Optimal Voltage and Electrical Pulse Conditions for Electrical Ablation to Induce Immunogenic Cell Death (ICD)

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Abstract

Electrical ablation (EA) is a non-thermal ablation technique that causes less damage to the tissue surrounding the targeted area than conventional thermal ablation. EA induces immunogenic cell death (ICD) and can potentially synergize with cancer immunotherapy. In this study, the optimal voltage and pulse conditions for the induction of ICD were identified. Finite element analysis was used to estimate the ablation zone under a variety of voltage and pulse conditions. Cancer cells were cultured in vitro under two- and three-dimensional conditions to assess cell viability under different voltage and pulse conditions. Additionally, the expression of damage-associated molecular pattern (DAMP) markers of ICD was measured to identify an optimal voltage condition. Tumor volume, body weight, and survival after EA treatment were measured in vivo to identify the optimal pulse conditions. The optimal conditions to induce ICD by EA identified in this study are suitable to be combined with cancer immunotherapy.

Graphical abstract

We report that electrical ablation (EA) induces immunogenic cell death (ICD) and can potentially synergize with cancer immunotherapy. The optimized voltage and pulse conditions in this study will generate great synergy when used in combination with nanomedicine in future immunotherapy.

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Introduction

Electrical ablation (EA) is a treatment that induces tumor cell death electrical stimulation in a localized region of tissue [1]. Irreversible electroporation (IRE) is a type of EA that was introduced by Davalos et al. and approved by the US Food and Drug Administration (FDA) for the treatment of pancreatic ductal adenocarcinoma (PDAC) [2]. EA has been clinically applicated to treat tumors in the liver, prostate and pancreas [3]. EA, a non-thermal ablation modality, is preferred over other techniques, such as radiofrequency ablation (RFA), high-intensity focused ultrasound (HIFU), percutaneous laser ablation (PLA) because it causes less damage to the blood vessels and lymphatic tissues near the tumor [4], [5], [6]. Therefore, EA causes fewer side effects and can be used in combination with other therapeutic modalities.

Combinations of EA and chemotherapy or immunotherapy have been investigated [7], [8]. In a PDAC model, EA significantly improved the intra-tumoral delivery of gemcitabine [9]. Interestingly, the delivery of anti-cancer agents that are encapsulated into nanoparticles to tumor sites has also been improved by EA [10], [11]. These reports suggest that the effect of EA is multifaceted: EA causes the death of cancer cells not only directly but also indirectly by increasing the efficiency of drug delivery. EA combined with anti-programmed cell death protein 1 (anti-PD1) immune checkpoint blockade significantly prolonged survival in mice with orthotopic PDAC tumors harboring an oncogenic mutation in the Kirsten rat sarcoma (KRAS) gene [12]. EA combined with allogeneic natural killer (NK) cell immunotherapy significantly increased the median values for progression-free survival and overall survival in stage III in patients with unresectable pancreatic cancer [13]. Based on these promising results, the use of EA in combination with cancer immunotherapies to treat refractory cancers is expected to increase.

Elucidating the effects of EA on the immune system of patients is an active area of study. During EA treatment, not only tumor cells are killed, but tumor antigens and various immunogenic factors, such as damage-associated molecular pattern (DAMP) molecules that can induce an immune response are also released from the tumor cells [14], [15]. Three representative DAMP molecules are calreticulin (CRT) [16], high-mobility group box 1 protein (HMGB1) [17], and adenosine triphosphate (ATP) [18]. DAMPs are endogenous adjuvants that can increase tumor immunogenicity and improve a patient’s response to therapy [19]. Moreover, metastatic tumors in untreated regions have been eliminated by a systemic immune response elicited by immunogenic cell death (ICD) [20], [21], [22].

In this study, we identified the optimal voltage and pulse conditions for eliciting effective ICD in response to EA (Scheme 1). The ablation zone induced by EA under a variety of voltage and pulse conditions was simulated using the finite element method (FEM). The cytotoxicity induced by electroporation was measured in two- and three-dimensional (2D and 3D) in vitro cell culture models, and the expressions of DAMP molecules CRT, HMGB1, and ATP were measured to identify conditions that optimally induced ICD in vitro. In vivo models of small and large tumors were then used to assess tumor growth, body weight, and survival over time in response to EA treatment.

Section snippets

FEM simulation

We simulated the ablation zone generated in response to a variety of EA treatment conditions with COMSOL Multiphysics 5.4. Ablation occurs where the applied electrical energy density (w) exceeds a critical value (wc) [23]. The relationship between the electrical field (E) and the electrical energy density is described by Eq. (1) [23]. Therefore, we calculated the critical electrical field (Ec), which is related to the wc, by using Eq. (2).w=E2nΔtσEc=wcnΔtσwhere n is the number of pulses

FEM simulation

We performed FEM simulations to identify a set of optimal EA conditions. The area of an ablation zone was defined as the area in which the electrical potential exceeds the critical value. Values were determined for two tissue diameters (3 and 6 mm) using a range of voltages and 8, 40, or 80 pulses (Fig. 1a, c). The ablation zone area increases with the number of pulses and applied voltage. The ablation zone is saturated at 500 V for 3-mm-diameter tissue samples, whereas it is saturated at

Conclusions

Cancer immunotherapy has become a cancer treatment paradigm. EA is a non-thermal ablation technique that is expected to be synergistic in combination with cancer immunotherapy. In this study, we investigated the electrical conditions under which EA induces ICD. The ablation zone generated by various voltage levels and numbers of EA pulses was assessed by FEM simulation. Cell viability after EA treatment was measured using LLC cells cultured under 2D and 3D conditions. We evaluated the

Conflict of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the National Research Foundation (NRF) of Korea. The grant was funded by the Ministry of Science and ICT (MSIT), Republic of Korea (2018R1C1B6001120, 2019R1A6A1A03032888, 2020M2D9A3094208, and 2020R1A2B5B03002344).

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