Fig 1: ADAM10 inhibition after MI reduces neutrophil chemotaxis-associated gene expression and CX3CL1 serum levels.a Principal components analysis (PCA) of transcriptomes of sham-operated (Sham, n = 3), GI254023X (A10i, n = 4) and DMSO (n = 4) treated mice 3 days after myocardial infarction. Principal components were computed for each sample using gene expression. The first two principal components (PC1 and PC2) are shown. The percent of variance explained by each component is reported in parentheses. b Volcano plot representing the expression changes of all genes. Significantly down- and upregulated genes (FDR < 0.01) are colored navy and magenta, respectively. Genes that do not show significant expression changes are colored gray. Examples of neutrophil chemotaxis-associated genes are labeled with gene names. P-values were calculated using the R packages DESeq2 R (v1.30.1)52 and IHW (1.18.0)53. Functional annotation clustering for the gene ontology (GO) terms c biological process and d molecular function of genes with significantly decreased expression upon A10i treatment (Fisher exact test with Benjamini-Hochberg posttest). e, f Gene set enrichment analysis of genes with significantly decreased expression upon A10i treatment performed on GO terms. Gene set enrichment analysis was performed using the GSEA tool (v4.1.0) of the Broad Institute55 and the DESeq2 R package (v1.30.1)52. g In-depth analysis of the significantly downregulated genes of the indicated functional annotation clusters. h Quantification of CXCL1 (n = 4 vs. 5, P = 0.380952), CXCL5 (n = 8 vs. 8, P = 0.633877), CXCL16 (n = 10 vs. 8, P = 0.572604), CX3CL1 (n = 9 vs. 9, P = 0.00399), IL-1ß (n = 8 vs. 8, P = 0.000155) and IL-6 (n = 6 vs. 5, P = 0.452381), serum levels in A10i and DMSO treated mice 3 days after MI (mean ± SEM, ns not significant, **P < 0.01, ***P < 0.001, two-tailed Mann–Whitney test). i Western blot analysis and j, k quantification of j CX3CL1 and k IL-1ß in heart tissue lysates of sham-operated (Sham) and LAD-ligated (MI) mice 3 days after infarction (n = 5, mean ± SEM, ns not significant, *P < 0.05, **P < 0.01, Kruskal–Wallis test with Dunn’s posttest, exact P-values are provided in the Source Data file). l Western blot analysis and m quantification of CX3CL1 in heart tissue lysates from patients with ischemic cardiomyopathy (ICM) and non-failing controls (NF) (n = 6, mean ± SEM, **P = 0.0031, two-tailed t test). Source data are provided as a Source Data file.
Fig 2: Characterization of liver metastasis and neutrophils infiltration.(A) Representative immunostaining of PanCK and CK19 on liver sections from Pten/Vdr(i)pe-/- mice 12 months AGI. Scale bar 250 μm (large view) and 50 μm (zoom). n = 3 mice. (B) Representative gates for flow cytometry analysis of neutrophils in blood from Pten(i)pe-/- mice 9 months AGI. n = 8. (C) CXCL5 levels determined in protein extracts from DLVPs of Pten/VdrL2/L2, Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice 9 months AGI. n = 3 Pten/VdrL2/L2, n = 5 Pten(i)pe-/- and n = 6 Pten/Vdr(i)pe-/- mice. The boxes extend from the 25th to 75th percentiles, the lines represent the median, and the whiskers go down to the smallest up to the largest value. ns: not significant.
Fig 3: VDR-loss in PTEN-deficient PECs impacts the tumor microenvironment.(A) t-distributed Stochastic Neighbor Embedding (t-SNE) plot of cells from dissociated prostates of Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice, 3 months AGI (n = 1 prostate per genotype). (B) Dot plot representing the expression of cell lineage markers per cluster. (C) KEGG database annotation of the DEGs in the luminal C cluster. FDR: false discovery rate. (D) Heatmap representing the DEGs between FACS-isolated luminal cells of Pten(i)pe-/- and Pten/Vdr(i)pe-/- DLVPs, 3 months AGI, determined by a Wald test corrected for multiple testing using the Benjamini and Hochberg method (n = 3 mice per condition). (E) Venn diagram of DEGs identified by scRNA-seq (Luminal C cluster) and by bulk RNA-seq (FACS-isolated luminal cells). (F) KEGG database annotation of the DEGs obtained by bulk RNAseq analysis. NES: normalized enriched score. (G) Volcano plot of DEGs between luminal cells in DLVPs of Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice, 3 months AGI, determined by a Wald test corrected for multiple testing using the Benjamini and Hochberg method (n = 3 mice per condition). Gray dots ns, p ≥ 0.05. (H) CXCL5 levels determined in protein extracts from DLVPs of Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice 3 months AGI. The dotted line represents the mean of CXCL5 levels in protein extracts from DLVPs of age-matched Pten/VdrL2L2 mice. The boxes extend from the 25th to 75th percentiles, the lines represent the median, and the whiskers go down to the smallest up to the largest value. n = 5 mice/group. The p value determined by Student’s t-test is indicated. (I) Scatter plots of circulating levels of 25(OH)D3 and neutrophils in the serum of PCa patients (n = 77). Estimated linear regression line (black), coefficient of determination (R2), p values and Pearson correlation coefficient (rp) are indicated. (J) Flow cytometry analysis of alive (DAPI negative) Ly-6Ghigh/Ly-6Clow neutrophils quantified in DLVPs 1 and 3 months AGI. n = 8 Pten/VdrL2/L2, Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice 3 months AGI, 4 Pten(i)pe-/- mice 1 month AGI and 6 Pten/Vdr(i)pe-/- mice 1 month AGI. The boxes extend from the 25th to 75th percentiles, the lines represent the median, and the whiskers go down to the smallest up to the largest value. The p value determined by two-way ANOVA with Tukey’s post-hoc are indicated. ns, p ≥ 0.05. The dotted line represents the mean of Ly-6Ghigh/Ly-6Clow neutrophils quantified in DLVPs of age-matched Pten/VdrL2L2 mice. Representative immunostaining of Ly-6G (magenta) on DLVP sections from Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice 1 (K) and 3 (L) months AGI. Scale bar = 100 μm. n = 3 mice. DAPI (blue) stains the nuclei. (M) KEGG database annotation of the DEGs in the T cell cluster. FDR: false discovery rate. (N) Flow cytometry analysis of alive (DAPI-) CD8 + /PD1 + T cells quantified in DLVPs of Pten(i)pe-/- and Pten/Vdr(i)pe-/- mice, 3 months AGI represented as fold of Pten/VdrL2/L2- mice. The boxes extend from the 25th to 75th percentiles, the lines represent the median, and the whiskers go down to the smallest up to the largest value. n = 5 mice/group. The dotted line represents the mean of PD-1 +/CD8 + T cells quantified in DLVPs of age-matched Pten/VdrL2L2 mice. The p value determined by Student’s t-test is indicated. (O) Dot plot representing the expression of Cxcl5, Cd274 and Pdcd1 determined by sc-RNA seq. Source data are available online for this figure.
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