Consistent with this view, a significant increase in ADCC against USPC was detected after exposure of effector cells to low doses of IL-2 in vitro for a brief time (i.e., for 5 h). significantly higher expression compared to normal endometrial cells ( 0.001). High surface expression of EpCAM was found in 83% (5 out of 6) of the USPC cell lines tested by flow cytometry. EpCAM-positive cell lines were found highly sensitive to MT201-mediated ADCC Extreme Drug Resistance (EDR) assay (Oncotech Inc. Irvine, CA) (22, data not shown). Briefly, tissue was mechanically minced to portions no larger than 1C3 mm3 in an enzyme solution made of 0.14% collagenase type I (Sigma) and 0.01% DNAse (Sigma, 2000 KU/mg) in RPMI 1640, and incubated in the same solution in a magnetic stirring apparatus for 1 hour at room temperature. Enzymatically dissociated cells were then washed twice in RPMI-1640 with 10% fetal bovine serum (FBS) and maintained in RPMI supplemented with 10% FBS, 200 g/ml penicillin and 200 g/ml streptomycin at 37C, 5% CO2 in 75-cm2 tissue culture flasks or Petri dishes (Corning, NY). 48C72 hrs after seeding on plasticware, non-adherent cells and contaminant inflammatory cells were gently removed from the culture by multiple washing with phosphate-buffered saline (PBS). The epithelial purity of the NEC and USPC cell lines was evaluated by immunocytochemical staining with antibody against pan-cytokeratin as previously described (13,14). Only cell cultures composed of at least 99% epithelial cells were retained for flow cytometry experiments EpCAM immunohistochemistry of cell blocks obtained from primary USPC cell lines cultured in vitro Cell cultures from six primary USPC cell lines were trypsinized and cells were suspended in Cytorich fixative (Richard Allen Scientific, Kalamazoo, MI), then centrifuged for 5 min at 2650 rpm. The supernatant was pipetted without disturbing the cell button. Four drops of human plasma and four drops of thromboplastin (Simplastin? Excel, Biomerieux, Durham, NC) were added to resuspend the cell button. The specimens were set MK-8745 aside until a clot formed (generally 5 minutes). The clot was then placed in a meshbag, fixed in 10% buffered formalin and processed as per routine histological technique. MK-8745 EpCAM immunohistochemical stains were performed on 5 m sections of the paraffin-embedded cell blocks. After pretreatment with 10 mM citrate buffer at pH 6.0 using a steamer, the slides were incubated with anti ESA/EpCAM MAb (Clone MOC-31) (Neomarkers/Thermo Scientific, Fremont, CA). The DAKO EnVision? kit was used for secondary detection and the reaction was visualized by DAB chromogen (DAKO, Carpinteria, CA). Rabbit polyclonal to ADAMTS3 The reactions were scored (0 to 3+) as described above. Appropriate positive and negative controls were used with each case. Flow cytometry Adecatumumab (i.e., human recombinant IgG1 MK-8745 antibody MT201, kindly provided by Micromet AG, Munich, Germany) was used for our flow cytometry and ADCC studies. Clinical grade MT201 was produced by the manufacturer in CHO cells and formulated in phosphate-buffered saline at 10 mg/mL. Briefly, six freshly established uterine serous tumor cell lines obtained from the above described patients who experienced progression on chemotherapy were stained by MT201. A FITC-conjugated goat anti-human F(ab1)2 immunoglobulin was used as a secondary MK-8745 reagent (BioSource International, Camarillo, CA). Analysis was conducted with a FACScalibur instrument using cell Quest software (Becton Dickinson). ADCC measurement A standard 5-h MK-8745 chromium (51Cr) release assay was performed to measure the cytotoxic reactivity of Ficoll-Hypaque separated peripheral blood lymphocytes (PBL) obtained from several healthy donors against all 6 USPC target cell lines. The release of 51Cr from pre-loaded target cells was measured as evidence of tumor cell lysis, after exposure of tumor cells to varying concentrations of MT201 (ranging from 0.5 g/ml to 100 g/ml). Controls included the incubation of target cells alone, with PBL, or mAb separately. The chimeric anti-CD20 IgG1 mAb rituximab (Rituxan, Genentech, CA) was used as antibody isotype control for MT201 in all bioassays. ADCC was calculated as the percentage of killing of target cells observed with mAb plus effector cells, compared to the 51Cr release from target cells incubated in the absence of mAb or effector cells..