Electrolytic ablation enables cancer cell targeting through pH modulation

Abstract

Minimally invasive ablation strategies enable locoregional treatment of tumors. One such strategy, electrolytic ablation, functions through the local delivery of direct current without thermal effects, facilitating enhanced precision. However, the clinical application of electrolytic ablation is limited by an incompletely characterized mechanism of action. Here we show that acid and base production at the electrodes precipitates local pH changes causing the rapid cell death that underlies macroscopic tumor necrosis at pH > 10.6 or < 4.8. The extent of cell death can be modulated by altering the local buffering capacity and antioxidant availability. These data demonstrate that electrolytic ablation is distinguished from other ablation strategies via its ability to induce cellular necrosis by directly altering the tumor microenvironment. These findings may enable further development of electrolytic ablation as a curative therapy for primary, early stage tumors.

Fig. 1

Model system: HCC cells embedded in low melting temperature agarose with viability assessment using a dual reporter assay. a The cell-encapsulation matrix was prepared as a 1:1 mixture of Huh-7 HCC cells and 3% low melting temperature agarose. b Staining with a pair of fluorescent viability reporters demonstrates that the encapsulation procedure does not impair cell viability (green fluorescence). Scale bar = 5 mm. c Boiling of the preparation resulted in complete cell death (red fluorescence (recolored to appear magenta) in the absence of green fluorescence). Scale bar = 5 mm

Fig. 2

Use of a cell encapsulation assay enables the spatial resolution of electrolytic ablation-induced changes in temperature, transmembrane potential, and pH. a Electrolytic ablation was performed by the application of direct current between a nitinol cathode (left) and platinum anode (right) held in place by a 3D-printed spacer sitting atop the cell encapsulation matrix that was cast in a 6 cm tissue culture dish. b Electrolytic ablation performed in this assay led to the observation of cell death in the regions surrounding the cathode (Ca) and anode (An). Scale bar = 5 mm. c, d 10× magnification brightfield image with and without fluorescence reporter overlay at an increased cell density of 3 × 10⁶ cells mL⁻¹ highlights changes at the border of the ablation zone. Scale bar = 400 μm. e A custom insert was 3D-printed to sit atop the encapsulation assay, which was prepared in a 6 cm dish. The insert holds the electrodes at a spacing of 1.5 cm and enables precise measurements of temperature, pH, and voltage potential (relative to the midpoint between the two electrodes). f Temperature measurement sites (circles). g pH and voltage potential measurement sites (triangles)

Fig. 3

Measurement of voltage and pH in the encapsulation assay suggests the generation of acid and base as the mechanism of death following electrolytic ablation. a 3D surface plot of 2D linear interpolation of voltage potential measurements. b 2D linear interpolation contour plot of transmembrane potential calculated from voltage potential measurements via linear interpolation of the gradient magnitude across the width of an HCC cell. None of the regions of the assay reached the threshold voltage of 1 V necessary for electroporation. c 3D surface plot of 2D linear interpolation of pH measurements. d 2D linear interpolation contour plot of pH measurements recorded surrounding the cathode and anode, revealing basic changes surrounding the cathode [Ca] and acidic changes surrounding the anode [An]

Fig. 4

Modification of the buffering capacity confirms a pH-dependent mechanism of cell death in electrolytic ablation. a, d, g Viability images in the region surrounding the cathode and anode following electrolytic ablation with HEPES concentrations of 10, 50, and 200 mM. Scale bar = 5 mm. b, e, h pH contour maps in the region surrounding the cathode and anode following electrolytic ablation with HEPES concentrations of 10, 50, and 200 mM. c, f, i pH contour maps overlaid upon viability images in the region surrounding the cathode and anode following electrolytic ablation with HEPES concentrations of 10, 50, and 200 mM, respectively. Scale bar = 5 mm. j Comparison of the total area of cell death in the three conditions reveals a decreasing area of cell death with increased assay buffering capacity (n = 4 for all tests; [ANOVA] F: 75.96 on 2 and 9 DF, p < 1 × 10⁻⁵; [10 mM v 50 mM] t: 4.39 on 4.59 DF, Bonferroni adjusted p < .05; [10 mM v 200 mM] t: 11.36 on 3.10 DF, Bonferroni adjusted p < .01; [50 mM v 200 mM] t: 11.75 on 3.34 DF, Bonferroni adjusted p < .01). k Comparison of total charge deposition in the three conditions reveals a decreasing quantity of charge deposition with increased buffering capacity (n = 3 for all tests; [ANOVA] F: 72.38 on 2 and 6 DF, p < 1 × 10⁻⁴; [10 mM v 50 mM] t: −0.25 on 3.25 DF, Bonferroni adjusted p: 1.0; [10 mM v 200 mM] t: 9.36 on 3.48 DF, Bonferroni adjusted p < 0.01; [50 mM v 200 mM] t: 12.95 on 3.95 DF, Bonferroni adjusted p < 0.001)

Fig. 5

By permitting diffusion or using multiple cathodes, precise ablation geometries may be achieved with electrolytic ablation. a An increased area of cell death was observed after allowing 60 min of diffusion following electrolytic ablation (dashed white lines indicate ablation margin immediately after treatment; t: 7.32 on 3.4 DF, p < 0.01). Scale bar = 5 mm. b A multi-cathode design allows the prescription of the volume in which cell death occurs as demonstrated by performing electrolytic ablation at 10 V for 10 s resulting in cell death surrounding the nine electrodes. Scale bar = 5 mm