Page 110 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 5
and 1% Bovine Serum Albumin fraction V pH 7.4, 10-9 M [111In]SB3 was added. The cells were incubated at 37°C in triplicates for 60 minutes, incubation was stopped by removal of the medium and rinsing with cold PBS twice. Thereafter, the cells were incubated for 10 minutes with acid wash buffer (50 mM Glycin- HCl/100 mM NaCl, pH 2.8). The supernatant was collected (membrane fraction), the cells were rinsed with acid wash buffer and collected in the same tube. Cells were lysed with 0.1 M NaOH and collected in another tube (internalized cell fraction). The amount of activity in the membrane- and cell fraction was measured on an automatic gamma counter.
Animal model
Healthy or xenografted Balb c nu/nu male mice (Janvier) were used in this study. To obtain xenografted mice, animals were injected subcutaneously in the right shoulder with PC-3, 3×106 cells in Matrigel (total volume: 150 μL, solution: 66% RPMI, 33% Matrigel) per animal. Prior to tumor cell inoculation the PC-3 cells were cultured in RPMI medium supplemented with 10% fetal bovine serum, 5,000 IU/mL penicillin and 5,000 μg/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2. PC295 is an androgen-dependent patient derived cell line with high GRPR-expression. Xenografts were generated by subcutaneous implantation of the PC295 human prostate tumor as previously described and were stored at -80°C until further use [35]. All culture supplies were purchased at Gibco.
Competition binding assay
For the competition binding assay, fresh frozen PC295 xenograft tissue slices (10 μm) were incubated in triplicate with 80 μL 5 × 10-10 M [125I]Tyr4- Bombesin, with tracer only or with an increasing concentration (10-12 to 10-6 M) of unlabeled SB3 for 1 h and exposed to super resolution phosphor screens (PerkinElmer) for 48 h. The phosphor screens were read using the Cyclone (PerkinElmer) and quantified using OptiQuant software. Binding of unblocked [125I]Tyr4-bombesin to the tissue slices was set at 100% and the percentage of binding relative to the unblocked tissue sample was calculated for the blocked tissue sections. These values were used to determine the IC50 value of unlabeled SB3.
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