Hedgehog Pathway Inhibition in Liver Cancer Model Figure 6. Hh pathway inhibition decreases in liver tumor volume by magnetic resonance imaging (MRI) and induces histologic features of tumor involution. A. Representative cross-sectional image of intrahepatic tumor as identified by contrast-enhanced MRI following GDC-0449 treatment. B. Representative MRI images of sagittal, coronal, and oblique perspectives used for volumetric analysis. C. Change in tumor volume were quantified by computerize generated volumetric measurements and graphed as Mean6SD (*p,0.05). D. Micrographs of representative histologic changes induced by, GDC-0449, treatment. Top panel shows tumor necrosis; middle panel demonstrates fatty degeneration; bottom panel show vacuolated tumor cells with pyknotic nuclei. doi:10.1371/journal.pone.0023943.g006 stimulate autocrine-paracrine synthesis of Hh ligands [20,42]. Relative to age/gender matched healthy control mice, aged mdr2- deficient mice with ductular reactions and liver fibrosis demon- strated higher hepatic mRNA levels of PDGF-b, suggesting a mechanism by which the fibrogenic repair response itself might perpetuate excessive hepatic accumulation of Hh ligands. The latter concept is supported by evidence that inhibiting Hh signaling with GDC-0449 reduced accumulation of myofibroblasts and ductular-type progenitors, decreased expression of PDGF-b, and suppressed hepatic expression Hh ligands in mice that remained at risk for hepatocyte injury due to genetic deficiency of Mdr2. Chronic over-production of Hh ligands has important patho- biological implications because mice that generated increased Hh ligands also demonstrated excessive activation of the Hh pathway in their livers. This was evidenced by larger numbers of cells with nuclear staining for the Hh-regulated transcription factor, Gli2, and enhanced expression of various Hh-target genes, including Gli1 and osteopontin. In such animals, we showed that treatment with a highly specific antagonist of Smoothened was able to PLoS ONE | www.plosone.org 9 September 2011 | Volume 6 | Issue 9 | e23943