Fig. 4.

Gene expression analysis of tumor-infiltrating immune cells and ECs. (A) Venn diagram of genes differentially expressed (up- and down-regulated compared to control IgG) according to RNA-seq in CD8⁺, F4/80⁺ (MØ), CD4⁺, and CD31⁺ cells sorted from orthotopic MMTV-PyMT tumors at day 5 posttreatment (4 mice per cell type per treatment). (B) Heatmaps of genes differentially and uniquely expressed (from A) after anti-VEGFA/Ang2/CD40 treatment in CD8⁺ cells (Left, 164 genes) and macrophages (Right, 368 genes). Expression of these genes is shown across the 4 treatment groups. (C and D) Pathway analysis scores for gene transcripts in CD31⁺, CD4⁺, CD8⁺, and F4/80⁺ cells, according to bulk RNA sequencing. (E) The qPCR analysis of Il12, Il1b, Cxcl9, and Cxcl10 from whole-tumor lysates of MMTV-PyMT mice at day 5 posttreatment. Data indicate the mean fold change over control (IgG treatment) after normalization to the average of Hprt and Gapdh housekeeping genes. Each data point represents one mouse. Data indicate mean values ± SEM. Statistical analyses by 1-way ANOVA with Tukey’s correction for multiple comparisons (black) or pairwise Student’s t test (red) unless otherwise indicated in Dataset S2. The number of mice employed in each experiment is reported in Dataset S2.