Publications by authors named "Anne Rix"

34 Publications

Regorafenib enhances anti-PD1 immunotherapy efficacy in murine colorectal cancers and their combination prevents tumor regrowth.

J Exp Clin Cancer Res 2021 Sep 13;40(1):288. Epub 2021 Sep 13.

Institute for Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Background: Patients with advanced colorectal cancer (CRC) have a poor prognosis. Combinations of immunotherapies and anti-angiogenic agents are currently being evaluated in clinical trials. In this study, the multikinase inhibitor regorafenib (REG) was combined with an anti-programmed cell death protein 1 (aPD1) antibody in syngeneic murine microsatellite-stable (MSS) CT26 and hypermutated MC38 colon cancer models to gain mechanistic insights into potential drug synergism.

Methods: Growth and progression of orthotopic CT26 and subcutaneous MC38 colon cancers were studied under treatment with varying doses of REG and aPD1 alone or in combination. Sustained effects were studied after treatment discontinuation. Changes in the tumor microenvironment were assessed by dynamic contrast-enhanced MRI, and histological and molecular analyses.

Results: In both models, REG and aPD1 combination therapy significantly improved anti-tumor activity compared with single agents. However, in the CT26 model, the additive benefit of aPD1 only became apparent after treatment cessation. The combination treatment efficiently prevented tumor regrowth and completely suppressed liver metastasis, whereas the anti-tumorigenic effects of REG alone were abrogated soon after drug discontinuation. During treatment, REG significantly reduced the infiltration of immunosuppressive macrophages and regulatory T (Treg) cells into the tumor microenvironment. aPD1 significantly enhanced intratumoral IFNγ levels. The drugs synergized to induce sustained M1 polarization and durable reduction of Treg cells, which can explain the sustained tumor suppression.

Conclusions: This study highlights the synergistic immunomodulatory effects of REG and aPD1 combination therapy in mediating a sustained inhibition of colon cancer regrowth, strongly warranting clinical evaluation in CRC, including MSS tumors.
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http://dx.doi.org/10.1186/s13046-021-02043-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8436536PMC
September 2021

Molecular magnetic resonance imaging of Alpha-v-Beta-3 integrin expression in tumors with ultrasound microbubbles.

Biomaterials 2021 08 27;275:120896. Epub 2021 May 27.

Institute for Experimental Molecular Imaging, RWTH Aachen University, 52074, Aachen, Germany. Electronic address:

Microbubbles (MB) are used as ultrasound (US) contrast agents and can be efficiently targeted against markers of angiogenesis and inflammation. Due to their gas core, MB locally alter susceptibilities in magnetic resonance imaging (MRI), but unfortunately, the resulting contrast is low and not sufficient to generate powerful molecular MRI probes. Therefore, we investigated whether a potent molecular MR agent can be generated by encapsulating superparamagnetic iron oxide nanoparticles (SPION) in the polymeric shell of poly (n-butylcyanoacrylate) (PBCA) MB and targeted them against αβ integrins on the angiogenic vasculature of 4T1 murine breast carcinomas. SPION-MB consist of an air core and a multi-layered polymeric shell enabling efficient entrapment of SPION. The mean size of SPION-MB was 1.61 ± 0.32 μm. Biotin-streptavidin coupling was employed to functionalize the SPION-MB with cyclic RGDfK (Arg-Gly-Asp) and RADfK (Arg-Ala-Asp) peptides. Cells incubated with RGD-SPION-MB showed enhanced transverse relaxation rates compared with SPION-MB and blocking αβ integrin receptors with excess free cRGDfK significantly reduced RGD-SPION-MB binding. Due to the fast binding of RGD-SPION-MB in vivo, dynamic susceptibility contrast MRI was employed to track their retention in tumors in real-time. Higher retention of RGD-SPION-MB was observed compared with SPION-MB and RAD-SPION-MB. To corroborate our MRI results, molecular US was performed the following day using the destruction-replenishment method. Both imaging modalities consistently indicated higher retention of RGD-SPION-MB in angiogenic vessels compared with SPION-MB and RAD-SPION-MB. Competitive blocking experiments in mice further confirmed that the binding of RGD-SPION-MB to αβ integrin receptors is specific. Overall, this study demonstrates that RGD-SPION-MB can be employed as molecular MR/US contrast agents and are capable of assessing the αβ integrin expression in the neovasculature of malignant tumors.
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http://dx.doi.org/10.1016/j.biomaterials.2021.120896DOI Listing
August 2021

Change of Apoptosis and Glucose Metabolism in Lung Cancer Xenografts during Cytotoxic and Anti-Angiogenic Therapy Assessed by Annexin V Based Optical Imaging and F-FDG-PET/CT.

Contrast Media Mol Imaging 2021 10;2021:6676337. Epub 2021 Apr 10.

