64Cu-DOTATATE PET/MRI for Detection of Activated Macrophages in Carotid Atherosclerotic Plaques

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PET quantification
Anatomical co-registration of MR and PET was carefully checked by matching anatomical landmarks like salivary glands. The carotid artery was analyzed slice by slice in every subject. The PET data was fused with the simultaneously acquired axial MR images (T1, T2, and proton density weighted). A free-hand region of interest (ROI) was drawn to include plaque, vessel wall and lumen starting at the carotid bifurcation proceeding caudally and cranially (not including the external carotid artery) to include the entire plaque. The mean standardized uptake value (SUV mean ) corrected for injected dose, patient weight and time to acquisition were calculated for each region of interest. For calculation of target-to-background (TBR) values the, SUV mean for each slice was normalized to blood pool activity (SUV mean ) obtained from a ROI placed in the ipsilateral jugular vein resulting in TBR values for subsequent calculations.

Ex vivo PET/CT
For the ex vivo acquisition, one extirpated plaque underwent PET/CT imaging 23 hours after injection of 64 Cu-DOTATATE using a Siemens Inveon MM PET/CT small animal scanner (Siemens Molecular Imaging, Knoxville, USA). The specimen was PET-scanned for 90 minutes with a 350-650 keV energy window and 3.432 ns coincidence window. The PET list mode data was reconstructed using an OP-MAP iterative algorithm, applying 18 iterations and 2 subsets, with a requested resolution of 1.0 mm. The CT scan was done using x-ray voltage and current of 50 kVp and 500 mA, respectively. Projections were acquired over 360 degrees using 720 steps, with an exposure time of 2 seconds and 1 second of settle time. The CCD detector was using 4096 by 4032 pixels and a binning factor of 2. The CT projections were reconstructed into an image using filtered back-projection resulting in an isotropic voxel size of 21.7 µm. The images were coregistered and processed using Siemens Inveon Research Workplace version 4.1 (Siemens Molecular Imaging, Knoxville, USA).

Tissue RNA extraction
Tissue conservation and cDNA production for real-time qPCR analysis was performed as earlier described 3-5 . Briefly; plaque tissue secured by carotid endarterectomy was sliced immediately at bedside in the operating theater into 3 mm slices caudally (common carotid artery) and cranially (internal carotid artery) oriented from the vessel bifurcature. A custom made knife with blades spaced evenly at 3 mm intervals was used to ensure consistency of slice thickness. The slices corresponded to the PET/MR imaging modality and were immediately conserved in RNAlater ® (cat. # AM7021, Ambion (Europe) Limited, Cambridgeshire, United Kingdom) in 1.5 mL PCR grade Eppendorf tubes for subsequent RNA extraction (n=61 slices). One plaque was conserved in toto for ex vivo scanning prior to slicing and RNA extraction. Additionally a piece of superior thyroid artery was secured as reference tissue from every patient. Subsequently tissue was stored at 5°C for 24 hours before RNAlater ® was drained and the tissue transferred to -80°C storage.
RNA extraction was performed in a specially assigned RNA laboratory and procedures were executed in a fume cupboard. Each tissue slice and reference tissue was freeze fractured in toto using a cryoPREP™ impactor (Covaris Europe, Brighton, UK) and TRI Reagent® (cat. # TR118, Molecular Research Center Inc., Cincinnati, USA) was used for RNA isolation in a protocol modified from the manufacturer's instructions to accommodate freeze fracture: Each sample was transferred from Eppendorf tubes into PCR grade tissue bags (cat. # TT1XT, Covaris Inc., Europe, Brighton, UK) cooled in liquid Nitrogen and freeze fractured using the impactor. The pulverized sample was transferred to fresh 1.5 mL PCR grade Eppendorf tubes and 1000 µL TRI Reagent® added along with 100 µL of BCP (cat. # BP151, Molecular Research Center Inc., Cincinnati, USA). The samples were stirred vigorously using a vortex mixer for 15 seconds and left to incubate at room temperature for 15 minutes. Subsequently the samples were centrifuged at 12.000 g for 15 minutes at 4°C leaving an upper aqueous phase containing RNA, an interphase containing DNA and a lower (red) phase containing protein. The upper phase (450 µL) was carefully transferred to a fresh 1.5 mL Eppendorf tube and an equal volume (450 µL) of isopropanol was added for RNA precipitation. The samples were mixed gently by hand and left to incubate for 10 minutes at room temperature. Centrifugation at 12.000 g at 4°C for 8 minutes was followed by careful removal of the supernatant leaving an RNA pellet. The pellet was then washed 2 times using 1 mL of RNasefree 75% ethanol alternating with centrifugation at 7.500 g at 4°C for 5 minutes before a final centrifugation at 13.000 g at 4°C for another 5 minutes where the remaining supernatant could be removed and pellet left to air dry for a minimum of 20 minutes. Finally 20 µL of molecular grade water (cat. # 2900136, 5-Prime GmbH, Hamburg, Germany) was added and the RNA reconstituted by vigorous mixing for 15 seconds using a vortex mixer. The RNA was stored at -80°C until further processing.

