Marra et al. BMC Cancer 2010, 10:129 http://www.biomedcentral.com/1471-2407/10/129 cancer immune response [11]. Deletion of PSCA gene does not appear to interfere with normal development as PSCA knockout mice are viable. Additionally, cross- ing of the PSCA knockout mice with prostate tumor models driven by large T antigen did not increase pri- mary tumor formation [12,13]. Here the biological role of human PSCA was evalu- ated using RNA interference and microarray analysis. To establish a pharmacologic control over gene expres- sion a shRNA against PSCA was identified and expressed under the control of dox in a lentivirus sys- tem [14]. Microarray analysis was utilized to identify genes coregulated with PSCA in tumor xenografts. Methods Cell culture and generation of lentivirus vectors The 293T and SW780 cell lines were cultured in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal calf serum. The lentivirus system for condi- tional gene suppression with dox-inducible shRNAs uti- lized has been described previously[14]. Briefly, in the absence of dox, tTR-KRAB repressor binds to tetO and suppresses H1-mediated shRNA transcription, thus allowing normal expression of the cellular target gene. In the presence of 10 μg/ml of dox, tTR-KRAB cannot bind to tetO and hence shRNAs are produced, leading to downregulation of PSCA. The green fluorescent pro- tein (GFP) cDNA contained in the shRNA vector pro- vides a monitoring device, as it is switched on by dox treatment and GFPis expressed. All recombinant lenti- viruses were produced by transient transfection in 293T cells. Briefly, 293T cells were cotransfected with 20 μg of pLUTHM-shPSCA3 plasmid, 15 μg of pCMV- ΔR8.91, and 5 μg of pMD2G-VSVG by calcium phos- phate precipitation. After 16 h medium was changed, and recombinant lentivirus vectors were harvested 48 h later. FACS analysis was conducted as previously described [15]. Viability assay Cell viability was monitored using the CellTiter-Blue Via- bility. The assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin); 1 × 103 SW780-shPSCA and SW780- shControl cells +/- Dox were plated in a 96 well plate in parallel with the parental cell line SW780. Cells were incubated at 37°c for 96 hrs, and fluorescence was subse- quently monitored using a plate-reading fluorometer. Tumor models Six-week-old female CD-1 nude mice were purchased from Charles River Laboratories and maintained in accordance to Guidelines for the Care and Use of Laboratory Animals in IRBM’s animal facility. This Page 2 of 9 study, was submitted and approved by the IRBM ethical committee . Mice were injected subcutaneously (sc) in the right flank with 2 × 106 SW780-shPSCA cells resus- pended into 100 μL phosphate-buffered saline (PBS) and Matrigel (1:1). Mice received 5% sucrose only or 5% sucrose plus 0.2 mg/ml of dox for control and knock- down cohorts, respectively. All water bottles were chan- ged 3 times per week. Tumors were measured with calipers and mice weighed twice per week. At the end of the dosing study, or as indicated, appropriate tumor samples were taken. Microarray experiments Total RNA from cell in culture was isolated with RNA- zolB and then dissolved in RNase-free water. 25 μg of total RNA was treated with DNase using the Qiagen RNase-free DNase kit and RNeasy spin columns. Then RNA was dissolved in RNase-free water to a final con- centration of 0.2 μg/μl. cRNA was generated using T7 RNA polymerase on 5 μg of total RNA and labeled with Cy5 or Cy3 (Cy Dye, Amersham Pharmacia Biotech). From each sample, 5 μg of labeled-RNA were co-hybri- dized with 5 μg of a reference RNA (pool of two untreated SW780 cell lines). Labeled cRNAs were frag- mented to an average size of 50-100 nucleotides by heating the samples to 60°C with 10 mM of zinc chlor- ide and then adding an hybridization buffer containing 1 M NaCl, 0.5% sodium sarcosine, 50 mM MES, pH6.5, and formamide to a final concentration of 30%. The final volume was 3 ml at 40°C. Samples were hybridized on a customized Agilent 44 k array containing ~40,000 unique probes mapping to ~21,000 human genes. Each sample was hybridized in duplicate with fluor reversal to systematically correct for dye bias. After hybridization, slides were washed and scanned using a confocal laser scanner (Agilent Tech- nologies). The raw intensities obtained after scanning were quantified, background-corrected and lowness nor- malized. A weighted average ratio was computed for dye-swapped pairs of hybridizations. Tumors were col- lected in RNA later and processed for RNA extraction as described previously [16]. Samples were hybridized on a customized Affymetrix array containing ~38,000 probes mapping to ~21,000 human genes [17]. Hierarchical clustering The microarray dataset was filtered before clustering in order to select the 2,000 most variable probes. probes with an absent call in more than 12 samples out of 14 were removed the 2,000 probes with the higher inter- quartile range were retained for subsequent analysis. Probes were hierarchically clustered using an average linkage algorithm based on Pearson correlation coefficients.