2C). The hepatocyte marker, CYP3A4, was down-regulated in EpCAM+ cells and not detected in CD90+ cells, compared with EpCAM− CD90− cells. POU5F1 and BMI1 were equally up-regulated in both EpCAM+ and CD90+ cells, compared with EpCAM− CD90− cells. EpCAM and CD90 were independently and distinctively expressed in different cellular lineages, so we evaluated the staining of EpCAM and CD90 separately and analyzed the clinicopathological characteristics of surgically resected

HCC cases. HCCs were regarded marker positive if ≥5% positive staining was detected in a given area. The existence of EpCAM+ cells selleck (≥5%) was characterized by poorly differentiated morphology and high serum AFP values with a tendency for portal vein invasion, whereas the existence of CD90+ cells (≥5%) was associated with poorly differentiated morphology and a tendency for large tumor size (Supporting Tables 2 and 3). Notably, the existence of CD90+ cells was associated with a high incidence of distant organ metastasis, including lung, bone, and adrenal gland, within 2 years after surgery, whereas EpCAM+ cell abundance appeared unrelated to distant organ metastasis. We evaluated the characteristics of EpCAM+ or CD90+ cells in seven representative HCC cell lines. Morphologically, all EpCAM+ cell

lines (HuH1, Ivacaftor chemical structure HuH7, and Hep3B) showed a polygonal, epithelial cell shape, whereas three of four CD90+ cell lines (HLE, HLF, and SK-Hep-1) showed a spindle cell shape (Fig. 3A). EpCAM+ cells were detected in 11.5%, 57.7%, and 99.6% of sorted HuH1, HuH7, and Hep3B cells, respectively. A small CD90+ cell population (0.66%) was observed in PLC/PRL/5, whereas 91.3%, 10.8%, and 59.0% of CD90+ cells were detected in HLE, HLF, and SK-Hep-1, respectively. Compared with primary HCCs, only EpCAM+ or CD90+ cells were detected in liver cancer cell lines under normal culture conditions (Fig. 3B), suggesting that these cell lines contain a relatively pure cell population most likely obtained by clonal selection through the establishment process. A class-comparison analysis with univariate t tests and a global permutation test (×10,000) of microarray data

yielded two main gene clusters up-regulated in EpCAM+ cell lines (HuH1, HuH7, selleck inhibitor and Hep3B) (cluster I, 524 genes) or in CD90+ cell lines (HLE, HLF, and SK-Hep-1) (cluster II, 366 genes) (Fig. 3C). PLC/PRL/5 showed intermediate gene-expression patterns between EpCAM+ and CD90+ cell lines using this gene set. Pathway analysis indicated that the genes enriched in cluster II were mainly associated with blood-vessel morpho- and angiogenesis (Fig. 3D). By contrast, the enriched genes in cluster I were significantly associated with known hepatocyte functions (P < 0.01) (Fig. 3E). In addition, we identified that the enriched genes in cluster II were significantly associated with neurogenesis, skeletal muscle development, and EMT.