(A) A biodistribution study showing the accumulation of ⁶⁸Ga-citrate in normal mouse tissues and subcutaneous U87 MG tumors at 2, 4, and 6 hours after injection. Tumor-bearing nu/nu mice (n = 6/time point) received ~400 μCi ⁶⁸Ga-citrate via tail vein. Peak radiotracer uptake was observed in the tumors at 4 hours after injection. The data were reproduced in 2 independent animal cohorts, and the cumulative data are represented in the figure. (B) Ex vivo biodistribution data from selected tissues showing the effect on ⁶⁸Ga-citrate biodistribution due to co-administration of an anti-TFRC antibody that disrupts the interaction between Tf and TFRC. Intact male nu/nu mice bearing subcutaneous U87 MG tumors (n = 4–7/treatment arm) received ⁶⁸Ga-citrate (~400 μCi/mouse) or ⁶⁸Ga-citrate (~400 μCi/mouse) 24 hours after administration of 100 μg DF1535, a monoclonal antibody (IgG) that binds an extracellular epitope on TFRC and disrupts Tf uptake into cells in vitro. The biodistribution data were collected 4 hours after injection of ⁶⁸Ga-citrate. Approximately 50% of the ⁶⁸Ga-citrate accumulation in the tumors was suppressed by DF1513 (^#P < 0.01), underscoring that ⁶⁸Ga-citrate localizes to tumors by binding Tf in situ. Moreover, radiotracer accumulation was higher in the blood pool of mice treated with DF1513, consistent with a model in which ⁶⁸Ga-citrate exists in the blood bound to a large biomolecule (MW of Tf, ~80 kDa). Last, radiotracer uptake was competed with DF1513 in the bone (*P < 0.01). Overall, these data show that ⁶⁸Ga accumulates in tumors within 4 hours after injection in a TFRC-dependent fashion. Statistically significant differences were calculated using an unpaired, 2-tailed Student’s t test. The data were reproduced in an additional animal cohort. Horizontal lines are placed to bridge the treatment arms for which a Student’s t test was applied to determine statistical significance.