Systematically evaluating DOTATATE and FDG as PET immuno-imaging tracers of cardiovascular inflammation

In recent years, cardiovascular immuno-imaging by positron emission tomography (PET) has undergone tremendous progress in preclinical settings. Clinically, two approved PET tracers hold great potential for inflammation imaging in cardiovascular patients, namely FDG and DOTATATE. While the former is a widely applied metabolic tracer, DOTATATE is a relatively new PET tracer targeting the somatostatin receptor 2 (SST2). In the current study, we performed a detailed, head-to-head comparison of DOTATATE-based radiotracers and [18F]F-FDG in mouse and rabbit models of cardiovascular inflammation. For mouse experiments, we labeled DOTATATE with the long-lived isotope [64Cu]Cu to enable studying the tracer’s mode of action by complementing in vivo PET/CT experiments with thorough ex vivo immunological analyses. For translational PET/MRI rabbit studies, we employed the more widely clinically used [68Ga]Ga-labeled DOTATATE, which was approved by the FDA in 2016. DOTATATE’s pharmacokinetics and timed biodistribution were determined in control and atherosclerotic mice and rabbits by ex vivo gamma counting of blood and organs. Additionally, we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [64Cu]Cu-DOTATATE and [18F]F-FDG. To evaluate differences in the tracers’ cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [64Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUVmax 1.3, IQR, 1.2–1.4, N = 4) and remote myocardium (SUVmax 0.7, IQR, 0.5–0.8, N = 4, p = 0.0286), and with respect to controls (SUVmax 0.6, IQR, 0.5–0.7, N = 4, p = 0.0286), than [18F]F-FDG PET. In atherosclerotic mice, [64Cu]Cu-DOTATATE PET aortic signal, but not [18F]F-FDG PET, was higher compared to controls (SUVmax 1.1, IQR, 0.9–1.3 and 0.5, IQR, 0.5–0.6, respectively, N = 4, p = 0.0286). In both models, [64Cu]Cu-DOTATATE demonstrated preferential accumulation in macrophages with respect to other myeloid cells, while [18F]F-FDG was taken up by macrophages and other leukocytes. In a translational PET/MRI study in atherosclerotic rabbits, we then compared [68Ga]Ga-DOTATATE and [18F]F-FDG for the assessment of aortic inflammation, combined with ex vivo radiometric assays and near-infrared imaging of macrophage burden. Rabbit experiments showed significantly higher aortic accumulation of both [68Ga]Ga-DOTATATE and [18F]F-FDG in atherosclerotic (SUVmax 0.415, IQR, 0.338–0.499, N = 32 and 0.446, IQR, 0.387–0.536, N = 27, respectively) compared to control animals (SUVmax 0.253, IQR, 0.197–0.285, p = 0.0002, N = 10 and 0.349, IQR, 0.299–0.423, p = 0.0159, N = 11, respectively). In conclusion, we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [18F]F-FDG for the evaluation of inflammation in small animal models of cardiovascular disease. Our results support further investigations on the use of DOTATATE to assess cardiovascular inflammation as a complementary readout to the widely used [18F]F-FDG.

we performed in vivo PET/CT experiments in mice with atherosclerosis, mice subjected to myocardial infarction and control animals, using both [ 64 Cu]Cu-DOTATATE and [ 18 F]F-FDG. To evaluate differences in the tracers' cellular specificity, we performed ensuing ex vivo flow cytometry and gamma counting. In mice subjected to myocardial infarction, in vivo [ 64 Cu]Cu-DOTATATE PET showed higher differential uptake between infarcted (SUV max  Cardiovascular disease is the principal cause of morbidity and mortality worldwide 1 . In the past two decades, pre-clinical and clinical studies [2][3][4][5][6] have uncovered inflammation's critical role in atherosclerotic plaque formation and the onset of cardio-and cerebrovascular events. As cardiovascular inflammation is rapidly developing into a therapeutic target, quantitative positron emission tomography (PET) inflammation imaging of the heart and vasculature is rapidly gaining momentum.
