Abstract
Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is resistant to immune checkpoint blockade due to its intrinsic immunosuppressive properties. The fibroinflammatory stroma, particularly inflammatory cancer associated fibroblasts (CAFs), are a key determinant of the immune microenvironment in PDAC, however therapies aimed at broad-based targeting of PDAC stroma have been decidedly unsuccessful. Recently, interleukin-1α (IL-1) has emerged as a critical mediator of CAF activation and orchestrator of immune suppression in PDAC. We sought to explore the effects of IL-1 blockade on reprogramming the immune microenvironment and enhancing susceptibility to checkpoint inhibition through suppression of inflammatory stromal signaling in PDAC. Methods: Ptf1acre/+;LSL-KrasG12D/+; Tgfbr2flox/flox (PKT) mice were treated with the recombinant IL-1 receptor antagonist (anakinra, 50 mg/kg IP BID) or vehicle control. Single-cell cDNA library generation was performed using the 10× Chromium System. Expression matrices were processed using the R package Seurat 3.0 and clustered using AUC-based scoring algorithm. Immunophenotyping experiments were performed on single-cell suspensions using the Cytoflex platform. For survival studies, PKT mice were treated with vehicle, anti-PD1 antibody (200 μg/twice weekly), anakinra, or combination until moribund. Tissues were further processed for downstream qPCR and histologic analyses. Human pancreatic stellate cell (hPSC) and immortalized CAF cell lines procured from patient-derived xenografts were utilized for in vitro mechanistic studies. Results: Single-cell analysis revealed that anakinra treatment in PKT mice reduced levels of numerous pro-inflammatory CAF cytokines, including Cxcl1 and Il6, within CAF subclusters. Confirmatory qPCR of bulk tumor samples revealed a significant reduction in Cxcl1 and Il6 gene transcription and a global reduction in stromal fibrosis with anakinra treatment. IL-1 inhibition selectively reduced alternatively-activated macrophage (CD11b+F480+CD206+) and PMN-MDSC (CD11b+Ly6G+) populations within PDAC tumors. Combination treatment of anakinra with anti-PD1 antibody reduced tumor weight, increased intratumoral levels of effector memory (CD8+CD44+CD62L-) and activated cytotoxic (CD8+CD107+) T cells, and improved overall survival in PKT mice. In vitro studies demonstrated that conditioned media from PDAC tumor cells significantly activated Cxcl1 and Il6 gene transcription in hPSCs and CAFs in an IL-1α-dependent manner. IL-1-mediated transcription of these cytokines was abrogated by selective inhibition of both the p38 MAPK and NFκB cascades, demonstrating a coordinated effort between these signaling pathways in modulating the IL-1-induced stress response in stromal cells. Conclusions: These findings provide important mechanistic data and compelling pre-clinical evidence to explore IL-1 inhibition in combination with immune checkpoint blockade in PDAC patients. Citation Format: Austin R. Dosch, Samara Singh, Xizi Dai, Siddharth Mehra, Anna Bianchi, Iago De Castro Silva, Supriya Srinivasan, Nagaraj Nagathihalli, Jashodeep Datta, Nipun B. Merchant. Single-cell transcriptomic analysis reveals interleukin-1 inhibition suppresses inflammatory cancer-associated fibroblast signaling and improves the immune response in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-049.