CD133+CD24lo defines a 5-Fluorouracil-resistant colon cancer stem cell-like phenotype

Abstract

The chemotherapeutic agent 5-Fluorouracil (5-FU) is the most commonly used drug for patients with advanced colon cancer. However, development of resistance to 5-FU is inevitable in almost all patients. The mechanism by which colon cancer develops 5-FU resistance is still unclear. One recently proposed theory is that cancer stem-like cells underlie colon cancer 5-FU resistance, but the phenotypes of 5-FU-resistant colon cancer stem cells are still controversial. We report here that 5-FU treatment selectively enriches a subset of CD133+ colon cancer cells in vitro. 5-FU chemotherapy also increases CD133+ tumor cells in human colon cancer patients. However, sorted CD133+ colon cancer cells exhibit no increased resistance to 5-FU, and CD133 levels exhibit no correlation with colon cancer patient survival or cancer recurrence. Genome-wide analysis of gene expression between sorted CD133+ colon cancer cells and 5-FU-selected colon cancer cells identifies 207 differentially expressed genes. CD24 is one of the genes whose expression level is lower in the CD133+ and 5-FU-resistant colon cancer cells as compared to CD133+ and 5-FU-sensitive colon cancer cells. Consequently, CD133+CD24lo cells exhibit decreased sensitivity to 5-FU. Therefore, we determine that CD133+CD24lo phenotype defines 5-FU-resistant human colon cancer stem cell-like cells.

Keywords: 5-Fluorouracil; CD133; CD24; colon cancer stem cells.

Figure 1. Enrichment of CD133⁺ cancer stem-like cells is linked to 5-FU resistance of human colon cancer

A. Scheme of establishment of 5-FU-resistant colon cancer cell lines. Human colon carcinoma cell lines SW620 and LS411N were cultured in the presence of increasing 5-FU concentrations as indicated to establish 5-FU-resistant SW620-5FU-R and LS411N-5FU-R cell lines, respectively. B. The parent (LS411N and SW620) and the respective 5-FU-resistant human colon carcinoma cells (LS411N-5FU-R and SW620-5FU-R) were cultured in the presence of 5-FU at the indicated doses for 3 days and analyzed for growth using MTT assays. C and D. The parent and 5-FU-resistant cell lines were analyzed for cell surface CD133 and CD44 protein levels by flow cytometry. Left panels are plots of CD133 and CD44 levels, and the right panels are quantification of cell surface CD133 and CD44 protein levels of the indicated cell lines.

Figure 2. 5-FU chemotherapy enriches CD133⁺ cancer cells in human colon cancer patients

Tumor tissues from human colon cancer patients without prior 5-FU therapy (n=5) and with prior 5-FU therapy (n=5) were stained with CD133-specific antibody. Brown color indicates CD133 protein expression, with counterstaining by hematoxylin in blue. Shown are representative images of colon carcinoma tissues from each of the five patients without prior 5-FU therapy (U1-5) and with 5-FU therapy (F1-5). Shown is CD133 staining intensity. Red arrows indicate CD133⁺ cells.

Figure 3. CD133⁺ colon cancer cells and 5-FU sensitivity

CD133⁺ and CD133⁻ cells were sorted from LS411N A. and SW620 B. cells, respectively. The cells were stained with CD133- and CD44-specific antibodies and analyzed by flow cytometry (top panel). The two subsets of cells were cultured in the presence of 5-FU at the indicated concentrations for 3 days and analyzed for viability by MTT assays.

Figure 4. Genome-wide gene expression profiles of colon cancer stem-like cells

A. Scheme of genome-wide gene expression analysis. LS411N-CD133⁺ cells were compared to LS411N-5FU-R cells and SW620-CD133⁺ cells were compared to SW620-5FU-R cells by DNA microarray analysis. Shown are phenotypes of the two pairs of cells. B. Analysis of the differentially expressed genes between CD133⁺ cells and 5-FU-R cells. Genes whose expression levels are changed by at least 2 fold (either up-regulated or down-regulated) were selected. The ratios are LS411N-CD133⁺/LS411N-5FUR and SW620-CD133⁺/SW620-5FU-R. The number of differentially expressed genes of each pair and the commonly differentially expressed genes of the two pairs of cells are shown. C. The commonly differentially expressed genes in two pairs of cells as shown in A and B (n=207) was selected. Cluster 3.0 program was used to analyze the gene expression patterns in a one-dimensional hierarchical clustering to generate gene dendrograms based on the pair-wise calculation of the Pearson coefficient of normalized fluorescence ratios as measurements of similarity and linkage clustering. The clustered data were loaded into TreeView program and displayed by the graded color scheme. Genes that have known functions in stem cell maintenance (CD24, LGR5, and SOX2), cell proliferation, and death (PCNA, CASP3, MLKL, DUSP5, DUSP6, NFKB2, and HDAC5), and for immune response (IL17RB and IL20RB) are indicated. Red columns indicate genes whose expression level is higher in LS411N-CD133⁺ and SW620-CD133⁺ cells as compared to LS411N-5FU-R and SW620-5FU-R cells, respectively. Green columns indicate genes whose expression level is higher in LS411N-5FU-R and SW620-5FU-R cells as compared to LS411N-CD133⁺ and SW620-CD133⁺ cells, respectively. The color bar at the bottom panel represents the level of differential expression. The number above the bar indicates the fold changes.

Figure 5. CD133⁺CD24^lo cells phenotypically resemble 5-FU-resistant human colon carcinoma cells

A. CD133 and CD24 expression profiles in subsets of colon cancer cells. The indicated four subsets of LS411N and SW620 cells were stained with CD133- and CD24-specific antibodies and analyzed by flow cytometry. Shown are representative images of one of two experiments. B. CD24 protein levels as shown in A were plotted as to show mean fluorescent intensity (MFI). The MFI of the four subsets of LS411N and SW620 cells were quantified and presented at the right. Column: mean; Bar:SD.

Figure 6. CD133⁺CD24^lo define a 5-FU-resistant human colon carcinoma cell phenotype

The indicated four subsets of LS411N and SW620 cells were cultured in the presence of 5-FU at the indicated concentrations for 3 days and analyzed for viability by MTT assays. Cell resistance to 5-FU is expressed as % cell viability.

Figure 7. CD24^lo human colon carcinoma cells exhibit less sensitivity to 5-FU and a mesenchymal phenotype

A. LS411N and SW620 cells were sorted into CD24^hi and CD24^lo cells. The sorted cells were then cultured in the presence of 5-FU at the indicated concentrations for 3 days and analyzed by MTT assay. Cell sensitivity to 5-FU is expressed as % cell viability. B. The sorted CD24^hi and CD24^lo cells were analyzed by real-time PCR for vimentin mRNA level. b (beta)-actin was used as normalization control.

Figure 8. CD133⁺CD24^lo human colon carcinoma cells express higher level of ALDH

The indicated four subsets of LS411N and SW620 cells were analyzed for ALDH enzyme level using the ALDEFLUOR Kit and analyzed by flow cytometry. ALDH protein levels were expressed as MFI. Column: mean; Bar: SD.