Hypoxia Enhances Tumor Stemness by Increasing the Invasive and Tumorigenic Side Population Fraction BIKUL DAS, a,b,c RIKA TSUCHIDA, b DAVID MALKIN, b,c,d GIDEON KOREN, c,e SYLVAIN BARUCHEL, b,c HERMAN YEGER a,f,g a Developmental Biology and Stem Cell Program, b Division of Hematology and Oncology, Department of Pediatrics, and f Division of Pathology, Department of Paediatric Laboratory Medicine, The Hospital for Sick Children, Toronto, Ontario, Canada; c Institute of Medical Sciences and Departments of d Medical Biophysics, e Clinical Pharmacology, and g Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Key Words. Side population cells • Hypoxia • Oct-4 expression levels • SDF-1 • Stemness • Tumor stem cell ABSTRACT Although advances have been made in understanding the role of hypoxia in the stem cell niche, almost nothing is known about a potentially similar role of hypoxia in maintaining the tumor stem cell (TSC) niche. Here we show that a highly tumorigenic fraction of side population (SP) cells is localized in the hypoxic zones of solid tumors in vivo. We first identified a highly migratory, invasive, and tumorigenic fraction of post- hypoxic side population cells (SPm [hox] fraction) in a diverse group of solid tumor cell lines, including neuroblastoma, rhab- domyosarcoma, and small-cell lung carcinoma. To identify the SPm (hox) fraction, we used an “injured conditioned medium” derived from bone marrow stromal cells treated with hypoxia and oxidative stress. We found that a highly tumorigenic SP fraction migrates to the injured conditioned medium in a Boy- den chamber. We show that as few as 100 SPm (hox) cells form rapidly growing tumors in vivo. In vitro exposure to hypoxia increases the SPm (hox) fraction significantly. Quantitative real- time polymerase chain reaction and immunofluorescence stud- ies showed that SPm (hox) cells expressed Oct-4, a “stemness” gene having a potential role in TSC maintenance. In nude mice xenografts, SPm (hox) cells were localized to the hypoxic zones, as demonstrated after quantum dot labeling. These results suggest that a highly tumorigenic SP fraction migrates to the area of hypoxia; this migration is similar to the migration of normal bone marrow SP fraction to the area of injury/hypoxia. Furthermore, the hypoxic microenvironment may serve as a niche for the highly tumorigenic fraction of SP cells. STEM CELLS 2008;26:1818 –1830 Disclosure of potential conflicts of interest is found at the end of this article. INTRODUCTION Hypoxic microenvironments play an important role in the traffick- ing of normal stem cells, including bone marrow (BM)-derived side population (SP) cells. The BM-derived SP cells are localized in their hypoxic niche to maintain their quiescent and undifferen- tiated state [1–3]. During stress, quiescent hematopoietic stem cells (HSCs) are mobilized/expanded in the hypoxic zone [2, 3] and then migrate to the area of injury for repair and regeneration [4]. The trafficking of bone marrow-derived endothelial progenitor cells from their niche in the bone marrow to an area of injury may rely on a hypoxia gradient [5, 6]. Exposure of stromal cells to hypoxia may increase the tissue level of stromal derived factor-1 alpha (SDF-1), leading to the migration of BM-derived SP cells, in- cluding endothelial progenitor cells [6]. Tumor hypoxia is characterized by zones of chronic and intermittent hypoxia [7]. The latter, an in vivo process of hyp- oxia-reoxygenation, may mimic “injury/stress” and therefore may serve as a niche for the highly tumorigenic fraction of SP cells and also the tumor stem cell (TSC) fraction. Recently, SP cells obtained from glioma, breast, prostate, neuroblastoma, hepatocellular carcinoma, ovarian carcinoma, and gastrointestinal tumor cell lines [8 –16] have shown TSC- like properties, including a high degree of tumorigenicity. Our preliminary study showed that the SP fraction is enriched in TSC-like cells and increases following exposure to hypoxia/ reoxygenation (Das B, Baruchel S, Yeger H. Hypoxia increases the population of stem cell-like side population cells in neuro- blastoma. Third Annual Meeting of the International Society for Stem Cells Research. San Francisco, June 23, 2005). Considering that tumor SP fractions have many similarities to their normal counterpart, including high self-renewal capac- ity, expression of the “stemness” gene Oct-4 and high tumori- genic (repopulation) capacity [8], the tumor SP fraction may also migrate to the hypoxic/ischemic regions of a tumor (Fig. Author contributions: B.D.: conception and experimental design, collection, analysis and interpretation of data, collaborative efforts, manuscript writing and revisions, journal correspondence; R.T.: collection and assembly of data, critical review of the manuscript; D.M.: financial support, provision of study material, critical review of the manuscript; G.K.: administrative support; S.B.: administrative support, critical review of the manuscript; H.Y.: financial and administrative support, data analysis and interpretation, critical revision of the manuscript. Correspondence: Herman Yeger, Ph.D., Division of Pathology, Department of Pediatric Laboratory Medicine, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Telephone: 416-813-5958; Fax: 416-813-5974; e-mail: hermie@sickkids.ca; or Bikul Das, M.B.B.S., Ph.D., Developmental Biology and Stem Cell program, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Telephone: 416-813-5937; Fax: 416-813-5327; e-mail: bikul.das@utoronto.ca Received August 30, 2007; accepted for publication April 24, 2008; first published online in STEM CELLS EXPRESS May 8, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0724 CANCER STEM CELLS S TEM CELLS 2008;26:1818 –1830 www.StemCells.com at University of Toronto on August 6, 2008 www.StemCells.com Downloaded from