Using ¹⁸F-ML-10 PET/CT imaging to detect atherosclerosis lesions and apoptotic processes in mice

Introduction

Acute myocardial infarction (AMI) represents a major global health challenge, accounting for high mortality rates and substantial disability (1). The rupture of atherosclerotic plaques represents a critical pathological event that can precipitate AMI and sudden cardiac death (2). Apoptosis, a genetically programmed cell death process, serves essential functions in both physiological homeostasis and pathological conditions (3,4). Notably, there is increasing evidence of its significant involvement in atherosclerosis progression (5,6), particularly in the development of plaque vulnerability (7).

In the initial phase of apoptosis, characteristic alterations occur at the cellular membrane level, collectively termed the apoptotic membrane imprint. These changes include the externalization of phosphatidylserine leading to membrane leaflet acidification, persistent membrane depolarization, irreversible disruption of intracellular pH homeostasis, and activation of phospholipid scrambling mechanisms (3,8). The Aposense platform comprises a series of novel small-molecule probes specifically designed to target the unique molecular signature of apoptotic cells. These compounds selectively accumulate in apoptotic cells through interactions with characteristic apoptotic features, including scramblase activation, irreversible membrane depolarization, and cellular acidification (9). 2-(5-fluoropentyl)-2-methylmalonic acid (ML-10), a low molecular weight (206 Da) Aposense compound, has been successfully radiolabeled with fluorine-18 ([¹⁸F]fluoride) for positron emission tomography (PET) imaging applications (9). In a landmark 2011 study by Höglund et al., [18F] fluoride 2-(5-fluoro-pentyl)-2-methylmalonic acid (¹⁸F-ML-10) was first administered to healthy human volunteers, demonstrating favorable pharmacokinetic properties, including excellent dosimetry, biodistribution, stability, and safety profiles, establishing its potential as a clinical PET tracer (10). Subsequently, the use of ¹⁸F-ML-10 in monitoring apoptotic activity across numerous disease models has been widely investigated. Bao et al. showed that ¹⁸F-ML-10 PET/computed tomography (CT) has potential value in predicting the radiosensitivity of nasopharyngeal carcinoma (11). Jouberton et al. showed that ¹⁸F-ML-10 PET imaging enables the visualization of apoptotic cells in triple-negative breast cancer models; however, they also found that this radiotracer could not reliably quantify the enhanced apoptotic response induced by paclitaxel chemotherapy in the murine system (12).

Cohen et al. reported that the ¹⁸F-ML-10 accumulated in cells presenting apoptosis-specific membrane alterations could be used to distinguish between apoptotic and necrotic cells (9). The non-invasive imaging of the apoptotic burden in atherosclerotic plaques represents a promising strategy for the identification of high-risk lesions. In a seminal study by Hyafil et al., ¹⁸F-ML-10 PET imaging was performed in a rabbit balloon-injury model, and radiotracer uptake was found to be strongly correlated with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL)-positive areas, a gold-standard marker of apoptosis (8). Fischer et al. showed that ¹⁸F-ML-10 could be used to detect cardiac apoptosis after transient left anterior descending ligation in mice (13). However, its performance in imaging human atherosclerosis remains unclear.

The primary objective of this study was to investigate the ability of ¹⁸F-ML-10 PET/CT imaging to detect apoptotic cells in atherosclerotic plaques in a murine model. We present this article in accordance with the ARRIVE reporting checklist (available at https://qims.amegroups.com/article/view/10.21037/qims-24-1874/rc).

Methods

Ethical statement

All the animal experiments were performed under a project license granted by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. All the experimental procedures were conducted in strict accordance with the institutional ethical guidelines and relevant national regulations for laboratory animal welfare. A protocol was prepared before the study, but it was not registered.

¹⁸F-ML-10 synthesis

¹⁸F-ML-10 was synthesized via the nucleophilic fluorination of the ML-10 tosylate precursor (Figure 1A). Briefly, [¹⁸F]fluoride (produced via cyclotron irradiation) was reacted with ML-10 tosylate dissolved in anhydrous acetonitrile (AcCN) at 90 ℃ for 15 minutes in accordance with established protocols (14). After the reaction was complete, it was first left to cool down, and a methanol solution of sodium hydroxide (3 mol/L, 0.5 mL) was then added for hydrolysis, after which it was neutralized with hydrochloric acid (1 mol/L, 3 mL).

