Hedgehog Pathway Inhibition in Liver Cancer Model Figure 3. GDC-0449 treatment reduces fibrosis in Mdr22/2 mice. A. Immunohistochemical staining for a-SMA (top panel) and Sirius red (bottom panel) in sections of non-tumor liver from representative age-matched Mdr22/2 and wildtype mice (106). B. Pooled Hepatic hydroxyproline content of 2–52 wk-old wildtype (WT) and age-matched Mdr22/2 mice (n = 3–5/group). Results in Mdr22/2 mice were normalized to that of age- matched WT mice and graphed as fold change. Data are displayed as mean +/2 SD (*p,0.05) C. Non-tumor liver sections stained for a-SMA (top panel, 206) and Sirius red (bottom panel, 106) in representative DMSO- and GDC- treated Mdr22/2 mice. D. Heptic hydroxyproline content of DMSO- and GDC- treated mice (n = 9/group). Results in GDC-0449-treated mice were normalized to that of DMSO vehicle-treated mice and graphed as fold change. Data are displayed as Mean +/2 SEM (*p,0.05). doi:10.1371/journal.pone.0023943.g003 necropsy findings: Only 56% of GDC-0449-treated mice had visible liver tumor nodules, compared to 80% of the DMSO mice (Table 1). Furthermore, both MRI and necropsies showed a decreased number of metastasis in GDC-0449-treated mice compared to vehicle-treated controls (Table 1). Histological analysis of H&E-stained liver sections was also performed on all animals from both cohorts. If tumor nodules were not grossly visible or greater than 10 mm in size, the samples were excluded from analysis. Microscopic tumor nodules in GDC-0449 treated animals demonstrated increased rates of hemorrhagic infarct (20% vs 0%; Figure 6D, top panel), microvesicular steatosis (40% vs 0%, Figure 6D, middle panel), acidophilic necrosis and degenerative cytoplasmic changes (70% vs 40%, Figure 6D, bottom panel) in comparison to tumors from vehicle treated animals. Thus, findings on MRI, necropsy, and liver histology were consistent and demonstrated that Hh pathway inhibition caused significant regression of primary and metastatic HCC. Discussion We studied Mdr22/2 mice to determine whether or not activation of the Hh pathway contributes to hepatocarcinogenesis PLoS ONE | www.plosone.org 6 September 2011 | Volume 6 | Issue 9 | e23943