Imaging of Atherosclerosis with [64Cu]Cu-DOTA-TATE: a Translational Head-to-Head Comparison Study with 2-[18F]FDG, and Na[18F]F in Rabbits

doi:10.21203/rs.3.rs-2337832/v1

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Imaging of Atherosclerosis with [⁶⁴Cu]Cu-DOTA-TATE: a Translational Head-to-Head Comparison Study with 2-[¹⁸F]FDG, and Na[¹⁸F]F in Rabbits

https://doi.org/10.21203/rs.3.rs-2337832/v1

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published 07 Jun, 2023

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Atherosclerosis is a chronic inflammatory disease of the larger arteries that may lead to cardiovascular events. Identification of patients at highest risk of cardiovascular events is challenging, but molecular imaging using positron emission tomography (PET) may prove useful.

The aim of this study was to evaluate and compare head-to-head three different PET tracers. Furthermore, tracer uptake is compared to gene expression alterations of the arterial vessel wall.

Methods

Male New Zealand White rabbits (control group; n=10, atherosclerotic group; n=11) were used for the study. Vessel wall uptake was assessed with the three different PET tracers: 2-[¹⁸F]FDG (inflammation), Na[¹⁸F]F (microcalcification), and [⁶⁴Cu]Cu-DOTA-TATE (macrophages), using PET/Computed Tomography (CT). Tracer uptake was measured as standardized uptake value (SUV), and arteries from both groups were analyzed ex vivo by autoradiography, qPCR, histology, and immunohistochemistry.

Results

In rabbits, the atherosclerotic group showed significantly higher uptake of all three tracers compared to the control group 2-[¹⁸F]FDG: SUV_mean 1.50 ± 0.11 vs. 1.23 ± 0.09, p = 0.025; Na[¹⁸F]F: SUV_mean 1.54 ± 0.06 vs. 1.18 ± 0.10, p = 0.006; and [⁶⁴Cu]Cu-DOTA-TATE: SUV_mean 2.30 ± 0.27 vs. 1.65 ± 0.16; p = 0.047. Of the 102 genes analyzed, 52 were differentially expressed in the atherosclerotic group compared to the control group and several genes correlated with tracer uptake.

Conclusion

In conclusion, we demonstrated the diagnostic value of [⁶⁴Cu]Cu-DOTA-TATE and Na[¹⁸F]F for identifying atherosclerosis in rabbits. The two PET tracers provided information that could not be obtained with 2-[¹⁸F]FDG.

Health sciences/Cardiology/Cardiovascular biology/Cardiovascular diseases/Vascular diseases/Atherosclerosis

Health sciences/Medical research/Pre clinical studies

PET/CT

Molecular Imaging

Atherosclerosis

[64Cu]Cu-DOTA-TATE

Na[18F]F

2-[18F]FDG

Atherosclerosis is a chronic inflammatory disease of the arterial vessel wall, characterized by the accumulation of lipids and fibrous elements[1]. As one of the leading causes of cardiovascular morbidity and mortality, better treatments and optimized diagnostic tools are warranted.

In time, atherosclerosis may progress into vulnerable plaques and, ultimately, lead to plaque rupture[2, 3]. The vulnerable plaque is characterized by intraplaque hemorrhage, inflammatory cell infiltration, neovessel formation of the vasa vasorum, and development of a thin fibrous cap[4]. During the progression of atherosclerosis, calcification occurs. The inflammatory processes involved in the progression of atherosclerosis are essential targets for visualizing and treating atherosclerosis.

