Proof of concept of a multimodal intravital molecular imaging system for tumour transpathology investigation.

BACKGROUND: Transpathology highlights the interpretation of the underlying physiology behind molecular imaging. However, it remains challenging due to the discrepancies between in vivo and in vitro measurements and difficulties of precise co-registration between trans-scaled images. This study aims to develop a multimodal intravital molecular imaging (MIMI) system as a tool for in vivo tumour transpathology investigation. METHODS: The proposed MIMI system integrates high-resolution positron imaging, magnetic resonance imaging (MRI) and microscopic imaging on a dorsal skin window chamber on an athymic nude rat. The window chamber frame was designed to be compatible with multimodal imaging and its fiducial markers were customized for precise physical alignment among modalities. The co-registration accuracy was evaluated based on phantoms with thin catheters. For proof of concept, tumour models of the human colorectal adenocarcinoma cell line HT-29 were imaged. The tissue within the window chamber was sectioned, fixed and haematoxylin-eosin (HE) stained for comparison with multimodal in vivo imaging. RESULTS: The final MIMI system had a maximum field of view (FOV) of 18 mm × 18 mm. Using the fiducial markers and the tubing phantom, the co-registration errors are 0.18 ± 0.27 mm between MRI and positron imaging, 0.19 ± 0.22 mm between positron imaging and microscopic imaging and 0.15 ± 0.27 mm between MRI and microscopic imaging. A pilot test demonstrated that the MIMI system provides an integrative visualization of the tumour anatomy, vasculatures and metabolism of the in vivo tumour microenvironment, which was consistent with ex vivo pathology. CONCLUSIONS: The established multimodal intravital imaging system provided a co-registered in vivo platform for trans-scale and transparent investigation of the underlying pathology behind imaging, which has the potential to enhance the translation of molecular imaging.

A Semi Rigid Novel Hydroxamate AMPED-Based Ligand for 89Zr PET Imaging.

In this work, we designed, developed, characterized, and investigated a new chelator and its bifunctional derivative for 89Zr labeling and PET-imaging. In a preliminary study, we synthesized two hexadentate chelators named AAZTHAS and AAZTHAG, based on the seven-membered heterocycle AMPED (6-amino-6-methylperhydro-1,4-diazepine) with the aim to increase the rigidity of the 89Zr complex by using N-methyl-N-(hydroxy)succinamide or N-methyl-N-(hydroxy)glutaramide pendant arms attached to the cyclic structure. N-methylhydroxamate groups are the donor groups chosen to efficiently coordinate 89Zr. After in vitro stability tests, we selected the chelator with longer arms, AAZTHAG, as the best complexing agent for 89Zr presenting a stability of 86.4 ± 5.5% in human serum (HS) for at least 72 h. Small animal PET/CT static scans acquired at different time points (up to 24 h) and ex vivo organ distribution studies were then carried out in healthy nude mice (n = 3) to investigate the stability and biodistribution in vivo of this new 89Zr-based complex. High stability in vivo, with low accumulation of free 89Zr in bones and kidneys, was measured. Furthermore, an activated ester functionalized version of AAZTHAG was synthesized to allow the conjugation with biomolecules such as antibodies. The bifunctional chelator was then conjugated to the human anti-HER2 monoclonal antibody Trastuzumab (Tz) as a proof of principle test of conjugation to biologically active molecules. The final 89Zr labeled compound was characterized via radio-HPLC and SDS-PAGE followed by autoradiography, and its stability in different solutions was assessed for at least 4 days.

Effective rational humanization of a PASylated anti-galectin-3 Fab for the sensitive PET imaging of thyroid cancer in vivo.

The lack of a non-invasive test for malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Human galectin-3 (hGal3) has emerged as a promising target for medical TC imaging and diagnosis because of its exclusive overexpression in malignant thyroid tissues. We previously developed a human-chimeric αhGal3 Fab fragment derived from the rat monoclonal antibody (mAb) M3/38 with optimized clearance characteristics using PASylation technology. Here, we describe the elucidation of the hGal3 epitope recognized by mAb M3/38, X-ray crystallographic analysis of its complex with the chimeric Fab and, based on the three-dimensional structure, the rational humanization of the Fab by CDR grafting. Four CDR-grafted versions were designed using structurally most closely related fully human immunoglobulin VH/VL regions of which one-employing the acceptor framework regions of the HIV-1 neutralizing human antibody m66-showed the highest antigen affinity. By introducing two additional back-mutations to the rodent donor sequence, an affinity toward hGal3 indistinguishable from the chimeric Fab was achieved (KD = 0.34 ± 0.02 nM in SPR). The PASylated humanized Fab was site-specifically labelled with the fluorescent dye Cy7 and applied for the immuno-histochemical staining of human tissue sections representative for different TCs. The same protein was conjugated with the metal chelator Dfo, followed by radiolabelling with 89Zr(IV). The resulting protein tracer allowed the highly sensitive and specific PET/CT imaging of orthotopic tumors in mice, which was confirmed by quantitative analysis of radiotracer accumulation. Thus, the PASylated humanized αhGal3 Fab offers clinical potential for the diagnostic imaging of TC.