RNA quality and concentration
The RNA quality was assessed using the Agilent 2100 Bioanalyzer and associated reagents (cat. # G2938-80023, Agilent Technologies Inc., Santa Clara, CA, USA) to assign RNA integrity values (RINs) 6 . The system utilizes chip-based microfluidic technology providing electrophoretic separation in an automated and reproducible manner of up to 12 samples pr chip. RNA samples (1 µL pr sample) were separated in the channels of the chip according to molecular weight and detected by laser induced fluorescence detection of an added dye. Electropherograms for each sample was constructed for visualization of RNA quality. The "Agilent RNA 6000 Nano Assay Protocol -Edition April 2007" was used for preparing and assigning RIN values to all samples. In preparation for analysis 2 µL of samples and 1.2 µL of ladder (cat. # 5067-1529, Agilent Technologies Inc., Santa Clara, CA, USA) were denatured at 70°C for 2 minutes.
Briefly: RNA 6000 Nano gel matrix (550 µL) was pipetted into a spin filter and centrifuged at 1500 g for 10 minutes at room temperature and divided into 65 µL aliquots. Concentrated RNA 6000 Nano dye was stirred for 10 seconds and 1 µL added to the 65 µL of the RNA 6000 Nano gel matrix. The gel-dye mix was then vigorously stirred using a vortex mixer and subsequently centrifuged at 13.000 g for 10 minutes at room temperature. A 6000 Nano chip (cat. # 5067-1511, Agilent Technologies Inc., Santa Clara, CA, USA) for determination of total eukaryotic RNA was placed in the chip priming station and loaded with gel-dye mix for priming of the chip using the plunger system of the priming station. Once primed, the sample and ladder wells were all loaded with 5 µL of RNA 6000 Nano Marker whereas 1 µL of sample and 1µL of ladder was added to the wells marked "sample" and "ladder" respectively. The chip was stirred in a custom vortexer for 1 minute at 2400 rpm and the analyses were performed on the Bioanalyzer immediately thereafter.
RNA concentrations from the isolation procedure were measured using the Nanodrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) prior to dilution for the cDNA synthesis step.

cDNA synthesis
Using the AffinityScript™ QPCR cDNA Synthesis Kit (cat. # 600559 Stratagene, La Jolla, CA, USA), 0.3 ng of total RNA was reverse transcribed to cDNA for subsequent real-time qPCR analysis of whole-slice gene expression. In short: A mixture containing 7 µL (0.3 ng) RNA, 10 µL first strand master mix (2x), 2.45 µL Oligo (dT) primer (100 ng/µL), 0.55 µl random primer (100 ng/µL) and 1 µL of Stratascript™ RT/RNase Block Enzyme Mixture were prepared and run on a Mastercycler gradient RT-PCR machine (Eppendorf AG, Hamburg, Germany) utilizing a program of 5 minutes of primer annealing at 25 °C, 15 minutes of cDNA synthesis at 42 °C and finally termination for 5 minutes at 95 °C. Subsequently cDNA was stored at -20 °C until qPCR analysis.

Quantitative real-time PCR
Optimal housekeeping genes were identified as described previously 3-5 . Briefly; we tested both reference and diseased tissue, using a commercially available panel of 12 housekeeping genes (cat. # A101, Version 1.3 TATAA Biocenter AB, Göteborg Sweden) and found 60S acidic ribosomal protein P0 (RPLP, gene ID: NM_001002) and peptidylprolyl isomerase A (PPIA, gene ID: NM_021130) to be optimal using the geNorm algorithm embedded in the qBase PLUS software (Biogazelle NV, Zwijnaarde, Belgium) 7 . Taqman ® based gene expression assays were designed using Beacon Designer™ 7.90 (PREMIER Biosoft, Palo Alto, CA, USA) and analyzed using Mx3000P ® or Mx3005P™ real-time PCR systems (Stratagene, La Jolla, CA, USA). Five genes of interest; cluster of differentiation 68 (CD68, gene ID: NM_001251) cluster of differentiation 163 (CD163, gene ID: NM_004244), matrix metalloproteinase 9 (MMP9, gene ID: NM_004994), interleukin 18 (IL-18, gene ID: NM_001562),cathepsin K (CTSK, gene ID: NM_000396) and tumor necrosis factor α (TNFα, gene ID: NM_000594.3) were analyzed. The thermal profile of the TaqMan ® assays consisted of an initial denaturization step at 95°C for 10 minutes followed by 40 cycles of denaturization at 95°C for 15 seconds and finally combined annealing and elongation for one minute at 60°C. Gene expression data analysis was performed in qBase PLUS incorporating a modified delta-delta-Cq model (2 ∆∆Cq ) and multiple reference genes 8, 9 . Inter-run calibration (three different samples replicated on every plate in each run) was used to eliminate plate-to-plate variation between runs. For assay and analysis details, see Tables I and II. All primers and Taqman ® probes were purchased at Sigma (Sigma-Aldrich, St. Louis, MO, USA).

