Microsecond Pulsed Electric Fields: An Effective Way to Selectively Target and Radiosensitize Medulloblastoma Cancer Stem Cells

Abstract

Purpose: Cancer stem cells constitute an endless reserve for the maintenance and progression of tumors, and they could be the reason for conventional therapy failure. New therapeutic strategies are necessary to specifically target them. In this context, microsecond pulsed electric fields have been selected to expose D283Med cells, a human medulloblastoma cell line resulted to be rich in cancer stem cells, and normal human astrocytes. Methods: We analyzed in vitro different endpoints at different times after microsecond pulsed electric field exposure, such as permeabilization, reactive oxygen species generation, cell viability/proliferation, cell cycle, and clonogenicity, as well as the expression of different genes involved in cell cycle, apoptosis, and senescence. Furthermore, the response of D283Med cells exposed to microsecond pulsed electric fields was validated in vivo in a heterotopic mouse xenograft model. Results: Our in vitro results showed that a specific pulse protocol (ie, 0.3 MV/m, 40 ms, 5 pulses) was able to induce irreversible membrane permeabilization and apoptosis exclusively in medulloblastoma cancer stem cells. In the surviving cells, reactive oxygen species generation was observed, together with a transitory G2/M cell-cycle arrest with a senescenceassociated phenotype via the upregulation of GADD45A. In vivo results, after pulsed electric field exposure, demonstrated a significant tumor volume reduction with no eradication of tumor mass. In conjunction, we verified the efficacy of electric

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

“…PEF-5 exposure gives rise to an increase in the transmembrane potential, which causes structural membrane defects and permeability disorder. However, PEF-5 is unable to generate irreversible membrane permeabilization, similar to that observed in MB cells [ 34 ], in both GBM cultures, which resulted in a lower level of cell death, demonstrating the existence of a PEF-5 toxicity threshold value, higher in U87-GBM than in D283-MB cells. This threshold appears to depend on specific cellular characteristics that may play a key role in predicting the cell response to PEF exposure [ 12 ].…”

“…In particular, three upregulated genes, i.e., Hyaluron synthase 2 ( HSA2 ), proline rich protein 2A ( SPRR2A ) and 2D ( SPRR2D ), were the same as U87 ML and NS cells (box in Figure 3 b). These genes have been described as components of the plasmatic membrane, having a role in membrane integrity, cell survival and inflammatory responses even post radiation and stressful environmental conditions [ 34 , 35 , 36 , 37 ].…”

“…These results again suggested a strong ability of PEF-5 to modify the stem phenotype of CSCs, decreasing their self-renewal capacity ( Figure 5 a–d). As radiosensitivity increases with cell differentiation status, we tested the effectiveness of a combined protocol, irradiating U87 ML and U87 NS cells with increasing IR doses (2, 5, 8 Gy), 3 h after PEF-5 exposure, as suggested in our previous work [ 34 ]. As shown in Figure 5 a–d, the combined treatment of PEF-5 and IR was able to reduce clone formation as a function of delivered doses, suggesting an additive effect between these two physical agents.…”

“…In this context, we demonstrated that a specific signal of this class, named PEF-5 (i.e., 5 electric pulses lasting 40 µs, pulse amplitude 0.3 MV/m, repeated at 1 Hz), irreversibly affected medulloblastoma (MB) CSCs, inducing PEF-surviving cells to better respond to a subsequent exposure to ionizing radiation (IR) [ 34 ].…”

Pulsed Electric Fields Alter Expression of NF-κB Promoter-Controlled Gene

“…We knew from previous reports that pulsed electric fields of nanosecond-duration impact intracellular pathways, processes, and expression of different genes [ 39 , 43 , 50 , 51 , 52 , 53 , 54 , 55 , 56 ], but the fact that a similar effect can also be achieved by microsecond-duration pulsed electric fields is a new discovery. Microsecond-duration pulsed electric fields, meanwhile, were better known for their plasma membrane pore opening effects [ 57 , 58 , 59 ]. Low basal activity of the NF-κB-controlled system observed during our study in Hep-2c cells and its significant inducibility after pulsed electric fields application would allow to further analyze its use in primary cervical cancer cell cultures or even in an in situ setting.…”