Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 1. Expression of TIM3 and TGFβRII and their ligands in prostate cancer: (A) Expression of GAL9 in prostate cancer and normal prostate tissues in the GEPIA 2 database; (B) Expression of TGF-β in prostate cancer and normal prostate tissues in the GEPIA 2 database; (C) Correlation between the expression of TIM3 and TGFβRII, GAL9, and TGF-β in the GEPIA 2 database; (D) IHC of GAL9 in prostate cancer in the HPA database; (E) IHC of TGF-β in prostate cancer in the HPA database; (F) The left panel shows unsupervised graph-based clustering of all samples visualized by UMAP delineated by cell type, and the right panel shows the expression of TIM3 and TGFβRII in different cell populations in the single-cell sequencing results; (G) Expression of TIM3 and TGFβRII in T cells in the single-cell sequencing results (the left panel is a scatter plot, and the right panel is a violin plot); (H) Coexpression of TIM3 and TGFβRII in T cells in the single-cell sequencing results, where 1 (blue) represents T cells that coexpress TIM3 and TGFβRII, and 0 (gray) represents other T cells.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Expressing, Sequencing

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 2. Preparation and phenotypic characterization of DT-PSMA-CAR-T cells: (A) Gene structure of PSMA-CAR and DT-PSMA-CAR; (B) Expansion of the UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells 14 days after electroporation; (C) The positivity rate of CAR in the PSMA-CAR-T group and DT-PSMA-CAR-T group, after magnetic bead sorting, assessed by flow cytometry; (D) Expression of TIM3 and TGFβRII on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (E) Expression of CD4 and CD8 on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays; (F) Expression of CD45RO on the three groups of T cells assessed by flow cytometry before performing in vitro killing assays.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Electroporation, Flow Cytometry, Expressing, In Vitro

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 4. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different concentrations of TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 5. Analysis of the resistance of DT-PSMA-CAR-T cells to immune suppression caused by TIM3 and TGFβRII activation in vitro was performed by tumor lysis assays: (A and B) UTD group, PSMA-CAR-T group, and DT-PSMA-CAR-T group T cells were cocultured with LNCAP cells in the presence of 20 ng/ml TGF-β and 10 μg/ml TIM3 activating monoclonal antibody; (C and D) Under an effector-to-target ratio of 5:1, the three groups of T cells were cocultured with LNCAP cells in the presence of different combinations of TGF-β and TIM3 activating monoclonal antibody; (E) Cytokine secretion was detected in the supernatant of all experimental groups in (C).

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Activation Assay, In Vitro, Lysis

Journal: International immunopharmacology

Article Title: Dominant negative TGFβ receptor II and truncated TIM3 enhance the antitumor efficacy of CAR-T-cell therapy in prostate cancer.

doi: 10.1016/j.intimp.2023.110807

Figure Lengend Snippet: Fig. 6. DT-PSMA-CAR-T cells can inhibit the growth of NSG mouse xe nografts in an environment activated by TIM3 and TGFβRII: (A) The timeline of animal experiments: 1 × 106 GAL9- PSMA-PC3 cells were subcutaneously injected; two weeks later, 5 × 106 UTD T cells, PSMA-CAR-T cells, or DT- PSMA-CAR-T cells were infused per mouse via the tail vein; and biolumi nescence imaging was performed weekly; (B) The location and tumor burden of the xenografts displayed by bioluminescence imaging; (C) Quanti tative results of tumor burden obtained by Aniview100.

Article Snippet: Untransduced (UTD) T cells, PSMA-CAR-T cells, and DT-PSMA-CAR- L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 L. Tang et al. International Immunopharmacology 124 (2023) 110807 T cells were co-cultured with prostate cancer cells at ratios of 1:1, 5:1, and 10:1 for 24 h.The T-cell culture medium was supplemented with appropriate concentrations (0 ng/ml, 5 ng/ml, 20 ng/ml, and 50 ng/ml) of TGF-β (P01137, Suzhou Novoprotein Scientific) and/or (0 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) TIM3 activating monoclonal antibodies (MAB23651, R&D Systems) from the day before coculture to the end of coculture.

Techniques: Injection, Imaging

Journal: International Journal of Oncology

Article Title: Discovery of the late autophagy inhibitor FZU-0045-053 and its anti-breast cancer and immunomodulatory effects

