Oct4, Nanog and Sox2 expression in mouse embryonic stem cells [42]. In summary, the findings reported in this study showed, for the first time, that ARCaPM and PC3 PDGF-D cells having EMT phenotype shared cellular and molecular characteristics of stem cells or cancer stem-like cells. Moreover, miR-200 and let-7 played a critical role in linking EMT phenotype with stem cell signatures by regulating the expression of Lin28B and Notch1. Therefore, we believe that these models would be useful in screening drug libraries for finding newer agents that could be useful in selective killing of EMT-type or cancer stem-like cells in prostate cancer in the future consistent with a similar approach that showed a great success in breast cancer [43]. Interestingly, recent studies have shown that some natural agents could up-regulate miR-200 and reverse the EMT phenotype [44,45]. Thus, in future studies we will use these natural agents to eliminate the stem-like cells by reversing EMT phenotypic cells to MET phenotype for assessing whether natural agents could be useful for the prevention of prostate cancer recurrence and metastasis. We firmly believe that strategies by which one could either reverse the EMT to MET phenotype or could selectively kill EMT-phenotypic cells or cancer stem-like cells would become a novel approach for the prevention of tumor progression and/or treatment of CRPC and its metastasis for which newer therapies are urgently needed. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Any animal found unhealthy or sick were promptly euthanized. The protocol was approved by the Committee on the Ethics of Animal Experiments of Wayne State University institutional Users Animal Care Committee (Permit Number: A-10-03-08). Cell lines and culture condition Generation of stable cell lines over-expressing PDGF-D was accomplished by transfection of PC3 cells with the corresponding empty vector pcDNA3 Neo or pcDNA3-PDGF-D:His as previously described [31] and referred to as PC3 Neo, or PC3 PDGF-D cells, respectively throughout this manuscript. The PC3 Neo or PC3 PDGF-D cell lines were cultured in RPMI 1640 medium with 2 mmol/L glutamine, 25 mmol/L Hepes (Invitro- gen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS), 50 units/ml Penicillin, and 50 mg/ml Streptomycin. ARCaPE and ARCaPM cells were purchased from Novicure Biotechnology (Birmingham, AL). ARCaPE and ARCaPM were established from bone tumor tissue derived from ARCaP cells. ARCaPE with cobblestone morphology expressed higher E- cadherin and cytokeritin 18, 19, which were associated with typical epithelial cell characters, and concomitant with lower level of vimentin expression. In contract, ARCaPM with spindle-shape fibrobastic morphology expressed more genes associated with mesenchymal markers such as increased vimentin and N- cadherin expression, and these cells also acquired drug-resistant phenotype [46]. These cells were cultured in Prostate Epithelial Cell Medium (MCaP) supplied by Novicure Biotechnology, and the medium was supplemented with 5% fetal bovine serum (FBS), 50 units/ml Penicillin, and 50 mg/ml Streptomycin. All cells were maintained in a 5% CO2-humidified atmosphere at 37uC, and genotypically characterized to support the authenticity of these cells, which was consistent with its origin. Stem Cell Feature in EMT Cells Research reagents and antibodies Antibodies against slug, Oct4, Sox2, Nanog and Lin28B were purchased from Cell Signaling Technology (Beverly, MA). Antibody against EZH2 was purchased from BD Biosciences (Bedford, MA). Antibody to vimentin was acquired from Abcam (Cambridge MA). Antibodies against ZEB1, ZEB2, Stat3, E- cadherin, jagged2, delta1, delta3, delta4, Notch1, Notch2, and Notch3 were obtained from Santa Cruz (Santa Cruz, CA). Goat anti-rabbit IgG (H + L)-HRP conjugate and goat anti-mouse IgG (H + L)-HRP conjugate were obtained from Bio-Rad (Reinach, BL,). Antibody to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Affinity BioReagents (Golden, CO). DAPT (N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenyl- glycine t-Butyl Ester) was purchased from EMB Bioscience (San Diego, California). Clonogenic assay PC3 Neo, PC3 PDGF-D, ARCaPE and ARCaPM cells were collected after trypsinization, and re-suspended in the complete medium. Single cell suspensions were plated in regular 10 cm in diameter Petri dishes at the clonal density of 1,000 cells per dish. After 2–3 weeks of culture, colonies were fixed with 4% paraformaldehyde for 10 min, stained with crystal violet for additional 10 min, and washed with 1X PBS. The colonies were photographed. The colony numbers were counted using software image analysis program Scion Image downloaded from NIH website (http://www.scioncorp.com). Particle Analysis program was used for counting the colony numbers. Data was presented as relative colony number of PC3 Neo or ARCaPE designated as unit value of one. Soft agar assay PC3 Neo and PC3 PDGF-D cells were harvested and suspended in culture medium. To make the bottom layer, 1 ml of 0.5% agarose (Invitrogen) was added to 6-well plates, and allowed to gel at room temperature. To prepare the top layer (0.25% agarose), 500 `ıl of 0.5% agarose was mixed with 500 `ıl cell suspension containing the 5000 cells. This mixture were overlaid above the bottom layer and allowed to solidify at room temperature. An additional 2 ml of culture medium was added after solidification to the top layer, and cells were incubated for 3 weeks at 37uC. After three weeks of growth, the colonies were photographed (40 X). The colony numbers were counted under a phase contrast microscope (40 X). Data was presented as colony numbers per field. Self-renewal assay Single cell suspensions of PC3 Neo, PC3 PDGF-D, ARCaPE, ARCaPM and PC3 PDGF-D cells transfected with Sox2, Nanog, Oct4, Lin28B or control siRNA as well as miR-200 and let-7, were plated on ultra low adherent wells of 6-well plate (Corning, Lowell, MA) at 1000 or 2000 cells/well in DMEM/F12 (Invitrogen) supplemented with B27 and N2 (Invitrogen). Single cell status was confirmed under microscope. Fresh medium was added every 3–4 days. After 3 or 6 days, spheres termed here as ‘‘prostaspheres’’ (P1) numbers were counted under microscope and the proportion of sphere-generating cells was calculated by dividing the number of cells seeded by the number of prostaspheres. Prostaspheres were photographed and the size of the prostaspheres in diameter was measured using software image analysis program Scion Image downloaded from NIH website. To confirm that prostaspheres were the progeny of individual cells rather than the aggregation of the multiple cells, the prostaspheres (P1) were collected by PLoS ONE | www.plosone.org 11 August 2010 | Volume 5 | Issue 8 | e12445