Platelet activating factor (PAF) promotes immunosuppressive neutrophil differentiation within tumors
Chronic inflammatory milieu in the tumor microenvironment (TME) leads to the recruitment and differentiation of myeloid-derived suppressor cells (MDSCs). Polymorphonuclear (PMN)-MDSCs, which are phenotypically and morphologically defined as a subset of neutrophils, cause major immune suppression in the TME, posing a significant challenge in the development of effective immunotherapies. Despite recent advances in our understanding of PMN-MDSC functions, the mechanism that gives rise to immunosuppressive neutrophils within the TME remains elusive. Both in vivo and in vitro, newly recruited neutrophils into the tumor sites remained activated and highly motile for several days and developed immunosuppressive phenotypes, as indicated by increased arginase 1 (Arg1) and dcTrail-R1 expression and suppressed anti-cancer CD8 T cell cytotoxicity. The strong suppressive function was successfully recapitulated by incubating naïve neutrophils with cancer cell culture supernatant in vitro. Cancer metabolite secretome analyses of the culture supernatant revealed that both murine and human cancers released lipid mediators to induce the differentiation of immunosuppressive neutrophils. Liquid chromatography–mass spectrometry (LC-MS) lipidomic analysis identified platelet activation factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) as a common tumor-derived lipid mediator that induces neutrophil differentiation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2), the PAF biosynthetic enzyme, is upregulated in human pancreatic ductal adenocarcinoma (PDAC) and shows an unfavorable correlation with patient survival across multiple cancer types. Our study identifies PAF as a novel lipid-driven mechanism of MDSC differentiation in the TME, providing a potential target for cancer immunotherapy. Overall design: Total RNA from neutrophils were isolated using RNeasy plus mini kit (Qiagen). Total RNA was preamplified with SMARTer Ultra Low Input kit v4 per manufacturer's protocol (Clontech). CDNA quality and quantity were measured using Qubit fluorometer (Life Technologies) and Agilent Bioanalyzer 2100. CDNA was used to generate Illumina-compatible sequencing libraries using NexteraXT library preparation kit (Illumina). The amplified libraries were hybridized to Illumina flow cell and sequenced using NextSeq 550 squener (Illumina). Reads were aligned with STAR-2.7.0 and reads quantified with Subread-1.6.4. DESeq2-1.26.0 was used to normalize count matrix and assess differential expression with adjusted Pvalue <0.05. Heatmap was created using pheatmap (1.0.12) using DESeq2-normalized counts. Heat map representations were produced using RStudio (1.3.1093) and GraphPad Prism (v10).