doi: 10.3892/ijo.2025.5823

Figure Lengend Snippet: Compound 053 modulates co-stimulatory and co-inhibitory molecules on T cell surfaces. (A) The protein levels of 4-1BB, OX40 and CD40L after 5 μ M ADM, GEM (80 μ M), 053 (5 μ M) or CQ (30 μ M) treatment were analyzed by western blotting. (B) The protein levels of TIM3, PD-1 and VISTA after 5 μ M ADM, GEM (80 μ M), 053 (5 μ M) or CQ (30 μ M) treatment were analyzed by western blotting. (C) After T cells were treated with 053 (0-10 μ M) for 24 h, ICOS expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (D) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of ICOS expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (E) After T cells were treated with 053 (0-10 μ M) for 24 h, OX-40 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (F) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of OX-40 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (G) After T cells were treated with 053 (0-10 μ M) for 24 h, 4-1BB expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (H) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of 4-1BB expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (I) After T cells were treated with 053 (0-10 μ M) for 24 h, TIM3 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (J) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of TIM3 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. (K) After T cells were treated with 053 (0-10 μ M) for 24 h, PD-1 expression on T cell surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (L) T cells were co-cultured with MDA-MB-231 cells at a 1:1 ratio and treated with 053 (0-10 μ M) for 24 h, followed by flow cytometric analysis of PD-1 expression on T cell surfaces. The bar graph shows quantitative analysis of relative fluorescence intensity. Data are presented as mean ± SEM (n=3). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test to compare all experimental groups against a single control group. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. 0 μ M 053. 053, FZU-0045-053; ADM, adriamycin; 4-1BB, tumor necrosis factor receptor superfamily member 9; OX40, tumor necrosis factor receptor superfamily member 4; CD40L, CD40 ligand; ICOS, inducible T-cell co-stimulator; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin-domain containing-3; VISTA, V-domain Ig suppressor of T cell activation; GEM, gemcitabine; CQ, chloroquine.

Article Snippet: The sections were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-Ki67 antibody (cat. no. GB111499-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-LC3 antibody (cat. no. 81004-1-RR; 1:1,000; Proteintech Group, Inc), anti-p62 antibody (cat. no. GB11239-1-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L1 antibody (cat. no. GB150059-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L2 antibody (cat. no. 27406-1-AP; 1:4,000; Proteintech Group, Inc), anti-CD3 antibody (cat. no. GB11014-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD4 antibody (cat. no. GB11064-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD8 antibody (cat. no. GB11068; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-4-1BB/CD137 antibody (cat. no. A2025; 1:200; ABclonal Biotech Co., Ltd), anti-CD134/OX40 antibody (cat. no. 32621-1-AP; 1:500; Proteintech Group, Inc), anti-PD1 antibody (cat. no. GB153744-100; 1:400; Wuhan Servicebio Technology Co., Ltd) and anti-TIM3 antibody (cat. no. 11872-1-AP; 1:1,000; Proteintech Group, Inc).

Techniques: Western Blot, Expressing, Flow Cytometry, Fluorescence, Cell Culture, Control, Activation Assay

Journal: International Journal of Oncology

Article Title: Discovery of the late autophagy inhibitor FZU-0045-053 and its anti-breast cancer and immunomodulatory effects

doi: 10.3892/ijo.2025.5823

Figure Lengend Snippet: Compound 053 enhances the in vivo antitumor effect of GEM in the 4T1 xenograft model. (A) Mouse treatment plan. (B) H&E staining of heart, liver, spleen, lung and kidney. Scale bar, 100 μ m. (C) Tumor morphology of mouse removed at 21 days of treatment. (D) The tumor size was measured with a caliper every 3 days and the volume was calculated (n=5). (E) Protein levels of CD3, CD4, CD8, 4-1BB, OX40, PD-1 and TIM3 were detected using immunohistochemical staining in tumor tissues of mice after 21 days of treatment. Nuclei are localized in blue and target proteins are localized in brown. Scale bar, 100 μ m. (F) Body weight was measured every 3 days (n=5). 053, FZU-0045-053; GEM, gemcitabine; 4-1BB, tumor necrosis factor receptor superfamily member 9; OX40, tumor necrosis factor receptor superfamily member 4; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin-domain containing-3.

Article Snippet: The sections were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-Ki67 antibody (cat. no. GB111499-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-LC3 antibody (cat. no. 81004-1-RR; 1:1,000; Proteintech Group, Inc), anti-p62 antibody (cat. no. GB11239-1-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L1 antibody (cat. no. GB150059-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L2 antibody (cat. no. 27406-1-AP; 1:4,000; Proteintech Group, Inc), anti-CD3 antibody (cat. no. GB11014-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD4 antibody (cat. no. GB11064-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD8 antibody (cat. no. GB11068; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-4-1BB/CD137 antibody (cat. no. A2025; 1:200; ABclonal Biotech Co., Ltd), anti-CD134/OX40 antibody (cat. no. 32621-1-AP; 1:500; Proteintech Group, Inc), anti-PD1 antibody (cat. no. GB153744-100; 1:400; Wuhan Servicebio Technology Co., Ltd) and anti-TIM3 antibody (cat. no. 11872-1-AP; 1:1,000; Proteintech Group, Inc).

