Figure 3.

IFN-stimulated, Ly6E^(hi) neutrophils mark response to αPD1 in 4T1 breast cancer

10X scRNA-seq was performed on GR1⁺ cells obtained from parental (4T1_P) (non-responsive) and mutagenized (4T1_M) (responsive) 4T1 breast cancer tumors (n = 3 mice pooled/group). (A) UMAP plot of 2886 filtered, GR1⁺ neutrophils (4T1_P = 681 cells, 4T1_M = 2185 cells), with cells colored based on differential abundance score. Two significantly enriched, cellular neighborhoods (dotted lines) are highlighted (see also Figure S2C). The top 10, most significant marker genes of each neighborhood are listed (FDR < 0.001, log₂ fold-change > 1.5). Monocytic cells (not shown) were discarded from the analysis (see: Figure S2).

(B) Trajectory analysis for 12 distinct, GR1⁺ granulocytic clusters. Solid black line = trajectory lineages, which form the basis of the pseudotemporal ordering as inferred by partition-graph based abstraction (PAGA). Black arrows = simplified RNA-velocity (for raw data, see Figure S2D).

(C) Top: A histogram of binned cell frequencies as a function of aligned pseudotime. Smoothed distributions, generated by loess regression, are overlaid. Significance was assessed by means of two-sample KS-test. Bottom: A heatmap of normalized, binned enrichment scores for all gene modules that display a significant association with pseudotime (FDR < 0.01). Only gene-modules common to both lineages are shown.

(D) Boxplots showing the concentration of IFNγ, TNFα and IFNα within untreated 4T1 tumor lysates (n = 4-5 mice/group).

(E) Binned, normalized expression of Ly6E. Data were imputed for visual clarity.

(F and G) Frequencies of Ly6E^(hi) neutrophils, as determined by flow cytometry (n = 5-10 mice/group), in 4T1 tumors (F); and the blood of 4T1 bearing mice (G); For the gating strategy see Figure S3A. In (D, F, and G), significance was assessed by means of a one-way Mann-Whitney test (NS, p > 0.01; ^∗, p < 0.01; ^∗∗, p < 0.001, ^∗∗∗, p < 0.0001).