We further explored gene- and protein-expression patterns as well as tumorigenic capacity of sorted cells isolated from 15 primary HCCs and 7 liver cancer cell lines in an attempt to identify the molecular portraits of each cell type. 5-FU, fluorouracil; Abs, antibodies; AFP, alpha-fetoprotein; check details CK-19, cytokeratin-19; CSC,

cancer stem cell; DNs, dysplastic nodules; EMT, epithelial mesenchymal transition; EpCAM; epithelial cell adhesion molecule; FACS, fluorescent-activated cell sorting; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; HSCs, hepatic stem cells; IF, immunofluorescence; IHC, immunohistochemistry; IR, immunoreactivity; MDS, multidimensional scaling; NBNC, non-B, non-C hepatitis; NOD/SCID, nonobese diabetic, severe combined immunodeficient; NT, nontumor; OV-1, ovalbumin 1; qPCR, quantitative real-time polymerase chain reaction; SC, subcutaneous; Smad3, Mothers against decapentaplegic homolog 3; TECs, tumor epithelial cells; TGF-β, transforming growth factor beta; T/N, tumor/nontumor; VECs, vascular endothelial cells; VM, vasculogenic mimicry; VEGFR, vascular endothelial growth factor receptor. HCC samples were obtained with informed consent from patients who had undergone radical resection at the Liver Center in Kanazawa University Hospital (Kanazawa, Japan), and tissue acquisition procedures Afatinib manufacturer were approved by the ethics committee of Kanazawa University. A total of 102 formalin-fixed

and paraffin-embedded HCC samples, obtained from 2001 to 2007, were used for IHC analyses. Fifteen fresh HCC samples were obtained between tuclazepam 2008 and 2012 from surgically resected specimens and an autopsy specimen and were used

immediately to prepare single-cell suspensions and xenotransplantation (Table 1). Seven hepatic stromal tumors (three cavernous hemangioma, two hemangioendothelioma, and two angiomyolipoma) were formalin fixed and paraffin embedded and used for IHC analyses. Additional details of experimental procedures are available in the Supporting Information. We first evaluated the frequencies of three representative CSC markers (EpCAM+, CD90+, and CD133+ cells) in 12 fresh primary HCC cases surgically resected by FACS (representative data shown in Fig. 1A). Clinicopathological characteristics of primary HCC cases are shown in Table 1. We noted that frequency of EpCAM+, CD90+, and CD133+ cells varied between individuals. Abundant CD90+ (7.0%), but almost no EpCAM+, cells (0.06%, comparable to the isotype control) were detected in P2, whereas few CD90+ (0.6%), but abundant EpCAM+, cells (17.5%) were detected in P4. Very small populations of EpCAM+ (0.09%), CD90+ (0.04%), and CD133+ cells (0.05%) were found in P12, but they were almost nonexistent in P8, except for CD90+ cells (0.08%) (Fig. 1A). We further evaluated the expression of EpCAM, CD90, and CD133 in xenografts obtained from surgically resected samples (P13 and P15) and an autopsy sample (P14).