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PURPOSE: Electrochemotherapy, clinically established for treating (sub)cutaneous tumors, has been standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy (ESOPE). Due to common side effects of chemotherapeutic drugs, recent advances focus on non-cytotoxic agents, like calcium, to induce cell death (calcium electroporation). Therefore, this study aims to determine the efficacy of electrochemotherapy with bleomycin or cisplatin, or calcium electroporation on human hepatocellular carcinoma cells (HepG2) in vitro using the ESOPE protocol. METHODS: HepG2 cell viability was measured with a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after electrochemotherapy with the chemotherapeutic drugs bleomycin or cisplatin (0-20 µM), or after calcium electroporation (0-20 mM), to determine its efficacy on HepG2 cells in vitro using the ESOPE protocol (8 rectangular pulses, 1000 V/cm, 100 µs) compared to non-electroporated drug treatment. RESULTS: Cell viability was significantly lower in electroporated samples, compared to their non-electroporated controls (27-75% difference). Electrochemotherapy with bleomycin and calcium electroporation, reached (almost) complete cell death (- 1 ± 3% and 2.5 ± 2%), in the lowest concentration of 2.5 µM and 2.5 mM, respectively. Electrochemotherapy with 2.5 µM cisplatin, significantly decreased cell viability to only 68% (± 7%). CONCLUSION: Electrochemotherapy with bleomycin or cisplatin, or calcium electroporation were more effective in reducing the HepG2 cell viability in vitro using the ESOPE protocol compared to the non-electroporated drug treatments alone. When comparing electrochemotherapy, HepG2 cells are more sensitive to bleomycin than cisplatin, in similar concentrations. Calcium electroporation has the same effectiveness as electrochemotherapy with bleomycin, but calcium potentially has a better safety profile and several treatment advantages.

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Uveal melanoma (UM) represents a rare tumor of the uveal tract and is associated with a poor prognosis due to the high risk of metastasis. Despite advances in the treatment of UM, the mortality rate remains high, dictating an urgent need for novel therapeutic strategies. The current study introduces the first in vivo analysis of the therapeutic potential of calcium electroporation (CaEP) compared with electrochemotherapy (ECT) with bleomycin in a patient-derived xenograft (PDX) model based on the chorioallantoic membrane (CAM) assay. The experiments were conducted as monotherapy with either 5 or 10 mM calcium chloride or 1 or 2.5 µg/mL bleomycin in combination with EP or EP alone. CaEP and ECT induced a similar reduction in proliferative activity, neovascularization, and melanocytic expansion. A dose-dependent effect of CaEP triggered a significant induction of necrosis, whereas ECT application of 1 µg/mL bleomycin resulted in a significantly increased apoptotic response compared with untreated tumor grafts. Our results outline the prospective use of CaEP and ECT with bleomycin as an adjuvant treatment of UM, facilitating adequate local tumor control and potentially an improvement in metastatic and overall survival rates.

