Cell and Developmental Biology Commons^™

Synergistic Effects Of Nanosecond Pulsed Plasma And Electric Field On Inactivation Of Pancreatic Cancer Cells In Vitro, Edwin A. Oshin, Zobia Minhas, Ruben M. L. Colunga Biancatelli, John D. Catravas, Richard Heller, Siqi Guo, Chunqi Jiang

Bioelectrics Publications

Nanosecond pulsed atmospheric pressure plasma jets (ns-APPJs) produce reactive plasma species, including charged particles and reactive oxygen and nitrogen species (RONS), which can induce oxidative stress in biological cells. Nanosecond pulsed electric field (nsPEF) has also been found to cause permeabilization of cell membranes and induce apoptosis or cell death. Combining the treatment of ns-APPJ and nsPEF may enhance the effectiveness of cancer cell inactivation with only moderate doses of both treatments. Employing ns-APPJ powered by 9 kV, 200 ns pulses at 2 kHz and 60-nsPEF of 50 kV/cm at 1 Hz, the synergistic effects on pancreatic cancer cells (Pan02) …

Identification Of Proteins Involved In Cell Membrane Permeabilization By Nanosecond Electric Pulses (Nsep), Giedre Silkuniene, Uma Mangalanathan, Alessandra Rossi, Peter A. Mollica, Andrei G. Pakhomov, Olga N. Pakhomova

Bioelectrics Publications

The study was aimed at identifying endogenous proteins which assist or impede the permeabilized state in the cell membrane disrupted by nsEP (20 or 40 pulses, 300 ns width, 7 kV/cm). We employed a LentiArray CRISPR library to generate knockouts (KOs) of 316 genes encoding for membrane proteins in U937 human monocytes stably expressing Cas9 nuclease. The extent of membrane permeabilization by nsEP was measured by the uptake of Yo-Pro-1 (YP) dye and compared to sham-exposed KOs and control cells transduced with a non-targeting (scrambled) gRNA. Only two KOs, for SCNN1A and CLCA1 genes, showed a statistically significant reduction in …

Modification Of The Tumor Microenvironment Enhances Anti-Pd-1 Immunotherapy In Metastatic Melanoma, Guilan Shi, Megan Scott, Cathryn G. Mangiamele, Richard Heller

Bioelectrics Publications

Resistance to checkpoint-blockade treatments is a challenge in the clinic. Both primary and acquired resistance have become major obstacles, greatly limiting the long-lasting effects and wide application of blockade therapy. Many patients with metastatic melanoma eventually require further therapy. The absence of T-cell infiltration to the tumor site is a well-accepted contributor limiting immune checkpoint inhibitor efficacy. In this study, we combined intratumoral injection of plasmid IL-12 with electrotransfer and anti-PD-1 in metastatic B16F10 melanoma tumor model to increase tumor-infiltrating lymphocytes and improve therapeutic efficacy. We showed that effective anti-tumor responses required a subset of tumor-infiltrating CD8⁺ and CD4 …

Electroporation And Cell Killing By Milli- To Nanosecond Pulses And Avoiding Neuromuscular Stimulation In Cancer Ablation, Emily Gudvangen, Vitalii Kim, Vitalij Novickij, Federico Battista, Andrei G. Pakhomov

Bioelectrics Publications

Ablation therapies aim at eradication of tumors with minimal impact on surrounding healthy tissues. Conventional pulsed electric field (PEF) treatments cause pain and muscle contractions far beyond the ablation area. The ongoing quest is to identify PEF parameters efficient at ablation but not at stimulation. We measured electroporation and cell killing thresholds for 150 ns–1 ms PEF, uni- and bipolar, delivered in 10- to 300-pulse trains at up to 1 MHz rates. Monolayers of murine colon carcinoma cells exposed to PEF were stained with YO-PRO-1 dye to detect electroporation. In 2–4 h, dead cells were labeled with propidium. Electroporation and …

In Vivo Metabolic Analysis Of The Anticancer Effects Of Plasma-Activated Saline In Three Tumor Animal Models, Miao Qi, Dehui Xu, Shuai Wang, Bing Li, Sansan Peng, Qiaosong Li, Hao Zhang, Runze Fan, Hai-Lan Chen, Michael G. Kong

