Fig 1: RRBP1 inhibition promotes antitumor immunity via the CXCL10-CXCR3 axis in BC. (A) ScRNA-seq data showed the CXCR3 expression of CD8+T cells in shNC and shRRBP1 groups. (B) The correlation between CXCR3 expression and CXCL10 expression or activated CD8+ T cell based on 571 patients from TCGA-BLCA cohort and GSE13507 cohorts. (C) MB49 cells were co-cultured with CD8+ T cells, and tumor cells were stained with crystal violet. (D) Evaluation of the effect of genetic inhibition of RRBP1 on the cytotoxicity of CD8+ T cells in vitro conditioned culture model. (E) Schematic diagram of in vitro CD8+ T-cell migration assays. (F) The number of CD8+ T cells passing through the membrane of a Transwell system was analyzed by flow cytometry. (G–I) C57BL/6 mice were subcutaneously injected with 5×105 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice received intraperitoneal injection of either vehicle or anti-CXCL10 when the tumor volume reached a calculated average of 100 mm3. The tumor sizes (G), volumes (H), and weights (I) were measured. (J) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. (K) Flow cytometric analysis of tumor-infiltrating CD8+ T cells, CXCR3+ CD8+ T cells, IFN-γ+ CD8+ T cells or GZMB+ CD8+ T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test (D, F, I, K) and two-way ANOVA with Tukey’s multiple comparison test (H). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; RRBP1, ribosomal-binding protein 1; scRNA-seq, single-cell RNA sequencing; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry; BLCA, bladder urothelial carcinoma.
Fig 2: Single-cell RNA sequencing reveals the difference of CD8+ T-cell subgroup. The UMAP plot of CD8+ T cells subpopulation, color-coded by cell cluster and cell type. (A) The expression of markers in each CD8+ T cells subpopulation. (B) Bar plot showed the proportion of CD8+ T cells subpopulation in the shNC and shRRBP1 groups. (C) The percentage of each CD8+ T-cell clusters in shNC and shRRBP1 groups. (D) Heatmap showed the differentially activated pathway among all the CD8+ T-cell clusters. (E) The differentially expressed genes in CD8+ T cells between shNC and shRRBP1 groups. (F) KEGG analysis for differentially expressed genes showed the enrichment of immune-associated pathways. (G, H) mIHC and flow cytometric analysis displayed the tumor-infiltrating IFN-γ+ or GZMB+ CD8+ T cells in shNC or shRRBP1 tumor tissues. Scale bar: 20 µm. (I–K) C57BL/6 mice were subcutaneously injected with 5×105 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Isotype control (IgG) or anti-mouse CD8 antibody administered on days –6, –3, and –1 before tumor challenge, with the same dose repeated on days 7, 9 and 11 after tumor challenge. Tumor sizes (I), volumes (J), and weight (K) were measured. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test (H, K) and two-way ANOVA with Tukey’s multiple comparison test (J). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. ANOVA, analysis of variance; GZMB, Granzyme B; IFN, interferon; TEX, exhausted T cells; UMAP, Uniform Manifold Approximation and Projection; mIHC, multiplex immunohistochemistry; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Fig 3: RRBP1 inhibition enhances response to anti-PD-L1 therapy in BC. (A–D) The protein expression of surface PD-L1 was analyzed in BC cells or tumor tissues by flow cytometry after RRBP1 inhibition and was shown as the mean fluorescence intensity. (E–G) C57BL/6 mice were subcutaneously injected with 5×105 stable MB49 cells (shNC or shRRBP1 cells) (n=6). Tumor-bearing mice were received intraperitoneal injection of either vehicle or anti-PD-L1 antibody when the tumor volume reached a calculated average of 100 mm3. The tumor sizes (E), volumes (F), and weights (G) were measured. (H) Representative images of IHC and mIHC staining for CD8, CXCR3, CXCL10, IFN-γ, GZMB in different tumor tissues. (I) Flow cytometric analysis of tumor-infiltrating CD8+ T cells, CXCR3+ CD8+ T cells, IFN-γ+ CD8+ T cells or GZMB+ CD8+ T cells in distinct tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using unpaired two-tailed t-test (B, D, G, I) and two-way ANOVA with Tukey’s multiple comparison test (F). The data presented represent on one or three independent experiments. *p<0.01, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BC, bladder cancer; GZMB, Granzyme B; IFN, interferon; PD-L1, programmed death-ligand 1; RRBP1, ribosomal-binding protein 1; IHC, immunohistochemistry; mIHC, multiplex immunohistochemistry.
Fig 4: Tumor-intrinsic RRBP1 inhibition triggers antitumor immunity. (A) Representative images of IHC staining for RRBP1 and CD8+ T cells in BC samples. (B) The correlation between RRBP1 expression and CD8+ T-cell infiltration was analyzed based on 96 patients from in-house BC cohort. Scale bar: 50 µm. (C) Representative images of IHC staining for RRBP1 expression in PD, SD, PR, and CR samples. Scale bar: 50 µm. (D) Bar plot showed the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. (E) Volcano plot of RNA-seq data for shNC or shRRBP1 tumors (n=3). Differentially expressed genes were identified with the threshold of |log2 (fold change) | >1 and FDR<0.05. (F) GSEA for DEGs showed the activation of immune-associated pathways in shRRBP1 tumors in the RNA-seq data. (G) Representative images of IHC and mIHC staining for RRBP1 and CD8+ T cells in shNC, shRRBP1, control or radezolid tumor tissues. Expression levels of the indicated proteins were displayed. Scale bar: 20 µm. (H, I) Flow cytometry showed the percentages of CD8+ T cells in CD3+ cells in shNC, shRRBP1, control or radezolid tumor tissues. Data are represented as mean means±SD. Statistical analysis was performed using Spearman correlation analysis (B), unpaired two-tailed t-test (I). ****p<0.0001. BC, bladder cancer; CR, complete response; FDR, false discovery rate; progressive disease; PR, partial response; PD-L1, programmed death-ligand 1; RNA-seq, RNA sequencing; RRB1, ribosomal-binding protein 1; SD, stable disease; IHC, immunohistochemistry; GSEA, gene set enrichment analysis; DEGs, differentially expressed genes; mIHC, multiplex immunohistochemistry.
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