Multicellular Tumour Spheroids Exposure to Pulsed Electric Field: A Combined Experimental and Mathematical Modelling Study Highlighting Temporal Dynamics of DAMP Release and Accelerated Regrowth at Intermediate Field Intensities

TL;DR

How spheroid fate depends on pulse strength is demonstrated and the importance of accounting for quiescent cells when designing electroporation-based therapies is emphasized, demonstrating how spheroid fate depends on pulse strength.

Abstract

Electroporation is increasingly used as a percutaneous ablation technique for tumours located near vital structures. Although effective, tumour regrowth may still occur. At the same time, in vitro studies on cell monolayers have shown that electroporation can trigger immunogenic cell death (ICD) through the release of damage-associated molecular patterns (DAMPs). These molecules can stimulate the immune system and could counteract tumour regrowth. To fully exploit electroporation, two key questions must be addressed: (1) what dynamics drive tumour regrowth, and (2) how ICD unfolds in space and time within three-dimensional cellular structures, which better mimic in vivo conditions than 2D cultures. Here, we combine in vitro experiments with a hybrid individual-based/continuous computational model to explore tumour spheroid regrowth and ICD potential under different pulse intensities. Experiments quantify spheroid viability, growth rate, and the release of ATP and HMGB1. In parallel, the hybrid model predicts the dynamics of proliferative, quiescent, and necrotic cells. Both approaches show that cell death and DAMP release scale with pulse intensity. The model, validated against experimental data, further highlights the dual role of quiescent cells: some die and free space and resources, while others survive and resume proliferation. Together, these findings demonstrate how spheroid fate depends on pulse strength and emphasize the importance of accounting for quiescent cells when designing electroporation-based therapies.

Figures (17)

• Figure 1: Analysis of GFP-expressing Hepa 1-6 spheroids growth after electroporation treatment (A) Representative fluorescence micrographs of spheroids at Day 0 (D0), Day 2 (D2) and Day 4 (D4) after treatment at varying electric field intensities. GFP channel is displayed in green. The yellow outlines correspond to viable (GFP) zone boundaries used for further analysis. Scale bar is 300 µ m. (B) Spheroid viability over time based on GFP-positive areas. The values obtained at Day 0 correspond to the first acquisition after treatment. (C) Spheroid growth speed based on viability curves from Day –2 to Day 1 (white bar) or after treatment with from Day 0 onwards, as per applied protocol (0, 500, 1500 or 2500 V/cm). Statistical significance was assessed using one-way ANOVA followed by a Dunnett’s multiple comparison test, in comparison to the 0 V/cm condition. *** $p < 0.001$. Data are shown as the mean ± SEM from three independent experiments (N=3), each including at least 6 spheroids per condition ($n\geq 6$).
• Figure 2: Spheroid cell death after pulse treatment. (A) General assessment of cell death irrespective of the death pathway: representative bright field and fluorescence micrographs of spheroids one day after treatment. Dead cells internalized propidium iodide and are represented in red. Scale bar is 200 µ m. (B) Cell death in spheroids one day after treatment, based on mean propidium iodide staining inside the spheroids. Statistical significance was assessed using an one-way ANOVA followed by a Bonferroni’s multiple comparison test, in comparison to the 0 V/cm condition. *** $p < 0.001$. (C) Representative brightfield and fluorescence micrographs of spheroids 0, 3 and 6 hours after treatment. Caspase 3/7 staining is in yellow. Scale bar is 300 µ m. (D) Apoptosis induction based on caspase 3/7 activation, relative to the 0 V/cm condition. Statistical significance was assessed using a two-way ANOVA followed by a Bonferroni multiple comparison test, in comparison to the 0 V/cm condition. * $p < 0.05$, **$p < 0.001$, **** $p < 0.0001$. Data are shown as the means $\pm$ SEM from three independent experiments, each including 6 spheroids per condition.
• Figure 3: DAMP release following electroporation treatment. (A) ATP level in supernatant measured 10 minutes after pulse treatment, or after lysis of pools of 3 spheroids (relative to ATP level at 0 V/cm). ATP release luminescence was normalized on luminescence from the controls of each experiment. Statistical significance was assessed using an one-way ANOVA followed by a Dunnett’s multiple comparison test, in comparison to the 0 V/cm condition. * $p < 0.05$, *** $p < 0.001$. Data are shown as the mean ± SEM from three independent experiments (N=3) each condition consisting of three pools of three spheroids, including 9 spheroids per experiment. (B) HMGB1 level in supernatant measured 3 and 6 hours after pulse treatment of a pool of 27 spheroids (relative to HMGB1 level at 2500 V/cm). Statistical significance was assessed using an one-way ANOVA followed by a Dunnett’s multiple comparison test, in comparison to the 2500 V/cm condition of the same timeframe. ** $p < 0.01$, *** $p < 0.001$, **** $p < 0.0001$. Data are shown as the mean ± SEM from three independent experiments (N=3), each condition consisting of a pool of 27 spheroids.
• Figure 4: Schematic summary of the in vitro experiments. In control (untreated) spheroids, a proliferation gradient was observed over time, characterized by an increasing size of spheroids and the formation of non-viable core in the center. On day 0, treated spheroids underwent pulse application at varying intensities. As a summary of the observations of our experimental study, the dynamics of the control and 500 V/cm, as well as 1500 V/cm and 2500 V/cm treated spheroids are schematized. At 500 V/cm the effects were comparable to the control (0 V/cm), while 1500 V/cm pulse intensity induces an almost complete spheroid ablation, with the survival of residual cells leading to increased spheroid regrowth at later timepoints, in comparison to the control. 2500 V/cm pulse intensity leads to a complete loss of viability in the large majority of treated spheroids. The amount and timepoints of ATP and HMGB1 release vary depending on pulsing conditions.
• Figure 5: Control scenario: tumour growth without electroporation effects. (A) Time evolution of the proliferative, quiescent and necrotic cell number, as well as the time evolution of the spheroid area in control conditions. These results correspond to the average and $+/-$ standard deviation over 3 simulations. The black markers highlight average (scatter points) and standard deviation (error bars) of the experimental data that are used to carry out model calibration. (B) Example of the spatial distribution of proliferative, quiescent and necrotic cells at different times of the simulation. Black (resp white) = 100 (resp. 0) % of proliferative/quiescent/necrotic cells.