For functional characterization and treatment monitoring, non-invasive molecular imaging modalities are very useful. Molecular imaging is specialized to visualize specific cellular components or metabolic pathways. Positron emission tomography (PET) provides valuable information on different molecular processes[5]. The most commonly used PET tracer in a clinical setting is 2-[¹⁸F]FDG[6]. It accumulates in metabolically active cells such as the vascular macrophages found in the inflamed plaque. 2-[18F]FDG is useful to both quantify and track inflammation in atherosclerosis[7–11]. Unfortunately, the physiological uptake of 2-[¹⁸F]FDG by cardiomyocytes challenges the visualization of inflammation in the coronary arteries[12]. These limitations can be eliminated using other PET tracers that more specifically target inflammatory cells. The PET tracer [⁶⁴Cu]Cu-DOTA-TATE detects somatostatin receptor 2 (SSTR2) expression on the surface of activated macrophages. Hence, it has been proven useful for assessing vascular inflammation[13–15]. Another PET tracer, Na[¹⁸F]F, has been used for characterization of early vascular vessel wall microcalcifications[16–18]. It is suggested that Na[¹⁸F]F uptake can serve as a non-invasive biomarker in risk stratification of increased cardiovascular risk[19]. The three PET tracers have separately been investigated. However, a head-to-head comparison of the tracers and correlation to the underlying gene expression alterations in the vessel wall, has to the best of our knowledge not been undertaken.

In this study, we aimed to assess and characterize key molecular components of the atherosclerotic process in a rabbit model. Three PET tracers were used for targeting inflammation and microcalcification in the arteries. Ultimately, the PET uptakes were compared to gene expression alterations in the arterial vessel wall.

All animal experiments were performed in accordance with appropriate approvals from the Animal Research Committee of the Danish Ministry of Justice (license number: 2016-15-0201-00831) and the study was conducted by following the ARRIVE and AVMA guidelines. All methods were performed in accordance with the relevant guidelines and regulations.

Rabbits

Twenty-one male SPF New Zealand White rabbits (11 weeks old) were randomized into a control group (n = 10) and an atherosclerotic group (n = 11) (KB Lidköping Kaninfarm, Sweden). The rabbits were single housed. Atherosclerosis was induced by a combination of diet and denudation of the abdominal and thoracic aorta. The atherosclerotic group were fed a high cholesterol diet (HCD) containing 0.30% cholesterol, two weeks prior to the first surgery. After eight weeks and the remining study period, rabbits were shifted to 0.15% HCD. The control group was fed a normal chow diet (see study outline Fig. 1).

Under aseptic conditions, the atherosclerotic group underwent denudation by firstly localizing the femoral artery in one of the extremities. Then introducing a 4F-Fogarty embolectomy catheter into the artery before advancing the balloon approximately 20 cm. A repetitive action was performed by inflating the balloon and retracting it three times to ensure vascular injury. Finally, the muscle layers and skin were closed with absorbable and non-absorbable sutures, respectively. The procedure was repeated after three weeks on the contralateral extremity. Infection prophylaxis enrofloxacin (15mg/kg) was injected intramuscularly before surgery.

Post-operative care consisted of the analgesic buprenorphine (0.01–0.05 mg/kg, subcutaneous injection (s.c.)) in combination with ketoprofen (3mg/kg, intramuscular injection (i.m.)), administered on the day of surgery.

Euthanization of the rabbits was an initial sedation by an injection of ketamine (35mg/kg) and xylazine (5mg/kg) i.m. followed by a lethal injection of penobarbital (140mg/kg) i.v. following the AVMA guidelines for the Euthanasia of Animals.

Small animal PET imaging and image analysis

All the rabbits (n = 21) were baseline scanned on a dedicated small animal PET/Computed Tomography (CT) system (Inveon, Siemens Medical Systems, PA, USA) with the tracer 2-[¹⁸F]FDG after which the rabbits were randomized into the two groups. Scans were performed as a one-bed position scan covering the abdominal aorta. The scans were performed 3 weeks prior to surgery of the atherosclerotic group and twenty weeks post-surgery, terminal scans were performed on both groups with different tracers: i) 2-[¹⁸F]FDG, ii) Na[¹⁸F]F, and iii) [⁶⁴Cu]Cu-DOTA-TATE. All three tracers circulated for 60 minutes prior to PET imaging with an acquisition time of 1,200 sec. The radioactive dose for each tracer was: 123.4 ± 15.6 MBq (2-[¹⁸F]FDG), 116.0 ± 10.2 MBq (Na[¹⁸F]F), and 84.41 ± 5.78 MBq ([⁶⁴Cu]Cu-DOTA-TATE). The 3 scans were performed in a randomized order to ensure ex vivo autoradiography of all 3 tracers for both groups. To allow physical decay of the tracers, a gap of at least 24 hours following tracer injection with ¹⁸F-labeled compounds (physical half-life of ¹⁸F: 110 min) and 72 hours following [⁶⁴Cu]Cu-DOTA-TATE injection (physical half-life of ⁶⁴Cu: 12.7 h) was ensured.

