All animal work was conducted in accordance with the UK Home Office, Animals (Scientific Procedures) Act 1986 under Project Licence P606320FB. Male Jax^TM apolipoprotein E-knockout mice (ApoE^−/−; B6.129P2-Apoetm1Unc/J; Charles River, UK) were used for experiments at 14 weeks of age. Male C57BL6/J mice (Charles River, UK) were used at 8 weeks of age for baseline biodistribution studies. All animals were part of the Jackson Laboratories Genetic Stability Programme to limit cumulative genetic drift. Mice were maintained at 21 °C with a 12-hour light/dark cycle and 50% to 70% humidity in GM500 individually ventilated cages (Techniplast, Italy) and fed standard chow diet and Hydropac water ad libitum. All mice were provided with a housing dome and two chew sticks as environmental enrichment.

For the AngII AAA model, 14-week-old male ApoE^−/− mice (Jackson Laboratories) received human AngII (Sigma A9525) infusions at 750 ng·kg⁻¹·min⁻¹ via Alzet^® 1002 (14 days) or 1004 (28 days) osmotic mini-pumps that were implanted subcutaneously by posterior neck incision under recovery isoflurane anaesthesia, as described previously.19 ApoE^−/− controls received saline infusions via identical pumps.

[¹⁸F]FLT was prepared from fluorine-18 fluoride produced using an on-site 7.5-MeV ABT Biomarker Generator cyclotron and purified using an in-house-developed microfluidic electrochemical cell for electrode trapping, through which irradiated target water (0.5 mL, oxygen-18 H₂O) containing ca. 1 GBq (27.03 mCi) of fluorine-18 fluoride was pumped at 0.2 mL·min⁻¹ while applying a 20 V potential, and the cell was flushed with 2 mL of MeCN at 1 mL·min⁻¹ with no potential applied. Subsequently, 0.4 mL of a solution containing KHCO₃ (30 mM) and K222 (37 mM) in MeCN was pumped through the cell at 40°C at 0.1 mL·min⁻¹. Ten mg of fluorothymidine precursor 3′-N-Boc-5′-dimethoxytrityl-3′-O-nosyl-thymidine was added to the released solution to perform a radio-labeling reaction at 100 °C for 10 minutes. After the fluorination, the unreacted fluorine-18 fluoride was trapped on the neutral alumina cartridge (light) and reaction mixture treated for 5 minutes at room temperature with an equivalent volume of 2 N HCl solution. The mixture was then neutralized with a stoichiometric amount of 8 N NaOH solution and purified by semi-preparative high-performance liquid chromatography on an ACE 5 C18 10 × 250 5A column eluted with 35% acetonitrile in water (both 0.1% trifluoroacetic acid) (flow rate= 4.7 mL·min⁻¹, R_t= 12 minutes). The high-performance liquid chromatography fraction containing partially protected product was diluted two to three-fold with water and passed over an HBL Oasis C18 cartridge. Trapped product was eluted with 0.5 mL of ethanol and 3 mL of diethyl ether and dried at 60 °C under an inert gas stream. The heat applied for drying generated the fully deprotected [¹⁸F]FLT (confirmed by analytical high-performance liquid chromatography with a cold fluorine-19 FLT standard), which was re-dissolved in 10% ethanol/phosphate-buffered saline solution, filtered through a 0.22-µm filter for sterility, and delivered for animal administration. Tracer preparation started with 1.0 to 1.3 GBq (27.03 to 35.14 mCi) of cyclotron-produced fluorine-18 fluoride and yielded 40 ± 8 MBq (1.08 ± 0.22 mCi) (n.d.c.) [¹⁸F]FLT intravenous-injectable formulation in 136 ± 15 minutes (n = 5) (RCY (decay-corrected)= 8% ± 2%).

Mice were induced with 5% isoflurane/oxygen (v/v) anaesthesia before maintenance at 2% at 1 L·min⁻¹. Mice were cannulated in the tail vein using a bespoke catheter before being placed into an imaging cell where temperature and respiration were monitored (Minerve, France). [¹⁸F]FLT was injected with the following means ± standard deviations (SDs) of activity in 200 µL of 0.9% saline solution (Aqupharm No1, Animalcare Ltd., York, UK) through the pre-cannulated lateral tail vein at the beginning of a 90-minute dynamic imaging sequence: 7.27 ± 2.89 MBq (0.20 ± 0.08 mCi) (14-day scans; n = 12) and 10.08 ± 1.77 MBq (0.27 ± 0.05 mCi) (28-day scans; n = 7). Images were acquired using the Super Argus (Sedecal) small-animal PET/CT scanner, with animals placed prone and the field-of-view centered on the abdominal aorta. Mice were maintained at 1% anaesthesia during scanning, with temperature and respiration monitored throughout. Following the PET scan, a CT image was acquired for anatomic co-registration (40 kV, 140 µA, 360 projections, 8 shots). PET images were histogrammed into 15 2-seconds, 2 15-seconds, 4 60-seconds, 1 300-seconds, and 8 600-seconds frames and reconstructed using the three-dimensional ordered subsets expectation maximization algorithm with 2 iterations and 16 subsets with attenuation correction, yielding voxel dimensions of 0.39 × 0.39 × 0.78 mm³.

