Fig. 6: Copper chelation facilitates infiltration and N1-polarization of neutrophils via copper mobilization to exert an anti-tumor response.

a Heatmap comparing the average expression of genes associated with copper metabolism across treatment arms within immune cell clusters. b Heatmap of log₂FC in expression between treatment arms for genes associated with migration and extravasation within the neutrophils cluster. Heatmap of log₂FC in expression for genes associated with (c) N1 anti-tumorigenic or (d) N2 pro-tumorigenic neutrophil phenotypes from control vs. TEPA-treated Th-MYCN tumors within the neutrophils cluster. Data presented in (a–d) were obtained from single-cell RNA sequencing with relevant cell values averaged and scaled. e Gene set enrichment analysis shows top pathways relatively enriched in TEPA-treated neutrophils. The “N1_ANTI_TUMOR NEUTROPHILS” signature was constructed using the N1-associated genes listed in (b). f IncuCyte cell imaging of neuroblastoma cell line SK-N-BE(2)-C transfected with a plasmid encoding tGFP-tagged MT1X protein following 24 h of TEPA treatment (10× objective). Representative image obtained from one independent experiment. Scale bar, 100 µm. g Concentration of copper in conditioned media before and after 30 min incubation with naive neutrophils isolated from healthy donors. Data are presented as mean ± SEM, n = 4/condition, biological replicates (healthy donors), three independent experiments. Significance was calculated using a two-tailed paired t-test with p value displayed in the figure. h qRT-PCR analysis for the expression of genes in human neutrophils associated with intracellular copper (MT1X), migration (S100A8) and pro-inflammatory activation (ISG15) obtained after 30 min incubation in conditioned media as per (e). Data are presented as mean, n = 2 biological replicates (healthy donors), one independent experiment. i Transwell migration assay of neutrophils towards untreated or TEPA-treated SK-N-BE(2)-C cells. Migrated neutrophils were counted using flow cytometry and percentage transmigration was calculated relative to input cells. Data are presented in a violin plot, n = 2 biological replicates (healthy donors) in triplicate, two independent experiments. Data minima and maxima values are as indicated, the median (solid line), and the first and third quartiles (dotted horizontal lines). Significance was calculated using an ordinary one-way ANOVA with Tukey’s post-hoc test with p-value displayed in the figure. j Antibody-dependent cytotoxicity assay against the Kelly neuroblastoma cell line using neutrophils isolated from healthy donors in the presence of anti-GD2 antibody (1 µg/ml) with caspase 3/7 activity quantified after 8 h. Data are presented as mean ± SEM, n = 3/condition, biological replicates (healthy donors) in triplicate, one independent experiment. Significance was calculated using a two-tailed Mann–Whitney U test with p-value displayed in figure. Abbreviations: FC fold change, tGFP turbo green fluorescent protein. Source data are provided as a Source Data file.