High-throughput lipidomic profiles sampled with electroporation-based biopsy differentiate healthy skin, cutaneous squamous cell carcinoma, and basal cell carcinoma.

BACKGROUND: The incidence rates of cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma (BCC) skin cancers are rising, while the current diagnostic process is time-consuming. We describe the development of a novel approach to high-throughput sampling of tissue lipids using electroporation-based biopsy, termed e-biopsy. We report on the ability of the e-biopsy technique to harvest large amounts of lipids from human skin samples. MATERIALS AND METHODS: Here, 168 lipids were reliably identified from 12 patients providing a total of 13 samples. The extracted lipids were profiled with ultra-performance liquid chromatography and tandem mass spectrometry (UPLC-MS-MS) providing cSCC, BCC, and healthy skin lipidomic profiles. RESULTS: Comparative analysis identified 27 differentially expressed lipids (p < 0.05). The general profile trend is low diglycerides in both cSCC and BCC, high phospholipids in BCC, and high lyso-phospholipids in cSCC compared to healthy skin tissue samples. CONCLUSION: The results contribute to the growing body of knowledge that can potentially lead to novel insights into these skin cancers and demonstrate the potential of the e-biopsy technique for the analysis of lipidomic profiles of human skin tissues.

Irreversible electroporation promotes a pro-inflammatory tumor microenvironment and anti-tumor immunity in a mouse pancreatic cancer model.

Pancreatic cancer is a significant cause of cancer-related mortality and often presents with limited treatment options. Pancreatic tumors are also notorious for their immunosuppressive microenvironment. Irreversible electroporation (IRE) is a non-thermal tumor ablation modality that employs high-voltage microsecond pulses to transiently permeabilize cell membranes, ultimately inducing cell death. However, the understanding of IRE's impact beyond the initiation of focal cell death in tumor tissue remains limited. In this study, we demonstrate that IRE triggers a unique mix of cell death pathways and orchestrates a shift in the local tumor microenvironment driven, in part, by reducing the myeloid-derived suppressor cell (MDSC) and regulatory T cell populations and increasing cytotoxic T lymphocytes and neutrophils. We further show that IRE drives induce cell cycle arrest at the G0/G1 phase in vitro and promote inflammatory cell death pathways consistent with pyroptosis and programmed necrosis in vivo. IRE-treated mice exhibited a substantial extension in progression-free survival. However, within a span of 14 days, the tumor immune cell populations reverted to their pre-treatment composition, which resulted in an attenuation of the systemic immune response targeting contralateral tumors and ultimately resulting in tumor regrowth. Mechanistically, we show that IRE augments IFN- Î³ signaling, resulting in the up-regulation of the PD-L1 checkpoint in pancreatic cancer cells. Together, these findings shed light on potential mechanisms of tumor regrowth following IRE treatment and offer insights into co-therapeutic targets to improve treatment strategies.

Comparing chemical transfection, electroporation, and lentiviral vector transduction to achieve optimal transfection conditions in the Vero cell line.

BACKGROUND: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells. METHODS: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells. RESULTS: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 104 Vero cells. CONCLUSION: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.

Importance of the electrophoresis and pulse energy for siRNA-mediated gene silencing by electroporation in differentiated primary human myotubes.

