Extended Data Fig. 6: Evaluation of gene signatures related to adenosine signaling pathways across multiple cell types, including SPP1^hi-TAMs, in human and mouse prostate cancers, and functional validation of the role of soluble factors in SPP1^hi-TAM-mediated immunosuppression.

(a) UMAP plots showing enrichment scores for the “Hypoxia” gene signatures across myeloid cells in both human and mouse prostate cancers. (b) Heatmaps depicting the normalized expression levels of ADORA2A and ADORA2B transcripts in the indicated CD8⁺ T cells and NK cells across different disease stages in patients. (c) UMAP plots showing enrichment scores for the “Adenosine Signaling Sig” gene signatures across myeloid cells in both human and mouse prostate cancers. (d) Plots depict the correlations between enrichment scores for the gene signatures “cDC1” (left) or “EEF1A1^hi-TAMs” (right) and enrichment scores for “Adenosine Signaling Sig” across different disease stages in patient samples. Localized disease, HSPC, and mCRPC are in gray, blue, and red, respectively. The best-fit lines are displayed, and individual patient samples are represented by circles. (e) Schematic illustration of transwell assays in which CD8⁺ T cells, with or without α-CD3/28 stimulation, are cultured in the presence or absence of FACS-isolated Spp1^hi-TAMs in opposite chambers to determine whether Spp1^hi-TAMs suppress T-cell proliferation via soluble factors. (f) Quantification of T-cell proliferation in the red-indicated chamber 3 days after culture initiation (P = 0.02, 0.002, and <0.001 for comparisons between the left vs. middle, middle vs. right, and left vs. right, respectively, as shown in the schematic in (e)). Results were normalized to the proliferation of activated T cells cultured alone in the top chamber of the inserts within each experiment. Bars show the mean + SEM from 3 independent experiments, each indicated by a distinct color; symbols represent averages of 2-3 technical replicate wells. Red lines indicate the normalized mean of activated CD8⁺ T cells cultured alone. (g) UMAP plot showing enrichment scores for the “Adenosine Signaling Sig” gene signatures across major cell types in patients. (h) Heatmap depicting the normalized expression levels of ENTPD1, NT5E, and CD38 transcripts in the indicated major cell types in patients. (i) Heatmaps depicting the normalized expression levels of CD38 transcripts in the indicated myeloid subsets both in humans and mice. (j) Heatmaps depicting the normalized expression levels of ENTPD1 and NT5E transcripts in the indicated tumor-associated macrophages across different disease stages in patients. Statistical significance was determined by (d) simple linear regression analyses, and (f) a repeated measures one-way ANOVA with the Sidak correction; P-values: *<0.05, **P < 0.01, ***<0.001. ns, not significant.