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BACKGROUND: Disruption of Ca2+ homeostasis after calcium electroporation (CaEP) in tumors has been shown to elicit an enhanced antitumor effect with varying impacts on healthy tissue, such as endothelium. Therefore, our study aimed to determine differences in Ca2+ kinetics and gene expression involved in the regulation of Ca2+ signaling and homeostasis, as well as effects of CaEP on cytoskeleton and adherens junctions of the established endothelial cell lines EA.hy926 and HMEC-1. METHODS: CaEP was performed on EA.hy926 and HMEC-1 cells with increasing Ca2+ concentrations. Viability after CaEP was assessed using Presto Blue, while the effect on cytoskeleton and adherens junctions was evaluated via immunofluorescence staining (F-actin, α-tubulin, VE-cadherin). Differences in intracellular Ca2+ regulation ([Ca2+]i) were determined with spectrofluorometric measurements using Fura-2-AM, exposing cells to DPBS, ionomycin, thapsigargin, ATP, bradykinin, angiotensin II, acetylcholine, LaCl3, and GdCl3. Molecular distinctions were identified by analyzing differentially expressed genes and pathways related to the cytoskeleton and Ca2+ signaling through RNA sequencing. RESULTS: EA.hy926 cells, at increasing Ca2+ concentrations, displayed higher CaEP susceptibility and lower survival than HMEC-1. Immunofluorescence confirmed CaEP-induced, time- and Ca2+-dependent morphological changes in EA.hy926's actin filaments, microtubules, and cell-cell junctions. Spectrofluorometric Ca2+ kinetics showed higher amplitudes in Ca2+ responses in EA.hy926 exposed to buffer, G protein coupled receptor agonists, bradykinin, and angiotensin II compared to HMEC-1. HMEC-1 exhibited significantly higher [Ca2+]i changes after ionomycin exposure, while responses to thapsigargin, ATP, and acetylcholine were similar in both cell lines. ATP without extracellular Ca2+ ions induced a significantly higher [Ca2+]i rise in EA.hy926, suggesting purinergic ionotropic P2X and metabotropic P2Y receptor activation. RNA-sequencing analysis showed significant differences in cytoskeleton- and Ca2+-related gene expression, highlighting upregulation of ORAI2, TRPC1, TRPM2, CNGA3, TRPM6, and downregulation of TRPV4 and TRPC4 in EA.hy926 versus HMEC-1. Moreover, KEGG analysis showed upregulated Ca2+ import and downregulated export genes in EA.hy926. CONCLUSIONS: Our finding show that significant differences in CaEP response and [Ca2+]i regulation exist between EA.hy926 and HMEC-1, which may be attributed to distinct transcriptomic profiles. EA.hy926, compared to HMEC-1, displayed higher susceptibility and sensitivity to [Ca2+]i changes, which may be linked to overexpression of Ca2+-related genes and an inability to mitigate changes in [Ca2+]i. The study offers a bioinformatic basis for selecting EC models based on research objectives.

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Despite recent advancements in the diagnosis and treatment of uveal melanoma (UM), its metastatic rate remains high and is accompanied by a highly dismal prognosis, constituting an unmet need for the development of novel adjuvant therapeutic strategies. We established an in vivo chick chorioallantoic membrane (CAM)-based UM xenograft model from UPMD2 and UPMM3 cell lines to examine its feasibility for the improvement of selection of drug candidates. The efficacy of calcium electroporation (CaEP) with 5 or 10 mM calcium chloride (Ca) and electrochemotherapy (ECT) with 1 or 2.5 µg/mL bleomycin in comparison to monotherapy with the tested drug or electroporation (EP) alone was investigated on the generated UM tumors. CaEP and ECT showed a similar reduction of proliferation and melanocytic expansion with a dose-dependent effect for bleomycin, whereas CaEP induced a significant increase of the apoptosis and a reduction of vascularization with varying sensitivity for the two xenograft types. Our in vivo results suggest that CaEP and ECT may facilitate the adequate local tumor control and contribute to the preservation of the bulbus, potentially opening new horizons in the adjuvant treatment of advanced UM.

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BACKGROUND: The development of new perioperative treatment modalities to activate the immune system in colorectal cancer might have a beneficial effect on reducing the risk of recurrence after surgery. Calcium electroporation is a promising treatment modality that potentially modulates the tumor microenvironment. The aim of this study was to evaluate the safety of the procedure in the neoadjuvant setting in localized left-sided colorectal cancer (CRC). METHODS: The study included patients with potentially curable sigmoid or rectal cancer with no indication for other neoadjuvant treatment. Patients were offered calcium electroporation as a neoadjuvant treatment before elective surgery. Follow-up visits were conducted on the preoperative day before elective surgery, POD2, POD14, and POD30, with an evaluation of adverse events, impact on elective surgery, clinical examination, and quality of recovery. RESULTS: Endoscopic calcium electroporation was performed as an outpatient procedure in all 21 cases, with no procedure-related complications reported. At follow-up, five adverse events were registered, two of which were classified as serious adverse events. Surgery was performed as planned in 19 patients (median time to surgery, 8 days), and the final two patients underwent surgery with a delay due to adverse events (14 and 33 days). No significant impact on the quality of recovery scores nor inflammatory markers were seen before and after calcium electroporation, nor baseline and POD30. CONCLUSIONS: Endoscopic calcium electroporation is a safe and feasible procedure in patients with potentially curable CRC. The study showed limited side effects and limited impact on the following elective surgical resection.

