Abstract:

Objective

To construct a co-culture model of gastric cancer organoids and CLDN18.2-chimeric antigen receptor T （CAR-T） cells， and to investigate the relationship between CAR-T cytotoxicity and clinicopathological factors， especially the intensity of CLDN18.2 expression.

Methods

Clinical and pathological data were collected from 73 patients with gastric cancer admitted in the Seventh Affiliated Hospital of Sun Yat-sen University from September 2022 to August 2023. CLDN18.2 expression in tumor tissues was detected using immunohistochemical staining. Gastric cancer organoids were established and the CLDN18.2 expression profile was verified by both immunohistochemical and immunofluorescence staining. CLDN18.2-CAR-T cells were developed and co-cultured with 12 gastric cancer organoid models.CAR-T cell cytotoxicity was evaluated using lactic dehydrogenase （LDH） release assays， enzyme linked immunosorbent assay （ELISA） for cytokine detection， and fluorescence imaging of cell killing. Univariate and correlation analyses were performed to explore the relationship between CLDN18.2 expression and CAR-T cell cytotoxicity.

Results

Organoids retained the expression characteristics of CLDN18.2 in the primary tumors. Co-culture experiments demonstrated that CAR-T cells killed the organoids effectively.Univariate analysis of clinicopathological factors confirmed that CLDN18.2 expression intensity was a factor influencing CAR-T cell cytotoxicity. Correlation analysis indicated significantly positive correlation between cytotoxicity and CLDN18.2 expression level （r=0.73， P=0.007）.

Conclusion

A successful co-culture model was constructed for in vitro validation of CAR-T therapeutic efficacy and development of highly active CLDN18.2-targeted CAR-T cells.

Key words: Stomach neoplasms, Organoids, CLDN18.2, CAR-T, Receptors，chimeric antigen