Room Temperature Al18 F Labeling of 2-Aminomethylpiperidine-Based Chelators for PET Imaging.

Positron emission tomography (PET) is a non-invasive molecular imaging technology that is constantly expanding, with a high demand for specific antibody-derived imaging probes. The use of tracers based on temperature-sensitive molecules (i. e. Fab, svFab, nanobodies) is increasing and has led us to design a class of chelators based on the structure of 2-aminomethylpiperidine (AMP) with acetic and/or hydroxybenzyl pendant arms (2-AMPTA, NHB-2-AMPDA, and 2-AMPDA-HB), which were investigated as such for {Al18 F}2+ -core chelation efficiency. All the compounds were characterized by HPLC-MS analysis and NMR spectroscopy. The AlF-18 labeling reactions were performed under various conditions (pH/temperature), and the radiolabeled chelates were purified and characterized by radio-TLC and radio-HPLC. The stability of labeled chelates was investigated up to 240 min in human serum (HS), EDTA 5 mM, PBS and 0.9 % NaCl solutions. The in vivo stability of [Al18 F(2-AMPDA-HB)]- was assessed in healthy nude mice (n=6). Radiochemical yields between 55 % and 81 % were obtained at pH 5 and room temperature. High stability in HS was measured for [Al18 F(2-AMPDA-HB)]- , with 90 % of F-18 complexed after 120 min. High stability in vivo, rapid hepatobiliary and renal excretion, with low accumulation of free F-18 in bones were measured. Thus, this new Al18 F-chelator may have a great impact on immuno-PET radiopharmacy, by facilitating the development of new fluorine-18-labeled heat-sensitive biomolecules.

Development of a high affinity Anticalin® directed against human CD98hc for theranostic applications.

Enhanced amino acid supply and dysregulated integrin signaling constitute two hallmarks of cancer and are pivotal for metastatic transformation of cells. In line with its function at the crossroads of both processes, overexpression of CD98hc is clinically observed in various cancer malignancies, thus rendering it a promising tumor target. Methods: We describe the development of Anticalin proteins based on the lipocalin 2 (Lcn2) scaffold against the human CD98hc ectodomain (hCD98hcED) using directed evolution and protein design. X-ray structural analysis was performed to identify the epitope recognized by the lead Anticalin candidate. The Anticalin - with a tuned plasma half-life using PASylation® technology - was labeled with 89Zr and investigated by positron emission tomography (PET) of CD98-positive tumor xenograft mice. Results: The Anticalin P3D11 binds CD98hc with picomolar affinity and recognizes a protruding loop structure surrounded by several glycosylation sites within the solvent exposed membrane-distal part of the hCD98hcED. In vitro studies revealed specific binding activity of the Anticalin towards various CD98hc-expressing human tumor cell lines, suggesting broader applicability in cancer research. PET/CT imaging of mice bearing human prostate carcinoma xenografts using the optimized and 89Zr-labeled Anticalin demonstrated strong and specific tracer accumulation (8.6 ± 1.1 %ID/g) as well as a favorable tumor-to-blood ratio of 11.8. Conclusion: Our findings provide a first proof of concept to exploit CD98hc for non-invasive biomedical imaging. The novel Anticalin-based αhCD98hc radiopharmaceutical constitutes a promising tool for preclinical and, potentially, clinical applications in oncology.

Development of a Chimeric Antigen-Binding Fragment Directed Against Human Galectin-3 and Validation as an Immuno-Positron Emission Tomography Tracer for the Sensitive In Vivo Imaging of Thyroid Cancer.

Background: The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for in vivo imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. Methods: The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of Escherichia coli and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to in vitro characterization and in vivo validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS200 was conjugated with deferoxamine (Dfo), labeled with 89Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of 89Zr-Dfo-PAS200-Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. Results: The αGal3-Fab-PAS200 revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The in vivo Gal3-targeting by the 89Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. Conclusions: The chimeric αGal3 89Zr-Dfo-PAS200-Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.

Assessment of alphavbeta3 integrin expression after myocardial infarction by positron emission tomography.

