Abstract 653: Tumor cell-macrophage crosstalk drives immune suppression in pancreatic cancer
Authors: Mehra Siddharth , Garrido Vanessa , Singh Samara , Silva Iago De Castro , Zhou Zhiqun , Srinivasan Supriya , Nivelo Luis Alberto , Bianchi Anna , Adams Andrew , Amirian Haleh , Morey Lluis , Yuguang Ban , Villarino Alejandro , Datta Jashodeep , Merchant Nipun , Nagathihalli Nagaraj 
Published: April 2023

Abstract
Background: Pancreatic ductal adenocarcinoma (PDAC) due to its unique genetic makeup and immunosuppressive tumor microenvironment (TME) produce a lack of response to current therapies. Macrophages, constitute a large innate immune subset and play a vital role in establishing an immune-suppressive microenvironment. Previously, we have identified Cyclic AMP Response Element Binding protein 1 (CREB) as a tumor cell-intrinsic oncogenic factor that promotes disease aggressiveness, poor survival, and immune suppression. Based on these, we sought to determine CREB mediated mechanisms of tumor-macrophage cross talk in driving immunosuppressive phenotype in PDAC.
Methods: We have generated a genetically engineered mouse model (GEMM) of pancreas-specific CREB deletion (CREBfl/fl) in LSL-KrasG12D/+; Trp53 R172H/+; Pdx1Cre/+ (KPC) mice. CRISPR/CAS9-based genomic editing was utilized to ablate CREB (CREBKO) in KPC tumor cells. RNA-sequencing analysis was performed in KPC CREBKO tumor cells. ChIP-qPCR analysis was performed in KPC tumor cells. Orthotopic tumor implantation of these cells was performed in the pancreata of mice. Immunophenotyping was accomplished to assess changes in the immune subsets with CREB deletion in vivo. Additionally, these tissues were also processed for single-cell RNA (scRNA) transcriptomics analysis to evaluate the impact of CREB deletion on different cellular constituents within the TME.
Results: CREB deletion in the KPC GEMM led to a significant reduction in the primary tumor burden, liver metastases, and improved overall survival compared to wild-type KPC. In assessing the immune repercussions of CREB deletion, we observed a decreased infiltration of tumor-promoting CD11b+ F4/80+ CD206+ tumor-associated macrophages (TAMs) and a concomitant increase in the antigen-presenting M1-like macrophages (F4/80+MHC-IIhighCD86high). Additionally, scRNA sequencing analysis within the macrophage compartment in CREBKO tumors revealed significant enrichment of M1 hallmark signaling pathways. Further, CREB ablation in these tumors facilitated increased infiltration of activated effector memory CD8+ T cells and resulted in enhanced adaptive immune response. RNA transcriptomic-based analysis of CREBKO tumor cells revealed downregulation of Leukemia inhibitory factor (LIF) as of the top targets. Mechanistically, ChIP qPCR analysis after CREB1 pulldown confirmed its occupancy on LIF promoter region. Further, on exploring the role of CREB regulated LIF on immune subsets, incubation of macrophages with CREBWT conditioned media in the presence of LIF neutralizing antibody or blocking its receptor (LIFR) decreases polarization of macrophages towards M2 like phenotype.
Conclusion: These findings broaden our understanding of the tumor cell-intrinsic role of CREB in fostering immunosuppressive profile via LIF by promoting skewness of TAMs towards M2 like state in PDAC.
Citation Format: Siddharth Mehra, Vanessa Garrido, Samara Singh, Iago De Castro Silva, Zhiqun Zhou, Supriya Srinivasan, Luis Alberto Nivelo, Anna Bianchi, Andrew Adams, Haleh Amirian, Lluis Morey, Ban Yuguang, Alejandro Villarino, Jashodeep Datta, Nipun Merchant, Nagaraj Nagathihalli. Tumor cell-macrophage crosstalk drives immune suppression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 653.