Journal: Oncology Reports

Article Title: High expression of eIF4A1 promotes angiogenesis through the NF-κB/VEGFA pathway and predicts poor prognosis in gastric cancer

doi: 10.3892/or.2025.8951

Figure Lengend Snippet: eIF4A1 is upregulated in gastric cancer tissues. (A) Pan-cancer analysis of eIF4A1 expression was performed using UALCAN. (B) Expression of eIF4A1 in gastric cancer tissues and in peripheral normal tissues from patients with gastric cancer was assessed using Home For Researchers database. (C) Expression of eIF4A1 in late-stage GC tissues, early-stage GC tissues and in peripheral normal tissues. (D) Statistics of eIF4A1 expression in different types of gastric tissues. (E) eIF4A1 protein in GC tissues and normal gastric tissues: (a) Well-differentiated adenocarcinoma, (b) papillary adenocarcinoma, (c) poorly-differentiated adenocarcinoma, (d) mucinous adenocarcinoma, (e) CG, (f) pericarcinomatous mucosa tissue, (g) IM and (h) HGIN. *P<0.05, ***P<0.001, ****P<0.0001. CG, chronic gastritis; eIF4A1, eukaryotic translation initiation factor 4A; HGIN, high-grade intraepithelial neoplasia; IM, intestinal metaplasia; LGIN, low-grade intraepithelial neoplasia.

Article Snippet: Lentiviruses containing eIF4A1 cDNA (pLV3-CMV-eIF4A1-CopGFP-Puro) or short hairpin (sh)RNA against eIF4A1 (pLV3-U6-eIF4A1-shRNA-CopGFP-Puro) were produced by GeneCopoeia, Inc, as well as their controls (NC and shNC).

Techniques: Expressing

Journal: Oncology Reports

Article Title: High expression of eIF4A1 promotes angiogenesis through the NF-κB/VEGFA pathway and predicts poor prognosis in gastric cancer

doi: 10.3892/or.2025.8951

Figure Lengend Snippet: Survival curves confirming the prognostic value of eIF4A1 and TNM stage in GC. (A) High eIF4A1 expression was associated with overall survival in patients with GC. (B) TNM stage was associated with the overall survival of patients with GC. GC, gastric cancer; TNM, Tumor-Node-Metastasis.

Article Snippet: Lentiviruses containing eIF4A1 cDNA (pLV3-CMV-eIF4A1-CopGFP-Puro) or short hairpin (sh)RNA against eIF4A1 (pLV3-U6-eIF4A1-shRNA-CopGFP-Puro) were produced by GeneCopoeia, Inc, as well as their controls (NC and shNC).

Techniques: Expressing

Journal: Oncology Reports

Article Title: High expression of eIF4A1 promotes angiogenesis through the NF-κB/VEGFA pathway and predicts poor prognosis in gastric cancer

doi: 10.3892/or.2025.8951

Figure Lengend Snippet: Dysregulation of eIF4A1 affects the angiogenic activity of gastric cancer cells. (A) Western blotting detected the expression of eIF4A1 in cells after lentivirus infection. (B) Cell Counting Kit 8 assay assessed the proliferation of HUVECs treated with CM derived from specific cells. The optical density at 0 h was set at 100%. (C) Representative images (left) and quantification (right) of wound healing assay of HUVECs treated with CM derived from specific cells. (D) Representative images (left) and quantification (right) of Transwell assay of HUVECs treated with CM derived from specific cells. (E) Representative images (left) and quantification (right) of angiogenesis assay of HUVECs treated with CM derived from specific cells. (F) Representative image of the tumors. (G) Growth curves of the tumors and (H) weight of the tumors. (I) Representative images (left) and quantification (right) of CD31 staining in tissues from a subcutaneous xenograft tumor model. # P<0.05 vs. NC; & P<0.05 vs. shNC; *P<0.05. CM, conditioned media; eIF4A1, eukaryotic translation initiation factor 4A; HUVECs, human umbilical cord endothelial cells; MVD, microvessel density; NC, negative control; sh, short hairpin.