AIMS: The purpose of this study was to determine the feasibility of a new positron emission tomography (PET) imaging approach using an (18)F-labelled alpha(v)beta(3) integrin antagonist ((18)F-Galacto-RGD) to monitor the integrin expression after myocardial infarction. METHODS AND RESULTS: Male Wister rats were subjected to 20 min transient left coronary artery occlusion followed by reperfusion. Autoradiographic analysis and in vivo PET imaging were used to determine myocardial (18)F-Galacto-RGD uptake at different time points following reperfusion. RESULTS: PET imaging and autoradiography demonstrated no significant focal myocardial (18)F-Galacto-RGD uptake in non-operated control rats and at day 1 after reperfusion. However, focal accumulation in the infarct area started at day 3 (uptake ratio = 1.91 +/- 0.22 vs. remote myocardium), peaked between 1 (3.43 +/- 0.57) and 3 weeks (3.43 +/- 0.95), and decreased to 1.96 +/- 0.40 at 6 months after reperfusion. Pretreatment with alpha(v)beta(3) integrin antagonist c(-RGDfV-) significantly decreased tracer uptake, indicating the specificity of tracer uptake. The time course of focal tracer uptake paralleled vascular density as measured by CD31 immunohistochemical analysis. CONCLUSION: Regional (18)F-Galacto-RGD accumulation suggests up-regulation of alpha(v)beta(3) integrin expression after myocardial infarction, which peaks between 1 and 3 weeks and remains detectable until 6 months after reperfusion. This new PET tracer is promising for the monitoring of myocardial repair processes.

Reproducibility and Comparability of Preclinical PET Imaging Data: A Multicenter Small-Animal PET Study.

The standardization of preclinical imaging is a key factor to ensure the reliability, reproducibility, validity, and translatability of preclinical data. Preclinical standardization has been slowly progressing in recent years and has mainly been performed within a single institution, whereas little has been done in regards to multicenter standardization between facilities. This study aimed to investigate the comparability among preclinical imaging facilities in terms of PET data acquisition and analysis. In the first step, basic PET scans were obtained in 4 different preclinical imaging facilities to compare their standard imaging protocol for 18F-FDG. In the second step, the influence of the personnel performing the experiments and the experimental equipment used in the experiment were compared. In the third step, the influence of the image analysis on the reproducibility and comparability of the acquired data was determined. Distinct differences in the uptake behavior of the 4 standard imaging protocols were determined for the investigated organs (brain, left ventricle, liver, and muscle) due to different animal handling procedures before and during the scans (e.g., fasting vs. nonfasting, glucose levels, temperature regulation vs. constant temperature warming). Significant differences in the uptake behavior in the brain were detected when the same imaging protocol was used but executed by different personnel and using different experimental animal handling equipment. An influence of the person analyzing the data was detected for most of the organs, when the volumes of interest were manually drawn by the investigators. Coregistration of the PET to an MR image and drawing the volume of interest based on anatomic information yielded reproducible results among investigators. It has been demonstrated that there is a huge demand for standardization among multiple institutions.

Galectin-3 Targeting in Thyroid Orthotopic Tumors Opens New Ways to Characterize Thyroid Cancer.

Preoperative characterization of thyroid nodules is challenging since thyroid scintigraphy fails to distinguish between benign and malignant lesions. Galectin-3 (gal-3) is expressed in well-differentiated and in undifferentiated thyroid cancer types but not in normal thyrocytes and benign thyroid lesions. Herein, we aimed to validate gal-3 targeting as a specific method to detect non-radioiodine-avid thyroid cancer in thyroid orthotopic tumor models. Methods: Papillary (BcPAP) and anaplastic (CAL62 and FRO82-1) thyroid carcinoma cell lines were characterized via Western blot and polymerase chain reaction for gal-3 and sodium-iodide symporter (NIS) expression. An 89Zr-labeled F(ab')2 antigal-3 was generated and characterized for binding versus 125I on 2- and 3-dimensional cell cultures. The thyroid carcinoma cells were inoculated into the left thyroid lobe of athymic nude mice, and the orthotopic tumor growth was monitored via ultrasound and fluorescence molecular tomography. Head-to-head PET/CT comparison of 124I versus 89Zr-deferoxamine (DFO)-F(ab')2 antigal-3 was performed, followed by biodistribution studies and immunohistochemical analysis for gal-3 and NIS expression. Results: The thyroid carcinoma cells investigated were invariably gal-3-positive while presenting low or lost NIS expression. 89Zr-DFO-F(ab')2 antigal-3 tracer showed high affinity to gal-3 (dissociation constant, ∼3.9 nM) and retained immunoreactivity (>75%) on 2-dimensional cell cultures and on tumor spheroids. 125I internalization in FRO82-1, BcPAP, and CAL62 was directly dependent on NIS expression, both in 2-dimensional and tumor spheroids. PET/CT imaging showed 89Zr-DFO-F(ab')2 antigal-3 signal associated with the orthotopically implanted tumors only; no signal was detected in the tumor-free thyroid lobe. Conversely, PET imaging using 124I showed background accumulation in tumor-infiltrated lobe, a condition simulating the presence of non-radioiodine-avid thyroid cancer nodules, and high accumulation in normal thyroid lobe. Imaging data were confirmed by tracer biodistribution studies and immunohistochemistry. Conclusion: A specific and selective visualization of thyroid tumor by targeting gal-3 was demonstrated in the absence of radioiodine uptake. Translation of this method into the clinical setting promises to improve the management of patients by avoiding the use of unspecific imaging methodologies and reducing unnecessary thyroid surgery.