Images were analyzed as fused PET/CT images using the Inveon Research Workspace 4.2 software (Siemens Medical Systems, PA, USA) by drawing Regions of Interests (ROIs) on the abdominal aorta, from the iliac bifurcation to the right renal artery. A volume sphere was created from all the ROIs drawn, volume of interest (VOI). The mean Standardized Uptake Values (SUV_mean) were calculated as the tracer uptake in the VOI divided by decay corrected injected activity and divided by the weight of the rabbit. Results are reported as the average SUV for the whole aortic segment (SUV_mean) for best comparison to gene expression results.

Ex vivo analysis

Immediately following the last scan, rabbits were euthanized, and segments of the aorta were selected for specific ex vivo analysis. Autoradiography was performed for all three tracers with the aortic arch and half of the descending thoracic aorta (n = 4 for each tracer). The thoracic part of the aorta was chosen for autoradiography, because the tissue gets squeezed during exposure in the cassette, and we wanted to preserve the morphology in the abdominal part where PET/CT imaging was performed.

The thoracic part, part of the abdominal aorta, and the aortic arch were fixed in 4% formalin for 24 hours before being sectioned into 2-3mm pieces and embedded in paraffin. A series of axial sections (4µm thickness) were obtained and selected for histology and immunohistochemistry (IHC).

For general morphological characterization, a Hematoxylin & Eosin (H&E) stain was performed and a Von Kossa stain for calcifications.

For IHC, RAM 11 (Monoclonal Mouse Anti-Rabbit Macrophage, Clone RAM11, Agilent DAKO, USA) was used for assessing macrophage infiltration. Envision FLEX DAB + Substrate Chromogen System (Agilent, DAKO, USA) was used for exposing immunoreactivity together with hematoxylin as counterstain.

Real-time Reverse Transcription - Polymerase Chain Reaction

All reagents and kits were purchased from QIAGEN (Hilden, Germany). Total RNA was extracted with TRIzol® Reagent and reverse-transcribed into cDNA using the RT² First Strand Kit. Two different arrays were used: Rabbit wound healing array (QIAGEN, PANZ-121ZA-RT² Profiler PCR Array) and a custom array (QIAGEN, CLAN32799A – Custom RT² PCR Array). Plates were read on the Mx3000P real-time PCR system (Stratagene, CA, USA). The results were analyzed using the online software GeneGlobe (QIAGEN). Gene expression levels of genes-of-interest (GOI) were normalized to the level of the reference genes ACTA2, ACTB, GAPDH, LDHA, and non-POU domain containing octamer-binding-like (LOC100346936). The data was analyzed using the 2-deltaCt method.

Statistical analysis

Statistical analysis of the in vivo data was obtained in GraphPad Prism version 8 (GraphPad Software Inc., USA).

An unpaired two-tailed t-test was performed to compare the tracer uptake between groups at the different time points and compare fold regulation of genes. Correlation between gene expression levels were analyzed using, a Pearson’s correlation. P-values less than 0.05 were considered significant.

All data are presented as mean ± Standard Error of the Mean (SEM).

Data Availability

The datasets generated and analyzed during the current study are available in the GEO repository, https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE220754.

The characteristic differences between the atherosclerotic and control groups were assessed by molecular imaging, histology, IHC, and gene expression analyses.

First, to confirm that atherosclerosis developed in the model, 2-[¹⁸F]FDG scans were performed at baseline and endpoint 20 weeks following the last denudation of the atherosclerotic group.

