Supplementary Figure 4: Intratumoral neutrophils show elevated expression levels of type I IFN-stimulated genes upon CSF-1R blockade.

(a) Hierarchical clustering of the top 400 variable genes between neutrophils isolated from tumors of KEP mice treated with cisplatin/control ab (n=3 biologically independent samples) and cisplatin/anti-CSF-1R (n=4 biologically independent samples). Mice were sacrificed one day after second cisplatin injection. FC: ≥ 1,5; unadjusted p-value: ≤ 0,05. Statistical analysis was performed using two-way ANOVA. (b) Gene set enrichment analysis (GSEA) of Egr2 target genes obtained from RNA-Seq data in tumor-infiltrating neutrophils of anti-CSF-1R-treated KEP mice compared to control ab-treated KEP mice (n=4 animals/group). Enrichment scores were calculated using a weighted Kolmogorov–Smirnov-like statistic. (c) Network visualization of GOEA of the top 100 up-regulated and top 100 down-regulated genes (cisplatin/anti-CSF-1R vs. cisplatin/control ab neutrophils; FC: 1,5, unadjusted p-value: ≤ 0.05) using BiNGO and EnrichmentMap. Red and blue nodes represent the positively and negatively enriched GO-terms, respectively. Node size represents corresponding enrichment p-values (FDR corrected p-value: ≤ 0.05). The genes used as input for the BiNGO analysis are derived from the 2-way ANOVA model. The enrichment score was calculated with a hypergeometric statistical test, multiple testing correction was performed with the Benjamin & Hochberg FDR correction. (d) Top three canonical pathways identified using ingenuity pathway analysis (IPA) enriched in neutrophils isolated from cisplatin/anti-CSF-1R-treated tumors (n=4 biologically independent samples) compared to neutrophils from cisplatin/control ab-treated tumors (n=3 biologically independent samples). Statistical analysis was performed with standard IPA software statistics.