Animals with failed or paravenous radiotracer injection (2/5 in the saline control group, 2/5 in the 14-day AngII AAA group, and 1/4 in the 28-day AngII AAA group) were excluded from the analysis. Three-dimensional isocontour regions of interest (ROIs) incorporating the supra-renal abdominal aortic area (between the renal arteries and diaphragm) were manually drawn on the reconstructed images using AMIDE v1.0.4 software. The aortic ROI was constructed between the kidneys and anterior to the anterior border of the vertebral column based on the CT images including the most prominent PET signal in the 80 to 90-minute timeframe. For saline controls, the aortic ROI was constructed between the kidneys, superior to the bladder, and anterior to the anterior border of the vertebral column based on the CT images. These ROIs had dimensions of 1.08 mm³ and contained 15 voxels (see Supplementary Figure 1 for example visual representation of ROIs). Incorporating mouse weights and injected radioactivity doses, the output statistics revealed the maximum and mean standardized uptake values (SUVs-) across all frames. These values were used to generate time-activity curves for ROIs in the abdominal aorta (as shown in Supplementary Figure 2), spleen, kidneys, and urinary bladder. The maximum SUV (SUV_max) was chosen as the metric of choice to quantify the aortic ROI to avoid partial volume effects of the adjacent kidneys and bladder, where high [¹⁸F]FLT signals were observed consistently.

Murine aortae were fixed in situ by perfuse fixation with 10 mL of phosphate-buffered saline followed by 5 mL of 4% paraformaldehyde in terminally anaesthetized animals and then fixed at 4 °C for 48 hours before they were embedded in Cellwax (Cellpath Ltd., UK) using a Leica EG1150H embedding station. Four-µm thickness sections were cut onto Plus Frost slides (Solmedia, UK) using a Leica RM2235 microtome. Proliferating cells were stained with rabbit anti-mouse Ki67 antibody (ab15580, Abcam; 1:750 dilution) for 1 hour and Menapath Polymer HRP secondary antibody (Menarini Diagnostics Ltd., Winnersh, UK) for 30 minutes. Slides were counterstained with haematoxylin and imaged using the Aperio^® AT2 (Leica Biosystems, Wetzlar, Germany) digital pathology slide scanner with × 20 maximal magnification. All quantitative analyses were performed using ImageJ 1.51k software.20 For quantification of the Ki67 staining, the proportion of Ki67-positive nuclei was determined in three 50-µm² ROIs and averaged for each animal.

An independent cohort of animals was used to harvest tissue at baseline and after 28-day saline, 14-day AngII, and 28-day AngII infusions. Aortic tissue (supra-renal abdominal aortic segment only) was harvested under terminal isoflurane anaesthesia, snap-frozen in liquid nitrogen after phosphate-buffered saline perfusion, and stored at − 80 °C until use. Proteins were extracted using cell extraction buffer (FNN0011, Invitrogen) supplemented with Protease Inhibitor Cocktail (P8340, Sigma). The DC Protein assay kit (5000112, Bio-Rad) was used to quantify isolated proteins. The average yield was more than 3 mg of protein per aorta. Western blotting was performed for TK-1, a key enzyme expressed during DNA synthesis and the substrate for [¹⁸F]FLT, and the specific carriers responsible for the transport of both [¹⁸F]FLT and thymidine across the cell membrane: equilibrative nucleoside transporter (ENT)-1 and -2 and concentrative nucleoside transporter (CNT)-1 and -3. For each protein, blots were conducted as per the manufacturer’s suggestions using 100 µg of protein per well. Antibodies and dilutions used were anti-TK-1, 1:500 (GTX113281; GeneTex, CA, USA); ENT-1, 1:1000 (ab135756); ENT-2, 1:1000 (ab181192); CNT-1, 1:500 (ab192438); CNT-3, 1:500 (ab223085); and β-actin, 1:1000 (Ab8226; all Abcam, Cambridge, UK). Proteins were detected using SuperSignal West Femto Maximum Sensitivity enhanced chemiluminescent substrate (ThermoFisher Scientific, Loughborough, UK). ImageJ 1.51k software was used to quantify the optical intensity of the bands relative to that of corresponding β-actin bands.20

