Fig. 1: Profiling the immune cell infiltration in prostate cancer models.

a, Experimental scheme for the profiling of the composition of the immune microenvironment by multiparametric flow cytometry and scRNA-seq in the prostate of transgenic Pten^pc−/−Trp53^pc−/− (tumor) and healthy (nontumor) mice. b, FACS analysis of macrophages in Pten^pc−/−Trp53^pc−/− transgenic prostate compared to nontumor tissue. Quantification of immune infiltrating cells (n = 4 nontumor-bearing mice and n = 6 Pten^pc−/−Trp53^pc−/− mice). Total macrophages were gated on CD45⁺ cells. The percentages of CD206⁻MHCII⁺, CD206⁺MHCII^+/− and ARG1-expressing macrophages were gated on F4/80⁺CD11b⁺. c, Uniform manifold approximation and projection (UMAP) of CD45⁺ cells in Pten^pc−/−Trp53^pc−/− transgenic prostate. Fourteen clusters characterized by lineage-specific and cluster-enriched genes were identified by integrated analysis. d, UMAP of scRNA-seq data from macrophages from Pten^pc−/−Trp53^pc−/− transgenic prostate (n = 2). e, Trajectory analysis of macrophages using Monocle3 inference methods. f, FACS analysis of macrophages in murine prostate orthotopically injected with Pten^−/−Trp53^−/− cells compared to macrophages in nontumor tissue (n = 4 mice per group). g, GSEA showing downregulated biological pathways (pathways down) and upregulated biological pathways (pathways up) in TAMs. The size of each dot indicates the number of enriched genes relative to the pathway of interest. The fraction of genes represents the proportion of the total number of genes in the pathway that were significantly enriched. h, Heat map illustrating all the differentially expressed genes according to bulk mRNA-seq from nonconditioned macrophages (untreated (Untr.), left) and macrophages exposed to conditioned media from Pten^−/−Trp53^−/− cells (CM-tr., right). i, Volcano plot showing differentially expressed genes in CM-tr. macrophages compared to Untr. Genes are colored according to their log₂FC value (blue, log₂FC ≤ −0.5; red, log₂FC ≥ 0.5). j, Proliferation of CD8⁺ T cells exposed to supernatant from Untr. and CM-tr. macrophages: bar graph shows the number of divisions (n = 3 per group). k, Scratch assay: graph shows the quantification of distance (μm) covered by tumor cells over time after exposure to supernatant from Untr. or CM-tr. macrophages (n = 9 per group). l, FACS analysis of macrophages upon exposure to Pten^−/−Trp53^−/− conditioned media: percentages of cells were gated on F4/80⁺CD11b⁺ cells (n = 5 per group). Statistical analyses were performed using two-tailed unpaired Student’s t-test. Values are presented as the mean ± s.e.m. Schematic in a created using BioRender.com. NK, natural killer; DC, dendritic cells; PMN, polymorphonuclear neutrophils; Mono-Mac, monocyte–macrophages; Inflam-Macs, inflammatory macrophages; Angio-Macs, angiogenic macrophages; LA-Macs, lipid-laden macrophages; RTM-Macs; Reg-Macs, Cl6 regulatory macrophages; INF-Macs, macrophages defined by Cl3 interferon-related genes; LPS, lipopolysaccharide; ECM, extracellular matrix; Pos., positive; NA, not applicable; MFI, mean fluorescence intensity; NS, not significant.