from implantation of tumor cells taken directly from patients and not passaged in vitro, CSC biology depends on the latter form of xenografts. Although the relevance of these, and all, xenografts to patient tumors has been questioned [5–7], transplantation of human cells into mice is the only model system that does not expose human cells to an aphysiological ex vivo environment comprised of tissue culture plastic and culture medium and which can recapitulate the cellular heterogeneity of a primary tumor in vivo. Another topic of controversy at the foundation of the CSC hypothesis is the proposition that complete heterogeneity of a tumor can result from a single cell [8,9]. Disregarding the stromal, endothelial, and hematopoietic elements recruited and incorporated into the tumor, it has been suggested that TG and NTG populations arise from different cells and not from a single CSC [9]. If CSC can indeed generate the cellular heterogeneity observed in patient tumors, and mouse xenografts composed of cells strictly passaged in vivo best maintain characteristics of tumors in patients, then the focus of efforts in the cancer biology field should be squarely on these cells and their microenvironmental niche. Chemotherapeutic strategies that target rapidly dividing cells have principally been used to treat tumors of epithelial origin. While often effective at debulking tumor mass, these agents have largely failed to eradicate disease [10]. A reason often attributed to this failure is that subsets of cells gain resistance to therapy through genetic mutation and natural selection. While this conjecture may hold true, particularly in a setting of prolonged treatment, one tenet of the ‘‘cancer stem cell hypothesis’’ posits that the cells responsible for tumor recurrence may inherently be more resistant to tumor debulking agents through any one of a number of mechanisms; thereby explaining refractory tumor growth follow- ing these treatments [11]. In support of this hypothesis, resistance to radiation can result from elevated expression of DNA damage response genes, as is the case for CD133+ glioblastoma stem cells [12]. In analogy to hematopoietic stem cells, solid tumor CSC have been proposed to exhibit high level expression of multidrug transporter family genes, such as ABCG2 and ABCB5, likely resulting in more efficient efflux of chemotherapeutic drugs [13– 15]. CSC may also enter the cell cycle less frequently, allowing them to resist toxicity by drugs that target highly proliferative cells. Evidence for chemoresistance by stem-like cells in epithelial cell lines and xenogeneic tumor-derived cells has been presented [16– 19]; however, these studies have either utilized cell lines adapted to tissue culture and/or do not assess altered tumor-initiating cell frequency in vivo following chemotherapy. By definition, tumor- initiating cells are enumerated retrospectively, therefore altered CSC frequencies post-therapy are best demonstrated by serial transplantation. Cyclophosphamide (CPA) and Irinotecan are agents that target proliferating cells and are commonly used chemotherapeutic agents in the treatment of solid tumors. Through different mechanisms, both act to inhibit DNA replication resulting in the slowing or inhibition of cell division and resulting in apoptosis. Resistance to CPA has been suggested to result from high cytoplasmic aldehyde dehydrogenase (ALDH) enzyme activity: particularly that of ALDH1 and ALDH3 [20,21], which oxidize and inactivate the bioactive metabolic byproduct of CPA, aldophosphamide/4-hydroxycyclophosphamide (4-HC) [22,23]. ALDH1, in particular, may play a major role in CPA resistance, as it’s Km for CPA is ,52 mM, whereas that of other ALDH family members with CPA catabolic activity, ALDH3 (ALDH3A1) and SSDH (ALDH5A1), are 10-fold lower (Km.520 mM) [24]. Though many tissues are relatively resilient in the wake of CPA treatment [25], only hematopoietic and neural stem cells have been demonstrated to contain high ALDH activity in reconstituting CoCSC Resist Chemotherapy transplantation studies following isolation of these cell populations [26–28]. Because colorectal cancer likely arises from colon stem or progenitor cells, it is tempting to hypothesize that similar mechanisms may render CoCSC resistant to CPA. Cancer stem cell populations have now been prospectively identified from various tumors of epithelial origin, including the breast, colon and prostate [19,29–33]. In all colorectal tumors we have investigated to date, tumors can be successfully transplanted using small numbers (e.g. ,1,000) of cells phenotypically positive for both epithelial-specific antigen (i.e. ESA or EpCAM) and CD44 [30]. In some tumors, isolation of cells positive for CD166, in addition to ESA and CD44, further enriches for colorectal cancer stem cells (CoCSC), allowing for efficient tumorigenesis with as few as 200 ESA + CD44+CD166+ cells [30]. Not only does this phenotype identify CoCSC in xenograft tumors, but tumorigenesis can be initiated from primary tumor samples with a small number of ESA + CD44+CD166+ cells. The reproducible identification of CoCSC using ESA and CD44 facilitates not only therapeutic efficacy studies across all patient-derived xenografts studied to date, but also facilitates dissection of the molecular pathways involved in tumorigenesis and resistance to therapy. Here we demonstrate that human ESA+CD44+ CoCSC generate adenocarcinomas that resemble parental tumors in both their phenotype and histology upon serial transplant in a xenograft setting and are enriched in tumors following classical chemother- apeutic regimens intended to shrink tumors. We further demonstrate both in vitro and in vivo that resistance to CPA is mediated, at least in part, by ALDH1 enzyme activity and that resistance to other chemotherapeutic agents (e.g. Irinotecan) is not likely attributed to this mechanism. To the best of our knowledge, this is the first functional demonstration that a human epithelial CSC population, as defined by serial transplantation, is enriched in residual solid tumors following chemotherapy. We further demonstrate that tumor heterogeneity in the form of a complex adenocarcinoma composed of TG and NTG populations can be generated by a single cell. Finally, the experimental methods used here provide a platform for assessing the efficacy of novel agents targeting solid tumor stem cells. Results CoCSC are enriched in residual tumors following CPA administration Early passage human xenograft tumors generated from cells never expanded in vitro appear to closely resemble those in patients and thus may serve as an excellent model by which to study cancer. We recently demonstrated that the subpopulation of human colorectal tumor cells with an ESA + CD44+ phenotype is uniquely responsible for generating heterogeneous adenocarcinomas in a xenograft setting (Figure 1A & B)[30]. Human ESA+CD44+ cells from xenogeneic colorectal tumors can be further subdivided based on CD166 expression, resulting in enrichment for tumorigenic (TG) cells among the CD166+ population. In contrast, all remaining cancer cells of human origin appear unable to initiate tumor growth and are referred to as non-tumorigenic (NTG). Having identified colorectal cancer stem cells (CoCSC) capable of fueling xenogeneic tumor growth, we sought to address a lingering question in the field regarding whether CSC are preferentially resistant to chemother- apeutic drugs. Cyclophosphamide (CPA) is an alkylating agent whose meta- bolic byproduct, phosphoramide mustard, crosslinks DNA and induces apoptosis in rapidly dividing cells [22]. CPA is a commonly used chemotherapeutic drug in the treatment of various types of cancer, such as soft tissue sarcomas, breast cancer PLoS ONE | www.plosone.org 2 June 2008 | Volume 3 | Issue 6 | e2428