Figure 1 Probe preparation and pharmacokinetics. (A) Synthetic scheme of ¹⁸F-ML-10. (B) Radio-high-performance liquid chromatography of ¹⁸F-ML-10. (C) Blood kinetic of ¹⁸F-ML-10. (D) Biodistributions at 30, 60, 90, and 120 minutes after the administration of ¹⁸F-ML-10 to the control mice (n=5 for each time point). %ID/g, percentage of the injected dose per gram tissue; ¹⁸F-ML-10, [18F] fluoride 2-(5-fluoro-pentyl)-2-methylmalonic acid; AcCN, acetonitrile; Et, ethyl; HCl, hydrochloric acid; M, mol/L; NaOH, sodium hydroxide; TsO, p-toluene-sulfonyl.

The crude ¹⁸F-ML-10 product was initially purified using a C-18 Sep-Pak cartridge and an aluminum oxide column, after which, it underwent semi-preparative reversed-phase high-performance liquid chromatography (Phenomenex octadecylsilane column; mobile phase: H₂O/AcCN/trifluoroacetic acid [80:20:1, v/v/v]; flow rate: 10 mL/min). The AcCN and trifluoroacetic acid were removed by rotary evaporator under reduced pressure. The ¹⁸F-ML-10 was re-dissolved with 10% ethanol aqueous solution and transferred to the product bottle.

Blood kinetics and biodistribution of ¹⁸F-ML-10

To investigate the in-vivo blood kinetics and biodistribution of ¹⁸F-ML-10, five and 15 C57BL/6J (C57) mice (male, 8 weeks) were selected, respectively, and 5.55×10⁶ Bq of ¹⁸F-ML-10 was intravenously injected via the tail vein per mouse. To investigate the blood kinetics of the probe, serial blood samples from the five mice were dynamically collected via the tail vein after injection. The blood clearance kinetics of ¹⁸F-ML-10 were determined by fitting a mono-exponential decay model using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). For the biodistribution analysis, organs from five mice per time point were collected at 3, 6, and 12 hours post-injection. The tissues were weighed, and radioactivity was measured using a γ-counter (CRC-15R, Capintec Inc., Ramsey, NJ, USA). After decay correction, the results were expressed as the percentage of the injected dose per gram of tissue (%ID/g).

Establishment of atherosclerotic animal models

Male apolipoprotein E knockout (ApoE^−/−) and wild-type C57 mice (8 weeks old) were obtained from the Laboratory Animal Center of Peking University (Beijing, China). To induce atherosclerosis, the ApoE^−/− mice were fed a high-fat diet (21% fat, 0.15% cholesterol), while the C57 control mice received standard chow (4–6% fat, 0.02% cholesterol). Following 20 or 32 weeks of dietary intervention, atherosclerotic plaque development was confirmed before the experimental procedures. All the animals were housed under specific pathogen-free conditions with ad libitum access to food and water.

Micro-PET/CT imaging and data analysis

When the mice had been fed for 20 and 32 weeks, their weight was measured and recorded. Following preparation, ¹⁸F-ML-10 (37 MBq/2 µg per mouse) was intravenously administered into the tail vein of the five ApoE^−/− and five C57 mice. After a 60-minute uptake period, anesthesia was induced with 2% isoflurane (in 100% oxygen at 1 L/min flow rate) before the imaging procedures. Next, micro-PET/CT imaging was performed using the Inveon small-animal PET/CT scanner (Siemens Preclinical Solutions, Shanghai, China). PET imaging was initially conducted using the following acquisition parameters: energy window centered at 511 keV with a 30% width, a spatial resolution of 0.775 mm/pixel, a 128×128 matrix size, and 15-minute static acquisition per mouse. Immediately following PET, the CT scans were acquired in the same bed position using the following settings: 256×512 matrix, 80 kVp tube voltage, 0.5 mA current, and 500 ms exposure time per frame. Image reconstruction was performed using two-dimensional ordered subsets expectation maximization. The PET/CT data were transferred to PMOD software (version 3.2, Zürich, Switzerland) for image analysis. Volumes of interest (VOIs) were delineated specifically over regions of the thoracic aorta that exhibited significant radiotracer uptake in each mouse. Correspondingly, background regions of interest (ROIs) of similar size were drawn in the surrounding muscle tissue to provide a reference. The abdominal aorta displayed pronounced uptake of the high-concentration renal imaging agent, which led to substantial scatter and poor visualization, making it unsuitable for meaningful quantitative analysis. Thus, we did not analyze the radiotracer uptake in the abdominal aorta. For each VOI, we quantified both the maximum standardized uptake value (SUVmax) and the percentage of the injected dose maximum (%IDmax). To enable cross-animal comparisons, plaque-to-background (P/B) ratios were calculated by normalizing the plaque signal intensity to the adjacent background tissue values.