At baseline, 2-[¹⁸F]FDG PET/CT scans revealed no difference in uptake between the control group and the atherosclerotic group (SUV_mean 1.13 ± 0.07 vs SUV_mean 1.04 ± 0.088; p = 0.9). While 2-[¹⁸F]FDG significantly increased from baseline to termination in the atherosclerotic group (p = 0.005), only a small and non-significant increase was observed in the age-matched control group during the 20 weeks study period (p = 0.389). General characteristics for the atherosclerotic development in the arteries were established by histology (Fig. 2C).

Assessment of metabolic activity in plaque through 2-[ ¹⁸ F]FDG PET imaging

At termination of the study, we observed a significantly higher uptake of 2-[¹⁸F]FDG in the atherosclerotic group compared to the control group (SUV_mean 1.50 ± 0.112 vs.1.23 ± 0.089; p = 0.025; Fig. 2A). Higher accumulation of 2-[¹⁸F]FDG was also observed in the aortic arch of the atherosclerotic group by autoradiography (Fig. 2B). Accumulation of foam cells, cholesterol crystals, and narrowed lumens were found in the atherosclerotic arteries by H&E. The control group showed a thin and intact arterial wall (Fig. 2C).

Assessment of microcalcification through Na[ ¹⁸ F]F PET imaging

For assessment of microcalcifications within the arterial vessel wall, rabbits were scanned with Na[¹⁸F]F PET/CT. A significant difference in uptake was observed between the atherosclerotic group and the control group (SUV_mean 1.54 ± 0.057 vs SUV_mean 1.18 ± 0.099; p = 0.006; Fig. 3A). A heterogeneous and high accumulation of Na[¹⁸F]F was observed in the atherosclerotic group by autoradiography (Fig. 3B), confirming the PET findings. The presence and location of microcalcifications within the plaques are also shown by the Von Kossa staining (Fig. 3C).

Assessment of inflammation through [ ⁶⁴ Cu]Cu-DOTA-TATE PET imaging

Arterial inflammation was investigated by [⁶⁴Cu]Cu-DOTA-TATE. The [⁶⁴Cu]Cu-DOTA-TATE uptake was significantly higher for the atherosclerotic group compared to the control group (SUV_mean 2.30 ± 0.266 vs SUV_mean 1.65 ± 0.157; p = 0.047, Fig. 4A). Accumulation of [⁶⁴Cu]Cu-DOTA-TATE in the arteries of the atherosclerotic group was confirmed by autoradiography (Fig. 4B). Abundant macrophage infiltration within the plaques was evident from the RAM11 stain. For the control arteries no presence of macrophages was observed (Fig. 4C) confirming the low uptake observed on PET and autoradiography.

Transcriptome analysis and correlation with imaging results

Clustergram analysis separated the atherosclerotic rabbits from the control rabbits and identified two clusters with differentially expressed genes (Fig. 5). Of the 102 GOIs analyzed, 34 GOIs were significantly upregulated in the atherosclerotic group compared to the control group, and 18 GOIs were significantly downregulated in the atherosclerotic group compared to the control group. Generally, genes associated with cell surface receptors, extracellular matrix remodeling, and inflammatory cytokines and chemokines were upregulated, whereas the downregulated genes were associated with cell adhesion, and vessel wall atherogenic effects (GeneGlobe, QIAGEN). The most differentially expressed genes were considered in the analysis of correlation with the three PET tracers, irrespectively of the two groups (Table 1).

Table 1

**Correlation between GOIs and PET tracer.** Correlation coefficient for each GOI and the three PET tracers, with corresponding p-values. CTSK: Cathepsin K, GRB2: Growth Factor receptor-bound protein 2, IL-2: Interleukin 2, IL-10: interleukin 10, ITGAL: Integrin Alpha L, SCARF1: Scavenger receptor class f member 1, TGFB3: Transforming growth factor, beta 3, TIAM1: T-cell lymphoma invasion and metastasis 1, TLR4: Toll-like receptor 4, TNF: Tumor Necrosis Factor
	2-[¹⁸F]FDG		Na[¹⁸F]F		[⁶⁴Cu]Cu-DOTA-TATE
	Correlation Coefficient	p-value	Correlation Coefficient	p-value	Correlation Coefficient	p-value
CD36			0.71	0.0003
CD86			0.55	0.0094	0.49	0.0236
CD163			0.45	0.0033
CTSK			0.49	0.0409	0.50	0.0332
GRB2	0.60	0.008	0.66	0.0029	0.55	0.0174
IL2					-0.53	0.0235
IL10					0.54	0.0212
ITGAL			0.54	0.0113	0.47	0.0321
Transgelin			-0.58	0.0121	-0.65	0.0035
SCARF1			0.43	0.0498	0.45	0.0397
TGFB3			-0.72	0.0007	-0.56	0.0166
TIAM1			0.55	0.0183	0.56	0.0168
TNF	0.48	0.0446	0.48	0.0453