Originally developed as a cancer PET tracer, [ 18 F]F-fluorodeoxyglucose ([ 18 F]F-FDG) is also the most commonly used inflammation tracer in cardiovascular disease, both in the context of atherosclerosis and cardiac ischemia 7,8 . Nevertheless, several limitations are associated with [ 18 F]F-FDG's use for cardiovascular inflammation imaging. First and foremost, being a glucose analog, [ 18 F]F-FDG is taken up by metabolically active cells and is not necessarily specific for inflammatory cells. In fact, in vitro 9 and recent in vivo atherosclerosis studies 10 indicate that vascular [ 18 F]F-FDG signal might also originate from cells other than plaque macrophages, including non-immune cells. In the context of cardiac imaging, the absence of [ 18 F]F-FDG signal is indicative of cardiomyocyte loss after myocardial infarction 11 . Unfortunately, high [ 18 F]F-FDG background signal in the healthy heart makes it challenging to visualize inflammation in the coronaries or in the infarct itself [12][13][14] .
To surpass these limitations, alternative, inflammatory cell-specific PET tracers are being actively investigated for use in cardiovascular disease 12,[15][16][17] . [ 68 Ga]Ga-or [ 64 Cu]Cu-labeled DOTATATE is a somatostatin receptor type 2 (SST2)-binding, FDA approved, PET radiotracer used to identify and monitor SST2-positive neuroendocrine tumors 18,19 . Since SST2 receptors are also expressed on macrophages 20 , DOTATATE-based tracers have recently gained significant interest for the quantification of inflammation in cardiovascular disease mouse models 21 , in human plaques 20,22 and, more recently, in myocardial infarction 23,24 . However, the advantages and challenges of using DOTATATE-based radioligands to characterize and quantify cardiovascular inflammation, especially in comparison with the validated and widely used [ 18

Results
Study design. This study's aim was to thoroughly characterize and compare DOTATATE-based radiotracers and [ 18 F]F-FDG for the evaluation of inflammation in mouse and rabbit small animal models of cardiovascular disease (Fig. 1 Fig. 2A and Supplementary Table S1, C57Bl/6 N = 7 and Apoe −/− N = 6). In both groups, separation of blood fractions revealed that most of the tracer was in the plasma, which contained on average 83.4 ± 0.6 and 88.7 ± 0.9% of the radioactivity at 120 min after radiotracer injection, respectively (Fig. 2B, N = 5 per group). Density gradient separation of blood cells indicated that the tracer was taken up by mononuclear cells at early time-points, with progressive increase in polynuclear cells in control and atherosclerotic animals over the 120 min after injection    www.nature.com/scientificreports/ likely due to atherosclerosis-driven chronic inflammation. Two hours after injection, signal in the thoracic aorta was also significantly higher in atherosclerotic compared to control mice with medians of 2.1 (IQR, 2-2.4) and 1 (IQR, 0.7-1.1) %ID/g, respectively (p = 0.0079, Fig. 2F and Supplementary Fig. S3A; Supplementary Tables S2  and S3, N = 5 per group).       Fig. S2B). Based on pharmacokinetic analyses and dynamic imaging experiments ( Supplementary Fig. S4C, N = (Fig. 6A), we found a 1.6-fold higher SUV max in  www.nature.com/scientificreports/ 3.36 × 10 9 -6.79 × 10 9 , p < 0.0001), no increase in fluorescent HDL signal was observed when comparing athero 4mo and athero 7mo , suggesting no increase in macrophage burden over time (p = 0.5584, Fig. 6G, controls N = 11, athero 4mo N = 15 and athero 7mo N = 8). A weak, but significant (r = 0.3522; p = 0.0141), correlation was found between [ 18 F]F-FDG and [ 68 Ga]Ga-DOTATATE aortic SUV max , reflecting the partially overlapping cellular specificity of the two tracers ( Supplementary Fig. S4D).

