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.

Gene electrotransfer (GET) is recognized as a promising technique for the development of an efficient tool for gene therapy. Such a therapy would have applications in the treatment of a variety of genetic diseases, including cancer. However, despite its wide applicability, the technique is limited by the lack of understanding of the fundamental mechanism of electroporation as well as other important factors that directly or indirectly influence its success rate. In the current study, we analyzed the impact of low concentrations (0–1 mM) of Ca²⁺ on the process of DNA electrotransfer using flow cytometry. The results revealed that the presence of a CaCl₂ concentration as low as 0.25 mM decreased the efficiency of GET by ~1.5-fold and cell viability decreased by ~2–3-fold. In addition, we determined that the observed phenomenon of the decrease in pDNA electrotransfer due to the influence of Ca²⁺ was not the consequence of cell death but rather should be attributed to secondary mechanisms. The data presented in this study provide an insight into the importance of Ca²⁺ in the process of gene electrotransfer that may be directly applicable to in vivo settings.