Damage associated molecular patterns molecules analysis following electroporation of cells in vitro

Abstract

Background. Tumor cells can die via immunogenic cell death pathway, in which damage associated molecular patterns (DAMP) molecules are released from cells. These molecules activate cells involved in immune response. Both innate and adaptive immune response can be activated, causing a destruction of the remaining cancer cells. Activation of immune response is also an important component in effectiveness tumor treated by of irreversible electroporation (IRE) and electrochemotherapy (ECT). We thus explored, if and when specific DAMP molecules are released as a consequence of electroporation in vitro.

Material and methods. In this in vitro study, 100 μs long pulses were applied to a suspension of Chinese hamster ovary cells. The release of DAMP molecules – specifically: ATP, calreticulin, nucleic acids and uric acid was investigated at different time points after exposing cells to electric pulses of different amplitudes. The release of DAMPs was statistically correlated with permeabilization and survival curve, e.g. reversible and irreversible electroporation using Pearson product moment test.

Results. In general release of DAMPs is increasing with increasing pulse amplitude. Investigated DAMP molecules concentrations depend on time post pulse analysis. Most of them strongly correlate to cell death and much less to membrane permeabilization. We were unable to detect uric acid.

Conclusions.  Release of DAMPs caused by electroporation pulses alone is much more complex than initially thought. Some DAMP molecule show strong correlation to survival/irreversible electroporation and weak correlation to permeabilization/reversible electroporation, yet detection of others remains uncertain. Also, it seems that the stability of certain DAMPs is time dependent.