CD133 and Colorectal Cancer Figure 7. Plasticity of CD133 expression. SW480 was sorted into CD133 positive and negative populations which were then cultured separately. (a) Gating was set so that only the extreme populations were collected which, on immediate retesting, were shown to be very pure populations. (b) After prolonged culture, both of the sorted populations became biphasic. The ratios of the CD133+ and CD1332 cells became stable after three weeks and did not change after that (although they were not the same as the original parental cell line). doi:10.1371/journal.pone.0010714.g007 Plasticity of CD133 expression in cell lines The SW480 cell line was sorted into CD133+ and CD1332 populations which were maintained in standard tissue culture conditions and underwent regular analysis by flow cytometry. Immediate re-analysis showed that the CD133+ and CD1332 populations were, respectively, 97.6% and 99.9% pure (Figure 7a). Quantitative PCR showed transcriptional repression of CD133 in the CD1332 population and although CD133 mRNA was still detectable, it was only at 15% of the level seen in the CD133+ population (Figure 8). Prolonged culture resulted in both populations reverting to a bimodal profile. The CD133+ population, after 3 weeks, consisted of 70% CD133+ cells and 30% CD1332 cells, frequencies which remained stable thereafter for at least 3 months. The findings run counter to published reports in which pure populations of CD133+ cells revert to normal ratio of CD133+/2 populations [8]. The CD1332 cells developed a population of CD133+ cells which, after 3 weeks, reached 17% but did not increase thereafter (Figure 7b). Discussion The CD133+ population is variable in CRC cell lines We screened large numbers of cells from eight well established CRC cell lines by flow cytometry to quantify the size of the CD133+ population. We found that, in two cell lines (DLD1 and SW837) there were no detectable CD133+ cells, in two cell lines (Caco2 and HT29) the CD133+ population was .95% of the total tumour cells whilst the remaining cell lines had population sizes ranging from 32%–64%. Our data are in agreement with studies by letaet al. and Dalerba et al. [18,24] who reported similar levels of CD133 expression in the cell lines HT29, Lovo and no expression in DLD1. Our data are however discrepant with those of O’Brien and Ricci-Vitiani [8,9], although the cause of this discrepancy is unknown. One possibility is that our studies used established cell lines whereas the other studies used novel cell lines derived in their own laboratories. It is possible that prolonged culture may cause maintained changes in CD133 regulation although this in itself would suggest that CD133 expression is not a very good marker of CSCs. Differences in technique may provide another explanation although we used the same antibodies as O’Brien and Ricci-Vitiani and we kept incubation times with the primary antibody down to 15 minutes. Either way, our data have unequivocally shown that in 4/8 cell lines, the CD133+ population is either absent or comprises nearly the whole tumour cell population. These data together with other data presented below, lead us to conclude that CD133 is probably not a specific marker of stem cells in CRC. Only CD133 splice variant AC133-2 is expressed in colonic tissue Although a wide variety of splice variants have been described for CD133, only one (termed AC133-1 and which was first to be cloned) has been allocated a Reference Sequence number. This contains the full length coding sequence whilst a second splice variant (AC133-2, the second to be cloned) appears to splice out exon 4. Our analysis of 8 CRC cell lines and 10 samples of normal mucosa, using both end-point and real-time methodologies, identified only variant AC133-2. This would be consistent with the study by Yu et al. [25] in which AC133-2 was reported as being the splice variant which is present in many stem cell compartments whilst AC133-1 is limited to foetal brain and skeletal muscle. CD133 expression is associated with some features of the stem cell phenotype In order to evaluate the function of CD133, the cell line HT29 was tested after knockdown of CD133 by RNA interference. This method can also produce ‘‘off-target’’ effects and so a comple- mentary approach was used to separate SW480–a cell line with a CD133+ population of 40% - into pure CD133+ and CD1332 populations. Both types of experiments yielded similar results. Firstly, levels of CD133 did not appear to alter cell proliferation or apoptosis. There were however significant differences in the features which may be regarded as part of the stem cell phenotype. Thus high levels of CD133 were associated with increased clonogenicity and resistance to staurosporine induced apoptosis. The latter may be due to an innate resistance to apoptosis (possibly mediated by such reported associations as between CD133 expression and anti-apoptotic genes such as FLIP (Caspase 8 inhibitor) and Bcl-2 [26]. Alternatively, it may be due to enhanced cytoprotective strategies such as the ability to actively extrude toxic substances from the cytoplasm [3]. Another feature we found to be associated with CD133 expression was enhanced cell motility. Other studies have suggested a role for CD133 in cell motility since it is classically expressed in membrane protrusions [27,28]. In certain situations, such as embryogenesis and wound healing, stem cells need to acquire features of motility. In CSCs, features of enhanced motility may allow invasion and metastasis to occur–a notion supported by the study of Rappa et al which found CD133 expression was associated with metastasis in melanoma cells [29]. Our data do, however, contradict those of Horst et al. [30] who did not find that CD133 expression was associated with cell motility in Caco2. We Figure 8. mRNA level in sorted populations. Q-RT-PCR analysis of the sorted populations showed that there was transcriptional repression of CD133 in the CD1332 population (although low levels of mRNA were still detectable). doi:10.1371/journal.pone.0010714.g008 PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10714