Initial characterization of an 18F-labeled myocardial perfusion tracer.

UNLABELLED: PET allows for quantitative, regional myocardial perfusion imaging. The short half-lives of the perfusion tracers currently in use limit their clinical applicability. Here, the biodistribution and imaging quality of a new 18F-labeled myocardial perfusion agent (18F-BMS-747158-02) in an animal model are described. METHODS: The biodistribution of 18F-BMS-747158-02 was determined at 10 and 60 min after injection. The first-pass extraction fraction of the tracer was measured in isolated rat hearts perfused with the Langendorff method. Small-animal PET imaging was used to study tracer retention. RESULTS: The biodistribution at 10 min after injection demonstrated high myocardial uptake (3.1 percentage injected dose per gram [%ID/g]) accompanied by little activity in the lungs (0.3 %ID/g) and liver (1.0 %ID/g). The tracer showed a high and flow-independent myocardial first-pass extraction fraction, averaging 0.94 (SD = 0.04). PET imaging provided excellent delineation of myocardial structures. The heart-to-lung activity ratio increased from 4.7 to 10.2 between 1 and 15 min after tracer injection (at rest). Adenosine infusion (140 microg/kg/min) led to a significant increase in myocardial tracer retention (from 1.68 [SD = 0.23]) s(-1) to 3.21 [SD = 0.92] s(-1); P = 0.03). CONCLUSION: The observation of a high and flow-independent first-pass extraction fraction promises linearity between tracer uptake and myocardial blood flow. Sustained myocardial tracer uptake, combined with high image contrast, will allow for imaging protocols with tracer injection at peak exercise followed by delayed imaging. Thus, 18F-BMS-747158-02 is a promising new tracer for the quantitative imaging of myocardial perfusion and can be distributed to imaging laboratories without a cyclotron.

A new 18F-labeled myocardial PET tracer: myocardial uptake after permanent and transient coronary occlusion in rats.

UNLABELLED: Conventional myocardial perfusion PET tracers require onsite tracer production because of their short radioactive half-lives. To investigate the potential of a new (18)F-labeled pyridazinone analog ((18)F-BMS-747158-02), we characterized this tracer in a rat model of permanent and transient coronary occlusion using small-animal PET. METHODS: Myocardial (18)F-BMS-747158-02 distribution in healthy rats (n = 7), rats with transient (3-min) left coronary artery occlusion (n = 11), and rats with permanent left coronary occlusion (n = 11) was analyzed with a dedicated small-animal PET scanner. RESULTS: Normal hearts demonstrated intense and almost homogeneous tracer uptake throughout the left ventricle for more than 2 h. During permanent coronary occlusion, PET demonstrated perfusion defects, which remained unchanged (37.6% +/- 8.8%, 37.4% +/- 10.2%, and 36.2% +/- 9.8% left ventricle at 15, 45, and 115 min, respectively, after tracer injection). After transient ischemia, the induced defect size decreased significantly after reperfusion (16.2% +/- 9.3%, 6.0% +/- 6.5%, and 1.4% +/- 1.3% left ventricle). Tracer reinjection after transient ischemia resulted in normalization of the induced defect. CONCLUSION: Coronary occlusion yielded distinct myocardial (18)F-BMS-747158-02 uptake defects in the area of ischemia, which demonstrated normalization of activity after reperfusion and reinjection. These promising kinetic parameters may allow for assessment of flow using exercise-rest protocols similar to those used in combination with exercise and rest perfusion SPECT.

Preclinical Evaluation of the Hsp70 Peptide Tracer TPP-PEG24-DFO[89Zr] for Tumor-Specific PET/CT Imaging.