Serological biochemical analysis

After in-vivo micro-PET/CT imaging, each mouse was anesthetized. Blood samples were collected via left ventricular puncture using heparinized 1-mL syringes. Following 20-minute coagulation at room temperature, the samples were centrifuged at 2,500 ×g for 10 minutes to obtain serum. Serum lipid profiles, including triglycerides (TGs), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and very LDL (VLDL) cholesterol, were quantified using an automated biochemistry analyzer (Falcor 350, Menarini Diagnostics, Florence, Italy) in accordance with the manufacturer’s protocols.

Ex-vivo PET imaging of aortas

After the blood sample collection and post in-vivo PET/CT imaging, cardiac perfusion with saline and 4% paraformaldehyde was performed in the mice. Next, the entire aortas were dissected, and ex-vivo PET imaging for 20 minutes was performed using the same machine and same parameters as those for the in-vivo imaging. Following overnight fixation in 4% paraformaldehyde at 4 ℃, the aortic specimens were cryoprotected in 20% sucrose solution and stored at 4 ℃ until processing.

Oil-red-O staining of aortic atherosclerotic lesions

The lipid deposition in the atherosclerotic plaques was visualized using oil-red-O histochemical staining. Briefly, the dissected aortas were longitudinally opened and immersed in 60% propylene glycol for 10 minutes at room temperature for pre-treatment. The tissues were then stained with 0.5% oil red O (in 60% propylene glycol) for 30 minutes, followed by differential washing in 60% propylene glycol (5 minutes × 3 washes) to achieve optimal background clearance. The stained aortas were carefully mounted on a black velvet background, and imaged under standardized conditions using a digital camera with consistent lighting. The images were then transferred to Image Pro Plus (version 6.0, Media Cybernetics, Washington DC, USA) to calculate the percentages of the positively stained areas

Histopathological and immunohistochemical analyses

Following paraformaldehyde fixation, the aortic specimens were paraffin-embedded and sectioned at 5 µm thickness, focusing on thoracic regions with evident atherosclerotic plaques. Consecutive sections underwent hematoxylin and eosin (H&E) staining for morphological assessment; and activated caspase-3 immunohistochemistry and TUNEL assay (using a commercial apoptosis detection kit) for apoptotic cell identification. TUNEL-positive cells were identified by characteristic brown chromogen deposition. All the stained sections were imaged under consistent conditions using an Olympus BX35 microscope with a Nikon DS-Fil digital camera system (Tokyo, Japan). The quantitative analysis included the integrated optic density (IOD) normalized to the area for caspase-3 immunoreactivity, and the apoptosis index (AI; percentage of TUNEL-positive cells per field) for the apoptotic burden assessment, which were both performed using Image Pro Plus software.

Statistical analysis

The statistical analyses were performed using SPSS 22.0 (IBM Corp., Chicago, IL, USA) and GraphPad Prism 6.0 (GraphPad Software, San Diego, CA, USA). The continuous variables are expressed as the mean ± standard deviation. To determine the statistical significance of differences between groups, the Student’s t-test was used. The correlations between the P/B ratio on ¹⁸F-ML-10 PET/CT and the AI on the immunohistochemical TUNEL images, as well as the IOD/area on the caspase-3 staining of plaques from corresponding areas were examined by Pearson correlation analysis. Statistical significance was defined as a two-tailed P value less than 0.05.

Results

Probe radiochemistry, kinetic, and biodistribution

The radiochemical yield of ¹⁸F-ML-10 was 30–40% after decay correction at the end of synthesis. After purification, the specific activity of the final product was about 40 GBq/mmol, and the radiochemical purity was greater than 99% (Figure 1B). As good stability of ¹⁸F-ML-10 had been achieved in a previous study (15), a corresponding experiment was not performed in the current study. The in-vivo kinetic analysis revealed that the probe had a mean blood half-life of 12.86 minutes (R²=0.816; Figure 1C), suggesting rapid clearance from circulation. Biodistribution studies of ¹⁸F-ML-10 in control mice (expressed as the %ID/g) showed low background tracer uptake across all organs at 30, 60, 90, and 120 minutes post-injection (Figure 1D). Notably, the tracer concentrations fell below 0.5% ID/g by 60 minutes, further supporting its efficient systemic clearance.