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PURPOSE: In this study, we aim to investigate gene expression changes in tumor samples obtained from patients with esophageal cancer treated with calcium electroporation. Previously, local treatment with calcium electroporation has been shown to induce gene expression alterations, potentially contributing to a more tumor-hostile microenvironment. METHODS: In this sub-study of a phase I clinical trial, we included five patients with esophageal cancer treated with calcium electroporation. We compared cancer-associated gene expression patterns in tumor samples before and after treatment. Furthermore, we used linear support vector regression to predict the cellular composition of tumor samples. RESULTS: Using differential expression analysis, we identified the downregulation of CXCL14 and upregulation of CCL21, ANGPTL4, and CRABP2 genes. We also found a decreased predicted proportion of dendritic cells while the proportion of neutrophils was increased. CONCLUSION: This study provides evidence that calcium electroporation for esophageal cancer induces local transcriptional changes and possibly alters the cellular composition of the tumor microenvironment. The results are explorative, larger studies are needed to confirm and further correlate our findings with clinical outcomes.

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Introduction: In calcium electroporation (CaEP), electroporation enables the cellular uptake of supraphysiological concentrations of Ca2+, causing the induction of cell death. The effectiveness of CaEP has already been evaluated in clinical trials; however, confirmatory preclinical studies are still needed to further elucidate its effectiveness and underlying mechanisms. Here, we tested and compared its efficiency on two different tumor models to electrochemotherapy (ECT) and in combination with gene electrotransfer (GET) of a plasmid encoding interleukin-12 (IL-12). We hypothesized that IL-12 potentiates the antitumor effect of local ablative therapies as CaEP and ECT. Methods: The effect of CaEP was tested in vitro as well as in vivo in murine melanoma B16-F10 and murine mammary carcinoma 4T1 in comparison to ECT with bleomycin. Specifically, the treatment efficacy of CaEP with increasing calcium concentrations alone or in combination with IL-12 GET in different treatment protocols was investigated. We closely examined the tumor microenvironment by immunofluorescence staining of immune cells, as well as blood vessels and proliferating cells. Results: In vitro, CaEP and ECT with bleomycin reduced cell viability in a dose-dependent manner. We observed no differences in sensitivity between the two cell lines. A dose-dependent response was also observed in vivo; however, the efficacy was better in 4T1 tumors than in B16-F10 tumors. In 4T1 tumors, CaEP with 250 mM Ca resulted in more than 30 days of growth delay, which was comparable to ECT with bleomycin. In contrast, adjuvant peritumoral application of IL-12 GET after CaEP prolonged the survival of B16-F10, but not 4T1-bearing mice. Moreover, CaEP with peritumoral IL-12 GET modified tumor immune cell populations and tumor vasculature. Conclusions: Mice bearing 4T1 tumors responded better to CaEP in vivo than mice bearing B16-F10 tumors, even though a similar response was observed in vitro. Namely, one of the most important factors might be involvement of the immune system. This was confirmed by the combination of CaEP or ECT with IL-12 GET, which further enhanced antitumor effectiveness. However, the potentiation of CaEP effectiveness was also highly dependent on tumor type; it was more pronounced in poorly immunogenic B16-F10 tumors compared to moderately immunogenic 4T1 tumors.

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(1) Background: Calcium electroporation is a novel cancer treatment. It includes injecting calcium-solution and applying electric pulses to tumour tissue. Data on quality of life for patients with cutaneous metastases treated with calcium electroporation is limited. We evaluated quality of life in patients with skin metastases treated with calcium electroporation using qualitative interviews. (2) Methods: This investigation featured a subgroup from a non-randomised phase II study (CaEP-R) at Zealand University Hospital, Denmark, studying response to calcium electroporation in cutaneous metastasis (ClinicalTrials no. NCT04225767). Participants were interviewed at baseline before calcium electroporation treatment and after two months. Data was analysed phenomenologically; (3) Results: Interviews were conducted February 2020-November 2021. Nine patients were included, of which seven participated in both interviews. All seven patients expected treated tumours to disappear, symptom relief and minimal side effects. Most patients requested peer accounts. All patients found the treatment uncomfortable but acceptable; all thought their fears of electric pulses exceeded their experience. All would repeat the treatment if effective. Successful treatment had a positive effect on pain, symptomatic wounds, sleep, vigour and social inclination; (4) Conclusions: Calcium electroporation enhanced health-related quality of life by reducing symptoms and increasing social inclination. Peer accounts provide patients with a shortcut to confidence in treatment on top of doctors' recommendations.

