Stefan Wiehr

Pre-clinical PET/MR: technological advances and new perspectives in biomedical research.

IntroductionCombined PET/MRI allows for multi-parametric imaging and reveals one or more functional processes simultaneously along with high-resolution morphology. Especially in small-animal research, where high soft tissue contrast is required, and the scan time as well as radiation dose are critical factors, the combination of PET and MRI would be beneficial compared with PET/CT.DevelopmentIn the mid-1990’s, several research groups used different approaches to integrate PET detectors into high-field MRI. First, systems were based on optical fibres guiding the scintillation light to the PMT’s, which reside outside the fringe magnetic field. Recent advances in gamma ray detector technology, which were initiated mainly by the advent of avalanche photodiodes (APD’s) as well as the routine availability of fast scintillation materials like lutetium oxyorthosilicate (LSO), paved the way towards the development of fully magnetic-field-insensitive high-performance PET detectors.TechnologyCurrent animal PET/MR technologies are reviewed and pitfalls when engineering a full integration of a PET and a high-field MRI are discussed. Compact PET detectors can be integrated in small-bore, high-field MRI tomographs. Detailed performance evaluations have shown that the mutual interference between the two imaging systems could be minimized. The performance of all major MR applications, ranging from T1- or T2-weighted imaging up to echo-planar imaging (EPI) for functional MRI (fMRI) or magnetic resonance spectroscopy (MRS), could be maintained, even when the PET insert was built into the MRI and acquiring PET data simultaneously. Similarly, the PET system performance was not influenced by the static magnetic field or applied MRI sequences.ApplicationsInitial biomedical research applications range from the combination of functional information from PET with the anatomical information from the MRI to multi-functional imaging combining metabollic PET and MRI data.DiscussionCompared to other multi-modality approaches PET/MR offers a multitude of complementary function and anatomical information. The ability to obtain simultaneous PET and MRI data with this new imaging modality could have tremendous impact on small animal imaging research.

PET imaging of prostate cancer xenografts with a highly specific antibody against the prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is highly expressed by virtually all prostate cancers and is currently the focus of several diagnostic and therapeutic strategies. We have previously reported on the generation of several monoclonal antibodies (mAb) and antibody fragments that recognize and bind with high affinity to the extracellular domain of cell-adherent PSMA. This article reports the in vivo behavior and tumor uptake of the radiolabeled anti-PSMA mAb 3/A12 and its potential as a tracer for PET. Methods: The mAb 3/A12 was conjugated with the chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and radiolabeled with 64Cu. Severe combined immunodeficient mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were used for small-animal PET imaging. Mice with PSMA-negative DU 145 tumors served as controls. For PET studies, each animal received 20–30 μg of radiolabeled mAb corresponding to an activity of 7.6–11.5 MBq. Imaging was performed 3, 24, and 48 h after injection. After the last scan, the mice were sacrificed and tracer in vivo biodistribution was measured by γ-counting. Results: Binding of the mAb 3/A12 on PSMA-expressing C4-2 cells was only minimally influenced by DOTA conjugation. The labeling efficiency using 64Cu and DOTA-3/A12 was 95.3% ± 0.3%. The specific activity after 64Cu labeling was between 327 and 567 MBq/mg. After tracer injection, static small-animal PET images of mice with PSMA-positive tumors revealed a tumor-to-background ratio of 3.3 ± 1.3 at 3 h, 7.8 ± 1.4 at 24 h, and 9.6 ± 2.7 at 48 h. In contrast, no significant tracer uptake occurred in the PSMA-negative DU 145 tumors. These results were confirmed by direct counting of tissues after the final imaging. Conclusion: Because of the high and specific uptake of 64Cu-labeled mAb 3/A12 in PSMA-positive tumors, this ligand represents an excellent candidate for prostate cancer imaging and potentially for radioimmunotherapy.

