Bleomycin electrotransfer or irreversible electroporation between different cell lines: an investigation of the bystander effect

A total of almost 2 million cases of cancer are predicted to be diagnosed in 2022, which equates to approximately 5,250 new cases each day [1]. It is known that many cancer treatments have been developed, one of them being radiation therapy, which uses ionizing radiation. However, new, more effective methods are needed due to the inhomogeneous and difficult to predict tumor growth. Therefore, safe, effective and alternative methods of cancer treatment are being sought. One alternative is electrochemotherapy, a method of delivering anticancer drugs to tumor cells based on electrical impulses [2]. Electrochemotherapy is based on the phenomenon of electroporation, when cells in tumor tissues are exposed to electric fields and the transmembrane potential of their membranes increases. When the potential exceeds a critical value, hydrophilic pores are created in the cell membrane through which various water-soluble molecules, such as the anticancer drug bleomycin (BLM), can enter the cell. Electroporation can be either reversible or irreversible. After reversible electroporation, the cell membrane is restored, and the cells are not killed. Meanwhile, irreversible electroporation is used when cells exposed to electrical pulses are destroyed without damaging nearby blood vessels or healthy tissue [3]. The cancer treatment method affected by irreversible electroporation is called electroablation. Both of these electroporation-based methods are already applied in clinical practice, but the phenomenon of electroporation, the electrotransfer of various molecules, and the secondary processes caused by these processes have not been fully investigated. Bystander effect has been observed to be induced in ionizing radiation therapy. Although the effect has been observed for some time after radiotherapy, it has not been studied when cells are exposed to the electric fields applied during electrochemotherapy. To date, only a few publications have been found showing that the bystander effect is possible after electrochemotherapy or electroablation [4], [5]. Also, there are still no data on how the bystander effect affects different types of cells, and in particular, how this effect affects the viability of different cells among themselves. The study investigated the bystander effect after BLM electrotransfer and irreversible electroporation in vitro. Five cell lines are used for experiments: chinese hamster ovary cells (CHO-K1), human adenocarcinoma alveolar basal epithelial cells (A-549), human mammary carcinoma cells (MX-1), murine melanoma cells (B16-F10), mice murine mammary carcinoma cells (4T1). A similar methodology is used to induce the bystander effect after both irreversible electroporation methods. BLM (20 nM) electrotransfer is induced using a single 100 μs electrical pulse of 1400 V/cm amplitude. After irreversible electroporation, the cells are exposed to one electric pulse of 2800 V/cm amplitude lasting 100 μs. After electroporation, wait 10 min for cell pores to close. Cells are plated in a 6-well plate with the addition of 1 ml of RPMI culture medium and incubated for one or several days at 37°C. After incubation, the medium is centrifuged twice and transferred onto 400 cells completely unaffected by electric fields. After the colonies have grown, a colony test is performed using crystal violet dye. From the obtained results, we found that the cells were divided into three groups when studying the bystander effect: insensitive, moderately sensitive and highly sensitive. Insensitive cells include CHO-K1 cells, moderately sensitive cells include 4T1 and B16-F10 cells, and highly sensitive cells include MX-1 and A-549 cells. Interchanging the media of the five cells showed different viability results. When using CHO-K1 cell culture, no significant changes were observed compared to untreated media, but the effect of bystander media from different cells was separated into two groups: the first CHO-K1, MX1 and 4T1, and the second B16-F10 and A-549. Bystander medium from the first group slightly affected CHO-K1 cells, but the second group completely killed those CHO-K1 cells both after BLM electrotransfer and irreversible electroporation.