Article Snippet: Lentiviruses containing eIF4A1 cDNA (pLV3-CMV-eIF4A1-CopGFP-Puro) or short hairpin (sh)RNA against eIF4A1 (pLV3-U6-eIF4A1-shRNA-CopGFP-Puro) were produced by GeneCopoeia, Inc, as well as their controls (NC and shNC).

Techniques: Activity Assay, Western Blot, Expressing, Infection, Cell Counting, Derivative Assay, Wound Healing Assay, Transwell Assay, Angiogenesis Assay, Staining, Negative Control

Journal: Oncology Reports

Article Title: High expression of eIF4A1 promotes angiogenesis through the NF-κB/VEGFA pathway and predicts poor prognosis in gastric cancer

doi: 10.3892/or.2025.8951

Figure Lengend Snippet: eIF4A1 affects regulation of the TME and angiogenesis. Association between eIF4A1 expression and various component cells in the TME was detected using R package (4.3.3) in (A) GSE62254 , (B) GSE15459 and (C) GSE84426 . Part of the left panel was enlarged and presented in the right panel. (D) Correlations between eIF4A1 expression and CD31, VEGFA, NFKB1 and NFKB2. (E) mRNA levels of VEGF family genes were detected by reverse transcription-quantitative polymerase chain reaction. (F) Changes in VEGEF and NF-κB expression after eIF4A1 overexpression and knockdown were detected by western blotting. *P<0.05. eIF4A1, eukaryotic translation initiation factor 4A; NC, negative control; sh, short hairpin; TME, tumor microenvironment. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Lentiviruses containing eIF4A1 cDNA (pLV3-CMV-eIF4A1-CopGFP-Puro) or short hairpin (sh)RNA against eIF4A1 (pLV3-U6-eIF4A1-shRNA-CopGFP-Puro) were produced by GeneCopoeia, Inc, as well as their controls (NC and shNC).

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Over Expression, Knockdown, Western Blot, Negative Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: In vivo CRISPR-cas9 screening identifies ENO1 as an immunotherapy resistance modulator in BC. (A) Schematic of the in vivo genome-wide CRISPR cas9 screening to identify genes associated with anti-PD-L1 responsiveness. (B) The in vivo efficacy of anti-PD-L1 antibody in C57BL/6J mice with xenografts of MB49 cells. The mice were treated with control IgG or anti-PD-L1 antibody for 14 days, and average tumor sizes for each group are plotted. (C) The relative enrichment of genes in the genome-wide CRISPR cas9 screening for anti-PD-L1 responsiveness. Total 1083 genes were depleted in anti-PD-L1-treated tumors (Beta-score <-2). (D) Volcano plot of RNA-seq for progressive disease (PD) or complete response (CR) human BC tumor tissues ( n = 3 biologically independent samples per group). Differentially expressed genes were identified with the threshold of |log2(fold change)| >1 and false discovery rate (FDR) < 0.05. (E) On integrating the CRISPR cas9 screening and RNA-seq results, 14 genes (ENO1, ANLN, CCL4, CDK1, FABP3, KLRC1, LAMP3, NUAK2, PTPRCAP, PTPRZ1, RORC, SGPL1, TMEM141, USP2) were finally identified. (G) Representative images of IHC staining for ENO1 and CD8 + T in human BC samples. Scale bars: 50 μm (H) Correlation between protein levels of ENO1 and CD8 + T cell infiltration was determined by IHC staining ( P < 0.001, r =-0.4306, n = 96). (I) Representative images of IHC staining for ENO1 in progressive disease (PD), stable disease (SD), partial response (PR), and complete response (CR) samples. (J) Bar plot illustrating the response rates of anti-PD-L1 therapy. Blue bars represent CR/PR, Red bars represent PD/SD. Scale bars: 50 μm Two-tailed χ 2 test. (K , L) Tumor growth curves and tumor sizes of anti-PD-L1 resistant MB49 cells ( n = 6 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. ns, no significant. (M) Western blotting demonstrating ENO1 expression in anti-PD-L1 resistance and parental MB49 cells. (N) Kaplan-Meier overall survival (OS) curve for 195 BC patients in the IMvigor210 cohort stratified by high or low ENO1 expression. Two-sided log-rank test