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We report a case of successful treatment of cutaneous metastases in HER2-positive breast cancer with calcium electroporation (CaEP), in addition to trastuzumab, over a period of 5 years. CaEP is performed in local or general anesthesia, by injecting calcium chloride intratumorally and then electroporating cells in the area. Using a handheld needle electrode, a series of short, high-voltage electric pulses are delivered, which transiently permeabilizes cell membranes, causing toxic intracellular calcium levels. The treatment causes cancer cell death, while normal cells are less affected, making the treatment useful for local management of cutaneous lesions. This case presents a 66-year-old female, who had mastectomy surgery followed by adjuvant chemo- and radiotherapy for an ER-negative, HER2-positive breast cancer on her right side in 2003, and a mastectomy followed by endocrine therapy for an ER-positive, HER2 normal breast cancer on her left side in 2006. In 2015, the patient presented local cutaneous recurrence of the ER-negative, HER2-positive breast cancer. The patient was treated with trastuzumab alone, trastuzumab emtansine (TDM1), and a combination of trastuzumab and CaEP. TDM1 was found to have a slightly better effect on the cutaneous metastases than trastuzumab, but the side effects of TDM1 were not acceptable to the patient. The combination of continuous HER2-inhibition and intermittent CaEP, when needed, has been effective in keeping the cutaneous metastases under control for 5 years, and presumably more tolerable for the patient than chemotherapy. An interesting finding was local sparing of calcium electroporated skin from new recurrences, otherwise seen in the general area, which could be a sign of local immunity. This warrants further studies investigating local immunomodulation following CaEP. The patient reported appreciation of a treatment option without chemotherapy, and satisfaction with the outcome of the combination of HER2 inhibition and CaEP treatment. CaEP treatment is currently phase II treatment, and mechanisms and possible applications still need investigation. This novel anticancer treatment could potentially benefit many patients, due to its efficacy, low cost, and accessibility. This case provides observations, which may inspire future trials with CaEP for skin metastases of HER2-positive breast cancer.

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Calcium electroporation (CaEP) is a novel cancer therapy wherein high intracellular calcium levels, facilitated by reversible electroporation, trigger tumor necrosis. This study aimed to establish safety with CaEP within esophageal cancer. Patients with non-curable esophageal cancer were included at Copenhagen University Hospital Rigshospitalet in 2021 and 2022. In an outpatient setting, calcium gluconate was injected intratumorally followed by reversible electroporation applied with an endoscopic electrode. The primary endpoint was the prevalence of adverse events, followed by palliation of dysphagia. All patients were evaluated with CT and upper endoscopies up to two months after treatment. The trial was registered at ClinicalTrials.gov (NCT04958044). Eight patients were treated. One serious adverse event (anemia, requiring a single blood transfusion) and three adverse events (mild retrosternal pain (two) and oral thrush (one)) were registered. Initially, six patients suffered from dysphagia: two reported dysphagia relief and four reported no change. From the imaging evaluation, one patient had a partial response, three patients had no response, and four patients had progression. Six months after treatment, the patient who responded well was still in good condition and without the need for further oncological treatment. CaEP was conducted in eight patients with only a few side effects. This study opens the way for larger studies evaluating tumor regression and symptom palliation.

