of novel agents either alone or in combination with standard of care therapies. Closer scrutiny of both normal tissue-resident stem cells and CSC will lead to a better understanding of disease mechanisms and, ultimately, better therapies. Materials and Methods Xenograft Line Propagation Human colorectal tumor lines used in this study were obtained and passaged in mice as previously described [30]. Briefly, all tumors were initiated by subcutaneous implantation of either bulk tumor cells from frozen stocks or FACS purified cell populations into 6–8 week old NOD/SCID mice (Jackson Laboratories). Mice were anesthetized with Isoflurane or a single IP injection of 75– 100 mg/kg Ketamine and 5–10 mg/kg Xylazine. Of the 3 xenograft lines used in this study, two (UM-C4 & UM-C6) originated at the University of Michigan and one (OMP-C8) originated at OncoMed Pharmaceuticals Inc. All experiments were carried out under approved institutional IACUC guidelines and protocols. Tissue Disaggregation and Cell Preparation 3 Tumor tissue was minced into tiny fragments (,2 mm ), followed by enzymatic digestion with 300 u/mL Collagenase, 100u/mL Hyaluronidase, 0.5 mg/mL Dispase and 100 u/mL DNAseI (all obtained from Stem Cell Technologies; Vancouver, BC) for 1 hour at 37uC/5% CO2 with intermittent pipetting to disperse cells. Cells were then filtered sequentially through 70 mm and 40 mm screens, followed by a wash with excess FACS Buffer (16 Hanks Buffered Saline Solution [HBSS], 2% Heat-inactivated Fetal Calf Serum [FCS] and 25 mM HEPES [pH 7.4]). Red blood cells were lysed during a brief exposure to Ammonium Chloride and washed again with excess FACS buffer. Flow Cytometry and Cell Sorting All analyses and cell isolations were performed using freshly dispersed cell suspensions. Antibody staining was performed in FACS buffer for 30 minutes at 4uC at a density of 16107 cells/ mL. Antibodies used in this study include: anti-mouse H-2Kd (SF1-1.1; BD Pharmingen), anti-mouse CD45 (30-F11; BioLe- gend), anti-human ESA (HEA-125; Miltenyi Biotec), anti-mouse/ human CD44 (IM7; eBioscience), anti-human CD49f (GoH3; BD Pharmingen) and anti-human CD166-PE (105902; R&D Systems). The AldefluorTM reagent was purchased from StemCell Technol- ogies and used per manufacturer instructions. In all experiments, cells staining positively for murine lineage markers (mLin+; H-2Kd and murine CD45) were excluded during flow cytometry using Cy5.5PE-labelled antibodies. Dead cells were excluded using the viability dye DAPI and cell doublets and clumps were excluded using doublet discrimination gating. Cellular phenotype and viability of FACS purified cells was confirmed by serial flow cytometric analysis prior to injection for tumorigenicity studies. Purity was typically .99%. Tumorigenicity Experiments Following gentle centrifugation at 900 rpm65 min, cells were resuspended in 50 mL of FACS buffer per mouse and mixed 1:1 with Matrigel (BD Biosciences), followed by subcutaneous injection into the lower abdominal region while mice were under general anesthesia as described above. Only one tumor was initiated per mouse. Health was monitored daily and tumor growth was measured weekly using a digital caliper for up to 4 months. Animals were euthanized when tumors exceeded 1500 mm3 or the 120 day timepoint was reached. Statistical CoCSC Resist Chemotherapy analysis of tumor growth includes only those mice with palpable tumors (Mean6SEM). Chemotherapy Regimen Female mice were implanted with mLin2ESA+CD44+ cells (400 per mouse) while under anesthesia as described above. Once palpable, tumors were calipered twice weekly, and their length and width were used to calculate tumor volume based on the following formula: V = (length6[width])2/2. When tumors reached ,400 mm3, mice were randomized into either the control group, which received vehicle (sterile water), or the therapeutic group, which received CPA administered intraperitoneally at the dose of 38 mg/Kg twice a week. Irinotecan was diluted in PBS and similarly administered once weekly at a dose of 15 mg/kg. Immunohistochemistry Immunohistochemical studies were performed on formalin- fixed paraffin embedded tissues using monoclonal antibodies raised against Ki-67 (Vector Laboratories, catalog number VP- RM04) and c-Myc (DAKO, Catalog number M3570). Briefly, 4 mm-thick paraffin sections were deparaffinized and hydrated. Antigen retrieval was performed in 0.1M Tris-Cl, pH 9.0, using the Decloaking chamber from Biocare. Sections were then incubated with 3% hydrogen peroxide to block endogenous peroxidase activity and incubated with 1:50 dilution of primary antibody for 1 hr at room temperature, and then detected using ImmPRESS anti-mouse or anti-rabbit Ig (peroxidase) followed by VectorH NovaREDTM substrate for visualization (Vector Labora- tories). Hematoxylin was used for counter staining. TUNEL staining was performed using the In Situ Cell Death DetectionTM kit (Roche). For Immunofluorescence staining of ALDH1 (Abcam), frozen sections were fixed with 220uC cold methanol for 10 min, followed by a blocking step. Sections were incubated with ALDH1 at 1 ug/ml for 1 hr at room temperature and visualized with anti-mouse antibody conjugated with Alexa Fluor 488. Anti-hapten IgG was used as negative control and mounted with Prolong Gold DAPI containing antifade (Invitrogen). In Vitro Culture & Cytotoxicity Studies Following tissue disaggregation, as described above, cell suspensions were depleted of murine lineage cells using magnetic beads and plated in 96-well Primaria (BD Bioscience) plates at a density of ,20,000 cells per well in serum free Medium-D (3:1 low glucose DMEM:F-12 Media, B27 supplement, ITS-X, Pen/Strep [all from Invitrogen] and 0.5 mg/mL hydrocortisone [Stem Cell Technologies]), supplemented with 20 ng/mL bFGF and EGF, 5 u/mL Heparin and 16106 u/mL LIF. Plates were gently spun at 500 rpm for 5 min at room temperature following plating to promote attachment. When cultured in vitro for more than 7 days, media was changed weekly. For cytotoxicity studies, cells were cultured for 3–4 days at 37uC/5% CO2/5% O2, non-attached cells were removed, and medium was replaced with that containing 4-hydroxycyclopho- sphamide (4-HC; obtained from Dr. OM Colvin; Duke University Medical Center), diethylaminobenzaldehyde (DEAB; Sigma), Irinotecan or their respective vehicle. In studies using 4-HC, cells were exposed for only 4 hours, after which the medium was removed, cells were washed twice with PBS, and fresh Medium-D was added. In experiments involving ATRA, cells were pre- incubated overnight prior to exposure to DEAB and/or 4-HC. Cell viability was then assessed using CellTitre-Glo (Promega) either 20 hr post-treatment for 4-HC studies, or 1 week after addition of Irinotecan. PLoS ONE | www.plosone.org 11 June 2008 | Volume 3 | Issue 6 | e2428