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Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line.

ABSTRACT: The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133 (+) and CD133 (-) subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133 (+) clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133 (+) cells decreased from 99% to 80% in the sorted CD133 (+) population while rising from 5 to 10% in the sorted CD133 (-) population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133 (+) clonal cells lost their CD133 marker while about 10% of the CD133 (-) clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133 (+) cells in both of the CD133 (+) and CD133 (-) clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133 (+) and the CD133 (-) SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.

SUBMITTER: Feng JM

PROVIDER: S-EPMC3542230 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line.

Feng Jian-Ming JM Miao Ze-Hong ZH Jiang Yi Y Chen Yi Y Li Jia-Xin JX Tong Lin-Jiang LJ Zhang Jin J Huang Yi-Ran YR Ding Jian J

Cancer biology & therapy 20120906 14

The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133 (+) and CD133 (-) subsets. Flow cytometry revealed that CD133 was bimodally ...[more]

PMID: 22954703

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Project description:BackgroundIsolation and characterization of tumourigenic colon cancer initiating cells may help to develop novel diagnostic and therapeutic procedures.MethodsWe characterized a panel of fourteen human colon carcinoma cell lines and their corresponding xenografts for the surface expression of potential stem cell markers CD133, CD24, CD44, CDCP1 and CXCR4. In five cell lines and nine xenografts, mRNA expression of these markers was determined. Tumour growth behaviour of CD133+, CD133- and unsorted SW620 cells was evaluated in vivo.ResultsAll five putative stem cell markers showed distinct expression patterns in the tumours examined. Two patient-derived cell lines highly expressed CD133 (> 85% of positive cells) and three other cell lines had an expression level of about 50% whereas in long-term culture based models CD133 expression ranged only from 0 to 20%. In 8/14 cell lines, more than 80% of the cells were positive for CD24 and 11/14 were over 70% positive for CD44. 10/14 cell lines expressed CDCP1 on ≥ 83% of cells. CXCR4 expression was determined solely on 94 L and SW480.Analyses of the corresponding xenografts revealed a significant reduction of cell numbers expressing the investigated surface markers and showed single cell fractions expressing up to three markers simultaneously.Statistical analysis revealed that the CXCR4 mRNA level correlates negatively with the protein expression of CD133, CD44, CD24 and CDCP1 in cell lines and xenografts.A lower differentiation grade of donor material correlated with a higher CDCP1 mRNA expression level in the respective tumour model.In vivo growth behaviour studies of SW620 revealed significantly higher take rates and shorter doubling times in the tumour growth of CD133 positive subclones in comparison to the unsorted cell line or CD133 negative subclones.ConclusionsOur data revealed correlations in the expression of surface markers CD44 and CD24 as well as CD44 and CDCP1 and strongly suggest that CD133 is a stem cell marker within our colon carcinoma panel. Further studies will elucidate its role as a potential therapeutic target.

Project description:Experimental data indicate that colorectal cancer cells with CD133 expression exhibit enhanced tumorigenicity over CD133-negative (CD133-) cells. We hypothesized that CD133-positive (CD133+) cells, compared with CD133-, are more tumorigenic because they are more interactive with and responsive to their stromal microenvironment. Freshly dissected and dissociated cells from a primary colon cancer were separated into carcinoma-associated fibroblasts (CAF) and the epithelial cells; the latter were further separated into CD133+ and CD133- cells using fluorescence-activated cell sorter. The CD133+ cells formed large tumors in non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice, demonstrating the phenotypic cellular diversity of the original tumor, whereas CD133- cells were unable to sustain significant growth. Affymetrix gene array analyses using t-test, fold-change and multiple test correction identified candidate genes that were differentially expressed between the CD133+ vs CD133- cells. RT-PCR verified differences in expression for 30 of the 46 genes selected. Genes upregulated (+ vs - cells) included CD133 (9.3-fold) and CXCR4 (4-fold), integrin ?8 and fibroblast growth factor receptor 2. The CAF highly express the respective ligands: stromal-derived factor-1 (SDF-1), vitronectin and FGF family members, suggesting a reciprocal relationship between the CD133+ and CAF cells. SDF-1 caused an increase in intracellular calcium in cells expressing both CD133 and CXCR4, confirming functional CXCR4. The CD133+/CXCR4+ phenotype is increased to 32% when the cells are grown in suspension compared with only 9% when the cells were allowed to attach. In Matrigel 3-D culture, the CD133+/CXCR4+ group treated with SDF-1 grew more colonies compared with vehicle, as well as significantly larger colony sizes of tumor spheres. These data demonstrate proof of principle that the enhanced tumorigenic potential of CD133+, compared with CD133-, cells is due to their increased ability to interact with their neighboring CAF.

Project description:Colon cancer stem cells are believed to originate from a rare population of putative CD133+ intestinal stem cells. Recent publications suggest that a small subset of colon cancer cells expresses CD133, and that only these CD133+ cancer cells are capable of tumor initiation. However, the precise contribution of CD133+ tumor-initiating cells in mediating colon cancer metastasis remains unknown. Therefore, to temporally and spatially track the expression of CD133 in adult mice and during tumorigenesis, we generated a knockin lacZ reporter mouse (CD133lacZ/+), in which the expression of lacZ is driven by the endogenous CD133 promoters. Using this model and immunostaining, we discovered that CD133 expression in colon is not restricted to stem cells; on the contrary, CD133 is ubiquitously expressed on differentiated colonic epithelium in both adult mice and humans. Using Il10-/-CD133lacZ mice, in which chronic inflammation in colon leads to adenocarcinomas, we demonstrated that CD133 is expressed on a full gamut of colonic tumor cells, which express epithelial cell adhesion molecule (EpCAM). Similarly, CD133 is widely expressed by human primary colon cancer epithelial cells, whereas the CD133- population is composed mostly of stromal and inflammatory cells. Conversely, CD133 expression does not identify the entire population of epithelial and tumor-initiating cells in human metastatic colon cancer. Indeed, both CD133+ and CD133- metastatic tumor subpopulations formed colonospheres in in vitro cultures and were capable of long-term tumorigenesis in a NOD/SCID serial xenotransplantation model. Moreover, metastatic CD133- cells form more aggressive tumors and express typical phenotypic markers of cancer-initiating cells, including CD44 (CD44+CD24-), whereas the CD133+ fraction is composed of CD44lowCD24+ cells. Collectively, our data suggest that CD133 expression is not restricted to intestinal stem or cancer-initiating cells, and during the metastatic transition, CD133+ tumor cells might give rise to the more aggressive CD133(- )subset, which is also capable of tumor initiation in NOD/SCID mice.

Dataset Information

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line.

Publications

Characterization of the conversion between CD133+ and CD133- cells in colon cancer SW620 cell line.

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