Figure 2. In vivo antitumor efficacy and infiltration of MMP3-overexpressing MSLN CAR-T cells in a CAF-enriched xenograft model. (A) Schematic overview of CAF-enriched xenograft model establishment and CAR-T cell infusion. NXG mice were subcutaneously injected with H1299-MSLN cells mixed with CAFs (2×106 cells each). Mice were infused with MOCK cells, MSLN CAR-T cells, or MMP3-MSLN CAR-T cells (2×106 cells per mouse) on day 0. Tumor growth was monitored by BLI and caliper measurements. (B–C) Representative BLI images (B) and kinetics of BLI signal intensity (C) at indicated time points (n=5 per group). (D) Tumor volume curves calculated from caliper measurements (length×width2/2) (n=5 per group). (E) Body weight monitoring throughout the study (n=5 per group). (F) Schematic overview of immune infiltration analysis. Peripheral blood and tumor tissues were collected on days 4 and 8 post-CAR-T infusion, respectively, for flow cytometric analysis. (G–I) Flow cytometry analysis of CD3+T cells (G), CAR+T cells (H), and CD137+CAR T cells (I) in peripheral blood (n=3 per group). (J–L) Flow cytometry analysis of CD3+T cells (J), CAR+T cells (K), and CD137+CAR T cells (L) in tumor tissues (n=3 per group). (M) Representative H&E staining images of tumor tissues collected 8 days post CAR-T cell infusion. (N) Representative immunohistochemical staining images of CD3+T cell infiltration in tumor tissues collected 8 days post CAR-T cell infusion. All data are presented as mean±SEM. Statistical significance was determined using Mann-Whitney U test (C), two-way ANOVA with Tukey’s multiple comparisons test (D, E), and unpaired two-tailed Student’s t-test (G–L). *p<0.05, **p<0.01, ***p<0.001. ANOVA, analysis of variance; BLI, bioluminescence imaging; CAF, cancer-associated fibroblast; CAR, chimeric antigen receptor; FACS, fluorescence-activated cell sorting; IHC, immunohistochemistry; i.v., intravenous; MOCK, untransduced control; MMP, matrix metalloproteinase; MSLN, mesothelin; ns, not significant; s.c., subcutaneous.
