Two recent studies identified CD133 as a marker for stem cells in colorectal cancers (CRC) [8,9]. In these studies, CD133 expressing (CD133+) cells were isolated from primary CRCs and were shown to have the ability to form tumours as xenografts in nude mice; in contrast CD1332 cells from the same tumours were shown to have very limited ability to form xenografts and even that was attributed to contamination by CD133+ cells. Analysis of the xenografts that were formed showed that the size of CD133+ population was similar to that seen in the primary tumour and, furthermore, the ability to continuously propagate the xenografts was tightly associated with CD133 expression. Subsequent studies, however, have not validated these observations [18] and in one study it has been reported that, in fact, the CD1332 population has the greater colony forming ability [19]. Our study sought to further clarify the expression and the role of CD133 in CRC. Two approaches were used: (i) CD133 expression was functionally evaluated in HT29 after gene knockdown and (ii) SW480 underwent cell sorting into a CD133+ population and a CD1332 population followed by comparative functional analysis of the two populations. Materials and Methods Tissue culture, flow cytometry and fluorescence activated cell sorting Eight human colorectal cancer cell lines (Caco2, DLD1, HT29, Lovo, LS1034, SW480, SW620, SW837) were maintained as previously described [20]. Identity of all cell lines was confirmed by fingerprinting for mutations in KRAS, BRAF, PI3KCA, CDC4, TP53 and testing microsatellite instability. Evaluation of the size of the CD133 expressing (CD133+) population in each cell line was undertaken by flow cytometry using a phycoerythrin (PE) labelled antibody - CD133/1 (clone AC133/1, Miltenyi Biotec, UK). Cells were detached using non-enzymatic cell dissociation solution (Sigma) and approximately 56105 cells were incubated with antibody (diluted 1:100 in FACS wash (0.5% bovine serum albumin; 2 mM NaN3; 5 mM EDTA)) for 15 minutes at 4uC. An isotype and concentration matched PE labelled control antibody (Miltenyi Biotec, UK) was used and samples labelled with this antibody were used to set the gating levels. After three 5 minute washes with FACS wash, the cells were re-suspended and fixed in solution containing FACS wash with 1% formaldehyde. Determi- nation of percentage of CD133+ cells and sorting of cell lines into CD133+/CD1332 populations were performed on an Epics Altra flow cytometry machine (Beckman Coulter). The results were analyzed using WinMDi 2.9 computer software. For fluorescence activated cell sorting (FACS) of SW480, the cells were labelled using the same protocol but without the final fixation step. In order to evaluate plasticity of CD133 expression, sorted cells were maintained in culture for 3 weeks before undergoing re-evaluation. For all other experiments, the CD133+/CD1332 populations were tested immediately after sorting. RNA extraction, detection of splice variants and mRNA quantification Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Samples of normal colonic mucosa were collected with full approval of the local ethics committee (Oxford Research Ethics committee B, REC reference C02.310). Written informed consent was obtained from all patients whose samples were tested for CD133 expression and splice variants. RNA was extracted and complementary DNA (cDNA) was synthesized as previously described [20]. CD133 and Colorectal Cancer Two main splice variants have been described for CD133. The larger variant AC133-1 (NM_006017) was identified first and contains all exons [15]. The second variant, AC133-2, splices out exon 4 which is 27 base pairs long and encodes a 9 amino acid sequence in the first extracellular domain of CD133. Expression of splice variants was tested by RT-PCR using an upstream primer in exon 3 and a downstream primer in exon 5 (primer sequences available from the authors). This would produce a product of 199 base pairs from AC133-1 and 172 bp from AC133-2. End point PCR analysis was performed by viewing PCR products on agarose gels. However, given that preferential amplification of the smaller product may produce data artefacts, the study was repeated using real-time PCR (with Sybr green as the reporter dye) on the MX3005P thermal cycler (Stratagene, UK). Specificity of the PCR was validated by bidirectional direct sequencing. The presence of splice variants was tested in cDNA obtained from the CRC cell lines, the sorted CD133+ and CD1332 populations of SW480 as well as 10 samples of normal colonic mucosa Sorted CD133+/2 populations underwent analysis of mRNA levels by quantitative RT-PCR analysis using the standard curve method. Analysis was performed in triplicate for each sample and CD133 values were normalized to the housekeeping gene HPRT. Each reaction was performed in a final volume of 25 ml in a MX3005P thermal cycler (Stratagene, UK). The data for Q-PCR were analyzed using the MxPro-QPCR software. Gene knockdown Gene knockdown was achieved by transfecting HT29 cells (shown to have high CD133 expression) with CD1332specific small interfering RNA (siRNAs). Approximately 26105 cells were seeded in 2 ml DMEM media with 10% FBS in 6 well plates (Corning) 24 hours prior to transfection. Cells were transfected with CD133-specific stealth siRNA duplexes (sequence: 59-GAG- UCGGAAACUGGCAGAUAGCAAU-39, Invitrogen) at a final concentration of 33 nM using Lipofectamine 2000 (Invitrogen) in accordance with the manufacturer’s instructions. These are henceforth annotated as HT29CD1332. Controls consisted of cells transfected with scrambled control siRNA (sequence: 59-GAGG- GAACAGUCGGAUAGACCUAAU-39) and are henceforth an- notated as HT29ssc (scrambled siRNA control). Western blotting For Western blotting, lysates were prepared and quantified as previously described [20]. Samples (30 mg) were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) using a 10% resolving polyacrylamide gel and transferred onto a Hybond-P PVDF membrane (Amersham Bioscience). After blocking with 5% Bovine Serum Albumin (BSA)/0.1% TPBS (Tween20 in PBS solution) for 60 minutes, the membrane was then incubated over night at room temperature with CD133 (C24B9) Rabbit mAB (Cell Signaling) in a concentration of 1:1000. The membrane was washed with 0.1% TPBS 3 times for 5 minutes each then incubated for 1 h at room temperature with a horseradish peroxidase-linked secondary antibody (Sigma Aldrich) (1:10000, anti-rabbit IgG) diluted with 5% BSA/PBS containing 0.1% Tween20. After a further 3 washes, the membrane was visualized enhanced chemilu- minescence reagents (Thermo scientific) For a loading control, the monoclonal Anti-b-actin antibody (Sigma Aldrich) in a dilution of 1:2000 against the b-actin protein was used. Proliferation assays A time course assay for proliferation was performed after gene knockdown and after cell sorting. For HT29, each well of a 24 well plate was seeded with 104 cells 24 hours after transfection with PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10714