(D-129) Characterization of Lethal Pulsed Electric Fields for In Vitro Tumor Spheroids and Metastatic Invasion

Introduction:: Electroporation is a biophysical phenomenon where nanoscale hydrophilic pores are generated on the cellular plasma membrane when exposed to an applied external electric field. Irreversible electroporation (IRE) can produce a protracted or permanent nanopore formation, causing cell death[1]. A second generation of IRE, known as High-Frequency Irreversible Electroporation (H-FIRE), was developed to deliver shorter bipolar pulses in series of bursts to mitigate muscle contractions induced by IRE. Tumor spheroids, cancer cells cultured into a large sphere (.01-2cm), can mimic in vivo conditions due to their spatial organization and cell-ECM interactions during metastasis. Previously, tumor spheroids have been treated with electroporation to cause permeation for delivering chemotherapeutics [2] and high calcium concentrations [3] to assess cell death in a physiologically relevant model. Here, we aim to subject the tumor spheroids with lethal and sub-lethal pulse electric fields (PEFs) at varying pulse widths to identify the extent of cell death and metastatic inhibition.

Materials and Methods:: To investigate the PEF variations to induce cell death within tumor spheroids, human glioblastoma cells (U251) were transferred to an ultra-low attachment 96-well plate at 50,000 cells per well for spheroid formation. After 3 days, the spheroids were harvested and seeded onto collagen hydrogels that were crosslinked at a 1-mm thickness in a 4-well rectangular plate. Once the spheroid attached overnight or after 3 days, the spheroid-seeded hydrogels were treated with two stainless-steel flat plate electrodes held together using a custom-made electrode holder to induce a uniform electric field. Day of treatment was denoted as day 0. A custom pulse generator (VoltMed Inc, Blacksburg, VA, USA) was used to deliver 500-2000 V/cm of IRE or 1000-2500 V/cm of H-FIRE with 500 V/cm intervals. The biphasic PEFs had varying pulse durations of 1, 2, and 5 µs, but maintained similar interphase delays of 1 µs. The burst on-time and bursts delivered remained constant between IRE and H-FIRE at 100 µs over 100 bursts, respectively. Viability was measured using live/dead staining and an XTT viability assay at days 1, 3, and 5 post-treatments. Metastatic invasion from the tumor spheroids was tracked daily using bright-field imaging and analyzed using ImageJ.


Results, Conclusions, and Discussions:: The XTT viability and live/dead imaging indicated that was significantly more cell death at both increasing pulse widths and applied electric field. IRE and 5-1-5 achieved complete cell death above 2000 V/cm and 2500 V/cm, respectively. The XTT viability assay revealed that the longer pulse durations, such as IRE and the 5-1-5, caused deeper penetration within the tumor spheroids producing immediate stark decreases in viability as the voltage increased. Shorter pulse durations such as the 2-1-2 displayed marginal cell death until 2500 V/cm, while the 1-1-1 presented minimal cell death across all PEFs applied. Only the longer pulse durations at the highest voltages applied were consistently successful in tumor spheroid elimination and complete metastatic invasion elimination. With the spheroids grown for 3 days on the hydrogels, each treatment parameter displayed varying degrees of eradication of the migration from the tumor spheroid. The results further indicate that that the electric field inhibits tumor regrowth, with the migration of viable cells significantly less than the control. When the applied energy was ≤ 1000 V for IRE and ≤ 1500 V for the H-FIRE pulses with the inhibition varying over several days. Due to the presence of cancerous cells post-electroporation, adjuvant therapies such as chemotherapeutics or immunotherapies are optimal solutions to eradicate remaining malignancies. The inclusion of additional cell types to create a vibrant tumor microenvironment for post-ablation investigation and differences in cell death mechanisms.

Acknowledgements (Optional): :

References (Optional): : [1] Davalos, R.V. et al. Ann Biomed Eng. 2005, 33, 223-231

[2] Gibot, L. et al. Journal of Controlled Release. 2013, 167, 138-147

[3] Frandsen S. et al. PLoS One. 2015, 10(12)