CoCSC Resist Chemotherapy Figure 6. In vitro maintenance and expansion of CoCSC. Human ESA+CD44+CD166+ cells were plated in limiting dilution and cultured for fourteen days in serum-free maintenance conditions. Colorectal tumor colonies were then either analyzed for A) ESA expression by IHC, or B) ESA, CD44 and CD166 expression by flow cytometry. C) Cellular phenotype of single colony-derived tumors, showing human ESA+ cell subpopulations expressing CD44 and CD166. D) Tumor growth curves are shown for either 2,000 CoCSC phenotype cells or an equal number of cells with all other phenotypes (Other), which were isolated from in vitro colony-derived tumors (tumors/animals injected). Inset shows lentiviral insertion band obtained by inverse PCR of 1) human xenograft tumor cells, 2) ESA+CD44+CD166+ (CoCSC) cells or 3) ESA+CD442 (Other) cells isolated by FACS. Phenotypic and morphological analysis of single-cell derived tumors from serially transplanted CoCSC show that the diverse E) phenotype and F) histological makeup of xenogeneic colorectal tumors are maintained following brief in vitro culture in limiting dilution. Black bar = 100 mm. doi:10.1371/journal.pone.0002428.g006 difference in tumor volumes between CPA and vehicle-treated groups significantly differed by day 10 (n$5, P = 0.0061). Upon termination of the study, tumors were removed and the cellular phenotype was assessed to determine whether CoCSC frequency differed in tumors where ALDH1A1 gene expression was being suppressed by shRNA. Whereas control Luciferase-targeted shRNA tumors were significantly enriched for ESA+CD44+CD166+ cells in CPA- versus vehicle-treated mice (n$5, *P = 0.0002), both CoCSC and NTG cells from ALDH1A1- targeted shRNA containing tumor cells appeared equally sensitive to CPA, as the frequency of CoCSC was no different (Figure 8C). Of note, shRNA-mediated knockdown of ALDH1A1 in vivo, significantly reduced the frequency of CoCSC in residual tumors following CPA therapy (n$6, **P = 0.045). Discussion While recent decades have witnessed a revolution in therapeutic strategies yielding significant clinical responses measured in terms of tumor regression and disease-free survival, overall survival has failed to substantially improve. The recent identification of tumor cell subpopulations with the unique ability to fuel tumor growth (i.e. CSC) may shed light on the disconnect between response rates and overall survival. That is, therapies that fail to adequately target CSC populations, which represent a minority of most epithelial tumors, will fail to eliminate those cells capable of regenerating the tumor after therapy has ceased. Furthermore, survival of these long-lived cells in the presence of toxic therapeutic agents provides an ideal selective vice for additional mutations. Familiarity with both the means of resistance to a particular chemotherapeutic agent and the phenotypic identity of those cells that harbor resistance mechanisms should help facilitate the discovery of therapies better able to clear minimum residual disease and prolong overall survival. Although the cancer stem cell paradigm explains tumor heterogeneity, provides rationale for how genetic mutations might be accumulated over long time periods and suggests resistance to chemotherapeutics/radiation may be inherent and not acquired properties of specific tumor cell subpopulations in epithelial tumors [1,2], a few key tenets of this theory have not been supported experimentally. Having previously identified surface markers that reliably identify colorectal cancer stem cells (CoCSC)[30], we show here that colorectal tumors are enriched for CSC following chemotherapeutic regimens that halt, or at least slow, tumor growth. ALDH1 enzymatic activity, which is generally highest in CoCSC, appears to play a major role in mediating resistance to CPA, as its inhibition in vitro, and reduced expression in vivo, sensitizes colorectal tumor cells to the bioactive metabolite of CPA. This chemotherapeutic resistance mechanism does not appear universal; however, as altered cytotoxicity profiles were not observed with other chemotherapeutic agents (e.g. Irinotecan) en lieu of ALDH1 inhibition. ESA and CD44 demarcate the subpopulation of cells with tumorigenic ability in all colorectal tumors examined to date. Overexpressed in a number of epithelial tumors and suggested to be an important prognostic marker of tumor progression [44], CD166 (i.e. ALCAM) appears to further segregate TG from NTG PLoS ONE | www.plosone.org 8 June 2008 | Volume 3 | Issue 6 | e2428