Effects of Ultra-Short Pulsed Electric Field Exposure on Glioblastoma Cells

Abstract

Glioblastoma multiforme (GBM) is the most common brain cancer in adults. GBM starts from a small fraction of poorly differentiated and aggressive cancer stem cells (CSCs) responsible for aberrant proliferation and invasion. Due to extreme tumor heterogeneity, actual therapies provide poor positive outcomes, and cancers usually recur. Therefore, alternative approaches, possibly targeting CSCs, are necessary against GBM. Among emerging therapies, high intensity ultra-short pulsed electric fields (PEFs) are considered extremely promising and our previous results demonstrated the ability of a specific electric pulse protocol to selectively affect medulloblastoma CSCs preserving normal cells. Here, we tested the same exposure protocol to investigate the response of U87 GBM cells and U87-derived neurospheres. By analyzing different in vitro biological endpoints and taking advantage of transcriptomic and bioinformatics analyses, we found that, independent of CSC content, PEF exposure affected cell proliferation and differentially regulated hypoxia, inflammation and P53/cell cycle checkpoints. PEF exposure also significantly reduced the ability to form new neurospheres and inhibited the invasion potential. Importantly, exclusively in U87 neurospheres, PEF exposure changed the expression of stem-ness/differentiation genes. Our results confirm this physical stimulus as a promising treatment to destabilize GBM, opening up the possibility of developing effective PEF-mediated therapies.

Keywords: cancer stem cells (CSCs); glioma; neurospheres; pulsed electric field; transcriptomic analysis.

Figure 1

Analysis of stemness/differentiation markers in U87 ML and U87 NS. (a) Representative images of U87 cells grown in standard medium as a monolayer. (b) Neurospheres grown for 7 days in a selective medium. (c,d) CD133 and CD15 mRNA expression levels, considered stemness markers, and (e) β-III TUBULIN, a well-known marker of neuron differentiation in U87 ML and NS cells. Data are shown as relative expression compared to U87 ML. p values were determined using a two-tailed t test, **** p < 0.0001.

Figure 2

Membrane permeabilization, ROS production, cell survival and cell cycle. (a) Yo-pro-1 uptake at T = 0 h and at T = 3 h after PEF-5 exposure in U87 ML and U87 NS cells. (b) ROS levels after PEF-5 exposure at T = 0 h and T = 3 h. The histogram shows the ratio between the mean fluorescence intensity (MFI) of exposed and sham-exposed cells in both cell culture conditions. (c–e) Bar graphs of sham-exposed and exposed U87 ML and NS cells 24 h after PEF-5 exposure for cell death quantification (Trypan blue assay). (d–f) Cell proliferation evaluated at different time points (0, 24 and 48 h) after PEF-5 exposure in U87 ML and U87 NS. (g–j) Cell cycle phases in U87 ML and NS at 24 h after PEF-5 exposure. p values were determined using a two-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Transcriptomic analysis. (a) Heat maps displaying differentially expressed genes in U87 ML and NS cells. (b) Venn diagram depicting the number of significantly downregulated and upregulated genes in ML and NS U87 cells, as well as the name of commonly upregulated genes. (c,d) Bar graphs displaying the positive (red) and negative (blue) transcriptional enrichments in MSigDB gene sets of differentially expressed genes between sham-exposed and PEF5-exposed U87 ML and U87 NS cells. (e) Enrichment maps displaying transcriptional enrichments in cellular remodeling and inflammation processes in U87 ML cells and in DNA repair and RNA processing and post-transcriptional modification in U87 NS cells.

Figure 4

Cell cycle checkpoint, hypoxia/inflammation and stemness/differentiation markers. (a,b) Relative mRNA expression levels of genes associated with cell cycle, i.e., P53, P21, CYCLIN D1, GADD45, CDK2 and CDK4. (c,d) Relative mRNA expression levels of genes associated with hypoxia/inflammation processes, i.e., IL6, CCL20, COX2, iNOS and β-CATENIN. (e,f) Relative mRNA expression levels of genes associated with stemness/differentiation processes, i.e., NANOG, OCT4, SOX2, CD133, CD15 and β-III TUBULIN. p values were determined using a two-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

Survival clonogenic assay. (a,b) Representative images of primary and secondary neurospheres after PEF-5 exposure and IR treatments. (c,d) Quantitative analysis revealed a significant decrease in the clonogenic capacity between sham- and PEF-5 exposed cells, regardless of culture conditions. The combined treatment (PEF-5 + IR) reduced clone formation as a function of delivered radiation doses. p values were determined using a two-tailed t test. * p < 0.05; ** p < 0.01, **** p < 0.0001.

Figure 6

Invasion and migration assay. Representative images of transmigrated cells for (a) U87 ML and (b) U87 NS cells. (c,d) Percentage of invasiveness in U87 ML and NS cells evaluated 24 h after PEF-5 exposure and IR combined treatment. p values were determined using a two-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001.