SOX2 Expression Associates with Stem Cell State in Human Ovarian Carcinoma

The SRY-related HMG-box family of transcription factors member SOX2 regulates stemness and pluripotency in embryonic stem cells and plays important roles during early embryogenesis. More recently, SOX2 expression was documented in several tumor types including ovarian carcinoma, suggesting an involvement of SOX2 in regulation of cancer stem cells (CSC). Intriguingly, however, studies exploring the predictive value of SOX2 protein expression with respect to histopathologic and clinical parameters report contradictory results in individual tumors, indicating that SOX2 may play tumor-specific roles. In this report, we analyze the functional relevance of SOX2 expression in human ovarian carcinoma. We report that in human serous ovarian carcinoma (SOC) cells, SOX2 expression increases the expression of CSC markers, the potential to form tumor spheres, and the in vivo tumor-initiating capacity, while leaving cellular proliferation unaltered. Moreover, SOX2-expressing cells display enhanced apoptosis resistance in response to conventional chemotherapies and TRAIL. Hence, our data show that SOX2 associates with stem cell state in ovarian carcinoma and induction of SOX2 imposes CSC properties on SOC cells. We propose the existence of SOX2-expressing ovarian CSCs as a mechanism of tumor aggressiveness and therapy resistance in human SOC.

Longitudinal PET-MRI reveals [beta]-amyloid deposition and rCBF dynamics and connects vascular amyloidosis to quantitative loss of perfusion

The dynamics of β-amyloid deposition and related second-order physiological effects, such as regional cerebral blood flow (rCBF), are key factors for a deeper understanding of Alzheimers disease (AD). We present longitudinal in vivo data on the dynamics of β-amyloid deposition and the decline of rCBF in two different amyloid precursor protein (APP) transgenic mouse models of AD. Using a multiparametric positron emission tomography and magnetic resonance imaging approach, we demonstrate that in the presence of cerebral β-amyloid angiopathy (CAA), β-amyloid deposition is accompanied by a decline of rCBF. Loss of perfusion correlates with the growth of β-amyloid plaque burden but is not related to the number of CAA-induced microhemorrhages. However, in a mouse model of parenchymal β-amyloidosis and negligible CAA, rCBF is unchanged. Because synaptically driven spontaneous network activity is similar in both transgenic mouse strains, we conclude that the disease-related decline of rCBF is caused by CAA.

ImmunoPET/MR imaging allows specific detection of Aspergillus fumigatus lung infection in vivo

Significance Invasive pulmonary aspergillosis (IPA) is a frequently fatal lung disease of immunocompromised patients, and is being increasingly reported in individuals with underlying respiratory diseases. Proven diagnosis of IPA currently relies on lung biopsy and detection of diagnostic biomarkers in serum, or in bronchoalveolar lavage fluids. This study supports the use of immunoPET/MR imaging for the diagnosis of IPA, which is so far not used for diagnosis. The antibody-guided imaging technique allows accurate, noninvasive and rapid detection of fungal lung infection and discrimination of IPA from bacterial lung infections and general inflammatory responses. This work demonstrates the applicability of molecular imaging for IPA detection and its potential for aiding clinical diagnosis and management of the disease in the neutropenic host. Invasive pulmonary aspergillosis (IPA) is a life-threatening lung disease caused by the fungus Aspergillus fumigatus, and is a leading cause of invasive fungal infection-related mortality and morbidity in patients with hematological malignancies and bone marrow transplants. We developed and tested a novel probe for noninvasive detection of A. fumigatus lung infection based on antibody-guided positron emission tomography and magnetic resonance (immunoPET/MR) imaging. Administration of a [64Cu]DOTA-labeled A. fumigatus-specific monoclonal antibody (mAb), JF5, to neutrophil-depleted A. fumigatus-infected mice allowed specific localization of lung infection when combined with PET. Optical imaging with a fluorochrome-labeled version of the mAb showed colocalization with invasive hyphae. The mAb-based newly developed PET tracer [64Cu]DOTA-JF5 distinguished IPA from bacterial lung infections and, in contrast to [18F]FDG-PET, discriminated IPA from a general increase in metabolic activity associated with lung inflammation. To our knowledge, this is the first time that antibody-guided in vivo imaging has been used for noninvasive diagnosis of a fungal lung disease (IPA) of humans, an approach with enormous potential for diagnosis of infectious diseases and with potential for clinical translation.

