3F). By contrast, genes associated with mesenchymal lineages and EMT, such as KIT, TWIST1, CD44, and THY1, were strongly up-regulated in CD90+ cell lines. We investigated the tumorigenic capacity of EpCAM+ or CD90+ cells by subcutaneously (SC) injecting 1 × 105 sorted cells of four HCC cell lines (HuH1, HuH7, HLE, and HLF) into nonobese diabetic, severe combined immunodeficient (NOD/SCID) mice. We excluded Hep3B cells for the evaluation of tumorigenicity because almost 100% of cells were EpCAM positive. We further excluded SK-Hep-1 cells from the analysis because they potentially originated from endothelial cells.12

The highly tumorigenic capacities of selleck chemicals llc EpCAM+ and CD90+ cells were reproduced in HuH1, HuH7, and HLF cell lines, compared with marker-negative cells (Fig. 4A). However, HLE cells did not produce SC tumors, even 12 months after transplantation, in NOD/SCID mice. EpCAM+ cells

from HuH1 and HuH7 formed larger tumors more rapidly than CD90+ cells from HLF (Fig. 4B). IHC analyses indicated that EpCAM+ cells did not produce CD90+ cells and vice versa in these cell lines in vivo (Fig. 4C). CD90+ cells showed a high metastatic capacity, whereas EpCAM+ cells showed no metastasis to the lung when SC tumor volume reached approximately 2,000 (HuH1 and HuH7) or 700 mm3 (HLF) (Fig. 4D). The high metastatic capacity of PLC/PRL/5 cells, which contain a small population of CD90+ cells, was also confirmed after SC injection into NOD/SCID Epacadostat chemical structure mice (data not shown). CD90+ cells could divide MCE to generate both CD90+ and CD90− cells, and CD90+ cells showed a high capacity to invade and form spheroids with overexpression of TWIST1

and TWIST2, which are known to activate EMT programs in HLF cells (Supporting Fig. 2A-D). We next evaluated the tumorigenic/metastatic capacity of CD45− tumor cells using 12 fresh primary HCC specimens (P1-P12) that had been surgically resected (Table 2). We further evaluated the tumorigenicity of EpCAM/CD90 sorted cells obtained from xenografts derived from primary HCCs (Supporting Fig. 3A). Of these, we confirmed the tumorigenicity of cancer cells obtained from six primary HCCs after SC injection into NOD/SCID mice within 3 months after transplantation (Fig. 5A; Table 2; Supporting Fig. 3B). EpCAM+ cells derived from four HCCs (P4, P7, P13, and P14) showed highly tumorigenic capacities, compared with EpCAM− cells. CD90+ cells derived from two HCCs showed equal (P12) or more-tumorigenic capacities (P15), compared with CD90− cells. Tumorigenicity of EpCAM+ cells was observed in three hepatitis C virus (HCV)-related HCCs and an hepatitis B virus (HBV)-related HCC, whereas tumorigenicity of CD90+ cells was observed in two HBV-related HCCs (Tables 1 and 2).