ORIGINAL PAPER Journal of Pathology J Pathol 2010; 221: 379–389 Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/path.2733 Barrett’s oesophageal adenocarcinoma encompasses tumour-initiating cells that do not express common cancer stem cell markers Brechtje A Grotenhuis, 1,2 Winand NM Dinjens, 2 Bas PL Wijnhoven, 1 Petra Sonneveld, 2 Andrea Sacchetti, 2 Patrick F Franken, 2 Herman van Dekken, 2# Hugo W Tilanus, 1 J Jan B van Lanschot 1 and Riccardo Fodde 2 * 1 Department of Surgery, Josephine Nefkens Institute, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands 2 Department of Pathology, Josephine Nefkens Institute, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands *Correspondence to: Riccardo Fodde, Department of Pathology, Josephine Nefkens Institute, PO Box 2040, 300 CA Rotterdam, The Netherlands. e-mail: r.fodde@erasmusmc.nl # Current address: Department of Pathology, St. Lucas Andreas Hospital, Amsterdam, The Netherlands. Abstract Accumulating evidence has suggested that tumours have a hierarchical organization in which only the cancer stem cells (CSCs) have tumour-initiating properties. Several surface antigens have been employed to isolate CSCs from various malignancies, although not from oesophageal adenocarcinoma (EA). We tested whether Barrett’s oesophagus (BE) and EA might serve as a model for the CSC concept. In vivo assays were performed by transplantation of serially diluted bulk EA cells into NOD-SCID mice to establish the presence and frequency of tumour-initiating cells. These were found to be present as ca. 1 in 64 000 cells. The transplanted tumours fully recapitulated the primary lesions. Subsequently, a panel of previously established CSC markers was employed for immunohistochemistry. CD24, CD29 and CD44 showed heterogeneous staining in EA. Nuclear β-catenin accumulation increased during progression from metaplasia to dysplasia and was often observed in the basal compartment with CD24 and CD29 staining. However, the overall staining patterns were not such to clearly point out specific candidate markers. Accordingly, all markers were employed to sort the corresponding subpopulations of cancer cells and transplant them at low multiplicities in NOD-SCID mice. No increased tumour-initiating capacity of sorted EA cells was observed upon transplantation. These results indicate that tumour-initiating cells are present in EA, thus reflecting a hierarchical organization. However, antibodies directed against novel surface antigens are needed to detect subpopulations enriched for CSCs in EA by transplantation assays. Copyright  2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Keywords: oesophageal adenocarcinoma; Barrett’s oesophagus; cancer stem cells; tumour-initiating cells Received 12 April 2010; Accepted 9 May 2010 No conflicts of interest were declared. Introduction Oesophageal exposure to refluxed gastric contents (gastro-oesophageal reflux disease, GERD) represents the major risk factor in the replacement of squamous epithelial cells lining the oesophagus by an intestinal columnar epithelium, a condition known as Barrett’s oesophagus (BE) [1]. The intestinal metaplasia charac- teristic of BE features mucous glands and goblet cells [2,3] and is recognized as a premalignant condition [4,5]. It is generally accepted that the development of oesophageal adenocarcinoma (EA) follows a metapla- sia (BE) → dysplasia → carcinoma sequence charac- terized by specific genetic and epigenetic changes [6]. However, the cell of origin involved in the transition from normal squamous epithelium into intestinal meta- plasia has not yet been identified. Trans-differentiation of oesophagus-specific cell types into intestinal-like columnar epithelial cells as the result of GERD may explain the observed histological changes [7,8]. On the other hand, it is also plausible that resident stem cells are involved in this process. In this alternative scenario, BE develops when GERD damages the superficial lay- ers of the oesophageal squamous epithelium, thereby exposing the basal epithelial layers (where stem cells are thought to reside [9]) to tissue-damaging agents, thus triggering abnormal differentiation programmes and metaplastic changes [6,7]. Attempts towards the isolation and characterization of stem cells of the nor- mal ooesophageal epithelium have not been successful thus far, although more recently CD34 has been shown to represent a potential stem cell marker in the mouse oesophagus [10]. Patients with BE have a significantly higher risk of developing EA when compared to the general popula- tion [11,12]. The overall estimate of cancer incidence Copyright  2010 Pathological Society of Great Britain and Ireland. J Pathol 2010; 221: 379–389 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com