Extended Data Fig. 9: Analyses of the membrane protrusions of mouse liver cancer NK cells.
From: Tumors evade immune cytotoxicity by altering the surface topology of NK cells
a–f, A genetically engineered mouse model of liver cancer developed with weakened PTEN signaling and with an activated K-RAS signal. a, Images of the liver from liver cancer model and normal mice. b, Representative density plots showing the purity of sorted mouse NK cells. c, SEM showing the membrane protrusions of the mouse NK cells from normal liver, spleen, and liver cancer. Representative SEM imaging (left). The mean number of membrane protrusions and the mean length of membrane protrusions (SEM) in each sample are shown (middle and right). n = 5 per group. d, SEM showing the membrane protrusions of the mouse NK cells from liver cancer. Freshly purified NK cells were treated with sphingomyelinase inhibitors (GW4869; 1 μM) or DMSO (control) for 24 h. n = 5 per group. e, Mouse liver-cancer cells (K-RASG12DPTEN−/− primary liver caner cell and Hep1-6 cell line) were employed as targets in cytotoxicity assays with the indicated NK cells, assessed with real-time Cell Index measurements (xCELLigence). % cytolysis is shown. After treatment with GW4869 (1 μM), or DMSO (control) for 24 h, liver cancer NK cells were co-cultured with target cells for cytotoxicity assays. n = 4 per group. f, Two mouse liver cancer models were established by subcutaneous injection of liver cancer cells (K-RASG12DPTEN−/− primary liver caner cell and Hep1-6 cell line). Freshly purified normal spleen NK cells were treated with D609 or PBS (control) for 24 h, followed by treated with GW4869 (1 μM), or DMSO (control) for 24 h. n = 6 per group. g, Single-cell MS was employed to analyse membrane sphingomyelins (SMs) of purified mouse NK cells from normal liver, spleen, and liver cancer. Normalized intensities of membrane SMs are shown. n = 5 in each group. h, SEM showing the membrane protrusions of the mouse NK cells from normal spleen. Freshly purified NK cells were treated with D609 (25, 100, 400 μM) or PBS (control) for 24 h. Each dot represents one mouse. n = 5 in each group. Data are the mean ± s.d. Data were analysed by two-way ANOVA. Scale bar, 1 μm.
