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.

The effect of ionizing radiation on the homeostasis and functional integrity of murine splenic regulatory T cells.

OBJECTIVE: Radiotherapy affects antitumor immune responses; therefore, it is important to study radiation effects on various compartments of the immune system. Here we report radiation effects on the homeostasis and function of regulatory T (Treg) cells, which are important in down-regulating antitumor immune responses. METHODS: C57Bl/6 mice were irradiated with 2 Gy and alterations in splenic lymphocyte fractions analyzed at different intervals. RESULTS: Total CD4+ numbers showed stronger decrease after irradiation than CD4+Foxp3+ Tregs. Tregs were less prone to radiation-induced apoptosis than CD4+Foxp3- T cells. The ratio of CD4+Foxp3- and CD4+Foxp3+ fractions within the proliferating CD4+ pool progressively changed from 74:26 in control animals to 59:41 eleven days after irradiation, demonstrating a more dynamic increase in the proliferation and regeneration of the Treg pool. The CD4+Foxp3+ fraction expressing cell-surface CTLA4, an antigen associated with Treg cell activation increased from 5.3 % in unirradiated mice to 10.5 % three days after irradiation. The expression of IL-10 mRNA was moderately upregulated, while TGF-ß expression was not affected. On the other hand, irradiation reduced Treg capacity to suppress effector T cell proliferation by 2.5-fold. CONCLUSION: Tregs are more radioresistant, less prone to radiation-induced apoptosis, and have faster repopulation kinetics than CD4+Foxp3- cells, but irradiated Tregs are functionally compromised, having a reduced suppressive capacity.

Robust Recombinant Expression of Human Placental Ribonuclease Inhibitor in Insect Cells.

Ribonuclease inhibitors (RIs) are an indispensable biotechnological tool for the detection and manipulation of RNA. Nowadays, due to the outbreak of COVID-19, highly sensitive detection of RNA has become more important than ever. Although the recombinant expression of RNase inhibitors is possible in E. coli, the robust expression is complicated by maintaining the redox potential and solubility by various expression tags. In the present paper we describe the expression of RI in baculovirus-infected High Five cells in large scale utilizing a modified transfer vector combining the beneficial properties of Profinity Exact Tag and pONE system. The recombinant RI is expressed at a high level in a fusion form, which is readily cleaved during on-column chromatography. A subsequent anion exchange chromatography was used as a polishing step to yield 12 mg native RI per liter of culture. RI expressed in insect cells shows higher thermal stability than the commercially available RI products (mainly produced in E. coli) based on temperature-dependent RNase inhibition studies. The endotoxin-free RI variant may also be applied in future therapeutics as a safe additive to increase mRNA stability in mRNA-based vaccines.

[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.

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

Modulated electro-hyperthermia (mEHT), induced by 13.56 MHz radiofrequency, has been demonstrated both in preclinical and clinical studies to efficiently induce tumor damage and complement other treatment modalities. Here, we used a mouse xenograft model of human melanoma (A2058) to test mEHT (~42°C) both alone and combined with NK-cell immunotherapy. A single 30 min shot of mEHT resulted in significant tumor damage due to induced stress, marked by high hsp70 expression followed by significant upregulation of cleaved/activated caspase-3 and p53. When mEHT was combined with either primary human NK cells or the IL-2 independent NK-92MI cell line injected subcutaneously, the accumulation of NK cells was observed at the mEHT pretreated melanoma nodules but not at the untreated controls. mEHT induced the upregulation of the chemoattractant CXCL11 and increased the expression of the matrix metalloproteinase MMP2 which could account for the NK-cell attraction into the treated melanoma. In conclusion, mEHT monotherapy of melanoma xenograft tumors induced irreversible heat and cell stress leading to caspase dependent apoptosis to be driven by p53. mEHT could support the intratumoral attraction of distantly injected NK-cells, contributed by CXCL11 and MMP2 upregulation, resulting in an additive tumor destruction and growth inhibition. Therefore, mEHT may offer itself as a good partner for immunotherapy.

