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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by <t>Ella</t> kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor
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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by <t>Ella</t> kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor
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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by <t>Ella</t> kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor
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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by <t>Ella</t> kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor
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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by <t>Ella</t> kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor
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Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by Ella kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Preclinical evaluation of CAR-T cell immunotherapy with a fully human EpCAM-specific scFv against pancreatic cancer

doi: 10.1007/s00262-025-04267-x

Figure Lengend Snippet: Construction, activation and cytotoxic assays of EpCAM-specific CAR-T cells in vitro. A Representative flow cytometry histogram plots detailing 28 CAR and BB CAR expression on the surface of human primary T cells, as determined by staining with an anti-G4S antibody. B Flow cytometry results showing the dilution of proliferation staining dye Cell Trace CFSE. Untransduced T (UTD) and two CAR-T cells were stained with 10 μM Cell Trace CFSE and co-cultured with four EpCAM positive pancreatic adenocarcinoma cells at a 1:1 ratio for 5 days before analyzed by flow cytometry. C Expression of the activation marker CD69 on UTD and two CAR-T cells after co-culture with target cells at a 4:1 ratio for 24 h by flow cytometry. D Quantification of ( C ) showing the percentage of CD69 positive cells. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). E, F IFN- γ ( E ) and TNF- α ( F ) secretion levels of UTD and two CAR-T cells co-cultured with target cells at a 4:1 ratio for 24 h were measured by Ella kits. Data are presented as mean with SD of triplicate wells (unpaired t test; * P < 0.01, ** P < 0.001, *** P < 0.0001). G Cytotoxic activity of UTD and two anti-EpCAM CAR-T cells against GFP positive BxPC-3, Capan-2, CFPAC-1, AsPC-1 and hTERT–HPNE cells after 48 h of co-culture at E:T ratios of 4:1, 2:1, 1:1, 1:2 and 1:4. Data are presented as mean with SD of triplicate wells. N = 1 T-cell donor

Article Snippet: Measurement of the human IL-2, IL-6, IFN- γ and TNF- α levels were performed using the Ella immunoassay microfluidic platform (ProteinSimple) according to the manufacturer’s instruction.

Techniques: Activation Assay, In Vitro, Flow Cytometry, Expressing, Staining, Cell Culture, Marker, Co-Culture Assay, Activity Assay