Fig. 3. Cancer-cell softness impairs T-cell mediated cytotoxicity in vitro and in vivo.

a, Lysis percentage of B16F10 cancer cells pre-treated with Chol (softened) or PBS (native) and co-cultured with activated Pmel CD8⁺ T-cells at an effector:target (E:T) ratio of 10:1 for 5 h (n = 3). b, Relative membrane cholesterol levels of B16F10 cancer cells with ACAT1 knock-down (ACAT1 KD) and ACAT1 overexpression (ACAT1 OE) (n = 3). Native B16F10 cancer cells serve as a standard (100%). c, Cortical stiffness of native, ACAT1 KD, and ACAT1 OE B16F10 cells measured by the optical tweezer (n = 19 ~ 21 individual cells). In the violin plots, the middle solid line shows median, and lower and upper dash lines show 25^th and 75^th percentiles, respectively. d, Representative scatter plots for native, ACAT1 KD, and ACAT1 OE B16F10 cells by deformability cytometry analysis, and their 50%-density contour plots (the inner contours correspond to 95% event density) with iso-elasticity lines dividing the diagrams into areas of different stiffness. e, Lysis percentage of ACAT1 KD and ACAT1 OE B16F10 cancer cells after 5-h co-culture with Pmel CD8⁺ T-cells at an E:T ratio of 10:1 (n = 4). Data are one representative of at least two independent experiments with biological replicates (a, b, e). f-h, Mice bearing native, ACAT1 KD, or ACAT1 OE B16F10 tumours were treated with adoptive transfer of Pmel CD8⁺ T-cells (5 × 10⁶ per mouse), as outlined in the experimental scheme (f) (n = 5 and 10 animals for PBS- and ACT-treated groups, respectively). Shown are tumour growth curves (g) and survival curves (h) of pooled data from two independent experiments with biological replicates. P values were determined by unpaired Student’s t test in (a-c, e), two-way ANOVA in (g), or log-rank test in (h). Error bars represent SEM. ACAT1, acyl-CoA:cholesterol acyltransferase 1; PBS, phosphate-buffered saline; ACT, adoptive cell transfer; s.c., subcutaneous; i.v., intravenous; n.s., not significant.