High precision in vivo PET/CT imaging of solid tumors improves diagnostic credibility and clinical outcome of patients. An epitope of the oligomerization domain of Hsp70 is exclusively exposed on the membrane of a large variety of tumor types, but not on normal cells, and thus provides a universal tumor-specific target. Here we developed a novel PET tracer TPP-PEG24-DFO[89Zr] based on the tumor cell-penetrating peptide probe TPP, which specifically recognizes membrane Hsp70 (mHsp70) on tumor cells. The implemented PEG24 moiety supported tracer stability and improved biodistribution characteristics in vivo The K d of the tracer ranged in the low nanomolar range (18.9 ± 11.3 nmol/L). Fluorescein isothiocyanate (FITC)-labeled derivatives TPP-[FITC] and TPP-PEG24-[FITC] revealed comparable and specific binding to mHsp70-positive 4T1, 4T1+, a derivative of the 4T1 cell line sorted for high Hsp70 expression, and CT26 tumor cells, but not to mHsp70-negative normal fibroblasts. The rapid internalization kinetics of mHsp70 into the cytosol and the favorable biodistribution of the peptide-based tracer TPP-PEG24-DFO[89Zr] in vivo enabled a tumor-specific accumulation with a high tumor-to-background contrast and renal body clearance. The tumor-specific enrichment of the tracer in 4T1+ (6.2 ± 1.1%ID/g), 4T1 (4.3 ± 0.7%ID/g), and CT26 (2.6 ± 0.6%ID/g) mouse tumors with very high, high, and intermediate mHsp70 densities, respectively, reflected mHsp70 expression profiles of the different tumor types, whereas benign mHsp70-negative fibroblastic hyperplasia showed no tracer accumulation (0.2 ± 0.03%ID/g). The ability of our chemically optimized peptide-based tracer TPP-PEG24-DFO[89Zr] to detect mHsp70 in vivo suggests its broad applicability in targeting and imaging with high specificity for any tumor type that exhibits surface expression of Hsp70.Significance: A novel peptide-based PET tracer against the oligomerization domain of Hsp70 has potential for universal tumor-specific imaging in vivo across many tumor type. Cancer Res; 78(21); 6268-81. ©2018 AACR.

Characterization of normal and infarcted rat myocardium using a combination of small-animal PET and clinical MRI.

UNLABELLED: The combination of small-animal PET and MRI data provides quantitative in vivo insights into cardiac pathophysiology, integrating information on biology and morphology. We sought to determine the feasibility of PET and MRI for the quantification of ischemic injury in the rat model. METHODS: Fourteen healthy male Wistar rats were studied with 18F-FDG PET and cine MRI. Myocardial viability was determined in a transmural myocardial infarction model in 12 additional rats, using 18F-FDG PET and delayed-enhancement MRI with gadolinium-diethylenetriaminepentaacetic acid. All PET was acquired with a dedicated small-animal PET system. MRI was performed on a 1.5-T clinical tomograph with a dedicated small-animal electrocardiographic triggering device and a small surface coil. RESULTS: In normal rats, 18F-FDG uptake was homogeneous throughout the left ventricle. The lowest mean uptake of the 18F-FDG was found in the apical regions (79% +/- 6.0% of maximum) and the highest uptake was in the anterior wall (93% +/- 4.3 % of maximum). Myocardial infarct size as determined by histology correlated well with defects of glucose metabolism obtained with 18F-FDG PET (r = 0.89) and also with delayed-enhancement MRI (r = 0.91). Left ventricular ejection fraction in normal rats measured by cine MRI was 57% +/- 5.4% and decreased to 38% +/- 12.9% (P < 0.001) in the myocardial infarction model. CONCLUSION: Integrating information from small-animal PET and clinical MRI instrumentation allows for the quantitative assessment of cardiac function and infarct size in the rat model. The MRI measurements of scar can be complemented by metabolic imaging, addressing the extent and severity of ischemic injury and providing endpoints for therapeutic interventions.

In-depth Characterization of a TCR-specific Tracer for Sensitive Detection of Tumor-directed Transgenic T Cells by Immuno-PET.

