Figure 3. ALDH1 enzyme activity demarcates a subpopulation of CoCSC. A) Phenotypic profile of human ESA+ UM-C4 tumor cells for ALDH1 enzymatic activity. Gates demarcate tumor subpopulations isolated by FACS for tumorigenicity studies, wherein B) growth kinetics of CD44+ALDH+ (red circles), CD44+ALDH2 (orange boxes) and CD442ALDH+ (green triangles) populations are plotted. Measurements reflect only mice with palpable tumors. Data representative of n = 3 independent experiments. C) Taqman qRT-PCR data displaying relative expression of ALDH1A1 (1A1), ALDH3A1 (3A1) and ALDH5A1 (5A1) in tumorigenic (TG; black) versus non-tumorigenic (NTG; white) cells (n$2) CoCSC Resist Chemotherapy from 2 patient-derived xenogeneic colorectal tumor lines (UM-C4 & UM- C6). Data reflects Mean6SEM, is normalized versus GUSB and displayed relative to UM-C4 NTG expression for each gene. doi:10.1371/journal.pone.0002428.g003 colorectal tumor line (Figure 3B). Although tumors can arise from ESA+CD44+ALDH2 cells taken from the UM-C6, OMP-C5 and OMP-C8 tumor lines [30], an ALDH+ subset of TG ESA+CD44+ cells does exist in all xenogeneic colorectal tumor lines investigated to date (n = 6). In contrast, a subpopulation of CD442 cells with high ALDH activity also exists in all tumor lines, but these cells fail to generate tumors upon transplantation. A subset of ALDH enzymes can oxidize, and thus inactivate, the cytotoxic CPA metabolite 4-HC/aldophosphamide [23,35]. The presence of cytoplasmic ALDH1 and ALDH3, in particular, have been associated with CPA resistance in the A549 lung cancer cell line [20,21]. ALDH1 (encoded by ALDH1A1) is greater than 10- fold more efficient at catabolizing 4-HC/aldophosphamide than its related family members ALDH3 and SSDH: encoded by the ALDH3A1 and ALDH5A1 genes, respectively [24]. In TaqmanTM qRT-PCR analysis of UM-C4 and UM-C6 tumors, ALDH1A1 is generally expressed at higher levels than ALDH3A1 and ALDH5A1. ALDH1A1 gene expression is 2.8-fold higher in CoCSC than in NTG cells, and despite slight elevation of ALDH3A1 expression in CoCSC versus NTG populations (Figure 3C), its message is barely detectible in either tumor line. Additionally, whereas ALDH5A1 is slightly elevated in TG versus NTG cells from UM-C4 tumors (,1.5-fold), its expression is similar among these populations from UM-C6 tumors (Figure 3C). The preferential expression of ALDH1A1 in TG cells supports observed enzymatic activity measurements among the human ESA + CD44+ subpopulation, wherein the majority of these cells have high DEAB-sensitive, ALDH1 activity. When considered in conjunc- tion with the 10-fold higher proficiency of ALDH1 versus ALDH3 and SSDH at inactivating CPA intermediates [24], one might interpret these results to suggest that CoCSC resistance to CPA might be predominantly mediated by ALDH1 enzymatic activity. ALDH1A1, MYC, and MYB are enriched in residual TG cells post CPA therapy Since ALDH1A1 gene expression is elevated in TG versus NTG cells, ALDH1 enzymatic activity is disproportionably high in cells with the CoCSC phenotype, and this activity may mediate resistance to CPA, we next asked whether the frequency of ESA+CD44+ALDH+ cells was elevated in UM-C4, UM-C6 and OMP-C8 tumors from mice receiving CPA versus those being administered vehicle. Like the observed phenotypic increase in CD166+ cells (Figures 1E, S1B & S1C), the ALDH+ subpopulation of ESA + CD44+ cells was consistently higher in CPA- versus vehicle-treated control mice (67.666.3% versus 56.866.8%, respectively; n = 5, P,0.012 in paired t-test) (Figure 4A). A number of gene products have been deemed critical to normal colon development and are widely expressed in colorectal tumors [36–39]. Following the isolation of TG versus NTG populations from both vehicle- and CPA-treated UM-C4 tumors with .99% purity, expression of numerous genes with suggested ties to colorectal cancer was determined by TaqmanTM qRT- PCR. Those differentially expressed in TG versus NTG cells included not only ALDH1A1, as described above (Figure 3C), but also c-Myb (MYB) and c-Myc (MYC; Figure 4B). In contrast, genes associated with differentiation and a mesenchymal phenotype [40,41], such as Vimentin (VIM), were more highly expressed in NTG cells. PLoS ONE | www.plosone.org 5 June 2008 | Volume 3 | Issue 6 | e2428