Cancer stem cells (CSCs) are the subpopulation of cells within a tumor that can self-renew, differentiate into multiple lineages, and drive tumor growth. Here we describe a two-pronged approach for the identification and characterization of CSCs from colorectal cancer cell lines, using a Matrigel-based differentiation assay, and cell surface markers CD44 and CD24. About 20 to 30% of cells from the SW1222 cell line form megacolonies in Matrigel that have complex 3D structures resembling colonic crypts. The megacolonies' capacity to self-renew in vitro is direct evidence that they contain the CSCs. Furthermore, just 200 cells from SW1222 megacolonies initiate tumors in NOD/SCID mice. We also showed that CD44 + CD24 + cells enriched for colorectal CSCs in the HT29 and SW1222 cell lines, which can self-renew and reform all four CD44/CD24 subpopulations, are the most clonogenic in vitro and can initiate tumors in vivo. A single SW1222 CD44 + CD24 + CSC, when grown in Matrigel, can form large megacolonies that differentiate into enterocyte, enteroendocrine, and goblet cell lineages. The HCT116 line does not differentiate or express CDX1, nor does it contain subpopulations of cells with greater tumor-forming capacity, suggesting that HCT116 contains mainly CSCs. However, forced expression of CDX1 in HCT116 leads to reduced clonogenicity and production of differentiating crypt-containing colonies, which can explain the selection for reduced CDX1 expression in many colorectal cancers. In summary, colorectal cancer cell lines contain subpopulations of CSCs, characterized by their cell surface markers and colony morphology, which can self-renew and differentiate into multiple lineages.clonogenicity | matrigel | CD44 | goblet cells

Hypoxia is an important regulator of normal and cancer stem cell (CSC) differentiation. Colorectal CSCs from SW1222, LS180, and CCK81 colorectal cancer-derived cell lines are able to differentiate into complex 3D lumen-containing structures in normoxia, whereas in hypoxia, they form undifferentiated dense colonies that have reduced expression of the enterocyte differentiation marker CDX1, lack goblet cell formation, and have increased expression of BMI1 and activated Notch1. Hypoxia increases the clonogenicity of CSCs, which is cumulative as each round of hypoxia enriches for more CSCs. The hypoxic phenotype is reversible, because cells from hypoxic-dense colonies are able to reform differentiated structures when regrown in normoxia. We show that CDX1 is able to stimulate the generation of lumens even in hypoxia and has a negative feedback on BMI1 expression. Knockdown of CDX1 reduces lumen formation but does not affect goblet cell formation, suggesting that enterocytes and goblet cells form from different progenitor cells. Notch inhibition by dibenzazepine (DBZ) allowed CSCs to form goblet cells in both normoxia and hypoxia. Finally, we show that Hif1α, but not CA9, is an important mediator of the effects of hypoxia on the clonogenicity and differentiation of CSCs. In summary, hypoxia maintains the stem-like phenotype of colorectal cell linederived CSCs and prevents differentiation of enterocytes and goblet cells by regulating CDX1 and Notch1, suggesting that this regulation is an important component of how hypoxia controls the switch between stemness and differentiation in CSCs.