Genome and transcriptome profiles of CD133-positive colorectal cancer cells

Abstract

Colorectal carcinomas (CRC) might be organized hierarchically and contain a subpopulation of tumorigenic, putative cancer stem cells that are CD133 positive. We studied the biological and genetic characteristics of such cells in CRC cell lines and primary tumors. Three CRC cell lines were sorted in CD133 positive and negative fractions. The respective genetic aberration profiles were studied using array comparative genomic hybridization (aCGH) and expression profiling. Tumorigenicity for each cellular population was tested by injection into nude mice. Additionally, we compared CD133+ and CD133- cells of 12 primary colorectal tumors using laser capture microdissection and aCGH. Three of five CRC cell lines displayed both CD133+ and CD133- cells, but tumorigenicity of these subfractions did not differ significantly and aCGH revealed essentially identical genomic imbalances. However, 96 genes were differentially expressed between the two populations. Array comparative genomic hybridization analysis after laser capture microdissection of CD133+ and CD133- areas in primary colorectal tumors revealed genetic differences in 7 of 12 cases. The use of cell lines for studying genomic alterations that define cancer stem cell characteristics, therefore, seems questionable. In contrast, CD133+ cells in primary cancer samples showed a unique genomic aberration profile. In conclusion, our data suggest that CD133 positivity defines a genetically distinct cellular compartment in primary CRC, which potentially includes tumor initiating cells.

Figure 1

Immunohistochemistry of primary tissue sections from normal mucosa (A) and colorectal cancer (B) with an antibody against CD133 (fluorescein isothiocanate) and nuclear counterstaining (DAPI). While normal mucosa did not stain for CD133, a strongly CD133+ tumor (herein 85% CD133+ glands) shows apical luminal staining of the tumor cells and staining of intraglandular debris. Scale bars = 100 μm.

Figure 2

Evaluation of the tumor initiating potential of CD133+ (dotted line) and CD133− (solid line) cells of colon cancer cell lines on subcutaneous injection of cells into nude mice. Y-axis, tumor volume in mm³. X-axis, time after injection in weeks. A: HCT 116, 2000 cells. B: HCT 116, 20,000 cells. C: HT-29, 2000 cells. D: HT-29, 20,000 cells.

Figure 3

Unsupervised hierarchical clustering based on gene expression profiles of CD133+ and CD133− fractions from Caco-2, HCT 116, and HT-29 cell lines, data expressed using Euclidean metrics and the complete linkage algorithm. Samples cluster by cell line first. Only in the cell line Caco-2 were the different CD133 fractions clearly separated.

Figure 4

Supervised principal component analysis (PCA) of gene expression arrays using 96 genes up or down-regulated in CD133+ versus CD133− fractions for HCT 116 (n = 4), Caco-2 (n = 4), and HT-29 (n = 5). The supervised PCA shows a clear separation of CD133+ and CD133− along the first principal component for all samples, except one outlier (one HT-29 CD133− sample).

Figure 5

Network annotation of genes differentially expressed according to the CD133 status in HT-29 using ingenuity pathway analyses. Red, genes up-regulated in CD133+; green, genes down-regulated in CD133+. Dark red or green shade, genes with > threefold differential expression; light red or green shade, genes with lower difference in expression. All genes spotted were deregulated significantly (P < 0.0001).

Figure 6

Ideogram of array comparative genomic hybridization chromosomal gains and losses for HT-29. (A) CD133+ and (B) CD133− cells. The aberration profile looked identical.