Fig. 5. TGFβ1 activation promotes c-Myc positive tumor cells progression and metastasis in vivo.

a Study design. Inducible TGFβ1 (or EGFP) expressing HCC4-4 cells were delivered into mouse liver through intrasplenic injection. Food containing doxycycline (Dox food) was administered 3 days after implantation to induce TGFβ1 or EGFP expression. Three mice from each arm (EGFP or TGFβ1) were sacrificed 4 weeks after implantation, and then mice were sacrificed every 3 days one per group in parallel. b Numbers of mice showing tumors in each organ at the time of sacrifice from EGFP (N = 8) and TGFβ1 (N = 8) groups. The Y axis (tumor incidence) represents the number of mice from the EGFP or TGFβ1 group displaying neoplastic lesions in each organ. Tumors were examined macroscopically and microscopically by H&E staining. c Representative images of H&E and c-Myc immunohistochemical staining in EGFP and TGFβ1 groups in different organs. Black boxes on right lower corner denote enlarged views for better visualization. Scale bars: 200 μm for H&E, 100 μm for c-Myc. d Representative images of Ki67 immunohistochemical staining and quantification of Ki67 positive cell percentage in liver tumors from both groups. Student t test was applied for statistical analysis. P < 0.0001. Scale bars: 100 μm. e mRNA expression of TGFβ downstream target genes (Snai1 Twist1, and Zeb1) and epithelial marker genes (Cdh1 and Tjp1) in EGFP or TGFβ1 activated murine liver tumors. Student t-test was applied for statistical analysis.