Hepatic hydroxyproline assay The hydroxyproline content in whole liver specimens was quantified colorimetrically as previously reported [34]. GDC-0449 treatment In the initial, dose-finding cohort of animals, 16 Mdr22/2 mice (age 57–68 weeks) were assigned to treatment with vehicle control (DMSO, n = 5), 20 mg/kg GDC-0449 (n = 5), or 40 mg/kg GDC- 0449 (n = 6). Male-female ratios were balanced between the groups. GDC-0449 (Selleck Chemicals, Houston, TX) was freshly constituted daily in DMSO. All mice were given a daily intraperitoneal injection for 9 days. On day 10, animals were sacrificed. Samples were collected as above. A second cohort of 20 Mdr22/2 mice (age 51–59 weeks of age) were subjected to whole body magnetic resonance imaging to assess tumor burden and then pairs of mice with comparable tumor burdens were assigned to treatment with either DMSO (control, n = 10) or 40 mg/kg GDC-0449 (n = 10). Drugs were delivered as described above; MRI scanning was repeated after the 9th day of treatment; mice were sacrificed for necropsy and tissue harvest. Pathology H&E and sirius red staining of liver sections was evaluated by a board-certified pathologist. Representative sections of tumor and non-tumor tissue were examined. Tumor tissue was defined as grossly visible nodules that were at least 10 mm in size and resectable. Abdominal magnetic resonance imaging Animals were assigned a blinding code, which was maintained during magnetic resonance (MR) data acquisition and analysis. MR mouse liver imaging was performed on a 7T Bruker Biospec 70/30 horizontal bore system (Billerica, MA). Animals were lightly anesthetized under isofluorane with continuous monitoring and maintenance of physiological parameters throughout the imaging session (,60 min for each animal). Axial and coronal 2D T2-weighted fast spin echo images (TURBO-RARE, TE/TR = 11/4200 ms with 1 mm slice thick, matrix = 2566256 and FOV of 2.4 cm62.4 cm, 5 averages, 0.0 mm interslice gap) images were first obtained for screening purposes and supplemental anatomic information. For directed tumor volumetric analysis, 64 contiguous 500 mM thick 3D FSE proton density images biased towards T1 weighting (TURBO- RARE, TE/TR = 9/1500 ms, matrix = 2566256664 and FOV 2.2 cm62.2 cm62.2 cm, 25 minutes duration) were acquired. All imaging sequences were performed using respiratory gating. Volumetric analysis from MR data sets was performed in Osirix software, an open source image processing application developed and maintained by Pixmeo (Geneva, SUI). Liver tumors were manually segmented in each animal by a board-certified radiologist (blinded to treatment) post-treatment. Selected areas were reviewed for consis- tency on coronal and sagittal representations, and cross-correlated with axial 2D FSE images. For volumetry, two separate volume measurements were obtained for each lesion, with the average volume then taken. Interrater reliability (kappa value) was = 0.97. T1 and T2-weighted scanning sequences were performed. Statistical analyses Results expressed as mean6SD. Significance established using the Student’s t-test. Differences were considered significant when p,0.05. Results Progressive accumulation of Hh-responsive cells in Mdr22/2 mice Various forms of acute and chronic liver injury induce hepatic expression of Hh ligands and activation of Hh responsive cells. Hedgehog Pathway Inhibition in Liver Cancer Model Serum levels of AST and ALT were consistently higher in Mdr22/2 mice than age-matched controls (Figure S1), confirm- ing that the knockout mice had chronic liver injury. Thus, we surveyed livers from Mdr22/2 mice for Hh pathway activation. Compared to liver protein lysates from wild type controls, lysates from Mdr22/2 mice demonstrated an increase in Sonic hedgehog (Shh) and Indian hedgehog (Ihh) by Western blot analysis. This was apparent at the first time point examined (2 weeks after birth) and maintained throughout the lifespan of the animals (up to 64 weeks of age) (Figure 1A). Immunohistochemistry for the Hh- regulated transcription factor, Gli2, demonstrated progressive, age-related accumulation of cells with nuclear Gli2 staining in Mdr22/2 mice. Such Gli2-positive cells included stromal cells, as well as hepatocytic and ductular cells (Figure 1B–C). Together, these data suggest that the Hh pathway is activated in chronically injured livers of Mdr2-deficient mice, resulting in progressive expansion of various Hh-responsive cell populations. Treatment of Mdr22/2 mice with the Smoothened inhibitor, GDC-0449, is safe and decreases Hedgehog pathway signaling To determine the appropriate dose of GDC-0449, a pilot study was done with 16 aged (57–68 week old) Mdr22/2 mice. Animals were given a daily intraperitoneal injection of 20 mg/kg GDC-0449 (n = 5), 40 mg/kg GDC-0449 (n = 6), or DMSO vehicle control (n = 5) for 9 days and then sacrificed. There was no statistical difference in mortality between the control and high dose (40 mg/ kg) GDC-0449 groups. At sacrifice, liver/body weight ratios of mice in the three groups were also similar, suggesting that a relatively short course of systemic treatment with GDC-0449 at 40 mg/kg is well tolerated by mice with advanced liver disease and HCC (Figures S2A–B). Western blot analysis of liver lysates from this cohort of animals demonstrated that GDC-0449 treatment caused a decrease in Shh ligand levels at the higher dose, and Gli2 expression attenuation at both doses (Figure S2C). Hedgehog signaling inhibition with GDC-0449 abrogates effects of Hh signaling on target gene expression Based on the data acquired in our dose-finding study, a second cohort of Mdr22/2 mice (51–59 weeks of age) were assigned to treatment with vehicle (DMSO; n = 10) or 40 mg/kg GDC-0449 (n = 10) via daily i.p injection for 9 days. Overall survival between the two groups in the second cohort was equal, with no deaths in the DMSO treatment group and 1 death in the GDC-0449 treatment group secondary to iatrogenic injury. Both the liver parenchyma and tumors of treated mice showed decreased expression of the Hh pathway target Gli2 (Figures 2A–B). Real-time PCR analysis of resected tumors revealed that GDC-0449 treatment released the inhibitory effects of Hh signaling on PPARa˜, causing a significant increase in its gene expression (Figure 2C). Treatment with GDC- 0449 also caused a significant decrease in expression of Gli1, another Hh target gene (Figure 2D). Hedgehog signaling inhibition with GDC-0449 reduces liver fibrosis Mdr22/2 mice demonstrated progressive age-related increases in hepatic expression of alpha-smooth muscle actin (a-sma), a marker of myofibroblastic hepatic stellate cells (Figure 3A, top panel). This was accompanied by enhanced expression of pro-fibrogenic factors, such as transforming growth factor TGF-b and platelet derived growth factor PDGF-b (Figures S3A–B), and progressive fibrosis, as evidenced by Sirius red staining (Figure 3A, bottom panel) and quantification of the hepatic hydroxyproline content PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e23943