Theranostics Targeting Fibroblast Activation Protein in the Tumor Stroma: ⁶⁴Cu- and ²²⁵Ac-Labeled FAPI-04 in Pancreatic Cancer Xenograft Mouse Models

Preparation of ⁶⁴Cu-, ⁶⁸Ga-, and ²²⁵Ac-Labeled FAPI-04 Solutions

The FAPI-04 precursor was obtained from Heidelberg University on the basis of a material transfer agreement for collaborative research. ⁶⁴CuCl₂ dissolved in 0.1 M hydrochloride was purchased from Fuji Film Toyama Chemicals. In a micro tube, the ⁶⁴CuCl₂ solution (74 MBq, 0.035 mL), 0.2 M ammonium acetate (0.47 mL), 2% sodium ascorbate (0.5 mL), and 1 mM FAP-04 (0.028 mL) were added and reacted at 80°C for 1 h. A ⁶⁸Ge–⁶⁸Ga generator was purchased from iTG Isotope Technologies Garching GmbH. ⁶⁸Ga was eluted with a solution of 0.1 M hydrochloride from the generator. In a micro tube, the ⁶⁸Ga solution (64 MBq), 2.5 M sodium acetate (0.03 mL), 10% ascorbic acid (0.02 mL), and 1 mM FAPI-04 (0.03 mL) were added and reacted at 95°C for 20 min.

²²⁵Ac was obtained by milking from its grandparent nuclide ²²⁹Th via ²²⁵Ra (13). A dry residue containing ²²⁹Th and its descendant nuclides was dissolved with 8 M HNO₃ (0.5 mL) and was loaded onto 2 connected anion-exchange columns (Muromac 1 × 8, 100–200 mesh, NO₃⁻ form, ∼1-mL column volume). Then, 8 M HNO₃ (3 mL) was loaded onto the columns to elute ²²⁵Ra and ²²⁵Ac. For only the bottom column, 8 M HNO₃ (3 mL) was additionally loaded to completely strip ²²⁵Ra and ²²⁵Ac. ²²⁹Th on the top column was separately recovered with 2 M HCl (10 mL) and distilled water (5 mL). The 8 M HNO₃ effluent was diluted to 4 M HNO₃ and loaded onto a column filled with N,N,N′,N′-tetrakis-2-ethylhexyldiglycolamide branched resin (2-mL cartridge; Eichrom). After ²²⁵Ra was eluted with 4 M HNO₃ (6 mL), ²²⁵Ac was stripped with 0.05 M HNO₃ (10 mL). After evaporation to dryness, ²²⁵Ac was dissolved in a 0.2 M ammonium acetate solution (0.2 mL). The radioactivity of ²²⁵Ac was determined from the γ-ray emissions for ²²¹Fr (218 keV) and ²¹³Bi (440 keV), which were in radioactive secular equilibrium with its parent ²²⁵Ac, using a high-purity germanium detector (BE-2020; Canberra). In a micro tube, the ²²⁵Ac solution (130 kBq, 0.2 mL), 0.2 M ammonium acetate (0.1 mL), 7% sodium ascorbate (0.1 mL), and 1 mM FAPI-04 (0.3 mL) were added and reacted at 80°C for 2 h.

Radiochemical yields for the 3 products labeled with ⁶⁴Cu, ⁶⁸Ga, and ²²⁵Ac were analyzed by cellulose acetate electrophoresis. An aliquot of each product was spotted on a strip of cellulose acetate. The voltage applied to the strip was 133 V at 1 mA/cm in a solution of 0.06 M barbital buffer (pH 8.6) for 40 min. The strip was exposed to an imaging plate, and the radioactivity on the strip was analyzed using a bioimager (Typhoon7000; GE Healthcare). The radiochemical yields of ⁶⁴Cu-FAPI-04, ⁶⁸Ga-FAPI-04, and ²²⁵Ac-FAPI-04 were 85.0%–88.5%, 95.0%, and 94.7%–96.9%, respectively.

Preparation of Xenograft Models

PANC-1 and MIA PaCa-2 cells were obtained from American Type Culture Collection. The cells were maintained in culture medium (RPMI1640 with l-glutamine and phenol red [Fujifilm Wako Pure Chemical] for PANC-1 and Dulbecco modified Eagle medium [high glucose] with l-glutamine and phenol red for MIA PaCa-2) with 10% heat-inactivated fetal bovine serum and 1% penicillin-streptomycin. Male nude mice were purchased from Japan SLC Inc.. Animals were housed under a 12-h-light/12-h-dark cycle and given free access to food and water. Tumor xenograft models were established by the subcutaneous injection of human pancreatic cancer cells (PANC-1 or MIA PaCa-2, 1 × 10⁷ cells) suspended in 0.1 mL of phosphate-buffered saline and Matrigel (1:1; BD Biosciences) in nude mice (n = 20). All animal experiments were performed in compliance with the guidelines of the Institute of Experimental Animal Sciences. The protocol was approved by the Animal Care and Use Committee of the Osaka University Graduate School of Medicine. The criteria for euthanasia were as follows: first, animal showed signs of intolerable suffering; second, a significant decrease in activity or a marked decrease in food and water intake was observed; third, the tumor size reached 3 cm in diameter; and fourth, the observation period ended (after 51 d). Euthanasia was performed by deep anesthesia by isoflurane inhalation.

