Have a personal or library account? Click to login
Invasive properties of patient-derived glioblastoma cells after reversible electroporation in vitro Cover

Invasive properties of patient-derived glioblastoma cells after reversible electroporation in vitro

Radiology and Oncology
Volume 59 (2025): Issue 4 (December 2025)
By: Anja Blazic,  Bernarda Majc,  Metka Novak,  Barbara Breznik and  Lea Rems  
Open Access
|Dec 2025

Figures & Tables

FIGURE 1.

Overview of the experimental workflow for evaluating patient-derived glioblastoma (GB) cell behaviour before and after electroporation. Created with BioRender.com. (A) Five patient-derived glioblastoma cell lines, including cells from the tumour core (CORE), infiltrative rim (RIM), and a recurrent lesion (REC), were initially screened using a transwell invasion assay. Cells were plated on Matrigel-coated inserts and incubated for 24 hours. Invading cells migrating to the lower surface of the insert membrane were fixed, permeabilized, and stained with Hoechst (nuclei) and then immunostained for Ki-67 (a proliferation marker). The cells were subsequently imaged to quantify the number of invading and proliferating cells. (B) NIB140 CORE and NIB216 CORE were selected for further experiments with electroporation based on their invasive behaviour. Electric pulses of increasing electric field strength were applied to cells in electroporation cuvettes and the resulting membrane permeabilization and survival were quantified to generate characteristic response curves. Additionally, we assessed the metabolic activity of cells using MTS. Post-treatment invasion assay and fluorescence imaging was used to assess changes in invasive potential, with image analysis performed in ImageJ Fiji to quantify total and proliferating cell numbers based on nuclear segmentation and Ki-67 expression.
Overview of the experimental workflow for evaluating patient-derived glioblastoma (GB) cell behaviour before and after electroporation. Created with BioRender.com. (A) Five patient-derived glioblastoma cell lines, including cells from the tumour core (CORE), infiltrative rim (RIM), and a recurrent lesion (REC), were initially screened using a transwell invasion assay. Cells were plated on Matrigel-coated inserts and incubated for 24 hours. Invading cells migrating to the lower surface of the insert membrane were fixed, permeabilized, and stained with Hoechst (nuclei) and then immunostained for Ki-67 (a proliferation marker). The cells were subsequently imaged to quantify the number of invading and proliferating cells. (B) NIB140 CORE and NIB216 CORE were selected for further experiments with electroporation based on their invasive behaviour. Electric pulses of increasing electric field strength were applied to cells in electroporation cuvettes and the resulting membrane permeabilization and survival were quantified to generate characteristic response curves. Additionally, we assessed the metabolic activity of cells using MTS. Post-treatment invasion assay and fluorescence imaging was used to assess changes in invasive potential, with image analysis performed in ImageJ Fiji to quantify total and proliferating cell numbers based on nuclear segmentation and Ki-67 expression.

FIGURE 2.

Patient-derived glioblastoma (GB) cell lines display variable intrinsic invasive potential. (A) Transwell invasion assay was performed with non-treated cell lines to assess the intrinsic invasive potential of five GB cell lines derived from different tumour regions. NIB140 CORE showed the highest number of invading cells, followed by NIB216 CORE, whereas NIB220 RIM, NIB237 CORE, and NIB261 REC displayed significantly lower invasion. Statistical analysis was performed using ANOVA on ranks. Significant differences are indicated with asterisks (*); p < 0.05. The number of Ki-67 positive (proliferating) cells, shown in black at the base of each bar, was low in all tested cell lines (< 10 %). Data are presented as mean ± SD from at least 4-5 independent experiments. (B) Doubling times were determined based on cell growth curves plotted as log2(N/N0) versus time, where N0 is the number of seeded cells at time 0 h, and N is the number of cells at selected time points (hours). Linear regression was applied to each cell line (R2 values shown), and doubling time was calculated from the slope of the fitted line. NIB140 CORE and NIB216 CORE showed similar doubling time (40–41 h).
Patient-derived glioblastoma (GB) cell lines display variable intrinsic invasive potential. (A) Transwell invasion assay was performed with non-treated cell lines to assess the intrinsic invasive potential of five GB cell lines derived from different tumour regions. NIB140 CORE showed the highest number of invading cells, followed by NIB216 CORE, whereas NIB220 RIM, NIB237 CORE, and NIB261 REC displayed significantly lower invasion. Statistical analysis was performed using ANOVA on ranks. Significant differences are indicated with asterisks (*); p < 0.05. The number of Ki-67 positive (proliferating) cells, shown in black at the base of each bar, was low in all tested cell lines (< 10 %). Data are presented as mean ± SD from at least 4-5 independent experiments. (B) Doubling times were determined based on cell growth curves plotted as log2(N/N0) versus time, where N0 is the number of seeded cells at time 0 h, and N is the number of cells at selected time points (hours). Linear regression was applied to each cell line (R2 values shown), and doubling time was calculated from the slope of the fitted line. NIB140 CORE and NIB216 CORE showed similar doubling time (40–41 h).