Techniques: In Vivo, Staining, Immunohistochemical staining

Journal: International Journal of Oncology

Article Title: Discovery of the late autophagy inhibitor FZU-0045-053 and its anti-breast cancer and immunomodulatory effects

doi: 10.3892/ijo.2025.5823

Figure Lengend Snippet: Compound 053 modulates PD-L1 on tumor cells and co-stimulatory/co-inhibitory molecules on tumor-infiltrating T cells in the 4T1 xenograft model. (A) Flow cytometry was performed to analyze PD-L1 surface expression on tumor cells. The bar graph shows quantitative analysis of relative fluorescence intensity. (B) PD-1 expression on tumor-infiltrating T cells surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (C) TIM3 expression on tumor-infiltrating T cells surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (D) 4-1BB expression on tumor-infiltrating T cells surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (E) ICOS expression on tumor-infiltrating T cells surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. (F) OX-40 expression on tumor-infiltrating T cells surfaces was analyzed by flow cytometry. The bar graph shows quantitative analysis of relative fluorescence intensity. Data are presented as mean ± SEM (n=3). Statistical significance was determined by one-way ANOVA followed by Dunnett's multiple comparisons test to compare all experimental groups against a single control group. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 vs. 0 μ M 053. (G) Flow cytometry was used to analyze the differentiation of CD3 + CD4 + T cells and CD3 + CD8 + T cells. 053, FZU-0045-053; 4-1BB, tumor necrosis factor receptor superfamily member 9; OX40, tumor necrosis factor receptor superfamily member 4; ICOS, inducible T-cell co-stimulator; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin-domain containing-3; GEM, gemcitabine; PD-L1, programmed death-ligand 1.

Article Snippet: The sections were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-Ki67 antibody (cat. no. GB111499-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-LC3 antibody (cat. no. 81004-1-RR; 1:1,000; Proteintech Group, Inc), anti-p62 antibody (cat. no. GB11239-1-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L1 antibody (cat. no. GB150059-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L2 antibody (cat. no. 27406-1-AP; 1:4,000; Proteintech Group, Inc), anti-CD3 antibody (cat. no. GB11014-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD4 antibody (cat. no. GB11064-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD8 antibody (cat. no. GB11068; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-4-1BB/CD137 antibody (cat. no. A2025; 1:200; ABclonal Biotech Co., Ltd), anti-CD134/OX40 antibody (cat. no. 32621-1-AP; 1:500; Proteintech Group, Inc), anti-PD1 antibody (cat. no. GB153744-100; 1:400; Wuhan Servicebio Technology Co., Ltd) and anti-TIM3 antibody (cat. no. 11872-1-AP; 1:1,000; Proteintech Group, Inc).

Techniques: Flow Cytometry, Expressing, Fluorescence, Control

Journal: International Journal of Oncology

Article Title: Discovery of the late autophagy inhibitor FZU-0045-053 and its anti-breast cancer and immunomodulatory effects

doi: 10.3892/ijo.2025.5823

Figure Lengend Snippet: Mechanism of compound 053 regulating autophagy and immune response to inhibit breast cancer. 053 can inhibit autophagy and promote apoptosis of breast cancer cells. 053 also downregulated PD-L1 expression in breast cancer cells, while promoting T cell activation and proliferation by upregulating co-stimulatory molecules (4-1BB, OX40 and ICOS) and downregulating co-inhibitory molecules (TIM-3 and PD-1) on the surface of the T cells. (By Figdraw). 4-1BB, tumor necrosis factor receptor superfamily member 9; OX40, tumor necrosis factor receptor superfamily member 4; ICOS, inducible T-cell co-stimulator; PD-1, programmed cell death protein 1; TIM3, T-cell immunoglobulin and mucin-domain containing-3; PD-L1, programmed death-ligand 1.

Article Snippet: The sections were then blocked with 5% BSA for 1 h at room temperature and subsequently incubated overnight at 4°C with the following primary antibodies: Anti-Ki67 antibody (cat. no. GB111499-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-LC3 antibody (cat. no. 81004-1-RR; 1:1,000; Proteintech Group, Inc), anti-p62 antibody (cat. no. GB11239-1-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L1 antibody (cat. no. GB150059-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-PD-L2 antibody (cat. no. 27406-1-AP; 1:4,000; Proteintech Group, Inc), anti-CD3 antibody (cat. no. GB11014-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD4 antibody (cat. no. GB11064-100; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-CD8 antibody (cat. no. GB11068; 1:1,000; Wuhan Servicebio Technology Co., Ltd), anti-4-1BB/CD137 antibody (cat. no. A2025; 1:200; ABclonal Biotech Co., Ltd), anti-CD134/OX40 antibody (cat. no. 32621-1-AP; 1:500; Proteintech Group, Inc), anti-PD1 antibody (cat. no. GB153744-100; 1:400; Wuhan Servicebio Technology Co., Ltd) and anti-TIM3 antibody (cat. no. 11872-1-AP; 1:1,000; Proteintech Group, Inc).

Techniques: Expressing, Activation Assay