The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer

Abstract

CD133 is a membrane molecule that has been, controversially, reported as a CSC marker in colorectal cancer (CRC). In this study, we sought to clarify the expression and role of CD133 in CRC. Initially the size of the CD133−expressing (CD133+) population in eight well-described CRC cell lines was measured by flow cytometry and was found to range from 0% to >95%. The cell line HT29 has a CD133+ population of >95% and was chosen for functional evaluation of CD133 after gene knockdown by RNA interference. A time course assay showed that CD133 inhibition had no significant effect on cell proliferation or apoptosis. However, CD133 knockdown did result in greater susceptibility to staurosporine-induced apoptosis (p = 0.01) and reduction in cell motility (p<0.04). Since gene knockdown may cause “off-target” effects, the cell line SW480 (which has a CD133+ population of 40%) was sorted into pure CD133+ and CD133− populations to allow functional comparison of isogenic populations separated only by CD133 expression. In concordance with the knockdown experiments, a time course assay showed no significant proliferative differences between the CD133+/CD133− populations. Also greater resistance to staurosporine-induced apoptosis (p = 0.008), greater cell motility (p = 0.03) and greater colony forming efficiency was seen in the CD133+ population than the CD133− population in both 2D and 3D culture (p<0.0001 and p<0.003 respectively). Finally, the plasticity of CD133 expression in tumour cells was tested. Quantitative PCR analysis showed there was transcriptional repression in the CD133− population of SW480. Prolonged culture of a pure CD133− population resulted in re-emergence of CD133+ cells. We conclude that CD133 expression in CRCs is associated with some features attributable to stemness and that there is plasticity of CD133 expression. Further studies are necessary to delineate the mechanistic basis of these features.

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

“…The two subpopulations were maintained at an apparently stable ratio of about 40 to 60% (CD133 + vs. 18,22,23 For instance, the detectable CD133 with its specific antibody (AC133) was shown to vary in a cell cycle dependent manner in colon cancer and melanoma cell lines; 18 the closest data to ours are those showing that CD133 re-emerged in the purified CD133 -SW480 cells when persistently incubated in vitro. 23 In addition, the level of CD133 in cancer cells could be upregulated in hypoxia. 19 Those data, together with ours, collectively indicate that the CD133 -cells could indeed convert into the CD133 + cells, which has long been neglected.…”

“…First, the data of ours and the reported 23 showed that only a small fraction of the CD133 + cells or the CD133 -cells could change into the opposite and that there appear to exist some yet-to-beclarified balance mechanisms to maintain those two types of the cells at a proper, apparently stable proportion according to different kinds of tumor cells. The expression of CD133 in a mixed population of the CD133 + cells and the CD133 -cells is likely to be in a dynamic, changeable state though with an apparently stable proportion.…”

Rapid re-expression of CD133 protein in colorectal cancer cell lines in vitro and in vivo

“…43 Furthermore, CD133 knockdown was shown to result in greater susceptibility to staurosporine-induced apoptosis and reduced cell motility. 29 Taken together, these findings suggested that CD133 has a role in response to treatment and/or CSC behavior of HT29 cells. A literature review, however, also indicated considerable differences in CD133 expression levels in HT29 cultures in various laboratories.…”

“…18,22,[26][27][28][29][30] Because the fluorescence-staining procedure critically impacts the ), whereas 90.9 ± 6.4% of exponentially growing HT29 cells in serum-containing DMEM clearly expressed CD133 on the cell surface. The resulting sort layout and a representative reanalysis after separation of the subpopulations are shown in Figure 1b.…”

“…18,22,[26][27][28][29][30] Because CD133 expression was found to be elevated upon infection with Mycoplasma hyorhinis, 28 our cell lines were routinely tested negative for mycoplasma and verified for genetic origin to exclude artificial modifications. The 92-110 kDa glycoprotein CD133 (prominin-1) consists of an extracellular N-terminal domain with two large glycosylated extracellular loops, five transmembrane domains and a cytoplasmic C-terminus.…”