Purity of OC and hepatocyte fractions was determined by morphology analysis, histologic analysis of hematoxylin and eosin (H&E)-stained cytospins and expression analysis by quantitative polymerase chain reaction (qPCR). All images were acquired on a Leica DMLB microscope and processed using Photoshop CS5 (Adobe, Munich, Germany). Error bars represent standard deviation (SD) except where indicated. Pairwise comparisons between continuous data were done using unpaired two-tailed Student t test. AGEs advanced glycation

endproducts ALT alanine aminotransferase AST aspartate transaminase BMOL bipotential murine oval liver CDE choline deficient ethionine-supplemented diet CML N-carboxymethyllysine DEN diethylnitrosamine dKO Mdr2−/− Nutlin3a Rage−/− HCC hepatocellular carcinoma HMGB1 high mobility learn more group box 1 Mdr2 multidrug resistance protein 2 OC oval cells RAGE receptor for advanced glycation endproducts sRAGE soluble RAGE To define the role of RAGE in inflammation-driven tumor development, we crossed Rage−/− mice with the Mdr2−/− mouse strain.23, 25 Mdr2−/− Rage−/− double knockout (dKO) mice were viable and produced offspring in a Mendelian ratio. At 15 months of age, control, Rage,−/− Mdr2−/−, and dKO mice (n = 10 for each group) were sacrificed and livers were subjected to histological analysis. Control and Rage−/− livers

did not present any focal lesion, while Mdr2−/− mice had enlarged livers that developed multiple HCCs and dysplastic nodules (Fig.

1A, and data not shown). Pathological grading of tumors from Mdr2−/− mice ranged from well differentiated (G1), moderately (G2), up to poorly differentiated (G3), according to the Armed Forces Institute of Pathology grading system. In contrast, dKO mice developed mainly dysplastic nodules (Fig. Sinomenine 1A,B) and only two dKO mice exhibited a single HCC classified as moderately differentiated (G2). Interestingly, while the percentage of mice without any detectable lesion was comparable between Mdr2−/− (28%) and dKO (30%) mice, most Mdr2−/− mice (61%) developed HCCs, whereas the majority of dKO mice (50%) exhibited only premalignant dysplastic nodules (Fig. 1B). In particular, dKO mice showed fewer and smaller liver lesions that did not exceed 12 mm in diameter, whereas lesions in Mdr2−/− mice were bigger in size (up to 20 mm in diameter) and in number (Fig. 1C). Furthermore, dKO mice showed significantly less multifocal tumorigenesis compared to Mdr2−/− mice (Fig. 1D). In contrast, when mice were treated with DEN, which is an alkylating agent causing DNA strand breaks promoting mutations and subsequent HCC formation in a cirrhosis-free manner,28–30 we could not detect any significant difference in tumor number, size, and multiplicity between wildtype (WT) and Rage−/− mice at 12 months after injection (Supporting Fig. 1).