Biodistribution & PET/CT imaging

hFR-α knock-in female mice bearing TC-1-hFR-α tumours (n = 4 per group) were i.v. injected (25 µg; 15 MBq) with [¹⁸F]FB-2BD42 or [¹⁸F]FB-R3B23. Wildtype C57BL/6 female mice bearing TC-1-hFAP-α tumours were i.v. injected (25 µg; 15 MBq) with [¹⁸F]FB-4AH29 (n = 4) or [¹⁸F]FB-R3B23 (n = 3). One hour after injection, micro-PET/CT images were acquired (detailed information in SI), followed by dissections 1h10 or 1h30 post injection in mice bearing TC-1-hFR-α tumours and mice bearing TC-1-hFAP-α tumours, respectively. The timepoint discrepancies are due to differences in the preclinical study design of both tracers. Animals were dissected, and organ and tissue activities were counted against a standard of known activity with an automated gamma counter (Wizard 2 2480, PerkinElmer) and expressed as a percentage of injected activity per gram (%IA/g), corrected for decay. In vitro characterization of the tracers, affinity measurement by cell saturation assay (supplemental Fig. 2) and in vitro stability in plasma (supplemental Table 3), can be found in the SI.