BACKGROUND: Electrotransfection is based on application of high-voltage pulses that transiently increase membrane permeability, which enables delivery of DNA and RNA in vitro and in vivo. Its advantage in applications such as gene therapy and vaccination is that it does not use viral vectors. Skeletal muscles are among the most commonly used target tissues. While siRNA delivery into undifferentiated myoblasts is very efficient, electrotransfection of siRNA into differentiated myotubes presents a challenge. Our aim was to develop efficient protocol for electroporation-based siRNA delivery in cultured primary human myotubes and to identify crucial mechanisms and parameters that would enable faster optimization of electrotransfection in various cell lines. RESULTS: We established optimal electroporation parameters for efficient siRNA delivery in cultured myotubes and achieved efficient knock-down of HIF-1α while preserving cells viability. The results show that electropermeabilization is a crucial step for siRNA electrotransfection in myotubes. Decrease in viability was observed for higher electric energy of the pulses, conversely lower pulse energy enabled higher electrotransfection silencing yield. Experimental data together with the theoretical analysis demonstrate that siRNA electrotransfer is a complex process where electropermeabilization, electrophoresis, siRNA translocation, and viability are all functions of pulsing parameters. However, despite this complexity, we demonstrated that pulse parameters for efficient delivery of small molecule such as PI, can be used as a starting point for optimization of electroporation parameters for siRNA delivery into cells in vitro if viability is preserved. CONCLUSIONS: The optimized experimental protocol provides the basis for application of electrotransfer for silencing of various target genes in cultured human myotubes and more broadly for electrotransfection of various primary cell and cell lines. Together with the theoretical analysis our data offer new insights into mechanisms that underlie electroporation-based delivery of short RNA molecules, which can aid to faster optimisation of the pulse parameters in vitro and in vivo.

Microfluidic Electroporation Arrays for Investigating Electroporation-Induced Cellular Rupture Dynamics.

Electroporation is pivotal in bioelectrochemistry for cellular manipulation, with prominent applications in drug delivery and cell membrane studies. A comprehensive understanding of pore generation requires an in-depth analysis of the critical pore size and the corresponding energy barrier at the onset of cell rupture. However, many studies have been limited to basic models such as artificial membranes or theoretical simulations. Challenging this paradigm, our study pioneers using a microfluidic electroporation chip array. This tool subjects live breast cancer cell species to a diverse spectrum of alternating current electric field conditions, driving electroporation-induced cell rupture. We conclusively determined the rupture voltages across varying applied voltage loading rates, enabling an unprecedented characterization of electric cell rupture dynamics encompassing critical pore radius and energy barrier. Further bolstering our investigation, we probed cells subjected to cholesterol depletion via methyl-ß-cyclodextrin and revealed a strong correlation with electroporation. This work not only elucidates the dynamics of electric rupture in live cell membranes but also sets a robust foundation for future explorations into the mechanisms and energetics of live cell electroporation.

Advantages of pulsed electric field ablation for COPD: Excellent killing effect on goblet cells.

Mucus hypersecretion resulting from excessive proliferation and metaplasia of goblet cells in the airways is the pathological foundation for Chronic obstructive pulmonary disease (COPD). Clinical trials have confirmed the clinical efficacy of pulsed electric field ablation (PFA) for COPD, but its underlying mechanisms is poorly understood. Cellular and animal models of COPD (rich in goblet cells) were established in this study to detect goblet cells' sensitivity to PFA. Schwan's equation was adopted to calculate the cells' transmembrane potential and the electroporation areas in the cell membrane. We found that goblet cells are more sensitive to low-intensity PFA (250 V/cm-500 V/cm) than BEAS-2B cells. It is attributed to the larger size of goblet cells, which allows a stronger transmembrane potential formation under the same electric field strength. Additionally, the transmembrane potential of larger-sized cells can reach the cell membrane electroporation threshold in more areas. Trypan blue staining confirmed that the cells underwent IRE rate was higher in goblet cells than in BEAS-2B cells. Animal experiments also confirmed that the airway epithelium of COPD is more sensitive to PFA. We conclude that lower-intensity PFA can selectively kill goblet cells in the COPD airway epithelium, ultimately achieving the therapeutic effect of treating COPD.

An Explorative Study on Calcium Electroporation for Low-risk Basal Cell Carcinoma.

In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.

High-throughput mechanical phenotyping and transcriptomics of single cells.

