SUPPLEMENTAL METHODS
Pentixafor Synthesis
Material for synthesis of
68
Ga-Pentixafor (CPCR4.2) was provided by Scintomics
(Fürstenfeldbruck, Germany). Synthesis was performed using a cold mass of 15 nmol CPCR4.2,
and a 68Ge/68Ga-generator (Eckert&Ziegler, Braunschweig, Germany) connected to a Scintomics
GRP synthesis module.
Animal Model and Experimental Protocol
The local state authority approved all animal procedures (Niedersächsisches Landesamt
für Verbraucherschutz und Lebensmittelsicherheit).
Animal Care. Animals were maintained in accordance with the guiding principles of the
American Physiological Society. Male C57Bl/6 mice (n=53, Charles River, Sulzfeld, Germany;
23.5±2.1g) were housed in groups of 10 on a 14h/10h light/dark cycle with food (Altromin 1324
TPF, Lage, Germany) and water ad libitum.
Surgical Procedure. Mice underwent coronary artery ligation (n=40), sham surgery (n=8)
or no surgery (n=5). MI was induced by left anterior descending coronary artery (LAD) ligation.
Mice were pretreated with 2 mg/kg butorphanol (Pfizer) subcutaneously and anesthesia was
induced with 3–4% isoflurane (Baxter). After oral intubation, anesthesia was maintained with
1.5–2% isoflurane. In sham operated mice, the ligature around the LAD was not tied. Animals
were placed on a heating pad connected to a temperature controller (Föhr Medical Instruments)
to maintain rectal temperature at 37 °C during surgery.
Drug Interventions. A subgroup of infarct mice (n=18) was treated with the angiotensin
converting enzyme inhibitor enalapril (20mg/kg/d), administered orally, from 2d prior to surgery
until 7d after. A second subgroup (n=5) underwent acute CXCR4 blockade with intravenous
AMD3100 (50µg in 100µL) immediately prior to PET tracer injection.
Small Animal Positron Emission Tomography (PET)
Image Acquisition. Scans were performed at 3 and 7d after surgery, using an Inveon
DPET (Siemens). Under light anesthesia (isoflurane 2% at 1L/min oxygen), a catheter was
inserted into the lateral tail vein for tracer administration.
68
Ga-Pentixafor (9.6±1.7MBq) was
administered as a 0.10-0.15mL bolus. Mice were recovered from anesthesia, kept conscious for
40min, then re-anesthetized and placed in the scanner. A 10min static scan was acquired at
50min after pentixafor injection. Then,
18
F-FDG (8.6±2.1MBq) was injected via tail vein to
facilitate localization of the heart and infarct territory. After 30 min under continued isoflurane, a
second 10min static scan was performed. Low dose CT was acquired between PET acquisitions.
Data Processing. Attenuation-corrected PET data were reconstructed using an iterative
algorithm (3D OSEM), then analyzed using Inveon Research Workplace (Siemens) to define
regions of interest within infarct territory (apical anterolateral wall), remote myocardium (basal
posteroseptal wall), spleen, and vertebral bone marrow. Fused
18
F-FDG images were used to
distinguish viable myocardium from infarct region. Mean percent injected dose (%ID)/g values
were calculated from activity concentration. Infarct-to-remote myocardial activity ratios were
calculated to evaluate regional
68
Ga-pentixafor uptake. In non-infarcted mice, ratios were
calculated using the same regions (i.e. apical anterolateral and basal posteroseptal wall).
Ex Vivo Validation
Ex vivo Autoradiography. A subgroup of mice (n=8) was killed by cervical dislocation
after PET. Hearts were excised, rinsed and frozen under liquid nitrogen. Short axis sections
(10µm) were immediately taken using a cryostat, with alternate sections allotted for
autoradiography or histopathology. After 1 hour exposure, phosphor screens were scanned using
a Cyclone Storage Phosphor System (Packard). Autoradiographic images were analysed using
Quantity One software.
Histology. Masson trichrome staining was performed to identify collagen deposition.
CD68
immunostaining
identified
macrophages.
Ly6G
immunostaining
identified
polymorphonuclear leukocytes (consistent with granulocytes).
Flow Cytometry. In sham operated (n=5) and infarcted mice (n=8 at 3d, n=8 at 7d) with
and without enalapril treatment, CD45-expressing leukocytes in the heart were identified using
flow cytometry. Excised left ventricles were digested with collagenase D/dispase/DNase for
30min. The resulting cell suspension was filtered and incubated with rat anti-mouse
CD16/CD32. Anti-mouse CD45 antibody was then added for immunofluorescent labeling. Cells
were then analyzed with a LSR II flow cytometer (Becton Dickinson), and absolute number of
total leukocytes in left ventricles was determined by BD TruCOUNTTM tubes (BD Biosciences).
