Fig. 4. EE enhances β–AR signaling to control tumor via sympathetic nervous system.

A, B ELISA analysis of Epinephrine (EPI) and Norepinephrine (NE) in the serum and liver from normal mice A or DEN + CCl₄-treated mice B which were fed under Standard Environment (SE) and Enriched Enrichment (EE) conditions (A, level of serum EPI, SE: n = 11, EE: n = 7; level of serum NE, n = 9 for each group; level of liver EPI or NE, n = 4 for each group; B, level of serum EPI, SE: n = 9, EE: n = 8; level of serum NE, n = 6 for each group; level of tumor EPI or NE, SE: n = 6, EE: n = 10). All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test. C Representative images of immunostaining of β1-Adrenergic Receptor (AR), β2-AR, and β3-AR on liver tumors tissues from DEN + CCl₄-induced HCC mouse model. Original magnification 20 × 10, Scale bar, 100 μm. D Positive β1-AR, β2-AR, and β3-AR areas of liver tumor tissues were quantified by ImageJ analysis in DEN + CCl₄-induced HCC mouse model (n = 8 for each group). All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test. E Western blot assay for detecting the expression of β1-AR, β2-AR, β3-AR, and CCL2 in tumor tissue lysates from DEN + CCl₄-induced HCC model and subcutaneous Hepa1-6 tumors (n = 4 for each group). F Total tumor numbers (left) and numbers of tumor with diameter ø ≥ 3 mm (right) on livers from DEN + CCl₄-induced tumor-bearing mice. Mice were fed under SE or EE conditions with or without β-ARs blockade treatment (β-block, treated with SR59230A + propranolol). Vehicile+SE: n = 7, Vehicile+EE: n = 9, β-block+SE: n = 9, β-block+EE: n = 9. All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test. G, H Tumor volume of subcutaneous Hepa1-6 tumor-bearing mice G and H22 tumor-bearing mice H treated without (Vehicle, VEH) or with β-ARs blockade treatment (G, SE + VEH: n = 8, EE + VEH: n = 12, SE + β-block: n = 10, EE + β-block: n = 12; H, SE + VEH: n = 8, EE + VEH: n = 8, SE + β-block: n = 11, EE + β-block: n = 8). All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test with *p < 0.05, **p < 0.01, ***p < 0.001. I Tumor volume of subcutaneous Hepa1-6 tumors in mice injected with or without6-hydroxydopamine (6OHDA, 25 mg/kg) SE + VEH: n = 12, EE + VEH: n = 11, SE + 6OHDA: n = 11, EE + 6OHDA: n = 14. All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test with *p < 0.05, **p < 0.01, ***p < 0.001. J–L Percent of immune cells in tumors were detected by flow cytometry from Fig. 4GH, Fig. 4HI and Fig. 4IJ (J, n = 6 for each group; K, n = 6 for SE + VEH and EE + VEH group, n = 5 for SE + β-block and EE + β-block group; L, n = 10 for SE + VEH and EE + VEH group, n = 6 for SE + 6OHDA and EE + 6OHDA group). All data are presented as the mean ± SEM, and analyzed by two-tailed unpaired Student’s t test with *p < 0.05, **p < 0.01, ***p < 0.001.