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: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Large expansion of phenotypically defined sx-MDSCs comprised mainly of M-MDSC and PMN-MDSCs immediately after surgery (POD1). ( A ) Representative flow plots showing CD33 versus Lineage (CD3/CD56/CD19) for baseline (left) and POD1 (right) PBMCs. ( B ) Representative flow plots gated down on CD33 + Lin − cells showing CD15 versus CD14 expression. ( C ) Healthy controls (n=15), Baseline, and POD1 patients (n=44) proportion of CD33 + Lin − cells (left) and MDSC subsets (right). ( D ) The MFI of HLA-DR gated on M-MDSCs (left) and the proportion of M-MDSCs that are HLA-DR lo (right) in healthy controls, baseline and POD1 patients with cancer. ( E ) Representative histograms of common M-MDSC and PMN-MDSC markers, before and after surgery. Statistical analysis was performed using Wilcoxon matched pairs signed-rank test or Kruskal-Wallis with Dunn’s post test. **** p≤0.0001. FMO, fluorescence minus one control; HLA-DR, human leukocyte antigen - DR isotype; E-MDSC, early stage-MDSC; M-MDSC, monocytic-MDSC; MDSC, myeloid derived suppressor cell; sx-MDSC, surgery-induced MDSC, MFI; mean fluorescence instensity; PBMC, peripheral blood mononuclear cell; PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Fluorescence, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Sx-MDSCs suppress NK92-MI cytotoxicity. ( A ) Schematic of MDSC-NK92 co-culture suppression assay with CP450-labeled K562 target cells. ( B ) NK92-MIs were co-cultured at increasing ratios with CD33+ cells for 6 or 24 hours (in the absence of K562s) to assess CD33+ cell death by NK92-MI (left). Sx-MDSCs from POD1 patient samples (n=10) were co-cultured with NK92-MI cells and NK cytotoxicity was measured as % dead K562 (right). ( C ) % suppression was calculated as the reduction of NK cytotoxicity, normalized to NK cells alone (ratio=0). CD33+ cells isolated from baseline and patient-matched POD1 blood were co-cultured with NK92-MI and K562 targets to measure their suppressive capacity (n=5). ( D ) Representative images of sorted cells stained with Giemsa Wright. ( E ) Bulk Sx-MDSCs, M-MDSCs, PMN-MDSCs, and HDN from POD1 patients (n=3) co-cultured with NK92-MI to measure effect on NK cytotoxicity. The NK92-MI alone control group is indicated by the broken horizontal line. Statistical analysis used Kruskal-Wallis with Dunn’s post-test. ****p≤0.0001. HDN, high-density neutrophils; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Co-Culture Assay, Suppression Assay, Labeling, Cell Culture, Isolation, Staining, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: scRNA-seq of cryopreserved PBMCs before and after surgery reveals drastically altered monocyte/myeloid cell expression profiles on POD1. ( A ) Schematic showing PBMCs (cryopreserved) from six matched baseline and POD1 patients were processed for multiplexed scRNA-seq with the 10x Genomics Chromium platform. ( B ) Dot plot displaying the relative expression of the top three marker genes (x-axis) of each cluster (y-axis). ( C ) UMAP plot of scRNA-seq data. Each point corresponds to a single cell and is colored by cluster. ( D ) Identical UMAP embedding as in ( C ), with Baseline and POD1 cells labeled. ( E ) UMAP plot of the CD14+ and CD16+ monocyte population at baseline and POD1. ( F ) GSEA showing the NES of the top upregulated and downregulated pathways. All gene sets are significantly enriched (FDR<0.05). ( G ) UMAP plot showing activity of an M-MDSC gene set from Alshetaiwai et al ; in monocyte populations in Baseline and POD1 samples. ( H ) NMF plots of gene expression programs for NMF 1, 2, 3 and 4 (see for all NMF programs). ( I ) A heatmap of selected genes driving the various NMF gene expression programs (z-score transformed; ranked according to NMF1). FDR, false discovery rate; GSEA, gene set enrichment analysis; M-MDSC, monocytic-myeloid-derived suppressor cell; NES, normalized enrichment scores; NMF, non-negative matrix factorization; PBMC, peripheral blood mononuclear cell; POD1, postoperative day 1; scRNA-seq, single-cell RNA sequencing; UMAP, uniform manifold approximation and projection.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Expressing, Marker, Labeling, Activity Assay, Gene Expression, Transformation Assay, Derivative Assay, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K inhibitors reverse the suppressive effects of sx-MDSCs on NK cells. ( A ) A library of 147 small molecules covering the major cellular signaling pathways was screened (n=4 experiments) at 1 µM in the MDSC-NK suppression assay. Compounds that improved NK cell cytotoxicity (blue, left y-axis) by >50% from DMSO control (black dotted line), without impacting NK cell viability (red, right y-axis), were considered hits. LY294002 (purple circle) improved cytotoxicity in all screens and was the top hit in 3/4 screens. ( B ) LY294002 improves NKC (n=6). ( C ) Dose response of LY294002 and the effect on NKC and K562 viability. ( D ) pan-PI3K and ( E ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in NK92—MDSC suppression assays. ( F ) pan-PI3K and ( G ) PI3K-γ specific inhibitors (IPI-549, TG100-115) in MDSC suppression assays with primary healthy donor NK cells. Statistical analysis used one-way ANOVA with Dunnett’s multiple comparisons test. ***p<0.0001. ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; MDSC, myeloid-derived suppressor cell; NK, natural killer; sx-MDSC, surgery-induced MDSC.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Protein-Protein interactions, Suppression Assay, Control, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: PI3K pathway activation in postoperative MDSCs and functional impact of PI3K inhibition. ( A ) Cohort of differentially expressed genes from baseline versus POD1 MDSCs. ( B ) Box plot inference of PI3K pathway activity in baseline versus POD1 MDSCs. ( C ) Pathway diagram of PI3K signaling and Akt phosphorylation. Phosphorylation status of AKT at the T308 (left) and S473 residue (right) (n=5). ( D ) Effect of PI3K inhibitors on pAKT (T308) MFI (top), MDSC-NK suppression (middle), and NKC (bottom). ( E ) Effect of IPI-549 on POD1 expression of anti-inflammatory versus pro-inflammatory mRNA transcripts, normalized to baseline. Statistical analysis used Wilcoxon matched pairs signed-rank tests and one-way ANOVA with Dunnett’s multiple comparisons test (vs DMSO control). ****p>0.0001. ANOVA, analysis of variance; ARG1, arginase-1; CCL, CC chemokine ligand; DMSO, dimethyl sulfoxide; IL, interleukin; MDSC, myeloid-derived suppressor cell; mRNA, messenger RNA; MFI, mean fluorescence intensity; NK, natural killer; POD1, postoperative day 1; TGF, transforming growth factor; TNF, tumor necrosis factor.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Activation Assay, Functional Assay, Inhibition, Activity Assay, Phospho-proteomics, Residue, Expressing, Control, Derivative Assay, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Preventing surgery-induced natural killer cell suppression and metastases by inhibiting PI3K-gamma signaling in myeloid-derived suppressor cells

