CD44 identifies CICs. (A) Single-cell suspensions from dissociated patient colorectal tumors were sorted by FACS for high expression of CD44-PE. The highly positive population was gated in a range of 4 to 10% (depending on the percentage of the total positive cell population of each sample) of the tail of the positive cells. (B–D) CICs were analyzed by FACS: (B) the epithelial marker EpCAM-FITC; (C) the functional stem cell assay Aldefluor; and (D) the stem cell immunophenotype by CD133-PE. (E) Quantitative RT-PCR analysis of CIC markers including c-Myc, ALDH1, Sox9, Oct4, MSI1, BMI1, and CD44 was performed on CICs and non-CICs. Data are presented as mean ± SD (n = 3); *, P < 0.05 ; **, P < 0.01; ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed twice and representative data are shown. (F) Colorectal tumor specimens from patients were subjected to dissociation to single cells and FACS sorting for high expression of CD44. The frequency of CD44⁺ in each tumor is indicated in the CD44⁺ column of the table. CD44⁺ cells were sequentially analyzed for the expression of the epithelial marker EpCAM, the functional stem cell assay Aldefluor, and the stem cell immunophenotype CD133. The percentages of marker positive CD44⁺ cells obtained are listed in a tabular form. (G) Single-cell suspensions from dissociated patient colorectal tumors and cells cultured in stem cell medium were analyzed by FACS for the expression of CD44. The percentages obtained are listed in a tabular form. (H) Quantification of sphere-formation assay demonstrates that CICs have elevated tumorsphere formation efficiency. CICs and non-CICs from two samples were plated in 96-well plates for a period of 14 d. Data are presented as a percentage of wells containing tumorspheres compared to the total number of wells. Data are presented as mean ± SD (n = 2); ***, P < 0.001. Student’s t test was used to assess the significance. The experiment was performed two times and representative data are shown. (I) Limiting dilution assay demonstrates that CD44⁺ cells have elevated tumorsphere formation efficiency. CD44⁺ and CD44^– population from two samples were plated in limiting dilution (50, 10, 1 cell[s] per well) in 96-well plates in stem cell media. The presence of spheres was evaluated after 14 d. Data are presented as mean ± SD (n = 20); ***, P < 0.001. The likelihood ratio test was used to assess the significance. The experiment was performed two times and representative data are shown. (J) Representative images of tumors initiated from CICs subcutaneously implanted into immunocompromised mice with hematoxylin and eosin staining of tumor sections. Scale bar, 100 μm. (K) CICs were tumorigenic while 10 fold more non-CICs were unable to generate tumors in four independent specimens. Five mice were used per group.