Irreversible cell electroporation-induced release of DAMPs increases cancer cell resistance to electroporation

Irreversible electroporation (IRE) utilizes high-voltage pulses to permeabilize cell membranes and induce cancer cell death, a process that releases damage-associated molecular patterns (DAMPs) and triggers immune responses. This study investigated the impact of intracellular DAMPs, extracted following cell treatment with electric pulses and termed electroporation supernatant (EP SN), on the viability of cancerous (4T1) and noncancerous (CHO) cells using an in vitro model. Viability was assessed via MTT, flow cytometry, and clonogenic assays. Protein, RNA, and DNA extraction levels were quantified using the BCA assay, SDS-PAGE, RT-PCR, and PCR, while cell integrity was confirmed through Cell Mask. Results revealed that EP SN rich in DAMPs enhanced cell resilience post-electroporation, with a more pronounced protective effect observed in 4T1 cells. We confirmed that DAMP release resulted from pulsing-induced permeabilization rather than cell lysis. Analysis showed that up to 65% of intracellular protein and significant amounts of RNA were released in a pulse-dependent manner, whereas DNA extraction remained negligible. Interestingly, heat-inactivation of the EP SN did not diminish its protective effect, suggesting that the improved viability is not dependent on the biochemical activity of the DAMPs. Instead, the data suggest that macromolecules like proteins and RNA stabilize cell homeostasis through colloidal osmotic pressure or Gibbs-Donnan effects. Consequently, the presence of these DAMPs during treatment may increase the resistance of cancerous 4T1 cells compared to noncancerous CHO cells, posing a potential challenge for IRE efficacy. To counteract this resistance, concomitant use of anticancer drugs like bleomycin or cisplatin is necessary.