A number of different technologies have been developed to monitor in vivo the distribution of gene-modified T cells used in immunotherapy. Nevertheless, in-depth characterization of novel approaches with respect to sensitivity and clinical applicability are so far missing. We have previously described a novel method to track engineered human T cells in tumors using 89Zr-Df-aTCRmu-F(ab')2 targeting the murinized part of the TCR beta domain (TCRmu) of a transgenic TCR. Here, we performed an in-depth in vitro characterization of the tracer in terms of antigen affinity, immunoreactivity, influence on T-cell functionality and stability in vitro and in vivo. Of particular interest, we have developed diverse experimental settings to quantify TCR-transgenic T cells in vivo. Local application of 89Zr-Df-aTCRmu-F(ab')2-labeled T cells in a spot-assay revealed signal detection down to approximately 1.8x104 cells. In a more clinically relevant model, NSG mice were intravenously injected with different numbers of transgenic T cells, followed by injection of the 89Zr-Df-aTCRmu-F(ab')2 tracer, PET/CT imaging and subsequent ex vivo T-cell quantification in the tumor. Using this setting, we defined a comparable detection limit of 1.0x104 T cells. PET signals correlated well to total numbers of transgenic T cells detected ex vivo independently of the engraftment rates observed in different individual experiments. Thus, these findings confirm the high sensitivity of our novel PET/CT T-cell tracking method and provide critical information about the quantity of transgenic T cells in the tumor environment suggesting our technology being highly suitable for further clinical translation.

Noninvasive In Vivo Imaging and Biologic Characterization of Thyroid Tumors by ImmunoPET Targeting of Galectin-3.

The high prevalence of thyroid nodules in the adult population and the relatively low incidence of thyroid cancer make the preoperative identification of malignant lesions challenging. The ß-galactoside-binding protein galectin-3 is widely expressed in well-differentiated thyroid carcinomas, but not in normal thyrocytes and benign thyroid nodules. This molecule offers a candidate biomarker to improve thyroid cancer diagnosis. Here we report the development of an immunoPET approach for noninvasive imaging of thyroid cancer. The method employs a (89)Zr-labeled mAb to galectin-3, which shows high specificity and binding affinity in vitro Reliable and specific immunoPET imaging was obtained of thyroid cancer in vivo in murine xenograft models of human thyroid cancer. Our findings provide a method to improve the clinical management of patients with thyroid nodules while reducing unnecessary surgery and social costs. Cancer Res; 76(12); 3583-92. ©2016 AACR.

Noninvasive imaging of transgene expression by use of positron emission tomography in a pig model of myocardial gene transfer.

BACKGROUND: Radionuclide imaging of reporter gene expression may be useful for noninvasive monitoring of clinical cardiac gene therapy. Experience until now, however, has been limited to small animals. METHODS AND RESULTS: To evaluate feasibility in a clinically applicable setting, pigs were studied by conventional positron emission tomography (PET) 2 days after regional intramyocardial injection of control adenovirus or adenovirus carrying herpesviral thymidine kinase reporter gene (HSV1-tk). Myocardial blood flow was quantified by use of [13N]ammonia. Subsequently, kinetics of the reporter substrate [124I]-2'-fluoro-2'-deoxy-5-iodo-1-beta-d-arabino-furanosyluracil (FIAU) were assessed over a period of 2 hours. Areas infected with adenovirus expressing HSV1-tk showed significantly elevated FIAU retention during the first 30 minutes after injection. At later times, washout was observed, and retention was not different from that in areas infected with control virus or remote myocardium. Early in vivo FIAU uptake correlated with ex vivo images, autoradiography, and immunohistochemistry for reporter gene product after euthanasia. After intramyocardial injection of both adenoviruses, myocardial blood flow was mildly elevated compared with that in remote areas, consistent with histological signs of regional inflammation. CONCLUSIONS: In vivo quantification of regional myocardial transgene expression is feasible with clinical PET methodology, the radioiodinated reporter probe FIAU, and the HSV1-tk reporter gene. Radioactivity efflux after specific initial uptake was not observed previously in tumor studies, suggesting that tissue-specific differences in nucleoside metabolism influence reporter probe kinetics. By coregistering reporter gene expression with additional biological parameters such as myocardial blood flow, PET allows for noninvasive characterization of the success of cardiac gene transfer along with its functional correlates.

Delayed response of insulin-stimulated fluorine-18 deoxyglucose uptake in glucose transporter-4-null mice hearts.