Bioelectrics Publications

In recent years, the emerging technology of cold atmospheric pressure plasma (CAP) has grown rapidly along with the many medical applications of cold plasma (e.g., cancer, skin disease, tissue repair, etc.). Plasma-activated liquids (e.g., culture media, water, or normal saline, previously exposed to plasma) are being studied as cancer treatments, and due to their advantages, many researchers prefer plasma-activated liquids as an alternative to CAP in the treatment of cancer. In this study, we showed that plasma-activated-saline (PAS) treatment significantly inhibited tumor growth, as compared with saline, in melanoma, and a low-pH environment had little effect on tumor growth in …

Connexin Hemichannel Activation By S-Nitrosoglutathione Synergizes Strongly With Photodynamic Therapy Potentiating Anti-Tumor Bystander Killing, Chiara Nardin, Chiara Peres, Sabrina Putti, Tiziana Orsini, Claudia Colussi, Flavia Mazzarda, Marcello Raspa, Ferdinando Scavizzi, Anna Maria Salvatore, Francesco Chiani, Abraham Tettey-Matey, Yuanyuan Kuang, Guang Yang, Mauricio A. Retamal, Fabio Mammano

Bioelectrics Publications

In this study, we used B16-F10 cells grown in the dorsal skinfold chamber (DSC) preparation that allowed us to gain optical access to the processes triggered by photodynamic therapy (PDT). Partial irradiation of a photosensitized melanoma triggered cell death in non-irradiated tumor cells. Multiphoton intravital microscopy with genetically encoded fluorescence indicators revealed that bystander cell death was mediated by paracrine signaling due to adenosine triphosphate (ATP) release from connexin (Cx) hemichannels (HCs). Intercellular calcium (Ca²⁺) waves propagated from irradiated to bystander cells promoting intracellular Ca²⁺ transfer from the endoplasmic reticulum (ER) to mitochondria and rapid activation of …

Stobe Photography Mapping Of Cell Membrane Potential With Nanosecond Resolution, Allen S. Kiester, Bennett L. Ibey, Zachary N. Coker, Andrei G. Pakhomov, Joel N. Bixler

Bioelectrics Publications

The ability to directly observe membrane potential charging dynamics across a full microscopic field of view is vital for understanding interactions between a biological system and a given electrical stimulus. Accurate empirical knowledge of cell membrane electrodynamics will enable validation of fundamental hypotheses posited by the single shell model, which includes the degree of voltage change across a membrane and cellular sensitivity to external electric field non-uniformity and directionality. To this end, we have developed a high-speed strobe microscopy system with a time resolution of ~ 6 ns that allows us to acquire time-sequential data for temporally repeatable events (non-injurious …

Secretion Of Proteins And Antibody Fragments From Transiently Transfected Endothelial Progenitor Cells, Loree Heller, Reynald Thinard, Melanie Chevalier, Sezgi Arpag, Yu Jing, Ruth Greferath, Richard Heller, Claude Nicolau

Bioelectrics Publications

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-beta-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-beta-amyloid Fab protein functions in beta-amyloid aggregate solubilization.

Nanosecond Pulsed Electric Signals Can Affect Electrostatic Environment Of Protiens Below The Threshold Of Conformational Effects: The Case Study Of Sod1 With A Molecular Simulation Study, Elena Della Valle, Paolo Marracino, Olga Pakhomova, Micaela Liberti, Francesca Apollonio

Bioelectrics Publications

Electric fields can be a powerful tool to interact with enzymes or proteins, with an intriguing perspective to allow protein manipulation. Recently, researchers have focused the interest on intracellular enzyme modifications triggered by the application of nanosecond pulsed electric fields. These findings were also supported by theoretical predictions from molecular dynamics simulations focussing on significant variations in protein secondary structures. In this work, a theoretical study utilizing molecular dynamics simulations is proposed to explore effects of electric fields of high intensity and very short nanosecond duration applied to the superoxide dismutase (Cu/Zn-SOD or SOD-1), an important enzyme involved in the …

Nanopulse Stimulation (Nps) Induces Tumor Ablation And Immunity In Orthotopic 4t1 Mouse Breast Cancer: A Review, Stephen J. Beebe, Brittany P. Lassiter, Siqi Guo

Bioelectrics Publications

Nanopulse Stimulation (NPS) eliminates mouse and rat tumor types in several different animal models. NPS induces protective, vaccine-like effects after ablation of orthotopic rat N1-S1 hepatocellular carcinoma. Here we review some general concepts of NPS in the context of studies with mouse metastatic 4T1 mammary cancer showing that the postablation, vaccine-like effect is initiated by dynamic, multilayered immune mechanisms. NPS eliminates primary 4T1 tumors by inducing immunogenic, caspase-independent programmed cell death (PCD). With lower electric fields, like those peripheral to the primary treatment zone, NPS can activate dendritic cells (DCs). The activation of DCs by dead/dying cells leads to increases …