Establishment of animal models and evaluation

The ApoE^–/– mice had significantly higher body weights compared to the control mice at both 20 weeks (31.9±2.23 vs. 28.7±1.91 g; P<0.001) and 32 weeks (34.6±3.59 vs. 29.8±4.16 g; P<0.001) post-feeding (Figure 2A). Further, the serum lipid analysis revealed elevated TG, TC, LDL, and VLDL levels in the ApoE^–/– mice (P<0.05 for all; Figure 2B); however, the HDL levels did not differ significantly between the groups at either timepoint (20 weeks: P=0.082; 32 weeks: P=0.131). The atherosclerotic plaque burden, assessed by oil-red-O staining, was significantly greater in the ApoE^–/– mice at 20 weeks than the control mice (23.9%±11.31% vs. 1.9%±1.75%; P=0.0291) and further increased by 32 weeks (53.0%±9.69% vs. 1.6%±0.40%; P<0.001; Figure 2C,2D). These findings confirmed the successful induction of atherosclerosis in the ApoE^–/– mice, validating the model for subsequent investigations.

Figure 2 Data supporting the successful establishment of animal models of atherosclerosis. (A) Comparison of the body weight between the ApoE^–/– and control mice when fed for 20 and 32 weeks. The ApoE^–/– mice were significantly heavier than the control mice at both 20 and 32 weeks (P<0.001) after feeding. (B) Comparison of serological lipid profiles between the ApoE^–/– and control mice. (C) Typical images of oil-red-O staining of mice aortas at both 20 and 32 weeks after feeding. (D) Image analysis of oil-red-O staining showing the higher percentage of the plaque area in the ApoE^–/– mice than the control mice at both 20 weeks (23.9%±11.31% vs. 1.9%±1.75%; P=0.0291) and 32 weeks (53.0%±9.69% vs. 1.6%±0.40%; P<0.001). HDL, high-density lipoprotein; LDL, low-density lipoprotein; TC, total cholesterol; TG, triglyceride.

In-vivo micro-PET/CT displayed plaque full of apoptosis

Obvious ¹⁸F-ML-10 uptake was observed in the aortic area in the PET/CT images of the ApoE^–/– mice at both 20 and 32 weeks (Figure 3A), while no tracer uptake in the aortic area of the control group was observed. The quantitative analysis revealed significantly higher radiotracer uptake in the ApoE^–/– mice compared to the control mice, as demonstrated by all three quantitative parameters (i.e., the %IDmax, SUVmax, and P/B ratio). These differences were observed at both 20 and 32 weeks post-intervention. At 32 weeks, the ApoE^–/– mice had a significantly higher %IDmax, SUVmax, and P/B ratio than those of the control group with P values of 0.002, 0.001, and 0.001, respectively (n=5). ¹⁸F-ML-10 PET/CT imaging revealed significantly higher P/B ratios in the 32-week-old ApoE^−/− mice compared to their 20-week-old counterparts (Table 1). Specifically, the P/B ratio values of the ApoE^−/− mice were 2.28±0.20 at 32 weeks and 1.69±0.22 at 20 weeks (P=0.002, n=5). The plaque area increased significantly in the ApoE^−/− mice between 20 and 32 weeks (P=0.028), while the control mice showed no significant temporal changes (P=0.810).