Results were analyzed by FlowJo software (version 10).
Patients
Twelve consecutive patients (4 female, 8 male; 62±11years) underwent myocardial
perfusion single-photon emission computed tomography (SPECT) and pentixafor PET within 8
days after acute ST-elevation myocardial infarction. Ten patients additionally underwent cardiac
magnetic resonance imaging (CMR). All had been treated by percutaneous coronary intervention
and stenting within a median of 4 hours (range: 2-20) of symptom onset. The culprit vessel was
LAD in 6, LCX in 1 and RCA in 5 patients. Median CKmax was 1106U/l (range: 164-9223).
Angiographically, 4 had single-vessel, 4 two-vessel, and 4 triple-vessel disease. The mean time
from reperfusion to PET was 5±2 days (range 2-8 days). SPECT and CMR were performed
within 1 day of PET. Medication at the time of imaging consisted of ACE inhibitors (n=11) or
angiotensin receptor blocker (n=1), along with beta-blockers, aldosterone antagonists, lipid
lowering agents and platelet inhibitors.
All noninvasive imaging was obtained for clinical purposes, to determine successful
reperfusion, ventricular function and inflammatory burden. Patients gave written informed
consent prior to imaging. 68Ga-Pentixafor was used clinically according to §13.2b of the German
Pharmaceuticals Act (AMG). The local ethical committee was informed of data analysis for the
purpose of this study and agreed with the project.
Noninvasive Clinical Imaging
Perfusion Imaging.
99m
Tc-Sestamibi (357±35MBq) was injected intravenously at rest.
Electrocardiographically gated SPECT images were acquired for 7min using a solid-state
detector equipped SPECT system (GE Discovery 530c, GE Healthcare, Waukesha, WI) at 45min
after injection. Low-dose CT scans were obtained on a separate scanner (GE Discovery
NMCT670), realigned with the SPECT data using dedicated software, and used for attenuation
correction. Gated and nongated images were reconstructed with an iterative algorithm.
Pentixafor PET/CT. 68Ga-Pentixafor (92±24MBq) was injected intravenously. Static PET
images were acquired for 20min using a Biograph mCT 128 scanner (Siemens, Knoxville, TN),
starting at 60min after injection. A low-dose CT scan was acquired for attenuation correction.
Tomographic images were reconstructed using time-of-flight information and a resolution
recovery incorporating iterative algorithm. The injected
68
Ga-pentixafor activity resulted in a
mean effective dose of 1.4±0.4mSv.
CMR. Examinations were performed on a 1.5T scanner (Magnetom Avanto, Siemens).
Cine images of LV function were obtained using steady state free precession sequences. Extent
and severity of myocardial edema were determined by T2-weighted turboinversion recovery
magnitude sequences in corresponding slices. Late contrast enhancement (LE) was imaged by
phase-sensitive inversion recovery sequences, 10-15min after bolus injection of 0.15mmol/kg
gadolinium-DTPA (Gadobutrol, Bayer Healthcare).
Clinical Image Analysis
Perfusion and Function. Commercial software was used for creation of polar maps from
perfusion SPECT datasets. Infarct size was determined using a 60% threshold of maximal left
ventricular activity (Munich Heart software). Additionally, using automated contour detection,
left ventricular ejection fraction was determined from gated SPECT (4DM SPECT software).
Segmental Scoring. SPECT, PET and CMR images were visually analyzed using the
AHA 17-segment model and a previously established scoring system with a scale of 0-4. In brief,
wall motion was scored on cine CMR (normal/mild/moderate hypokinesia/akinesia/dyskinesia),
regional edema and late enhancement were scored based on transmurality (absent/<25%/2650%/51-75%/>75%
transmural),
SPECT
perfusion
defect
was
scored
by
severity
(normal/mild/moderate/severe reduction/absent uptake), and pentixafor uptake was assessed
relative to blood pool (below/equal/mildly/moderately/severely above). Microvascular
obstruction (MVO) in late enhancement CMR was assessed as present/absent. Global summed
scores were calculated for each parameter in each patient. Additionally, scores were compared
within segments.
Quantitative PET Analysis. For a multi-organ analysis, mean standardized uptake values
(SUV) were obtained for infarcted and remote myocardium, bone marrow of vertebral bodies,
spleen, liver and blood pool. Guided by CT and fusion images, spherical volumes of interest
(VOI, 5mm diameter) were placed in infarcted myocardium (hottest region), in remote
myocardium, in left atrial cavity for blood pool, and in bone marrow of three vertebral bodies.
Larger VOIs (30 mm) were placed in the spleen and liver.