Discussion
The use of SST2-specific PET radiotracers, and predominantly DOTATATE-based ligands, is rapidly gaining momentum for cardiovascular inflammation imaging 8,26,31 . In pre-clinical settings, visible uptake of SST2-specific PET radiotracers 15,21 including, but not limited to, DOTATATE has been previously demonstrated in mice with atherosclerosis. Yet, in selected studies, in vivo vascular imaging findings were confounded by concomitant uptake in nearby organs. Following successful retrospective analyses in cancer patients 32,33 , DOTATATE vascular uptake has been shown in the vasculature of patients with coronary and carotid atherosclerosis 20,34 , although other studies have question DOTATATE's ability to discriminate symptomatic lesions in humans 35 . Studies in patients with myocardial infarction 23 and a case report on cerebral stroke 36 40 . This feature is important when performing in vivo imaging and especially in the vasculature, whose signal can be easily contaminated by the blood pool because of partial volume errors. Timed biodistribution analysis in the mouse confirmed fast accumulation in the kidneys and gastrointestinal organs 41 , and, as expected, targeting of SST2-expressing organs, such as pancreas and adrenal gland. We further used the combination of in vivo PET/CT imaging and ex vivo radiometric and immunological assays to investigate [ 64 Cu]Cu-DOTATATE as an inflammation tracer in mice with atherosclerosis and mice subjected to myocardial infarction. For the myocardial infarction model, mice were imaged 3 days after LAD ligation surgery. This timeline was chosen to capture the peak of SST2-expressing inflammatory macrophage infiltration in the infarcted myocardium 26 , since SST2 is upregulated in inflammatory macrophages/ LPS-stimulated macrophages, while expression in other leukocytes is negligible 20,42,43 . A previous preclinical study 24 that investigated the use of [ 68 Ga]Ga-DOTATATE in mouse models of cardiac ischemia did not show significant radiotracer accumulation in the infarct. While we also generally observed low myocardial signal, our data suggests improved infarct delineation from healthy myocardium with [ 64 Cu]Cu-DOTATATE in comparison with [ 18 F]F-FDG, as confirmed by in vivo SUVmax measurements. The difference between our findings and previous studies employing [ 68 Ga]Ga-DOTATATE may be due to the lower positron range of [ 64 Cu]Cu (1 mm) with respect to [ 68 Ga]Ga (4 mm), which intrinsically lowers the occurrence of partial volume artifacts from the blood stream, while offering intrinsic better signal-to-noise and spatial resolution 19,22,44 . Our results are also in agreement with findings from a recent clinical study 23 ,where focal, infarct-related [ 68 Ga]Ga-DOTATATE signal in patients with MI was well-visualized thanks to the tracer very low physiological background tracer uptake, and was significantly higher than in the remote myocardium. In line with several clinical studies, as well as previous ex vivo autoradiography and in vivo analysis in Apoe −/− mice using [ 68 Ga]Ga-DOTATATE [20][21][22]33,34,41,45 , we confirmed accumulation of [ 64 Cu]Cu-DOTATATE in mouse aortic plaques. Pre-clinical studies applying different SST2-specific PET radiotracers, such as the antagonist [ 111 In]In-DOTA-JR11, in atherosclerotic Apoe −/− mice have also demonstrated visible tracer accumulation in plaques. However, the interpretation of in vivo imaging findings was hampered by thymus uptake 15 , making it challenging to use these different tracers in pre-clinical research settings. Our atherosclerosis mouse model also showed that [ 64 Cu]Cu-DOTATATE uptake was higher in the ascending aorta of atherosclerotic versus control animals, while no differences were detected for [ 18 F] F-FDG. While other studies in mice showed higher [ 18 F]F-FDG accumulation in atherosclerotic versus control animals 46,47 , a recent report indicated that the specific anesthesia regimen used, and periaortic fat uptake may significantly affect [ 18 F]F-FDG plaque signal 48 . [ 64 Cu]Cu-DOTATATE blood half-life was longer in atherosclerotic mice (and rabbits) compared to controls, a factor that may potentially confound vessel wall readings because of higher blood background signal. We hypothesize that this phenomenon may be attributed to lower renal clearance (due to impaired kidney function) in diseased animals. However, ex vivo gamma counting validated the higher aortic tracer accumulation in atherosclerotic versus healthy animals, thereby mitigating these concerns.
Unlike a recently published in vitro analysis that employed macrophages differentiated from an immortalized THP-1 cell line 49  www.nature.com/scientificreports/ might be attributed to methodological differences between the two studies, such as our studies being conducted in vivo, in mouse models, as opposed to in vitro, in cells derived from human blood, as well as the different cell isolation protocols. Our results are in line with a recent clinical study that confirmed high expression of SST2 in human M1 inflammatory macrophages, in comparison with other myeloid and immune cells. However, in the same study, glucose transporters 1 and 3 (GLUT1 and GLUT3) were found to be highly expressed by all immune cells, corroborating the lower cellular specificity of [ 18 F]F-FDG 20 .
In addition to the extensive mechanistic mouse work, we evaluated [ 68 Ga]Ga-DOTATATE in a rabbit model of atherosclerosis. Ex vivo quantification of aortic HDL accumulation (a marker of macrophage burden) by near-infrared fluorescence imaging was in line with the in vivo [ 68 Ga]Ga-DOTATATE readout, indicating no increased macrophage burden during atherosclerosis progression. These findings strengthen the notion that the two tracers report on inter-related but intrinsically different processes.