Histology
Recovered carotid plaque specimens were fixed in 10% neutrally buffered formalin for 24 hours and subsequently processed and paraffin embedded. A representative plaque was sectioned (4 µm), transferred to slides, deparaffinized and stained with Mayer's hematoxylin and eosin (cat. # 860213 and 854653, Region H Apothecary, Copenhagen, Denmark) before being dehydrated using ethanol and finally mounted with pertex.

Immunohistochemistry
Immunohistochemistry was performed to assess markers of vulnerability using mouse monoclonal antibodies against macrophages (CD68; dilution 1:100, clone PG-M1, Dako, Glostrup, Denmark # ab137867] respectively). Briefly: 4 µm sections was mounted on glass slides and placed at 40°C overnight. Next day, the temperature was increased for one hour to 60°C and the slides deparaffinized in shifts of xylen for 15 minutes followed by multiple shifts of ethanol in decreasing concentrations advancing the sections to demineralized water. Epitope demasking was performed using heat-induced epitope retrieval (HIER) in citrate buffer pH 6.0 for 15 minutes in a microwave oven followed by 30 minutes rest at room temperature and then immersion in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (cat. # P1379-25mL, Sigma-Aldrich, St. Louis, MO, USA) for five minutes. Subsequent reactions all took place at room temperature. After 10 minutes of immersion in PBS, the slides were transferred to humidity chambers where each section was covered with peroxidase blocker (cat.# S2023, Dako, Glostrup, Denmark) for eight minutes and then rinsed with three shifts of PBS every two minutes. The sections were blocked using 2% bovine serum albumin (cat. # A7906-100g, Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes followed by primary antibody diluted in 2% bovine serum albumin for samples and positive control tissue, whereas species-matched FLEX control (cat. # IS600 [rabbit] / IS750 [mouse], Dako, Glostrup, Denmark) was added to negative control samples. The samples were then incubated for one hour followed by rinse in three shifts of PBS every two minutes. Species matched secondary horse-radish peroxidase conjugated antibody was now added to the samples; EnVision FLEX (cat. # K4003 [rabbit] / K4001 [mouse], Dako, Glostrup, Denmark) which were left to incubate for another 40 minutes followed by rinse in two shifts of PBS for five minutes. The samples were developed using 3,3'-Diaminobenzidine (DAB) for 10 minutes (cat. # K3468, Dako, Glostrup, Denmark) followed by rinse in three shifts of PBS. The samples were transferred to soak in demineralized water for five minutes, counterstained by two minutes of Mayer's hematoxylin, soaked for five minutes in demineralized water, before being dehydrated in ethanol and finally mounted using pertex.
Slides were scanned using an Axio Scan.Z1 slide scanner (Carl Zeiss Microscopy GmbH, Jena, Germany) and subsequent image preparation was performed using a combination of Photoshop CS6 (Adobe Systems, San Jose, CA, USA) and Microsoft Powerpoint 2010 (Microsoft Corporation, Redmond, WA, USA).

Statistical analysis
Data was analyzed using a linear mixed model adjusting for serial correlation (AR1) predefined in repeated measurements from the same patient. Effects of explanatory variables (CD163, CD68, CTSK, IL-18, MMP9 and TNFα) were tested in univariate analyses. Signficiant explanatory variables (CD163, CD68) were thereafter included in a multivariate analysis. Gene expression and MR data are presented as mean ± SEM. Analyses were performed using SPSS 20 (IBM Corporation, Armonk, New York, USA) while Graphpad Prism 5 (Graphpad software Inc, La Jolla, CA, US) was used for illustrations. Normal distribution assumptions were tested using the Kolmogorov-Smirnov test. Furthermore in vivo PET data and gene expression analyses were compared using the Spearman correlation coefficient as appropriate. Early and late PET data were compared using paired samples t-test and Bland Altman statistics. Logarithmic transformation (log 2 )of the gene expression data was applied to obtain normal distribution. P<0.05 was considered statistical significant.     Table Position H  Table Position 0