Fig. 6

A combined strategy of DNase 1 plus HCQ/Aspirin against NETs effectively impaired HCC metastasis in a mice model. a Effect of DNase 1 and HCQ/Aspirin alone or in combination on HCC progression and spontaneous metastasis (n = 8 each) in a modified Hepa1-6 orthotopic model. Representative images of liver tumor with intrahepatic metastasis (IHM, pointed with a rightward arrow) and lung metastasis, and quantification were presented. b Representative images of H3cit and Ly6G expression and quantification. The expression of H3cit was stressed in red color by Adobe Photoshop software with identical procedure. Scare bar: 50 μm. c Representative fluorescence images of impaired NET formation from HCC-associated neutrophils treated with HCQ or Aspirin in vitro. Neutrophils were isolated from peripheral blood of Hepa1-6 orthotopically implanted mice, incubated for 4 h for NET formation in the presence of HCQ, Aspirin, or DNase 1, fixed and stained for DNA with SytoxGreen. Scare bar: 100 μm. d Impaired NET formation by Aspirin or HCQ plus DNase 1 treatment in vivo by serum MPO-DNA level. e System illustration of NETs to trap and fuel HCC metastasis potential through triggering an inflammatory response through TLR4/9-COX2 signaling. *P < 0.05; **P < 0.01. Data were presented as means ± SEM