Phenotypic characterization of human colorectal cancer stem cells

Abstract

Recent observations indicate that, in several types of human cancer, only a phenotypic subset of cancer cells within each tumor is capable of initiating tumor growth. This functional subset of cancer cells is operationally defined as the "cancer stem cell" (CSC) subset. Here we developed a CSC model for the study of human colorectal cancer (CRC). Solid CRC tissues, either primary tissues collected from surgical specimens or xenografts established in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice, were disaggregated into single-cell suspensions and analyzed by flow cytometry. Surface markers that displayed intratumor heterogeneous expression among epithelial cancer cells were selected for cell sorting and tumorigenicity experiments. Individual phenotypic cancer cell subsets were purified, and their tumor-initiating properties were investigated by injection in NOD/SCID mice. Our observations indicate that, in six of six human CRC tested, the ability to engraft in vivo in immunodeficient mice was restricted to a minority subpopulation of epithelial cell adhesion molecule (EpCAM)(high)/CD44+ epithelial cells. Tumors originated from EpCAM(high)/CD44+ cells maintained a differentiated phenotype and reproduced the full morphologic and phenotypic heterogeneity of their parental lesions. Analysis of the surface molecule repertoire of EpCAM(high)/CD44+ cells led to the identification of CD166 as an additional differentially expressed marker, useful for CSC isolation in three of three CRC tested. These results validate the stem cell working model in human CRC and provide a highly robust surface marker profile for CRC stem cell isolation.

Fig. 1.

EpCAM/CD44 expression profiles in primary CRC tumors and normal colonic tissues. Analysis of EpCAM/CD44 expression in primary tissues revealed similar profiles among primary CRC tumors (A–C) and normal colorectal epithelium (D–F). Both normal and malignant tissues contained two main cell subsets: EpCAM^high/CD44⁺ and EpCAM^low/CD44⁻. To minimize experimental variability and contributions of genetic background, primary tumors were compared with autologous normal mucosa and analyzed on the same day. EpCAM expression was analyzed by using the B29.1 anti-ESA monoclonal antibody (Biomeda, Foster City, CA). Percentages reported in flow plots indicate the percentage of cells contained within gate P5.

Fig. 2.

The EpCAM/CD44 expression profile of human CRC xenografts recapitulates that observed in primary CRC tumors. (A–D) Analysis of EpCAM/CD44 expression in human CRC xenografts grown in NOD/SCID mice confirmed the existence of two main cancer cell subsets: EpCAM^high/CD44⁺ and EpCAM^low/CD44⁻. Relative frequencies of the two subpopulations varied among different xenografts. EpCAM expression was analyzed by using the B29.1 anti-ESA monoclonal antibody (Biomeda). Percentages reported in flow plots indicate the percentage of cells contained within gate P5.

Fig. 3.

Reconstitution of parental EpCAM/CD44 expression profiles in tumors grown from sorted EpCAM^high/CD44⁺ cells. Analysis of tumors grown from injection of sorted EpCAM^high/CD44⁺ cells (B and E) showed reconstitution of parental expression profiles (A and D), including similar relative frequencies of EpCAM^high/CD44⁺ and EpCAM^low/CD44⁻ populations (UM-COLON#4; C). The capacity to form tumors in NOD/SCID mice was restricted to the EpCAM^high/CD44⁺ cell population (MICOL-69; F, arrow). No tumor growth was usually observed on injection of EpCAM^low/CD44⁻ cells on the opposite flank of the same animals (circled area). EpCAM expression was analyzed by using the B29.1 anti-ESA monoclonal antibody (Biomeda). Percentages reported in flow plots indicate the percentage of cells contained within gate P5.

Fig. 4.

Coexpression of CD44 and CD166 in human CRC xenografts. (A–D) Analysis of CD166 expression in human CRC xenografts revealed that CD166 was differentially expressed within cancer cell populations and that all analyzed tumors contained a distinct CD44⁺/CD166⁺ double-positive cell subset.

Fig. 5.

CD44/CD166 and EpCAM/CD166 expression profiles in normal and malignant primary colorectal tissues. Analysis of CD44/CD166 expression profiles in primary tissues confirmed CD44/CD166 coexpression in normal colorectal epithelium (E and G) and primary CRC tumors (C), with a profile similar to that of CRC xenografts (A). As expected, CD166⁺ cells were predominantly EpCAM^high (B, D, F, and H). EpCAM expression was analyzed by using the B29.1 anti-ESA monoclonal antibody (Biomeda). Percentages reported in flow plots indicate the percentage of cells contained within gate P9.