Pre-activated CR2/3–CAR-T ^VSV cells proliferated and exhibited strong metabolic and immunological activities in tumor microenvironment. (A) Diagram depicting transcriptome analysis of intra-tumor CAR-T cells. NCG mice bearing U87 intracranial xenograft were injected with B7H3–CAR-T^VSV (5 × 10⁶ cells) and CR2/3–B7H3–CAR-T^VSV (5 × 10⁶ cells). 16 h after the injection, CAR-T cells were isolated via magnetic bead-bound Ab against CD3. (B) GSEA for CAR-T cells isolated from A. The top 15 enriched pathways in CR2/3–B7H3–CAR-T^VSV group were presented compared with B7H3–CAR-T^VSV group (n = 3 biological replicates). (C–G) Flow cytometric analysis of intra-tumor CAR-T cells in A. The ratios of intra-tumor CAR-T cells (C), Ki67 expression (D), mitochondria mass (E), glucose uptake (F), and IFNγ expression (G) of CAR-T cells were analyzed (n = 4 biological replicates). (H) Scheme of the experimental design for analyzing the CAR-T persistence in vivo. CAR-T cells were labeled with DiR (5 µM) and then were loaded with VSV (MOI = 10). CAR-T cells were injected 7 days after tumor implantation. (I) Representative fluorescence images of tumor-bearing mice for measuring the distribution of DiR-labeled CAR-T cells. (J) Quantitative analysis of fluorescence images (n = 5 mice). (K) Diagram depicting generation of antigen heterogeneity glioma model and CAR-T treatment schedule. VSVΔ51 was combined with CAR-T cells in three different forms for virus delivery: (1) physical increase of viral adsorption onto CAR-T via a spin-infected protocol (CAR-T^{VSV-spin infected}), (2) separate delivery to CAR-T and virus (CAR-T + VSV i.v.), and (3) specific loading of virus and CR2/3-CAR (CAR-T^{VSV loading}). CAR-T cells and VSVΔ51 were i.v. injected at 7 days, and flow cytometry was performed at 14 days. (L) Kaplan–Meier survival curve of mice bearing U87^{20% B7H3-KO} intracranial tumors (n = 5 mice). (M and N) Monitoring of tumor progression via bioluminescence imaging of luciferase in U87 xenograft mice at indicated time. (M) Representative bioluminescence images of U87 intracranial tumor after treatment. (N) Quantitative analysis of tumor bioluminescence data (n = 5 mice per group). Data represent the mean ± SD from one of two independent experiments (C–G, J, L, and N). Statistics by unpaired two-tailed Student’s t test (C–G and J), log-rank Mantel–Cox tests (L), and one-way ANOVA with Tukey’s post hoc test (N). *, P < 0.05; **, P < 0.01; N.S., no significance.