Ionizing radiation (IR) is known to activate the immune system by releasing damage-associated molecular pattern (DAMP) molecules. To combine the IR with novel immunotherapies it is important to know how different irradiation doses influence the release of DAMPs and induce other molecular changes within the initial 48 h post-irradiation, a period preceding extensive cell death. Therefore, our aim was to determine the activation of most common markers of immunogenic cell death, calreticulin (CRT), HMGB1 and ATP and the changes in the expression of MHC I, MHC II and PD-L1 molecules to evaluate early post-IR events. We used three immunologically different mouse tumor cell lines, B16-F10, 4T1 and CT26, which form differently immunogenic tumor models in vivo. They were irradiated in vitro with doses at which 30 (IC30), 50 (IC50) or 70

Veterinary medicine serves as an important translational bridge between preclinical studies and human medicine, offering a valuable model for investigating naturally occurring diseases and generating data unattainable from purely experimental animal models. This is particularly relevant for evaluating novel cancer treatment approaches. Electrochemotherapy (ECT) and gene electrotransfer represent 2 such approaches in both human and veterinary clinical oncology. Current evidence indicates that ECT achieves excellent local ablative control of treated tumors; however, its principal limitation is that ECT does not reliably control untreated or metastatic disease. One strategy to overcome this limitation is combining ECT with the systemic immunomodulatory action of IL-12 gene electrotransfer as both modalities rely on the same platform technology, electroporation. In veterinary medicine, this combined approach has been successfully applied across various tumor types in dogs, demonstrating excellent local antitumor control alongside indications of systemic effects. These have been clinically associated with prolonged survival, regression of distant untreated tumors and lymph node metastases, and reduced invasive tumor growth. Immunological studies are also consistent with systemic immune activation, with flow cytometry analysis of peripheral blood demonstrating transient increases in circulating CD8+ cells following each therapeutic session. Further studies are needed to find the suitable biomarkers for selecting the appropriate patients for the selected treatments.

BACKGROUND:Electrochemotherapy (ECT) can be performed on tumors in patients that precedes tumor irradiation or on recurrent tumors after tumor irradiation. In this study we evaluated the feasibility, safety and effectiveness of the combined ECT and tumor irradiation (radiotherapy, RT) on different tumors in the head and neck region. PATIENTS AND METHODS:For the present cohort analysis, patients were selected from the Insp-ECT database, based on the following criteria: presence of head and neck lesions of any histological origin, treatment with both ECT and RT in any sequence, and a maximum interval of 12 months between the two treatments. The patients were treated with ECT followed by RT (ECT → RT) or RT followed by ECT (RT → ECT), in both cases because the first treatment resulted in incomplete response or subsequent relapse. In the case of ECT → RT the meantime interval was 5.7 months and in the case of RT → ECT 7.6 months. The minimal time interval between the treatments was 1 month. RESULTS:Treatment with ECT with bleomycin was feasible in all patients, treated either before or after RT. Included were 37 patients with the head and neck lesions. The combined treatment was performed predominantly on older population of patients with the age range from 50 to 96 years, median over 80 years. Importantly, in the group ECT → RT the major reason for RT was partial response (67%) after ECT. While, in the RT → ECT group the major reason for subsequent ECT was 58% of stable or progressive disease (failure to treatment). In both groups local symptoms decreased, like ulceration, odour and suppuration of tumors. In the ECT → RT group, the short-term response after 2 months was high, with 89% objective responses. In contrast, the RT → ECT group had a significantly lower rate, with 58% objective responses. At 24-month progression free survival (PFS) was 68% in the ECT → RT group and 20% in the RT → ECT group (p = 0.0101). The overall progression free survival Kaplan-Meier curves also differed significantly (log-rank p = 0.0232). CONCLUSIONS:The combined treatment of ECT and RT is feasible, safe, and effective in elderly patients. The findings suggest that ECT may have particular value as a neoadjuvant treatment before RT, especially for bulky, ulcerated, or symptomatic lesions in this population.

