5B). Morphologically, tumorigenic EpCAM+ cells showed this website an epithelial cell shape, whereas CD90+ cells showed a mesenchymal VEC shape (Fig. 5C and Supporting Fig. 3C). FACS analysis indicated that P12 HCC cells

showed abundant expression of vascular endothelial growth factor receptor (VEGFR) 1 and a vascular endothelial marker endoglin (CD105) (Fig. 5D). By contrast, P4 and P7 HCC cells did not express these vascular endothelial markers (data not shown). Lung metastasis was detected in NOD/SCID mice transplanted with P12 HCC cells, but not in mice transplanted with P4 and P7 HCC cells (Fig. 5E,F). Taken together, these results suggest that the tumorigenic and metastatic capability of primary HCC may depend on the presence of distinct EpCAM+ or CD90+ CSCs. EpCAM+ cells were associated with a high tumorigenic capacity with hepatic epithelial stem cell features, whereas CD90+ cells were related to the metastatic propensity with VEC PI3K Inhibitor Library features. We previously demonstrated that Wnt/β-catenin signaling

inhibitors could successfully attenuate the tumorigenic capacity of EpCAM+ CSCs in HCC.8, 10 To explore the potential molecular targets activated in CD90+ CSCs, we investigated the expression of the known VEC markers, CD105, VEGFR1 (encoded by FLT1), and c-Kit (encoded by KIT), in cell lines and showed that they were abundantly expressed in CD90+ cell lines, but not EpCAM+ cell lines (Fig. 6A). No expression of VEGFR2 was detected in this set of cell lines, suggesting that molecular reagents specifically targeting VEGFR2 may have no effects on CD90+ CSCs. CD44, a stem cell marker that functionally regulates redox status and is a potential target of CD90+ CSCs, was also abundantly expressed in CD90+ cell lines (Supporting Fig. 4A), consistent with previous data.5, 6-phosphogluconolactonase 13 No significant difference was detected in the expression of the hematopoietic marker, CD34, or ABCG2 between EpCAM+ and CD90+ cell lines (Supporting Fig. 4A). Among these molecular

targets, we focused on the characterization of c-Kit because the c-Kit tyrosine kinase inhibitor, imatinib mesylate, is readily available, is widely used for the treatment of gastrointestinal stromal tumor with activation of c-Kit, and may have potential antitumor activity against a subset of HCC.14 We explored the effect of imatinib mesylate on HCC cell lines and found that treatment with 10 μM reduced cell proliferation and spheroid formation in CD90+ cell lines, but had no effect on EpCAM+ cell lines (Supporting Fig. S4B,C). We further explored the effect of imatinib mesylate in vivo. Because EpCAM+ and CD90+ cells reside in the primary HCC, but not in established cell lines, we SC injected HuH7 and HLF cell lines to generate tumors organized by EpCAM+ and CD90+ CSCs.