High-resolution animal PET imaging of prostate cancer xenografts with three different 64Cu-labeled antibodies against native cell-adherent PSMA.

The prostate specific membrane antigen (PSMA) is expressed by virtually all prostate cancers and represents an ideal target for diagnostic and therapeutic strategies. This article compares the in vivo behavior and tumor uptake of three different radiolabeled anti‐PSMA monoclonal antibodies (mAbs) and corresponding F(ab)2 and Fab fragments thereof.

Preclinical and Translational PET/MR Imaging

Combined PET and MR imaging (PET/MR imaging) has progressed tremendously in recent years. The focus of current research has shifted from technologic challenges to the application of this new multimodal imaging technology in the areas of oncology, cardiology, neurology, and infectious diseases. This article reviews studies in preclinical and clinical translation. The common theme of these initial results is the complementary nature of combined PET/MR imaging that often provides additional insights into biologic systems that were not clearly feasible with just one modality alone. However, in vivo findings require ex vivo validation. Combined PET/MR imaging also triggers a multitude of new developments in image analysis that are aimed at merging and using multimodal information that ranges from better tumor characterization to analysis of metabolic brain networks. The combination of connectomics information that maps brain networks derived from multiparametric MR data with metabolic information from PET can even lead to the formation of a new research field that we would call cometomics that would map functional and metabolic brain networks. These new methodologic developments also call for more multidisciplinarity in the field of molecular imaging, in which close interaction and training among clinicians and a variety of scientists is needed.

Phosphoglycerate kinase 1 promoting tumor progression and metastasis in gastric cancer - detected in a tumor mouse model using positron emission tomography/magnetic resonance imaging.

Background/Aims: Tumor dissemination is frequent in gastric cancer and implies a poor prognosis. Cure is only achievable provided an accurate staging is performed at primary diagnosis. In previous studies we were able to show a relevant impact of increased phosphoglycerate kinase 1 expression (PGK1; a glycolytic enzyme) on invasive properties of gastric cancer in-vivo and in-vitro. Thus the aim of the present study was to evaluate the effect of enhanced PGK1 expression in gastric cancer employing magnetic resonance (MR)-imaging combined with positron emission tomography (PET), a recently emerging new high resolution imaging technique in a mouse model. Methods: A metastatic nude mouse model simulating human gastric cancer behavior by orthotopic tumor implantation was established. Mice were divided into one control group (n=5) and two experimental groups (n=30) divided by half in animals baring tumors from MKN45-cells and MKN45-cells with plasmid-mediated overexpression of PGK1. In the course of tumor growth MR-imaging and PET/MRI fusion was performed. Successively experimental animals were examined macroscopically and histopathologically regarding growth, metastasis and PGK1 expression. Results: Elevated PGK1 expression increased invasive and metastatic behavior of implanted gastric tumors significantly. MR/PET- imaging results in-vivoand subsequent ex-vivo findings concerning tumor growth and metastasis correlated excellently and could be underlined by concordant immuohistochemical PGK1 staining. Conclusion: Consistent in-vivo findings suggest that PGK1 might be crucially involved in gastric malignancy regarding growth and metastasis, which was also underlined by novel imaging techniques. Thus, PGK1 may be exploited as a prognostic marker and/or be of potential therapeutic value preventing malignant dissemination.

