Fig. 6: MLKL-driven necroptosis induces METs formation to promote tumour metastasis.

a 3D tumour spheroid-based invasion of co-cultured tumour cells; Scale bar: 100 μm. b 3D tumour spheroid-based migration of co-cultured tumour cells; Scale bar: 100 μm. c Expression of Fn1 in cancer cells projected onto the single-cell trajectories. d WB of EMT markers in indirectly co-cultured tumour cells treated with or without GW. e Incidence of liver metastasis in NOD-SCID orthotopic model. One million co-cultured tumour cells treated with saline or GW were injected, and mice were further treated with or without GW (100 µM in 50 μl, iv., 3×/week); mice were euthanized 4 weeks after injection; n = 8 mice in each group. f Representative immunofluorescence staining of CitH3 and MMP12 in macrophages indirectly co-cultured with tumour cells. g WB of MET markers in solo-cultured macrophages and co-cultured with macrophages treated with DMSO or MET inhibitor (Cl-amidine, 25 µg/ml). Macrophages were collected together with the CM as the sample. h The effect of GW treatment on MET-related protein levels was determined by WB analysis. i After indirectly coculturing with tumour cells for 72 h under different treatments, the macrophages were stained for extracellular nucleic acids with SYTOX-Orange (0.6 µM); Scale bar: 100 µm. j ELISA analysis of plasma CitH3 in C57BL/6 orthotopic model mice received different treatments: GW (100 µM in 50 µl, iv., 3×/week), anti-Ly6G (100 µg, iv., 3×/week), or macrophage depletion (clodronate liposomes, 200 µl, iv.); n = 3 mice for each group. k WB of EMT markers in co-cultured tumour cells treated with DMSO or MET inhibitor (Cl-amidine, 25 µg/ml). l Incidence of liver metastasis in NOD-SCID orthotopic model. One million tumour cells co-cultured under saline or MET inhibitor (Cl-amidine, 50 mg/kg) and the corresponding concentrated supernatant were injected. n = 8 mice in each group. mice were euthanized 4 weeks after injection. n = 3 biologically independent samples in (a–c, f–h, j, l). Unless specified otherwise, the data are presented as means ± SEM (error bar) and compared using the two-sided Student’s t test; *P < 0.05; **P < 0.01; and ***P < 0.001; ns, no significance. Source data are provided as a Source Data file.