Fig. 7
The TNF-α-VEGF-D axis is involved in lymphangiogenesis and lymph node metastasis (LNM) of GBC in vivo. a. Establishment of orthotopic xenograft models of GBC in nude mice. After anesthesia, the abdominal cavity of the nude mouse was opened, the gallbladder was exposed, and one of three NOZ cell lines (NOZ, LV-siNC, or LV-siVEGF-D) was injected into the cavity of gallbladder; the abdominal cavity was subsequently closed. b, c. After treatment with TNF-α (2 μg⁄ kg) twice a week for 3 weeks, the mice were dissected, and the tumors were excised. Infiltrative growth (green arrow), LNM (yellow arrow), ascites (red arrow) and hepatic metastasis (white arrow) were observed in the orthotopic xenograft models. LNM was further confirmed by H-E staining (C-2: 200×, C-3: 400×), and invasive tumor cells (black arrow) could be observed in the lymphoid follicles. d. Detection of lymphatic vessels (marked by LYVE-1 and indicated by blue arrows) in the orthotopic xenograft tumors was achieved by immunohistochemistry. e. Number of lymphatic vessel in the orthotopic xenograft tumors. TNF-α increased the number of lymphatic vessels in the NOZ and LV-siNC group, whereas the knockdown of VEGF-D decreased this effect (*P < 0.05)