New effective and less painful high frequency electrochemotherapy protocols: From optimization on 3D models to pilot study on veterinary patients

Abstract

Electroporation, a physical method that permeabilizes cell membranes, is increasingly used in cancer treatment. By enhancing the uptake of hydrophilic antitumor drugs, it boosts their cytotoxic effects and has proven effective in both human and veterinary medicine through electrochemotherapy. However, this treatment requires loco-regional or even general anesthesia, as electrical pulses cause muscle contractions and pain. Several clinical studies have demonstrated that application of high frequency pulses (above 5000 Hz) and short pulse duration (under 11 μs) causes much less discomfort to patients. In order to reduce the pain associated with contractions while maintaining the effectiveness of the treatment, we have developed new protocols using a high-frequency generator that delivers electric field pulses at a pulse repetition rate up to 2 MHz, associated to a multipolar electrode. In vitro tests on colorectal cancer cells were performed to assess the efficiency of cisplatin and bleomycin in inducing cell death. The efficiency obtained after one single treatment on both cell suspensions and on 3D multicellular spheroid models were similar to the ones obtained using ESOPE (European standard operating procedures for electrochemotherapy) protocol, which is currently used in clinics. In addition, as tumor cells die in an immunogenic cell death (ICD) mode and can release danger associated molecular patterns (DAMPs), major hallmarks of ICD were evaluated following the treatment by quantifying the apoptotic cell death, caspases 3/7 activation and key DAMPs. Subsequently, pilot studies on small number of conscious cats and horses under mild sedation confirmed that these protocols did not cause any noticeable muscle contractions and resulted in either partial or complete responses. New high-frequency electroporation protocols, described herein, show great promise in shifting electrochemotherapy into an effective and painless cancer treatment.

Keywords: Cells suspensions; Electrochemotherapy; Electropermeabilization; Electroporation; High-frequency; Multicellular spheroids; Pain.