Extended Data Fig. 8: The phenotype of intratumoral NK cells after blockade of SM catabolism.
From: Tumors evade immune cytotoxicity by altering the surface topology of NK cells
a, SEM image showing the membrane protrusions of purified intratumoral NK cells from liver cancer patients. Intratumoral NK cells were subjected to knockdown of an acidic SMase (si-ASM), of neutral SMases (si-NSMASE1/2/3), or control (si-mock). Each dot represents one patient sample. n = 5 samples. Scale bar, 1 μm. b–e, SEM image showing the membrane protrusions of purified peripheral and liver NK cells. Representative SEM imaging is presented. The mean number of membrane protrusions and the mean length of membrane protrusions in each sample are shown. Each dot represents one sample. n = 5 samples. Scale bar, 1 μm. f, Flow cytometry assaying proliferation (Ki67+) and apoptosis (annexin-V+) of intratumoral NK cells. g, Flow cytometry assaying the expression of IFN-γ, granzyme-B (GZMB), CD107a and perforin in intratumoral NK cells. n = 5 per group. f–h, Intratumoral NK cells were treated with GW4869 (1 μM), LCL521 (1 μM), or DMSO (control) for 24 h. h–i, (Related to Fig. 6g and i) human liver-cancer cells (HepG2) were employed as targets in cytotoxicity assays with indicated intratumoral cells by real-time Cell Index measurements (xCELLigence). % cytolysis is shown (n = 6 per group). Intratumoral NK cells cells were co-cultured with liver-cancer cells for cytotoxicity assays after treatment with GW4869 (1 μM) + anti-Tim3-blocking antibody (10 μg/mL), LCL521 (1 μM)+anti-Tim3-blocking antibody (10 μg/mL), GW4869 (1 μM), LCL521 (1 μM), anti-Tim3-blocking antibody (10 μg/mL), or DMSO (control) for 24 h. Data are the mean ± s.d. Data were analysed by two-way ANOVA.
