Cellular evaluation of receptor-binding radiotracers in a MR/PET compatible bioreactor

Objectives Three-dimensional (3D) cell and tissue culture bioreactors provide a novel platform to evaluate, in a controlled environment, cellular and tissue metabolism and function in the presence or absence of molecular stimuli or therapeutic intervention. An MR compatible bioreactor was previously developed for metabolic studies in cells and tissues using hyperpolarized 13C spectroscopy (Keshari, et al., Magn Reson Med 2010). The platform was extended to assess FDG metabolic response to treatment in prostate cancer cells (Keshari, et al., Prostate 2015). The objective of this study was to evaluate receptor-based radiotracers, in multiple cell lines simultaneously, using a MR/PET compatible bioreactor system.

Methods PC3 (PSMA-), LNCaP (PSMA++) and CWR22Rv1 (PSMA+) cells were grown to 80% confluency in DMEM. 15 million cells of each cell line were encapsulated in alginate (Keshari, 2010). Encapsulated cells were cultured in a custom-designed 5mm chamber MR/PET compatible bioreactor with a perfusion system for uninterrupted flow of 37°C medium continuously oxygenated with 95% Air/ 5% CO2. The perfusion system was set to simultaneously deliver media to four culture chambers. Dynamic PET imaging was performed on an Inveon MicroPET/CT system (Siemens). Fluorine-18 labeled fluorobenzamido(FB)-phosphoramidate ligands (FB-TG97, AH-TG97 and AH2-TG97; AH = aminohexyl) (Ganguly, et al., Nucl Med. Biol. 2015) with known high affinity for prostate specific membrane antigen (PSMA) were prepared by succinimidyl [18F]fluorobenzoate coupling to the corresponding amino-precursor. The bioreactor was perfused with medium for 10 min followed by 20 min with medium containing 5 µCi/mL of 18F-PSMA ligand then washed out for 20 min with clean medium. Blocking studies were performed by bioreactor perfusion with 15 µg/mL nonradioactive FB-AH-TG97 20 min prior to co-perfusion with 5 µCi/mL of 18F-PSMA ligand and 15 µg/mL nonradioactive FB-AH-TG97 followed by wash out with clean medium. Dynamic PET imaging data was reconstructed at 1 min intervals over the entire period of baseline, tracer wash in and wash out, for a total of 60 minutes per tracer experiment.

Results Uptake and retention, post wash out, was seen in the PSMA positive cell lines (LNCaP, CWR22Rv1) versus PSMA negative PC3 cells for all three 18F-PSMA tracers. Pre- and co-administration of non-radioactive FB-AH-TG97 in the medium with the 18F-PSMA tracers resulted in limited uptake and retention of the radiotracers confirming receptor mediated interaction with the cells. The cellular retention of 18FB-AH-TG97 was consistent with the receptor concentration. Doubling the number of CWR22Rv1 cells in the chamber resulted in two-fold increase in18FB-AH-TG97 retention. LNCaP cells retained nearly 5-fold more activity, consistent with the known 5-10 fold receptor concentration increase versus CWR22Rv1 cells.

Conclusions We have demonstrated the development of a multichamber MR/PET compatible bioreactor that may be used to perfuse radiotracers through small cell cultures. This platform was successfully applied to the assessment of PSMA binding radiotracer properties. Retention of the tracers was correlated with PSMA expression, binding and cell line receptor concentrations. The 3D MR/PET bioreactor platform may be more efficient than traditional radiotracer cell binding assays allowing the real-time monitoring of tracer uptake and retention as well as the direct comparison of tracer properties in multiple cell lines simultaneously.