Figure 3.

Dec and Aza pretreated CD44v6 CAR-T cells exhibit enhanced anti-tumor ability in vitro and in vivo. (A) Cytotoxicity of NT and CD44v6 CAR-T cells treated with Dec (0.05, 0.1 μM) (left, n=6) and Aza (0.5, 1 μM) (right, n=5) for 6 days against MV4-11 cells in an E:T ratio of 10:1 for 24 h. (B) Percentage of CD107a⁺ NT and CD44v6 CAR-T cells treated with Dec (0.05, 0.1 μM) (left, n=3) and Aza (0.5, 1 μM) (right, n=4) for 6 days against MV4-11 cells in an E:T ratio of 1:1 for 16 h. (C, D) Cytokine IFN-γ (C) and TNF-α (D) release of CD44v6 CAR-T cells (n=5) treated with Dec (0.05, 0.1 μM) and Aza (0.5, 1 μM) for 6 days against MV4-11 cells in an E:T ratio of 1:1 for 24 h. (E) Heatmap shows expression of elevated tumor necrosis factor and interleukin genes of CD44v6 CAR-T cells treated with Dec 0.1 μM and Aza 1 μM for 6 days in RNA-seq. (F) Schematic of Dec pretreated CD44v6 CAR-T treatment of MV4-11 cells xenografts mice. BALB/c-nu mice were subcutaneously injected with 5×10⁶ MV4-11-firefly luciferase (Luc⁺ MV4-11) cells on day 0. NT (n=5), CD44v6 CAR-T (n=5) and Dec pretreated CD44v6 CAR-T (dCAR-T, n=5) (5×10⁶) were intratumorally injected on day 10 and day 17. Tumor burden was analyzed by BLI on days 7, 14, 21 and 28. (G, H) Graph (G) and BLI images (H) showing the progress of tumor burden at the indicated time point. Luc⁺ MV4-11, luciferase-expressing MV4-11 cells; dCAR-T, Dec pretreated CD44v6 CAR-T; BLI, bioluminescence imaging. Data are depicted as the mean ± SD. *p<.05; **p<.01; ***p<.001; ****p<.0001; ns, not significant.