(AnnArbor, MI, USA) following the recommendations of the manufacturer. Animals 6-week-old male SCID mice were obtained from Charles River (Saint Germain sur l’Arbresle, France). Surgical Orthotopic Implantation and Castration Intraprostatic prostate cancer xenografts were established in SCID mice by surgical orthotopic implantation [36]. Mice were anesthetized by isoflurane inhalation and placed in the supine position. A lower midline abdominal incision was made and 20 mL tumor cell suspension (16106 cells) was injected into the dorsal lobe of the prostate. The incision was closed in two layers with 4/0 Dexon interrupted sutures. All procedures were done with a dissecting microscope. After anesthesia by isoflurane inhalation, testes were gently pushed into the scrotum and a 5 mm incision was made under. Each testis was surgically removed, and the spermatic cord and vascular plexus were tied with sterile suture to prevent hemorrhage. In sham controls, scrotal incision alone was performed. Autopsy, Histology and In Vivo Fluorescence Imaging One week after castration, all mice were anesthetized and euthanized by cervical dislocation for direct internal imaging. A long midline incision was made to access the abdominal and the thoracic cavities. The fluorescent primary tumor was removed en bloc with the seminal vesicles and a meticulous fluorescence-assisted exploration was conducted to establish the presence of periaortic nodal extension, as well as adrenal, liver, and lung metastasis. GFP fluorescence was detected with a Leica MZFL III fluorescence stereomicroscope (Leica Microsystems, Wetzlar, Germany). High- resolution 16-bit images (1.39261.040 pixels) were captured by a thermoelectrically cooled charge-coupled device camera (Cool- SNAP HQ, Roper Scientific, Evry, France). To visualize the whole tumor and lymph nodes or micrometastases, 68 and635 magnifications were used, respectively. Selective excitation was produced with a Mercury Arc Lamp (HBO, Osram, Munich, Germany) and a GFP filter (Leica). Color images were obtained using a Micro*color tunable RGB filter (CRI, Woburn, MA). The images were processed for contrast and brightness and the fluorescence was analysed with MetaVue 6.2 software (Princeton Instruments, Trenton, NJ). The fluorescent area of the tumors was defined as a region of interest (ROI). A manual definition was used to distinguish between the fluorescent tumor area from other dark tissues. The area (a) of a ROI and the small diameter (d) were used to assess tumor volume (v) using the formula v = a6d62/3. Immunohistochemistry Sections were incubated for 4 hrs with primary rabbit anti-SphK1 antibody (1:200) and revealed by the EnVision+ peroxidase kit and References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2008) Cancer statistics, 2008. CA Cancer J Clin 58: 71–96. 2. Zincke H, Oesterling JE, Blute ML, Bergstralh EJ, Myers RP, et al. (1994) Long- term (15 years) results after radical prostatectomy for clinically localized (stage T2c or lower) prostate cancer. J Urol 152: 1850–1857. 3. Shipley WU, Thames HD, Sandler HM, Hanks GE, Zietman AL, et al. (1999) Radiation therapy for clinically localized prostate cancer: a multi-institutional pooled analysis. JAMA 281: 1598–1604. 4. Pound CR, Partin AW, Eisenberger MA, Chan DW, Pearson JD, et al. (1999) Natural history of progression after PSA elevation following radical prostatec- tomy. Jama 281: 1591–1597. 5. Graff JR, Konicek BW, McNulty AM, Wang Z, Houck K, et al. (2000) Increased AKT activity contributes to prostate cancer progression by dramatically accelerating prostate tumor growth and diminishing p27Kip1 expression. J Biol Chem 275: 24500–24505. SphK1 and Androgen Signaling diaminobenzidine (DAB) chromogen substrate (Dako, Glostrup, Denmark). In the second reaction, sections were incubated with primary anti-Chromogranin A (clone LK2H10, Ventana Medical Systems, Tucson, AZ) in a dedicated staining system (Ventana) and revealed with anti-mouse immunoglobulins and alkaline phospha- tase-conjugated streptavidin. Alkaline phosphatase activity was then revealed with Fast Blue (BB salt; 10 mg/10 ml TBS 0.1M, pH 8.2). Statistical Analysis The statistical significance of differences between the means was evaluated using the unpaired Student’s t test or the one-way analysis of variance (ANOVA) test. The frequencies of metastasis between the two groups were compared using Fisher’s exact test. All statistical tests were two-sided and the level of significance was set at P,0.05. Calculations were performed using Instat (Graphpad Software, San Diego, CA). Supporting Information Figure S1 The progression of C4-2B to androgen-refractory state is associated with increase in SphK1 expression and activity. A, Chromogranin A (CgA) content was evaluated by immuno- blotting at the indicated times. B, SphK1 activity (left panel) was determined at the indicated times in C4-2B cells incubated in CSS conditions. Points, mean of five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001. Expression of SphK1, phospho-Akt, and Akt were analyzed by Western blotting at the indicated times. (right panel). Similar results were obtained in five independent experiments. C, expression of CgA and phospho-Akt (left panel) and SphK1 activity (right panel) were analyzed 10 days after treatment or not with 1 mM LY294002 or 200 nM wortmannin (WT) under charcoal stripped conditions. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: ***, P,0.001. D, Expression of CgA and phospho- Akt were analyzed by western blot. Columns, mean of five independent experiments; bars, SD. The two-tailed P values between the means are as follow: **, P,0.01. Found at: doi:10.1371/journal.pone.0008048.s001 (0.38 MB TIF) Table S1 Found at: doi:10.1371/journal.pone.0008048.s002 (0.05 DOC) MB Author Contributions Conceived and designed the experiments: AD LB MG BM OC. Performed the experiments: AD LB CM CM NP ND LN MG BM. Analyzed the data: AD LB CM CM NP ND MG JT GS PR BM OC. Wrote the paper: OC. 6. Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ (2001) Role of PI3K signaling in survival and progression of LNCaP prostate cancer cells to the androgen refractory state. Endocrinology 142: 4795–4805. 7. Shi XB, Ma AH, Tepper CG, Xia L, Gregg JP, et al. (2004) Molecular alterations associated with LNCaP cell progression to androgen independence. Prostate 60: 257–271. 8. Pfeil K, Eder IE, Putz T, Ramoner R, Culig Z, et al. (2004) Long-term androgen-ablation causes increased resistance to PI3K/Akt pathway inhibition in prostate cancer cells. Prostate 58: 259–268. 9. Lu S, Ren C, Liu Y, Epner DE (2006) PI3K-Akt signaling is involved in the regulation of p21(WAF/CIP) expression and androgen-independent growth in prostate cancer cells. Int J Oncol 28: 245–251. 10. Paweletz CP, Charboneau L, Bichsel VE, Simone NL, Chen T, et al. (2001) Re- verse phase protein microarrays which capture disease progression show activation of pro-survival pathways at the cancer invasion front. Oncogene 20: 1981–1989. PLoS ONE | www.plosone.org 12 November 2009 | Volume 4 | Issue 11 | e8048