Figure 8

In vivo tumorigenity: xenografts in balb/c nude mice. (A) Photographs of Large duct-type iCCA macroscopic tumour masses removed from the flank of mice 10 weeks after the injection of Large duct-type iCCA cells. Mice injected with pre-treated Large duct-type iCCA with Metformin 10 µM for 57 days (Pre-treated) did not developed a macroscopically evident tumour mass within subcutaneous tissues at the level of the injection point while mice injected with pre-treated Large duct-type iCCA with Metformin 0 µM for 57 days (Controls) cells did. See also supplementary Figure 4. (B) H&E stains on explanted tissues confirm the neoplastic nature of the subcutaneous mass in mice injected with Control Large duct-type iCCA (Control, panels on the left). Neoplastic cells infiltrate subcutaneous tissues including the inguinal lymph node (arrow). In mice injected with Pre-treated Large duct-type iCCA (panels on the right), subcutaneous tissue were collected from the injection point, using inguinal lymph nodes as reference. The microscopical examination of inguinal lymph node (arrow, upper panel) and subcutaneous tissues (lower panel) indicates the absence of tumour formation at microscopical level. Representative images for N = 6 independent experiments for mice injected with pre-treated Large duct-type iCCA with Metformin 10 µM for 57 days and N = 6 for iCCA untreated cells (Metformin 0 µM, Controls).