EpCAM-targeting CAR-T cells against hepatoma cells in vitro

Abstract

Objective To investigate the killing efficacy of chimeric antigen receptor T (CAR-T) cells targeting epithelial cellular adhesion molecule (EpCAM) in several hepatoma cell lines. Methods Flow cytometry was used to detect the expression of EpCAM in hepatoma cells (SK-hep1, Hep-G2, HuH7 and BEL-7402). After EpCAM -targeting CAR-T cells were constructed using lentiviral vectors with CAR genes, flow cytometry was used to detect the expression of EpCAM-CAR in the obtained cells, and to analyze the CD3/4/8 phenotype of CAR-T cells without transfection of lentiviral vectors. Then the effector cells and target cells were co-cultured, and LDH release assay and ELISA were used to detect the release of LDH and major cytokines in the supernatant. Results EpCAM was highly expressed in Hep-G2, HuH7 and BEL-7402 cells, but not in SK-hep1 cells. Flow cytometry showed that the expression rate of EpCAM-CAR was 43.21% in CAR-T cells after lentivirus infection. Flow cytometry also indicated that up to 95.1% of peripheral blood mononuclear cells (PBMCs) were CD3 positive, of which 61.40% were CD8+ T cells and 32.13% were CD4+ T cells. LDH release assay revealed that the obtained CAR-T cells had stronger direct killing ability on EpCAM-positive hepatoma cell lines Hep-G2, HuH7, and BEL-7402. ELISA showed that EpCAM-positive target cells Hep-G2, HuH7, and BEL-7402 induced CAR-T cells to release more IFN-γ and TNF-α. For SK-hep1 cells with negative EpCAM expression, IFN-γ and TNF-α releases were not significantly different between the CAR-T group and the control group, and the levels of these 2 cytokines werw increased with the increase of E ∶T ratio. Results Compared with ordinary CAR-T cells, EpCAM-targeting CAR-T cells have a stronger killing effect on EpCAM-positive hepatocellular carcinoma cells.

Keywords

• epithelial cellular adhesion molecule
• chimeric antigen receptor t cells
• hepatocellular carcinoma