cells), colorectal adenocarcinomas appear to contain both immature and mature colorectal cell lineages that are somewhat unstructured in their organization. Proto-oncogenes such as c- Myb not only coordinate normal development of the distal colon, but altered expression is commonly associated with hyperproli- feration of immature colorectal cells and overt cancer [37,45]. c- Myc has recently been demonstrated to mediate nuclear b- catenin-mediated tumorigenesis in the APC-deficient mouse model of intestinal neoplasia [46]. In fact, MYB appears to be a downstream target of c-Myc. Of significant interest in studies performed here is the observation that MYC expression is elevated in TG versus NTG cells from xenogeneic colorectal tumors, and that MYC levels are further increased in residual cells with the CoCSC phenotype. Conversely, MYC levels do not change, or are reduced in the NTG contingent of tumor cells, which themselves are progeny of CoCSC. These observations corroborate past studies demonstrating the association of the MYB and MYC proto- oncogenes with cancer, but underscore the distinction between TG versus NTG cells, in that those cells most resistant to therapy (i.e. CoCSC) also most resemble stem/progenitor cells in their phenotype and gene expression profiles. Establishing mechanisms of resistance to chemotherapeutic drugs can be difficult, especially with heterogeneous xenogeneic tumors. However, in vitro culture conditions that facilitate colony formation with an input of tumorigenic colorectal tumor cells have been established and offer a new approach to the characterization of underlying mechanisms of drug resistance. Culture of CoCSC in these conditions is herein demonstrated to generate morpho- logically and histologically diverse tumors from limiting dilutions of colorectal tumor cells. That is, minimally cultured individual colonies generated in vitro are able to generate tumors in vivo that resemble the parental adenocarcinomas from which they were obtained. We further show for the first time that tumor heterogeneity can result from a single CoCSC using classical lentiviral insertion site analysis. Because in vitro colonies are highly enriched for TG ESA + CD44+CD166+ cells, the fate of these cells in defined culture conditions can now be assessed without the dangers encompassing extended in vitro culture and passaging. Intracellular ALDH enzymes oxidize aldehydes to carboxylic acids and carry out various catabolic processes, including ethanol and amine catabolism and conversion of vitamin A to retinoic acid [23]. ALDH enzyme activity can also protect cells from the cytotoxic affects of CPA, as a subset of ALDH enzyme family members can catabolize the bioactive metabolite of CPA, aldophosphamide/4-HC [22]. Although ALDH1A1, ALDH3A1, and ALDH5A1 gene products can degrade biologically active CPA metabolites [20,21], DEAB appears to specifically inhibit ALDH1 [34]. Like hematopoiesis, intestinal epithelium is resilient following damage incurred during CPA therapy [25]. Hematopoietic stem cells (HSC) have high ALDH1 activity and can be isolated from bone marrow based on that unique trait [27,28]. Similarly, we suggest that stem/progenitor cells in the base of colon crypts, like CoCSC, may have high ALDH1 activity, thus providing protection against CPA-induced cytotoxicity and tissue ablation during therapy. As in vitro experiments suggest, inhibition of ALDH activity in vivo sensitizes tumors to CPA therapy; however, the half-life of DEAB in vivo is extremely short [42] and such studies cannot be done. Knock down of ALDH1A1 expression using a lentiviral-based shRNA approach in UM-C6 tumors demonstrated that CoCSC can be sensitized to CPA in vivo, as there was no noticeable enrichment of CoCSC in tumors from CPA- versus vehicle-treated mice. Furthermore, unlike for 4-HC in vitro, DEAB was unable to alter colorectal tumor cell sensitivity to Irinotecan, suggesting that the target of this inhibitor (ALDH1 CoCSC Resist Chemotherapy enzyme activity) plays an important role in resistance to CPA, and resistance to Irinotecan appears to involve another mechanism. Like DEAB, retinoic acid (a vitamin A/retinaldehyde metab- olite and product of ALDH activity) appears to decrease ALDH1 and ALDH3 protein levels via a feedback mechanism that can sensitize cells to CPA-induced cytotoxicity [23,43]. Retinoic acid, in the form of ATRA, is used with great success in the clinical setting for a subset of acute promyelocytic leukemia patients who have chromosomal translocations involving the retinoic acid receptor-a gene, RARa [47], but the use of retinoids in solid tumors has not been promising to date [48]. As we demonstrated here, either DEAB or ATRA alone sensitize colorectal tumor cells to 4-HC, and the combination of both appears synergistic. Because normal stem cell populations, such as HSC, neural stem cells and in all likelihood, intestinal stem cells, have high ALDH activity, its inhibition by ATRA as a pre-therapeutic regimen to CPA may also negatively impact normal stem cell populations. Nevertheless, as shown here, detailed study of rare tumor populations responsible for fueling tumor growth can provide mechanistic insights not only into tumorigenesis, but resistance mechanisms to common therapies. These inherent resistance mechanisms can include drug specific catabolic enzyme activity; such as that of ALDH1. Here we show that xenogeneic colorectal tumors investigated to date contain a subset of TG ESA + CD44+ cells with high ALDH activity, and that this subpopulation is enriched in xenogeneic tumors from mice treated with CPA. These observations are supported by qRT-PCR using TG or NTG cells isolated by FACS, which show that ALDH1A1 is the predominant cytoplasmic ALDH enzyme in colorectal tumors and its expression is further increased in residual tumor cells following therapy with CPA; consistent with the phenotypic increase in ALDH+ cells among the CoCSC phenotype. Importantly, however, ALDH1 activity alone does not confer tumorigenicity nor demarcate TG cells. When tumorigenicity of CD44+ versus CD442 ESA+ALDH+ cells is compared, only the CD44+ subset is able to initiate actively growing tumors. Secondly, extended inhibition of ALDH1 activity with DEAB in vitro does not appear to alter cell proliferation or survival, as its presence in Irinotecan combination studies for 7 days in vitro did not differ from control. Furthermore, initiation of tumorigenesis with ALDH1A1- versus Luciferase-targeted shRNA containing cells was identical, demonstrating that ALDH1 enzymatic activity is not requisite in the absence of CPA exposure. The advent of flow cytometry and cell sorting has revolutionized the study of developmental biology and disease, particularly in the hematopoietic system. Hematologic malignancies are among the best understood of the neoplastic diseases precisely because hematopoietic cells are easy to obtain and the in vivo and in vitro assays to determine the fate and potential of these cells have been developed. Similarly, the field of solid tumor biology has begun to enter an era where the cells responsible for fueling tumor growth can be identified, isolated, and their characteristics tested both in vivo and in vitro. Here we demonstrate for the first time that CoCSC are responsible for fueling both tumor growth and heterogeneity, and are enriched in residual tumors following chemotherapy. We also reveal that inherent resistance mechanisms differentially expressed within tumor subpopulations, such as ALDH1 enzy- matic activity in CoCSC, can explain the inability of chemother- apeutic agents to improve overall survival despite tumor regression. In addition to providing evidence supported by serial transplantation studies for a previously unsupported tenet of the ‘‘cancer stem cell hypothesis’’, we identify a major CSC-specific mechanism of resistance to a classical chemotherapeutic agent and establish experimental platforms both in vitro and in vivo for testing PLoS ONE | www.plosone.org 10 June 2008 | Volume 3 | Issue 6 | e2428