combining irreversible electroporation (IRE) with TLR3/9 agonists and PD-1 blockade for potent anti-tumor immunity

Cancer is a leading cause of death worldwide, taking nearly 10 million lives each year. CD8+ T cytotoxic lymphocytes (CTLs) play a critical role in human immunity against cancer. Irreversible electroporation (IRE) is a new cancer ablation technology that utilizes electric current to induce tumor cell apoptosis. Compared to other thermal ablative techniques, IRE is safer to use around sensitive structures such as blood vessels or nerves. Although IRE has been successful as a cancer ablation therapy, patients often die due to the recurrence of residual tumors. Therefore, it is critical to improve tumor ablation technology to achieve better therapeutic outcomes for cancer patients. Toll-like receptors (TLRs) are conserved pattern recognition receptors (PRRs) that recognize microbial compounds and stimulate innate and adaptive immune responses. TLR3 agonist Poly I:C (pIC) and TLR9 agonist CpG are known to stimulate strong CD4+ Th1 and CD8+ CTL responses and have been used in combination with other treatments to improve cancer immunotherapy. Programmed death 1 (PD-1) receptors are expressed on activated CTLs, and the interaction between PD-1 and its ligand (PDL-1) on activated CTLs leads to CTL exhaustion and inhibition of CTL-mediated anti-tumor immunity. PD-1 blockade by monoclonal antibodies against PD-1 or PDL-1 blocks their interaction, relieves T cell inhibition and enhances T cell responses. In this study, we developed a mouse model bearing large primary (300 mm3) and medium distant (100 mm3) EG7 lymphomas engineered to express ovalbumin (OVA) as a nominal tumor antigen. We established experimental protocols including IRE alone and IRE combined with TLR3/9 agonists (poly I:C/CpG) (IRE+pIC/CpG) or PD-1 blockade (IRE+PD-1 blockade) or both (IRE+Combo) to investigate the therapeutic effects on primary and distant EG7 tumors and conversional effects on an immunotolerant tumor microenvironment (TME). A dominant immunosuppressive TME is created by tumor cells to promote tumor progression and inhibit effective anti-tumor immune responses. We demonstrate that IRE alone stimulates very weak OVA-specific CD8+ T cell responses and does not inhibit primary tumor growth. IRE+pIC/CpG synergistically stimulates more efficient OVA-specific CD8+ T cell responses and inhibition of primary tumor growth than IRE+PD-1 blockade. IRE+pIC/CpG plays a major role in the modulation of immune cell profiles (ICP), but a minor role in the down-regulation of PDL-1 expression in the TME, and vice versa for IRE+PD-1 blockade. IRE+Combo cooperatively induces potent OVA-specific CD8+ T cell immunity and rescues exhausted intratumoral CD8+ T cell, leading to eradication of not only primary but also untreated concomitant distant tumors and lung metastases. IRE+Combo efficiently modulates ICPs, as evidenced by a reduction of immunotolerant type-2 macrophages (M2), myeloid-derived suppressor cells (MDSCs), plasmacytoid dendritic cells (pDCs) and CD4+Foxp3+ regulatory T (Treg) cells and an increase of immunogenic M1, CD169+ macrophages, type-1 conventional dendritic cells (cDC1) and CD8+ T cells, leading to conversion of immunotolerance in not only primary but also untreated distant TMEs. IRE+Combo also shows effective therapeutic results in two breast cancer models. Targeting immunotolerant subsets in the TME represent a future direction towards improved immunotherapy and IRE-ablated cancer therapy. Therefore, our IRE+Combo protocol capable of eradicating both primary and distant tumors as well as lung metastases via converting immunotolerant TME may become a promising strategy for cancer IRE-ablation therapy