The molecular system regulating cellular mechanical properties remains unexplored at single-cell resolution mainly due to a limited ability to combine mechanophenotyping with unbiased transcriptional screening. Here, we describe an electroporation-based lipid-bilayer assay for cell surface tension and transcriptomics (ELASTomics), a method in which oligonucleotide-labelled macromolecules are imported into cells via nanopore electroporation to assess the mechanical state of the cell surface and are enumerated by sequencing. ELASTomics can be readily integrated with existing single-cell sequencing approaches and enables the joint study of cell surface mechanics and underlying transcriptional regulation at an unprecedented resolution. We validate ELASTomics via analysis of cancer cell lines from various malignancies and show that the method can accurately identify cell types and assess cell surface tension. ELASTomics enables exploration of the relationships between cell surface tension, surface proteins, and transcripts along cell lineages differentiating from the haematopoietic progenitor cells of mice. We study the surface mechanics of cellular senescence and demonstrate that RRAD regulates cell surface tension in senescent TIG-1 cells. ELASTomics provides a unique opportunity to profile the mechanical and molecular phenotypes of single cells and can dissect the interplay among these in a range of biological contexts.

Electroporation of mRNA as a Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens and Functional Proteins.

Electroporation (EP) of mRNA into human cells is a broadly applicable method to transiently express proteins of choice in a variety of different cell types. We have spent more than two decades to optimize and adapt this method, first for antigen-loading of dendritic cells (DCs) and subsequently for T cells, B cells, bulk PBMCs, and several cell lines. In this regard, antigens were introduced, processed, and presented in context of MHC class I and II. Next to that, functional proteins like adhesion receptors, T-cell receptors (TCRs), chimeric antigen receptors (CARs), constitutively active signal transducers (i.e. caIKK), and others were successfully expressed. We have also established this protocol under full GMP compliance as part of a manufacturing license to produce mRNA-electroporated DCs and mRNA-electroporated T cells for therapeutic applications in clinical trials. Therefore, we here want to share our universal mRNA electroporation protocol and the experience we have gathered with this method. The advantages of the transfection method presented here are: (1) easy adaptation to different cell types; (2) scalability from 106 to approximately 108 cells per shot; (3) high transfection efficiency (80-99%); (4) homogenous protein expression; (5) GMP compliance if the EP is performed in a class A clean room; and (6) no transgene integration into the genome. The provided protocol involves: OptiMEM® as EP medium, a square-wave pulse with 500 V, and 4 mm cuvettes. To adapt the protocol to differently sized cells, simply the pulse time has to be altered. Thus, we share an overview of proven electroporation settings (including recovery media), which we have established for various cell types. Next to the basic protocol, we also provide an extensive list of hints and tricks, which, in our opinion, are of great value for everyone who intends to use this transfection technique.

Immune cell populations in the tumour environment following calcium electropora-tion for cutaneous metastasis: a histopathological study.

BACKGROUND AND PURPOSE: Calcium electroporation (CaEP) involves injecting calcium into tumour tissues and using electrical pulses to create membrane pores that induce cell death. This study assesses resultant immune responses and histopathological changes in patients with cutaneous metastases. PATIENTS/MATERIALS AND METHODS: The aimed cohort comprised 24 patients with metastases exceeding 5 mm. Tumours were treated once with CaEP (day 0) or twice (day 28). Biopsies were performed on days 0 and 2, with additional samples on days 7, 28, 30, 35, 60, and 90 if multiple tumours were treated. The primary endpoint was the change in tumour-infiltrating lymphocytes (TILs) two days post-treatment, with secondary endpoints evaluating local and systemic immune responses via histopathological analysis of immune markers, necrosis, and inflammation. RESULTS: Seventeen patients, with metastases primarily from breast cancer (14 patients), but also lung cancer (1), melanoma (1), and urothelial cancer (1), completed the study. Of the 49 lesions treated, no significant changes in TIL count or PD-L1 expression were observed. However, there was substantial necrosis and a decrease in FOXP3-expression (p = 0.0025) noted, with a slight increase in CD4+ cells but no changes in CD3, CD8, or CD20 expressions. Notably, four patients showed reduced tumour invasiveness, including one case of an abscopal response. INTERPRETATION: This exploratory study indicates that CaEP can be an effective anti-tumour therapy potentially enhancing immunity. Significant necrosis and decreased regulatory lymphocytes were observed, although TIL count remained unchanged. Several patients exhibited clinical signs of immune response following treatment.

