Cellular fragmentation underlies the immunogenicity of irreversible electroporation-mediated tumor cell killing.

Irreversible electroporation (IRE) is a focal ablative cancer therapy that destroys cells through membrane destabilization via pulsed electric fields. It also has the capacity to induce a systemic, anti-tumor immune response, thus acting as an in situ vaccine. Although many studies characterize the immunogenicity of focal therapies by their released biochemical constituents, here we show that the biophysical context of the presentation of these immunogenic signals is vital to understanding downstream immune functions. Compared to thermal ablation or cryoablation, IRE generates similar numbers of exosome-like particles (ELP, 50-200 nm) but significantly greater numbers of microparticles (MP, 200-1000 nm) and large debris particles (LDP, 2-6 μm) in both melanoma and pancreatic cancer cell lines. We show that LDPs contain antigen and tumor-associated DNA, which dendritic cells (DCs) internalize in greater proportions from IRE-treated cells compared to other treatments. For the submicron particles, we demonstrate both in vitro and in vivo that MPs induce greater T-cell proliferation and differentiation compared to ELPs on a per-particle basis. This novel biophysical analysis of the immunogenicity of IRE-treated cancer cells opens a new avenue toward improving the systemic immune response to focal ablation-based cancer immunotherapies via increasing cell fragmentation and particle generation.