Institute for Experimental Molecular Imaging, RWTH Aachen University, Aachen, Germany.

Methods: For apoptosis imaging, the near-infrared probe Annexin Vivo750 was used in combination with fluorescence molecular tomography and microcomputed tomography (FMT/CT). Glucose metabolism was assessed using F-FDG-PET/CT. Five groups of nude mice bearing lung cancer xenografts (A549) were investigated: (i) untreated controls and two groups after (ii) cytotoxic (carboplatin) or (iii) anti-angiogenic (sunitinib) treatment for four and nine days, respectively. Imaging data were validated by immunohistochemistry.

Results: In response to carboplatin treatment, an inverse relation was found between the change in glucose metabolism and apoptosis in A549 tumors. Annexin Vivo showed a continually increasing tumor accumulation, while the tumor-to-muscle ratio of F-FDG continuously decreased during therapy. Immunohistochemistry revealed a significantly higher tumor apoptosis (=0.007) and a minor but not significant reduction in vessel density only at day 9 of carboplatin therapy. Interestingly, during anti-angiogenic treatment there was an early drop in the tumor-to-muscle ratio between days 0 and 4, followed by a subsequent minor decrease (F-FDG tumor-to-muscle-ratio: 1.9 ± 0.4; day 4: 1.1 ± 0.2; day 9: 1.0 ± 0.2; =0.021 and =0.001, respectively). The accumulation of Annexin Vivo continuously increased over time (Annexin Vivo: untreated: 53.7 ± 36.4 nM; day 4: 87.2 ± 53.4 nM; day 9: 115.1 ± 103.7 nM) but failed to display the very prominent early induction of tumor apoptosis that was found by histology already at day 4 (TUNEL: =0.0036) together with a decline in vessel density (CD31: =0.004), followed by no significant changes thereafter.

Conclusion: Both molecular imaging approaches enable visualizing the effects of cytotoxic and anti-angiogenic therapy in A549 tumors. However, the early and strong tumor apoptosis induced by the anti-angiogenic agent sunitinib was more sensitively and reliably captured by monitoring of the glucose metabolism as compared to Annexin V-based apoptosis imaging.
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http://dx.doi.org/10.1155/2021/6676337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8057888PMC
April 2021

Corrigendum to "MR and PET-CT monitoring of tissue-engineered vascular grafts in the ovine carotid artery" [Biomaterials 216 (2019) 119228].

Biomaterials 2021 Apr 23;271:120723. Epub 2021 Feb 23.

Dept. of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute Aachen and Institute for Textiles Engineering (ITA), RWTH Aachen University, Forckenbeckstr. 55, 52074, Aachen, Germany. Electronic address:

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http://dx.doi.org/10.1016/j.biomaterials.2021.120723DOI Listing
April 2021

Molecular Ultrasound Imaging.

Recent Results Cancer Res 2020 ;216:509-531

Institute for Experimental Molecular Imaging Organization University Clinics, RWTH Aachen University, Forckenbeckstrasse 55, 52074 Aachen, Germany.

Contrast-enhanced ultrasound (CEUS) imaging is a valuable tool for preclinical and clinical diagnostics. The most frequently used ultrasound contrast agents are microbubbles. Besides them, novel nano-sized materials are under investigation, which are briefly discussed in this chapter. For molecular CEUS, the ultrasound contrast agents are modified to actively target disease-associated molecular markers with a site-specific ligand. The most common markers for tumor imaging are related to neoangiogenesis, like the vascular endothelial growth factor receptor-2 (VEGFR2) and αβ integrin. In this chapter, applications of molecular ultrasound to longitudinally monitor receptor expression during tumor growth, to detect neovascularization, and to evaluate therapy responses are described. Furthermore, we report on first clinical trials of molecular CEUS with VEGFR2-targeted phospholipid microbubbles showing promising results regarding patient safety and its ability to detect tumors of prostate, breast, and ovary. The chapter closes with an outlook on ultrasound theranostics, where (targeted) ultrasound contrast agents are used to increase the permeability of tumor tissues and to support drug delivery.
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http://dx.doi.org/10.1007/978-3-030-42618-7_15DOI Listing
September 2020

Influence of MRI Examinations on Animal Welfare and Study Results.

Invest Radiol 2020 08;55(8):507-514

From the Institute for Experimental Molecular Imaging.

Objectives: Magnetic resonance imaging (MRI) is considered to be well tolerated by laboratory animals. However, no systematic study has been performed yet, proving this assumption. Therefore, the aim of this study was to investigate the possible effects of longitudinal native and contrast-enhanced (CE) 1-T and 7-T MRI examinations on mouse welfare as well as 4T1 breast cancers progression and therapy response.