Figure 3 Validation of PET/CT imaging. Sagittal PET/CT images of an ApoE^–/– mouse after feeding for 32 weeks (A) and 20 weeks (D), respectively, and a control mouse (G) scanned at 1 hour after an injection of ¹⁸F-ML-10. VOIs were drawn in the place of the thoracic aorta with obvious tracer uptake in each mouse, while background ROIs of similar sizes were drawn in the surrounding muscle tissue. (B,E,H) Show the corresponding ex-vivo PET images of the aortas excised from the mice in (A,D,G), respectively, directly after in-vivo PET/CT imaging. The ex-vivo PET imaging was performed for 20 minutes using the same machine and same parameters as those used in the in-vivo imaging. The oil-red-O staining of these aortas are presented in (C,F,I), respectively. The red rectangle in (A) denotes atherosclerotic plaques with obvious accumulation of radioactivity. (J) Comparisons of quantifications of tracer uptake between mice. The Student’s t-test was used to compare the differences between the ApoE^–/– and control groups. At 32 weeks, the ApoE^–/– mice exhibited significantly higher radioactivity, as indicated by the plaque %IDmax, the SUVmax, and the P/B ratio. Specifically, the differences were statistically significant, with P values of 0.002 for the plaque %IDmax, 0.001 for the SUVmax, and 0.001 for the P/B ratio (n=5). %IDmax, percentage of the injected dose maximum; ¹⁸F-ML-10, [18F] fluoride 2-(5-fluoro-pentyl)-2-methylmalonic acid; P/B, plaque-to-background; PET/CT, positron emission tomography/computed tomography; ROI, region of interest; SUVmax, the maximum standardized uptake value; VOI, volume of interest.

Ex-vivo aortic PET imaging, oil-red-O staining, and immunohistochemistry results corroborated the in-vivo PET/CT findings

In the ex-vivo aortic PET images, radioactivity distribution along the whole aorta of the ApoE^–/– mice at 32 weeks was observed, and was most intense in the aortic arch and thoracic aorta (Figure 3B). Notably, the ApoE^–/– mice at 20 weeks showed relatively low radiotracer accumulation in the aortic images, while the control mice exhibited no detectable tracer signal. Representative in-vivo PET/CT images, ex-vivo aortic PET scans, and corresponding oil-red-O staining of the aortic specimens from the ApoE^–/– mice at 32 weeks are presented in Figure 3A-3C, while the results of the ApoE^–/– mice at 20 weeks are shown in Figure 3D-3F. Notably, at 32 weeks, the control mice showed no detectable tracer signal in either the in-vivo or ex-vivo PET/CT imaging, with corresponding negative oil-red-O staining results (Figure 3G-3I). The quantitative comparison of the radiotracer uptake of the ApoE^–/– mice and age-matched controls at 32 weeks is summarized in Figure 3J.

To investigate the plaque characteristics, histological analyses were performed on the aortic sections corresponding to the PET/CT-positive regions. The H&E staining revealed that the radiotracer-accumulating lesions were precisely colocalized with the atherosclerotic plaque morphology (Figure 4A,4B). Further, these regions exhibited extensive cellular apoptosis as demonstrated by the TUNEL staining (Figure 4C), which was correlated with elevated caspase-3 expression (Figure 4D). Notably, high radioactivity lesions normally displayed a high amount of cell apoptosis with higher expression of caspase-3, expressed as the AI and IOD/area measured by Image Pro Plus. The Pearson correlation analysis further confirmed the significant correlations between the P/B ratio of the lesions on PET/CT and the AI on immunohistochemistry TUNEL images (r=0.950, P<0.001; Figure 4E), as well as the IOD/area on caspase-3 staining (r=0.955, P<0.001; Figure 4F).

Figure 4 Pathological and immunohistochemical confirmation of in-vivo PET/CT imaging. (A) Sagittal ¹⁸F-ML-10 PET/CT image of an ApoE^–/– mouse, with radioactivity obviously accumulated in the aortic arch and thoracic aorta, distributed along the spinal column. (B) Hematoxylin and eosin staining (×100) of a tissue section sliced at the position marked in the red rectangle in (A). TUNEL (×200, C) and caspase-3 staining (× 400, D) selected at an area corresponding to the position marked in the red rectangle in (A,E,F) respectively, show Pearson correlation analyses between plaque radioactivity expressed as the P/B ratio and the AI on TUNEL, as well as the IOD/area on caspase-3 staining, in the corresponding area measured by immunohistochemistry. ¹⁸F-ML-10, [18F] fluoride 2-(5-fluoro-pentyl)-2-methylmalonic acid; AI, apoptosis index; IOD, integrated optic density; P/B, plaque-to-background; PET/CT, positron emission tomography/computed tomography; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling.

