Abstract
Abstract
Introduction: We have previously shown using the KPC (KrasLSL G12D/+; p53 r172H/+; Pdx1-Cre) mouse model of PDAC that sequential treatment with the DNA hypomethylating agents (HMA) followed by anti-PD-1 led to increased tumor necrosis, slowed tumor growth, increased tumor-infiltrating CD8+ cells, and significantly increased mean survival. However, acquired treatment resistance occurred, with emergence of a specific subtype of M2-polarized putatively immunosuppressive Chi3l3+ macrophages. In this study, we characterize the mechanism of polarization of these cells, define their function, and identify a potential therapeutic strategy to combine with epigentic therapy to prevent unfavourable macrophage polarization and improve the efficacy of epigenetic priming of immunotherapy against PDAC. Methods: Studies were conducted with primary macrophages (PM)e isolated from bone marrow (BMDM) or the peritoneum of 6-8 weeks mice and RAW264.7 cells were also used and exposed to conditioned media from primary KPC cell lines with and without previous treatment with the HMAs decitabine or azacytidine. The effect was compared with polarization by direct HMA, IFN-γ and IL-4. Gene set enrichment analysis was done on RNASeq data from myeloid cells and validated by RT-PCR. We used cytokine arrays and Western blot validation to identify secreted cytokines from KPC cells. Results: We profiled expression of RAW264.7 cells, BMDM and PMs following exposure to IL-4 or IFN-γ and identified a signature associated with each treatment (CD206, Fizz1, Tlr8, IGF1, Mgl2 associated with IL-4 and TNFα, CD86, CD64, CD40, NOS2 associated with IFN-γ). Direct exposure of myeloid cells to hypomethylating agents did not result in a significant polarization. We next used conditioned media of KPC cells to identify a hypomethylation specific effect and found a significant IL-4 like enrichment (Chi3l3, Arg1, Il4i1, Raet1a, Lgals4) and a HMA-specific myeloid signature (CD137, IL1a, Ccl2, Ccl7, Spp1) with treatment. To assess which cytokines might trigger this polarization, we employed cytokine arrays and identified a small number of candidate cytokines that are specific to hypomethylation induced polarization of myeloid cells, including CXCL1/2, CCL2, GM-CSF, FGF-21, IGFBP-6 and ICAM-1. Conclusions: Our results show that paracrine secretion of cytokines from cancer cells treated with HMA drugs, rather than direct effect of hypomethylating therapy on macrophages, is responsible for polarizing macrophages into an immunosuppressive subtype. We further identified several candidate cytokines secreted by cancer cells following hypomethylating therapy that may be relevant for this immunosuppressive polarization of macrophages and might be specific therapeutic targets in combination with hypomethylating therapy.
Citation Format: Arturo Orlacchio, Daniel Weissinger, Catherine Do, Benjamin Tycko, Diane M. Simeone, Tamas Gonda. Hypomethylating therapy induces a potential immuno-suppressive myeloid phenotype by altering cancer cell cytokine secretion in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C041.