OBJECTIVES: We sought to evaluate the time course of insulin-stimulated myocardial glucose uptake (MGU) in mice that had undergone ablation of glucose transporter-4 (GLUT4). BACKGROUND: The relative importance of GLUT4, the most abundant insulin-responsive glucose transporter, to modulate myocardial glucose metabolism is not well defined. METHODS: Myocardial glucose uptake was assessed at various time points after glucose (1 mg/g) and insulin (8 mU/g) injection in GLUT4-null (G4N) (n = 48) and wild-type (WT) (n = 48) mice with (18)F-2-deoxy-2-fluoro-d-glucose (FDG) using in vivo positron emission tomography (PET), in vitro gamma-counter biodistribution, and isolated, perfused hearts. RESULTS: Baseline assessment with PET imaging showed comparable MGU in G4N (0.66 +/- 0.12) and WT (0.67 +/- 0.11, p = 0.70) mice. Early after insulin injection, WT mice demonstrated a 3.5-fold increase in MGU (2.45 +/- 0.45, p = 0.03), whereas G4N mice presented no increase (1.11 +/- 0.24, p = 0.28). At 60 min, MGU was comparable in G4N (3.19 +/- 0.60) and WT (2.66 +/- 0.47, p = 0.28) mice. In vitro gamma-counter biodistribution evaluation confirmed in G4N mice a lack of MGU increase early after insulin, but a slow response over 120 min. The isolated, perfused hearts of G4N mice during short-term (15 min) insulin stimulation displayed no increase in MGU (0.08 +/- 0.01 ml/g/min), whereas WT mice presented a threefold increase (0.22 +/- 0.01 ml/g/min, p < 0.01). With long-term (60 min) insulin stimulation, similar MGU was found in G4N (0.31 +/- 0.02 ml/g/min) and WT (0.33 +/- 0.04 ml/g per min, p = 0.04) mice. CONCLUSIONS: The G4N mice displayed an increase of MGU in response to insulin similar to that of controls, but with a markedly delayed time response. Our findings underscore the important role of GLUT4 in the rapid adaptive response of myocardial glucose metabolism.

Myocardial kinetics of reporter probe 124I-FIAU in isolated perfused rat hearts after in vivo adenoviral transfer of herpes simplex virus type 1 thymidine kinase reporter gene.

UNLABELLED: Reporter gene imaging holds promise for noninvasive monitoring of cardiac gene therapy. We recently demonstrated that (124)I-labeled 2'-fluoro-2'-deoxy-5'-iodo-1beta-d-arabinofuranosyluracil ((124)I-FIAU) is suitable for PET of myocardial expression of herpes simplex virus type 1 thymidine kinase reporter gene (HSV1-tk). In contrast to previous studies in tumors, early specific uptake was followed by rapid washout. Myocardial kinetics of (124)I-FIAU are still poorly understood. This study aimed at a further investigation under controlled conditions using an isolated heart perfusion model. METHODS: Male Wistar rats underwent transthoracic regional injection of replication-defective adenovirus (2.5 x 10(9) plaque-forming units) containing either HSV1-tk (n = 16) or LacZ reporter gene (n = 15) into the inferior wall. Nonmanipulated rats (n = 5) served as further controls. Hearts were excised 2 d later and perfused according to the Langendorff technique with (124)I-FIAU-containing buffer (15 min, followed by 30 min of nonradioactive perfusion). Experiments were performed under baseline conditions and in the presence of thymidine (competitive substrate) or fludarabine (in vitro inhibitor of 5'-nucleotidase). Time-activity curves were acquired by external coincident detectors. The myocardial rate of (124)I-FIAU uptake (K(i)), clearance rate (K(o)), and volume of distribution (V(d) = K(i)/K(o)) were calculated. Subsequently, hearts were subjected to gamma-counting, followed by microtome slicing and autoradiography. RESULTS: The V(d) from Langendorff perfusion significantly correlated with final whole-heart tracer retention (r = 0.88, P = 0.019) and the autoradiographic area of regional myocardial activity (r = 0.89, P = 0.016). HSV1-tk hearts showed higher K(i) and V(d) of (124)I-FIAU compared with that of controls (P < 0.001) and detectable but slower washout compared with that of the LacZ group (P < 0.01). Addition of thymidine to the perfusate inhibited myocardial uptake of (124)I-FIAU by reducing V(d) and K(i) in HSV1-tk and LacZ hearts compared with the baseline. Addition of fludarabine did not result in the expected reduction of washout in HSV1-tk hearts due to inhibition of 5'-nucleotidases (which may dephosphorylate (124)I-FIAU monophosphate). It acted as an uptake inhibitor similar to thymidine, reducing V(d) in HSV1-tk hearts. CONCLUSION: Assessment of specific reporter probe kinetics after regional in vivo reporter gene transfer is feasible using the isolated perfused rat heart preparation. This model allows one to study the effects of pharmacologic interventions and may refine understanding of the reporter probe signal for in vivo imaging. Different nucleoside analogs significantly inhibit (124)I-FIAU uptake, emphasizing the importance of transporter mechanisms for reporter probe kinetics.

Comparison of 18F-Labeled Fluoroalkylphosphonium Cations with 13N-NH3 for PET Myocardial Perfusion Imaging.