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: In Vivo, CRISPR, Genome Wide, Control, RNA Sequencing, Immunohistochemistry, Two Tailed Test, Western Blot, Expressing

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: Tumor-intrinsic ENO1 deficiency inhibits tumorigenesis and triggers antitumor immunity. (A-C) Subcutaneous inoculation of WT or ENO1-KO cells into C57BL/6J mice ( n = 6 per group) followed by measurement of tumor sizes ( A ), volumes ( B ), and weights ( C ). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (D) Kaplan-Meier survival curves of C57BL/6J mice inoculated with WT or ENO1-KO MB49 cells. Tumor volumes exceeding 1500mm 3 were considered events ( n = 6 per group). Two-sided log-rank test. (E-H) In vivo bioluminescence imaging of a bladder orthotopic tumor model established by subcutaneously inoculating luciferase-labeled WT or ENO1 MB49 cells into the bladder of C57BL/6J mice ( n = 6 per group). The signals were measured on 20 days using an IVIS Spectrum In Vivo imaging system ( E ), and tumor sizes ( F ), volumes ( G) , and weight ( H ) were measured. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (I-K) Subcutaneous inoculation of MB49 cells into C57BL/6J mice followed by treatment with 8 mg/kg ENOblock every other day ( n = 6 per group). Tumor sizes ( I ), volumes ( J ), and weight (K) were measured. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (L) Volcano plot of RNA-seq data for WT or ENO1-KO tumors inoculated into C57BL/6J mice ( n = 3 biologically independent samples per group). Differentially expressed genes were identified with the threshold of |log2(fold change)| >1 and false discovery rate (FDR) < 0.05. (M) KEGG analysis for differentially expressed genes to show immune-related pathways. (N) GSEA revealing immune-associated pathways correlated with ENO1 expression in the RNA-seq dataset. (O) Heatmap of RNA-seq data for interleukin and chemokine family member expression. (P-R) Representative images of IHC and mIHC staining for ENO1 and CD8 + T cells in WT or ENO1-KO tumor tissues ( n = 6 per group). Expression levels of the indicated proteins displayed. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (S) Flow cytometric analysis of tumor-infiltrating CD8 + T cells in WT or ENO1-KO tumor tissues ( n = 6 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: In Vivo, Imaging, Luciferase, Labeling, In Vivo Imaging, RNA Sequencing, Expressing, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: Single-cell RNA sequencing reveals the difference of CD8 + T cell subgroups of ENO1-WT versus ENO1-KO tumors. (A) The umap plot displaying all cells isolated from tumor tissues from the WT and KO groups, with each color representing a distinct cell cluster and cell type. (B) Bar plot showing the proportion of each cell subpopulation in the WT and KO groups. (C) The t-SNE plot of CD45 + cells subpopulations, color coded by cell cluster and cell type. (D) Bar plot showing the proportion of each CD45 + cells subpopulations subpopulation in the WT and KO groups. (E) The umap plot of CD8 + T cells subpopulations, color coded by cell cluster and cell type. (F) Bar plot showing the proportion of each CD8 + T-cell clusters. (G) Heatmap of differentially activated pathway among all the CD8 + T-cell clusters. (H , I) Flow cytometric analysis and mIHC of tumor-infiltrating IFN-γ + or GZMB + CD8 + T cells in WT or ENO1-KO tumor tissues ( n = 6 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (K-M) 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 ( K ), volumes ( L) , and weight ( M ) were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean values ± SD. *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: RNA Sequencing, Isolation, Control, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: Tumor intrinsic ENO1 inhibits the function of CD8 + T cells via SPP1. (A) Volcano plot of RNA-seq data for CD8 + T cells that cocultured with ENO1-WT or ENO1-KO MB49 cells ( n = 3 biologically independent samples per group). Differentially expressed genes were identified with the threshold of |log2(fold change)| >1 and false discovery rate (FDR) < 0.05. (B) Genes encoding cytotoxic molecules (e.g., PRF1, TNF, CD8A, CD8B1, GZMA, GZMB, GZMC, GZMD, GZME, and GZMF) expression levels in CD8 + T cells. (C) KEGG enrichment analysis for differentially expressed genes to show immune-related pathways. (D) CellChat to was used to delineate cell-cell communications in the single-cell RNA sequencing dataset. (E) Apoptosis assay results of WT, ENO-KO, or ENO1-KO + oeSPP1 cells in the presence of mouse CD8 + T cells. n = 3 biologically independent samples per group. One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean values ± SD. (F) Flow cytometry analysis showed Ki67, IFN-γ, GZMB expression of mouse CD8 + T cells that were cocultured with WT, ENO-KO, ENO1-KO + oeSPP1 cells. n = 3 biologically independent samples per group. One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean values ± SD. (G) A schematic representation of a transwell coculture assay involving mouse CD8 + T cells combined with His-SPP1 MB49 cells. (H) Co-immunoprecipitation assays of CD8 + T cells were performed using anti-His antibody. n = 3 biologically independent samples per group. (I) Western blotting assay detected ITGA4 and ITGB1 protein expression in mouse CD8 + T