Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a A schematic of the setup that enables a fluorescence imaging of electroporated cell. Tungsten electrodes were positioned on the glass coverslip with seeded normal or cancer urothelial cells on the stable 45° angle between coverslip and electrodes. An 800 × 600 µm region of interest (ROI) was positioned such that its longer edge overlapped the line connecting the tips of the two electrodes. The ROI extended beyond the electrode gap on the side opposite the electrodes. ROI was divided into three equal subregions (subROI), each measuring 800 × 200 µm, by drawing two parallel lines, evenly spaced along the shorter axis of the ROI. b Calculated electric field distribution along the line perpendicular to the axis connecting the centers of both electrodes with 1 V applied between them. The ROI were placed in the electric field marked in ( c ). The time course of YP fluorescence measure as arbitrary units (a.u.) (mean ± the standard error of the mean (SEM)) in T24, UM-UC-3, SV-HUC-1 and HBLAK cells after electroporation by train of 200, 300-ns, 11.5 kV/cm at 10 Hz pulse in the solution with 1 µM YP and at different Ca 2+ concentration (marked in the titles) n = 18–20. The mean fluorescence was fitted with single-exponential curve (coefficients of determination (R²) > 0.95 for all tested conditions and cell lines). The comparison of area under curve (AUC) of YP fluoresce curves show highly significant differences between normal and cancer urothelial cells. Differences were assessed using one-way Welch’s ANOVA W (DFn; DFd). For 0 mM Ca²⁺: W(3, 36.07) = 56.15; for 2 mM Ca²⁺: W(3, 36.13) = 93.29; and for 5 mM Ca²⁺: W(3, 37.59) = 72.11. Dunnett’s T3 post hoc test was used to correct for multiple comparisons. Statistical significance is indicated as follows: ( p ≥ 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. d Violin plot showing the distribution of mean YP florescence intensity at 180 seconds (s) after the exposure in 0 mM, 2 mM and 5 mM concentration of Ca 2+ n = 18–20. Differences were assessed using one-way Welch’s ANOVA followed by Dunnett’s T3 post hoc test for multiple comparisons: for 0 mM Ca²⁺: W(3, 35.06) = 16.22; for 2 mM Ca²⁺: W(3, 35.62) = 31.60; and for 5 mM Ca²⁺: W(3, 37.66) = 13.98.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Imaging, Electroporation, Concentration Assay, Comparison

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Schematic representation of membrane resealing dynamics, visualized through changes in YP fluorescence. (1). Prior to electroporation, the cell membrane remains intact, preventing the uptake of the membrane-impermeant dye YP. (2) Following electroporation, transient nanopores form in the plasma membrane, enabling the entry of YP and water from the extracellular environment into the cell. The kinetics of dye uptake can be fitted with a single-phase exponential curve, reflecting the dynamics of membrane permeability. (3) As the membrane reseals, dye uptake ceases and the fluorescence signal reaches a plateau. This plateau indicates successful membrane recovery, with no further entry of YP. b Time course of YP dye uptake in urothelial cancer cells and normal urothelial cells under three different extracellular Ca²⁺ concentrations: 0, 2, and 5 mM. The uptake dynamics were fitted with a single-phase exponential model, and the time constants τ (in s) are indicated next to each curve. The fits showed a strong correlation, with R² exceeding 0.95 for all cell lines under all tested conditions, indicating robust exponential uptake kinetics. Data are presented as mean ± SEM, with n = 18–20 per condition. c Average time constants derived from the exponential fits, shown as mean ± SEM ( n = 18–20). Points represent individual measurements from independent experimental replicates. Differences between different Ca 2+ concentration for each cell line and electric field intensity were assessed using one-way Welch’s ANOVA for followed by Dunnett’s T3 post hoc test for multiple comparisons. Welch’s ANOVA results (W (DFn, DFd)) for comparisons across Ca²⁺ concentrations at 11.5 kV/cm, 10 kV/cm, and 7.5 kV/cm were as follows: For HBLAK: W(2.000, 35.04) = 1.125; W(2.000, 32.86) = 0.5627; W(2.000, 33.46) = 0.6787. For SV-HUC-1: W(2.000, 32.85) = 0.5254; W(2.000, 32.32) = 1.099; W(2.000, 33.62) = 1.085. For T24: W(2.000, 37.64) = 6.939; W(2.000, 36.92) = 14.82; W(2.000, 28.14) = 22.13. For UM-UC-3: W(2.000, 36.72) = 6.800; W(2.000, 36.92) = 14.82; W(2.000, 37.80) = 2.404.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Membrane, Fluorescence, Electroporation, Clinical Proteomics, Permeability, Derivative Assay, Concentration Assay

