Involvement of Mitochondria in the Selective Response to Microsecond Pulsed Electric Fields on Healthy and Cancer Stem Cells in the Brain

Abstract

In the last few years, pulsed electric fields have emerged as promising clinical tools for tumor treatments. This study highlights the distinct impact of a specific pulsed electric field protocol, PEF-5 (0.3 MV/m, 40 μs, 5 pulses), on astrocytes (NHA) and medulloblastoma (D283) and glioblastoma (U87 NS) cancer stem-like cells (CSCs). We pursued this goal by performing ultrastructural analyses corroborated by molecular/omics approaches to understand the vulnerability or resistance mechanisms triggered by PEF-5 exposure in the different cell types. Electron microscopic analyses showed that, independently of exposed cells, the main targets of PEF-5 were the cell membrane and the cytoskeleton, causing membrane filopodium-like protrusion disappearance on the cell surface, here observed for the first time, accompanied by rapid cell swelling. PEF-5 induced different modifications in cell mitochondria. A complete mitochondrial dysfunction was demonstrated in D283, while a mild or negligible perturbation was observed in mitochondria of U87 NS cells and NHAs, respectively, not sufficient to impair their cell functions. Altogether, these results suggest the possibility of using PEF-based technology as a novel strategy to target selectively mitochondria of brain CSCs, preserving healthy cells.

Keywords: CD133 protein; cancer stem cells (CSCs); filopodium-like protrusions; glioma; medulloblastoma; mitochondria dysfunction; normal human astrocyte; transcriptomics.

Figure 1

Ultrastructural analysis by SEM. Sham or PEF-5-exposed SEM images of (a,b) NHA, (c,d) D283, and (e,f) U87 NS cells. Filopodia protrusions disappeared in all analyzed cells 1 h after PEF-5 exposure, revealing visible pores on the surface of U87 NS cells, as indicated by the white arrowheads (f) and relative magnification (inset). (g) The graph shows the cell area fold increase of PEF-5-exposed cells with respect to relative sham-exposed cells (dotted line). p values were determined using a two-tailed t test; *** p < 0.001; **** p < 0.0001.

Figure 2

Cytofluorimetric quantification of CD133-positive cell and immunogold electron microscopy (IEM) analysis. Representative histograms of anti-CD133 staining and relative quantification of (a–c) sham- and PEF-5-exposed D283 cells and (d–f) U87 NS cells. IEM analysis in (g,h) sham-exposed D283 cells shows the normal localization of CD133 protein on plasma membrane protrusions, as indicated by the black arrowheads; (i,j) no filopodium-like protrusions and rare gold particles along the flat regions of the plasma membrane were observed in pulsed D283 cells 1 h after PEF-5 exposure. p values were determined using a two-tailed t test; * p < 0.05; ** p < 0.01.

Figure 3

TEM images of cytoskeleton distribution 1 h after PEF-5 exposure. TEM images show sham and pulsed (a–c) NHA, (d–f) D283, and (g–i) U87 NS cells. The colored area highlights the region where the cytoskeleton is distributed and densely packed, and in (c,f,i), the relative magnification shows fine details.

Figure 4

Mitochondrial morphological alteration assessment 1 h after PEF-5 exposure. (a–f) In TEM images, mitochondria are colored in red to highlight the difference between sham- and PEF-5-exposed (a,b) NHA, (c,d) D283, and (e,f) U87 NS cells. (g) The graph shows the percentage of morphologically damaged mitochondria per pulsed cell. (h) The graph shows the transversal mitochondrial area in pulsed cells compared with that of relative control cells (dotted line). p values were determined using a two-tailed t test; ** p < 0.01; **** p < 0.0001.

Figure 5

Mitochondrial dysfunction assessment. (a) The graph shows the intracellular ATP quantification in NHA, D283, and U87 NS cells 30 min after PEF-5 exposure. Intracellular ATP was calculated as luminescence fold change with respect to relative control for each cell type. (b–g) Cytometric analysis of JC-1 in untreated cells and after PEF-5 exposure. 50,000 events were acquired. FL2 (585/30)- FL1 (525/40) compensation was set to 40%. Densitometric analyses (b,c,e,f) are representative examples of biological triplicates. The color scale indicates the relative percentile. (d,g) Graphs show the mean red/green fluorescence intensity ratio ± SD in D283 and in U87 NS, respectively. p values were determined using a two-tailed t test; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6

Transcriptomic analysis, evaluated 24 h after PEF-5 exposure. (a,b) Venn diagrams show commonly enriched pathways from C5bp (biological process) gene sets. (c) Enrichment map displays the significantly (FDR q value < 0.05) enriched pathways (up- and downregulated) in PEF-5 treated NHA, D283, and U87 NS cells, relative to matched controls (C2cp MSigDB). GS: gene set; NES: Normalized Enrichment Score.