Material And Methods: Forty-seven healthy and 72 breast cancer-bearing mice (4T1) were investigated. One-Tesla (ICON) and 7-T (Biospec) MRI measurements were performed thrice per week under isoflurane anesthesia in healthy BALB/c mice for 4 weeks and 3 times within 2 weeks in tumor-bearing animals. Animal welfare was examined by an observational score sheet, rotarod performance, heart rate measurements, and assessment of fecal corticosterone metabolites. Furthermore, we investigated whether CE-MRI influences the study outcome. Therefore, hemograms and organ weights were obtained, and 4T1 tumor growth, perfusion, immune cell infiltration, as well as response to the multikinase inhibitor regorafenib were investigated. Statistical comparisons between groups were performed using analysis of variance and Tukey or Bonferroni post hoc tests.

Results: Mice showed no alterations in the observational score sheet rating, rotarod performance, heart rate, and fecal corticosterone metabolites (P > 0.05) after repeated MRI at both field strengths. However, spleen weights were reduced in all healthy mouse groups that received isoflurane anesthesia (P < 0.001) including the groups investigated by 1-T and 7-T MRI (P = 0.02). Neither tumor progression nor response to the regorafenib treatment was affected by isoflurane anesthesia or CE-MRI monitoring. Furthermore, immunohistological tumor analysis did not indicate an effect of isoflurane and MRI on macrophage infiltration of tumors, perfusion of tumor vessels, and apoptotic cell rate (P > 0.05).

Conclusions: Repeated MRI did not influence the welfare of mice and did not affect tumor growth and therapy response of 4T1 tumors. However, systemic immunological effects of isoflurane anesthesia need to be considered to prevent potential bias.
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http://dx.doi.org/10.1097/RLI.0000000000000669DOI Listing
August 2020

Multimodal and multiscale optical imaging of nanomedicine delivery across the blood-brain barrier upon sonopermeation.

Theranostics 2020 12;10(4):1948-1959. Epub 2020 Jan 12.

Institute for Experimental Molecular Imaging (ExMI), University Clinic and Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Aachen, Germany.

: The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. Sonopermeation, which relies on the combination of ultrasound and microbubbles, has emerged as a powerful tool to permeate the BBB, enabling the extravasation of drugs and drug delivery systems (DDS) to and into the central nervous system (CNS). When aiming to improve the treatment of high medical need brain disorders, it is important to systematically study nanomedicine translocation across the sonopermeated BBB. To this end, we here employed multimodal and multiscale optical imaging to investigate the impact of DDS size on brain accumulation, extravasation and penetration upon sonopermeation. : Two prototypic DDS, i.e. 10 nm-sized pHPMA polymers and 100 nm-sized PEGylated liposomes, were labeled with fluorophores and intravenously injected in healthy CD-1 nude mice. Upon sonopermeation, computed tomography-fluorescence molecular tomography, fluorescence reflectance imaging, fluorescence microscopy, confocal microscopy and stimulated emission depletion nanoscopy were used to study the effect of DDS size on their translocation across the BBB. : Sonopermeation treatment enabled safe and efficient opening of the BBB, which was confirmed by staining extravasated endogenous IgG. No micro-hemorrhages, edema and necrosis were detected in H&E stainings. Multimodal and multiscale optical imaging showed that sonopermeation promoted the accumulation of nanocarriers in mouse brains, and that 10 nm-sized polymeric DDS accumulated more strongly and penetrated deeper into the brain than 100 nm-sized liposomes. : BBB opening via sonopermeation enables safe and efficient delivery of nanomedicine formulations to and into the brain. When looking at accumulation and penetration (and when neglecting issues such as drug loading capacity and therapeutic efficacy) smaller-sized DDS are found to be more suitable for drug delivery across the BBB than larger-sized DDS. These findings are valuable for better understanding and further developing nanomedicine-based strategies for the treatment of CNS disorders.
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http://dx.doi.org/10.7150/thno.41161DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6993230PMC
February 2021

Performance of severity parameters to detect chemotherapy-induced pain and distress in mice.

Lab Anim 2020 Oct 29;54(5):452-460. Epub 2019 Oct 29.

Institute for Experimental Molecular Imaging, Medical Faculty, RWTH Aachen International University, Germany.