Tumor Cell Death After Electrotransfer Of Plasmid Dna Is Associated With Cytosolic Dna Sensor Upregulation, Katarina Znidar, Masa Bosnjak, Nina Semenova, Olga N. Pakhomova, Loree Heller, Maja Cemazar

Bioelectrics Publications

Cytosolic DNA sensors are a subgroup of pattern recognition receptors (PRRs) and are activated by the abnormal presence of the DNA in the cytosol. Their activation leads to the upregulation of pro-inflammatory cytokines and chemokines and can also induce cell death. The presence of cytosolic DNA sensors and inflammatory cytokines in TS/A murine mammary adenocarcinoma and WEHI 164 fibrosarcoma cells was demonstrated using real time reverse transcription polymerase chain reaction (RT-PCR), western blotting and enzyme-linked immunosorbent assay (ELISA). After electrotransfer of plasmid DNA (pDNA) using two pulse protocols, the upregulation of DNA-depended activator of interferon regulatory factor or Z-DNA binding …

Nano-Pulse Stimulation Ablates Orthotopic Rat Hepatocellular Carcinoma And Induces Innate And Adaptive Memory Immune Mechanisms That Prevent Recurrence, Brittany P. Lassiter, Siqi Guo, Stephen J. Beebe

Bioelectrics Publications

Nano-pulse stimulation (NPS), previously called nsPEFs, induced a vaccine-like effect after ablation of orthotopic N1-S1 hepatocellular carcinoma (HCC), protecting rats from subsequent challenges with N1-S1 cells. To determine immunity, immune cell phenotypes were analyzed in naïve, treated and protected rats. NPS provides a positive, post-ablation immuno-therapeutic outcome by alleviating immunosuppressive T regulatory cells (Treg) in the tumor microenvironment (TME), allowing dendritic cell influx and inducing dynamic changes in natural killer cells (NKs), NKT-cells and T-lymphocytes in blood, spleen and liver. NPS induced specific increases in NKs and NKT-cells expressing CD8 and activation receptors CD314-NKG2D and CD161 (NK1.1) in the TME …

Electrotransfer Of Different Control Plasmids Elicits Different Antitumor Effectiveness In B16.F10 Melanoma, Masa Bosnjak, Tanjo Jesenko, Urska Kamensek, Gregor Sersa, Jaka Lavrencak, Loree Heller, Maja Cemazar

Bioelectrics Publications

Several studies have shown that different control plasmids may cause antitumor action in different murine tumor models after gene electrotransfer (GET). Due to the differences in GET protocols, plasmid vectors, and experimental models, the observed antitumor effects were incomparable. Therefore, the current study was conducted comparing antitumor effectiveness of three different control plasmids using the same GET parameters. We followed cytotoxicity in vitro and the antitumor effect in vivo after GET of control plasmids pControl, pENTR/U6 scr and pVAX1 in B16.F10 murine melanoma cells and tumors. Types of cell death and upregulation of selected cytosolic DNA sensors and cytokines were …

Cytosolic Dna Sensor Upregulation Accompanies Dna Electrotransfer In B16.F10 Melanoma Cells, Katarina Znidar, Masa Bosnjak, Maja Cemazar, Loree C. Heller

Bioelectrics Publications

In several preclinical tumor models, antitumor effects occur after intratumoral electroporation, also known as electrotransfer, of plasmid DNA devoid of a therapeutic gene. In mouse melanomas, these effects are preceded by significant elevation of several proinflammatory cytokines. These observations implicate the binding and activation of intracellular DNA-specific pattern recognition receptors or DNA sensors in response to DNA electrotransfer. In tumors, IFN β mRNA and protein levels significantly increased. The mRNAs of several DNA sensors were detected, and DAI, DDX60, and p204 tended to be upregulated. These effects were accompanied with reduced tumor growth and increased tumor necrosis. In B16. F10 …

Dielectric Characterization Of Coastal Cartilage Chondrocytes, Michael W. Stacey, Ahmet C. Sabuncu, Ali Beskok

Bioelectrics Publications

BACKGROUND: Chondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy.

METHODS: Dielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are …