USS was performed under 5% recovery isoflurane anaesthesia before maintenance at 2% using the Vevo2100 high-resolution (30 µm), high-frequency pre-clinical µUSS system (Visualsonics, FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) with an MS-550D transducer at a 40-MHz frequency on a heated platform maintaining a core body temperature of 35 to 37 °C. Prior to imaging, the abdomen area was shaved and hair removed using depilatory cream. Imaging was performed using Aquasonic^® clear gel (Parker Labs). Transverse imaging was acquired with a motor along an 11.96-mm ROI from the right renal artery in a cranial direction with 157 frames at 0.076-mm intervals gated for respiration with electrocardiographic triggering 50 ms after the r-wave. Images were reconstructed and measured using Vevo Lab v1.7.0 (VisualSonics, FUJIFILM VisualSonics, Inc., Toronto, ON, Canada) to generate three-dimensional lumen volume measurements. For the co-registration analysis, bony landmarks on CT and USS were aligned and displayed using ImageJ 1.51k.

Animals were anaesthetised with 5% isoflurane with an oxygen flow rate of 2 L·min⁻¹. [¹⁸F]FLT in 100 µL of 0.9% saline solution (Aqupharm No1, Animalcare Ltd., York, UK) was injected into the tail vein at the following doses (mean ± SD): C57BL6/J study, 6.07 ± 2.57 MBq (0.16 ± 0.07 mCi); AngII AAA study, 0.25 ± 0.32 MBq (0.01 ± 0.01 mCi). For the AngII AAA ex vivo gamma counting study, we reduced the injected dose to allow more animals to be included in a single experiment with a single delivery of [¹⁸F]FLT. This was possible due to the high sensitivity of the Hidex gamma counter. Animals were recovered for 90 minutes and subsequently humanely culled by cervical dislocation under Schedule 1 of the UK (Scientific Procedures) Act 1986. Samples (blood, plasma, heart, spleen, kidneys, small and large intestines, supra-renal abdominal aorta, bone, and tail) were collected, weighed, and measured for radioactivity using a gamma counter (Hidex). Importantly, blood was removed from the aortic lumen before it was placed in the gamma counter. All ex vivo biodistribution data were decay-corrected, and radioactivity counts were expressed as the percentage of injected dose per gram of tissue (%ID/g).

Following a 90-minute delay for biodistribution and gamma counting, spleen, control aorta, and 14-day AngII AAA whole-organ samples were placed on a phosphor screen (PerkinElmer) covered with clear cellophane wrap. After overnight exposure, the imaging plates were scanned using the Cyclone Plus imager (PerkinElmer) at a resolution of 300 DPI. Images were processed using OptiQuant software (PerkinElmer).

All statistical analyses were performed using Origin 2017 software (OriginLab Corporation, Northampton, MA, USA). All graphs show the mean ± the standard error of the mean (SE) unless otherwise stated. Histology, USS, and some gamma counting results were analyzed using two-sampled t tests with Welch’s correction. PET/CT, western blotting, and some gamma counting results were analyzed using one-way analysis of variance with post hoc Bonferroni-Holm correction. The threshold for statistical significance was set at P < 0.05.

As expected, ApoE^−/− mice infused with AngII developed significant supra-renal aortic lumen dilatation compared to the saline-infused controls (Figure 1A). To test for cell proliferation in AngII AAA, we stained fixed supra-renal abdominal aortic tissue from ApoE^−/− mice infused with saline or AngII for 28 days with Ki67, which accumulates in cells throughout the S, G2, and M cell cycle phases.21 A significant increase in the proportion of Ki67-positive nuclei was observed in the aortic wall in AngII-infused mice compared to saline controls (P < 0.001, Figure 1B to C), and this was positively correlated with aortic volume (Figure 1D).

Twenty-two mice were enrolled in the PET/CT study: five in the saline control group and seventeen in the AngII infusion group. Within the first 14 days of the study, ten mice died from aortic rupture in the AngII-infused group (8 thoracic, 2 abdominal), leaving twelve mice (5 saline controls and 7 AngII-infused) for imaging. A further four mice (2 saline controls and 2 AngII-infused) were excluded due to paravenous radiotracer injection. The PET/CT images obtained from the remaining 8 animals (3 saline controls and 5 AngII-infused) suggested increased [¹⁸F]FLT uptake in abdominal aortic ROIs (as shown in Supplementary Figure 1) after 14- and 28-day AngII infusions compared to saline controls. Notably, the signal was more intense after 14 days of AngII infusion, corresponding to the active growth phase in the AngII AAA model, than that after 28 days, corresponding to the end of the growth phase (Figure 2A; Supplementary Figure 3A to C, Supplementary Videos 1 to 4). It is noteworthy that repeat imaging of the AngII-infused mice on day 28 was limited to 3 animals as 1 injection was paravenous and 1 animal died from aortic rupture at the time of radiotracer injection. Splenic [¹⁸F]FLT uptake was observed consistently. Uptake was observed in the kidneys from 0.5 to 90 minutes and urinary bladder from 2 to 90 minutes after scan initiation, per its predominant renal clearance.

