Co-inhibition of RAGE and TLR4 sensitizes pancreatic cancer to irreversible electroporation in mice by disrupting autophagy

Irreversible electroporation (IRE) is a local ablative treatment for patients with pancreatic cancer. During the IRE procedure, high-intensity electric pulses are released intratumorally to disrupt plasma membranes and induce cell death. Since the intensity of the pulsed electric field (PEF) can be decreased by the tumor microenvironment, some cancer cells are subjected to a sublethal PEF and may survive to cause tumor recurrence later. Autophagy activation induced by anticancer therapies is known to promote treatment resistance. In this study, we investigated whether autophagy is activated in residual cancer cells after IRE and assessed the roles it plays during tumor recurrence. Subcutaneous KPC-A548 or Panc02 murine pancreatic cancer cell line xenograft mouse models were established; once the tumors reached 7 mm in one dimension, the tumor-bearing mice were subjected to IRE. For in vitro sublethal PEF treatment, the pancreatic cancer cell suspension was in direct contact with the electrodes and pulsed at room temperature. We showed that autophagy was activated in surviving residual cells, as evidenced by increased expression of LC3 and p62. Suppression of autophagy with hydroxychloroquine (60 mg/kg, daily intraperitoneal injection) markedly increased the efficacy of IRE. We demonstrated that autophagy activation can be attributed to increased expression of high-mobility group box 1 (HMGB1); co-inhibition of two HMGB1 receptors, receptor for advanced glycosylation end products (RAGE) and Toll-like receptor 4 (TLR4), suppressed autophagy activation by upregulating the PI3K/AKT/p70 ribosomal S6 protein kinase (p70S6K) axis and sensitized pancreatic cancer cells to PEF. We prepared a polymeric micelle formulation (M-R/T) encapsulating inhibitors of both RAGE and TLR4. The combination of IRE and M-R/T (equivalent to RAGE inhibitor at 10.4 mg/kg and TLR4 inhibitor at 5.7 mg/kg, intravenous or intraperitoneal injection every other day) significantly promoted tumor apoptosis, suppressed cell cycle progression, and prolonged animal survival in pancreatic tumor models. This study suggests that disruption of HMGB1-mediated autophagy with nanomedicine is a promising strategy to enhance the response of pancreatic cancer to IRE.