A few of the analyzed genes correlated with the uptake of 2-[¹⁸F]FDG. However, most of the correlations were found between Na[¹⁸F]F and [⁶⁴Cu]Cu-DOTA-TATE (Table 1).

Cathepsin K (CTSK) showed a 5.40 fold upregulation (p < 0.00001) in the atherosclerotic group compared to the control group together with positive correlations with both Na[¹⁸F]F (r = 0.49, p = 0.0409) and [⁶⁴Cu]Cu-DOTA-TATE (r = 0.50, p = 0.0332) (see Table 1). The same trend was seen with the macrophage marker, CD86, displaying a 78-fold upregulation in the atherosclerotic group compared to the control group (p < 0.00001) (see Table 2). A 235-fold upregulation of integrin alpha L (ITGAL) was found in the atherosclerotic group compared to the control group as well as a positive correlation with Na[¹⁸F]F (r = 0.54, p = 0.0113) and [⁶⁴Cu]Cu-DOTA-TATE (r = 0.47, p = 0.0321).

Table 2

**Fold Upregulation in Atherosclerotic group compared to Control group.** CTSK: Cathepsin K, ITGAL: Integrin Alpha L, SCARF1: Scavenger receptor class f member 1, TIAM1: T-cell lymphoma invasion and metastasis 1. TNF: Tumor Necrosis Factor.
	Fold Upregulation	p-value
CD163	4.48	0.00218
CD36	13.70	< 0.00001
CD86	78.00	< 0.00001
CTSK	5.40	< 0.00001
ITGAL	235.00	< 0.00001
SCARF1	5.60	< 0.00001
TIAM1	29.90	< 0.00001
TNF	4.06	0.00251

In this study, we demonstrated the usefulness of [⁶⁴Cu]Cu-DOTA-TATE for assessing atherosclerosis in a rabbit model. We showed by PET/CT and autoradiography that [⁶⁴Cu]Cu-DOTA-TATE, 2-[¹⁸F]FDG, and Na[¹⁸F]F accumulate in atherosclerotic plaques. Our imaging results support our findings of pronounced morphological alterations in the arterial vessel wall and the upregulation of pro-atherogenic molecular markers in the atherosclerotic arteries.

2-[¹⁸F]FDG has been the common tracer used for PET imaging of atherosclerosis due to its ability to visualize metabolic activity in cells[14]. 2-[¹⁸F]FDG uptake is associated with macrophage differentiation, cell activation and cellular glucose metabolism[5, 20, 21]. However, due to high uptake of 2-[¹⁸F]FDG in the myocardial tissue and thus spillover, the usefulness of the tracer for investigating atherosclerosis in the coronary arteries is limited. As previously mentioned, [⁶⁴Cu]Cu-DOTA-TATE detects expression of SSTR2 on the surface of activated macrophages, which provides a more specific marker for inflammation, which can be utilized in atherosclerosis imaging. In the current study, we assessed tracer accumulation in the abdominal aorta, where spill-over effects are not hampering the quantification the same way as in the coronary arteries. Our results confirm, that the [⁶⁴Cu]Cu-DOTA-TATE tracer is as least as good as the [¹⁸F]FDG tracer for assessing atherosclerosis.