Discussion

Thrombosis caused by the rupture of atherosclerotic plaques is a common cause of acute cardiovascular events. The question of how to accurately assess the vulnerability of plaque before its rupture is a challenge facing modern cardiovascular physicians (16,17). Apoptosis from the smooth muscle cells and the infiltrated inflammatory cells plays a pivotal role in the development and rupture of atherosclerotic plaques (6,18). Thus, apoptosis provides a unique target point for identifying vulnerable atherosclerotic plaques. The identification of apoptosis-rich plaques may enable targeted therapeutic strategies, including aggressive risk factor modification and intensive pharmacotherapy, to enhance plaque stability and mitigate rupture risk in vulnerable patients.

PET is a reliable imaging method for revealing biological processes in atherosclerotic plaques (19). ¹⁸F-fluorodeoxyglucose (¹⁸F-FDG) PET is the only clinically viable method for assessing the vulnerability of plaque (20). However, ¹⁸F-FDG is only associated with plaque inflammation and lacks specificity, rendering its use in assessing atherosclerosis challenging, especially in coronary arteries due to the high uptake of myocardium (2,20). Further, the progress of atherosclerosis is a complicated process that involves macrophage-mediated inflammation (16,20), apoptosis (6,18), hypoxia (21), neoangiogenesis (22), etc. Consequently, developing more specific molecular PET tracers is crucial, as such agents would not only advance our understanding of atherogenesis but could also transform anti-atherosclerotic therapeutic strategies.

In our study, ¹⁸F-ML-10 was synthesized as a probe for imaging atherosclerosis. This probe has a small molecule, allowing for a quick clearance rate and excellent biodistribution. In addition, the labeling process is preferable. Using this probe, the in-vivo PET/CT imaging successfully detected atherosclerotic plaques enriched with apoptotic cells. These findings were further validated by the ex-vivo aortic PET imaging, oil-red-O staining, and apoptosis-related immunohistochemical analysis (e.g., caspase-3). Collectively, our results show the potential use of ¹⁸F-ML-10 in the non-invasive diagnosis of atherosclerosis and quantitative assessment of intraplaque apoptosis—a key determinant of plaque vulnerability. Our findings are consistent with those of Hyafil et al. (8), who employed ¹⁸F-ML-10 to detect apoptotic cells in atherosclerotic plaques in a rabbit model. In their study, plaque formation was induced via a four-month high-cholesterol diet combined with double-balloon aortic injury at 2 and 6 weeks post-diet initiation. PET imaging with ¹⁸F-ML-10 showed significantly higher uptake in the atherosclerotic aortas compared to the controls (average target to background ratio: 2.00±0.52 vs. 1.22±0.30, P<0.05). The ex-vivo analysis confirmed increased ¹⁸F-ML-10 accumulation in the atherosclerotic plaques, which was correlated with the presence of apoptotic cells detected by TUNEL staining. Although we used different animal models and imaging time points, our conclusions all indicate that ¹⁸F-ML-10 can be used for the apoptosis imaging of atherosclerotic plaques.

Several non-invasive molecular imaging probes have been developed to detect apoptotic cells in atherosclerotic plaques. Among these, ^99mTc-Annexin V has shown clinical promise; Kietselaer et al. demonstrated its significantly higher uptake in the vulnerable carotid plaques of patients suffering from transient ischemic attacks (7). Another emerging tracer, ^99mTc-duramycin, exhibited superior atherosclerotic plaque targeting abilities compared to ^99mTc-Annexin V in preclinical studies; however, further validation is needed before its clinical application (23). Both these tracers were only used for single-photon emission computed tomography (SPECT)/CT imaging, which has limited imaging resolution, compared to PET/CT imaging (24). In recent years, couples of apoptosis-targeting probes have been developed for imaging atherosclerosis. However, none of these probes have been successfully applied in clinical practice. The molecular properties of ¹⁸F-ML-10, including its low molecular weight, rapid renal clearance, and short blood half-life, facilitate its efficient diffusion into atherosclerotic plaques while minimizing non-specific background retention (8). These favorable characteristics may enable its application in clinical practice as an apoptotic tracer for the PET imaging of atherosclerotic plaques.

Conclusions

The current study establishes ¹⁸F-ML-10 micro-PET/CT as a valuable tool for both identifying and quantitatively assessing atherosclerotic plaques with high apoptotic cell content.

Acknowledgments

A portion of this work was previously presented at the 2018 Annual Meeting of the Society of Nuclear Medicine and Molecular Imaging (SNMMI) and published as an abstract in the Journal of Nuclear Medicine (JNM). In accordance with the policy of JNM, the authors retain the copyright and are permitted to publish the full article in another journal following the meeting year.