Live Imaging of Migrating Neurons and Glial Progenitors Visualized by in Utero Electroporation.

During cortical development, both neurons and glial cells are generated in the germinal zone near the lateral ventricle, migrate in the correct direction, and settle in their appropriate locations. This developmental process can be clearly visualized by introducing fluorescent protein-expression vectors via in utero electroporation. In this chapter, we describe labeling methods for migrating neurons and glial progenitors, as well as methods for slice culture, and time-lapse imaging.

Brain Sample Preparation After Performing in Utero Electroporation to Proliferating and Differentiated Cells in the Developing Mouse Neocortex.

In utero electroporation (IUE) enables labeling and manipulating specific types of cells by introducing DNA plasmids with desired promoters. After the surgery, mouse brains are fixed at any stage and analyzed after staining using specific antibodies. Here, we describe the flow of the IUE experiment from the preparation to microscopic observations.

Molecular Simulations Reveal the Free Energy Landscape and Transition State of Membrane Electroporation.

The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation were proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. The discrepancy originates from different geometries of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process, whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.

Biological autoluminescence enables effective monitoring of yeast cell electroporation.

The application of pulsed electric fields (PEFs) is becoming a promising tool for application in biotechnology, and the food industry. However, real-time monitoring of the efficiency of PEF treatment conditions is challenging, especially at the industrial scale and in continuous production conditions.  To overcome this challenge, we have developed a straightforward setup capable of real-time detection of yeast biological autoluminescence (BAL) during pulsing. Saccharomyces cerevisiae culture was exposed to 8 pulses of 100 µs width with electric field strength magnitude 2-7 kV cm-1. To assess the sensitivity of our method in detecting yeast electroporation, we conducted a comparison with established methods including impedance measurements, propidium iodide uptake, cell growth assay, and fluorescence microscopy. Our results demonstrate that yeast electroporation can be instantaneously monitored during pulsing, making it highly suitable for industrial applications. Furthermore, the simplicity of our setup facilitates its integration into continuous liquid flow systems. Additionally, we have established quantitative indicators based on a thorough statistical analysis of the data that can be implemented through a dedicated machine interface, providing efficiency indicators for analysis.

Homologous Overexpression of Tyrosinase in Trichoderma reesei and Its Application in Glycinin Cross-Linking.

Tyrosinase is capable of oxidizing tyrosine residues in proteins, leading to intermolecular protein cross-linking, which could modify the protein network of food and improve the texture of food. To obtain the recombinant tyrosinase with microbial cell factory instead of isolation tyrosinase from the mushroom Agaricus bisporus, a TYR expression cassette was constructed in this study. The expression cassette was electroporated into Trichoderma reesei Rut-C30 and integrated into its genome, resulting in a recombinant strain C30-TYR. After induction with microcrystalline cellulose for 7 days, recombinant tyrosinase could be successfully expressed and secreted by C30-TYR, corresponding to approximately 2.16 g/L tyrosinase in shake-flask cultures. The recombinant TYR was purified by ammonium sulfate precipitation and gel filtration, and the biological activity of purified TYR was 45.6 U/mL. The purified TYR could catalyze the cross-linking of glycinin, and the emulsion stability index of TYR-treated glycinin emulsion was increased by 30.6% compared with the untreated one. The cross-linking of soy glycinin by TYR resulted in altered properties of oil-in-water emulsions compared to emulsions stabilized by native glycinin. Therefore, cross-linking with this recombinant tyrosinase is a feasible approach to improve the properties of protein-stabilized emulsions and gels.

Effects of bipolar irreversible electroporation with different pulse durations in a prostate cancer mouse model.

