Objective:To construct pancreatic cancer cell lines expressing luciferase and mesothelin (MSLN), and evaluate the feasibility of using them as target cells in analyzing the cytotoxicity activity of immune cells.Methods:Lentiviral vectors expressing luciferase and MSLN genes were constructed, and pancreatic cancer cell lines were infected after lentivirus packaging. Single-cell clones were obtained by limited dilution following antibiotic screening, and the stable expression of the target genes were verified. These cells were used as target cells to detect the cytotoxicity of immune cells by real-time cell analysis (RTCA) and luciferase activity. Besides, these luciferase-expressing cells were transplanted into B-NDG mice to establish the animal models of pancreatic cancer, and in vivo optical imaging technology was used to detect the expression of luciferase and monitor the tumor growth in mice. The cytotoxicity of chimeric antigen receptor T (CAR-T) cells was verified in these animal models. Results:Three pancreatic cancer cell lines, panc-1-luc, panc-1-luc-MSLN and capan-2-luc, that could stably express luciferase and MSLN genes were successfully constructed. The expression of the reporter gene in these cells were high, and positively correlated with the number of cells. There were 95.6% of panc-1-luc-MSLN cells expressing MSLN. MSLN-CAR-T cells had specific killing effect on MSLN-positive panc-1-luc-MSLN cells and capan-2-luc cells, with the minimum killing rates of (70.00±18.19)% and (57.00±5.29)%, respectively. But they had no cytotoxicity to MSLN-negative panc-1-luc cells. RTCA results showed that MSLN-CAR-T cells were able to lyse all three pancreatic cancer cell lines, and the minimum killing rates were (56.33±7.64)%, (93.00±2.65)% and (26.33±28.15)%, respectively. The killing of target cells by NK-92MI cells was not depended on MSLN expression. The cytotoxicity in the mice models of pancreatic cancer was consistent with the results in vitro. The in vivo and in vitro test results suggested that the expression of luciferase by target cells could reflect the cytotoxicity of immune cells. Conclusions:This study establishes three pancreatic cancer cell lines stably expressing luciferase, which can be used to evaluate the cytotoxicity of immunotherapy products targeting tumor cells in vitro and in vivo.