SPP1-driven immunometabolic reprogramming of tumor-associated neutrophils in glioblastoma

Introduction: Tumor-associated neutrophils (TANs) in glioblastoma (GBM) are heterogeneous and poorly captured by the classical N1/N2 dichotomy. We characterized TAN states relative to peripheral blood neutrophils (PBNs) and wondered if a discrete, targetable program drives their pro-tumoral phenotype. Methods: Public scRNA-seq datasets were reanalyzed (GSM8380727, GSM8380728: TANs 15,000 cells; PBNs 10,000 cells; 36,601 genes). Standard Seurat workflow was applied (stringent QC; SCTransform; integration; PCA/UMAP; shared-nearest-neighbor clustering). Differential expression used Wilcoxon rank-sum with Bonferroni FDR (adj. p<0.05, |log2FC|>0.25). Module scoring examined prespecified programs: antigen presentation/co-stimulation, interferon/cytotoxicity, lipid/stress adaptation. Functional enrichment and protein-protein networks were assessed via GSEA and STRING. Results: Integration revealed six tumor-associated Neutrophil states beyond the classical N1/N2 paradigm. Among them, an SPP1+(osteopontin-high) (avg log2FC ~10.7; adj. p≈0) population was particularly transcriptionally associated with lipid processing genes (APOE, APOC1, APOC2) and chemokine master regulators (CCL3, CCL4) of immune cell recruitment and metabolic reprogramming. SPP1+ TANs featured repression of cytotoxicity pathways and induction of oxidative and lipid metabolism modules, pointing toward a shift from antimicrobial to tissue-remodeling and tumor-supporting activities. Network analysis revealed two large hubs: SPP1-APOE/APOC (lipid remodeling) and CCL3/CCL4 (immune signaling) that are bridged by metabolic stress genes (CTSB, EIF1B) into a cohesive immunometabolic circuit. Conclusion: Single-cell analysis characterizes GBM TAN heterogeneity and implicates an SPP1-centered, APC-like neutrophil axis bridging lipid regulation (APOE/APOCs) and chemokine signaling (CCL3/CCL4). This osteopontin-driven axis establishes a mechanistic basis for TAN-mediated tumor support and nominates SPP1 and its lipid-chemokine network as actionable targets to reprogram TANs for anti-tumor activity.This is among the first studies to define an osteopontin-driven immunometabolic axis in pro-tumoral neutrophils, establishing a mechanistic framework for future neutrophil-targeted immunotherapies in glioblastoma.