The field of oncology has witnessed remarkable progress with the integration of high-tech innovations in tumor ablation. Tumor ablation therapies, such as radiofrequency ablation (RFA), microwave ablation (MWA), cryoablation (Cryo), irreversible electroporation (IRE), and electrochemotherapy (ECT) have evolved beyond conventional boundaries, offering patients less invasive, highly targeted therapeutic options. Since it works across cancer histologies, tumor ablation is being integrated into cancer care at several levels. Tumor ablation is even considered curative in selected patients with small primary or secondary tumors, e.g. in the liver. Furthermore, it is central in treatment of oligometastatic disease or oligoprogression. Finally, ablation is widely used for symptomatic relief when tumors lead to symptoms affecting quality of life. Knowledge about ablative therapies and their inclusion at multidisciplinary team decision making enables effective and more personalized treatment for patients. This review synthesizes emerging applications of these therapies, focusing on artificial intelligence-driven personalization, robotic-assisted precision, and hybrid models combining ablation with systemic treatments, immunotherapy or targeted drug delivery. It also discusses the infrastructural, educational, regulatory, and ethical challenges that influence the clinical adoption of such treatments. Finally, the review presents strategic recommendations for integrating advanced ablation therapies into healthcare systems while ensuring equity, patient trust, and global accessibility.

This study investigated the antitumor efficacy of chemokine CCL5 gene therapy using gene electrotransfer (GET) in combination with radiotherapy (RT) in solid murine tumors CT26 and 4T1. In vitro, CT26 and 4T1 tumor cells transfected with plasmid DNA (pDNA) encoding CCL5 induced migration of RAW264.7 macrophages. In vivo, CCL5 overexpression achieved via GET of pDNA encoding CCL5 led to increased splenocyte infiltration in dorsal window chamber models. When combined with RT, GET of pDNA encoding CCL5 shifted the tumor cytokine profile toward a proinflammatory state, with elevated Ifn-γ, Cxcl9, Cxcl10, and Il-12α. Although CD8 + and CD4 + T cells were reduced post-treatment, due to radiation-induced cell death, the combination of GET of pDNA encoding CCL5 and RT significantly delayed tumor growth in both models. In 4T1 tumors, this delay was also significant compared to the equivalent treatment with GET of control pDNA. These findings support GET of pDNA encoding CCL5 combined with RT as a strategy to enhance immune-mediated tumor control.

Bleomycin electrosclerotherapy (BEST) is an emerging, minimally invasive treatment for treating slow-flow vascular malformations. Following intralesional administration of bleomycin, reversible electroporation is applied to enhance drug uptake and prolong local exposure due to a vascular lock effect and endothelial disruption. The procedure is based on electrochemotherapy principles, which have shown selective sensitivity of dysplastic and proliferative endothelium. Clinical experience indicates that BEST achieves effective lesion reduction with lower cumulative bleomycin doses and fewer treatment sessions compared to conventional Bleomycin sclerotherapy. This article presents a standardized protocol based on the current operating procedure (COP) developed by the International Network for Sharing Practices on Electrochemotherapy (InspECT) and collaborating centers. Sections cover patient selection, pre-treatment imaging, anesthesia considerations, lesion access, drug preparation, optimal dosing, electrode selection, electrical parameters, and post-procedural care. The protocol supports ultrasound-guided interventions and uses Cliniporator-validated European Standard Operating Procedures of Electrochemotherapy (ESOPE) pulse parameters. BEST is especially suitable for venous, lymphatic, capillary, and mixed slow-flow malformations where intralesional injection and safe electrode placement are possible. The method offers benefits for primary, resistant, or recurrent lesions and may significantly reduce disease burden. This protocol is intended to facilitate reproducible implementation of BEST across clinical centers and serves as a foundation for future refinement toward a fully validated standard operating procedure (SOP).