cells with or without recombinant mouse SPP1 protein treatment. n = 3 biologically independent samples per group. (J) Western blotting assay detected ITGA4 and ITGB1 protein expression in mouse CD8 + T cells cocultured with WT or ENO1-KO cells, with or without recombinant mouse SPP1 protein. n = 3 biologically independent samples per group. (K) Flow cytometric analysis of Ki67, IFN-γ and GZMB expression in siITGA4 or siITGB1 CD8 + T cells treated with recombinant mouse SPP1 protein. n = 3 biologically independent samples per group. (L) Flow cytometric analysis of Ki67, IFN-γ and GZMB expression in siITGA4 or siITGB1 CD8 + T cells cocultured with MB49 cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (M) IHC staining of Ki67 or CD8 expression in ENO1-KO or ENO1-KO + oeSPP1 tumors ( n = 5 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (N) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, IFN-γ + or GZMB + CD8 + T cells in ENO1-KO or ENO1-KO + oeSPP1 tumor tissues ( n = 5 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (O-Q) C57BL/6J mice ( n = 6 per group) were subcutaneously inoculated with WT or ENO1-KO cells. Mice received intraperitoneally treated with 10 mg/Kg SPP1 inhibitor every others day after tumor inoculation. Tumor sizes ( O ), volumes ( P ), and weight ( Q ) were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean values ± SD. (R-U) Representative images of IHC and mIHC staining for Ki67, CD8, IFN-γ, GZMB in different tumor tissues ( n = 6 per group). Expression levels of the indicated proteins displayed. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. (V) Flow cytometric analysis of tumor-infiltrating CD8 + T cells, IFN-γ + or GZMB + CD8 + T cells in distinct tumor tissues ( n = 6 per group). Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: RNA Sequencing, Expressing, Apoptosis Assay, Flow Cytometry, Co-culture Assay, Immunoprecipitation, Western Blot, Recombinant, Immunohistochemistry, Comparison, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: ENO1 promotes the polarization of M2 TAMs via SPP1 in TME. (A) The umap plot of M1 (antitumoral) and M2 (protumoral) macrophages from scRNA-seq data. (B) Bar plot presenting the proportion of M1 and M2 macrophages subpopulations. (C) Heatmap of differentially activated pathway between M1 and M2 macrophages. (D-G) Flow cytometric analysis and IHC staining of CD86 (M1 TAM-like marker) or CD206 (M2 TAM-like marker) expression in WT and ENO1-KO tumors ( n = 6 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (H) Flow cytometric analysis of CD206 expression in M1-polarized BMDMs cocultured with WT or ENO1-KO cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (I) The mRNA expression of M2 TAM-like marker gene (CD206, Arg1) in M1-polarized BMDMs cocultured with WT or ENO1-KO cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (J) Violin plot showing the SPP1 mRNA expression in CD11b + macrophage from WT versus ENO1-KO tumors. (K) The mRNA expression of M2 TAM-like marker gene (CD206, Arg1) in M1-polarized BMDMs cocultured with or without mouse SPP1 recombinant protein. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (L) The mRNA expression of M2 TAM-like marker gene (CD206, Arg1) and M1-like marker gene (CD86, IL-1α) in M1-polarized BMDMs cocultured with WT or ENO1-KO cells, with or without SPP1 overexpression. n = 3 biologically independent samples per group. One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean ± SD. (M , N) Flow cytometric analysis of CD206 expression in M1-polarized BMDMs cocultured with WT or ENO1-KO cells, with or without SPP1 overexpression or recombinant mouse SPP1 protein. n = 3 biologically independent samples per group. One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean ± SD. (O) Flow cytometric analysis of tumor-infiltrating CD206 + macrophages in ENO1-KO or ENO1-KO + oeSPP1 tumor tissues ( n = 5 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (P) A schematic representation of a transwell coculture assay involving ex vivo programmed M1-polarized BMDMs combined with Vector or His-SPP1 MB49 cells. (Q) Co-immunoprecipitation assays of M1-polarized BMDMs were performed using anti-His antibody. n = 3 biologically independent samples per group. (R) Immunofluorescence of M1-polarized BMDMs cells with a His antibody (green), a ITGA4 or ITGB1 antibody (red), and a DAPI antibody (blue). Scale bar: 25 μm. n = 3 biologically independent samples per group. (S) Western blotting assay detected ITGA4 and ITGB1 protein expression in M1-polarized BMDMs with or without recombinant mouse SPP1 protein treatment. (T) Western blotting assay detected ITGA4 and ITGB1 protein expression in M1-polarized BMDMs cocultured with WT or ENO1-KO cells, with or without recombinant mouse SPP1 protein. n = 3 biologically independent samples per group. (U) Flow cytometric analysis of CD206 expression in siITGA4 or siITGB1 M1-polarized BMDMs treated with recombinant mouse SPP1 protein. n = 3 biologically independent samples per group. (V) Flow cytometric analysis of CD206 expression in siITGA4 or siITGB1 M1-polarized BMDMs cocultured with MB49 cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: Immunohistochemistry, Marker, Expressing, Recombinant, Over Expression, Co-culture Assay, Ex Vivo, Plasmid Preparation, Immunoprecipitation, Immunofluorescence, Western Blot

