Fibroblast Activation Protein (FAP) is a transmembrane protein overexpressed in cancer-associated fibroblasts within the tumor microenvironment of several solid tumors. Due to its selective expression, FAP has become an attractive molecular target, leading to the development of high-affinity inhibitors such as FAPi-46. When conjugated with DOTA, FAPi-46 can be radiolabeled with gallium-68, enabling tumor imaging by PET/CT. The development of the radiolabeling process and the evaluation of biodistribution parameters in animal models is an essential step in translational research for the successful implementation of this radiopharmaceutical in routine clinical practice.

To describe preliminary results of the radiolabeling of FAPi-46 and biodistribution in animal model of non-small cell lung cancer (NSCLC).

[68Ga]GaCl3 was eluted from generator (68Ge/68Ga) with 6mL of 0.1M HCl and trapped on an SCXcartridge. The radionuclide was subsequently eluted with 0.5mL of a 5M NaCl/5.5N HCl solution (95:5, v/v) into a reaction vial containing 50µg of FAPi-46 dissolved in 1.5mL of acetate buffer (pH 4.5), 0.1mL of ethanol and 0.1mL of ascorbic acid (10mg/mL). The reaction mixture was heated at 95°C/15min. The product was purified using Sep-Pak C18 cartridge, preconditioned with EtOH and saline, eluted with 70% ethanol, sterile-filtered through a 0.22µm filter, and finally diluted with 6mL of saline. For biodistribution studies (CEUA-6249), male Balb/c nude mice (∼25g) were allocated into three groups: healthy controls (n=5) and tumor-bearing animals evaluated at 30 and 60 min post-injection (n=6/group). Tumors were induced by (sc) inoculation of 100 µL containing 5 × 10^6 A-549 cells in matrigel into right thigh, with tumor growth observed after 4–5 weeks. Animals received an activity ranging from 4.8–6.6 MBq. At the selected time points, organs were harvested, weighed, and measured using a gamma-counter. Results were expressed as mean % injected dose per gram of tissue (%ID/g). Additionally, values were normalized to allow relative comparison among organs, assuming the sum of all measured %ID/g as 100%. Analyzed tissues included blood, heart, spleen, liver, stomach, intestines, lungs, kidneys, tumor, contralateral muscle, and carcass. Tumor-to-kidney and tumor-to-muscle uptake ratios were calculated.

In healthy controls, the highest uptake was observed in kidneys (38.61%ID/g), followed by intestines (15.11%ID/g) and stomach (13.02%ID/g). In tumor-bearing animals at 30min post-injection, tumor uptake reached 18.52%ID/g, with concurrent distribution in kidneys (28.92%ID/g), lungs (8.36%ID/g), blood (7.09%ID/g), and muscle (2.15%ID/g). At 60min, tumor uptake was 15.40%ID/g, while kidneys, intestines, blood, and muscle presented values of 15.15%ID/g, 24.38%ID/g, 4.91%ID/g, and 3.18%ID/g, respectively. Tumor-to-background ratios reflected this distribution pattern: at 30min, tumor-to-muscle and tumor-to-blood ratios were 8.56±2.32 and 2.67±1.00; at 60min, these ratios were 5.58±2.01 and 3.26±1.09.

These findings indicate that although absolute tumor uptake decreases over time, tumor-to-blood contrast improves at later time points, supporting favorable imaging properties. Overall, the data demonstrate rapid systemic clearance, sustained tumor retention, and high tumor-to-background contrast, suggesting that [68Ga]Ga-FAPi-46 is a promising PET-tracer for tumors image, including NSCLC.