Bleomycin (BLM) is a cytotoxic antibiotic used in veterinary oncology, primarily in electrochemotherapy (ECT), a local ablative therapy where electric pulses increase drug uptake in tumours. BLM is administered intravenously or intratumourally, with the standard intravenous dose for dogs being 15,000 IU/m², followed by electric pulses 8-10 minutes later. This protocol is derived from human oncology and lacks extensive pharmacological data in dogs. We studied BLM pharmacokinetics in 29 dogs with various tumours treated with intravenous BLM and ECT between 2017 and 2023. Samples were collected from serum of 15 dogs, serum and tumours of 8 dogs, and tumours of 6 dogs. The mean volume of distribution (Vd) of BLM was 224.5 ± 75.02 ml/kg, clearance (CL) was 7.04 ± 2.05 ml/kg/min, and area under the curve (AUC) was 65.87 ± 2.11 µg·min/l. The half-life (t₁/₂) of BLM in dogs was 22.03 ± 0.88 minutes. No significant difference was found in tumour BLM concentrations between 8 minutes post-administration and 2 minutes after pulse completion. These results support the recommended 8-28-minute window for applying electric pulses following intravenous BLM administration and may indicate no need for dose adjustments based on body weight or age.

Harnessing the body’s own immune system to fight cancer is one of the most encouraging fields of cancer research today. Promising new cancer immunotherapies are currently being tested in preclinical and clinical trials, and a few have also reached regulatory approval. Likewise, the immunological properties of conventional therapies are being rediscovered and reinvestigated. It is now clear that the immune system is involved in antitumor action of most antitumor therapies. Special attention has been on different local ablative techniques, electrochemotherapy (ECT) being one of them. The main proposed mechanism of immunological involvement in the effectiveness of these therapies is their ability to induce an immunogenic form of cell death characterized by the release of tumor antigens from the therapy-killed cells in the context of a “danger signal,” which can lead to formation of immune response against the released tumor antigens. Hence, ECT can be seen as an in situ vaccine. But since no systemic antitumor effects have been reported after ECT in clinical practice so far, this means that ECT monotherapy is not sufficient to overcome the tumor-tolerating immunosuppressive microenvironment of the tumor. Therefore, combining ECT with immunotherapy could be an efficient way to cure both the ECT-treated nodules and any distant nodule. In this chapter, the hypothesis of ECT as in situ vaccine will be present, putative adjuvants that could be, or already were, tested in combination with ECT will be listed, and the first clinical data for different combinations of ECT and immunotherapy that support the concept will be presented.

Background Bleomycin electrosclerotherapy (BEST) is an emerging treatment option for vascular malformations (VMs), predominantly slow-flow venous malformations, with increasing use in other types of VMs. By combining application of bleomycin with electroporation, BEST enhances intracellular drug delivery and may improve treatment efficacy while allowing the use of lower drug doses. Although clinical evidence supporting its efficacy is growing, the biological mechanisms underlying these effects remain poorly understood. Key unresolved questions include endothelial responses to BEST, what are the dominant mechanisms of vascular injury and remodeling, and how hemodynamics and abnormal vessel architecture affect bleomycin distribution, pharmacokinetics, and effective dosing within the lesion. Although the clinical effects of BEST may be similar to the vascular disrupting effect of electrochemotherapy, it remains unclear whether these vascular mechanisms are in fact the same.Conclusions Understanding, how bleomycin is delivered, distributed, and retained within VM tissue, and how this interacts with endothelial susceptibility and electroporation efficiency, will be essential for defining optimal dosing strategies. Addressing these questions will require experimental approaches and physiologically relevant models capable of capturing the genetic, structural, and hemodynamic features of VMs. Such advances will be critical for elucidating the mechanisms of BEST and optimizing its clinical application.

