glacial colonization history of the species. Genetic differentiation among sites (FCT = 0.148) was an order of magnitude greater than between elevations within sites (FSC = 0.031), nevertheless high elevation populations consistently exhibited a stronger FSGS. Structural equation modelling revealed that elevation and, to a lesser extent, post-glacial colonization history, but not climatic and habitat variables, were the best predictors of FSGS across populations. These results suggest that high elevation habitats have been colonized more recently across the species range. Additionally, paternity analysis revealed a high reproductive skew among adults and a stronger FSGS in seedlings than in adults, suggesting that FSGS may conserve the signature of demographic changes for several generations. Our results emphasize that spatial patterns of genetic diversity within populations provide information about demographic history complementary to non-spatial statistics, and could be used for genetic diversity monitoring, especially in forest trees.

[The adjuvant killing effect of modulated electro-hyperthermia combined with chemotherapy on B16F10 melanoma cells]. / Modulált elektro-hipertermia sejtpusztító hatásának mechanizmusa és kemoterápiával való kölcsönhatása B16F10 melanóma-sejtvonalon.

Our aim was to detect the effect of modulated electro-hyperthermia (mEHT) on cell viability and to examine if hyperthermia can augment the cell killing effect of various chemotherapeutic agents. B16F10 melanoma cells were treated for 30, 60, 90 and 120 minutes with mEHT using LabEHY100 (OncothermTM). Cell viability was measured using MTT assay and apoptosis by annexin V/7-AAD staining using flow cytometry 24 hours post-treatment. For analyzing gene expression with qPCR cells were harvested after 60 minutes treatment. In combined protocols, cells were treated with paclitaxel (40 nM), dacarbazine (40 µM) or nutlin-3a (10 µM) after mEHT. mEHT induced nuclear translocation of p53 which in turn regulates pro- and anti-apoptotic gene expression accounting for decreased cell viability. In combination with chemotherapy, mEHT augmented the cell killing effect of dacarbazine or nutlin-3a but not that of paclitaxel determined 48 hours post-treatment. The sensitizing effect on chemotherapeutics demonstrate the efficiency of mEHT as an adjuvant modality in cancer treatment.

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

Modulated electro-hyperthermia (mEHT) is a novel complementary therapy in oncology which is based on the higher conductivity and permittivity of cancerous tissues due to their enhanced glycolytic activity and ionic content compared to healthy normal tissues. We aimed to evaluate the potential of mEHT, inducing local hyperthermia, in the treatment of pulmonary metastatic melanoma. Our primary objective was the optimization of mEHT for targeted lung treatment as well as to identify the mechanism of its potential anti-tumor effect in the B16F10 mouse melanoma pulmonary metastases model while investigating the potential treatment-related side effects of mEHT on normal lung tissue. Repeated treatment of tumor-bearing lungs with mEHT induced significant anti-tumor effects as demonstrated by the lower number of tumor nodules and the downregulation of Ki67 expression in treated tumor cells. mEHT treatment provoked significant DNA double-strand breaks indicated by the increased expression of phosphorylated H2AX protein in treated tumors, although treatment-induced elevation of cleaved/activated caspase-3 expression was insignificant, suggesting the minimal role of apoptosis in this process. The mEHT-related significant increase in p21waf1 positive tumor cells suggested that p21waf1-mediated cell cycle arrest plays an important role in the anti-tumor effect of mEHT on melanoma metastases. Significantly increased CD3+, CD8+ T-lymphocytes, and F4/80+CD11b+ macrophage density in the whole lung and tumor of treated animals emphasizes the mobilizing capability of mEHT on immune cells. In conclusion, mEHT can reduce the growth potential of melanoma, thus offering itself as a complementary therapeutic option to chemo- and/or radiotherapy.

[Preclinical and clinical investigation and development of electromagnetic oncological device - experience with solid tumors]. / Elektromágneses daganatterápiás készülék preklinikai és klinikai vizsgálatai, valamint mûszaki továbbfejlesztése: tapasztalatok szolid tumorokkal.

Our objective was to develop an electromagnetic tumor therapy device in a consortial cooperation between Semmelweis University and Oncotherm Ltd., to provide data and contribute to the development of the next generation of devices through preclinical, clinical and developmental modules via in vivo, in vitro studies, and patient treatments. Our numerous preclinical studies support the efficacy of mEHT. Clinical treatments were performed in 181 patients with inoperable and/or oligometastatic solid tumors. The protocols were developed, an international guideline was completed, and the planned steps of device development were realized. By optimizing previous selective RF techniques based on recent research findings, we can provide the most modern evidence-based treatment in the future.

Stress-Induced, p53-Mediated Tumor Growth Inhibition of Melanoma by Modulated Electrohyperthermia in Mouse Models without Major Immunogenic Effects.

Modulated electrohyperthermia (mEHT), an innovative complementary technique of radio-, chemo-, and targeted oncotherapy modalities, can induce tumor apoptosis and contribute to a secondary immune-mediated cancer death. Here, we tested the efficiency of high-fever range (~42 °C) mEHT on B16F10 melanoma both in cell culture and allograft models. In vivo, mEHT treatment resulted in significant tumor size reduction when repeated three times, and induced major stress response as indicated by upregulated cytoplasmic and cell membrane hsp70 levels. Despite the increased PUMA and apoptosis-inducing factor 1, and moderate rise in activated-caspase-3, apoptosis was not significant. However, phospho-H2AX indicated DNA double-strand breaks, which upregulated p53 protein and its downstream cyclin-dependent kinase inhibitors p21waf1 and p27kip. Combined in vitro treatment with mEHT and the p53 activator nutlin-3a additively reduced cell viability compared to monotherapies. Though mEHT promoted the release of damage-associated molecular pattern (DAMP) damage signaling molecules hsp70, HMGB1 and ATP to potentiate the tumor immunogenicity of melanoma allografts, it reduced MHC-I and melan-A levels in tumor cells. This might explain why the number of cytotoxic T cells was moderately reduced, while the amount of natural killer (NK) cells was mainly unchanged and only macrophages increased significantly. Our results suggest that mEHT-treatment-related tumor growth control was primarily mediated by cell-stress-induced p53, which upregulated cyclin-dependent kinase inhibitors. The downregulated tumor antigen-presenting machinery may explain the reduced cytotoxic T-cell response despite increased DAMP signaling. Decreased tumor antigen and MHC-I levels suggest that natural killer (NK) cells and macrophages were the major contributors to tumor eradication.