Keywords

1 Introduction

Calcium electroporation is a novel method for local tumor ablation, where electroporation is used as a physical delivery method that permeabilizes cell membranes, allowing calcium ions to enter the cells and induce cell death [1, 2]. Calcium is ubiquitous second messenger, involved in signaling of many different cellular pathways, including cell mobility, proliferation and cell death [3]. For that reason the cells maintain low levels of calcium ions in the cytosol. When cells are exposed to pulsed electric field, pores in the membrane allow flux of large quantities of calcium ions into the cell, causing severe disturbance in homeostasis [4]. To remove the excess of calcium in the cytosol, they pump the calcium out of the cell or in the intracellular storage compartments – endoplasmic reticulum and mitochondria. Cells are then exposed to energy depletion, most likely due to increased consumption and impaired production of ATP in the mitochondria, where formation of mitochondrial permeability pores occurs [5]. Other mechanisms that may be involved are leakage of reactive oxygen species from the mitochondrial matrix and activation of lipases and proteases [4,5,6,7,8]. It was shown in previous in vitro studies on different cell lines and spheroids, that calcium electroporation causes intracellular ATP depletion and reduction in cell viability [9,10,11]. However, in contrast to tumor cells, normal cells seem to be less sensitive to this disturbance, resulting in better survival [12]. Studies performed in vivo confirmed that calcium electroporation causes tumor necrosis and has vascular disruptive effects [2, 13]. Furthermore, first clinical studies with calcium electroporation executed on cutaneous metastases and on head and neck cancer showed similar effectiveness to electrochemotherapy with bleomycin, which is widely used for treatment of cutaneous tumors from different histologies [14, 15]. Furthermore, a case report of a patient with disseminated malignant melanoma was published, where systemic response after electrochemotherapy with bleomycin and calcium electroporation was observed [16]. Thus, calcium electroporation has a potential to be used as a substitute for bleomycin, when the use of chemotherapeutic drugs is contraindicated. The aim of this study was to investigate efficacy of calcium electroporation with increasing concentrations of calcium solution in two different murine tumor models.

2 Materials and Methods

2.1 Cell Lines and Animals

Murine melanoma cells B16F10 (American Type Culture Collection (ATCC), Manassas, VA, USA) were cultured in Advanced Minimum Assential Medium (AMEM; Thermo Fisher Scientific, Waltham, Massachusetts, USA), murine breast carcinoma cells 4T1 were cultured in Advanced RPMI 1640 (Thermo Fisher Scientific) in a humidified 5% CO2 atmosphere at 37 ℃. All media were supplemented with 5% fetal bovine serum (FBS, Thermo Fisher Scientific), 10 mM L-glutamine (Thermo Fisher Scientific), 50 mg/ml gentamicin (Krka, Novo mesto, Slovenia) and 100 U/ml penicillin (Sandoz, Holzkirchen, Germany).

Eight to ten-week old female C57BL/6NCrl and BALB/cAnNCrl (Charles River Laboratories, Wilmington, Massachusetts, USA) weighing 18–21 g were used in experiments. Mice were kept in quarantine for 2 weeks prior experiments. Throughout the quarantine and experiments mice were kept in a specific-pathogen-free housing conditions. All procedures were performed in compliance with the guidelines for animal experiments of the EU directive (2010/63/EU) and the permission from the Veterinary Administration of the Ministry of Agriculture, Forestry and Food of the Republic of Slovenia (permission no. U34401-1/2015/26). Permission was provided based on the approval of the National Ethics Committee for Experiments on Laboratory Animals, which is under the auspices of Ministry of Agriculture, Forestry and Food of the Republic of Slovenia. In the experimental groups between 6 and 20 mice were included due to the high biological variability of tumor response to calcium electroporation.

2.2 Tumor Induction

Cells were detached by trypsinisation and collected by centrifugation. Then, cells were resuspended in physiological saline solution in concentration of 1 × 107/ml for B16F10 cells and 5 × 106/ml for 4T1 cells. 100 µl of cell suspension was injected subcutaneously on the right flank of the C57Bl/6 (B16F10) and Balb/c (4T1) mice.

2.3 Calcium Electroporation in Vivo

When tumors reached size of −40 mm3 (after 6 to 9 days for B16F10 melanoma and after 8–11 days for 4T1 carcinoma), treatments were performed by intratumoraly injecting 40 µl of 50 mM, 168 mM or 250 mM CaCl2, respectively. In combined treatment groups, electric pulses (8 square-wave pulses, 1300 V/cm, 100 µs, 1 Hz) were delivered immediately after injection of CaCl2 solution by stainless-steel electrodes with a 6 mm distance between the electrodes. Good contact between electrodes and skin was ensured with the use of conductive gel. Electric pulses were generated by Electro CELL B10 HVLV pulse generator (LEROY biotech, Betatech, Saint-Orens-de-Gameville, France). Throughout the entire procedure mice were kept under isoflurane inhalation anesthesia.

