Figure 4. CoCSC with high ALDH1 activity are more prevalent following CPA therapy. A) Ratio of ALDH+ tumor cells among the human ESA+CD44+ population in tumors from vehicle-treated control or CPA-treated mice. Data reflects the paired Mean of 5 independent experiments using 3 different xenogeneic colorectal tumor lines (UM- C4, UM-C6 & OMP-C8) and n$5 mice per experiment. B) Taqman qRT- PCR data for the denoted genes using TG and NTG populations isolated from (V) vehicle- or CPA-treated UM-C4 tumors. Data represents Mean6SEM (n$2). C) Immunofluorescence or immunoperoxidase staining of frozen or formalin-fixed, paraffin embedded tumors for ALDH1 or c-Myc, respectively, from vehicle- and CPA-treated mice. Black bar = 100 mm. doi:10.1371/journal.pone.0002428.g004 Consistent with the elevated frequency of residual tumor cells expressing high levels of tumorigenicity-associated markers, such as CD166, relative expression of CoCSC-associated versus NTG- associated transcripts, such as ALDH1A1 and VIM, respectively, was further disparate in CPA- versus vehicle-treated control UM- C4 tumors (Figure 4B). Consistent with the hypothesis that ALDH1 may have a role in mediating resistance to CPA, ALDH1A1 gene expression was further elevated in residual cells with the CoCSC phenotype, but ALDH3A1 and ALDH5A1 were not (Figures 4B & S3). In addition, genes previously identified as highly expressed in the epithelial stem/progenitor cell compart- ment at the base of colon crypts and in neoplastic tissue (e.g. MYB & MYC) were more highly expressed in residual tumor CoCSC than the NTG population of cells that represent the bulk of the tumor, but which are generated by CoCSC and appear more susceptible to CPA-induced cytotoxicity. Similar expression CoCSC Resist Chemotherapy patterns were also observed in other xenogeneic tumor lines following CPA-treatment, including UM-C6 (Figure S3). Finally, gene expression differences in CPA- versus vehicle- treated control tumors were further correlated with protein expression by immunofluorescence and IHC. Intracellular levels of ALDH1 and nuclear c-Myc, for example, were more prevalent in CPA-treated tumors (Figure 4C). Interestingly, ALDH1 staining in CPA-treated tumors appeared to localize to the apical surface, where it may be hypothesized to intercept CPA metabolites as they enter the cell. In contrast, ALDH1 protein levels were lower and more diffuse in control tumors. Similarly, c-Myc was noticeably more concentrated in the nucleus of colorectal tumor cells of CPA- treated mice, consistent with the increased frequency of TG cells and elevated MYC expression within this resilient population. CoCSC resistance to chemotherapy is not specific to CPA To determine whether CoCSC also exhibit resistance to other important chemotherapeutic agents, we examined the impact of Irinotecan (a.k.a. SN-38) on CoCSC frequency. Experiments were initiated whereupon tumor bearing mice received weekly injections of 15 mg/kg Irinotecan. After a slight delay of approximately 1 week, tumor growth was halted by Irinotecan (Figure 5A). Upon tumor harvest two weeks after the initiation of chemotherapy, the percentage of remaining CoCSC was assessed by flow cytometry. In Irinotecan-treated tumors, the percentage of bulk tumor cells with the CoCSC phenotype was increased 61% versus control mice administered vehicle (Figure 5B). Next, the inherent tumorigenicity of ESA+CD44+CD166+ cells from vehicle- or Irinotecan-treated mice was tested in a serial transplant setting versus those cells that did not phenotypically classify as CoCSC. Similar take rates were observed with CoCSC from control- or Irinotecan-treated tumors in these transplants, and cells phenotypically classified as ‘‘Other’’ did not initiate tumors (data not shown). The increased frequency of CoCSC phenotype cells in residual tumors from mice administered Irinotecan and the inability of cells with other phenotypes to transplant disease, together suggest that CoCSC are also preferen- tially resistant to Irinotecan. CPA resistance is specific to cells with high ALDH1 activity Seeing that ESA+CD44+ tumor cells have high ALDH activity, the frequency of ESA+CD44+ALDH+ cells is increased in tumors from mice treated with CPA, and ALDH1A1 gene expression is elevated in CPA-resistant CoCSC, we therefore sought to determine whether resistance to CPA was mediated by ALDH1 enzyme activity. Because the ALDH1 specific inhibitor DEAB is highly unstable in vivo [42], in vitro culture conditions that support TG colorectal tumor cell expansion were established. Notably, the cellular phenotype of cells best able to establish colonies in vitro was ESA+CD44+CD166+ (also that of CoCSC), whereas FACS purified CD442 cells were unable to do so (data now shown). To validate that this culture system supports maintenance of TG cells, we sorted ESA + CD44+CD166+ CoCSC in limiting dilution into plates containing Mitomycin C-treated 3T3 or MEF feeder cells as support, depending on the tumor line used: UM-C4 or UM-C6, respectively. Colony formation frequency of CoCSC under these conditions was roughly similar to the TG cell frequency observed upon transplantation (,1:86633; n = 7 for UM-C4 and 1:5265; n = 3 for UM-C6). Colonies were cultured for 14 days without passaging prior to reimplantation into mice. Not only did cells maintain ESA, CD44 and CD166 expression during in vitro culture (Figures 6A & B), but the ability to generate heterogeneous tumors resembling parental tumors was conserved as well (Figure 6C). To validate that tumor-initiating cells in PLoS ONE | www.plosone.org 6 June 2008 | Volume 3 | Issue 6 | e2428