cytometer and DIVA software (BD Biosciences, Mountain View, CA). Live cells were gated on the basis of forward scatter/side scatter parameters and 25,000 events were acquired in each experiment with FL1 channel (log scale) for PKH67 and FL2 channel (log scale) for CMTMR. The synaptic transfers were deducted from the PKH67 mean fluorescence of gated CMTMR- labelled cells after 3 minutes and 3 hours of co-culture. The cell transfer assay was repeated with labelled P-gp. The experiment is representative of three independent experiences. Confocal microscopy PKH67-stained hospicells and CMTMR-labelled OVCAR3 cells were processed as follows. After contact for 3 minutes or 3 hours, cells were gently resuspended and plated on poly-L-lysine (Sigma-Aldrich, St Louis, MI) coated slides for 5 minutes at 37uC. After fixation with PBS containing 4% p-formaldehyde, cells were washed and directly mounted in PBS containing 90% glycerol and 2% 1-4-diazabicyclo (2.2.2) octane (DABCO, Sigma-Aldrich, St Louis, MI). Samples were examined using an LSM 410 confocal microscope (Carl Zeiss, Jena, Germany). Fluorescence Activated Cell Sorting (FACS) To determine MDR protein expression in Hospicells, they were seeded at a density of 86104 cells/well. Two days later, the cells were serum-starved for 24 hours, fixed in 3% paraformaldehyde, and permeabilized with 0.1% Triton X-100 in PBS. The pattern of MDR protein expression was assessed by Fluorescence Activated Cell Sorting (FACS) using the Intraprep permeabiliza- tion reagent kit (Beckman-Coulter). Isotype controls were provided by Chemicon International Inc. (Temecula, CA, USA). Protein expression was analysed by flow cytometry (EPICS Altra, Beck- man Coulter). The functionality of ABC proteins expressed by Hospicells was determined by flow cytometry using rhodamine (1026 mol/L) and cyclosporine (261026 mol/L) as probes. Their effect was expressed as a shift in the mfi of dye accumulation. For each sample, 5,000 events were collected. The experiment is representative of three independent experiences. Proliferation assay 96 wells plate were seeded with 20000 Hospicells. 5000 eGFP- OVCAR3 cells were then added. Culture were performed at 37uC in 5% CO2/95% air in RPMI medium (Mediatech Inc.) supplemented with 10% fetal bovine serum (FBS; Mediatech References 1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. (2008) Cancer statistics. CA Cancer J Clin 58: 71–96. 2. Bhoola S, Hoskins WJ (2006) Diagnosis and management of epithelial ovarian cancer. Obstet Gynecol 107: 1399–1410. 3. Eisenkop SM, Spirtos NM, Lin WC (2006) ‘‘Optimal’’ cytoreduction for advanced epithelial ovarian cancer: a commentary. Gynecol Oncol 103: 329–35. 4. Pfisterer J, Ledermann JA (2006) Management of platinum-sensitive recurrent ovarian cancer. Semin Oncol 33: S12–6. 5. Di Nicolantonio F, Mercer SJ, Knight LA, Gabriel FG, Whitehouse PA, et al. (2005) Cancer cell adaptation to chemotherapy. BMC Cancer 5: 78. 6. Wang E, Ngalame Y, Panelli MC, Nguyen-Jackson H, Deavers M, et al. (2005) Peritoneal and subperitoneal stroma may facilitate regional spread of ovarian cancer. Clin Cancer Res 111: 13–22. 7. Shostak A, Chakrabarti E, Hirszel P, Maher JF (1988) Effects of histamine and its receptor antagonists on peritoneal permeability. Kidney Int 34: 786–90. 8. Muijsken MA, Heezius HJ, Verhoef J, Verbrugh HA (1991) Role of mesothelial cells in peritoneal antibacterial defence. J Clin Pathol 44: 600–4. 9. Zhang XY, Pettengell R, Nasiri N, Kalia V, Dalgleish AG, et al. (1999) Characteristics and growth patterns of human peritoneal mesothelial cells: comparison between advanced epithelial ovarian cancer and non-ovarian cancer sources. J Soc Gynecol Investig 6: 333–340. 