Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: 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.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Immunohistochemistry, Expressing, RNA Sequencing, Activation Assay, Staining, Control, Flow Cytometry, Two Tailed Test, Binding Assay, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: 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 5×10 5 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.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Single Cell, RNA Sequencing, Expressing, Injection, Control, Two Tailed Test, Comparison, Multiplex Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: 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 5×10 5 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 mm 3 . 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.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Cell Culture, Staining, In Vitro, Migration, Membrane, Flow Cytometry, Injection, Two Tailed Test, Comparison, Binding Assay, Single Cell, RNA Sequencing, Immunohistochemistry, Multiplex Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Targeting RRBP1 reverses immune evasion and enhances immunotherapy efficacy via the CXCL10-CXCR3 axis in bladder cancer

doi: 10.1136/jitc-2025-013809

Figure Lengend Snippet: 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 5×10 5 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 mm 3 . 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.

Article Snippet: Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation, and CD8 + T cells were subsequently enriched using MagCellect Human CD8 + T Cell Isolation Kit (R&D Systems) and the MagCellect Mouse CD8 + T Cell Isolation Kit (R&D Systems) according to the manufacturer’s protocols.

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Injection, Staining, Two Tailed Test, Comparison, Binding Assay, Immunohistochemistry, Multiplex Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 inhibits immunosuppression induced by TGFβ or coculture with cancer cells or deficient costimulatory signal. a CD4 + IFNγ + or CD8 + IFNγ + of CD3 (%) in PBMCs with indicated treatment condition. Human PBMCs were treated with indicated condition for 24 h and analyzed by flow cytometry. (One-way ANOVA, Dunnett vs. anti-CD3 + TGFβ) b IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA. Human PBMCs were treated with indicated condition for 72 h. c The population of NKG2D + cells on CD56 dim or CD56 bright NK cells in human PBMCs compared to vehicle. NK2GD + cells (%) were assessed by flow cytometry. d The population of NKG2D + cells on NK92 cell lines (left) compared to vehicle and cellular cytotoxicity of K562 in NK92/K562 (E/T ratio = 2:1) coculture system (right). e Recovery of IFNγ secretion by indicated treatment condition. The amount of IFNγ in PBMC culture supernatant was measured with ELISA and calculated as relative ratio to anti-CD3/CD28 group. f Population of IFNγ-producing CD4 or CD8 T lymphocyte subsets in PBMC with indicated treatment condition analyzed by flow cytometry. Noted aCD3 indicates anti-CD3 stimulation, TU indicates TU2218, and V indicates vactosertib. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 (One-way ANOVA, Dunnett or Tukey, vs. anti-CD3 + TGFβ or anti-CD3/CD28 + TGFβ or TGFβ)

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: TU2218 induces antitumor activity in combination with anti-PD1 in B16F10 syngeneic model. a Experimental design of B16F10-bearing C57BL/6 mouse model. b Tumor volume at indicated time points. *** p ≤ 0.001 vs. TU2218, * p ≤ 0.05 vs. anti-PD1 (Two-way ANOVA) (left). Mean body weight at indicated time points (right). (c) MFI of VCAM-1 in CD31 + populations in tumor tissue. * p ≤ 0.05, ** p ≤ 0.01 vs. vehicle (One-way ANOVA, Tukey) (left). Percent of CD8 + IFNγ + T cells in tumors. * p ≤ 0.05 vs. vehicle (One-way ANOVA, Tukey) (right). Data are shown as mean ± SEM

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Activity Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: ALK5/VEGFR2 dual inhibitor TU2218 alone or in combination with immune checkpoint inhibitors enhances immune-mediated antitumor effects

doi: 10.1007/s00262-024-03777-4

Figure Lengend Snippet: The combination with TU2218 and anti-CTLA4 leads to the formation of immunological memory. a Tumor volume during drug treatment and additional cessation of drug treatment (left). Tumor volume after CT26-rechallenge in tumor-eradicated mice showing CR by anti-CTLA4 + TU2218 administration and age-matched naïve mice at indicated timepoint (middle) or endpoint (right). After tumor eradication or CT26-rechallenge, mice were observed without drug administration to evaluate the formation of antitumor immunological memory. Data are shown as mean ± SEM. *** p ≤ 0.001 vs. age-matched naïve mice group (Two-way ANOVA). b Representative images of mice with CT26-rechallenge at endpoint. c The relative population of CD4 + and CD8 + T effector memory subsets in spleen of mice with CT26-rechallenge at endpoint. The data were obtained in six mice per group * p ≤ 0.05, ** p ≤ 0.01 ( t -test, Two-tailed)

Article Snippet: Erythrocytes were lysed and single-cell suspensions were incubated with Fc block and stained with the following antibodies: anti-mouse CD31-FITC (clone 390, eBioscience), anti-mouse VCAM-1-APC (clone 429, Biolegend) anti-mouse CD45-BV510 (clone 30-F11, BD Bioscience), anti-mouse CD3-APC-Cy7 (clone 145-2C11, Invitrogen), anti-mouse CD8-BB515 (clone 53–6.7, BD Bioscience), and anti-mouse IFNγ-APC (clone XMG1.2, BD Bioscience).

Techniques: Two Tailed Test