2.4 Tumor Growth Assay

Mice were monitored every day and tumor growth was determined by measuring the tumor diameters every 2 to 3 days after treatment. Three mutually orthogonal tumor diameters were measured with Vernier caliper and volume was calculated using the following formula:

$$ {\text{V}} = {\text{length}} \times {\text{width}} \times {\text{height}} \times\uppi/6 $$
(1)

Mice that responded to the treatment with complete regression of the tumors were monitored for 100 days. After that, mice were re-challenged with the injection of the same type of tumor cells on the opposite flank and the outgrowth of tumors was checked.

2.5 Statistical Analysis

Statistical analysis and graphical representation was performed by GraphPad Prism software (GraphPad Holdings LLC, San Diego, CA, USA). Statistical evaluation was made by one-way-analysis of variance (One-way ANOVA). A P-value of less than 0.05 was considered statisticaly significant.

3 Results and Discussion

The anti-tumor effectiveness of calcium electroporation with regard to increasing concentrations of calcium solution was tested in two distinct solid murine tumor models, B16F10 melanoma and 4T1 breast carcinoma tumors. These two tumor models differ in many features, including histology, physiology, mutational burden and immunogenicity [17].

B16F10 tumor model is a highly metastatic and aggressive as demonstrated by the steep slope of growth curves and tumor doubling times (in Fig. 1, Table 1). It is known, that these tumors are poorly immunogenic with high mutational burden [17]. Whereas 4T1 tumor model has longer doubling time and shallow slope of growth curve, demosntrating its immunogenicity. In addition, this tumor model has low mutational burden and can spontaneously metastasize to multiple distant sites [18] (Fig. 2).

Fig. 1.
figure 1

Mean growth curves of B16F10 melanoma tumors (error bars represent standard error).

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Table 1. Doubling time in days of B16F10 melanoma tumors and 4T1 carcinoma tumors. Doubling times of tumors with complete regression were not included.
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Fig. 2.
figure 2

Mean growth curves for 4T1 carcinoma tumors (error bars represent standard error).

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In both tumor models injection of 50 mM and 168 mM calcium solution without electroporation has no visible effect on the tumor growth. In contrast, injection of 250 mM calcium solution prolonged tumor doubling times by at least factor of 2 in both tumor models compared to control untreated tumors. When combined with electric pulses, the tumor doubling time was further prolonged in both tumor models. Antitumor effectiveness was dose dependent in both tumor models, however in B16F10 the tumor doubling time was at the highest dose tested (250 mM calcium solution) only 6.2 days, demonstrating moderate effectiveness. On the other hand, 4T1 tumor model responded better to calcium electroporation as B16F10. Injection of 250 mM calcium solution alone to 4T1 tumors resulted in complete regression of 6/17 tumors (35% CR). The number of complete responses of tumors after 250 mM calcium electroporation was similar - 6/20 (30%) with no statistical difference between the two experimental groups. These mice were monitored for 100 days for the presence of possible tumor re-growth. After 100 days they were re-challenged with the same 4T1 cells injected into the opposite (left) flank and they all developed tumors, indicating that long term immune memory was not formed.

Morphologically, in both tumor models, the tumors that were treated either with electroporation, calcium alone or combination, developed swelling in the treated area which resolved in 2–3 days. Simultaneously, a visible tumor necrosis and formation of a crust developed (Fig. 3). Nevertheless, most tumors continued to grow from the tumor rim or beneath the crust. In 4T1 tumors that responded with complete regression, a good cosmetic effects were obtained, leaving only a small scar at the site of the tumor (Fig. 3B).

Fig. 3.
figure 3

Examples of morphological appearance of (A) B16F10 tumor 1 week after injection of calcium solution (left) or 1 week after calcium electroporation (right). (B) 4T1 tumor 1 week after calcium electroporation (left) and 2 weeks after the same treatment (right).

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4 Conclusions

In conclusion, our results demonstrated that calcium electroporation has dose dependent antitumor effectiveness. Furtheremore, it has tumor histotype specific efficiency. This needs to be further elucidated with regard to selection of therapeutic modality for a specific tumor type. Nevertheless, our results indicate that calcium electroporation is a safe method for treatment of the tumors from different histologies.