Extended Data Fig. 3: Analyses of binding and immunological synapses of human liver-cancer cells and NK cells.

a, SEM showing the immunological synapses of human liver-cancer cells and NK cells. Primary NK cells purified from resected tumors of liver-cancer patients, of liver NK cells from outside the tumor of these liver-cancer patients, and of NK cells isolated from peripheral blood of normal donors and were co-cultured with liver-cancer cells (HuH7 cell line) for 1, 2 and 4 h. The large cells on the left represent HuH7 cells. The small cells on the right represent NK cells. Scale bar, 0.5 μm. Data are representative of 9 samples. b, (related to Fig. 2c), TEM showing the interface of human liver-cancer cells and NK cells at low magnification. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 cells for 1, 2 or 4 h. The large cells are HepG2 cells; the small cells are NK cells. The red arrows guides the junction of human liver-cancer cells and NK cells. Scale bar, 1 μm. Data are representative of 5 samples in each group. c, Super resolution microscopy (Leica, STED) show the polymerized actin filaments (microclusters) of purified NK cells. Representative 3D images were shown. Scale bar, 1 μm. d, Fluorescence photobleaching recovery (FPR) was used to assess membrane fluidity (DiI membrane stain) based on CLSM imaging. Data are representative of 2 samples in each group. e, CLSM showing the binding of human liver-cancer cells and NK cells. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. The large cells on the left are HepG2 cells; the small cells on the right are NK cells. Green, F-actin staining. Scale bar, 0.5 μm. f, K562 cells were used as target cells in cytotoxicity assays with NK cells isolated from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood. % target cell death presents the percent of dead target cells among total target cells (7AAD + CFSE + ) assessed by flow cytometry. n = 3 samples per group. g–h, CLSM showing GZMB and CD107a stain in NK cells. NK cells freshly purified from liver cancer patients (tumor and liver tissue outside tumor) and normal donor peripheral blood and were co-cultured with HepG2 (liver cancer cell line) cells. Scale bar, 0.5 μm. Data are the mean ± s.d. Data were analyzed by two-way ANOVA.

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