Fig. 4

Col1-enriched cirrhotic-ECM initiated neutrophils to form NETs to shield tumor cells and impede T cell motivation. A, B Establish scheme (A) and quantification (B) of neutrophils chemotaxis to Col1-treated HCC cells in a Transwell system. Col1 and HCC cell were co-placed on the lower chamber, and CSFE-labeled neutrophils were placed on the upper chamber. C Representative images and quantification of SytoxgGreen-marked NETs formation in a co-culture system of neutrophils and HepG2 w/o ECM/ECM-Col/Col1. Scale Bar: 5 μm. D Scheme plot of the HepG2-neutrophil-T cell co-culture assay in dishes coated w/o ECM/ECM-Col/Col1. E Quantification of HepG2 survival by flow cytometry in assay in D. F Fluorescence images of NETs (SytoxGreen) shielding HepG2 cell (ER tracker blue) from adherent T cells (Dio red) w/o Col1. Scale Bar: 5 μm. G Representative fluorescence images of local spatial distribution of HCC cell (Hoechst), NETs (H3cit, green) and T cell (CD8 red) in Fig. 3H. Scale Bar: 5 μm. H Establish scheme (H) and quantification (I) of T cells chemotaxis to Col1-treated HCC cells in a Transwell system by flow cytometry. HCC cells were placed on the lower chamber of Transwell assay. PET membrane was coated with Col1/NETs/Col1+NETs. CSFE-labeled T cells were placed on the upper chamber. J IHC images of spatial distribution of intra-tumor T cell and neutrophils at the intra-tumor injection site of Col1 in Hepa1-6 subcutaneous model. Scale Bar: 200 μm. K, L Images of Tumor (K), and quantification (L) of Intra-tumor GZMB+CD8 T cell, IFN-γ+CD8 T cell and CD8 T cell by flow cytometry in Hepa1-6 orthotopic model with DMN-induced cirrhosis w/o GSK484. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001