Combined immunotherapy with TLR ligands in pancreatic cancer elicits strong anti-tumor response and boosts interferon-mediated killing of tumor cells

The incidence and the lethality of pancreatic ductal adenocarcinoma (PDAC) is alarmingly increasing in the last decades worldwide and novel therapies are urgently needed to face this malignancy. In this project, we investigated the therapeutic potential of poly(I:C) (pIC) and resiquimod (R848), two Toll-like receptor agonists, in different preclinical models of PDAC. We found that, when used in combination, pIC+R848 elicit a potent antitumoral response in all the in vivo models investigated, leading to tumor eradication in >95% of the treated mice and the development of long-lasting immune memory. We observed, at the cellular level, an increase in monocytes and neutrophils at the expenses of macrophages in the tumor microenvironment (TME) of pIC+R848-treated tumors, as well as an increase in the ratio of mature CD8 T cells and NK cells. We identified, by carrying out multiple depletion experiments, an uncommon central role of CD4 T cells in pIC+R848 immunotherapy, acting also on CD8 T cells to boost cancer regression. We showed that pIC+R848 triggers type I and II IFNs in the TME and that this induction is essential for tumor regression in our models. Interestingly, an upregulation of intratumoral IFN-related transcripts correlates with better overall survival also in an in-house cohort of PDAC patients. Single-cell RNA sequencing analyses performed on pIC+R848 treated tumors demonstrated that monocytes and macrophages are the main responders to these ligands and that pIC+R848 reshape this compartment and the functionality of these cells. Interestingly, while the global blocking of type I or type II IFN signaling inside the TME completely abolished the efficacy of pIC+R848, a residual activity of these cytokines was still present in a Ifnar1 or Ifngr1 KO background. We thus reasoned that IFNs exert a direct antitumoral activity on PDAC cancer cells and, while we did not observe such effect in vivo upon pIC+R848 therapy, the in vitro combination of IFN-g and IFN-b greatly inhibited cell growth compared to the single stimulation and deregulated a discrete set of genes involved in immune cell death. In conclusion, we introduce here a novel immunotherapy for PDAC based on the targeting of intratumoral myeloid cells and on the induction of type I and II IFNs, eventually directly killing cancer cells through a unique immune cell death program.