Fig 2: Inflammatory and histological changes after overexpression of ANG-2.(A) Representative H&E staining of sWAT sections (Magnification = 200×) from 5 week HFD/Dox 600 mg/kg fed Adipo-rtTA and Adipo-ANG2 mice. Bar = 60 µm. (B) Representative H&E staining of liver tissues (magnification = 200×) from both groups. Lipid droplets are evident in the liver sections. Bar = 60 µm.(C) Representative immunofluorescence staining picture of the macrophage marker F4/80 (Green, Magnification = 200×) in sWAT sections from both groups. Bar = 100 µm. (D and E). Inflammatory marker mRNA levels assessed by qPCR in sWAT of control and ANG-2 transgenic mice. General inflammatory markers: Adgre1 (F4/80), Il6 (IL-6), Csf1r (CD115), Itgam (CD11b); pro-inflammatory macrophage markers: Ly6c, Ifng, Tnfa, Nos2; anti-inflammatory macrophage markers: Mrc1 (gene symbol for CD206), Il10, Clec10a (gene symbol for CD301). (F) The relative content of anti-inflammatory macrophages (M2 type macrophages) in both sWAT (left panel) and eWAT (right panel) from control and ANG-2 overexpressing mice. For the flow cytometry analysis, anti-inflammatory macrophages are considered as the F4/80+/CD11b+/CD206+ population, and the relative content in the total macrophage population is shown. (G) Representative Evan’s Blue staining of sWAT from Adipo-rtTA and Adipo-ANG2 mice. Black arrow indicates positive staining of Evan’s Blue dye, reflecting enhanced leakage from blood vessels in sWAT. (H) Beiging adipose tissue marker Ucp1 analyzed through qPCR in sWAT and eWAT of control and ANG-2 transgenic mice. For the flow cytometry study, n = 3 mice per group. For all the other statistical graphs: n = 10 mice per group. Data are shown as mean ± s.e.m. *p<0.05, **p<0.01.DOI: http://dx.doi.org/10.7554/eLife.24071.009

Fig 3: GS inhibited pro-inflammatory macrophage recruitment. (A) Western blot analysis of CCR2 in liver tissues and statistical graph, n = 3. (B) Immunohistochemical staining of CCR and statistical graph, n = 4–6. (C) The phenotype of macrophages in liver tissues was measured by flow cytometry, n = 3. F4/80+CD11b+ was labeled to represent the macrophages, while CD86+CD206- macrophages were recognized as M1 phenotype, and CD86-CD206+ macrophages were recognized as M2 phenotype. (D) Double immuno-fluorescence staining of iNOS and CD11b. Results are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Uptake 4T1 exosome-like nanovesicles (ENVs) by Kupffer cells. (A) Schematic representation of 4T1 ENVs preparation from 4T1 exosomes. (B) Representative electron microscopic images of 4T1 exosomes (left) and 4T1 ENVs (right). Scale bar, 200 nm. (C) Size distribution of 4T1 exosomes and 4T1 ENVs using NanoSight NS300 (Westborough, MA) with flow speed at 0.03 mL per min. (D-E) Representative images of 4T1 breast primary tumors (D, top) and lungs (E, top, metastatic nodules indicated by arrows) from tumor-bearing BALB/c mice (n=5) at 21 d subjected to breast pad injection of 1x106 4T1 tumor cells along with 4T1 ENVs (0.5g/kg, body weight, n=5) or PBS via intravenous (i.v.) injection. Quantification of primary tumor volume and metastasis nodule numbers (> 1 µM) (D-E, bottom). NS, no statistical significance, paired t-test. (F) BALB/c mice were i.v. injected with DiR dye-labeled 4T1 ENVs (50 pmol per mouse, n=5) 24 h after administration of clodronate. Intensity of fluorescence of ENVs in mice before and after treatments showing representative images (left) and mean intensity of fluorescence presented (right, mean net intensity/sum intensity/area, clodronate/ENVs vs ENVs, * p<0.05, t-test). (G) Representative images of liver and lung from BALB/c mice receiving a single intravenous injection of 0.5 g/kg DiR dye-labeled 4T1 ENVs at 6 h (top); quantification of fluorescence density (middle); comparison of fluorescence intensities between the liver and lung (bottom). (H) PKH26-labeled (red) 4T1 ENVs located in liver Kupffer cells (F4/80+, green) from BALB/c mice treated with clodronate for 24 h following i.v. injection of ENVs, visualized with confocal microscopy, assessed 1 d after administration of clodronate. (I) 4T1 ENVs labeled with fluorescence PKH26 subjected to RAW264.7 cells for 24 h. After washing with PBS, 4T1 ENVs/PKH26-positive macrophage cells estimated by FACS. Numbers in quadrants indicate percent cells in each throughout. Data are representative of three independent experiments (error bars, s.e.m.). NS, no statistical significance, *P < 0.05 (two-tailed t-test).

Fig 5: Antitumor immune response induced by photo-irradiated aCD47/Ce6@PPG hydrogel.a Representative flow cytometry analysis of tumor-infiltrating CD4+ and CD8+ T cells gated on CD3+ T cells on day 8 after the surgery. Relative quantifications of CD8+ T cells (b) and CD4+ T cells (c) in tumors on day 8 after the surgery. d Relative quantification of mature DCs in tumor-draining lymph nodes on day 8 after the surgery. e Representative flow cytometry analysis of M1-like TAMs (CD80+) and M2-like TAMs (CD206+) gated on F4/80+CD11b+ cells in tumors on day 8 after the surgery. Relative quantifications of the proportions of M1-like TAMs (f) and M2-like TAMs (g) gated on F4/80+CD11b+ cells. h The ratio of M1/M2 in different groups. Cytokine levels of IL-6 (i), IFN-? (j), TNF-a (k) isolated from the plasma of the mice in different treatment groups on day 8 after the surgery. All data are shown as mean ± SEM (n = 3 mice per group). The comparison of two groups was followed by Student’s t test (two-tailed). Source data are provided as a Source Data file.