⁶⁴Cu-FAPI-04 PET Imaging and Analysis

Tumor xenograft mice (9 wk old; body weight, 22.5 ± 1.2 g) were investigated using a small-animal PET scanner (Siemens Inveon PET/CT) 3 wk after the implantation of PANC-1 (n = 12) and MIA PaCa-2 (n = 8) when tumor size reached approximately 1.2 cm in diameter (14). After the intravenous injection of ⁶⁴Cu-FAPI-04 (7.21 ± 0.46 MBq), dynamic scans (scan duration, 60 min) were acquired for PANC-1 mice (n = 4) and delayed PET scans (scan duration, 20 min) were acquired 2.5 h after injection for all mice (n = 20) under isoflurane anesthesia. Sinograms were generated in multiple time-frames in the dynamic PET scan (2 min × 30 frames) and in 1 frame in the delayed PET scan. All PET data were reconstructed by 2-dimensional ordered-subset expectation maximization (16 subsets, 4 iterations) with attenuation and scatter correction. Regional uptake of radioactivity was decay-corrected to the injection time and expressed as the SUV, which was corrected for the dose (MBq) and body weight (g). Ellipsoid sphere regions of interest were manually placed on the tumor, muscle, heart, liver, intestine, kidneys, and bladder of PET images with reference to the fused PET/CT images. SUV_mean was measured to obtain time–activity curves and static uptake in the delayed scan using PMOD (version 3.6).

Comparison of Uptake Between ⁶⁴Cu-FAPI-04 and ⁶⁸Ga-FAPI-04

Static scans 1 h after the injection of ⁶⁸Ga-FAPI-04 (3.6 ± 1.4 MBq) were performed using the same cohort of xenograft mice. Uptake rates were compared between ⁶⁴Cu-FAPI-04 and ⁶⁸Ga-FAPI-04 using PANC-1 or MIA PaCa-2 xenograft mice (n = 4 each).

Immunohistochemistry

Immunohistochemical staining was performed to confirm FAP expression in the tumor xenograft using a FAP-α antibody. After the animals were sacrificed by euthanasia, all tumor xenografts were resected and fixed with 4% paraformaldehyde (overnight, 4°C). The fixed tissues were immersed in 30% sucrose in phosphate-buffered saline (overnight, 4°C). Frozen sections of the samples were then incubated with anti-FAP, α-antibody (ab53066; Abcam). Immunohistochemistry was performed using the Dako EnVision + System–HRP Labeled Polymer Anti-Rabbit (K4003) (DAKO Corp.). Staining without the primary antibody was also performed to confirm its specificity as a negative control. The stained sections were analyzed by light microscopy (Keyence).

In Vitro Cellular Uptake Analysis

Cellular uptake was analyzed to confirm that the expression of FAP was not observed in the tumor cell itself but in the stroma of the tumor xenograft. C6 glioma cells were obtained from Riken BRC and used as a negative control for the FAP expression test. PANC-1 cells, MIA PaCa-2 cells, and C6 glioma cells were seeded onto 24-well plates (1 × 10⁵ cells per well) and cultured overnight. ⁶⁴Cu-FAPI-04 solution (40 kBq/250 μL) was added to each well and incubated for 10 min. Cells were washed twice with phosphate-buffered saline and collected in solutions after they were lysed with 0.1 N NaOH. The radioactivity of the collected solution was measured by AccuFlex γ7000 (Hitachi Aloka). The amounts of proteins were measured in a plate reader (iMark; Bio-Rad) using the bicinchoninic acid protein assay kit (Fujifilm Wako Pure Chemical Corp.).

Biodistribution and Treatment Effect of ²²⁵Ac-FAPI-04

²²⁵Ac-FAPI-04 (10 kBq) was injected into PANC-1 xenograft mice (n = 6, 3 wk after implantation; tumor size, 0.81 ± 0.27 cm³) to evaluate the whole-body biodistribution. Animals were sacrificed by euthanasia at 3 and 24 h after injection, and samples of major organs were collected after dissection. For the bone and muscle, part of the rear limb was collected. For the collection of bone marrow, 0.8 mL of saline was used for flushing. The radioactivity of each sample was measured using a 2480 Wizard² γ-Counter (Perkin Elmer). Radioactivity counts were normalized by calibration using the ²²⁵Ac standard solution. Excrement in the cage was also measured to calculate the excretion rate at 3 and 24 h after injection. At 3 h after injection, lung data were not available because of a technical problem. ICR mice (7 wk old, n = 3) were used as an alternative.

For radioligand therapy using an α-emitter, ²²⁵Ac-FAPI-04 (34 kBq/100 μL) was injected into PANC-1 xenograft mice via the tail vein (n = 6, 3 wk after implantation; tumor size, 0.98 ± 0.66 cm³). Tumor size was monitored by the elliptic sphere model calculation using the caliper and compared with that of control mice for up to 51 d (n = 6; tumor size at 3 wk after implantation, 0.85 ± 0.59 cm³). The equivalent dose (Gy) in the dosimetry of ²²⁵Ac was estimated according to a previous report (15). Residence times were calculated from the tumoral uptakes at 3 and 24 h after injection, and the area under the curve after 24 h was assumed to decrease with physical decay. Energy per decay (MeV/Bq·s) of ²²⁵Ac was estimated as 28.0 from the α-particle energy and the recoil energy, including the emission from all daughter nuclides.

Statistical Analysis

Comparisons between 2 groups were performed using the Mann–Whitney U test and SPSS (version 25.0; IBM Corp.), and a P value of less than 0.05 indicated statistical significance.