Extended Data Fig. 7: Assessment of CD39 and CD73 levels in Spp1^hi-TAMs in mouse prostate cancer, and investigation of the role of the IL-1R pathway in immunosuppression mediated by SPP1^hi-TAMs both in humans and mice.

(a) Heatmap depicting the normalized expression levels of Entpd1 and Nt5e transcripts in the indicated tumor-associated macrophages and monocytes across different disease stages in mice. (b-c) (b) Representative flow cytometry plots and (c) quantification of fold changes in the levels of cell surface CD73 expressed on Spp1^hi-TAMs from CRPC relative to HSPC. HSPC, CRPC, and isotype control stains are shaded in blue, red, and gray, respectively in (b). In (c), P = 0.35, 0.01, and 0.01 for CD163^hi-TAM, CX3CR1^hi-TAM, and Spp1^hi-TAM, respectively. Bars represent the mean + SEM from 4 independent experiments, each indicated by a distinct color; symbols represent individual mice from each experiment. The red line indicates a fold change of 1. (d) Pathways associated with inflammation, significantly enriched in SPP1^hi-TAMs compared to other myeloid cells in both human (red) and mouse (gray) prostate cancers, were identified using the Enrichr bioinformatics tool with GO Biological Process 2023 gene sets. (e) UMAP plots showing enrichment scores for the “Tumor-promoting Inflammation” gene signatures across myeloid cells in both human and mouse prostate cancers. (f) Plot depicting the correlation between enrichment scores for the gene signatures “SPP1^hi-TAMs” and enrichment scores for “Tumor-promoting Inflammation Sig” across different disease stages in patient samples. Localized disease, HSPC, and mCRPC are in gray, blue, and red, respectively. The best-fit line is displayed, and individual patient samples are represented by circles. (g) Bar plots showing decreased suppression of activated splenic CD8⁺ T cells when co-cultured with Spp1^hi-TAMs in the presence of an anti-IL-1R antibody (10 μg/ml) compared to isotype-treated cultures (P = 0.01). Bars show mean + SEM from 3 independent experiments, each indicated by a distinct color; symbols represent averages of 2-3 technical replicate wells. (h) Bar plots showing the percentage change in suppression of activated splenic CD8⁺ T cells mediated by Spp1^hi-TAMs in the presence of either ciforadenant (a A2AR inhibitor; 10 μM), anti-IL-1R antibody, or a combination of both. Bars show mean - SEM from 3 independent experiments, each indicated by a distinct color; symbols represent averages of 2-3 technical replicate wells. Statistical significance was determined by (c, g) two-sided one sample t-tests, (d) Fisher’s exact tests or the hypergeometric tests with the Benjamini-Hochberg correction, and (f) simple linear regression analysis; P-values: *<0.05. ns, not significant.