38 mg/kg CPA, twice weekly. Within 15 days of randomization and administration, tumor growth was noticeably retarded in the CPA-treated animals (Figures 1C and Figure S1A). Following euthanization, tumors were removed for histological and pheno- typic analysis. Although the percentage of proliferating tumor cells (i.e. Ki-67+) did not appear altered, there was noticeably more cell death (i.e. TUNEL+ cells; Figure 1B) and ESA+CD44+ cells were more frequent in tumors from CPA-treated mice (Figure 1D). The trend towards more frequent ESA+CD44+ cells in CPA-treated tumors generally held true independent of tumor volume and was more pronounced at higher CPA doses (e.g. 38 mg/kg versus 25 mg/kg; data not shown). Enrichment of ESA+CD44+CD166+ cells was even more striking and particularly evident when CD166 expression was analyzed on residual human ESA + CD44+ cells (Figures 1E, S1B & C). Similar results were observed with both xenogeneic tumor lines investigated. To determine whether the phenotypic increase in CoCSC following CPA administration correlated with an authentic rise in TG cell frequency, we serially transplanted 4 sets of mice with bulk UM-C4 tumor cells using a limiting dilution approach and scored them as positive or negative for tumor growth after three months. Mice with palpable tumors at 90 days were kept alive to determine whether tumor growth would continue beyond 200 mm3, as rare palpable masses can occur, but which contain murine stromal elements and no detectable human cells upon analysis at the termination of the study (data not shown). Based on Poisson distribution statistics, TG cells were .2.2-fold more frequent in CPA-treated tumors than in mice administered vehicle (P = 0.0016; Table 1 & Figure 2A). Importantly, the cellular phenotype of these serially transplanted secondary tumors were identical to the parental tumor cells used to initiate the study (Figure 2B), demonstrating that intervening treatment and serial transplantation of cells in limiting dilution did not alter either the tumorigenic potential of CoCSC nor their capacity to generate heterogeneous tumors containing predominantly non-tumorigenic (i.e. CD442) cells. Because TG cells were more frequent and tumor cell populations from CPA-treated animals were enriched for phenotypes associated with CoCSC, we next isolated CoCSC phenotype cells from UM-C4 tumors of vehicle- versus CPA- treated mice and asked whether these cells inherently differed in their ability to generate secondary tumors. Serial transplantation of 50 ESA + CD44+ or ESA + CD44+CD166+ cells from tumors in CPA- versus vehicle-treated control mice resulted in secondary tumors with roughly equal frequency (Table 1), which when considered in conjunction with previous observations suggest that CoCSC are more frequent within tumors exposed to CPA Table 1. Tumors/animals injected (%) (100%) 18/18 (94%) (61%) (10%) (50%) (70%) CPA 19/19 13/18 12/20 2/6 8/10 (100%) (100%) (72%) (60%) (33%) (80%) Cell type injected # cells Control Bulk Tumor Cells mLin2ESA+CD44+ mLin2ESA+CD44+CD166+ doi:10.1371/journal.pone.0002428.t001 1500 500 167 56 50 50 18/18 16/17 11/18 2/20 3/6 7/10 CoCSC Resist Chemotherapy Figure 2. Tumorigenic UM-C4 cells are enriched in residual tumors following CPA administration. A) Limiting dilution analysis of unfractionated UM-C4 tumor cells was used to calculate TG cell frequency using Poisson distribution statistics (695% confidence level; * P = 0 . 0 0 1 6 ) . B ) P e r c e n t a g e o f h u m a n E S A + C D 4 4 + a n d ESA+CD44+CD166+ cells in secondary tumors arising from residual tumorigenic cells transplanted serially in limiting dilution following vehicle or CPA treatment regimens. doi:10.1371/journal.pone.0002428.g002 chemotherapeutic regimens and are unaffected in their ability to fuel tumor growth. CoCSC have high expression of ALDH1A1 In searching for additional CoCSC markers, we previously used a tool that can identify hematopoietic and neural stem cell populations: ALDH enzyme activity [30]. Using the AldefluorTM reagent, which undergoes a shift in fluorescence following enzymatic cleavage by ALDH enzymes, and the ALDH1-specific inhibitor diethylaminobenzaldehyde (DEAB) [34], a large sub- population of ESA + CD44+ cells from both UM-C4 and UM-C6 tumor lines was determined to have high ALDH activity (Figure 3A) [30]. Similar observations were also made in other patient-derived xenograft colorectal tumor lines (Figure S2). Furthermore, when ESA + CD44+ cells were subdivided based on ALDH activity and isolated by FACS, ALDH+ cells were tumorigenic in all cases investigated [30]. Of note, tumorigenicity is strictly conferred by ESA+CD44+ALDH+ cells in the UM-C4 PLoS ONE | www.plosone.org 4 June 2008 | Volume 3 | Issue 6 | e2428