isolated and grown at 37uC in 5% CO2/95% air in RPMI medium (Mediatech Inc.) supplemented with 10% fetal bovine serum (FBS; Mediatech Inc.) and 5% glutamine. Human ovarian cancer cells (OVCAR3), a fibroblast cell line (CCD-976SK) and human bone marrow endothelial cells (HBMEC) were obtained from American Type Culture Collection (ATCC) and were maintained as instructed. The Tissue Micro Array were constructed from surgical specimens obtained in the Institut Claudius Regaud, the patients gave a general written informed consent and the use of these specimens was authorized by the IRB of the Institut Claudius Regaud. Immunohistochemistry Stromal cells were pelleted and fixed in paraffin. Immunohis- tochemistry was performed on 4 mm-thick routinely processed paraffin sections. The following antibodies were used to charac- terize stromal cells: cytokeratin (KL1, Beckman Coulter), vimentin (V9, Beckman Coulter), CD45 (2b11 and PD7/26, Dako), CD20 (L26, Dako), CD3 (SP7, Neomarkers), CD68 (KP1 and PG-M1, Dako), CD 34 (OBend10, Dako), CD-10 (clone 56C6, Novocastra, Newcastle, UK), S100 protein (polyclonal, Dako), myeloperox- ydase (polyclonal, Dako), CD166 (polyclonal, Dako), CD146 (polyclonal, Dako) and epithelial membrane antigen (E29, Dako). The antibodies were used according to the manufacturers’ instructions. Antibodies against ABC proteins (QCRL1, QCRL3, MRP2, MRP3, MRP5, LRP, BXP21, and BXP34) and the anti-P- gp (CD243) antibody linked to phycoerythrin (PE) were provided by Immunotech (Marseille). To assess clinical relevance of the presence of Hospicells we retrospectively reviewed the correlation between their presence and chemoresistance. Using a tissue micro- array instrument (Beecher Instruments, Alphelys), we removed two representative areas of the tumor from paraffin-embedded tissue blocks that had been prepared at the time of resection. Tissue micro-arrays containing the tissue cores were then cut into 5-um sections for staining with Harris’s hematoxylin and immunohistochemical staining. 29 patients treated by neo- adjuvant chemotherapy (e.g. six cycles of carboplatine and taxol) and secondary surgery in our cancer center between January 2000 and January 2005 were included in this preliminary study. Staging was assessed according to the International Federation of Gynecology and Obstetrics (FIGO) classification. Chemosensitiv- ity was defined as achieving complete clinical response after completion of the first-line chemotherapy associated to a pathologic complete response defined as no evidence of disease after pathologic examination of all specimens at the time of secondary surgery. Chemoresistant was defined as patients with histologically confirmed residual disease at the time of secondary surgery. This is a more rigorous definition of resistance as compared with conventional clinical criteria. It has already been used by others and recognizes that absence of a pathologic complete response signifies the presence of a chemoresistant cellular population. Each specimen was examined for the Hospicells infiltrates within the tumor. The densities of these stromal infiltrates were scored independently (EM, AR) on a 0 to 10 scale combining a CD10 immunoblotting and morphological alaysis. All vascular-like structures were excluded from the evaluation. The clinical data were independently retrieved by another investigator (JC). Electron microscopy Ovarian cancer aggregates were cultured for 48 hours. Cells were subsequently washed with the PBS and fixed for 45 min in 30% formaldehyde +5% glutaraldehyde. Thereafter, the cells were centrifuged, treated with 50 mM ammonium chlorate, dehydrated Oncologic Trogocytosis and enveloped in the Epoxy resin at low temperature at polymerization conditions. The micro sections (600–800 Au) were performed and colored with uranyl acetate and lead and visualized on a Philips CM 10 electron microscope [51]. Primary Hospicells and OVCAR3 cultures were performed and analyzed to establish the microscopic characteristics of the two different cell types and identify each one of them among the aggregates. Adhesion assay Culture plates were coated with Hospicells, fibroblasts or HBMEC (Human Bone Marrow Endothelial Cells) up to 70% confluency. OVCAR3 cells overexpressing enhanced green fluorescent protein (eGFP) were added. Gentle washing after 1 hour eliminated non-adherent cells. Optical density was measured. The experiment is representative of three independent experiences. Matrigel cultures Ninety six wells plates pre-coated with matrigel (Becton- Dickinson, Le Pont de Claix, France) were used and matrigel was allowed to polymerize for 1–2 hours at 37uC. Hospicells were cultivated on Matrigel for 24 hours before eGFP-Ovcar3 were seeded onto the matrigel-coated wells. Following a 24 hours incubation periods at 37uC, intercellular interactions were assessed using an inverted microscope fitted with a digital camera (Nikon- Diafot). Chemoresistance assay Hospicells were first grown to 60% confluency in 96-well flat- bottomed tissue-culture plates. OVCAR3 cells over-expressing e- GFP were plated at a density of 20,000 cells/well and co-cultured for 24 hours with the hospicells before addition of 22.2 mM carboplatin or 1.4 mM paclitaxel [52]. The effect of these cytotoxic agents was assessed using a quantitative sulphorhodamine B (SRB) colorimetric assay, as described previously [53]. The same experiment was also performed using a transwell co-culture system in which Hospicells were cultured in the bottom chamber and OVCAR3 cells were cultured in the upper membrane. Both types of experiments were repeated after addition of 50 mM verapamil. To confirm the potential role of Hospicells we isolated aggregates from ascitis of non-previously treated patients with ovarian peritoneal carcinosis. We cultured these aggregates on RPMI media supplemented by calf fetal serum (10%) and antibiotics. Carboplatin and paclitaxel treatment were performed using the same conditions as in vitro. The effect of chemotherapy was assessed by conventional microscopy. The experiment is representative of three independent experiences. Cell transfer (‘‘oncologic trogocytosis’’) assay The assay was carried out as previously described [54]. Cancer cells or Hospicells were stained with the lipophilic dye PKH67 (Sigma-Aldrich) or with the cytoplasmic Cell Tracker Orange CMTMR (5-(and-6)-(4-chloromethyl-benzoyl-amino-tetramethyl- rhodamine)) (Molecular Probes, Eugene, Oregon, USA) according to the manufacturer’s instructions. The PKH67-labelled cells were then co-cultured for 3 hours with CMTMR-labelled cells in 96- well round-bottomed tissue-culture plates at a density of 66105 cells in 120 ml of complete RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Culture plates were centrifuged for 1 min at 700 rpm to promote cell contact and left for 1 hour at 37uC in 5% CO2. Cells were then washed twice with PBS containing 0.5 mM EDTA and analysed at two time-points (3 minutes and 3 hours) by flow cytometry using a LSRII PLoS ONE | www.plosone.org 11 December 2008 | Volume 3 | Issue 12 | e3894