Single-cell Atlas Reveals a Pro-metastatic RELB+ Neutrophil-Myeloid Subset Underlying Lymph Node Metastasis in EGFR-wildtype LUAD

Fasheng LiXiao YangAng LiYutao PangHongfei ZhangLiyao Lin^*Dong Wu^*

• Affiliated Hospital of Guangdong Medical University, Zhanjiang, China

Background: Lymph node metastasis is a critical event in the progression of EGFR wildtype lung adenocarcinoma (LUAD), a subtype lacking effective targeted therapies and associated with poor prognosis. The specific myeloid cell subsets and molecular mechanisms driving LNM in this context remain poorly understood. Methods: To elucidate the cellular drivers of LNM, we performed an integrated analysis of multi-cohort single-cell RNA sequencing data from EGFR-wildtype LUAD patients and bulk transcriptomic data from The Cancer Genome Atlas (TCGA). Pathological lymph node status from the bulk RNA-seq cohort was mapped to the single-cell transcriptomes using the UCell algorithm. Myeloid cell heterogeneity was analyzed via sub-clustering, and the transcription factor regulon activity was inferred using pySCENIC. The functional role of the key regulator RELB was validated using siRNA-mediated knockdown in neutrophil-tumor cell co-culture systems, assessed by qPCR, proliferation, clonogenic, migration, and invasion assays. A RELB signature score was constructed based on its target genes and validated in independent cohorts for prognostic, immunotherapeutic, and drug sensitivity assessment. Results: Myeloid cells were significantly enriched in the LNM group. The process of sub-clustering identified a pro-metastatic subset of ELANE-positive neutrophils that was specifically expanded in LNM samples, linked to advanced N stage, higher clinical stage, and shorter overall survival. RELB was identified as a central regulator of ELANE+Neu through transcription factor analysis, showing significantly heightened regulon activity in LNM. Using a neutrophil-tumor cell co-culture system, we found that RELB knockdown in neutrophils attenuated inflammatory signaling in tumor cells and subsequently reduced their proliferative, clonogenic, migratory, and invasive capacities. A high score of the RELB signature was a strong predictor of poor prognosis and was associated with pro-tumorigenic pathways, and the creation of an immunosuppressive microenvironment. Additionally, RELB scores were associated with lower tumor mutational burden, poorer response to immunotherapy, different drug sensitivity patterns, and increased expression of several antibody-drug conjugate targets. Conclusion: The study highlights a pro-metastatic ELANE+ neutrophil subpopulation, with RELB acting as its primary transcriptional regulator. The RELB signature may serve as a biomarker for prognosis and treatment response prediction, indicating a potential target for precision treatment in EGFR wildtype LUAD.