Fig 1: 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+, IFNγ+CD8+, and TNF-α+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, IFNγ+CD8+ CAR-T, and TNFα+CD8+ CAR-T cells among all three groups (P > 0.05).
Fig 2: 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 CD3ζ 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 3: Association between MSLN expression and prognosis of ovarian cancer. (A) Correlations between the expression of MSLN and the overall survival rate in different stages of ovarian cancer. (B, C) Kaplan-Meier analysis of the relationship between MSLN and overall survival rate of ovarian cancer at different histological levels in TCGA. (D–G) Kaplan-Meier analysis of the relationship between MSLN and overall survival at different stages of ovarian cancer in TCGA. (H–J) Kaplan-Meier analysis of the relationship between MSLN and overall survival at different grades of ovarian cancer in TCGA.
Fig 4: MSLN is highly expressed in chemotherapy-resistant cell lines of ovarian cancer. (A, B) Heat maps of the top 50 differential genes in ovarian cancer cisplatin resistance/cisplatin sensitivity and oxaliplatin resistance/oxaliplatin sensitivity based on data from GEO database. (C) Venn diagram of differential genes for cisplatin resistance and oxaliplatin resistance. (D) Western blot shows the expression of MSLN in A2780 and A2780DDP and statistical analysis of gray value. (E) Effect of MSLN on cisplatin sensitivity (IC50) in ovarian cancer patients was assessed by Genomics of Drug Sensitivity in Cancer (GDSC). **, p < 0.01.
Fig 5: Correlation of MSLN with immune cell infiltration and immune checkpoint in ovarian cancer. (A) Immune cell infiltration level in the high MSLN expression group and low MSLN expression group in TCGA cohort. (B) Correlations between MSLN and Immune cell infiltration in ovarian cancer is shown in lollipop charts; The larger the circle, the stronger the correlation. (C, D) Expression level of immune checkpoint (C) and HLA molecule (D) in the high MSLN expression group and low MSLN expression group in TCGA cohort. *, p< 0.05; **, p<0.01; ***, p<0.001.
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