Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: UBE2C knockdown suppresses bladder cancer progression and CTLA‐4 expression. (A) qRT‐PCR analysis of UBE2C expression in T24 cell. (B) Western blot analysis of UBE2C expression in T24 cell. (C) Transwell assay for T24 cell invasion ability. Scale bars: 100 μm. (D) CCK‐8 assay for T24 cell viability. (E) Flow cytometry for T24 cell apoptosis levels. (F) Western blot analysis of Ki‐67 and γ‐H2AX expression in tumor tissue from C57 mice harvested at the experimental endpoint (day 21 postinoculation). (G) Spearman correlation analysis between UBE2C and CTLA‐4 using TCGA‐BLCA database. (H) IHC analysis of CD8 + T cell infiltration in BCa tissue from patients. Scale bars: 100 μm. (I) Flow cytometry analysis of CD3 + CD8 + T cell proportion in tumor tissue. (J) Flow cytometry analysis of CTLA‐4 expression in tumor tissue. *si‐NC versus si‐UBE2C. * p < 0.05, *** p < 0.001.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Western Blot, Transwell Assay, CCK-8 Assay, Flow Cytometry

Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: The UBE2C/CTLA‐4 axis drives T cell exhaustion in the bladder cancer microenvironment. (A) CTLA‐4 expression in T cells. (B) UBE2C expression in T24 cells and CTLA‐4 expression in T cells. (C) The expression levels of T cell exhaustion‐related proteins. (D) T cell viability. (E) T cell apoptosis. (F–H) ELISA assay for IFN‐γ (F), TNF‐α (G), and TGF‐β (H) levels in the supernatant. *si‐NC versus si‐UBE2C, # si‐UBE2C versus si‐UBE2C + oe‐CTLA‐4. ## p < 0.01, ### p < 0.001, and *** p < 0.001.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: CTLA‐4 overexpression reverses the inhibition of tumor growth mediated by UBE2C knockdown. (A) Mouse diagram. (B) Mouse tumor tissue. (C) Mouse weight. (D) Mouse tumor volume. (E) Mouse tumor weight. *shNC versus shUBE2C or shNC + anti‐CTLA‐4, # shUBE2C versus shUBE2C + AAV‐CTLA‐4. ### p < 0.001, ** p < 0.01 and *** p < 0.001. N = 3.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Over Expression, Inhibition, Knockdown

Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: Knocking down UBE2C inhibits bladder cancer growth by alleviating CTLA‐4‐mediated immune suppression. (A, B) Immunohistochemistry (A) and Western blot (B) analysis of UBE2C and CTLA‐4 expression in tumor tissues. Scale bars: 100 μm. (C) T cell exhaustion protein expression in tumor tissues. (D–F) ELISA analysis of IFN‐γ (D), TNF‐α (E), and TGF‐β (F) levels in mouse blood. *shNC versus shUBE2C or shNC + anti‐CTLA‐4, # shUBE2C versus shUBE2C + AAV‐CTL A‐4. ### p < 0.001 and *** p < 0.001. N = 3.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Immunohistochemistry, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: UBE2C promotes the expression of CTLA‐4 by regulating CD147 and induces T cell exhaustion. (A, B) CD147 expression in T24 cells (A) and mouse tumor tissue (B). (C) CD147 expression in T24 cells. (D, E) T cell apoptosis. (F) T cell proliferation activity. (G) UBE2C expression in T24 cells and CTLA‐4 expression in T cells. (H) T cell exhaustion proteins. (I–K) ELISA assay for IFN‐γ (I), TNF‐α (J), and TGF‐β (K) levels in cell culture supernatants. (L) ELISA detection of soluble CD147 (sCD147) levels in the conditioned medium. (M) Western blot analysis of exosomal markers CD63 and CD9 in isolated extracellular vesicles. (N) Western blot analysis of CD147 expression in tumor‐derived exosomes. *si‐NC versus si‐UBE2C or si‐CD147, # si‐UBE2C versus si‐UBE2C + oe‐CD147. ### p < 0.001 and *** p < 0.001.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Expressing, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Western Blot, Isolation, Derivative Assay

Journal: Cancer Science

Article Title: UBE 2 C ‐Mediated CD 147‐ CTLA ‐4 Axis Promotes T Cell Exhaustion and Immunosuppressive Microenvironment in Bladder Cancer

doi: 10.1111/cas.70430

Figure Lengend Snippet: UBE2C upregulates cellular CD147 and enhances its release into the tumor microenvironment both as soluble CD147 (sCD147) and packaged within tumor‐derived exosomes. These CD147‐bearing signals induce T‐cell exhaustion, marked by elevated CTLA‐4 expression and reduced secretion of effector cytokines (IFN‐γ, TNF‐α, TGF‐β), ultimately diminishing CD8 + T cell abundance and antitumor immunity.

Article Snippet: One to two weeks after inoculation, the mice were randomly grouped as shNC ( N = 3), shNC + anti‐CTLA‐4 ( N = 3), shUBE2C ( N = 3), and shUBE2C + AAV‐CTLA4 ( N = 3). shNC + anti‐CTLA‐4 mice were treated with anti‐mouse CTLA‐4 (9D9) Antibody (HY‐ P99132 , MCE, USA; 200 μg per mouse) The shUBE2C + AAV‐CTLA‐4 group received CD8 promoter‐specific AAV‐CTLA‐4 treatment (Genomeditech; 30 μg per mouse), and the administration method was peritumoral injection at d7, d10, and d13, while the remaining groups of mice were injected with the same dose of sterile PBS solution.

Techniques: Derivative Assay, Expressing