For quantification, SUV_max and mean SUVs (SUV_mean) were used to construct time-activity curves for 0 to 90-minute aortic uptake post-[¹⁸F]FLT administration. Uptake in the saline control aortic ROIs remained consistently low throughout each scan. Meanwhile, all time-activity curves of the AngII AAA ROIs exhibited an initial peak corresponding to the bolus injection in the vena cava, followed by a plateau and late peak in the final 10 minutes of the scan (Supplementary Figure 2, Video 2). All subsequent SUV analyses were based on tracer uptake in the 80 to 90-minute timeframe.

Quantification and statistical analysis in all manually drawn ROIs revealed the following maximum SUVs (mean ± standard error of the mean): 0.007 ± 0.002, saline control; 0.31 ± 0.03, 14-day AngII AAA; 0.20 ± 0.05, 28-day AngII AAA. Significantly greater [¹⁸F]FLT uptake was observed in 14-day (P < 0.001) and 28-day (P < 0.001) AngII AAA compared to saline controls. The signal significantly decreased between 14- and 28-day AngII AAA (P < 0.01) (Figure 2B). Similar results were observed for SUV_mean (Figure 2B).

When USS was performed just before [¹⁸F]FLT PET/CT and the images were co-registered using bony landmarks, the observed PET signal appeared to overlay the remodeled and thickened aortic wall at the site of the aneurysm (Figure 2C).

Next, using an independent cohort of mice, we harvested the aortae at baseline and after saline and 14- and 28-day AngII infusions. We then extracted protein and performed western blotting for TK-1, the [¹⁸F]FLT substrate. We observed significantly increased TK-1 expression following 14-day AngII infusion compared to controls (P < 0.001) and significantly decreased TK-1 expression between 14- and 28-day AngII infusions (P < 0.001) (Figure 2D; uncropped blots in Supplementary Figure 4A), in keeping with the pattern of [¹⁸F]FLT activity observed during the PET/CT experiments.

To further confirm that the [¹⁸F]FLT PET/CT signal was reflective of proliferation, we treated AngII AAA mice with vehicle or the anti-proliferative agent imatinib by oral gavage for 3 days after the initial PET/CT scan and then repeated the imaging. The signal in the aortic ROI diminished after imatinib treatment but was unchanged following vehicle treatment (Supplementary Figure 5).

Many [¹⁸F]FLT transporters are upregulated in cancer, aiding efficient [¹⁸F]FLT accumulation in proliferative cells.22,23 We observed significantly increased ENT-1, ENT-2, CNT-1, and CNT-3 expression in 14-day AngII AAA than in saline control aortae (P < 0.001 for all) and in 28-day AngII AAA (P < 0.001 for all). Any differences between baseline and saline control aortae were insignificant (Figure 3A to D; full uncropped blots in Supplementary Figure 4B to E).

As expected, baseline [¹⁸F]FLT biodistribution revealed high splenic uptake and low aortic and heart uptake. The aorta exhibited comparable uptake to the heart, while the spleen exhibited greater uptake relative to the heart (P = 0.69 vs P < 0.001) (Figure 4A); we therefore used the heart and spleen as negative and positive control tissues, respectively, for the AngII AAA study.

Three-dimensional USS demonstrated the expected aortic lumen volume increase following 14-day AngII infusion compared to saline-infused controls (15.34 ± 0.80 mm³ vs 11.16 ± 0.73 mm³, P < 0.01, Figure 4B). To confirm the origin of the aortic [¹⁸F]FLT signal, we performed ex vivo gamma counting of the aorta and spleen normalized to the heart following PET/CT (to control for mouse-to-mouse variability). We observed significantly increased [¹⁸F]FLT counts in aortic tissue from 14-day AngII-infused mice compared to saline controls (relative to the heart: P < 0.05, Figure 4C), consistent with our PET/CT and TK-1 western blotting data. The counts in 14-day AngII AAA were comparable to those observed in the spleen (%ID/g relative to the heart, mean ± standard error of the mean): 2.09 ± 0.32 and 2.19 ± 0.21, respectively. Ex vivo autoradiography further supported these data (Figure 4C). We observed a significant positive correlation between aortic volume and [¹⁸F]FLT uptake by gamma counting (Pearson’s r = 0.71, P < 0.01) (Figure 4D). The in vivo PET signal was also positively correlated with the ex vivo gamma counting signal when performed in the same animals (Figure 4E).