10. Casey RC, Burleson KM, Skubitz KM, Pambuccian SE, Oegema TR, et al. (2001) Beta1-integrins regulate the formation and adhesion of ovarian carcinoma multicellular spheroids. Am J Pathol 159: 2071–80. Oncologic Trogocytosis Inc.) and 5% glutamine. Proliferation was evaluated daily by evaluating the fluorescence of each well using a fluorescent reader (Wallac). Each condition was done in triplicate. The experiment is representative of three independent experiences. Statistical Analysis Descriptive statistics were calculated for baseline demographic and clinicopathologic characteristics. Associations between CD10 immunoreactivity and clinicopathologic features were assessed by the chi-square test. For other experiments Student-t, Fisher exact or chi-square test were performed as appropriate. All p-values are two-sided with statistical significance evaluated at the 0.05 alpha level. Ninety-five percent confidence intervals (95% CI) were calculated to assess the precision of the obtained estimates. All analyses were performed in SAS Version 9.1 (SAS Institute, Inc., Cary, North Carolina) and Stata Version 8.0 (Stata Corporation, College Station, Texas). Mean6SEM are shown on the graphs. Supporting Information Figure S1 Proliferative effect of Hospicells on OVCAR3 cells. In-vitro proliferation assay. Co-culture of Hospicells and eGFP- OVCAR3 cells. 96 wells plate were seeded with 20000 Hospicells. 5000 eGFP-OVCAR3 cells were then added. Culture were performed at 37uC in 5% CO2/95% air in RPMI medium supplemented with 10% Fetal Calf Serum. Proliferation was assessed daily using a fluorescent plate reader. (Representative of 3 different experiments). Found at: doi:10.1371/journal.pone.0003894.s001 (0.32 MB TIF) Figure S2 Acquisition of Pgp by e-GFP-OVCAR3 cells when incubated with low PgP expressing Hospicells. Intercellular transfer of labelled P-gp on co-culture of Hospicells with low expression of Pgp and eGFP-OVCAR3 cells for 5 hours. As displayed there was no acquisition of PgP by eGFP-OVCAR3 cells. Found at: doi:10.1371/journal.pone.0003894.s002 (1.98 MB TIF) Author Contributions Conceived and designed the experiments: ART MP JJF JPM EPL JS. Performed the experiments: ART MM MP AMF AS ED EM RL JC JB FD MM. Analyzed the data: MP ED EM RL JC JB DQ FD JJF JS. Contributed reagents/materials/analysis tools: BC EPL GF. Wrote the paper: ART BC DQ JPM GF. 11. Gardner MJ, Jones LM, Catterall JB, Turner GA (1995) Expression of cell adherence molecules on ovarian tumour cell lines and mesothelial cells, in relation to ovarian cancer metastasis. Cancer Lett 91: 229–34. 12. Jones LM, Gardner MJ, Catterall JB, Turner GA (1995) Hyaluronic acid secreted by mesothelial cells: a natural barrier to ovarian cancer cell adherence. Clin Exp Metastasis 13: 373–80. 13. Burleson KM, Hansen LK, Skubitz AP (2004) Ovarian carcinoma spheroids disaggregate on type I collagen and invade live human mesothelial cell monolayers. Clin Exp Metastasis 21: 685–697. 14. Burleson KM, Casey RC, Skubitz KM, Pambuccian SE, Oegema TR, et al. (2004) Ovarian carcinoma ascites spheroids adhere to extracellular matrix components and mesothelial cell monolayers. Gynecol Oncol 93: 170–181. 15. Sako A, Kitayama J, Yamaguchi H, Kaisaki S, Suzuki H, et al. (2003) Vascular endothelial growth factor synthesis by human omental mesothe- lial cells is augmented by fibroblast growth factor-2: possible role of mesothelial cell on the development of peritoneal metastasis. J Surg Res 115: 113–120. 16. Wilson A (1989) Mesothelial cells stimulate the anchorage-independent growth of human ovarian tumour cells. Br J Cancer 59: 876–82. 17. Kishikawa T, Sakamoto M, Ino Y, Kubushiro K, Nozawa S, et al. (1995) Two distinct patterns of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models. Invasion Metastasis 15: 11–21. 18. Trimble EL, Wright J, Christian MC (2001) Treatment of platinum-resistant ovarian cancer. Expert Opin Pharmacother 2: 1299–306. PLoS ONE | www.plosone.org 12 December 2008 | Volume 3 | Issue 12 | e3894