Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy Scott J. Dylla1*, Lucia Beviglia1, In-Kyung Park1, Cecile Chartier1, Janak Raval1, Lucy Ngan 1 , Kellie Pickell1, Jorge Aguilar1, Sasha Lazetic1, Stephanie Smith-Berdan 1 , Michael F. Clarke2, Tim Hoey1, John Lewicki1, Austin L. Gurney1 1 OncoMed Pharmaceuticals Inc., Redwood City, California, United States of America, 2 Stanford Institute for Stem Cell Biology & Regenerative Medicine, Stanford University, Palo Alto, California, United States of America Abstract Background: Patients generally die of cancer after the failure of current therapies to eliminate residual disease. A subpopulation of tumor cells, termed cancer stem cells (CSC), appears uniquely able to fuel the growth of phenotypically and histologically diverse tumors. It has been proposed, therefore, that failure to effectively treat cancer may in part be due to preferential resistance of these CSC to chemotherapeutic agents. The subpopulation of human colorectal tumor cells with an ESA+CD44+ phenotype are uniquely responsible for tumorigenesis and have the capacity to generate heterogeneous tumors in a xenograft setting (i.e. CoCSC). We hypothesized that if non-tumorigenic cells are more susceptible to chemotherapeutic agents, then residual tumors might be expected to contain a higher frequency of CoCSC. Methods and Findings: Xenogeneic tumors initiated with CoCSC were allowed to reach ,400 mm3, at which point mice were randomized and chemotherapeutic regimens involving cyclophosphamide or Irinotecan were initiated. Data from individual tumor phenotypic analysis and serial transplants performed in limiting dilution show that residual tumors are enriched for cells with the CoCSC phenotype and have increased tumorigenic cell frequency. Moreover, the inherent ability of residual CoCSC to generate tumors appears preserved. Aldehyde dehydrogenase 1 gene expression and enzymatic activity are elevated in CoCSC and using an in vitro culture system that maintains CoCSC as demonstrated by serial transplants and lentiviral marking of single cell-derived clones, we further show that ALDH1 enzymatic activity is a major mediator of resistance to cyclophosphamide: a classical chemotherapeutic agent. Conclusions: CoCSC are enriched in colon tumors following chemotherapy and remain capable of rapidly regenerating tumors from which they originated. By focusing on the biology of CoCSC, major resistance mechanisms to specific chemotherapeutic agents can be attributed to specific genes, thereby suggesting avenues for improving cancer therapy. Citation: Dylla SJ, Beviglia L, Park I-K, Chartier C, Raval J, et al. (2008) Colorectal Cancer Stem Cells Are Enriched in Xenogeneic Tumors Following Chemotherapy. PLoS ONE 3(6): e2428. doi:10.1371/journal.pone.0002428 Editor: D. Gary Gilliland, Brigham and Women’s Hospital, United States of America Received November 29, 2007; Accepted May 8, 2008; Published June 18, 2008 Copyright: 2008 Dylla 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: MFC is a founder and member of the paid advisory board of OncoMed Pharmaceuticals Inc., and has an equity position in the company. All other authors are employees of OncoMed Pharmaceuticals Inc., a biotechnology company focused on therapeutic targeting of Cancer Stem Cells that has applied for patents related to this study. Funding for these studies was obtained via private financing; however, these parties had no role in the decision to perform, analyze or publish the results. Competing Interests: MFC is a founder and member of the paid advisory board of OncoMed Pharmaceuticals Inc., and has an equity position in the company. All other authors are employees of OncoMed Pharmaceuticals Inc., a biotechnology company that has applied for patents related to this study. * E-mail: mnscott11@yahoo.com Introduction The presence of diverse cell populations in normal and neoplastic tissue has long been recognized. While normal tissue structure and function is facilitated by diverse cell types, generated during development and continually replaced to maintain homeostasis, cancer is generally characterized by disorganized overproliferation. Because genetic material is propagated over extended periods of time due to the self-renewal properties of stem cells, the compounding mutations required for tumorigenesis have been hypothesized to arise in these rare cells and not their more numerous progeny, which have a finite lifespan once committed to differentiation. Like normal tissue-resident stem cells that support the cellular hierarchy comprising a particular tissue over the lifespan of an individual, cancer stem cells (CSC) are defined by their ability to self-renew indefinitely, while maintaining their ability to generate both tumorigenic (TG) and non-tumorigenic (NTG) cells [1]. Unlike in normal development, however, neoplastic progenitor cell populations can gain self-renewal capabilities, thereby also fulfilling the definition of a CSC [2,3]. Ultimately, demonstration of the self-renewal and differentiation capabilities that define a stem cell, both normal and neoplastic, can be confirmed by serial transplant studies that enable discrimination of cells possessing self-renewal ability versus those capable of numerous, though finite, non self-renewing cell divisions [4]. The CSC paradigm rests on the foundation that tumor heterogeneity can be generated by a single CSC. Because traditional cell lines and xenografts do not recapitulate the cellular and morphological heterogeneity observed in xenografts arising PLoS ONE | www.plosone.org 1 June 2008 | Volume 3 | Issue 6 | e2428