The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer Tarek M. A. Elsaba1,2, Luisa Martinez-Pomares3, Adrian R. Robins3, Simon Crook1, Rashmi Seth1, Darryl Jackson 1 , Amy McCart4, Andrew R. Silver4, Ian P. M. Tomlinson 5 , Mohammad Ilyas1,6* 1 Division of Pathology, School of Molecular Medical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, United Kingdom, 2 Department of Pathology, South Egypt Cancer Institute, Assiut University, Assiut, Egypt, 3 Institute of Infection, Immunity and Inflammation, School of Molecular Medical Sciences, Queen’s Medical Centre, University of Nottingham, Nottingham, United Kingdom, 4 Centre for Academic Surgery, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, London, United Kingdom, 5 Molecular and Population Genetics, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom, 6 Nottingham Digestive Diseases Centre–Biological Research Unit, Queen’s Medical Centre, Nottingham, United Kingdom 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 CD1332expressing (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 CD1332 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+/CD1332 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 CD1332 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 CD1332 population of SW480. Prolonged culture of a pure CD1332 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. Citation: Elsaba TMA, Martinez-Pomares L, Robins AR, Crook S, Seth R, et al. (2010) The Stem Cell Marker CD133 Associates with Enhanced Colony Formation and Cell Motility in Colorectal Cancer. PLoS ONE 5(5): e10714. doi:10.1371/journal.pone.0010714 Editor: Mikhail V. Blagosklonny, Roswell Park Cancer Institute, United States of America Received February 9, 2010; Accepted April 27, 2010; Published May 19, 2010 Copyright: 2010 Elsaba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by the University of Nottingham and CR-UK. Tarek Elsaba is a recipient of a PhD scholarship which has been provided by the Egyptian government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Mohammad.Ilyas@nottingham.ac.uk Introduction Recent years have seen the emergence of the ‘‘cancer stem cell hypothesis’’ which postulates that a minority population of cells within a tumour consists of cancer stem cells (CSC) [1,2]. This population is purportedly responsible for generating the bulk of the tumour which consists of cells in varying degrees of differentiation. The hierarchy of a tumour is thus thought to be similar to the tissue from which the tumour originates and CSCs are deemed neoplastic counterparts of stem cells in the normal tissue. In this respect, CSCs would be expected to have a stem cell-like phenotype (generally referred to as ‘‘stemness’’). This is characterised by features such as limitless replicative ability, multipotency and resistance to apoptosis [3]. The stem cell phenotype may also include cyto-protective strategies such as ability to actively extrude dangerous substances from the cell–a feature which may be the basis of resistance to chemotherapeutic agents [3,4]. In parallel with the emergence of the cancer stem cell hypothesis, there has been a growing interest in the isolation and study of CSCs. A number of studies claim to have isolated CSCs from several different tumour types such as brain [5,6], breast [7], colon [8,9], hepatocellular carcinoma [10] and pancreatic cancer [11]. These studies have used putative CSC markers to separate stem cells from differentiating cells within a tumour. One common method of separation is the dye elimination method (i.e. side population [12]) although identification of a number of cell surface markers (such as CD24, CD44, CD166, and integrins) has allowed use of fluorescence activated cell sorting (FACS) to isolate CSCs [13]. One marker consistently reported as a stem cell marker in tumours of differing origins is CD133 (also known as Prominin 1). The CD133 gene (PROM1) maps to chromosome 4p15 and encodes a 120 kD transmembrane glycoprotein (ENSG00000007062). The CD133 protein is pentaspan cell surface receptor although neither its ligand nor its secondary messengers are known. It was first recognized as a surface marker for haematopoietic stem cells [14,15]. Later, it was used to recognize cancer stem cells in many solid tumours arising in, for example, breast [13], pancreas [16] and liver [17]. PLoS ONE | www.plosone.org 1 May 2010 | Volume 5 | Issue 5 | e10714