Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

The immunosuppressive microenvironment that is shaped by hepatic metastatic pancreatic ductal adenocarcinoma (PDAC) is essential for tumor cell evasion of immune destruction. Neutrophils are important components of the metastatic tumor microenvironment and exhibit heterogeneity. However, the specific phenotypes, functions and regulatory mechanisms of neutrophils in PDAC liver metastases remain unknown. Here, we show that a subset of P2RX1-negative neutrophils accumulate in clinical and murine PDAC liver metastases. RNA sequencing of murine PDAC liver metastasis-infiltrated neutrophils show that P2RX1-deficient neutrophils express increased levels of immunosuppressive molecules, including PD-L1, and have enhanced mitochondrial metabolism. Mechanistically, the transcription factor Nrf2 is upregulated in P2RX1-deficient neutrophils and associated with PD-L1 expression and metabolic reprogramming. An anti-PD-1 neutralizing antibody is sufficient to compromise the immunosuppressive effects of P2RX1-deficient neutrophils on OVA-activated OT1 CD8+ T cells. Therefore, our study uncovers a mechanism by which metastatic PDAC tumors evade antitumor immunity by accumulating a subset of immunosuppressive P2RX1-negative neutrophils.


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No data were excluded from analysis.
Experiments involving in vitro cell studies and most of animal studies were repeated three times to ensure reproducibility. All attempts at replication were successful.
All mice and cells were randomly assigned to experimental groups.
Investigators were blinded to group allocation for mouse experiments. Also, quantification of tumor parameters by histological analyses was performed in a blinded fashion. For in vitro experiments, investigators were not blinded in conducting experiments as they needed to know which groups to treat with which drugs. Where possible, researchers were blinded during data analysis. Note that full information on the approval of the study protocol must also be provided in the manuscript.

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The purchased cell lines were validated by the commercial vendors, and FC1199 cell line was validated by Professor Jing Xue using short tandem repeat (STR) profiling.
All cell lines were tested negative for mycoplasma contamination.
No misidentified cell line was used in the study.
No wild animals were used in this study.
No field-collected samples were used in the study.
Animal experiments were approved by Institutional Animal Care and Use Committee of East China Normal University.
This information is shown in supplementary table 1. 20 cases of PDAC liver metastases and adjacent livers tissues were obtained from Ren Ji hospital from January 2015 to June 2018. All patients had not received radiotherapy, chemotherapy, hormone therapy or other related anti-tumor therapies before surgery.
The study was approved by the Research Ethics Committee of Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University. Written informed consent was provided before enrollment.