FIGURE 3.

Permeabilization and survival of NIB140 CORE and NIB216 CORE glioblastoma (GB) cell lines in response to H-FIRE pulses resulting in different electric field strengths. (A) The percentage of permeabilized cells was assessed by propidium iodide (PI) uptake 3 minutes after pulse delivery (presented as •). The percentage of viable cells was assessed by PI assay 24 hours after pulse delivery (presented as ▴). (B) Cell survival was assessed by metabolic MTS assay 24 hours after pulse delivery. Data are presented as mean ± SD from at least three independent experiments. Solid lines are least-square fits to sigmoid curves. Statistically significant differences (p < 0.05) between cell lines at specific electric field strengths were tested using Student’s t-test and are indicated by asterisks (*). Data for NIB140 CORE and NIB216 CORE are shown in blue and pink, respectively.
Permeabilization and survival of NIB140 CORE and NIB216 CORE glioblastoma (GB) cell lines in response to H-FIRE pulses resulting in different electric field strengths. (A) The percentage of permeabilized cells was assessed by propidium iodide (PI) uptake 3 minutes after pulse delivery (presented as •). The percentage of viable cells was assessed by PI assay 24 hours after pulse delivery (presented as ▴). (B) Cell survival was assessed by metabolic MTS assay 24 hours after pulse delivery. Data are presented as mean ± SD from at least three independent experiments. Solid lines are least-square fits to sigmoid curves. Statistically significant differences (p < 0.05) between cell lines at specific electric field strengths were tested using Student’s t-test and are indicated by asterisks (*). Data for NIB140 CORE and NIB216 CORE are shown in blue and pink, respectively.

FIGURE 4.

Electroporation enhances the invasion potential of patient-derived glioblastoma (GB) cell lines in a cell type-dependent manner. Invasion was assessed 24 hours after electroporation using H-FIRE pulses resulting in electric field strength of 1 kV/cm. (A-B) Box-and-whisker plots showing the number of invading cells in NIB140 CORE (A) and NIB216 CORE (B) in sham-treated (grey) and electroporated samples (blue or pink). Each group represents a separate biological replicate (REP1–REP3), with 2–3 technical replicates per biological replicate. The horizontal line within each box represents the median, and whiskers indicate the full range of values. (C) Relative increase in the number of invading cells in electroporated samples compared to sham controls. Data are presented as mean ± SD from three biological replicates. (D) Percentage of Ki-67–positive (proliferating) cells in sham-treated and electroporated samples. Values remained below 10 % across all conditions, demonstrating that the observed increase in invasion was not due to increased proliferation. (E) Representative masks obtained after thresholding images of Hoechst-stained NIB140 CORE invading cells, showing increased invasion following electroporation.
Electroporation enhances the invasion potential of patient-derived glioblastoma (GB) cell lines in a cell type-dependent manner. Invasion was assessed 24 hours after electroporation using H-FIRE pulses resulting in electric field strength of 1 kV/cm. (A-B) Box-and-whisker plots showing the number of invading cells in NIB140 CORE (A) and NIB216 CORE (B) in sham-treated (grey) and electroporated samples (blue or pink). Each group represents a separate biological replicate (REP1–REP3), with 2–3 technical replicates per biological replicate. The horizontal line within each box represents the median, and whiskers indicate the full range of values. (C) Relative increase in the number of invading cells in electroporated samples compared to sham controls. Data are presented as mean ± SD from three biological replicates. (D) Percentage of Ki-67–positive (proliferating) cells in sham-treated and electroporated samples. Values remained below 10 % across all conditions, demonstrating that the observed increase in invasion was not due to increased proliferation. (E) Representative masks obtained after thresholding images of Hoechst-stained NIB140 CORE invading cells, showing increased invasion following electroporation.

