Fig. 4: Chronic exposure to tumor-derived G-CSF alters ^Gneutrophil ageing and promotes accumulation of tissue‑toxic CD62L^low subpopulations.

a Mice were injected subcutaneously with MOPC or ^GMOPC cells (n = 5 mice in each group). On day 14, animals were sacrificed, lungs were collected, viable Ly6G⁺ neutrophils (gating strategy Fig. S3a) were isolated and analyzed by proteomics. Volcano plot of proteins differentially expressed in ^Gneutrophils according to SNR > 1, highlighting upregulated proteins involved in the regulation of neutrophil ageing and apoptosis. b–k Isolated viable Ly6G⁺ lung ^Gneutrophils stratified by CD62L surface expression were evaluated in vitro in the absence or presence of Pseudomonas aeruginosa (MOI 10). b Decreased apoptosis in ^Gneutrophils (isolated from n = 4 mice) in comparison to lung neutrophils from MOPC-bearing (n = 4) and tumor-free mice (n = 4). c Reduced Casp3 gene expression in ^Gneutrophils (isolated from n = 7 mice) in comparison to lung neutrophils from MOPC-bearing (n = 4) and tumor-free mice (n = 4). d Accumulation of CD62L^{low G}neutrophils in lungs in comparison to lung neutrophils from MOPC-bearing and tumor-free mice (n = 8 mice in each group). e Increased degranulation of CD62L^{low G}neutrophils in comparison to CD62L^{high G}neutrophils (isolated from n = 4 mice) (SSC signal). f Elevated Mmp9 release by CD62L^{low G}neutrophils in comparison to CD62L^{high G}neutrophils (isolated from n = 3 mice). g Increased cytotoxicity of CD62L^{low G}neutrophils in comparison to CD62L^{high G}neutrophils (isolated from n = 3 mice) toward epithelial tumor cells. h Reduced phagocytic capacity of CD62L^{low G}neutrophils in comparison to CD62L^{high G}neutrophils (isolated from n = 10 mice). i Representative image showing reduced NET release by CD62L^{low G}neutrophils (orange, histone 1; blue, DAPI; scale bar 100 µm). j Decreased proportion of NET‑producing cells among CD62L^{low G}neutrophils (n = 4) in comparison to CD62L^{high G}neutrophils (isolated from n = 5 mice). k Reduced NET length released by CD62L^{low G}neutrophils (n = 61 NETs) in comparison to CD62L^{high G}neutrophils (n = 104 NETs). l G‑CSF release by HNSCC (in tumor‑conditioned medium) negatively correlates with CD62L expression on tumor‑associated neutrophils (n = 12 tumors). m In HNSCC patients, oral G‑CSF concentrations negatively correlate with CD62L expression on salivary CD66b⁺ viable neutrophils (n = 29 oral rinses). n–p Circulating neutrophils were isolated from healthy donors, CD62L^high and CD62L^low subsets were compared. n Elevated spontaneous ROS production in CD62L^low neutrophils in comparison to CD62L^high neutrophils isolated from n = 4 healthy volunteers. o Reduced NET generation in CD62L^low neutrophils in comparison to CD62L^high neutrophils isolated from n = 4 healthy volunteers. p Reduced phagocytic capacity of CD62L^low neutrophils in comparison to CD62L^high neutrophils isolated from n = 4 healthy volunteers. G‑CSF granulocyte colony‑stimulating factor, Co control tumor‑free mice, MOPC mice bearing non-G‑CSF‑producing tumors, ^GMOPC mice bearing G‑CSF‑producing tumors, Casp3 caspase‑3, SSC side scatter, Mmp9 matrix metalloproteinase, NETs neutrophil extracellular traps, ROS reactive oxygen species, CFU colony‑forming units. Data are shown as means with individual values (biological replicates). Statistical tests: Kruskal–Wallis with Bonferroni correction for multiple‑group comparisons and two-sided Mann–Whitney for two‑group comparisons (b–l); Spearman correlation (m); paired Student’s t‑test (n–p). *p < 0.05, **p < 0.01, ***p < 0.001. Source data and exact p-values are provided as a Source Data file.