CR2/3–CAR-T cells efficiently delivered VSVΔ51 to GBM cells in vitro and in vivo. (A) Representative cytometry plots and MFI analysis of VSVΔ51 release from CR2/3–B7H3–CAR-T cells after incubating with U87 cells for 30 min; VSVΔ51 was stained using CY7. (B) Generation of recombinant VSVΔ51 to express luciferase (VSV-luc) for measuring the distribution in vivo and replication of virus in vitro. (C) Luciferase activity was measured in LN229, U87, and U251 cells, when cocultured with the B7H3–CAR-T or CR2/3–B7H3–CAR-T cells preloading with the VSV-luc (n = 4 biological replicates). (D–F)In vivo evaluation for viral delivery of B7H3–CAR-T model. (D) Diagram depicting monitoring of the in vivo virus distribution via bioluminescence imaging of luciferase in U87 xenograft mice. CAR-T or VSV was i.v. injected. (E) The bioluminescence imaging of luciferase in U87 xenograft mice at indicated time points from two independent experiments. Two independent experiments were conducted to assess viral signals at indicated time points. (F) Quantitative radiance of mice was analyzed (n = 5 mice). (G) H&E staining and immunofluorescence analysis of U87 intracranial xenograft. The mouse brain tissue sections for H&E staining and immunofluorescence were consecutive sections. H&E staining revealed the site of brain tumor, while immunofluorescence staining showed the distribution of the viral protein VSV-G. (H) mRNA level of VSV-G in U87 intracranial xenograft was measured (n = 5 mice). Data represent the mean ± SD from three independent experiments (A), four independent experiments (C), and one representative from two independent experiments (E and H). Statistics by one-way ANOVA with Tukey’s post hoc test (A and F) and unpaired two-tailed Student’s t test (C and H). *, P < 0.05; **, P < 0.01; N.S., no significance.