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.

Extracellular vesicles mediate low dose ionizing radiation-induced immune and inflammatory responses in the blood.

PURPOSE: Radiation-induced bystander effects (RIBE) imply the involvement of complex signaling mechanisms, which can be mediated by extracellular vesicles (EVs). Using an in vivo model, we investigated EV-transmitted RIBE in blood plasma and radiation effects on plasma EV miRNA profiles. MATERIALS AND METHODS: C57Bl/6 mice were total-body irradiated with 0.1 and 2 Gy, bone marrow-derived EVs were isolated, and injected systemically into naive, 'bystander' animals. Proteome profiler antibody array membranes were used to detect alterations in plasma, both in directly irradiated and bystander mice. MiRNA profile of plasma EVs was determined by PCR array. RESULTS: M-CSF and pentraxin-3 levels were increased in the blood of directly irradiated and bystander mice both after low and high dose irradiations, CXCL16 and lipocalin-2 increased after 2 Gy in directly irradiated and bystander mice, CCL5 and CCL11 changed in bystander mice only. Substantial overlap was found in the cellular pathways regulated by those miRNAs whose level were altered in EVs isolated from the plasma of mice irradiated with 0.1 and 2 Gy. Several of these pathways have already been associated with bystander responses. CONCLUSION: Low and high dose effects overlapped both in EV-mediated alterations in signaling pathways leading to RIBE and in their systemic manifestations.

[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.

Extracellular Vesicles Mediate Radiation-Induced Systemic Bystander Signals in the Bone Marrow and Spleen.

Radiation-induced bystander effects refer to the induction of biological changes in cells not directly hit by radiation implying that the number of cells affected by radiation is larger than the actual number of irradiated cells. Recent in vitro studies suggest the role of extracellular vesicles (EVs) in mediating radiation-induced bystander signals, but in vivo investigations are still lacking. Here, we report an in vivo study investigating the role of EVs in mediating radiation effects. C57BL/6 mice were total-body irradiated with X-rays (0.1, 0.25, 2 Gy), and 24 h later, EVs were isolated from the bone marrow (BM) and were intravenously injected into unirradiated (so-called bystander) animals. EV-induced systemic effects were compared to radiation effects in the directly irradiated animals. Similar to direct radiation, EVs from irradiated mice induced complex DNA damage in EV-recipient animals, manifested in an increased level of chromosomal aberrations and the activation of the DNA damage response. However, while DNA damage after direct irradiation increased with the dose, EV-induced effects peaked at lower doses. A significantly reduced hematopoietic stem cell pool in the BM as well as CD4+ and CD8+ lymphocyte pool in the spleen was detected in mice injected with EVs isolated from animals irradiated with 2 Gy. These EV-induced alterations were comparable to changes present in the directly irradiated mice. The pool of TLR4-expressing dendritic cells was different in the directly irradiated mice, where it increased after 2 Gy and in the EV-recipient animals, where it strongly decreased in a dose-independent manner. A panel of eight differentially expressed microRNAs (miRNA) was identified in the EVs originating from both low- and high-dose-irradiated mice, with a predicted involvement in pathways related to DNA damage repair, hematopoietic, and immune system regulation, suggesting a direct involvement of these pathways in mediating radiation-induced systemic effects. In conclusion, we proved the role of EVs in transmitting certain radiation effects, identified miRNAs carried by EVs potentially responsible for these effects, and showed that the pattern of changes was often different in the directly irradiated and EV-recipient bystander mice, suggesting different mechanisms.

RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay).

PURPOSE: In the framework of the 'Realizing the European Network of Biodosimetry' (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. MATERIALS AND METHODS: Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. RESULTS: The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. CONCLUSION: The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.

RENEB accident simulation exercise.