To confirm the development of atherosclerosis over time we used [¹⁸F]FDG PET/CT at baseline in addition to the terminal scans. We found a significant increase in 2-[¹⁸F]FDG uptake in the atherosclerotic group from baseline to last scan, while no significant increase was seen in the age-matched control rabbits. Thus, confirmed by non-invasive imaging with [¹⁸F]FDG PET/CT, that vascular inflammation was only detectable at advanced age following the dietary and surgical intervention and did not develop spontaneously in the rabbits.

A few clinical studies have reported successful use of [⁶⁸Ga]Ga-DOTA-TATE in assessing the atherosclerotic burden in patients[14, 22, 23]. In the present study, we used [⁶⁴Cu]Cu-DOTA-TATE, which binds to the same target but due to a four-fold lower positron range of [⁶⁴Cu] compared to [⁶⁸Ga] (≈1 mm vs. 4 mm), [⁶⁴Cu] provides a much better spatial resolution of particular value when evaluating thin structures as the vessel wall. Collectively, studies using DOTA-TATE PET tracers all found the tracer uptake to be correlated with the presence of calcifications and with macrophage markers[14, 15, 22].

Apart from inflammation, other processes contribute to the pathogenesis of atherosclerosis. Following plaque inflammation, osteogenic differentiation gives rise to microcalcification development driven by mineral metabolic activity. Such microcalcification may indicate early and more unstable stages of atherosclerosis, which could affect a potential plaque rupture caused by destabilization of the fibrous cap surrounding the plaque[24]. Calcifications can be detected by CT at later stages. Conversely, studies have indicated, that Na[¹⁸F]F PET can detect microcalcifications, thus identify the disease at an earlier stage. Late stage calcification is, however, inversely correlated with Na[¹⁸F]F-uptake[25]. The uptake of Na[¹⁸F]F in early microcalcifications is correlated to the healing response of atherosclerotic plaques. Thus Na[¹⁸F]F is associated with inflammation and high-risk plaque[26]. In this study, the rabbits with atherosclerosis showed high uptake of Na[¹⁸F]F in the arteries, possibly indicating early-stage metabolic active mineralization. Figure 2A shows particular lesions with higher uptake of Na[¹⁸F]F, which confirm the presence of microcalcifications and atherosclerosis, while no calcifications were evident on the CT scans of the atherosclerotic rabbits.

We validated the PET/CT findings with histology and IHC. The stains confirmed the infiltration of macrophages surrounding the foam cells and cholesterol crystals, and the presence of microcalcifications. Autoradiographic findings from the thoracic aorta were also confirmed by histology and IHC, and no pronounced difference in atherosclerotic burden was found between the abdominal and thoracic part of the aorta.

Comparing the gene expression of atherosclerotic arteries to control arteries revealed upregulated genes associated with atherosclerosis through various pathways. The most upregulated genes were chosen for a correlation analysis with the three tracers used in this study. Interestingly, only a few of the genes correlated with 2-[¹⁸F]FDG, whereas several genes correlated with [⁶⁴Cu]-Cu-DOTA-TATE and Na[¹⁸F]F accumulation. Previously, CTSK have been found to be associated with FDG-uptake in carotid plaque in humans and not associated with [⁶⁴Cu]-Cu-DOTA-TATE uptake[27]. Contrarily, our findings show a positive correlation between CTSK and [⁶⁴Cu]-Cu-DOTA-TATE and Na[¹⁸F]F, yet no correlation with 2-[¹⁸F]FDG. We confirmed that [⁶⁴Cu]-Cu-DOTA-TATE correlates to macrophage infiltration with a positive correlation with CD86. Surprisingly [⁶⁴Cu]-Cu-DOTA-TATE did not correlate with the marker of activated macrophages CD163 as previously found by our group[15]. Interestingly, a positive correlation was found between the macrophage markers CD36, CD86, and CD163 and Na[¹⁸F]F, indicating Na[¹⁸F]F activity correlates not only with microcalcification, but also macrophage infiltration[28].