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: M2 TAMs inhibits the function of CD8 + T cells via SPP1. A , B) Flow cytometry analysis showed Ki67, IFN-γ, GZMB expression of mouse/human CD8 + T cells that were cocultured with M2-polarized BMDM ( A ) or M2-polarized THP-1 cells (B). n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (C) The ELISA assay detected the concentration of SPP1 in the coculture system. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (D , E) Flow cytometry analysis showed Ki67, IFN-γ, GZMB expression of mouse/human CD8 + T cells that were cocultured with siSPP1 M2-polarized BMDM or M2-polarized THP-1 cells with or without recombinant spp1 protein. n = 3 biologically independent samples per group. One-way ANOVA with Dunnett’s multiple comparisons test. Data are presented as mean values ± SD. (F) The ELSA assay detected SPP1 concentration in the supernatant of M2-polarized BMDM (A) or M2-polarized THP-1 cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (G-I) C57BL/6J mice ( n = 6 per group) were subcutaneously co-injected with MB49 cells and M2-ploraized BMDM. Mice received intraperitoneally treated with 10 mg/Kg SPP1 inhibitor every other day after tumor inoculation. Tumor sizes ( G ), volumes ( H ), and weight ( I ) were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean values ± SD. (K-N) Representative images of mIHC and IHC staining for SPP1, CD206, CD8, IFN-γ, GZMB in WT or ENO1-KO tumor tissues ( n = 6 per group). Expression levels of the indicated proteins displayed. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. (O) Flow cytometric analysis of tumor-infiltrating CD206 + cells, CD8 + T cells, IFN-γ + or GZMB + CD8 + T cells in distinct tumor tissues ( n = 6 per group). Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Recombinant, Injection, Comparison, Immunohistochemistry