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Electroporation-based antitumor therapies, including bleomycin electrotransfer, calcium electroporation, and irreversible electroporation, are very effective on directly treated tumors, but have no or low effect on distal nodules. In this study, we aimed to investigate the abscopal effect following calcium electroporation and bleomycin electrotransfer and to find out the effect of the increase of IL-2 serum concentration by muscle transfection. The bystander effect was analyzed in in vitro studies on 4T1tumor cells, while abscopal effect was investigated in an in vivo setting using Balb/c mice bearing 4T1 tumors. ELISA was used to monitor IL-2 serum concentration. We showed that, similarly to cell treatment with bleomycin electrotransfer, the bystander effect occurs also following calcium electroporation and that these effects can be combined. Combination of these treatments also resulted in the enhancement of the abscopal effect in vivo. Since these treatments resulted in an increase of IL-2 serum concentration only in mice bearing one but not two tumors, we increased IL-2 serum concentration by muscle transfection. Although this did not enhance the abscopal effect of combined tumor treatment using calcium electroporation and bleomycin electrotransfer, boosting of IL-2 serum concentration had a significant inhibitory effect on directly treated tumors.

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Electrochemotherapy (ECT) is the combination of transient pore formation following electric pulse application with the administration of cytotoxic drugs, which enhances the cytotoxic effect of the applied agent due to membrane changes and permeabilization. Although EP represents an established therapeutic option for solid malignancies, recent advances shift to the investigation of non-cytotoxic agents, such as calcium, which can also induce cell death. The present study aims to evaluate the cytotoxic effect, the morphological changes in tumor spheroids, the effect on the cell viability, and the cell-specific growth rate following calcium electroporation (CaEP) in uveal melanoma (UM) 2D monolayer cell cultures as well as in 3D tumor spheroid models. The experiments were conducted in four cell lines, UM92.1, Mel270, and two primary UM cell lines, UPMD2 and UPMM3 (UPM). The 2D and 3D UM cell cultures were electroporated with eight rectangular pulses (100 µs pulse duration, 5 Hz repetition frequency) of a 1000 V/cm pulse strength alone or in combination with 0.11 mg/mL, 0.28 mg/mL, 0.55 mg/mL or 1.11 mg/mL calcium chloride or 1.0 µg/mL or 2.5 µg/mL bleomycin. The application of calcium chloride alone induced an ATP reduction only in the UM92.1 2D cell cultures. Calcium alone had no significant effect on ATP levels in all four UM spheroids. A significant decrease in the intracellular adenosine triphosphate (ATP) level was documented in all four 2D and 3D cell cultures for both CaEP as well as ECT with bleomycin. The results suggest a dose-dependent ATP depletion with a wide range of sensitivity among the tested UM cell lines, control groups, and the applied settings in both 2D monolayer cell cultures and 3D tumor spheroid models. The colony formation capacity of the cell lines after two weeks reduced significantly after CaEP only with 0.5 mg/mL and 1.1 mg/mL, whereas the same effect could be achieved with both applied bleomycin concentrations, 1.0 µg/mL and 2.5 µg/mL, for the ECT group. The specific growth rate on day 7 following CaEP was significantly reduced in UM92.1 cell lines with 0.5 and 1.1 mg/mL calcium chloride, while Mel270 showed a similar effect only after administration of 1.1 mg/mL. UM92.1 and Mel270 spheroids exhibited lower adhesion and density after CaEP on day three in comparison to UPM spheroids showing detachment after day 7 following treatment. CaEP and bleomycin electroporation significantly reduce cell viability at similar applied voltage settings. CaEP may be a feasible and inexpensive therapeutic option for the local tumor control with fewer side effects, in comparison to other chemotherapeutic agents, for the treatment of uveal melanoma. The limited effect on normal cells and the surrounding tissue has already been investigated, but further research is necessary to clarify the effect on the surrounding tissue and to facilitate its application in a clinical setting for the eye.

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Gynecological carcinomas affect an increasing number of women and are associated with poor prognosis. The gold standard treatment plan is mainly based on surgical resection and subsequent chemotherapy with cisplatin, 5-fluorouracil, anthracyclines, or taxanes. Unfortunately, this treatment is becoming less effective and is associated with many side effects that negatively affect patients' physical and mental well-being. Electroporation based on tumor exposure to electric pulses enables reduction in cytotoxic drugs dose while increasing their effectiveness. EP-based treatment methods have received more and more interest in recent years and are the subject of a large number of scientific studies. Some of them show promising therapeutic potential without using any cytotoxic drugs or molecules already present in the human body (e.g., calcium electroporation). This literature review aims to present the fundamental mechanisms responsible for the course of EP-based therapies and the current state of knowledge in the field of their application in the treatment of gynecological neoplasms.