TNF is an activated M1 macrophage related cytokine and is involved in systemic inflammation. In our study, we found TNF to positively correlate with both 2-[¹⁸F]FDG and Na[¹⁸F]F, which confirm atherosclerosis in the arteries of the rabbits, as well as inflammation in the plaque. These results are in line with a previous study finding a positive correlation in inflamed atherosclerotic plaques between TNF and 2-[¹⁸F]FDG[29].

IL-10 is an anti-inflammatory cytokine with deactivating properties in macrophage modulation and protects against atherosclerosis. It was upregulated in the atherosclerotic group by 14-fold and positively correlated with [⁶⁴Cu]-Cu-DOTA-TATE. A predictor of atherosclerosis progression, ITGAL, was also found to correlate with both [⁶⁴Cu]-Cu-DOTA-TATE and Na[¹⁸F]F. Of all the genes investigated, ITGAL was most differentially expressed between the atherosclerotic group and the control group. ITGAL expression is a signature for inflammatory monocytes, which together with other immune cells accumulates and become atherosclerotic plaque[30, 31].

In the current study, we found a trend of several of the atherosclerotic process biomarkers we investigated, correlated with both Na[¹⁸F]F and [⁶⁴Cu]Cu-DOTA-TATE. Both PET tracers showed positive correlations with genes associated with macrophages and microcalcification, thus providing information of the disease progression.

The gene expression is performed ex vivo and demands an invasive approach. Molecular imaging by PET/CT provides a non-invasive approach. Furthermore, PET/CT scans can be performed at various time points to achieve an accurate image of the present disease progression, without harming the patient or animal included in the study.

A limitation of the rabbit model is the lack of progression into intraplaque hemorrhage, plaque rupture, and infarction. Another limitation is the use of only male rabbits in the study; however, this was chosen to avoid the potential effect of hormonal changes in female rabbits. Future prospective clinical trials are needed to confirm that both [⁶⁴Cu]Cu-DOTA-TATE and Na[¹⁸F]F predicts plaque vulnerability.

We demonstrated the value of [⁶⁴Cu]Cu-DOTA-TATE and Na[¹⁸F]F for assessment of atherosclerosis. The results obtained are distinct from those obtained with 2-[¹⁸F]FDG. Key inflammatory biomarkers, such as CD36, CD86 and ITGAL, were upregulated in the atherosclerotic rabbits and correlated with tracer accumulation of [⁶⁴Cu]Cu-DOTA-TATE and Na[¹⁸F]F, confirming the ability and value of these tracers for visualization of the atherosclerotic process.

Andreas Kjaer is an inventor/holds IPR on ⁶⁴Cu-DOTA-TATE for human use. No other potential conflicts of interest relevant to this article exist.

Author Contributions

CEG: Investigation, Methodology, Data Collection, Formal Analysis, Writing – original draft, Writing – review & editing, SFP: Investigation, Conceptualization, Data Collection, Writing – review & editing CC: Investigation, AD: Formal Analysis, Writing – review & editing, TE: Investigation, Writing – review & editing, TB: Supervision, Methodology, Data Collection, Formal analysis, Validation, Writing – Original draft, Writing – review & editing, AK: Supervision, Conceptualization, Writing – review & editing, Project administration, Funding Acquisition. All authors reviewed the manuscript.

Financial Disclosure: This project received funding from the European Union’s Horizon 2020 research and innovation program under grant agreements no. 670261 (ERC Advanced Grant) and 668532 (Click-It), the Lundbeck Foundation, the Novo Nordisk Foundation, the Innovation Fund Denmark, the Danish Cancer Society, Arvid Nilsson Foundation, the Neye Foundation, the Research Foundation of Rigshospitalet, the Danish National Research Foundation (grant 126) - PERSIMUNE, the Research Council of the Capital Region of Denmark, the Danish Health Authority, the John and Birthe Meyer Foundation and Research Council for Independent Research. Andreas Kjaer is a Lundbeck Foundation Professor.

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No competing interests reported.

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published 07 Jun, 2023

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Imaging of Atherosclerosis with [⁶⁴Cu]Cu-DOTA-TATE: a Translational Head-to-Head Comparison Study with 2-[¹⁸F]FDG, and Na[¹⁸F]F in Rabbits

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