Biphasic, time-dependent neutrophil biology in glioblastoma revealed by in vivo survival and flow cytometry with single-cell transcriptomic corroboration

Introduction: Tumor-associated neutrophils (TANs) are abundant in glioblastoma (GBM), yet their functions are phase-dependent. We investigated whether TANs are differentially transcribed from peripheral blood neutrophils (PBNs), if they acquire antigen-presenting functions in situ, and if neutrophil depletion timing influences survival and intratumoral immunity Methods: Transcriptomics: Integrated analysis of publicly deposited recent human GBM data sets (bulk RNA-seq and scRNA-seq; Seurat standard pipeline, stringent QC, integration/UMAP, Wilcoxon DE). Prespecified modules: co-stimulation (CD83/CD86/CD40/ICOSLG), MHC-II (HLA-DRB3/A, DPA1/DPB1), and antigen-processing/chaperones (CD74, CALR, PSME2, HLA-DMA/DMB). In vivo survival: Orthotopic CT2A and GL261; anti-Ly6G (1A8) or isotype on two schedules, (day −1 pre-implantation through ≥day 14) and delayed (start day +8). Kaplan-Meier/log-rank; Cox models (HR, 95% CI). Ex vivo flow (day 8): Bead-normalized spectral cytometry; CD45+ leukocytes; CD11b+ myeloids (Ly6G+ neutrophils; Ly6C^hi monocytes), F4/80+ TAMs with MHC-II, CD3+/CD8+ T cells. Two-sided Mann-Whitney; FDR where indicated. Ly6G epitope masking identified; depletion confirmed by CD11b+SSC^hi back-gating and weekly blood counts. Results: Transcriptomics: TANs vs PBNs showed coherent upregulation of APC/co-stimulatory programs (CD83/CD86/CD40/ICOSLG; HLA-DR/DP; CD74/CALR/PSME2/HLA-DMA/DMB), in line with dendritic-like, non-cytotoxic TAN states. Survival (biphasic): Previous anti-Ly6G impaired outcomes, CT2A (n=9/arm) median 17 vs 23 days; HR 1.95 (1.10-3.46), p=0.018. Subsequent depletion abrogated this penalty, CT2A HR 1.23 (0.68-2.22), p=0.49; Flow (day 8): Early-depleted tumors contained more Ly6C^hi monocytes (of CD45+: 29% vs 18%, p=0.006), fewer CD8+ T cells (3.2% vs 6.1%, p=0.011), and lower CD8:Ly6C^hi ratio (0.11 vs 0.36, p=0.004). Directional but non-significant aggregates: CD11b+ myeloids (68% vs 55%, p=0.07), CD3+T cells (9% vs 14%, p=0.09), reduced MHC-II on F4/80+ TAMs (p=0.08), reduced CD11c⁺MHC-II^hi APCs (q≈0.12). Conclusion: Human TANs take up APC/co-stimulatory programs, and timing is critical: pre-implantation neutrophil depletion imposes a myeloid-skewed, antigen-poor, T-cell-depleted setting, augments survival, while late targeting diminishes this. These observations favor phase-specific TAN modulation. maintenance or re-education of early TANs and suppression of late suppressive programs selectively, to regulate biomarker-based GBM immunotherapies.