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://qims.amegroups.com/article/view/10.21037/qims-24-1874/rc

Data Sharing Statement: Available at https://qims.amegroups.com/article/view/10.21037/qims-24-1874/dss

Funding: This research was supported in part by the National Natural Science Foundation of China (Nos. 81701730, 81471706, and 81671735), Shanghai Science and Technology Project (No. 17511104201), and the National Key Research and Development Program of China (No. 2022YFC2406902).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://qims.amegroups.com/article/view/10.21037/qims-24-1874/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All animal experiments were performed under a project license granted by the Institutional Animal Care and Use Committee of Zhongshan Hospital, Fudan University. All the experimental procedures were conducted in strict accordance with the institutional ethical guidelines and relevant national regulations for laboratory animal welfare.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

References

1. Martin SS, Aday AW, Allen NB, Almarzooq ZI, Anderson CAM, Arora P, et al. 2025 Heart Disease and Stroke Statistics: A Report of US and Global Data From the American Heart Association. Circulation 2025;151:e41-e660. [PubMed]
2. Joshi NV, Vesey AT, Williams MC, Shah AS, Calvert PA, Craighead FH, Yeoh SE, Wallace W, Salter D, Fletcher AM, van Beek EJ, Flapan AD, Uren NG, Behan MW, Cruden NL, Mills NL, Fox KA, Rudd JH, Dweck MR, Newby DE. 18F-fluoride positron emission tomography for identification of ruptured and high-risk coronary atherosclerotic plaques: a prospective clinical trial. Lancet 2014;383:705-13. [Crossref] [PubMed]
3. Reshef A, Shirvan A, Akselrod-Ballin A, Wall A, Ziv I. Small-molecule biomarkers for clinical PET imaging of apoptosis. J Nucl Med 2010;51:837-40. [Crossref] [PubMed]
4. Wang X, Feng H, Zhao S, Xu J, Wu X, Cui J, Zhang Y, Qin Y, Liu Z, Gao T, Gao Y, Zeng W. SPECT and PET radiopharmaceuticals for molecular imaging of apoptosis: from bench to clinic. Oncotarget 2017;8:20476-95. [Crossref] [PubMed]
5. De Saint-Hubert M, Bauwens M, Deckers N, Drummen M, Douma K, Granton P, Hendrikx G, Kusters D, Bucerius J, Reutelingsperger CP, Mottaghy FM. In vivo molecular imaging of apoptosis and necrosis in atherosclerotic plaques using microSPECT-CT and microPET-CT imaging. Mol Imaging Biol 2014;16:246-54. [Crossref] [PubMed]
6. Figg NL, Bennett MR. Quantification of Apoptosis in Mouse Atherosclerotic Lesions. Methods Mol Biol 2015;1339:191-9. [Crossref] [PubMed]
7. Kietselaer BL, Reutelingsperger CP, Heidendal GA, Daemen MJ, Mess WH, Hofstra L, Narula J. Noninvasive detection of plaque instability with use of radiolabeled annexin A5 in patients with carotid-artery atherosclerosis. N Engl J Med 2004;350:1472-3. [Crossref] [PubMed]
8. Hyafil F, Tran-Dinh A, Burg S, Leygnac S, Louedec L, Milliner M, Ben Azzouna R, Reshef A, Ben Ami M, Meilhac O, Le Guludec D. Detection of Apoptotic Cells in a Rabbit Model with Atherosclerosis-Like Lesions Using the Positron Emission Tomography Radiotracer [18F]ML-10. Mol Imaging 2015;14:433-42.
9. Cohen A, Shirvan A, Levin G, Grimberg H, Reshef A, Ziv I. From the Gla domain to a novel small-molecule detector of apoptosis. Cell Res 2009;19:625-37. [Crossref] [PubMed]
