A Comprehensive Review of Calcium Electroporation -A Novel Cancer Treatment Modality

Abstract

Calcium electroporation is a potential novel anti-cancer treatment where high calcium concentrations are introduced into cells by electroporation, a method where short, high voltage pulses induce transient permeabilisation of the plasma membrane allowing passage of molecules into the cytosol. Calcium is a tightly regulated, ubiquitous second messenger involved in many cellular processes including cell death. Electroporation increases calcium uptake leading to acute and severe ATP depletion associated with cancer cell death. This comprehensive review describes published data about calcium electroporation applied in vitro, in vivo, and clinically from the first publication in 2012. Calcium electroporation has been shown to be a safe and efficient anti-cancer treatment in clinical studies with cutaneous metastases and recurrent head and neck cancer. Normal cells have been shown to be less affected by calcium electroporation than cancer cells and this difference might be partly induced by differences in membrane repair, expression of calcium transporters, and cellular structural changes. Interestingly, both clinical data and preclinical studies have indicated a systemic immune response induced by calcium electroporation. New cancer treatments are needed, and calcium electroporation represents an inexpensive and efficient treatment with few side effects, that could potentially be used worldwide and for different tumor types.

Keywords: calcium electroporation; clinical trial; in vitro; in vivo; veterinary study.

Figure 1

Calcium electroporation. (A) Calcium is injected in the tumor causing a high extracellular calcium concentration. (B) Immediately after the injection, the tumor is electroporated using an electrode (e.g. needle electrode) causing transient permeabilisation of the cell membrane allowing passage of calcium into the cell (C) causing cancer cell death (D).

Figure 2

Cellular calcium homeostasis. Calcium is tightly regulated to maintain the low intracellular calcium concentration. (1) Calcium can enter the cell through calcium channels. (2) Inside the cells calcium is chelated by proteins. (3) Mitochondria and endoplasmic reticulum store calcium where transport is facilitated by transporters including the sarco-endoplasmic reticulum calcium ATPase (SERCA). (4) Calcium is extruded from the cell by the ATP dependent plasma membrane calcium ATPase (PMCA) and the sodium calcium exchanger (NCX) and the sodium calcium potassium exchanger (NCKX). (5) Calcium electroporation induces high intracellular concentrations of calcium by permeabilisation of the plasma membrane in the presence of high extracellular calcium concentrations.

Figure 3

Normal and cancer cell response to calcium electroporation. Calcium electroporation induces different response in normal cell spheroids and cancer cell spheroids where cancer cell spheroids decrease in size while no change in size is seen in the normal cell spheroids. However, intracellular ATP level is depleted in both normal and cancer cell spheroids for up to 72 hours, which the normal cells are able to survive. Adapted from Frandsen, Gibot et al., 2015 [85].

Figure 4

Proposed mechanism of action published in 2012. Calcium electroporation of cells induces influx of calcium and sodium and loss of ATP and potassium due to the concentration gradients (1). This may lead to increased ATP consumption (2) by the calcium ATPase and sodium potassium ATPase trying to re-establish the calcium homeostasis as well as loss of ATP production (3) since increased intracellular calcium can induce the opening of permeability transition pores (PTP) in the mitochondria membrane dissipating the proton gradient. This leads to ATP depletion which has been observed after calcium electroporation as well as necrotic cell death. Other cellular effects (4) such as increased activity of lipases and proteases as well as increased concentration of reactive oxygen species (ROS) may also be involved. From Frandsen et al., 2012 [18].

Figure 5

Systemic immune response after calcium electroporation. A patient with cutaneous metastases from malignant melanoma was treated with electrochemotherapy using intravenous bleomycin. Four months after treatment of some of the multiple metastases, new metastases appeared. Two of these were treated (A), one with calcium electroporation and one with electrochemotherapy using intratumoral bleomycin, both with complete response within 6 months. Nine months after this treatment complete levelling of all cutaneous metastases appeared (B) as well as no or limited signs of previously enlarged lymph nodes on PET/CT scan (C,D). Adapted from Falk et al., 2018 [19].