Electrochemotherapy (ECT) combined with gene electrotransfer of interleukin-12 (IL-12 GET) has shown promising results in the treatment of canine mast cell tumors (MCTs). However, when tumors are located in anatomically challenging sites, there is a need for the use of a single-needle electrode to access the tumor. Therefore, this case report aimed to evaluate the feasibility and effectiveness of using a bipolar single-needle electrode in a dog with an MCT on the nasal planum. The 2-cm3 tumor was treated with the simultaneous intratumoral administration of cisplatin (CDDP) and a plasmid encoding canine IL-12, followed by the application of electric pulses using a bipolar single-needle electrode. In addition, the dog was treated with a tyrosine kinase inhibitor. We observed a reduction in microvessel density 6 months after treatment, along with a decreased number of proliferating cells. These combined interventions resulted in long-term local tumor control for 18 months before recurrence occurred. Importantly, our results demonstrated that the bipolar single-needle electrode approach was both feasible and effective. In conclusion, using IL-12 GET with a bipolar single-needle electrode for the treatment of MCTs is a practical and effective therapeutic option, particularly for tumors located in difficult-to-treat anatomical sites.

Electrochemotherapy (ECT) is a local ablative treatment and in veterinary oncology, evidence supporting ECT efficacy is mainly derived from case reports and small case series, underscoring the need for standardized, multicentric data collection comparable to human oncology registries. The primary aim of this study was to establish VetInspECT, a veterinary clinical registry adapted from the human InspECT platform, to enable standardized reporting and multicentre data collection for ECT. The secondary aim was to evaluate antitumor effectiveness using data from the single centre that initiated the registry. VetInspECT was designed to systematically record patient characteristics, tumor features, treatment parameters, and follow-up outcomes in animals treated with ECT. Retrospective data spanning from 2012 to 2024 from feline patients treated at a Slovenian center were entered into the database. Squamous cell carcinoma (SCC) was the most frequently treated tumor (66

BACKGROUND:Electroporation-Based Treatments and Therapies (EBTTs) - electrochemotherapy, irreversible electroporation, pulsed field ablation, and gene electro-transfer - use short, high-voltage electric pulses to permeabilize cell membranes, enabling non-thermal tissue ablation and enhanced local drug delivery. Although EBTTs have been used clinically for over three decades, primarily in oncology, their adoption remains limited compared to thermal ablation modalities. In contrast, cardiac pulsed field ablation (PFA) has seen rapid adoption since the first FDA device approval in December 2023. CONCLUSIONS:This expert opinion paper reviews the current clinical landscape of EBTTs in oncology and cardiac arrhythmia treatments, and focuses on emerging non-oncological and non-cardiac applications, including endoscopic pulsed electric field therapy for type 2 diabetes, bronchial rheoplasty for chronic bronchitis, bleomycin electrosclerotherapy for vascular malformations, nanosecond PFA for benign thyroid nodules, and electroporation-mediated gene delivery - and identifies key barriers to wider adoption: insufficient randomized evidence, lack of validated treatment planning tools, fragmented reimbursement, and limited standardization. We outline measures to facilitate clinical translation.

Platinum-based chemotherapeutics remain a crucial cancer treatment; however, their clinical efficacy is limited by systemic toxicity and acquired drug resistance. The present study evaluates the cytotoxic and mechanistic effects of two platinum(II) complexes of 5,7-dibromo-8-hydroxyquinoline (complex A and complex B) combined with electroporation (electrochemotherapy, ECT) in human colorectal (HT29), breast (MCF7), and ovarian (IGROV-1 and cisplatin-resistant IGROV-1/RDDP) carcinoma cell lines. Both complexes exhibited dose-dependent cytotoxicity that was significantly enhanced by ECT. Complex B showed the highest efficacy in HT29 cells, while MCF7 cells were more responsive to cisplatin-based ECT. Apoptosis was the predominant mode of cell death, supported by Annexin V/7-AAD flow cytometry, with minimal necrosis. Cell cycle analysis indicated treatment-dependent arrest at the G₀/G₁ or G₂/M phase. Importantly, both complexes, particularly complex B, retained strong activity in cisplatin-resistant IGROV-1/RDDP cells. Increased intracellular and DNA-bound platinum levels following ECT confirmed enhanced drug uptake. ECT also significantly inhibited tumor spheroid growth in 3D models. These findings support further preclinical evaluation of these complexes, especially complex B, as promising candidates for ECT-based cancer therapy.