FIGURE 5.

Transcriptomic differences between electroporated and shamtreated NIB140 CORE and NIB216 CORE cell lines. RNA transcriptome analysis was performed in cells harvested 24 hours after electroporation. (A) The gene expression levels analysis is presented through co-expression Venn diagrams showing the overlap in expressed genes between sham-treated (CTRL, 0 V) and electroporated (EP, 1 kV/cm) samples of each cell line, and between shamtreated NIB140 CORE and NIB216 CORE. (B) The differential gene expression analysis is presented through volcano plots. Red and green points represent significantly upregulated and downregulated genes, respectively (p < 0.05), while blue points indicate non-significant changes. Genes were classified as differentially expressed, if they met the threshold of |log2FoldChange| > 0.0.
Transcriptomic differences between electroporated and shamtreated NIB140 CORE and NIB216 CORE cell lines. RNA transcriptome analysis was performed in cells harvested 24 hours after electroporation. (A) The gene expression levels analysis is presented through co-expression Venn diagrams showing the overlap in expressed genes between sham-treated (CTRL, 0 V) and electroporated (EP, 1 kV/cm) samples of each cell line, and between shamtreated NIB140 CORE and NIB216 CORE. (B) The differential gene expression analysis is presented through volcano plots. Red and green points represent significantly upregulated and downregulated genes, respectively (p < 0.05), while blue points indicate non-significant changes. Genes were classified as differentially expressed, if they met the threshold of |log2FoldChange| > 0.0.

FIGURE 6.

Functional enrichment analysis of differentially expressed genes following electroporation. (A) Gene ontology (GO) enrichment analysis of significantly upregulated (right) and downregulated (left) genes in NIB140 CORE cells 24 hours after electroporation. (B) GO enrichment analysis for NIB216 CORE. Dot size reflects the number of genes contributing to each GO term, while colour intensity indicates statistical significance (adjusted p-value, padj). The GeneRatio represents the proportion of differentially expressed genes associated with each GO term relative to the total number of input genes. Selected invasion-relevant categories are highlighted in bold. GO terms include biological processes, molecular functions, and cellular components.
Functional enrichment analysis of differentially expressed genes following electroporation. (A) Gene ontology (GO) enrichment analysis of significantly upregulated (right) and downregulated (left) genes in NIB140 CORE cells 24 hours after electroporation. (B) GO enrichment analysis for NIB216 CORE. Dot size reflects the number of genes contributing to each GO term, while colour intensity indicates statistical significance (adjusted p-value, padj). The GeneRatio represents the proportion of differentially expressed genes associated with each GO term relative to the total number of input genes. Selected invasion-relevant categories are highlighted in bold. GO terms include biological processes, molecular functions, and cellular components.
DOI: https://doi.org/10.2478/raon-2025-0058 | Journal eISSN: 1581-3207 | Journal ISSN: 1318-2099
Journal RSS Feed
Language: English
Page range: 535 - 550
Submitted on: Aug 11, 2025
Accepted on: Sep 29, 2025
Published on: Dec 16, 2025
Published by: Association of Radiology and Oncology
In partnership with: Paradigm Publishing Services
Publication frequency: 4 issues per year
Keywords:
electroporation,
high-frequency electric pulses,
glioblastoma,
patient-derived cells,
invasion
Related subjects:
Medicine,
Clinical medicine,
Internal medicine,
Haematology, oncology,
Radiology

© 2025 Anja Blazic, Bernarda Majc, Metka Novak, Barbara Breznik, Lea Rems, published by Association of Radiology and Oncology
This work is licensed under the Creative Commons Attribution 4.0 License.

Previous articleVolume 59 (2025): Issue 4 (December 2025)Next article