Neutrophil infiltration associated genes on the prognosis and tumor immune microenvironment of lung adenocarcinoma

Abstract

The neutrophils exhibit both anti-tumor and pro-tumor effects in cancers. The correlation between neutrophils and tumor development in lung adenocarcinoma (LUAD) is still uncertain, possibly due to a lack of specific neutrophil infiltration evaluation methods. In this study, we identified 30 hub genes that were significantly associated with neutrophil infiltration in LUAD through data mining, survival analysis, and multiple tumor-infiltrating immune cells (TICs) analysis, including TIMER, CIBERSORT, QUANTISEQ, XCELL, and MCPCOUNTER. Consensus clustering analysis showed that these 30 hub genes were correlated with clinical features in LUAD. We further developed a neutrophil scoring system based on these hub genes. The neutrophil score was significantly correlated with prognosis and tumor immune microenvironment (TIME) in LUAD. It was also positively associated with PD-L1 expression and negatively associated with tumor mutational burden (TMB). When combined with the neutrophil score, the predictive capacity of PD-L1 and TMB for prognosis was significantly improved. Thus, the 30 hub genes might play an essential role in the interaction of neutrophils and LUAD, and the neutrophil scoring system might effectually assess the infiltration of neutrophils. Furthermore, we verified the expression of these 30 genes in the LUAD tumor tissues collected from our department. We further found that overexpressed TNFAIP6 and TLR6 and downregulated P2RY13, SCARF1, DPEP2, PRAM1, CYP27A1, CFP, GPX3, and NCF1 in LUAD tissue might be potentially associated with neutrophils pro-tumor effects. The following in vitro experiments demonstrated that TNFAIP6 and TLR6 were significantly overexpressed, and P2RY13 and CYP27A1 were significantly downregulated in LUAD cell lines, compared to BEAS-2B cells. Knocking down TNFAIP6 in A549 and PC9 resulted in the upregulation of FAS, CCL3, and ICAM-1, and the downregulation of CCL2, CXCR4, and VEGF-A in neutrophils when co-culturing with the conditioned medium (CM) from LUAD cells. Knocking down TNFAIP6 in LUAD also led to an elevated early apoptosis rate of neutrophils. Therefore, overexpressed TNFAIP6 in LUAD cancer cells might lead to neutrophils "N2" polarization, which exhibited pro-tumor effects. Further research based on the genes identified in this pilot study might shed light on neutrophils' effects on LUAD in the future.

Keywords: LUAD; bioinformatics analysis; neutrophil infiltration; survival analysis; tumor associated neutrophil; tumor immune microenvironment.