Expression, regulation, function and clinical significance of B7-H6 on neutrophils in human gastric cancer

Neutrophils are conspicuous components of gastric cancer (GC) tumors, increasing with tumor progression and poor patient survival. However, the phenotype, regulation, function and clinical relevance of neutrophils in human GC are presently unknown. We used flow cytometry analyses to examine levels and phenotype of neutrophils in samples from 50 patients with GC. Kaplan-Meier plots for patient survival were performed using the log-rank test, and multivariate analysis of prognostic factors for patient survival was performed using the Cox proportional hazards model. Neutrophils were isolated, stimulated and/or cultured for regulation and function assays. We found that GC patients showed a significantly higher neutrophil infiltration in tumors, and that neutrophil infiltration was positively associated with tumor progression but negatively correlated with patient survival. Most tumor-infiltrating neutrophils showed an activated CD54⁺ phenotype and expressed high level B7-H6. Tumor tissue culture supernatants from GC patients inhibited neutrophil apoptosis and induced the expression of CD54 and B7-H6 on neutrophils in time-dependent and dose-dependent manners. Intratumoral CD54⁺ neutrophils and B7-H6⁺ neutrophils positively correlated with increased G-CSF detection ex vivo; and in vitro both G-CSF and tumor-derived G-CSF induced the expression of CD54 and B7-H6 on neutrophils via NF-κB signaling pathway activation. Furthermore, blockade of B7-H6 promoted the apoptosis of tumor-infiltrating and tumor-conditioned neutrophils, and shortened their lifespan. Importantly, intratumoral B7-H6⁺ neutrophils increased with tumor progression and predicted poor patient survival. Our results illuminate a novel mechanism of B7-H6 expression on tumor-activated neutrophils in GC, and also suggest B7-H6⁺ neutrophils would be novel potential biomarkers in GC.