Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy with limited effective treatments options. PDAC remains resistant to immunotherapy due to its immunosuppressive tumor microenvironment and dense desmoplastic stroma，which are attributed to an abundance of cancer associated fibroblasts (CAFs) and infiltrating immunocytes. Macrophages release extracellular traps which are composed of a backbone of extracellular DNA fibers containing histones and cytotoxic proteases such as myeloperoxidase, elastase, and matrix metalloproteinase in order to exert local immunoregulation. However, the role of macrophages extracellular traps (METs) in PDAC is at present unclear.

Aims & Methods

METs expression was evaluated in human PDAC tissues using Immunofluorescence co-localization of citrullinated histone H3 (H3cit) and CD68. Patients cohorts were classified into METs-High and METs-Low groups based on their numbers of postive cells. The collagen deposition and infiltrating immune cell populations were detected by Immunohistochemistry in both human and mice PDAC tissues. Cytokine production in human serum was assayed using the Olink proteomic platform. The level of C-C motif chemokine ligand 11 (CCL11) and double-stranded DNA (dsDNA) in serum and conditioned medium were determined by ELISA. Pancreatic orthotopic transplantation tumors were constructed using panc02 cell line and organoids derived from KrasG12D/+; Trp53R172H/+; Pdx1Cre (KPC) mouse PDAC tissue. Western blot and q-PCR were utilized to validate the expression levels of CAFs and METs markers across different groups. T cell activation, macrophage phenotype and programmed death-ligand 1 (PD-L1) level in mouse pancreatic tumors were evaluated by flow cytometry.

Results

The METs level was elevated in PDAC compared to paracancerous tissues and it was significantly linked to poor patient survival, advanced tumor grade and stage, fewer infiltrating CD8+ T-cells, increased CD86+ macrophages, and increased α-SMA+ CAFs. CCL11, secreted by CAFs, was a key upstream signaling molecule for METs formation. Mechanistically, CCL11 binded to chemokine receptor 5 (CCR5) on the surface of macrophages and promoted PAD2 expression, facilitating MET formation. Maraviroc, a CCR5 inhibitor, effectively suppressed this process and increased the expression of PD-L1 and the infiltration of CD8+ T cells in PDAC tissue. In vivo, maraviroc effectively enhanced the anti-tumor effects of immunotherapy.

Conclusion

CAFs promoted the formation of METs via CCL11/CCR5 axis and further contributed to the formation of an immunosuppressive microenvironment in PDAC. Through the inhibition of METs formation by antagonizing CCR5 reshaped the immune microenvironment of PDAC and enhanced the sensitivity of immunotherapy.