To summarize, in this study we present a detailed, head-to-head comparison of the novel SST2-specific tracer DOTATATE and the validated metabolic tracer [ 18 F]F-FDG for the immune evaluation of inflammation in small animal models of cardiovascular disease. Our encouraging results support DOTATATE's use to assess cardiovascular inflammation, as a complementary readout to the widely used [ 18 F]F-FDG PET.

High-performance liquid chromatography (HPLC) and radio-HPLC.
High-performance liquid chromatography (HPLC) was performed on a Shimadzu (Kyoto, Japan) HPLC system equipped with two LC-10AT pumps and an SPD-M10AVP photodiode array detector. Radio-HPLC was performed using a Lablogic (Tampa, FL) Scan-RAM Radio-TLC/HPLC detector. Reverse phase chromatography was performed using a Waters Atlantis T3 column, 100, 5 µm, 4.6 mm × 250 mm (Waters, Milford, MA) with an acetonitrile to water gradient from 5 to 95% acetonitrile over 20 min at a flow rate of 1.0 mL min −1 .
Animal experiments. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the Icahn School of Medicine at Mount Sinai and followed National Institutes of Health guidelines for animal welfare. This study is reported in accordance with ARRIVE guidelines.
Mouse model. Animals were housed under a constant room temperature at 25 ± 2 °C and 50 ± 5% humidity with a 12-h daylight period and 12-h darkness period, with free access to water. For the mouse atherosclerosis model, 8-weeks old female Apoe −/− mice (N = 51) were purchased from Jackson Laboratories (Bar Harbor, ME) and, after a 48 h acclimatization period, were fed a Western Diet (42% Kcal from fat TD88137, Envigo, Huntingdon, UK) for 12 weeks. 16-20 weeks old female C57Bl/6 controls (N = 67) were kept on regular chow diet. The myocardial infarction group consisted 16-20 weeks old female C57Bl/6 mice (N = 18) purchased from Jackson Laboratories and subjected to ligation of the left anterior descending artery, as previously described 50 .
Rabbit model. For the rabbit studies, 3 months old male New Zealand White (NZW) rabbits (N = 47) were purchased from Charles River Laboratories (Wilmington, MA). Randomly assigned control rabbits (N = 11) were kept on chow diet and imaged with PET/MR. Among these some animals were euthanized for biodistribution assays (N = 6) or used for time-activity characterization of aorta signal (N = 3). For the atherosclerosis model, remaining rabbits (N = 36) were placed on Western diet (initial 8 weeks on regular chow enriched with 0.3% cholesterol and 4.7% coconut oil, and the remaining time period on 0.15% enriched cholesterol diet from Research diets, Inc. Brunswick, NJ). In order to induce atherosclerotic lesions, these rabbits were subjected to two endothelial denudations of the aorta through the right and left femoral artery at 2 and 6 weeks after diet initiation, respectively, as described previously 27 . Briefly, femoral artery angioplasty was performed under fluoroscopic guidance using a 4F-Fogarty embolectomy catheter. Once catheter is inserted, catheter is positioned in the thoracic descending aorta region. The balloon was then inflated to 2 atm. Catheter is then pulled back over the entire length of the aorta down to the iliac bifurcation, and the pullback procedure is repeated for two additional times. Catheter is then removed, and femoral artery is ligated. Rabbits with atherosclerosis were imaged 4 months after diet initiation (N = 36). Among these animals some were euthanized for ex vivo biodistribution analyses (N = 9), used for time-activity characterization of aorta signal (N = 3) or near infra-red imaging (N = 27). The remaining atherosclerotic rabbits were fed a Western diet for an additional 3 months (N = 9) to advance atherosclerosis. At the end of the 3-month period, these rabbits were imaged and euthanized. www.nature.com/scientificreports/ Myocardial infarction surgery in mice. Myocardial infarction in mice was induced by permanent ligation of the left anterior descending (LAD) coronary artery of female C57Bl/6 mice (N = 18). Briefly, animals were anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) and intubated using an endotracheal intubation kit from Braintree Scientific (Braintree, MA). Left-sided thoracotomy and pericardial incision were performed. A 7-0 Silk suture was used to occlude the LAD. Incisions were closed with a 5-0 Silk suture. Infarcted animals were treated with 0.1 mg/kg of buprenorphine every 12 h and used 3 days after the surgery.  Fig. S1A) to evaluate the tracer's biodistribution. After euthanasia, an insulin syringe was used to collect blood samples from the right ventricle. Blood samples were used for the establishment of pharmacokinetics profile. Mice were then perfused with 20 mL of phosphate-buffered saline (PBS) and tissues of interest (heart, aorta, bone marrow, spleen, liver, stomach, bladder, kidneys, intestines, lungs, adrenal gland, muscles and pancreas) were harvested. Tissues were blotted for gamma counting on a Wizard2 2480 automatic gamma counter (Perkin Elmer, Waltham, MA). Values were corrected for decay and normalized to tissue weight to express radioactivity concentration as percentage injected dose per gram (%ID/g). Blood half-life was calculated by measuring blood radioactivity over time for 120 min and data were fitted using a two-phase decay non-linear regression using GraphPad Prism v8.4.3 (Supplementary Table S1). To investigate the tracer's distribution within different blood compartments, after gamma counting, 100 μL of blood were spun down at 2000 g for 15 min at 4 °C. Plasma (supernatant) and cells (pellet) were gamma counted and expressed as percentage of total activity. The remainder of the blood was separated using Lymphoprep density gradient medium (Nycomed Pharma, Zurich, Switzerland) following manufacturer's instructions. Percentage of activity in mononuclear and polynuclear cells was calculated.