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Electroporation is a phenomenon of transient or irreversible permeabilization of the cell membrane after pulsed electric field treatment. Fluorescent probes are frequently used to assess the extent of permeabilization, however, as an alternative, a D-luciferin oxidation-based method can be used. In this work, we have used sequences of a microsecond (1.3 kV/cm × 100 µs) and nanosecond (12.5 kV/cm × 100 ns) pulses to trigger various levels of cell permeabilization and assessed the differences in the response using a conventional fluorescent probe (YO-PRO-1 (YP)) and D-luciferin oxidation methodology. The nanosecond pulses (n = 5-100) have been delivered with 1 kHz repetition frequency, and the results were compared with 1 MHz protocols. Additionally, the effects of extracellular Ca2+ have been assessed. Various concentrations of CaCl2 (2, 5, and 10 mM) have been used, and it was shown that the bioluminescence of the cells after electroporation depends on extracellular calcium concentration. It was shown that the changes in bioluminescence signal could be used as a marker of cell membrane permeabilization on par with YP assay when calcium is added and thus, effectively employed for analysis of electroporation phenomenon in vitro both for nanosecond and microsecond pulses.

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Ion attachment can modify stability and structure of phospholipid bilayers. Of particular importance is the interaction of phospholipids with divalent cations, such as calcium ions playing an important role in numerous cellular processes. The aim of our study was to determine effects of calcium ions on phospholipid membranes employing two cell membrane analogues, liposomes and planar lipid bilayers, and for the first time the combination of two instrumental setups: gas-phase electrophoresis (nES GEMMA instrumentation) and electrical (capacitance and resistance) measurements. Liposomes and planar lipid bilayers consisted of phosphatidylcholine, cholesterol and phosphatidylethanolamine. Liposomes were prepared from dried lipid films via hydration while planar lipid bilayers were formed using a Mueller-Rudin method. Calcium ions were added to membranes from higher concentrated stock solutions. Changes in phospholipid bilayer properties due to calcium presence were observed for both studied cell membrane analogues. Changes in liposome size were observed, which might either be related to tighter packing of phospholipids in the bilayer or local distortions of the membrane. Likewise, a measurable change in planar lipid bilayer resistance and capacitance was observed in the presence of calcium ions, which can be due to an increased rigidity and tighter packing of the lipid molecules in the bilayer.

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BACKGROUND: According to the National Cancer Registry, 3486 people (1744 men and 1742 women) were diagnosed with pancreatic adenocarcinoma in Poland in 2018, resulting in 4908 deaths (2396 men and 2512 women). The only chance of successful treatment is through surgical resection, which is possible in only 20-30% of patients (stage I, II and some stage III cases).The remaining 70-80% of patients are those with stage III and IV disease, for whom resection is not possible. Mean survival in these patients is approx. 10.4 months (stage III). In the recent decade, an innovative method called electroporation, which involves destabilization of the cell membrane, has been established. This process can be reversible (RE) or irreversible (IRE), and leads to cell death. The ability to change membrane permeability has led to the development of novel methods involving electrochemotherapy (ECT) and calcium electroporation (CaEP) to treat solid tumors. OBJECTIVES: In this study, both ECT and CaEP will be used to treat pancreatic cancer patients with poor prognosis. For each patient, the best "therapeutic moment" for the procedure will be selected based on the therapeutic protocol. MATERIAL AND METHODS: Patients will receive reversible and irreversible electroporation (control arm-group A), CaEP (active arm-group B), or ECT with intravenous and intratumoral administration of bleomycin (active arm-group C) randomized 1:1:1. RESULTS: The primary endpoints will be progression-free survival (PFS) and patients' quality of life (QOL) assessed using the EORTC-PAN 26 scale. Secondary endpoints will be patient overall survival (OS), body weight, pain level, and levels of biomarkers such as Ca 19-9. CONCLUSION: The Irreversible Electroporation, ELectrochemotherapy and Calcium electroporation (IREC) study is necessary to examine the safety and efficiency of irreversible electroporation, electrochemotherapy and calcium electroporation in pancreatic cancer treatment.

