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

Stine Krog Frandsen, Laure Gibot, Moinecha Madi, Julie Gehl, Marie-Pierre Rols, Stine Krog Frandsen, Laure Gibot, Moinecha Madi, Julie Gehl, Marie-Pierre Rols

Abstract

Background: 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.

Methods: 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).

Results: 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.

Conclusion: 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.

Conflict of interest statement

Competing Interests: Two of the authors on this paper, Stine Krog Frandsen and Julie Gehl, have submitted a patent (Therapeutic applications of calcium electroporation to effectively induce tumour necrosis; EU #11195435.0 and USA #6L/579,775). This does not alter the authors' adherence to all PLOS ONE policies on sharing data and materials.

Figures

Fig 1. Spheroid size.
Fig 1. Spheroid size.
Size measurements of human normal dermal fibroblast (A), colon cancer (B), bladder cancer (C), and metastatic breast cancer (D) spheroids. Left panel: representative fluorescence microscopy images of a spheroid just after electroporation (8 pulses of 100 μs, 1000 V/cm, and 1 Hz) in buffer containing propidium iodide (PI) to visualize electropermeabilized cells and light microscopy images of another spheroid just before treatment (LM). Right panel: Growth curves of the spheroids after treatment with respectively 168 mM calcium (Ca), 1 mM bleomycin (Bleo), electroporation (EP), 168 mM calcium electroporation (Ca EP), electrochemotherapy using 1 mM bleomycin (Bleo EP), and untreated controls (Control). Measurements performed before treatment and at day 2, 3, and 4. Spheroid size is normalized to the size before treatment, means +/- SD, n = 5–6.
Fig 2. Live/dead staining.
Fig 2. Live/dead staining.
Live/dead staining with Calcein-AM and EthD-1 of human normal dermal fibroblast, colon cancer, and bladder cancer spheroids 4 days after treatment with 168 mM calcium (Ca), 1 mM bleomycin (Bleo), electroporation (EP; 8 pulses of 100 μs, 1000 V/cm, and 1 Hz), 168 mM calcium electroporation (Ca EP), or electrochemotherapy using 1 mM bleomycin (Bleo EP), and of untreated controls (Control). Upper panels are calcein-AM staining (living cells), middle panels are EthD-1 staining (dead cells), and lower panels are merged images of living (green) and dead (red) cells.
Fig 3. Intracellular ATP level.
Fig 3. Intracellular ATP level.
Intracellular ATP measurements of human normal dermal fibroblast, colon cancer, bladder cancer, and breast cancer spheroids 1, 4, 24, and 72 hours after treatment with 168 mM calcium (Ca), electroporation (EP; 8 pulses of 100 μs, 1000 V/cm, and 1 Hz), 168 mM calcium electroporation (Ca EP), high voltage EP (8 pulses of 100 μs, 5000 V/cm, and 1 Hz), and of untreated controls (Control). Means + SD, n = 3–5 (n = 2 for blank). Please note the difference in the y-axes.

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