In vivo PET/CT imaging in mice.
For the PET/CT experiments, mice were fasted for 12 h before radiotracer injection and anesthetized with xylazine (10 mg/kg) and ketamine (100 mg/kg) through intraperitoneal injection prior to radiotracer administration. , and subsequently imaged on a high resolution (700 µm) Mediso nanoScan PET/CT scanner (Mediso, Budapest, Hungary). CT scan was acquired at 50 kVp and 300 ms exposure per projection. eXIA160 (Binitio, Ontario, Canada) was used as a contrast agent to improve imaging of the vasculature by intravenous administration of 100 μl per mouse 5 min prior to CT acquisition 51 . PET acquisition time was 40 min. Reconstruction was performed using TeraTomo 3D reconstruction engine, for 8 iterations and 6 subsets per iteration for both tracers. The voxel size was isotropic at 0.3 mm. Immediately after the PET/CT scan, animals were euthanized for ex vivo assays.
Flow cytometry. After PET imaging, mouse hearts and aortas were harvested and collected in PBS-filled tubes. Aortas were minced and digested with an enzymatic digestion solution containing liberase TH (4 U/ml) (Roche, Basel, Switzerland), DNase I (40 U/ml) and hyaluronidase (60 U/ml) in PBS. Heart tissue was minced and digested using an enzymatic digestion solution containing DNase I (60 U/ml), collagenase type I (450 U/ ml), collagenase type XI (125 U/ml) and hyaluronidase (60 U/ml) in PBS. Samples were treated with the respective enzymatic solution for 60 min at 37 °C. All enzymes were purchased from Sigma-Aldrich (St. Louis, MO). Samples were then passed through a 70 μm filter, washed and prepared for antibody staining and flow sorting. Cellular fragments and debris were gated out of the analysis by utilizing forward and side angle light scatter signal. Macrophages were identified as Ly6G − (Clone 1A8, PE/Cy7), CD11b + (Clone M1/70, PE), CD11c − (Clone N418, PerCP/Cy5.5) and F4/80 + (Alexa Fluor ® 647, Clone BM8) from Biolegend (San Diego, CA). The remaining CD11b + cells were identified as Ly6G lo , CD11b hi , CD11c hi . Data were acquired on a FACS Aria flow sorter (BD Biosciences, East Rutherford, NJ) and analyzed using FlowJo v10.0.7 (Tree Star, Ashland, OR). Sorted cells were gamma counted and activity per cell values were calculated.
Autoradiography. Tissues (heart and aorta) were placed in a film cassette against a phosphorimaging plate (BASMS-2325, Fujifilm) at − 20 °C to determine the regional radioactivity distribution. Exposure time was optimized to dose, tracer, and tissue uptake differences. Tissues from the heart of [ 18 F]F-FDG injected animals were exposed for 5 min, [ 18 F]F-FDG infused aortas were exposed for 30 min and tissues from animals in the [ 64 Cu]Cu-DOTATATE group were exposed for 60 min. The plates were read at a pixel resolution of 25 μm with a Typhoon 7000IP plate reader (GE Healthcare, Pittsburgh, PA). Images were analyzed using ImageJ software v1.52 (Madison, WI).