10. Höglund J, Shirvan A, Antoni G, Gustavsson SÅ, Långström B, Ringheim A, Sörensen J, Ben-Ami M, Ziv I. 18F-ML-10, a PET tracer for apoptosis: first human study. J Nucl Med 2011;52:720-5. [Crossref] [PubMed]
11. Bao X, Yang Z, Wang S, Zheng Y, Wang M, Gu B, Zhang J, Zhang Y, Zhang Y. The preclinical study of predicting radiosensitivity in human nasopharyngeal carcinoma xenografts by 18F-ML-10 animal- PET/CT imaging. Oncotarget 2016;7:20743-52. [Crossref] [PubMed]
12. Jouberton E, Schmitt S, Chautard E, Maisonial-Besset A, Roy M, Radosevic-Robin N, Chezal JM, Miot-Noirault E, Bouvet Y, Cachin F. [18F]ML-10 PET imaging fails to assess early response to neoadjuvant chemotherapy in a preclinical model of triple negative breast cancer. EJNMMI Res 2020;10:2.
13. Fischer M, Zacherl MJ, Olivier J, Lindner S, Massberg S, Bartenstein P, Grawe F, Ziegler S, Brendel M, Lehner S, Boening G, Todica A. Detection of apoptosis by [18F]ML-10 after cardiac ischemia-reperfusion injury in mice. Ann Nucl Med 2023;37:34-43.
14. Reshef A, Shirvan A, Waterhouse RN, Grimberg H, Levin G, Cohen A, Ulysse LG, Friedman G, Antoni G, Ziv I. Molecular imaging of neurovascular cell death in experimental cerebral stroke by PET. J Nucl Med 2008;49:1520-8. [Crossref] [PubMed]
15. Dewkar GK, Sundaresan G, Lamichhane N, Hirsch J, Thadigiri C, Collier T, Hartman MC, Vaidyanthan G, Zweit J. Microfluidic radiosynthesis and biodistribution of [18 F] 2-(5-fluoro-pentyl)-2-methyl malonic acid. J Labelled Comp Radiopharm 2013;56:289-94.
16. Stone GW, Maehara A, Lansky AJ, de Bruyne B, Cristea E, Mintz GS, Mehran R, McPherson J, Farhat N, Marso SP, Parise H, Templin B, White R, Zhang Z, Serruys PW. PROSPECT Investigators. A prospective natural-history study of coronary atherosclerosis. N Engl J Med 2011;364:226-35. [Crossref] [PubMed]
17. Fleg JL, Stone GW, Fayad ZA, Granada JF, Hatsukami TS, Kolodgie FD, Ohayon J, Pettigrew R, Sabatine MS, Tearney GJ, Waxman S, Domanski MJ, Srinivas PR, Narula J. Detection of high-risk atherosclerotic plaque: report of the NHLBI Working Group on current status and future directions. JACC Cardiovasc Imaging 2012;5:941-55. [Crossref] [PubMed]
18. Johnson JL, Baker AH, Oka K, Chan L, Newby AC, Jackson CL, George SJ. Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis. Circulation 2006;113:2435-44. [Crossref] [PubMed]
19. Tarkin JM, Dweck MR, Evans NR, Takx RA, Brown AJ, Tawakol A, Fayad ZA, Rudd JH. Imaging Atherosclerosis. Circ Res 2016;118:750-69. [Crossref] [PubMed]
20. Sammartino AM, Falco R, Drera A, Dondi F, Bellini P, Bertagna F, Vizzardi E. Vascular inflammation and cardiovascular disease: review about the role of PET imaging Int J Cardiovasc Imaging 2023;39:433-40. [Crossref] [PubMed]
21. Nie X, Laforest R, Elvington A, Randolph GJ, Zheng J, Voller T, Abendschein DR, Lapi SE, Woodard PK. PET/MRI of Hypoxic Atherosclerosis Using 64Cu-ATSM in a Rabbit Model. J Nucl Med 2016;57:2006-11. [Crossref] [PubMed]
22. Ng SJ, Lau HC, Naseer R, Sandhu S, Raynor WY, Werner TJ, Alavi A. Atherosclerosis Imaging: Positron Emission Tomography. PET Clin 2023;18:71-80. [Crossref] [PubMed]
23. Hu Y, Liu G, Zhang H, Li Y, Gray BD, Pak KY, Choi HS, Cheng D, Shi H. A Comparison of [99mTc]Duramycin and [99mTc]Annexin V in SPECT/CT Imaging Atherosclerotic Plaques. Mol Imaging Biol 2018;20:249-59.
24. Matter CM, Stuber M, Nahrendorf M. Imaging of the unstable plaque: how far have we got? Eur Heart J 2009;30:2566-74. [Crossref] [PubMed]