cholangitis with accompanying portal inflammation, ductular proliferation and portal fibrosis. Liver injury manifests shortly after birth and hepatocellular carcinomas emerge spontaneously between 50 to 60 weeks of age [8,9,10,11,12,13,14]. Unlike xeno- graft models that are widely utilized to examine mechanisms of- and treatments for- HCC, Mdr22/2 mice provide a model that parallels the natural evolution of HCC on a background of chronic inflammation, liver injury and fibrosis [15]. Gene expression analyses in Mdr2-deficient mice and hetero- zygote controls have demonstrated robust and sustained induction of multiple adaptive mechanisms that control cellular responses related to oxidative stress, inflammation, lipid metabolism, and proliferation, prompting speculation that these processes contrib- ute to hepatocarcinogenesis [16]. Although not formally assessed by earlier studies of Mdr2-deficient mice, another pathway that might play a role in fibrosis-associated hepatocarcinogenesis is Hedgehog (Hh), because Hh signaling has been implicated in both fibrogenic repair of liver injury and HCC. The Hh pathway is an evolutionarily conserved signaling pathway that is activated when Hh ligands (Sonic hedgehog and Indian hedgehog) bind to Patched (Ptc), a transmembrane receptor that is expressed on the surface of Hh-responsive cells. Upon ligand binding, Ptc is inactivated, relieving its repression of Smoothened (Smo), a trans-membrane protein that mediates Hh signaling inside the cell. Smo activation culminates in the nuclear localization of Gli-family transcription factors, Gli1, Gli2, and Gli3, which, in turn, regulate downstream gene expression. The pathway is quiescent in normal liver [17,18], but becomes re- activated as a repair mechanism in chronic liver injury [19,20, 21,22,23]. Hh ligands promote the growth and viability of myofibroblasts, the accumulation of which leads to abnormal liver repair, fibrosis and eventual cirrhosis [24,25]. Hh ligands also serve as viability and proliferative factors for liver epithelial progenitors, and expansion of this compartment has been linked to the formation and maintenance of hepatocellular carcinomas [26]. The possibility that Hh pathway activation contributes to hepatocarcinogenesis is supported by the fact that Sonic hedgehog (Shh) ligand expression is noted in approximately 60% of human HCCs, and expression of the Hh-regulated genes, Gli1 and Ptc, occurs in 50% of human tumors [27,28,29,30]. Based on these observations, we postulated that Hh pathway activation contributes to the pathogenesis of both liver fibrosis and fibrosis-associated HCC. To test this hypothesis, we treated aged Mdr2-deficient mice with the Hh pathway inhibitor, GDC-0449, a small-molecule inhibitor that binds to Smoothened (SMO). This agent was selected because of its human safety profile in phase 1 trials, as well as its effectiveness in solid organ tumors like basal cell carcinoma (BCC) and medulloblastoma [31,32,33]. Our aims were to determine whether or not mice with advanced liver disease and HCCs would tolerate Hh pathway inhibition and experience improvements in liver fibrosis and/or tumor burden. Methods Mice Mdr22/2 mice were a gift from Dr. Detlef Schuppan (Beth Israel Deaconess Medical Center, Boston, MA). Age matched FVB/NJ wild type mice were obtained from Jackson Laboratory (Bar Harbor, ME). All mice were housed with a 12-h light-dark cycle and given water and standard chow ad libitum. At ages, 2, 4, 12, 36 52 and 62-weeks, mice were sacrificed under general anesthesia. Liver weight and body weight were recorded, serum and liver tissue were collected. Animal studies were approved by the Institutional Animal Care and Use Committee as governed by the National Institute of Hedgehog Pathway Inhibition in Liver Cancer Model Health’s ‘‘Guide for the Care and Use of Laboratory Animals’’, Duke University Animal Welfare Assurance Number A3195-01. Serum AST/ALT determination Serum aspartate aminotransferase (AST) and alanine amino- transferase (ALT) were measured using kits commercially available from Biotron Diagnostics (66-D and 68-D respectively; Hemet, CA) according to the manufacturers’ instructions. Western Blot Total protein was extracted from snap frozen whole liver tissue in RIPA buffer. Samples were pooled by age (2–4 samples per age group) except as noted. Western blot analysis was performed as previously described. Membranes were probed with the following primary antibodies: Sonic hedgehog (sc-9024; Santa Cruz Biotechnology, Santa Cruz, CA), Indian hedgehog (ab39634; Abcam), b-actin (sc-47778, Santa Cruz Biotechnology), Gli2 (18- 732-292462, Genway, San Diego, CA). All antibodies were diluted 1:1000 and were incubated at 4uC overnight. Western blot images were acquired using a FluorChem HD2 digital darkroom system (Cell Biosciences, Santa Clara, California). Immunohistochemistry 4 mm formalin-fixed, paraffin-embedded samples were dewaxed and rehydrated. To evaluate tissue architecture, slides were stained with hematoxylin and eosin (H&E) and Sirius red per standard protocol. For immunohistochemistry, slides were incubated in 3% hydrogen peroxide/methanol. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer or 0.25% pepsin (K19; Invitrogen, Carlsbad, CA) for 10 minutes. Sections were blocked (Dako Envision, Carpinteria, CA) and incubated with primary antibodies overnight at 4uC: glioblastoma-2 (Gli-2; 18-732-292462; 1:2000; Genway, San Diego, CA); cytokeratin 19 (Troma-III; Hybridoma Bank, Iowa City, IA; 1:500); a-fetoprotein (AFP) (Dako, 1:1000); Indian hedgehog (Abcam; 1:750, Cambridge, MA); Polymer-HRP anti-rabbit (K4003; Dako) or anti-mouse (K40011; Dako) or MACH3 mouse AP polymer kit (MP530, Biocare Medical, Concord, California, USA) were used as secondary antibodies. 3,39- Diaminobenzidine (DAB) Substrate Chromogen System (K3466; Dako) and/or Ferangi Blue (Biocare) was employed in the detection procedure. Omitting primary antibodies from the reactions elimi- nated staining which demonstrated staining specificity. Images were acquired on an Olympus IX71 (Tokyo, Japan) inverted microscope using the DP2-BSW (Olympus) image acquisition software system. Quantitative Real-Time Reverse Transcription PCR RNA was isolated from whole liver, as well as from resected tumor specimens had standard TriZol extraction as has been previously described [24]. A table of primers has been included in the Supplementary Materials. Morphometry Formalin-fixed liver and tumor sections were stained for CD44 and osteopontin as described above, and were quantified by morphometric analysis with MetaView software (Universal Imaging, Downington, PA). A minimum of 5 randomly selected 106 or 206 fields/section were evaluated for each mouse. Quantitative Immunohistochemical Analysis Formalin-fixed liver and tumor sections were costained for Gli2 and CK19. A minimum of 5 ductular regions, 206 fields per section, were evaluated for each mouse by counting the number of cells co-labelled. PLoS ONE | www.plosone.org 2 September 2011 | Volume 6 | Issue 9 | e23943