Page 150 - Cellular Imaging in Regenerative Medicine, Cancer and Osteoarthritis

Chapter 7
Radiolabeled tracers
Two SSTR2 agonists, DOTA-Tyr3-octreotate, further referred to as DOTA-TATE (BioSynthema, Saint Louis, USA) and DTPA-octreotide (Mallinckrodt Medical, Petten, The Netherlands), and the SSTR2 antagonist DOTA-JR11 (kindly provided by Dr. Helmut Maecke) were used. The tracers were radiolabeled with Indium-111 (Covidien, Zaltbommel, The Netherlands) as previously described (29, 30). Molar activity was 200-300 MBq/nmol. Radiochemical yield was >99% and radiochemical purity was >90% for each tracer.
Binding of SSTR2 tracer
The binding of the radiotracer to SSTR2 present on macrophages was established in vitro. Human monocytes were seeded in 6-well plates at a density of 5x105 cells/cm2, and subsequently cultured for 3 days with IFNγ and TNFα, as described in the previous paragraph. After 3 days of stimulation, the macrophages were washed with PBS (Gibco, Carlsbad, USA) and were incubated with 4x10-9 M [111In]In-DOTA-TATE. To determine specific binding the cells were also incubated with 4x10-9 M [111In]In-DOTA-TATE plus 10-6 M DOTA- TATE for 1 h at 37˚C. Cellular uptake of the tracer was stopped by removing the supernatant and washing twice with cold PBS. The cells were lysed with 0.1 M NaOH and the amount of radioactivity present in the samples was counted in a gamma counter (1480 WIZARD automatic gamma counter, PerkinElmer, Turku, Finland).
In vitro autoradiography was performed on human OA synovium cryosections (10 μm). The sections were incubated for 1 hour with 80 μL 10−9 M [111In]In- DOTA-JR11 with or without excess (10−6 M) unlabeled DOTA-TATE to determine non-specific binding. After incubation, the excess radiotracer was removed and the sections were exposed to super resolution phosphor screens (Packard Instruments Co., Meriden, USA) for 7 days. Binding of the radiotracers to SSTR2 containing areas in the sections was quantified using Optiquant (Packard Instruments Co., Groningen, The Netherlands) and expressed as density light units/mm2 (DLU/mm2). Sections of human H69 tumour, an SSTR2-positive xenograft, were used as positive control. All sections were stained with hematoxylin and eosin to determine the localisation of cell dense areas where the macrophages can be found.
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