Additional file 1 of KMT9 drives T cell exclusion and dysfunction by promoting PMN-MDSCs infiltration and ARG1 expression in prostate cancer

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Supplementary Material 1: Supplemental Fig. 1: KMT9 controls expression of PMN-MDSC-chemoattractants in PCa. A, Table depicting numbers and clinical features of human normal prostate and prostate tumour samples in the TCGA-PRAD cohort. B, Box-Whisker plots with min to max values showing KMT9β mRNA expression in healthy human prostate (blue, n=52) and prostate tumour samples (red, n=500). P value obtained by unpaired Student’s test. C, Kaplan-Meier progression-free survival analysis for patients with prostate tumours expressing high (red, n=240) or low (blue, n=241) levels of KMT9β mRNA. HR = hazard ratio. A-C, Data were extracted from TCGA-PRAD. D, Box-Whisker plots with min to max values displaying Kmt9a expression in human prostate tumours with different genetic aberrations. The number of tumour samples (n) is indicated. E, Representative view of Ctrl and Kmt9a KO mice prostates. F-H Ctrl and Kmt9a KO mice prostates were haematoxylin stained (f) or analysed by immunohistochemistry (G) and Western blot (H) using anti-KMT9a antibody. Rabbit (r) IgG (G) and GAPDH (h) served as controls. I, J, Western blot analysis of Pten/Trp53 KO prostate using the indicated antibodies. GAPDH served as control. K, L, Four colour multiplex immunofluorescence staining (K) and box-Whisker plots with min and max values representing signal intensity quantification for the indicated proteins (L) in epithelial cells in Ctrl prostates and tumours cells in Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice prostates. Monolayers of epithelial cells in normal prostates (Ctrl) are indicated by arrows. Areas of tumour cells in Pten/Trp53 KO and Pten/Trp53/Kmt9a KO are delimited by dashed line. Scale bars represent 40 μM. P values obtained by one-way ANOVA test are indicated. M-O, Analysis of protein expression of the indicated chemokines (M) by Western blot (N) of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice prostates using a proteome chemokine array. Western blot signal intensities were quantified (O). p, UMAP plot showing the different cell populations identified by scRNA-seq in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice prostates. Q, R, Dot blots showing expression levels of markers used to identify the different cells population in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates by scRNA-seq. S, Dot blot showing expression levels of Kmt9a in the different cell populations identified by scRNA-seq in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates. T, Dot plot showing expression levels of Kmt9a in luminal cells of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates identified by scRNA-seq. U, V, Violin plots showing expression levels of Pten (U) and Trp53 (V) in luminal cells of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates. W, Loss of genes in Pten/Trp53 KO and Pten/Trp53/Kmt9a KO prostate luminal cells was validated by Western blot using the indicated antibodies. GAPDH and extracts of normal prostate luminal cells (Ctrl) were used as controls. X, diagram showing the number of genes identified by ChIP-seq in Pten/Trp53 KO cells displaying KMT9a localization at gene promoters. Y, Expression of NLS-KMT9a in Pten/Trp53/Kmt9a KO prostate luminal cells transduced with pLenti-NLS-KMT9a was verified by Western blot analysis using the indicated antibodies. a-Tubulin served as control. Z, AA, Analysis of protein expression of the indicated chemokines by Western blot (Z) of Ctrl, Pten/Trp53 KO, Pten/Trp53/Kmt9a KO, and Pten/Trp53/Kmt9a KO/NLS-KMT9a mouse prostate luminal cells using a proteome chemokine array. Western blot signal intensities were quantified (AA). Data represent means +/- s.d. (O, AA). P values obtained by unpaired Student T test (B, D) and by two-way ANOVA test (L) are indicated. Supplemental Fig. 2: KMT9 regulates composition and spatial distribution of immune cell populations within the TIME. A, B, Multiparametric flow cytometry analysis of blood leukocytes