Abstract 2452: Generation of hepatocellular carcinomas with cancer stem cell properties from primary mouse hepatocytes
Authors: Agnes Holczbauer, Valentina M. Factor, Jens U. Marquardt, David Kleiner, Daekwan Seo, Chiara Raggi, Mitsuteru Kitade, Jesper B. Andersen, Marian Durkin, Snorri S. Thorgeirsson
Journal: Cancer Research (2011)

Abstract BACKGROUND: Current evidence suggests that hepatocellular carcinoma (HCC), the most common adult primary liver cancer, comprises biologically distinct subgroups including the recently identified hepatic progenitor cell- and cholangiocarcinoma (CC)-like subtypes. These observations support the notion that HCCs can be derived from different cells within the lineage from adult liver stem cell to hepatocyte. AIM: We addressed two questions: (1) does the transforming potential of oncogenes depend on the stage of hepatocytic differentiation and (2) is the tumor phenotype determined by the stage of differentiation at which transformation occurs. METHODS: Fetal (ED16.5) hepatocytes were purified by magnetic sorting and adult (3-month) hepatocytes were isolated by collagenase perfusion from C57BL/6 mice. Cells were transduced with lentiviral vectors carrying oncogenic H-Ras and SV40 large T (LT) together with GFP and mCherry fluorescent reporters. Double positive cells were sorted and injected into the spleen of NOD/SCID mice. Tumors larger than 3 mm derived from the transduced fetal (n=20) and adult hepatocytes (n=20) were macrodissected and subjected to histopathology evaluation, immunohistochemistry and transcriptome profiling. Cancer stem cell traits were examined by FACS analysis of lineage specific and cancer stem cell associated markers, side population and tumor sphere formation assays using cell lines established from fetal and adult hepatocyte-derived tumors. RESULTS: Both fetal and adult hepatocytes co-transduced with oncogenic H-Ras/SV40LT produced liver tumors with similar growth characteristics and gave rise to lung metastases with 80% (8/10) and 63% (5/8) frequency, respectively. Histologically, fetal and adult hepatocyte-derived tumors were moderately-poorly differentiated HCCs with CC-like phenotype. All tumor cell lines were 100% positive for CK18, CK19, EpCam, A6, CD44, CD49f and CD29 and contained SP fraction (1-2%) as measured by FACS analysis regardless of the cell origin. CD133 was expressed at lower and variable levels in fetal (26.0±7.2%) and adult (46.1±9.1%) tumor cell lines. Both fetal and adult tumor cells possessed a sphere forming capacity which was maintained within 5 sphere generations. Unsupervised hierarchical clustering separated the fetal and adult tumors after log2 transformation by using corresponding normal samples as reference. CONCLUSION: Our results suggest that the activation of certain oncogenic pathways may have more significant effect on the tumor phenotype than the stage of hepatocytic differentiation at which the transformation occurs. Oncogenic H-Ras and SV40LT are capable of inducing a cancer stem cell-like state in mature hepatocytes that displays characteristics of hepatic stem cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2452. doi:10.1158/1538-7445.AM2011-2452