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c-Met-targeted chimeric antigen receptor T cells inhibit human serous ovarian cancer cell SKOV-3 in vitro

DU Na-Na¹, ZHANG Yan-Jun², LI Yan-Qiu¹, ZHANG Lu², AN Ran³, ZHEN Xiang-Cheng⁴, MIN Jing-Ting⁴, LI Zheng-Hong^1,5,*

¹School of Basic Medicine, Bengbu Medical University, Bengbu 233030, China；²School of Life Sciences, Bengbu Medical University, Bengbu 233030, China；³Public Basic College, Bengbu Medical University, Bengbu 233030, China；⁴Clinic Medical College, Bengbu Medical University, Bengbu 233030, China；⁵Anhui Province Key Laboratory of Tumor Evolution and Intelligent Diagnosis and Treatment, Bengbu 233030, China

Abstract

The study aimed to construct the second and third generation chimeric antigen receptor T cells (CAR-T) targeting the cmesenchymal- epithelial transition factor (c-Met) protein, and observe their killing effect on human serous ovarian cancer cell SKOV- 3. The expression of MET gene in ovarian serous cystadenocarcinoma, the correlation between MET gene expression and the abundance of immune cell infiltration, and the effect of MET gene expression on the tissue function of ovarian serous cystadenocarcinoma were analyzed by bioinformatics. The expression of c-Met in ovarian cancer tissues and adjacent tissues was detected by immunohistochemical staining. The second and third generation c-Met CAR-T cells, namely c-Met CAR-T(2G/3G), were prepared by lentivirus infection, and the cell subsets and infection efficiency were detected by flow cytometry. Using CD19 CAR-T and activated T cells as control groups and A2780 cells with c-Met negative expression as Non target groups, the kill efficiency on SKOV-3 cells with c-Met positive expression, cytokine release and cell proliferation of c-Met CAR-T(2G/3G) were explored by lactate dehydrogenase (LDH) release, ELISA and CCK-8 respectively. The results showed that MET gene expression was significantly up-regulated in ovarian cancer tissues compared with normal tissues, which was consistent with the immunohistochemistry results. However, in all pathological stages, there was no obvious difference in MET expression and no correlation between MET gene expression and the race and age of ovarian cancer patients. The second generation and third generation c-Met CAR-T cells were successfully constructed. After lentivirus infection, the proportion of CD8+ T cells in c-Met CAR-T(2G) was upregulated, while there was no significant change in the cell subsets of c-Met CAR-T(3G). The LDH release experiment showed that the kill efficiency of c-Met CAR-T(2G/3G) on SKOV-3 increased with the increase of effect-target ratio. When the effect-target ratio was 20:1, the kill efficiency of c-Met CAR-T(2G) reached (42.02 ± 5.17)% (P < 0.05), and the kill efficiency of c-Met CAR-T(3G) reached (51.40 ± 2.71)% (P < 0.05). ELISA results showed that c-Met CAR-T released more cytokine compared to CD19 CAR-T and activated T cells (P < 0.05). Moreover, the cytokine release of c-Met CAR-T(3G) was higher than c-Met CAR-T(2G) (P < 0.01). The CCK-8 results showed that after 48 h, the cell number of c- Met CAR-T(2G) was higher than that of c-Met CAR-T(3G) (P < 0.01). In conclusion, both the second and third generation c-Met CAR-T can target and kill c-Met-positive SKOV-3 cells, with no significant difference. c-Met CAR-T(2G) has stronger proliferative ability, and c-Met CAR-T(3G) releases more cytokines.

Key words: ovarian cancer; c-Met; CAR-T; bioinformatics

Received: 　　Accepted:

Corresponding author: 李正红　　E-mail:

DOI: 10.13294/j.aps.2025.0002

Citing This Article：

DU Na-Na, ZHANG Yan-Jun, LI Yan-Qiu, ZHANG Lu, AN Ran, ZHEN Xiang-Cheng, MIN Jing-Ting, LI Zheng-Hong. c-Met-targeted chimeric antigen receptor T cells inhibit human serous ovarian cancer cell SKOV-3 in vitro. Acta Physiol Sin 2025; 77 (2): 241-254 (in Chinese with English abstract).