Synergistic immunotherapeutic effects of irreversible electroporation and CAR-NK cell therapy against hepatocellular carcinoma.

Chimeric antigen receptor natural killer (CAR-NK) cell therapy has emerged as a promising immunotherapeutic modality with potent cytotoxicity and a favorable safety profile. However, its therapeutic efficacy is often limited by poor infiltration into tumors and the profoundly immunosuppressive tumor microenvironment (TME). In hepatocellular carcinoma (HCC), one of the leading causes of cancer-related mortality, this suppressive TME severely compromises the function of CAR-NK cells. To overcome this limitation, we developed a combinatorial strategy integrating irreversible electroporation (IRE), a clinically approved nonthermal ablation modality capable of reshaping the TME, with glypican-3 (GPC3)-targeted CAR-NK cells generated via 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-functionalized lipid nanoparticle (LNP)-mediated gene delivery. IRE promoted immunogenic cell death and reprogrammed the TME through the release of damage-associated molecular patterns and chemokines, notably CX3CL1, thereby enhancing NK cell infiltration. Moreover, IRE-treated HCC cells exhibited heightened susceptibility to NK-mediated cytotoxicity through elevated intracellular reactive oxygen species, establishing a mechanism of immune sensitization. When combined with LNP-engineered GPC3-specific CAR-NK cells, this approach elicited synergistic antitumor activity, as demonstrated by superior tumor lysis in vitro and robust tumor regression in various HCC models without systemic toxicity. By coupling TME remodeling of IRE with the precision and safety of LNP-engineered CAR-NK cells, we propose a durable, clinically actionable treatment paradigm to overcome resistance in solid tumors, such as HCC.