Tccsup

HEK293 cells, metastatic bladder cancer cell lines (TCC-SUP and 5637) and Natural Killer (NK)-92 cells were from DSMZ (Germany). Metastatic melanoma cell line (MelC), originating from human metastatic melanoma specimens, was obtained from the Department of Therapeutic Research and Medicines Evaluation, Pharmacogenetics (ISS Rome, Italy). Metastatic colon cancer cell line (HT29) was obtained from Mario Negri Institute of Milan (Italy). Human breast cancer (MDA-MB-231) cell line was from ATCC. HEK 293 cells were cultured in DMEM medium supplemented with 10% of FBS (Sigma), 1mM L-glutamine (Carlo Erba), glucose and 1mM non-essential amino acids (Gibco). MelC and 5637 cells were cultured in RPMI 640 medium with 10% of FBS and 1mM L-glutamine. TCC-SUP cells were grown in DMEM medium with 20% of FBS and 1mM L-glutamine. NK-92 cells were cultured in α MEM medium with 10% FBS and 2mM L-glutamine and IL-2 (75 IU/ml, R&D system). HT29 cells were grown in McCoy’s medium with 10% FBS and 1mM L-glutamine. MDA-MB-231 cells were cultured in DMEM medium (Corning) with 10% of FBS (Sigma) and 1X GlutaMAX (Corning). All cells were grown at 37°C in a 5% CO₂ humidified incubator.

RT112, UMUC-3 (ATCC/LGC Promochem GmbH, Wesel, Germany) and TCCSUP (DSMZ, Braunschweig, Germany) bladder carcinoma cells were grown and subcultured in RPMI 1640, 10% fetal calf serum (FCS), 20 mM HEPES-buffer, 1% glutamax and 1% penicillin/streptomycin (all: Gibco/Invitrogen; Karlsruhe, Germany). RT112 is an invasive (pathological stage T2) moderately differentiated (grade 2/3) model of human bladder cancer, whereas TCCSUP is a transitional cell carcinoma, grade 4. UMUC-3 represents a high grade 3, invasive bladder cancer. Subcultures from passages 7–24 were used.

RT112, UMUC-3 (ATCC/LGC Promochem GmbH, Wesel, Germany) and TCCSUP (DSMZ, Braunschweig, Germany) bladder carcinoma cells were grown and subcultured in RPMI 1640, 10% fetal calf serum (FCS), 20 mM HEPES-buffer, 1% glutamax and 1% penicillin/streptomycin (all: Gibco/Invitrogen; Karlsruhe, Germany). Subcultures from passages 7–24 were selected for experimental use. Human endothelial cells (HUVEC) were isolated from human umbilical veins and harvested by enzymatic treatment with dispase (Gibco/Invitrogen). HUVEC were grown in Medium 199 (M199; Biozol, Munich, Germany), supplemented with 10% FCS, 10% pooled human serum, 20 µg/ml endothelial cell growth factor (Boehringer, Mannheim, Germany), 0.1% heparin, 100 ng/ml gentamycin and 20 mM HEPES-buffer (pH 7.4). Subcultures from passages 2–6 were selected for experimental use. HUVEC were used in the study. The institutional ethics committee of the Goethe-University Hospital, Frankfurt, Germany, approved the investigation and waived the need for consent, since HUVEC were used anonymously for in vitro assays with no link to patient data.