One of current applications of electroporation is electrochemotherapy and electroablation for local cancer treatment. Both of these electroporation modalities share some similarities with radiation therapy, one of which could be the bystander effect. In this study, we aimed to investigate the role of the bystander effect following these electroporation‑based treatments. During direct CHO‑K1 cell treatment, cells were electroporated using one 100 µs duration square wave electric pulse at 1400 V/cm (for bleomycin electrotransfer) or 2800 V/cm (for irreversible electroporation). To evaluate the bystander effect, the medium was taken from directly treated cells after 24 h incubation and applied on unaffected cells. Six days after the treatment, cell viability and colony sizes were evaluated using the cell colony formation assay. The results showed that the bystander effect after bleomycin electrotransfer had a strong negative impact on cell viability and cell colony size, which decreased to 2.8% and 23.1%, respectively. On the contrary, irreversible electroporation induced a strong positive bystander effect on cell viability, which increased to 149.3%. In conclusion, the results presented may serve as a platform for further analysis of the bystander effect after electroporation‑based therapies and may ultimately lead to refined application of these therapies in clinics.

Electrochemotherapy is an e cient method for the local treatment of cutaneous and subcutaneous metastases, but its e cacy 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 e ect, but its applications are limited because of possible side e ects, 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 e ect of bleomycin-based electrochemotherapy. With appropriately selected concentrations of bleomycin and plasmid DNA, it is possible to achieve e cient cell transfection while killing cells via the cytotoxic e ect 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 e cient bleomycin mediated cell killing.