of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice. Percentages of blood myeloid cells (CD45+ CD11b+) (A) and neutrophils (CD45+ CD11b+ Ly6G+) (B) are shown. Data represent means +s.d. n, number of mice analysed. C, D, Representative flow cytometry plots of peripheral blood myeloid cells (C) and neutrophils (D) in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice. E-I, Representative flow cytometry plots of prostate tumour infiltrating myeloid cells (CD45+ CD11b+) (E), M-MDSCs (CD45+ CD11b+ Ly6G- Ly6Chi) and PMN-MDSCs (CD45+ CD11b+ Ly6G+ Ly6Clo) (F), CD45+ CD3+ CD4+ CD25+ cells (G), cytotoxic T cells (CD45+ CD3+ CD8a+) (H), activated cytotoxic T cells (CD45+ CD3+ CD8a+ GZMB+) (I). J, QRT-PCR analysis showing relative mRNA levels of the indicated genes in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice prostates. Data represent means +s.d. n=3 represents the number of biologically independent samples. K, Cross-correlation matrix for markers and corresponding cell types identified by single-cell phenotyping in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates. L, UMAP plot showing the cell populations identified by single -cell phenotyping in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates. M, Dendrogram and unsupervised hierarchical clustering heatmap of cell types identified by single-cell spatial phenotyping in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice prostates. N, Distribution of normal epithelial, tumour epithelial, and proliferating cells in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates unravelled by single-cell phenotyping. Cell types and antibodies used for staining are indicated. Scale bars represent 100 µm. O, Bar graphs representing distribution and percentages of normal and tumour epithelial cell populations, proliferating cells, and fibroblast in Ctrl (n=5), Pten/Trp53 KO (n=5), and Pten/Trp53/Kmt9a KO (n=3) prostates identified by single-cell spatial phenotyping. n, represents the number of prostate samples analysed. Data represent means +s.d. P values were obtained by one-way ANOVA test (A, B, J) and by two-way ANOVA test (O). Supplemental Fig. 3: Kmt9a loss combined with CXCR2 inhibition increases inhibition of prostate tumour growth. A, Dot blot showing expression levels of Cxcr2 in different cell populations identified in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9aKO prostates. B, Bar graphs representing distribution and percentages of PMN-MDSCs and CXCR2+ PMN-MDSCs in Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO prostates identified by flow cytometry (n=4). n, represent the number of prostates analysed. C, Schematic protocol for treatment of mice with tamoxifen (Tam) and the CXCR2 inhibitor SB225002. D, Overall weight of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice after treatment with SB225002 or vehicle. n=4 mice per condition. Data represent means +/- s.d. E, Representative images of prostates of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice treated with vehicle or SB225002. Prostate structures of Ctrl, Pten/Trp53 KO, and Pten/Trp53/Kmt9a KO mice treated with vehicle or SB225002 revealed by staining with anti-SMA and anti-Pan-KRT antibodies. Scale bars represent 1000 µm. F, Bar graphs representing the percentages of CD8a+ T cells in Pten/Trp53 KO and Pten/Trp53/Kmt9a KO prostates of mice treated with vehicle or SB225002 identified by single-cell spatial phenotyping. n, represents the number of prostate samples analysed. Supplemental Fig. 4: KMT9-regulated expression of ARG1 impairs T cell cytotoxicity in PCa. A-F, Growth of Pten/Trp53 KO (A,D), Pten/Trp53/Kmt9a KO (B, E), and Pten/Trp53/Kmt9a KO/NLS-KMT9a (C, F) prostate tumour cells alone or cocultured in presence of autologous (A-C) or syngeneic (D-F) T cells activated with anti-CD3/CD28 beads. n=3 represents the number of biologically independent samples. Data represent means +/- s.d. P values were obtained by two-way ANOVA test. G, ChIP-seq tracks showing KMT9a at representative genes in Pten/Trp53 KO mouse prostate tumour cells. Supplemental Fig. 5: Kmt9a loss combine

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