PET imaging and biodistribution studies

PET imaging was performed in the metastatic mice model at 4–5 weeks post-inoculation when the average luminescence in the metastatic areas was in the range of 10⁷-10⁸. For the subcutaneous xenograft mice model, PET imaging was performed when the tumor sized exceeded 1 cm. The FTC133 and AR42J formed 1–2 cm tumors within 2 weeks post-inoculation, while BCPAP - in 3–4 weeks and TT – in 4–5 weeks. Each mouse received a dose of ~100 μCi of each radiolabeled SST analog (⁶⁸Ga-DOTA-TATE, ⁶⁸Ga-DOTA-JR11, ⁸⁶Y-DOTA-EB-TATE) consecutively via tail vein injection. DOTA-TATE (TATE supplied by CSBio) and DOTA-JR11 (JR11 provided by IPSEN®), as agents characterized by a short half-life, were labeled with ⁶⁸Ga with a half-life of 67.7 min. DOTA-EB-TATE (EB-TATE patented by NIH and licensed by Molecular Targeting Technologies Inc.), an agent with a long half-life in the blood due to a reversible binding to albumin²⁵, required radiolabeling with ⁸⁶Y, since this isotope is characterized by a longer half-life of 14.7 h. These pharmacokinetics and pharmacodynamics formed the rationale for the particular order of consecutive injections for each mouse with ⁶⁸Ga-DOTA-TATE and ⁶⁸Ga-DOTA-JR11, injected 4 h apart (~4 half-lives apart), followed by DOTA-EB-TATE as the last SST analog in all mice (Supplemental Figure 8A). Randomization to injection of the DOTA-EB-TATE as the first agent is futile since it would necessitate performing the remaining PET/CTs after approximately 4 days (~4 half-lives). Significant tumor growth could take place during this delay, rendering the imaging results incomparable. The static PET imaging for ⁶⁸Ga-DOTA-TATE and ⁶⁸Ga-DOTA-JR11 was done at 60 min post-injection, while for ⁸⁶Y-DOTA-EB-TATE imaging was performed at 16–24 h post-injection per the previously published pharmacokinetic and pharmacodynamic studies (13, 24). The PET scans were acquired on Inveon (Siemens) scanners. The PET images were analyzed using ASIPro (Siemens) to plot regions of interest (ROI) and calculate standard uptake value (SUV). The SUV was corrected for injected radioactivity dose, decay, branching, and mouse weight. After the last scan, mice were euthanized and tumor, heart, lungs, liver, spleen, and kidneys were collected to perform biodistribution studies. The counts per minute (CPM) readings were normalized by the weight of the tissues and biodistribution data are presented as percentages of the injected dose per gram of tissue (%ID/g).