Induction of Bystander and Abscopal Effects after Electroporation-Based Treatments.

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.

Ca2+ roles in electroporation-induced changes of cancer cell physiology: From membrane repair to cell death.

The combination of Ca2+ ions and electroporation has gained attention as potential alternative to electrochemotherapy. Ca2+ is an important component of the cell membrane repair system and its presence directly influences the dynamics of the pore cycle after electroporation which can be exploited for cancer therapies. Here, the influence of Ca2+ concentration is investigated on small molecule electrotransfer and release of Calcein from 4T1, MX-1, B16F10, U87 cancer cells after cell exposure to microsecond electric pulses. Moreover, we investigated simultaneous molecule electrotransfer and intracellular calcium ion influx when media was supplemented with different Ca2+ concentrations. Results show that increased concentrations of calcium ions reduce the electrotransfer of small molecules to different lines of cancer cells as well as the release of Calcein. These effects are related with an enhanced membrane repair mechanism. Overall, we show that the efficiency of molecular electrotransfer can be controlled by regulating Ca2+ concentration in the electroporation medium. For the first time, the cause of cancer cell death in vitro from 1 mM CaCl2 concentrations is related to the irreversible loss of Ca2+ homeostasis after cell electroporation. Our findings provide fundamental insight on the mechanisms of Ca2+ electroporation that might lead to improved therapeutic outcomes.

Sudden Cell Death Induced by Ca2+ Delivery via Microbubble Cavitation.

Intracellular calcium ion delivery via sonoporation has been validated to be a substitute for classical chemotherapy. However, the mechanism behind calcium sonoporation remains unclear to this day. To elucidate the role of calcium in the process of sonoporation, we aimed to investigate the influence of different calcium concentration on cell membrane permeabilization and cell viability after sonoporation. In this study, we present experimental evidence that extracellular calcium plays a major role in cell membrane molecular transport after applying ultrasound pulses. Ultrasound-microbubble cavitation in the presence of different calcium concentration affects fundamental cell bio-physio-chemical conditions: cell membrane integrity, metabolic activity, and colony formation. Corresponding vital characteristics were evaluated using three independent viability tests: propidium iodide assay (20 min-3 h), MTT assay (48 h), and cell clonogenic assay (6 d). The results indicate instant cell death, as the level of cell viability was determined to be similar within a 20 min-48 h-6 d period. Inertial cavitation activities have been determined to be directly involved in calcium delivery via sonoporation according to high correlation (R2 > 0.85, p < 0.01) of inertial cavitation dose with change in either cell membrane permeabilization, metabolic activity, and colony formation efficiency. In general, calcium delivery via sonoporation induces rapid cell death, occurring within 20 min after treatment, that is the result of ultrasound mediated microbubble cavitation.

Extracellular-Ca2+-Induced Decrease in Small Molecule Electrotransfer Efficiency: Comparison between Microsecond and Nanosecond Electric Pulses.

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.

A Novel Method for Controlled Gene Expression via Combined Bleomycin and Plasmid DNA Electrotransfer.

Electrochemotherapy is an efficient method for the local treatment of cutaneous and subcutaneous metastases, but its efficacy as a systemic treatment remains low. The application of gene electrotransfer (GET) to transfer DNA coding for immune system modulating molecules could allow for a systemic effect, but its applications are limited because of possible side effects, e.g., immune system overactivation and autoimmune response. In this paper, we present the simultaneous electrotransfer of bleomycin and plasmid DNA as a method to increase the systemic effect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve efficient cell transfection while killing cells via the cytotoxic effect of bleomycin at later time points. We also show the dynamics of both cell electrotransfection and cell death after the simultaneous electrotransfer of bleomycin and plasmid DNA. Therefore, this method could have applications in achieving the transient, cell death-controlled expression of immune system activating genes while retaining efficient bleomycin mediated cell killing.