doi: 10.1136/jitc-2025-013304

Figure Lengend Snippet: Blockade of PI3Kγ signaling in sx-MDSCs reduces NK cell suppression and metastatic disease in mouse models of surgical stress. ( A ) Ex vivo effect of PI3K inhibitors on pAKT (T308) phosphorylation in splenic MDSCs from C57Bl/6 mice (n=4). ( B ) Effect of preoperative in vivo administration of PI3K inhibitors on pAKT (T308) phosphorylation measured on POD1 in splenic MDSCs (n=4). ( C ) Ex vivo effect of PI3K inhibitors on MDSC-NK %suppression (n=3). ( D ) Effect of preoperative in vivo administration of PI3K-γ inhibitors on sx-MDSC suppressive capacity. ( E ) Adoptive transfer of representative images of lungs is shown. One-way ANOVA with Holm-Sidak or two-way ANOVA with Dunnett’s multiple comparisons tests were performed. ****p<0.0001. ANOVA, analysis of variance; M-MDSC, monocytic-MDSC; MDSC, myeloid-derived suppressor cell; MFI, mean fluorescence intensity; NK, natural killer; sx-MDSC, surgery-induced MDSC, PMN-MDSC, polymorphonuclear-MDSC; POD1, postoperative day 1.

Article Snippet: Sx-MDSCs were isolated using the MDSC Isolation Kit (Miltenyi #130-094-538) from surgically stressed mice on POD1.

Techniques: Ex Vivo, Phospho-proteomics, In Vivo, Adoptive Transfer Assay, Derivative Assay, Fluorescence