Efficacy of chimeric antigen receptor T-cell with programmed cell death-1 knockdown targeting folate receptor alpha in killing hepatoma cells

Junye WEN; Junqi ZHANG; Hang REN; Haiqiang ZHANG; Xueshuai YE

doi:10.12449/JCH250619

Volume 41 Issue 6

Jun. 2025

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Article Navigation > Journal of Clinical Hepatology > 2025 > 41(6): 1128-1134

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Efficacy of chimeric antigen receptor T-cell with programmed cell death-1 knockdown targeting folate receptor alpha in killing hepatoma cells

DOI: 10.12449/JCH250619

1.
Department of Hepatobiliary Surgery，Hebei General Hospital，Shijiazhuang 050000，China
2.
Office of Discipline Development and Graduate Education，The Affiliated Hospital/Clinical Medical College of Hebei University of Engineering，Handan，Hebei 056002，China
3.
Department of Gastrointestinal Surgery，The Second Hospital of Hebei Medical University，Shijiazhuang 050000，China

Research funding:

Natural Science Foundation of Hebei Province (H2024402005)

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Corresponding author: YE Xueshuai， yexueshuai@hebeu.edu.cn （ORCID： 0009-0000-3665-3394）
Received Date: 2024-11-04
Accepted Date: 2025-01-21
Published Date: 2025-06-25

Abstract

Abstract

Objective To investigate the ability of chimeric antigen receptor T-cell with programmed cell death-1 （PD-1） knockdown （si-PD-1 CAR-T） targeting folate receptor alpha （FRα） to eliminate hepatoma cells. Methods The bioinformatics database TCGA was used to analyze the expression level of FRα antigen in liver cancer tissue and normal liver tissue and the association between FRα expression and the survival of liver cancer patients. The mRNA encoding the CAR structure targeting FRα antigen and the small interfering RNA （siRNA） targeting the PD-1 gene were transduced into T cells using an electroporator to prepare FRα-CAR-T and si-PD-1-CAR-T cells. Flow cytometry was used to analyze the expression efficiency of FRα-CAR and the knockdown efficiency of PD-1. Hepatoma cell lines JHH-1 and Hep-G2 were cultured in vitro， and flow cytometry was used to analyze the expression of FRα on the surface of tumor cells. With FRα-CAR-T， si-PD-1 CAR-T， and mock vector-transduced T cells （Mock T） used as effector cells and with JHH-1 and Hep-G2 cells as target cells， CCK-8 assay was used to measure the killing efficiency of effector cells against target cells at different effector-to-target ratios （1∶1， 2.5∶1，5∶1，10∶1，20∶1）. ELISA was used to measure the secretion of interferon gamma （IFN-γ） and interleukin-2 （IL-2） in the supernatants from co-cultures of effector and target cells （10∶1）. The independent-samples t test was used for comparison of normally distributed continuous data between two groups， while a one-way analysis of variance was used for comparison between multiple groups， and the SNK test was used for further comparison between two groups. The Kaplan-Meier method was used for comparison of survival differences. Results The analysis of the TCGA database showed that there was a significant increase in the expression level of FOLR1 in liver cancer tissue， and liver cancer patients with high expression of FOLR1 had a significantly shorter overall survival than those with low expression （P=0.013）. After transduction of mRNA into T cells， the expression rate of FRα-CAR reached 89.8% in CAR-T and 84.7% in si-PD-1 CAR-T cells， and co-transfection with mRNA and siRNA could downregulate PD-1 in T cells and maintain a low expression state for at least 7 days. The expression rate of FRα antigen was 100% in JHH-1 cells， while it showed negative expression in Hep-G2 cells. CCK-8 assay showed that the killing efficiency of si-PD-1-CAR-T against JHH-1 cells was significantly higher than that against FRα-CAR-T cells （P<0.05）. ELISA showed that compared with Mock T cells， FRα-CAR-T cells co-cultured with JHH-1 cells showed significant increases in the secretion of IL-2 （1 032.50±135.90 pg/mL vs 50.26±7.87 pg/mL，P<0.001） and IFN-γ （1 430.56±184.20 pg/mL vs 89.05±11.26 pg/mL，P<0.001）， and in addition， the release levels of IFN-γ and IL-2 after co-culture of si-PD-1-CAR-T and JHH-1 cells were significantly higher than the release level of FRα-CAR-T （P<0.05）. Conclusion FRα is a potential target for liver cancer treatment， and PD-1 knockdown in T cells can significantly enhance the in vitro killing activity of FRα-CAR-T cells.
- Carcinoma， Hepatocellular,
- Receptors， Chimeric Antigen,
- Folate Receptor 1,
- Programmed Cell Death 1 Receptor

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Efficacy of chimeric antigen receptor T-cell with programmed cell death-1 knockdown targeting folate receptor alpha in killing hepatoma cells

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Efficacy of chimeric antigen receptor T-cell with programmed cell death-1 knockdown targeting folate receptor alpha in killing hepatoma cells

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