Research Article: Calcium Electroporation: Evidence for Differential Effects in Normal and Malignant Cell Lines, Evaluated in a 3D Spheroid Model

Date Published: December 3, 2015

Publisher: Public Library of Science

Author(s): Stine Krog Frandsen, Laure Gibot, Moinecha Madi, Julie Gehl, Marie-Pierre Rols, Boris Rubinsky.

http://doi.org/10.1371/journal.pone.0144028

Abstract

Calcium electroporation describes the use of high voltage electric pulses to introduce supraphysiological calcium concentrations into cells. This promising method is currently in clinical trial as an anti-cancer treatment. One very important issue is the relation between tumor cell kill efficacy–and normal cell sensitivity.

Using a 3D spheroid cell culture model we have tested the effect of calcium electroporation and electrochemotherapy using bleomycin on three different human cancer cell lines: a colorectal adenocarcinoma (HT29), a bladder transitional cell carcinoma (SW780), and a breast adenocarcinoma (MDA-MB231), as well as on primary normal human dermal fibroblasts (HDF-n).

The results showed a clear reduction in spheroid size in all three cancer cell spheroids three days after treatment with respectively calcium electroporation (p<0.0001) or electrochemotherapy using bleomycin (p<0.0001). Strikingly, the size of normal fibroblast spheroids was neither affected after calcium electroporation nor electrochemotherapy using bleomycin, indicating that calcium electroporation, like electrochemotherapy, will have limited adverse effects on the surrounding normal tissue when treating with calcium electroporation. The intracellular ATP level, which has previously been shown to be depleted after calcium electroporation, was measured in the spheroids after treatment. The results showed a dramatic decrease in the intracellular ATP level (p<0.01) in all four spheroid types—malignant as well as normal. In conclusion, calcium electroporation seems to be more effective in inducing cell death in cancer cell spheroids than in a normal fibroblast spheroid, even though intracellular ATP level is depleted in all spheroid types after treatment. These results may indicate an important therapeutic window for this therapy; although further studies are needed in vivo and in patients to investigate the effect of calcium electroporation on surrounding normal tissue when treating tumors.

Partial Text

Electroporation or electropermeabilization is a method to generate transient permeabilization of the cell membrane by applying short, intense electric pulses [1]. The method can be used to facilitate transport of normally non-permeant ions or molecules into most cell types [2]. This method is used in the clinic in combination with chemotherapeutic drugs (electrochemotherapy) where the effect of the drug is increased drastically [2–5]. Due to common side effects of most chemotherapeutic drugs including bleomycin [6]; it is appealing to be able to use non-toxic molecules or ions that will induce cell death, when they are internalized into the cells in high concentration by electroporation. Calcium is a tightly regulated ubiquitous second messenger that is involved in regulation of transcription, metabolism, proliferation, apoptosis, necrosis, and many other cellular processes [7–9]. Calcium electroporation, where calcium is introduced into the cell cytosol by electroporation, has previously been shown to efficiently induce cell death in vitro and in vivo associated with acute ATP depletion [10], using electroporation parameters similar to those used for electrochemotherapy [11]. Calcium electroporation is currently in clinical trial (ClinicalTrials.gov ID- NCT01941901) for cutaneous metastasis where the response of the treatment will be compared with the response of electrochemotherapy using bleomycin, which is a clinically used anti-cancer treatment [12,13]. Calcium electroporation has been proven efficient in cell death of cancer cells but the effect on a broader range of tumor cell lines and on normal cells has not previously been investigated. We have tested the effect of calcium electroporation and electrochemotherapy using bleomycin in four different human cell spheroids (three cancer cell lines and one normal primary cell type) in order to investigate any difference in sensitivity between the different cell types. We chose using 3D spheroids as an in vivo-like model of tumors, a system which has previously been used for electroporation and electrochemotherapy studies [14,15].

Calcium electroporation seemingly had no effect on the size of the human fibroblast spheroids compared with untreated controls, but efficiently reduced the size of the three different cancer cell spheroids compared with untreated controls (p<0.0001 three days after treatment), at least as efficiently as electrochemotherapy using bleomycin (p<0.0001 three days after treatment; Fig 1). Although the effect of calcium electroporation appears to be superior to the effect of electrochemotherapy using bleomycin in two of the cancer cell spheroids (HT29 and SW780), this could be due to the chosen concentrations (168 mM CaCl2 as previously used in vivo [10] and 1 mM bleomycin as previously used in spheroids [15]).   Source: http://doi.org/10.1371/journal.pone.0144028