Fig 2: CD11b+ CD86+ IL10+ tumor-associated macrophage was dominate subtype in tumor tissue after bevacizumab continuous treatment.a Representative H&E staining image of tumor sections from mice treated with bevacizumab and PBS at 8-week. Scale bar, 100 µm. b Representative image and quantitative evaluation of tumor sections from mice treated with bevacizumab and PBS stained for CD68 in each tumor of mice at 8-week (IOD/Area). n = 5. Scale bar, 100 µm. *P < 0.05 by t test. Error bars represent s.d. c Representative image and quantitative evaluation of tumor sections from mice treated with bevacizumab and PBS stained for CD86 at 8-week. Scale bar, 100 µm. *P < 0.05 by t test. Error bars represent s.d. d Representative image and quantitative evaluation of tumor sections from mice treated with bevacizumab and PBS stained for CD206 at 8-week. Scale bar, 100 µm. *P < 0.05 by t test. Error bars represent s.d. e Representative image and quantitative evaluation of tumor sections from mice treated with bevacizumab and PBS stained for IL10 at 8-week. Scale bar, 100 µm. *P < 0.05 by t test. Error bars represent s.d. f Macrophages were detected as CD11b+ CD86+ or CD11b+ CD86+ cells, respectively. Each sample contained tumor tissues from three mice. g Representative analysis of the single-cell suspended from PBS or bevacizumab-treated tumor tissues. Macrophages were detected as CD11b+ CD86+ IL10+ cells. Each sample contained tumor tissues from three mice. h Representative images of double staining for CCL1 and nucleus. Scale bar, 50 µm.

Fig 3: (a–e) Markers associated with inflammation induced renal fibrosis. mRNA expression of Il1a, Il1b, Il6, Tnfa and Il10 in renal cortical tissues of WT and DCM mice administered NAC or saline (n = 6–8). Data are mean ± SEM. *P < 0.05, **P < 0.01 vs saline treated WT mice. +++ P < 0.001 vs saline treated DCM mice. Il1a = interleukin 1 alpha, Il1b = interleukin 1 beta, Il6 = interleukin 6, Tnfa = tumor necrosis factor alpha, Il10 = interleukin 10. Other abbreviations are as for Fig. 1.

Fig 4: Continuous treatment with bevacizumab induces acquired resistance and cytokines change in vivo.a Schematic of experimental design for bevacizumab resistance model development. b Tumor growth volume over time following subcutaneous transplant of tumor tissues into Balb/c nude mice. n = 5 for each condition. *P < 0.05 by two-way ANOVA test. Error bars represent s.d. c Representative images of sections from tumors at the time points of 4-week and 8-week stained for CD31 and VEGF A. Scale bar, 100 µm. d Quantitative evaluation of CD31 and VEGF A in each tumor of mice (IOD/Area). **P < 0.01; ***P < 0.001 by t test. Error bars represent s.d. e qRT-PCR of IL1ß, IL6, IL10, HIF1a, TGFß, TNFa, VEGF, HMGB1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL8, CCL9, CCL11, CCL22, CCL24, CXCL10, CXCL16 mRNA levels in tumor tissues of 4-week, 8-week and 13-week. ***P < 0.001; ****P < 0.0001 by t test. Error bars represent s.d.

Fig 5: Sorafenib treatment increases intratumoral hypoxia and alters HCC microenvironment towards an immune-resistant state in mouse models.A, B orthotopic Hepa 1-6 and Hepa 1c1c7 tumors were treated with sorafenib or vehicle control for three weeks, then collected protein lysates for western blot (A) or tissue samples for Pimonidazole staining (B). C, D infiltrating immune cells in orthotopic Hepa 1-6 and Hepa 1c1c7 tumors treated with sorafenib or vehicle control were evaluated by flow cytometry. CD3+CD4+CD25+FoxP3+ Treg, CD11b+Gr-1-Ly6C-F4/80+ TAM, CD11b+Gr-1+Ly6CintLy6G+ PMN-MDSC, CD11b+Gr-1+Ly6ChighLy6G- M-MDSC and CD3+CD4-CD8+ Cyto T cells were evaluated. E relative expression of PD-L1, TGFB1, IL10, and IL13 in orthotopic Hepa 1-6 and Hepa 1c1c7 tumors was evaluated by qRT-PCR. F protein expression of PD-L1 in orthotopic Hepa 1-6 and Hepa 1c1c7 tumors was evaluated by western blot. All assays were done with at least three repeats. Data were shown as mean ± s.d., *P < 0.05.