Electrochemotherapy (ECT) is a well-established local cancer treatment that combines the administration of chemotherapeutic drugs with the application of short electric pulses to enhance intracellular drug uptake. Over the past 3 decades, ECT has become an accepted therapeutic modality in both human and veterinary oncology for the treatment of a wide range of superficial and deep-seated tumors. Although favorable response rates have been reported across many tumor types, treatment outcomes remain variable among patients and neoplasms. Currently, patient selection is based primarily on clinical and pathological factors, while validated predictive biomarkers are lacking. This review summarizes the evidence regarding factors influencing ECT response in veterinary oncology, including tumor histotype, size, local invasiveness, clinical stage, previous treatments, immunohistochemical and molecular markers, and tumor blood perfusion. Available data indicate that smaller tumors, lower disease burden, and earlier intervention are associated with improved outcomes, whereas recurrent and advanced-stage tumors show reduced responsiveness. Future research integrating molecular profiling, immune characterization, functional imaging, and liquid biopsy approaches may facilitate the development of predictive biomarkers and support a more personalized application of ECT in veterinary cancer patients.

BACKGROUND:In recent years, various gene therapy strategies have been developed for cancer treatment. One of these strategies is electroporation-based delivery of therapeutic transgenes - gene electrotransfer (GET). Electrochemotherapy and GET have been combined in several contemporary preclinical and veterinary studies. In most cases, two different pulse protocols are used, each for a specific treatment. The aim of our current study was to test whether the standard pulse protocol used in daily clinical practice for electrochemotherapy can also be used for effective GET. MATERIALS AND METHODS:Experiments were performed in vitro in a tumor (B16F10) and two normal tissue cell lines (C2C12 myoblasts and L929 fibroblasts). Four different GET protocols, three using monopolar electric pulses and one bipolar electric pulses, were tested for the GET of plasmid DNA, which codes for green fluorescent protein in vitro. In addition, two GET protocols were chosen for in vivo tumor and muscle transfection. RESULTS:Two GET protocols using monopolar electric pulses of different voltages delivered at 1 Hz transfected B16F10 tumor cells significantly better than normal cells. GET4 protocol, which uses monopolar electric pulses at 5 kHz, again transfected the B16F10 tumor cells significantly better, but the difference to the C2C12 myoblast cells was not significant. Compared with other GET protocols, GET3 using bipolar electric pulses at 1 Hz was significantly less effective. Both the GET2 (1 Hz) and GET4 (5 kHz) protocols resulted in similar tumor transfection efficiencies, whereas only the GET4 protocol was effective for muscle transfection in vivo. CONCLUSIONS:Our study demonstrated the efficient transfection of tumors and muscles with the GET4 pulse protocol, which is used clinically for electrochemotherapy. The use of this protocol could enable simultaneous electrochemotherapy and GET of the therapeutic gene in one session, which will significantly shorten the procedure and thus will be more tolerable for patients.

BACKGROUND:Laser speckle contrast imaging (LSCI) is an emerging imaging modality that enables noninvasive visualization and assessment of tissue perfusion and microcirculation. In this article, we evaluated LSCI in imaging perfusion in clinical oncology through a systematic review of the literature. METHODS:The inclusion criterion for the literature search in PubMed, Web of Science and Scopus electronic databases was the use of LSCI in clinical oncology, meaning that all animal, phantom, ex vivo, experimental, research and development, and purely methodological studies were excluded. RESULTS:Thirty-six articles met the inclusion criteria. The anatomic locations of the neoplasms in the selected articles were brain (5 articles), breasts (2 articles), endocrine glands (4 articles), skin (12 articles), and the gastrointestinal tract (13 articles). CONCLUSIONS:While LSCI is emerging as an appealing imaging modality, it is crucial for more clinical sites to initiate clinical trials. A lack of standardized protocols and interpretation guidelines are posing the most significant challenge.