Fig. 2

Distinct immune milieu in different phenotypes and immature myeloid cells are enriched in resistant tumors. a Schematic for tumor dissociation, isolation of tumor infiltrated leukocytes and Drop-seq based scRNA sequencing. Ctrl, vehicle-treated control; Ab, anti-HER2/Neu antibody; Pal, CDK4/6 inhibitor Palbociclib; TILs, tumor infiltrated leukocytes. b Clustering of 1444 TILs derived from Ctrl, APP, and APR tumors and t-SNE plot colored by clusters (left) and annotation of TIL-clusters on t-SNE plot colored by treatment groups (right). c Abundance of each cluster (as clustered and annotated in b) in TILs with indicated treatment. d Analysis of immune cell populations among CD45⁺ TILs by mass cytometry. e Representative images and quantification of CD3 and CD8 immunofluorescence staining for Ctrl, APP, and APR tumors. Arrows indicate CD3 and CD8 double-positive cells. Scale bar, 50 μm. f Single-cell transcriptional analysis of tumor-produced cytokines and chemokines. The size of each circle reflects the percentage of cells in a treatment group where the gene is detected, and the color intensity reflects the average expression level within each treatment group. g Schematic for tumor dissociation, isolation of CD45⁺ TILs and Gr1⁺ cells and scRNA-seq. h t-SNE plot of scRNA-seq data containing 1153 CD45⁺ TILs and 1318 Gr1⁺ cells. i Overlay of marker genes for Gr1⁺ cell population identified from experiment of g on t-SNE plots derived from b and g, respectively. j Violin plot showing distribution of ssGSEA enrichment score among TIL-clusters identified from b. MDSCs signature generated by experiment as shown in g was used for enrichment analysis. k Growth of APR tumors by adding Gr1 antibody and ICB. APR tumors were transplanted to recipient MMTV-neu mice and treated with Ab + Pal first to acquire the resistance phenotype (left) and then adding IgG (n = 6), anti-Gr1 (n = 5) or anti-Gr1+ICB (n = 7) treatments were followed (right). Each point represents a single cell in b, h, i, j. Error bars represent SD in d and SEM in e. P-value in d, e, k by one-way ANOVA with Tukey’s test