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Representative images of monolayers of HBLAK, SV-HUC-1, UM-UC-3, and T24 cells stained with Phalloidin and Hoechst 33342 dyes to visualize actin filaments (red) and nuclei (blue), respectively. b Morphological features of the cells area measurements were obtained from 18 randomly selected ROIs (each 250 µm × 250 µm) containing approximately 40–80 stained cells. The average of measured morphological feature of cells within each ROI was treated as an independent data point for statistical analysis, resulting in n = 18. Data were visualized using violin plots, and statistical comparisons between cell lines were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc testing. Welch’s ANOVA revealed statistically significant differences between cell lines in average cell area (W(3, 37.28) = 46.01), major axis length (W(3, 37.46) = 38.88), and minor axis length (W(3, 37.32) = 49.28). No significant differences were observed in the form factor (W(3, 36.34) = 0.98). c Plot of the electroporation threshold electric field as a function of pulse duration for cells of varying radii. The external electric field (E, in V/m) necessary to reach electroporation threshold was calculated for the cathode-facing pole of a spherical cell, assuming a membrane charging time constant (τ) of 2 µs. See main text for methodological details. d Representative images and violin plots showing the average cell area and form factor of single cells across 9 tissue microarrays (TMAs), with each point representing the mean value per TMA ( n = 9). The dataset includes 47 normal urothelial tissue samples, 66 samples from primary urothelial carcinomas, and 56 samples from lymph node metastases. Statistical comparison of mean urothelial cell area across normal urothelial tissue, urothelial carcinoma, and lymph node metastasis was performed using weighted one-way ANOVA, with the number of tissue sections per array used as weights and the Tukey adjustment for multiple comparisons. Pairwise comparisons using weighted marginal means revealed significantly larger mean cell areas in normal urothelial tissue compared to both cancer and lymph node tissue (F(2, 24) = 13.42) as well as lower form factor of normal urothelial cells, (F(2, 24) = 6.39).

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Staining, Electroporation, Membrane, Comparison

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Spheroids and PDOs were positioned between two stainless steel electrodes. YP uptake was monitored via fluorescence imaging for 10 min post-exposure to nsPEFs. Fluorescence images were pseudocolored to enhance visualization. b Representative pseudocolored images of spheroids and PDOs before and after nsPEFs exposure. c Time-course analysis of YP fluorescence uptake in spheroids derived from normal urothelial cells and urothelial cancer cells, under different extracellular Ca²⁺ concentrations. The spheroids were observed for 10 min following exposure. Data were shown as mean ± SEM ( n = 9–11). Statistical comparisons of the AUC of fluorescence between normal urothelial SV-HUC-1 spheroids and cancer RT4 spheroids with PDO were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons: for 0 mM Ca²⁺, W(4, 28.14) = 28.34, for 2 mM Ca²⁺, W(4, 20.46) = 38.53 and for 5 mM Ca²⁺, W(4, 18.09) = 53.60. d Quantification and correlation of single-cell size in PDOs and YP fluorescence after exposure. The mean cell size was calculated based on H&E-stained slides of spheroids. Statistical comparisons of single cell area between spheroids and PDOs were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test for multiple comparisons (W(4, 787.4) = 10.74). The correlation between mean single-cell size in spheroids and YO-PRO-1 fluorescence intensity 10 min after nsPEF exposure in 2 mM Ca²⁺ was assessed by linear regression analysis. The analysis included data from 45 spheroids and PDOs derived from RT4, SV-HUC-1, PDO 154, PDO 270, and PDO 319. Each dot represents a single spheroid ( n = 45).

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Fluorescence, Imaging, Derivative Assay, Staining

Journal: Communications Biology

Article Title: Nanosecond pulsed electric fields induce cell-size-dependent selective permeabilization of urothelial cancer cells

doi: 10.1038/s42003-025-09432-7

Figure Lengend Snippet: a Changes in the 2D projected area of spheroids and PDOs (mean ± SEM, n = 9–11) following nsPEFs exposure in solutions containing 0 mM, 2 mM, or 5 mM Ca²⁺. Statistical comparisons of the area under the curve (AUC) for 2D projected area changes over time were performed using Welch’s ANOVA, followed by Dunnett’s T3 post hoc test to compare normal urothelial SV-HUC-1 spheroids with cancer RT4 spheroids and PDOs in different Ca 2+ concentrations: for 0 mM Ca²⁺, W(4, 21.73) = 835.0; for 2 mM Ca²⁺, W(4, 19.38) = 485.5; and for 5 mM Ca²⁺, W(4, 19.46) = 326.2. b Mechanical properties of spheroids of normal SV-HUC-1 and cancer RT4 cells were evaluated using AFM force mapping, conducted at 25 points (5 × 5 grid) across the spheroid surface at defined time intervals post-exposure. Young’s modulus values were calculated from force–distance curves by fitting them to the Hertz model. Values obtained from a single spheroid were averaged to yield one representative value per spheroid. c Comparison of Young’s modulus (“stiffness”) between SV-HUC-1 and RT4 spheroids in 0 mM and 2 mM Ca²⁺ solutions, both before and 10 minutes after exposure to nsPEFs ( n = 9–11). Data are presented as mean ± SEM. Statistical comparisons were performed using two-way ANOVA with Geisser–Greenhouse correction for unequal variability: Before exposure: effect of Ca²⁺, F(1, 32) = 3.315; effect of cell line, F(1, 32) = 5.963. After exposure: effect of Ca²⁺, F(1, 34) = 0.104; effect of cell line, F(1, 34) = 23.24. d Young’s modulus values were normalized to the pre-exposure baseline and the mean values were fitted using a single-exponential nonlinear regression model (mean ± SEM, n = 9–11): R² for the exponential fits were 0.85 and 0.99 for SV-HUC-1 spheroids, and 0.95 and 0.94 for RT4 spheroids under 0 mM and 2 mM Ca²⁺ conditions, respectively. Points represent individual measurements from independent experimental replicates. For statistical comparisons at defined time points, repeated measures two-way ANOVA with Geisser–Greenhouse correction was applied, followed by Šidák’s multiple comparisons test. In 0 mM Ca²⁺: effect of time, F(2.235, 33.53) = 48.26; effect of cell type, F(1, 15) = 15.22. In 2 mM Ca²⁺: effect of time, F(1.665, 26.65) = 55.19; effect of cell type, F(1, 16) = 3.252.

Article Snippet: T24, UM-UC-3, RT4 and SV-HUC-1 cell line were acquired from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Comparison