The influence of bystander effect to the viability of directly unaffected cells after electroporation based treatments

Cancer research is one of the leading research topics worldwide. Therefore, alternative anticancer treatments are being developed alongside to the conventional ones. One of most promising novel anticancer therapies are electrochemotherapy (ECT) and electroablation [1]. Both therapies are based on the electroporation (EP) phenomenon. Such process occurs as a result of electric field triggered increase of transmembrane potential (TP). After reaching threshold level of TP the formation of transient electropores is initiated. If the electric field triggered TP does not deviate far from the EP threshold, then cells reseal the pores and not die, thus reversible electroporation (RE) is in action. If the TP greatly exceeds the EP threshold irreversible electroporation (IRE) is induced and cells die. This is the main mechanism of electroablation anticancer therapy. ECT is based on (RE), when created transient hydrophilic electropores act as a bridge for various water-soluble molecules, such as the anticancer drug bleomycin (BLM). If BLM is replaced to Ca+2 ions, then this EP based treatment is termed calcium electroporation (CaEP). All EP based anticancer therapies are local i.e., a well-defined tumour area of treatment effect is present. Naturally, there will be a part of the tissue in between area of affected and unaffected areas of the tumour. The effect of such area in EP based anticancer therapies is not defined well. However, in radiotherapy as another local anticancer treatment method, the therapeutic effect of such area is termed as bystander effect [2]. Although the effect has been observed for some time after radiotherapy [3], it has not been studied when cells are exposed to the electric fields applied during ECT, CaEP or electroablation. However, studies used different viability methods, hence it is hard to compare the published data [4,5]. Therefore, here we present results that are evaluated using clonogenic (ability to form colonies), flow cytometry (real number of cells that remain after the treatment), and Alamar blue (metabolic activity) assays. 4T1 (mouse breast cancer) cells were used in the experiments. A single electrical pulse with an amplitude of 1400 V/cm and a duration of 100 μs was used to electrotransfer CaCl2 and the anticancer drug BLM. A single electrical pulse with an amplitude of 2800 V/cm and a length of 100 μs was used to accomplish irreversible electroporation. Following electroporation, the cells were cultured in a 24-well plate with 0.2 ml of RPMI growth media for 48 hours. The medium is collected after incubation and centrifuged twice. Finally, the Bystander effect was created by adding the conditioned media on the untreated cells. To imitate the expressed molecule gradient the conditioned media was diluted with fresh media (FM) in a way that conditioned media had 10, 30, 50, 70, 90 and 100 % of total applied media. Depending on experiment this media was put on cells as conditioned media. In the case of Alamar Blue assay, conditioned media (200 μl) was put on 8000 cells, then incubated for 48 hours. Afterwards, 20 μl of Alamar Blue dye was put on the conditioned media, incubated for two hours and then fluorescence was measured. In the case of flow cytometry assay conditioned media (200 μl) was put on 15000 cells, then incubated for 48 hours. Then cells were detached from the surface with TrypLE and measured with flow cytometry. In the case of clonogenic assay conditioned media (2000 μl) was put on 400 cells that were put in 40 mm petri dish 24 hours prior to addition of conditioned media. After 6 days colonies were stained, imaged and ability to form colonies was determined. Results show that CaEP, ECT has similar tendencies when measuring ability to form colonies, real number of cells that remain after the treatment and metabolic activity. Nevertheless, when conditioned media used form ECT and CaEP was diluted in ratio (9 CM to 1 FM) cell number and colony formation decreased to around 60 %, yet metabolic activity stayed at 100 %. However, the main differences occurred with IRE at diluted ratios 7 CM to 3 FM, 9 CM to 1 FM and 10 CM to 0 FM the viabilities were accordingly 55,653,81%, 17,440,71% and 24,961,36% with flow cytometry assay, 81,40 3,92 %, 64,99 7,09 % and 2,36 0,73 % with clonogenic assay, and 96,634,02%, 71,923,92% and 56,20 5,19 % with Alamar Blue assay. Conclusions. Here we have showed that bystander effect has decreased the ability to form colonies, real number of cells that remain after the treatment and metabolic activity with similar tendencies after CaEP, ECT. However, the same viability measurements do not correlate when bystander effect is triggered by IRE.