Vegfr2

Immunohistochemical (IHC) staining of HLA class I, TTK, URLC10, KOC1, VEGFR1 and VEGFR2 antigens of the ESCC and adjacent normal tissues were investigated using the serial sections of formalin-fixed, paraffin-embedded biopsy samples, as described previously [25 (link)]. The primary antibodies used in this study were as follows: HLA class I (Cosmo Bio. Co.Ltd. Tokyo, Japan), URLC10 (Imagenex, San Diego, CA.), TTK (Novus Bio. LLC. Littleton, CO.), KOC1 (Santacruz Bio. Inc. Dallas, Texas), VEGFR1 (Novus Bio. LLC.) and VEGFR2 (R&D Systems Inc., Minneapolis, MN). Immunoreaction was detected using the following secondary antibody systems: Simple Stain MAX-PO kit (Nichirei Bioscience, Tokyo, Japan) for HLA class I, URLC10, KOC1 and TTK; and CSA-II Biotin-free Tyramide Single Amplification System (Dako Inc., Carpinteria, CA) for VEGFR1 and VEGFR2, according to the instructions of the manufacturer. The intensity of staining was evaluated using the following criteria: strong positive staining of more than 80% (+++), positive staining of 50-80% (++); positive less than 50% (+); and no appreciable staining in tumor cells (-).

Fresh tissue (50 mg) was washed with saline (10% heparin), then soaked in saline for ~40 min. With ophthalmic scissors the tissue was cut into small pieces ~1 mm³ and digested with 1 ml trypsin at 37°C for 15 min, gentle agitation every 3 min during the process. Digestion was terminated with 2 ml 10% fetal calf serum (Clonetics, Cambrex, MD, USA). Large clumps of tissue and connective tissue were removed using a 200-mesh filter and the cell suspension was collected. The solution was centrifuged at low speed (1,300 × g) for 10 min, the cells were collected and the supernatant was discarded. The precipitate was washed with 1 ml phosphate-buffered saline (PBS) and resuspended in 200 μl PBS buffer containing CD34 (BD Biosciences, San Diego, CA, USA), CD133 (Miltenyi Biotec, Bergisch Gladbach, Germany), VEGFR-2 (R&D Systems Inc., Minneapolis, MN, USA) and 5 μl monoclonal fluorescent antibody. The mixture was incubated for 30 min according to the manufacturer’s recommendations (BD Biosciences). Samples were fixed in 1% formaldehyde and analyzed using a FACSCalibur flow cytometer (BD Biosciences). The control and experimental groups used the same processing methods.

Human peripheral blood mononuclear cells (PBMCs) were separated and collected by density gradient centrifugation with Histopaque-1077 (Sigma) and seeded on fibronectin (Gibco) precoated 6-well culture plates. The EGM-2 MV BulletKit (Lonza Clonetics) complete medium was used and changed every three days. The adherent cells were passaged at approximately 80% confluence after two weeks of culture. Third to fourth passage cells were used for the subsequent experiments.
For EPC identification, cells were immunofluorescently stained using antibodies against endothelial markers CD34 (Epitomics) and VEGFR-2 (R&D Systems) as well as stem/progenitor marker CD133 (Origene). Additionally, cells were identified by flow cytometry after staining with the above three antibodies. Dual staining for acetylated low density lipoprotein (Dil-AcLDL, Molecular Probes) uptake and lectin from Ulex europaeus (FITC-UEA-I, Sigma) binding was also used for EPC confirmation [20 (link)].