Oncologic Trogocytosis Figure 4. Chemoresistance induced by Hospicells. A. Induced chemoresistance when Hospicells co-cultured with OVCAR3 cells over-expressing e-GFP are treated with 22.2 mM carboplatin and/or 1.4 mM paclitaxel (* p.0.05). Fibroblasts, HBMEC and OVCAR3 cells were used as controls. B. Same assay using a transwell co-culture system. C. Reversal of chemoresistance by treating the co-culture by 50 mM verapamil. Cell density is expressed as the optical density (OD), means with standard deviations (6 replicates were used per experiments. The experiments were performed 3 times). doi:10.1371/journal.pone.0003894.g004 EOC cells can capture Hospicells’ fragments when cells are in direct contact as in EOCA. As shown on the FACS and confocal analysis the acquisition of PKH67 was not due to the diffusion of the fluorescent dye in the culture media. MDR proteins and membrane transfer Transmembrane drug transporters that extrude anti-tumor agents from the cells have an important role in the multi drug resistance mechanisms (MDR). Indeed over-expression of the ATP binding cassette transporters such as ABCB1 (MDR1) has been directly implicated in resistance to a broad spectrum of chemotherapeutic agents in vitro including paclitaxel and in some studies carboplatin [24,25]. As co-culture with Hospicells seemed to induce chemoresistance of the OVCAR3 cells we examined whether the Hospicells were expressing MDR proteins. As displayed in Figures 7-A to C, the presence of P-gp, LRP, MRP and BCRP proteins was assessed using immunofluorescence technique. The levels of MDR expression was also assessed using FACS using different Hospicells from 5 different patients. Figure 7- D displays the pattern of expression of the MDR proteins on the Hospicells isolated initially in this study. Several MDR proteins were mostly expressed on the Hospicells including the P-gp family and the Lung resistance Protein (LRP). We completed this observation by determining the functionality of the MDR proteins expressed by Hospicells by flow cytometry using different probes. Figure 7-E shows the result for P-gp using rhodamine as a probe, the ability of the cells to excrete rhodamine is observed and we also demonstrated that this ability was inhibited by the use of cyclosporine. So far we displayed the acquisition of chemoresistance through intercellular interactions between the Hospicells and the ovarian cancer cells. We also demonstrated that Hospicells expresses functional MDR proteins. Having demonstrated that OVCAR3 cells and Hospicells can exchange membranes, thus enabling OVCAR3 cells to acquire functional MDR proteins, we performed the control experiment of co-culturing OVCAR3 cells with cloned Hospicells that expressing a low level of MDR proteins. This did not lead to acquisition of MDR proteins by OVCAR3 cells (Figure S2). However after 5 hours of co-culturing OVCAR3 cells with high P-gp expressing Hospicells, 10,2% of the OVCAR3 cell population was found to have acquired a P-gp+ phenotype. This expression remained stable after 18 hours of co-culture (Figure 7F). Clinical correlation A total of 29 ovarian carcinomas were assessed histopatholog- ically for a Hospicells infiltrate within the tumor using a CD10 Figure 5. Effect of chemotherapy on freshly isolated ECOA. ECOA were isolated and cultured 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. A and C. ECOA freshly isolated in culture before treatment. B. Day 3 after treatment by carboplatin (22.2 mM). D. Day 3 after treatment by paclitaxel (1.4 mM). doi:10.1371/journal.pone.0003894.g005 PLoS ONE | www.plosone.org 6 December 2008 | Volume 3 | Issue 12 | e3894