Insertion of CR2/3 domain slightly reduced the tumor-killing capacity of CAR-T cells, while significantly improved the viral delivery of VSVΔ51 to tumor cells. (A–C) CAR expression efficiency of EGFR–CAR-T model (A), B7H3–CAR-T (B), and IL13Rα2–CAR-T (C). The transfection efficiency of CAR was evaluated by detecting the expression of the G4S linker and reporter gene truncated CD19 in CAR-T cells. A representative experiment is shown, repeated at least twice with similar results. (D–F) Cell cytotoxicity was measured by LDH release assay of EGFR–CAR-T model (D), B7H3–CAR-T (E), and IL13Rα2–CAR-T (F). U87 cells were cocultured with CAR-T cells at different ratios for 24 h, and then the LDH release from U87 cells was measured. The cell cytotoxicity experiments were performed independently three times (n = 3 biological replicates). (G) Luciferase activity was measured in coculture glioma cells with IL13Rα2–CAR-T preloaded with the VSV-luc (n = 4 biological replicates). (H and I)In vivo evaluation for viral delivery of EGFR–CAR-T model. (H) Diagram depicting the monitoring of the in vivo virus distribution via bioluminescence imaging of luciferase in U87 xenograft mice. (I) Monitoring of the in vivo virus distribution via bioluminescence imaging of luciferase in U87 xenograft mice. The mice were injected with the EGFR–CAR-T^VSV-luc, EGFR^VSV-luc, or VSV-luc (n = 5 mice). Data represent the mean ± SD from three independent experiments (D–F), four independent experiments (G), and one representative from two independent experiments (I). Statistics by two-way ANOVA with Bonferroni post hoc test (D–F) and unpaired two-tailed Student’s t test (I). *, P < 0.05; **, P < 0.01; N.S., no significance.