Thus, three independent FACS analyses during the last 7 months have shown that virtually the entire population of CR4 cells remains undifferentiated, and the majority of cells express high levels of CD133, CD44, CD166, EpCAM and Lgr5. In particular, about 65% of cells coexpressed high levels of CD133 and CD44. Importantly, about 20% of CR4 cells are positive for marker of metastatic Betamipron activity, CXCR4. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express several key markers of pluripotency, including Sox-2, Oct3/4 and c-Myc. NSC 14613 Nuclear localization of these markers shown by ICC was confirmed by western blotting, which has demonstrated their expression in the nuclear protein fraction and their absence in the cytoplasmic one. We have found that the CD133 + fraction of the CR4 cells express higher ratios of several markers of pluripotency compared to the unsorted CR4 cells. Thus, FACS analysis has shown that more than a quarter of the CD133 + population expressed Sox-2 and 10% of the population were positive for c-Myc. In contrast, unsorted CR4 cells and CD133-negative fraction expressed lower levels of these pluripotency markers. Colocalization analysis has shown that only cells with the highest expression of CD133 usually have nuclear staining for the pluripotancy markers. High proportion of the Sox-2positive cells and its nuclear localization was also confirmed by IHC of the NOD/SCID mice tumor xenografts. Importantly, the CR4 cells, similarly to the prostate PPT2 CICenriched cell line, did not express the two major regulators of apoptosis and tumor suppressor genes, p53 and p21. In addition, the nuclear fraction of the CR4 cells grown on type I collagen in stem cell medium as separate holoclones strongly expressed phosphorylated p65, which means that NFkB is constitutively overexpressed in colorectal CICs. In contrast, unphosphorylated p65 was located predominantly in the cytoplasmic fraction. All of the above may partially explain the high tumorigenic and clonogenic capacities and exceptional drug resistance of this CIC-enriched cell line. In particular, CR4 cells are highly tolerant to treatment with commonly used cytotoxic drugs such as Paclitaxel or Taxol. Thus, after 72-hour treatment in concentration range from 10 nM up to 10 mM, CR4 cells have shown little or no cytotoxicity; moreover, they often increased their proliferation in response to lower doses of the paclitaxel.