Figure 5. CoCSC phenotype cells are also enriched following Irinotecan treatment. A) Upon randomization to normalize treatment groups at 400 mm3 at day 43, once weekly administration of vehicle (green boxes) or 15 mg/kg Irinotecan (red triangles) commenced and tumors were measured twice weekly. Growth curves representing the Mean6SEM are shown. B) Percentage of human ESA+CD44+CD166+ CoCSC phenotype cells in residual tumors is shown. Data reflects n$4 mice per treatment group. doi:10.1371/journal.pone.0002428.g005 tumors originating from single in vitro colonies both resembled parental tumors and were derived from a single cell, UM-C4 cells were transduced with a lentivirus carrying GFP-Luciferase, tumors were generated, and single colonies obtained from ESA+CD44+CD166+ cells were transplanted into mice. Upon harvest of these tumors derived from a single colony, either cells with the CoCSC phenotype or all other tumor cells with differing phenotypes were isolated by FACS, and tumorigenicity and clonality studies were performed. An input of 2,000 FACS purified cells resulted in a 100% take frequency with CoCSC phenotype cells, whereas human cells with all other phenotypes generated only 1 tumor (of 10 mice transplanted); likely resulting from rare contaminating CoCSC (Figure 6D). Of note, tumor heterogeneity can result from a single cell, as lentiviral transduction studies show that both ESA+CD442 (i.e. NTG) and ESA + CD44+CD166+ (i.e. CoCSC) cells have identical lentiviral insertion sites (Figure 6D inset), but only CoCSC are able to propagate tumors. Phenotypic and histological analysis of these serially transplanted tumors, which originated from single in vitro colonies, show that the ability to generate adenocarcinomas similar to parental UM-C4 tumors is maintained during short-term in vitro culture in these conditions (Figures 6E & F). To test whether CPA resistance is mediated by ALDH1 enzyme activity, UM-C4 or UM-C6 colorectal tumor cell colonies CoCSC Resist Chemotherapy established in vitro over the course of 3–4 days were then exposed to the bioactive CPA-metabolite, 4-HC, for 4 hours without or including various concentrations of the ALDH1-specific inhibitor, DEAB. In the absence of DEAB, the EC50 of 4-HC was ,60 mg/ mL (Figure 7A). Inhibition of ALDH1 activity resulted in increased susceptibility to 4-HC mediated cell death, as the EC50 was shifted to 43 mg/mL and 6 mg/mL in the presence of 30 mM and 100 mM DEAB, respectively. Like DEAB, all-trans retinoic acid (ATRA) has been demonstrated to reduce ALDH enzyme activity; however, it does so by reducing ALDH1 and ALDH3 protein levels [43]. Like DEAB, pre-treatment with 1 mM ATRA sensitized colorectal tumor colonies to 4-HC (n = 3; P,0.015) (Figure 7B). Surprisingly, the combination of ATRA pre-treatment and concurrent DEAB exposure synergized to facilitate increased chemosensitivity (n = 3; P = 0.003). Preferential ALDH1A1 gene expression in CoCSC and sensitization of colorectal tumor cells to 4-HC by inhibiting ALDH1, together strongly suggest that ALDH1 enzyme activity mediates CPA resistance. The above observation raised the question as to whether high ALDH1 enzyme activity also confers resistance to Irinotecan. We next established colonies of colorectal tumor cells and exposed them to serial dilutions of Irinotecan for one week in the presence or absence of 75 mM DEAB; a concentration known to potently inhibit ALDH1 activity. In contrast to 4-HC, the unaltered cytotoxicity profile of Irinotecan despite the presence of DEAB (Figure 7C) suggests that inherent resistance of CoCSC to Irinotecan occurs by a mechanism other than ALDH1 enzyme activity. Of note, neither DEAB nor an equal volume of its vehicle (1:200 dilution of 95% Ethanol) significantly reduced proliferation or survival during extended culture (7 days), suggesting that ALDH1 activity is only critical in a chemotherapeutic setting. ALDH1 is a major mediator of CoCSC resistance to CPA The above in vitro studies strongly suggest that the ALDH1A1 gene product, ALDH1, mediates resistance to CPA. To determine the importance of ALDH1 to CoCSC tumorigenicity and resistance to CPA, we transduced UM-C6 tumor cells with shRNA targeting ALDH1A1. UM-C6 tumors were chosen because of their high ALDH1A1 expression and relatively low expression of other ALDH family members (e.g. ALDH3A1 & ALDH5A1; see Figure 4C). Cells successfully transduced with either an ALDH1A1- targeted shRNA or Luciferase-targeted shRNA control vector were isolated by FACS and implanted subcutaneously to generate tumors. TaqmanTM qRT-PCR studies of UM-C6 tumor cells verified that ALDH1A1 gene expression was reduced 88% versus Luciferase-targeted shRNA transduced control cells, whereas neither ALDH3A1 nor ALDH5A1 gene expression were altered (Figure 8A). After tumors reached roughly 175 mm3, tumors were randomized within each group and then either treated with vehicle or 38 mg/kg of CPA, twice weekly. By ten days post-randomiza- tion, tumors in vehicle-treated mice from both groups had almost doubled in size. Of note, the growth of ALDH1A1-targeted shRNA transduced cells did not differ significantly from controls (not shown). Consistent with observations described above, control Luciferase-targeted shRNA transduced tumors undergoing a regimen of CPA were retarded in their growth versus vehicle- treated controls (Figure 8B; open circles). In agreement with results observed in vitro with the ALDH1 inhibitor, DEAB, shRNA targeting of ALDH1A1 gene expression appeared to sensitize tumors to CPA in vivo, as tumor growth essentially stopped, whereas ALDH1A1-targeted shRNA cells treated with vehicle continued to grow (Figure 8B; black triangles). Compared to vehicle-treated, ALDH1A1-targeted shRNA containing tumors, the PLoS ONE | www.plosone.org 7 June 2008 | Volume 3 | Issue 6 | e2428