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In this study, irreversible electroporation (IRE), electrochemotherapy (ECT), and calcium electroporation (CaEP) techniques were investigated as new strategies for human pancreatic cancer. Qualification of the patients, best "therapeutic moment" for each patient, safety, and complications after procedures were examined. In this pilot study were included 13 patients in this study, which were operated on in different pancreatic cancer stages. Patients underwent IRE or ECT with intravenous admission of cisplatin or electroporation with calcium intratumoral administration. The IRE procedure was safe for the patients. Medium overall survival for IRE, IRE + CTH, and IRE + CaCl2 was respectively: 16, 29.5, and 19 months comparing to 10 months in control chemotherapy (CTH) group. Thus, IRE, ECT, and CaEP can be effective strategies for pancreatic cancer treatment.

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Estrogens (Es) play a significant role in the carcinogenesis and progression of ovarian malignancies. Depending on the concentration, Es may have a protective or toxic effect on cells. Moreover, they can directly or indirectly affect the activity of membrane ion channels. In the presented study, we investigated in vitro the effectiveness of the ovarian cancer cells (MDAH-2774) pre-incubation with 17ß-estradiol (E2; 10 µM) in the conventional chemotherapy (CT) and electrochemotherapy (ECT) with cisplatin or calcium chloride. We used three different protocols of electroporation including microseconds (µsEP) and nanoseconds (nsEP) range. The cytotoxic effect of the applied treatment was examined by the MTT assay. We used fluorescent staining and holotomographic imaging to observe morphological changes. The immunocytochemical staining evaluated the expression of the caspase-12. The electroporation process's effectiveness was analyzed by a flow cytometer using the Yo-Pro™-1 dye absorption assay. We found that pre-incubation of ovarian cancer cells with 17ß-estradiol may effectively enhance the chemo- and electrochemotherapy with cisplatin and calcium chloride. At the same time, estradiol reduced the effectiveness of electroporation, which may indicate that the mechanism of increasing the effectiveness of ECT by E2 is not related to the change of cell membrane permeability.

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Electroporation-a transient electric-field-induced increase in cell membrane permeability-can be used to facilitate the delivery of anticancer drugs for antitumour electrochemotherapy. In recent years, Ca2+ electroporation has emerged as an alternative modality to electrochemotherapy. The antitumor effect of calcium electroporation is achieved as a result of the introduction of supraphysiological calcium doses. However, calcium is also known to play a key role in membrane resealing, potentially altering the pore dynamics and molecular delivery during electroporation. To elucidate the role of calcium for the electrotransfer of small charged molecule into cell we have performed experiments using nano- and micro-second electric pulses. The results demonstrate that extracellular calcium ions inhibit the electrotransfer of small charged molecules. Experiments revealed that this effect is related to an increased rate of membrane resealing. We also employed mathematical modelling methods in order to explain the differences between the CaCl2 effects after the application of nano- and micro-second duration electric pulses. Simulation showed that these differences occur due to the changes in transmembrane voltage generation in response to the increase in specific conductivity when CaCl2 concentration is increased.

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Sarcoids are common equine skin tumors where the risk of recurrence after treatment is high, and better treatment options are warranted. Calcium electroporation is a novel anti-cancer treatment where lethally high calcium concentrations are introduced into the cells by electroporation, a method where short high-voltage pulses induce transient permeabilization of the cell membrane. This study investigated the safety and long-term response of calcium electroporation on sarcoids. Thirty-two sarcoids in eight horses were included. The study suggested that calcium electroporation is a safe and feasible treatment for sarcoids, including inoperable sarcoids. Horses were treated once (2/8) or twice (6/8) under general anesthesia, where sarcoids were injected with 220 mM calcium chloride followed by electroporation with 8 pulses of 100 µs, 1 kV/cm, and 1 Hz. Biopsies were taken prior to treatment. The sarcoid size was monitored for 12-38 weeks after the first treatment. Complete response was observed in 22% (6/27) of treated sarcoids, and partial response in 22% (6/27), giving a 44% total response. Treatment efficacy did not appear to be related to location, type, or size. In all non-biopsied lesions, a complete response was seen (4/4). In conclusion, in this small study, 44% of sarcoids responded with 22% of sarcoids disappearing.