Abstract
Neutrophils are prominent components of gastric cancer (GC) tumors and exhibit distinct phenotypes in GC environment. However, the phenotype, regulation, and clinical relevance of neutrophils in human GC are presently unknown. Here, immunohistochemistry, real-time PCR, and flow cytometry analyses were performed to examine levels and phenotype of neutrophils in samples from 41 patients with GC, and also isolated, stimulated, and/or cultured neutrophils for in vitro regulation assays. Finally, we performed Kaplan-Meier plots for overall survival by using the log-rank test to evaluate the clinical relevance of neutrophils and their subsets. In our study, neutrophils in tumor tissues were significantly higher than those in nontumor tissues and were positively associated with tumor progression but negatively correlated with GC patient survival. Most intratumoral neutrophils showed an activated CD54+ phenotype and expressed high-level immunosuppressive molecule B7-H4. Tumor tissue culture supernatants from GC patients induced neutrophils to express CD54 and B7-H4 in both time-dependent and dose-dependent manners. Locally enriched CD54+ neutrophils and B7-H4+ neutrophils positively correlated with increased granulocyte-macrophage colony-stimulating factor (GM-CSF) detection ex vivo, and in vitro GM-CSF induced the expression of CD54 and B7-H4 on neutrophils in a time-dependent and dose-dependent manner. Moreover, GC tumor-derived GM-CSF activated neutrophils and induced neutrophil B7-H4 expression via Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathway activation. Furthermore, higher intratumoral B7-H4+ neutrophil percentage/number was found in GC patients with advanced tumor node metastasis stage and reduced overall survival following surgery. Our results illuminate a novel regulating mechanism of B7-H4 expression on tumor-activated neutrophils in GC, suggesting that functional inhibition of these novel GM-CSF-B7-H4 pathways may be a suitable therapeutic strategy to treat the immune tolerance feature of GC.
Highlights
Gastric cancer (GC), with 5-year survival of less than 40%, is one of the leading causes of tumor death in many lessdeveloped countries [1]
It is accepted that Helicobacter pylori infection is associated with GC [2, 3], the pathogenesis of GC is presently unknown and its development and prognosis are closely associated with the infiltrating immune cells in the GC environment [4]
It has been reported that the Janus kinase (JAK)-signal transducer and activator of transcription 3 (STAT3) signaling pathways are activated in GC cells [20] and GC-associated macrophages [21] by IL-6
Summary
Gastric cancer (GC), with 5-year survival of less than 40%, is one of the leading causes of tumor death in many lessdeveloped countries [1]. As to the infiltrating neutrophils in the GC environment, there are studies by using immunohistochemistry showing the relationships between high tumor-infiltrating neutrophils and poor prognosis of GC patients [8]. These studies on peripheral and infiltrating neutrophils together suggest that neutrophils may play pathological roles in GC. As to GC, it has been shown that B7-H4 expression on gastric tumor tissues [17], circulating monocytes [18], or regulatory T cells [19] predicts poor survival of patients suffering from GC. B7-H4 expression on human primary neutrophils and its regulatory pathway as well as its clinical relevance in GC has not yet been explored
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