1C and data not shown). ZEB treatment increased the frequency of SP-derived tumor

spheres relative to non-SP in all cell lines (Fig. 1D,E). Similar effects were observed using fluorescence-based colony-forming assays (data not shown). Thus, epigenetic modulation amplified sphere-forming and clonogenic potential of SP cells, suggesting relative enrichment of CSCs within the SP fraction. In support of this, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed up-regulation of CSCs (ABCG2, CD133, GPC3, and c-KIT) and stemness (OCT4, NANOG, SOX2) associated genes in ZEB-treated SP cells as compared with non-SP cells, albeit to a different degree (Supporting Fig. 1). Limiting

dilution analysis confirmed a higher frequency of tumor-initiating cells within SP fractions from Huh7, WRL68, and KMCH compared with non-SP cells at 8 weeks after subcutaneous selleck transplantation into NOD/SCID mice, although by 10 weeks the differences became less pronounced in untreated cells (Table 1). ZEB remarkably increased the number of tumor-initiating cells in SP fractions (seven-fold, P = 6.12 × 10−5) (Table 1). As few as 100 ZEB-treated SP cells produced tumors, whereas 1,000 non-SP Selleckchem Obeticholic Acid cells gave rise to fewer (KMCH, WRL68) or no tumors (Huh7) (Supporting Table 2). Selected animals injected with non-SP cells from Huh7 and KMCH were followed over 20 weeks after transplantation without tumor growth. Limiting dilution analysis performed at 10 weeks revealed a 14.8-fold increase in tumor-initiating capacity of SP cells compared with non-SP (P = 5.45 × 10−6). Histologically, tumors derived from SP and non-SP cells were similar, and recapitulated features of the parental tumors Orotidine 5′-phosphate decarboxylase regardless of ZEB treatment (Supporting Fig. 2). In addition, ZEB-treated SP cells were analyzed for self-renewal potential. Cells were re-isolated from Huh7 and KMCH

xenograft-tumors established from 100 SP-ZEB cells, propagated in short-term cultures, treated with ZEB for 3 days, and FACS-sorted for SP and non-SP fractions before retransplanting into secondary recipients. In both cell lines, SP cells not only sustained tumorigenic potential in serial transplantations but also increased progressively in frequency (Fig. 2A,B). KMCH secondary tumors developed with a shorter latency. Conversely, the corresponding non-SP cells showed either a dramatic increase in tumor latency and a decrease in tumor incidence (KMCH) or no tumor growth at all (Huh7) (Fig. 2C,D). Together, these data show that ZEB significantly enhanced the tumorigenic potential of SP cells while reducing it in non-SP cells. To provide evidence that the CSC-enriching effect of ZEB was due to inhibition of DNMT-1, we determined the protein levels of DNMT-1, as well as DNMT-3a and DNMT-3b, both in SP and non-SP cells.