UNLABELLED: Despite substantial advances in the diagnosis of cardiovascular disease, there is a need for 18F-labeled myocardial perfusion agents for the diagnosis of ischemic heart disease because current PET tracers for myocardial perfusion imaging have a short half-life that limits their widespread clinical use in PET. Thus, 18F-labeled fluoroalkylphosphonium derivatives (18F-FATPs), including (5-18F-fluoropentyl)triphenylphosphonium cation (18F-FPTP), (6-18F-fluorohexyl)triphenylphosphonium cation (18F-FHTP), and (2-(2-18F-fluoroethoxy)ethyl)triphenylphosphonium cation (18F-FETP), were synthesized. The myocardial extraction and image quality of the 18F-FATPs were compared with those of 13N-NH3 in rat models. METHODS: The first-pass extraction fraction (EF) values of the 18F-FATPs (18F-FPTP, 18F-FHTP, 18F-FETP) and 13N-NH3 were measured in isolated rat hearts perfused with the Langendorff method (flow velocities, 0.5, 4.0, 8.0, and 16.0 mL/min). Normal and myocardial infarction rats were imaged with small-animal PET after intravenous injection of 37 MBq of 18F-FATPs and 13N-NH3. To determine pharmacokinetics, a region of interest was drawn around the heart, and time-activity curves of the 18F-FATPs and 13N-NH3 were generated to obtain the counts per pixel per second. Defect size was analyzed on the basis of polar map images of 18F-FATPs and 13N-NH3. RESULTS: The EF values of 18F-FATPs and 13N-NH3 were comparable at low flow velocity (0.5 mL/min), whereas at higher flows EF values of 18F-FATPs were significantly higher than those of 13N-NH3 (4.0, 8.0, and 16.0 mL/min, P<0.05). Myocardium-to-liver ratios of 18F-FPTP, 18F-FHTP, 18F-FETP, and 13N-NH3 were 2.10±0.30, 4.36±0.20, 3.88±1.03, and 0.70±0.09, respectively, 10 min after injection, whereas myocardium-to-lung ratios were 5.00±0.25, 4.33±0.20, 7.98±1.23, and 2.26±0.14, respectively. Although 18F-FATPs and 13N-NH3 sharply delineated myocardial perfusion defects, defect size on the 13N-NH3 images was significantly smaller than on the 18F-FATP images soon after tracer injection (0-10 min, P=0.027). CONCLUSION: 18F-FATPs exhibit higher EF values and more rapid clearance from the liver and lung than 13N-NH3 in normal rats, which led to excellent image quality in a rat model of coronary occlusion. Therefore, 18F-FATPs are promising new PET radiopharmaceuticals for myocardial perfusion imaging.

Evaluation of cardiac beta-adrenoreceptors in the isolated perfused rat heart using (S)-11C-CGP12388.

UNLABELLED: (S)-(11)C-CGP12388 ((11)C-CGP12388) was recently developed as an in vivo PET tracer for the evaluation of cardiac beta-adrenergic receptors. The purpose of this study was to evaluate the myocardial kinetics of (11)C-CGP12388 using the perfused rat heart model. METHODS: Normal rat hearts were cannulated for retrograde perfusion according to the Langendorff method. Studies were performed using constant coronary flow rates of 12 mL/min (high flow: n = 6) and 6 mL/min (low flow: n = 6). Beta-adrenergic-blocking studies were also done using propranolol (blocking: n = 6). Two bolus injections of (11)C-CGP12388 were administered at a 25-min interval, and time-activity curves were measured using bismuth germanate detectors. The beta-adrenergic receptor density (B(max)) and total distribution volume (DV(tot)) were estimated using compartmental modeling. After the experiment, B(max) in vitro was measured for all hearts using (3)H-CGP12177, and the values were compared with the B(max) estimated in isolated hearts. RESULTS: DV(tot) was significantly lower in the blocking group than in the high-flow group (P < 0.01), and there was no significant difference in DV(tot) between the high- and the low-flow groups. B(max) values estimated from (11)C-CGP12388 kinetics were 5.05 +/- 0.90 pmol/g under the high-flow model and 5.20 +/- 0.63 pmol/g under the low-flow model. The B(max) results in isolated hearts correlated significantly with the measured in vitro B(max) values (r(2) = 0.69; P < 0.001). CONCLUSION: Beta-adrenoreceptor density in the isolated rat heart can be quantified using (11)C-CGP12388 and a 2-injection protocol. The binding of the tracer was flow independent, with low nonspecific binding. These results suggest that (11)C-CGP12388 is a promising PET tracer that may be applicable to human studies.