Assessment of PET Tracer Uptake in Hormone-Independent and Hormone-Dependent Xenograft Prostate Cancer Mouse Models

The pharmacokinetics of 18F-fluorodeoxythymidine (FLT), 18F-FDG, 11C-choline, and 18F-fluoroethylcholine (FEC) in 2 hormone-independent (PC-3, DU145) and 2 hormone-dependent (CWR22, PAC120) prostate cancer xenograft mouse models were evaluated by PET and compared by immunohistochemistry. Further investigation was performed to determine whether PET can detect early changes in tumor metabolism after androgen ablation therapy through surgical castration. Methods: PET was performed on 4 consecutive days. In addition, the CWR22 and PAC120 tumor models were surgically castrated after the baseline measurement and imaged again after castration. The tracer uptake was analyzed using time–activity curves, percentage injected dose per volume (%ID/cm3), and tumor-to-muscle ratio (T/M). Results: Regarding the hormone-independent prostate tumor models, 18F-FLT showed the best T/M and highest %ID/cm3 in PC-3 (2.97 ± 0.63 %ID/cm3) and DU145 (2.06 ± 0.75 %ID/cm3) tumors. 18F-FDG seemed to be the tracer of choice for delineation of the PC-3 tumors but not for the DU145 tumors. Using 11C-choline (PC-3: 1.33 ± 0.29 %ID/cm3, DU145: 1.60 ± 0.27 %ID/cm3) and 18F-FEC, we did not find any significant uptake in the tumors, compared with muscle tissue. Regarding the hormone-dependent prostate tumor models, the CWR22 model showed a highly significant (P < 0.01) decrease in tumor 18F-FDG uptake from 4.11 ± 1.29 %ID/cm3 to 2.19 ± 1.45 %ID/cm3 after androgen ablation therapy. However, the 18F-FLT, 11C-choline, or 18F-FEC tracers did not provide sufficient uptake or reliable information about therapy response in CWR22 tumors. The PAC120 model showed a significant increase in 18F-FLT tumor uptake (P = 0.015) after androgen ablation therapy. The accumulation of 18F-FEC (before: 2.32 ± 1.01 %ID/cm3, after: 1.36 ± 0.39 %ID/cm3) was found to be the next highest after 18F-FDG (before: 2.45 ± 0.93 %ID/cm3, after: 2.18 ± 0.65 %ID/cm3) in PAC120 tumors before castration and is better suited for monitoring therapy response. Conclusion: This comprehensive study in 2 hormone-dependent and 2 hormone-independent prostate tumor mouse models shows that 18F-FLT and 18F-FDG are the most appropriate tracers for delineation of PC-3, DU145 (except 18F-FDG), and CWR22 tumors, but not for PAC120 tumors. 18F-FEC and 11C-choline, in particular, revealed insufficient T/M ratio in the prostate tumor models. The results may indicate that radiolabeled choline and choline derivatives compete with a high concentration of the precursor dimethylaminoethanol, resulting in reduced uptake in small-rodent tumor models, a hypothesis that is currently under investigation in our laboratory.

Assessment of murine brain tissue shrinkage caused by different histological fixatives using magnetic resonance and computed tomography imaging

Especially for neuroscience and the development of new biomarkers, a direct correlation between in vivo imaging and histology is essential. However, this comparison is hampered by deformation and shrinkage of tissue samples caused by fixation, dehydration and paraffin embedding. We used magnetic resonance (MR) imaging and computed tomography (CT) imaging to analyze the degree of shrinkage on murine brains for various fixatives. After in vivo imaging using 7 T MRI, animals were sacrificed and the brains were dissected and immediately placed in different fixatives, respectively: zinc-based fixative, neutral buffered formalin (NBF), paraformaldehyde (PFA), Bouin-Holland fixative and paraformaldehyde-lysine-periodate (PLP). The degree of shrinkage based on mouse brain volumes, radiodensity in Hounsfield units (HU), as well as non-linear deformations were obtained. The highest degree of shrinkage was observed for PLP (68.1%, P < 0.001), followed by PFA (60.2%, P<0.001) and NBF (58.6%, P<0.001). The zinc-based fixative revealed a low shrinkage with only 33.5% (P<0.001). Compared to NBF, the zinc-based fixative shows a slightly higher degree of deformations, but is still more homogenous than PFA. Tissue shrinkage can be monitored non-invasively with CT and MR. Zinc-based fixative causes the smallest degree of brain shrinkage and only small deformations and is therefore recommended for in vivo ex vivo comparison studies.