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: ENO1 enhances the stability of SPP1 mRNA in BC cells. (A) Correlation analysis between ENO1 expression and SPP1 expression in the TCGA and GEO datasets. Two-tailed Spearman correlation is reported. (B) Representative images of IHC staining for ENO1 and SPP1 in human BC samples. Scale bars: 50 μm. (C) Correlation between protein levels of ENO1 and SPP1 was determined by IHC staining. Two-tailed Spearman correlation is reported. (D) Kaplan-Meier curves of overall survival for BC patients stratified by ENO1 and SPP1 levels in the TCGA and GEO datasets. Data were analyzed using the log-rank test. (E) Kaplan-Meier curves of overall survival for BC patients stratified by ENO1 and SPP1 expression levels in our in-house cohort. Data were analyzed using the log-rank test. (F-I) Knockdown of ENO1 significant inhibit the SPP1 mRNA expression and protein expression in MB49 cells and T24 cells. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (J , K) IHC staining of SPP1 expression in WT and ENO1-KO tumors ( n = 6 per group). Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (L) Distribution of ENO1-binding peaks across SPP1 by integrative genomics Viewer. (M , N) RIP analyses of MB49 cells or T24 cells were performed with an anti-ENO1 antibody followed by qPCR analyses with primer against SPP1 mRNA. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (O) Luciferase with the WT or mutated 3’UTR site in the SPP1 gene was transfected into shNC or shENO1 T24 cells Relative luciferase activity was measured. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (P) Luciferase with the WT or mutated 3’UTR site in the SPP1 gene was transfected into WT or ENO1-KO MB49 cells Relative luciferase activity was measured. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. (Q , R) Knockdown of ENO1 to detect the SPP1 mRNA expression levels in MB49 cells or T24 cells that treated with actinomycin D for the indicated time. n = 3 biologically independent samples per group. Two-side unpaired Student’s t-test. Data are presented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: Expressing, Two Tailed Test, Immunohistochemistry, Knockdown, Binding Assay, Luciferase, Transfection, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Tumor-intrinsic ENO1 inhibition promotes antitumor immune response and facilitates the efficacy of anti-PD-L1 immunotherapy in bladder cancer

doi: 10.1186/s13046-025-03464-x

Figure Lengend Snippet: ENO1 is a therapeutic target for cancer immunotherapy. (A-C) C57BL/6J mice ( n = 6 per group) were subcutaneously inoculated with WT or ENO1-KO cells. Mice received intraperitoneally treated with 200 µg anti-PD-L1 or IgG on days 7, 9, 11, 13, and 15 after tumor inoculation. Tumor volumes ( A ), weight ( B ), and mice survival time ( C ) were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean values ± SD. Tumor volumes exceeding 1500mm3 were considered events. Two-sided log-rank test. (D-F) In vivo bioluminescence imaging of bladder orthotopic tumor model. Luciferase-labeled WT or ENO1 MB49 cells were subcutaneously inoculated into the bladder of C57BL/6J mice ( n = 6 per group). Mice were intraperitoneally treated with 200 µg anti-PD-L1 or IgG on days 7, 9, 11, and 13 after tumor inoculation. The signals were measured on 18 days using an IVIS Spectrum In Vivo imaging system ( D ), and tumor volumes ( E ) and mice survival time were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. (G-I) MB49 cells were subcutaneously inoculated into C57BL/6J mice and then treatment with ENOblock and anti-PD-L1 ( n = 6 per group). Tumor volumes ( G ), weight ( H ) and mice survival time ( I ) were measured. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. Tumor volumes exceeding 1500mm3 were considered events. Two-sided log-rank test. (J-L) Representative images of IHC and mIHC staining for Ki67, CD8, IFN-γ, GZMB in different tumor tissues ( n = 6 per group). Expression levels of the indicated proteins displayed. Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. (M) Flow cytometric analysis of tumor-infiltrating CD4 + T cells, CD8 + T cells, IFN-γ + or GZMB + CD8 + T cells in distinct tumor tissues ( n = 6 per group). Two-way ANOVA with Tukey’s multiple comparison test. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: Short hairpin RNA (shRNA) constructs targeting ENO1 was purchased from GeneChem Co., Ltd.

Techniques: Comparison, In Vivo, Imaging, Luciferase, Labeling, In Vivo Imaging, Staining, Expressing