PURPOSE: The RENEB accident exercise was carried out in order to train the RENEB participants in coordinating and managing potentially large data sets that would be generated in case of a major radiological event. MATERIALS AND METHODS: Each participant was offered the possibility to activate the network by sending an alerting email about a simulated radiation emergency. The same participant had to collect, compile and report capacity, triage categorization and exposure scenario results obtained from all other participants. The exercise was performed over 27 weeks and involved the network consisting of 28 institutes: 21 RENEB members, four candidates and three non-RENEB partners. RESULTS: The duration of a single exercise never exceeded 10 days, while the response from the assisting laboratories never came later than within half a day. During each week of the exercise, around 4500 samples were reported by all service laboratories (SL) to be examined and 54 scenarios were coherently estimated by all laboratories (the standard deviation from the mean of all SL answers for a given scenario category and a set of data was not larger than 3 patient codes). CONCLUSIONS: Each participant received training in both the role of a reference laboratory (activating the network) and of a service laboratory (responding to an activation request). The procedures in the case of radiological event were successfully established and tested.

Age-related effects of X-ray irradiation on mouse hippocampus.

Therapeutic irradiation of pediatric and adult patients can profoundly affect adult neurogenesis, and cognitive impairment manifests as a deficit in hippocampal-dependent functions. Age plays a major role in susceptibility to radiation, and younger children are at higher risk of cognitive decay when compared to adults. Cranial irradiation affects hippocampal neurogenesis by induction of DNA damage in neural progenitors, through the disruption of the neurogenic microenvironment, and defective integration of newborn neurons into the neuronal network. Our goal here was to assess cellular and molecular alterations induced by cranial X-ray exposure to low/moderate doses (0.1 and 2 Gy) in the hippocampus of mice irradiated at the postnatal ages of day 10 or week 10, as well as the dependency of these phenomena on age at irradiation. To this aim, changes in the cellular composition of the dentate gyrus, mitochondrial functionality, proteomic profile in the hippocampus, as well as cognitive performance were evaluated by a multidisciplinary approach. Our results suggest the induction of specific alterations in hippocampal neurogenesis, microvascular density and mitochondrial functions, depending on age at irradiation. A better understanding of how irradiation impairs hippocampal neurogenesis at low and moderate doses is crucial to minimize adverse effects of therapeutic irradiation, contributing also to radiation safety regulations.

Growth Differentiation Factor-15 (GDF-15) is a potential marker of radiation response and radiation sensitivity.

We have investigated the importance of GDF-15 (secreted cytokine belonging to the TGF-ß superfamily) in low and high dose radiation-induced cellular responses. A telomerase immortalized human fibroblast cell line (F11hT) was used in the experiments. A lentiviral system encoding small hairpin RNAs (shRNA) was used to establish GDF-15 silenced cells. Secreted GDF-15 levels were measured in culture medium by ELISA. Cell cycle analysis was performed by flow cytometry. The experiments demonstrated that in irradiated human fibroblasts GDF-15 expression increased with dose starting from 100mGy. Elevated GDF-15 expression was not detected in bystander cells. The potential role of GDF-15 in radiation response was investigated by silencing GDF-15 in immortalized human fibroblasts with five different shRNA encoded in lentiviral vectors. Cell lines with considerably reduced GDF-15 levels presented increased radiation sensitivity, while a cell line with elevated GDF-15 was more radiation resistant than wild type cells. We have investigated how the reduced GDF-15 levels alter the response of several known radiation inducible genes. In F11hT-shGDF-15 cells the basal expression level of CDKN1A was unaltered relative to F11hT cells, while GADD45A and TGF-ß1 mRNA levels were slightly higher, and TP53INP1 was considerably reduced. The radiation-induced expression of TP53INP1 was lower in the silenced than in wild type fibroblast cells. Cell cycle analysis indicated that radiation-induced early G2/M arrest was abrogated in GDF-15 silenced cells. Moreover, radiation-induced bystander effect was less pronounced in GDF-15 silenced fibroblasts. In conclusion, the results suggest that GDF-15 works as a radiation inducible radiation resistance increasing factor in normal human fibroblast cells, acts by regulating the radiation-induced transcription of several genes and might serve as a radiation-induced early biomarker in exposed cells.