Volume 142, Issue 5, Pages 1229-1239.e3 (May 2012) Disruption of Trp53 in Livers of Mice Induces Formation of Carcinomas With Bilineal Differentiation  Sarah–Fee Katz, André Lechel, Anna C. Obenauf, Yvonne Begus–Nahrmann, Johann M. Kraus, Eva M. Hoffmann, Johanna Duda, Parisa Eshraghi, Daniel Hartmann, Birgit Liss, Peter Schirmacher, Hans A. Kestler, Michael R. Speicher, K. Lenhard Rudolph  Gastroenterology  Volume 142, Issue 5, Pages 1229-1239.e3 (May 2012) DOI: 10.1053/j.gastro.2012.02.009 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 p53 deletion as a single genetic lesion leads to formation of liver carcinoma with bilineal differentiation features. (A) Tumor-free survival curve. Liver tumor formation was analyzed in mice with liver-specific homozygous (n = 94) and heterozygous p53 deletion (n = 47) and in germline p21 knockout mice (n = 22). (B) Spectrum of the histologically analyzed tumors. Liver carcinoma with hepatocytic and cholangiocytic differentiation (HCC/CC) (n = 51), liver carcinoma with only hepatocytic features (HCC) (n = 7), lymphoma (n = 1), and sarcoma (n = 5). (C) Macroscopic liver tumor in p53−/− mice. (D) H&E-stained sections of mixed differentiated p53−/− liver tumors showing hepatocytic differentiation (white arrow, left panel), ductular structure (red arrow, right panel), and stromal cells (black arrow, right panel). (E) H&E staining of an advanced liver tumor (left panel) and a macroscopically tumor-free liver carrying an early, microscopic liver tumor (right panel). (F) Representative immunofluorescence of the hepatocytic (albumin) and the cholangiocytic marker (K19) in p53−/− liver tumors with bilineal differentiation (left panel). Representative immunohistochemistry of the oval cell marker A6 in p53−/− liver tumors of bilineal differentiation (right panel). Scale bars = 100 μm. Gastroenterology 2012 142, 1229-1239.e3DOI: (10.1053/j.gastro.2012.02.009) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 p53 deletion prolongs self-renewal of bipotent progenitor cells in adult mouse liver. (A) Representative fluorescence-activated cell sorting plot diagrams of nonparenchymal liver cells. Squares indicate the gates for sorting of LPCs (CD13+, CD133+, CD49f+). (B and C) Immunofluorescence of colonies derived from single LPCs for cholangiocytic (K19) and hepatocytic (albumin) markers demonstrating colonies with (B) cholangiocytic and (C) bilineal differentiation. Scale bars = 100 μm. (D and E) Bar graphs show the colony-forming capacity of LPCs depicting the percentage of cells that can form (D) cholangiocytic colonies or (E) bilineal differentiated colonies. Gastroenterology 2012 142, 1229-1239.e3DOI: (10.1053/j.gastro.2012.02.009) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 p53 deletion induces proliferation of hepatocytes and induces genomic instability of LPCs and differentiated hepatocytes. (A) Bromodeoxyuridine-positive hepatocytes after continuous bromodeoxyuridine labeling. (B) Chromosomal aberrations were analyzed by aCGH in single, freshly isolated LPCs, differentiated hepatocytes, and primary colonies derived from single LPCs from 8- to 10-month-old mice. The bar graphs of the denoted genotypes summarize genomic imbalances. (C) Percentage of single freshly isolated cells (noncultured) and colonies derived from single LPCs (cultured) with chromosomal imbalances. Gastroenterology 2012 142, 1229-1239.e3DOI: (10.1053/j.gastro.2012.02.009) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 p53 deletion leads to transformation of liver cells. (A) Tumor-free survival curve of immunocompromised (NOD-scid IL2Rgnull) mice that underwent transplantation with LPC cultures with bilineal differentiation from 2- to 3-month-old p53−/− (n = 28/46), p53+/+ (n = 1/64), p53+/− (n = 11/40), or p21−/− (n = 0/40) donors. (B) Tumor-free survival curve of recipient mice that underwent transplantation with LPC cultures with bilineal (n = 28/46) and cholangiocytic differentiation (n = 14/28), or hepatocyte cultures with bilineal differentiation (n = 32/48) from 2- to 3-month-old p53−/− donors. (C) Tumor-free survival curve of recipient mice that underwent transplantation with LPC cultures with bilineal differentiation from 2- to 3-month-old (n = 28/46) and 8- to 10-month-old (n = 80/88) p53−/− donors. (D) H&E staining of the tumors in recipient mice revealed a similar morphology compared with the primary liver tumors with bilineal differentiation in p53−/− mice. (E) Immunofluorescence showing expression of hepatocytic (albumin) and cholangiocytic marker (K19) in a tumor. Scale bars = 100 μm. (F) Ideogram summarizes genomic imbalances (gains, red; losses, green) in tumors derived from p53−/− LPCs with bilineal differentiation. Gastroenterology 2012 142, 1229-1239.e3DOI: (10.1053/j.gastro.2012.02.009) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Rb pathway analysis in bilineal liver carcinoma. (A) Hierarchical clustering (Euclidean distance, average linkage) gene expression profiles from primary p53−/− liver tumors, DEN-induced p53+/+ liver tumors, and p53−/− and p53+/+ noncancerous liver. The y-axis gives the height (distance of log2 expression values) at which 2 nodes of the dendrogram are merged. (B) Histogram showing significance of differentially regulated genes of an Rb pathway gene set (red line: t*) between p53−/− liver tumors and DEN-induced liver tumors compared with randomly selected gene sets. The test statistic was the absolute value of the median Spearman correlation coefficient from the genes in the gene set to the class label. Blue bars show the distribution of the test statistic under the null hypothesis. The dotted line (t0.95) and red line (t*) show the critical value for the 95% percentile and the value of the test statistic for the Rb pathway gene set. (C) Heat map of expression profiles for Rb pathway genes for p53−/− and DEN-induced liver tumors (green, up-regulation compared with the mean of expression values of that gene; red, down-regulation). Gastroenterology 2012 142, 1229-1239.e3DOI: (10.1053/j.gastro.2012.02.009) Copyright © 2012 AGA Institute Terms and Conditions