Irreversible electroporation (IRE) is a non-thermal ablation technique for local tumor treatment known to be influenced by pulse duration and voltage settings, affecting its efficacy. This study aims to investigate the effects of bipolar IRE with different pulse durations in a prostate cancer mouse model. The therapeutic effectiveness was assessed with in vitro cell experiments, in vivo tumor volume changes with magnetic resonance imaging, and gross and histological analysis in a mouse model. The tumor volume continuously decreased over time in all IRE-treated groups. The tumor volume changes, necroptosis (%), necrosis (%), the degree of TUNEL-positive cell expression, and ROS1-positive cell (%) in the long pulse duration-treated groups (300 µs) were significantly increased compared to the short pulse duration-treated groups (100 µs) (all p < 0.001). The bipolar IRE with a relatively long pulse duration at the same voltage significantly increased IRE-induced cell death in a prostate cancer mouse model.

Ex vivo gene editing and cell therapy for hereditary tyrosinemia type 1.

BACKGROUND: We previously demonstrated the successful use of in vivo CRISPR gene editing to delete 4-hydroxyphenylpyruvate dioxygenase (HPD) to rescue mice deficient in fumarylacetoacetate hydrolase (FAH), a disorder known as hereditary tyrosinemia type 1 (HT1). The aim of this study was to develop an ex vivo gene-editing protocol and apply it as a cell therapy for HT1. METHODS: We isolated hepatocytes from wild-type (C57BL/6J) and Fah-/- mice and then used an optimized electroporation protocol to deliver Hpd-targeting CRISPR-Cas9 ribonucleoproteins into hepatocytes. Next, hepatocytes were transiently incubated in cytokine recovery media formulated to block apoptosis, followed by splenic injection into recipient Fah-/- mice. RESULTS: We observed robust engraftment and expansion of transplanted gene-edited hepatocytes from wild-type donors in the livers of recipient mice when transient incubation with our cytokine recovery media was used after electroporation and negligible engraftment without the media (mean: 46.8% and 0.83%, respectively; p=0.0025). Thus, the cytokine recovery medium was critical to our electroporation protocol. When hepatocytes from Fah-/- mice were used as donors for transplantation, we observed 35% and 28% engraftment for Hpd-Cas9 ribonucleoproteins and Cas9 mRNA, respectively. Tyrosine, phenylalanine, and biochemical markers of liver injury normalized in both Hpd-targeting Cas9 ribonucleoprotein and mRNA groups independent of induced inhibition of Hpd through nitisinone, indicating correction of disease indicators in Fah-/- mice. CONCLUSIONS: The successful liver cell therapy for HT1 validates our protocol and, despite the known growth advantage of HT1, showcases ex vivo gene editing using electroporation in combination with liver cell therapy to cure a disease model. These advancements underscore the potential impacts of electroporation combined with transplantation as a cell therapy.

The Induction of Combined Hyperthermal Ablation Effect of Irreversible Electroporation with Polydopamine Nanoparticle-Coated Electrodes.

Irreversible electroporation (IRE) is a prominent non-thermal ablation method widely employed in clinical settings for the focal ablation therapy of solid tumors. Utilizing high-voltage, short-duration electric pulses, IRE induces perforation defects in the cell membrane, leading to apoptotic cell death. Despite the promise of irreversible electroporation (IRE) in clinical applications, it faces challenges concerning the coverage of target tissues for ablation, particularly when compared to other thermal ablation therapies such as radiofrequency ablation, microwave ablation, and cryoablation. This study aims to investigate the induced hyperthermal effect of IRE by applying a polydopamine nanoparticle (Dopa NP) coating on the electrode. We hypothesize that the induced hyperthermal effect enhances the therapeutic efficacy of IRE for cancer ablation. First, we observed the hyperthermal effect of IRE using Dopa NP-coated electrodes in hydrogel phantom models and then moved to in vivo models. In particular, in in vivo animal studies, the IRE treatment of rabbit hepatic lobes with Dopa NP-coated electrodes exhibited a two-fold higher increase in temperature (ΔT) compared to non-coated electrodes. Through a comprehensive analysis, we found that IRE treatment with Dopa NP-coated electrodes displayed the typical histological signatures of hyperthermal ablation, including the disruption of the hepatic cord and lobular structure, as well as the infiltration of erythrocytes. These findings unequivocally highlight the combined efficacy of IRE with Dopa NPs for electroporation and the hyperthermal ablation of target cancer tissues.