Fig 1: Generation and Characterization of ACAT-1-Inhibited Anti-MLSN CAR-T Cells(A) Schematic representation of MSLN-targeted constructs with a CD28 and 4-1BB costimulatory domain, a CD3? domain, and an ACAT-1 DNA suppression sequence. (B) Quantitative real-time RT-PCR profile showing ACAT-1 mRNA levels in CAR-T-1847, CAR-T-1848, and CAR-T-2598 cells. (C) Western blot image showing the ACAT-1 protein level in CAR-T-1847, CAR-T-1848, and CAR-T-2598 cells. (D) Flow cytometry profile showing the transduction efficiencies. (E) LDH profile of different anti-MLSN CAR-T constructs.
Fig 2: The Phenotype of Anti-MLSN CAR-T Cells(A–H) Flow cytometry profile showing the (A and B) transduction efficiencies of CAR-T cells and the (C–H) percentage distribution of CD8+, IFN?+CD8+, and TNF-a+CD8+ targeting MSLN CAR-T cells. Representative results from one of six patients are shown. (A and B) The transduction efficiencies of CAR-T-1847 and CAR-T-1848 cells were significantly lower than that of CAR-T-2598 cells (P < 0.001). (C–H) There were no significant differences in the percentages of CD8+ CAR-T, IFN?+CD8+ CAR-T, and TNFa+CD8+ CAR-T cells among all three groups (P > 0.05).
Fig 3: ACAT-1 Inhibition Enhanced the Targeting MSLN CAR-T Antitumor FunctionTargeting MSLN CAR-T cells were cocultured with target cells at different E:T ratios for 18 h. (A–C) Cell lysis of cocultures was assessed with a standard LDH release assay at E:T ratios of (A) 5:1, (B) 8:1, and (C) 10:1. (D) Profile showing the dose-dependent lysis of MSLN-K562 cells by targeting MSLN CAR-T cells. (E–K) Profiles showing the results of the cytokine release assay. (B and C) CAR-T-1847 cells showed a higher cytotoxic effect at E:T ratios of 8:1 and 10:1. (B) CAR-T-1848 cells also showed a higher cytotoxic effect at an E:T ratio of 8:1. (D) Dose-dependent lysis of MSLN-K562 cells by targeting MSLN CAR-T cells was bserved. (E and J) CAR-T-1847 and CAR-T-1848 cells induced higher levels of IL-2 (P < 0.05) and IFN? (P < 0.01) production compared to that by CAR-T-2598 cells.
Fig 4: ACAT-1 Inhibition Increased the Targeting MSLN CAR-T Cell Potency against MSLN-BxPC-3-Luci Xenografts In Vivo(A and B) Tumor bioluminescence images of mice transplanted with MSLN-BxPC-3-Luci tumor cells at the indicated time points. (C and D) Comparison of the tumor bioluminescence intensities between the CAR-T and control groups. Bioluminescence imaging showed that sustained tumor regression was observed for CAR-T-1847 cells compared to the Blank and CAR-T-2598 groups at day 33 (P < 0.05). (E and F) Comparison of the tumor volume between the CAR-T and control groups. CAR-T-1847 group showed a significantly reduced tumor volume compared to that of the CAR-T-2598 group (120.29 ± 98.04 mm3 vs. 379.18 ± 63.19 mm3; P < 0.05).
Fig 5: MSLN Overexpression in Human PC Patients(A) Representative micrographs at ×20 and ×40 original magnification showing MSLN-positive PC cells. MSLN-positive tumor glands are indicated by the arrow. (B) MSLN expression in NC, ad-PC, and PC tissues. (C) ELISA profile showing the relative level of circulating soluble MSLN in patient serum samples. Each symbol represents a patient sample.
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