In-depth single-cell and bulk-RNA sequencing developed a NETosis-related gene signature affects non-small-cell lung cancer prognosis and tumor microenvironment: results from over 3,000 patients.

Background: Cell death caused by neutrophil extracellular traps (NETs) is known as NETosis. Despite the increasing importance of NETosis in cancer diagnosis and treatment, its role in Non-Small-Cell Lung Cancer (NSCLC) remains unclear.

Methods: A total of 3298 NSCLC patients from different cohorts were included. The AUCell method was used to compute cells' NETosis scores from single-cell RNA-sequencing data. DEGs in sc-RNA dataset were obtained by the Seurat's "FindAllMarkers" function, and DEGs in bulk-RNA dataset were acquired by the DESeq2 package. ConsensusClusterPlus package was used to group patients into different NETosis subtypes, and the Enet algorithm was used to construct the NETosis-Related Riskscore (NETRS). Enrichment analyses were conducted using the GSVA and ClusterProfiler packages. Six distinct algorithms were utilized to evaluate patients' immune cell infiltration level. Patients' SNV and CNV data were analyzed by maftools and GISTIC2.0, respectively. Drug information was obtained from the GDSC1, and predicted by the Oncopredict package. Patient response to immunotherapy was evaluated by the TIDE algorithm in conjunction with the phs000452 immunotherapy cohort. Six NRGs' differential expression was verified using qRT-PCR and immunohistochemistry.

Results: Among all cell types, neutrophils had the highest AUCell score. By Intersecting the DEGs between high and low NETosis classes, DEGs between normal and LUAD tissues, and prognostic related genes, 61 prognostic related NRGs were identified. Based on the 61 NRGs, all LUAD patients can be divided into two clusters, showing different prognostic and TME characteristics. Enet regression identified the NETRS composed of 18 NRGs. NETRS significantly associated with LUAD patients' clinical characteristics, and patients at different NETRS groups showed significant differences on prognosis, TME characteristics, immune-related molecules' expression levels, gene mutation frequencies, response to immunotherapy, and drug sensitivity. Besides, NETRS was more powerful than 20 published gene signatures in predicting LUAD patients' survival. Nine independent cohorts confirmed that NETRS is also valuable in predicting the prognosis of all NSCLC patients. Finally, six NRGs' expression was confirmed using three independent datasets, qRT-PCR and immunohistochemistry.

Conclusion: NETRS can serves as a valuable prognostic indicator for patients with NSCLC, providing insights into the tumor microenvironment and predicting the response to cancer therapy.