CoCSC Resist Chemotherapy Figure 7. ALDH1 enzyme inhibition sensitizes colorectal tumor cells to CPA in vitro. In vitro cell viability measurements of human colorectal tumor cells following 4 hours of exposure to A) varying concentrations of 4-HC and/or the ALDH1-specific inhibitor DEAB, or B) 20 mg/mL 4-HC in the presence or absence of 75 mM DEAB and/or 1 mM ATRA. C) Cell viability measurements following 7 days of exposure to varying concentrations of Irinotecan in the presence or absence of 75 mM DEAB. All data is expressed as the Mean6SEM of triplicate measurements and is normalized versus vehicle-treated controls. All data is representative of n$2 independent experiments using either UM-C4 or UM-C6 tumor cells. *P,0.015. **P = 0.003. doi:10.1371/journal.pone.0002428.g007 cells when used in combination with ESA and CD44 [30]. Consistent with the hypothesis that TG cells are more resistant to chemotherapy and the association between CD166 expression and poor outcome, the tumorigenic CD166+ subset of ESA+CD44+ cells appeared more resilient to not only CPA, but also Irinotecan. Like the normal colon crypt, which is predominantly composed of two different cell lineages (i.e. absorptive colonocytes and goblet Figure 8. Knockdown of ALDH1A1 gene expression sensitizes tumors to CPA in vivo. UM-C6 tumor cells transduced with Luciferase- (black) or ALDH1A1-targeted shRNA (white) were A) assessed by TaqmanTM qRT-PCR for relative expression of ALDH1A1, ALDH3A1 and ALDH5A1, or B) transplanted into mice at 400 cells/mouse to initiate tumors. Following randomization when tumors reached a Mean volume 3 of 175 mm , mice were treated twice weekly with either vehicle or 38 mg/kg CPA. The Mean difference in tumor volume between CPA- and vehicle-treated mice is plotted for Luciferase shRNA control (open circles) or ALDH1A1 shRNA-containing (triangles) cells. C) Percentage of human ESA+CD44+CD166+ CoCSC phenotype cells in residual tumors is shown. Data reflects n$5 mice per treatment group. doi:10.1371/journal.pone.0002428.g008 PLoS ONE | www.plosone.org 9 June 2008 | Volume 3 | Issue 6 | e2428