Neutrophils recruited by NKX2-1 suppression via activation of CXCLs/CXCR2 axis promote lung adenocarcinoma progression

Background: Lung adenocarcinoma (LUAD) progression is dependent on the immune tumor microenvironment through paracrine signaling. Lineage-specific transcription NKX2-1 is a well-developed pathology marker to define LUAD with progressive impact in patients. However, the involvement of NKX2-1 in modeling the tumor immune microenvironment is still unclear. Here, we demonstrated that NKX2-1-low tumors expedite tumor progression in LUAD through increased tumor-promoting neutrophils. Method: Single-cell RNA sequencing and Visium in situ capturing profiling were used to characterize the infiltration of neutrophils in NKX2-1-low LUAD tumors at high resolution. Clinical relevance of NKX2-1 expression and disease status were evaluated by immunohistochemical analysis of LUAD tissue arrays and the overall survival analysis was performed by TCGA databases. qRT-PCR validated the level of CXC chemokines, and proinflammatory genes in LUAD cells, meanwhile chemokine array confirmed the secretion of chemokines. Mechanistically, ATAC-seq was used to confirm the modulatory role of NKX2-1 on the chromatin accessibility of CXC chemokine genes. Results: NKX2-1 downregulation was observed in high-grade LUAD with increased neutrophil recruitment and infiltration. NKX2-1 knockdown promoted the expression and secretion of CXCL1, CXCL2, CXCL3, and CXCL5 in LUAD cells. Mechanistically, ATAC-seq revealed the restrictive regulation of NKX2-1 on the promoters of CXCL1, CXCL2 and CXCL5. Single-cell RNA sequencing and Visium in situ capturing revealed the infiltrated neutrophil had strong cell-cell communication through the activation of CXCLs/CXCR2 signaling with increased tumor growth and vice versa when inhibited with CXCR2 antagonist SB225002. Conclusion: This study revealed that NKX2-1 negatively regulates the infiltration of tumor-promoting neutrophils by suppressing CXCLs/CXCR2-dependent mechanisms. Hence, targeting CXCR2 in NKX2-1-low tumors is a potential antitumor therapy that improves LUAD patient outcomes. In order to examine the suppressive function of NKX2-1 in the advancement of lung adenocarcinoma, we generated H1975 and HCC827 cell lines with depleted NKX2-1 expression using shRNA technology. We then performed gene expression profiling analysis including bulk, single cell, and spatial RNA sequencing. In order to identify the specific DNA region affected by the presence of NKX2-1, ATAC-seq analysis was performed on H1975 cells under conditions of NKX2-1 knockdown and control. To examine the impact of cytokines on neutrophils, we conducted a co-culture experiment involving HL60 neutrophil cells with either control or NKX2-1 knockdown H1975 cells, followed by bulk RNA sequencing analysis.