According to European Union directive 2010/63/EU a severity classification of experimental procedures performed on laboratory animals is mandatory. This includes a prospective evaluation of all interventions performed within the experiment, as well as an assessment of the actual burden of each animal during the experiment. In this regard, the evaluation and scoring of defined criteria regarding the health state of animals could help to early identify deteriorations in animal health and facilitate the application of humane endpoints. This article discusses the applicability of an adapted score sheet in BALB/cAnNRj mice receiving either cisplatin, doxorubicin or busulfan, three chemotherapeutic agents with different toxicological profiles and longitudinal non-invasive molecular imaging. The health state was investigated by score sheets documenting general state, body weight, spontaneous behaviour and treatment specific parameters (e.g. anaemia, neurotoxicity, persistent diarrhoea). Although blood and serum analyses clearly indicated various organ damage, most scoring parameters except for body weight did not report on the deceasing animal health state. Thus, there is need for more sensitive observational parameters to judge the animal's health state and welfare.
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http://dx.doi.org/10.1177/0023677219883327DOI Listing
October 2020

Assessment of Chemotherapy-Induced Organ Damage with Ga-68 Labeled Duramycin.

Mol Imaging Biol 2020 06;22(3):623-633

Institute for Experimental Molecular Imaging -Center for Biohybrid Medical Systems -CBMS, Medical Faculty, RWTH Aachen University, Forckenbeckstr. 55, 52074, Aachen, Germany.

Purpose: Evaluation of [Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity.

Procedures: Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters.

Results: In vitro experiments confirmed specific binding of [Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology.

Conclusion: [Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.
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http://dx.doi.org/10.1007/s11307-019-01417-3DOI Listing
June 2020

Non-invasive Imaging and Modeling of Liver Regeneration After Partial Hepatectomy.

Front Physiol 2019 17;10:904. Epub 2019 Jul 17.

Institute for Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, Aachen, Germany.

The liver has a unique regenerative capability upon injury or partial resection. The regeneration process comprises a complex interplay between parenchymal and non-parenchymal cells and is tightly regulated at different scales. Thus, we investigated liver regeneration using multi-scale methods by combining non-invasive imaging with immunohistochemical analyses. In this context, non-invasive imaging can provide quantitative data of processes involved in liver regeneration at organ and body scale. We quantitatively measured liver volume recovery after 70% partial hepatectomy (PHx) by micro computed tomography (μCT) and investigated changes in the density of CD68 macrophages by fluorescence-mediated tomography (FMT) combined with μCT using a newly developed near-infrared fluorescent probe. In addition, angiogenesis and tissue-resident macrophages were analyzed by immunohistochemistry. Based on the results, a model describing liver regeneration and the interactions between different cell types was established. analysis of liver volume regeneration over 21 days after PHx by μCT imaging demonstrated that the liver volume rapidly increased after PHx reaching a maximum at day 14 and normalizing until day 21. An increase in CD68 macrophage density in the liver was detected from day 4 to day 8 by combined FMT-μCT imaging, followed by a decline towards control levels between day 14 and day 21. Immunohistochemistry revealed the highest angiogenic activity at day 4 after PHx that continuously declined thereafter, whereas the density of tissue-resident CD169 macrophages was not altered. The simulated time courses for volume recovery, angiogenesis and macrophage density reflect the experimental data describing liver regeneration after PHx at organ and tissue scale. In this context, our study highlights the importance of non-invasive imaging for acquiring quantitative organ scale data that enable modeling of liver regeneration.
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http://dx.doi.org/10.3389/fphys.2019.00904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6652107PMC
July 2019

MR and PET-CT monitoring of tissue-engineered vascular grafts in the ovine carotid artery.

Biomaterials 2019 09 27;216:119228. Epub 2019 May 27.

Dept. of Biohybrid & Medical Textiles (BioTex), Institute of Applied Medical Engineering (AME), Helmholtz Institute Aachen and Institute for Textiles Engineering (ITA), RWTH Aachen University, Forckenbeckstr. 55, 52074, Aachen, Germany. Electronic address:

The modification of biomaterials to comply with clinically employed monitoring techniques is a promising strategy to support clinical translation in regenerative medicine. Here, multimodal imaging of tissue-engineered vascular grafts (TEVG) was enabled by functionalizing the textile scaffold with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The resulting MR-imageable grafts (iTEVG) were monitored non-invasively throughout their whole life-cycle, from initial quality control to longitudinal functional evaluation in an ovine model for up to 8 weeks. Crucial features such as the complete embedding of the textile mesh in the developing tissue and the grafts' structural stability were assessed in vitro using 1T-, 3T- and 7T-MRI scanners. In vivo, the grafts were imaged by 3T-MRI and PET-CT. Contrary to unlabeled constructs, iTEVG could be delineated from native arteries and precisely localized by MRI. USPIO labeling neither induced calcifications, nor negatively affected their remodeling with respect to tissue-specific extracellular matrix composition and endothelialization. Functionality was confirmed by MR-angiography. F-FDG uptake (assessed via PET-CT) indicated only transient post-surgical inflammation. In conclusion, USPIO-labeling enables accurate localization of TEVG and opens up opportunities for multimodal imaging approaches to assess transplant acceptance and function. Thereby, it can support clinical decision-making on the need for further pharmacological or surgical interventions.
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http://dx.doi.org/10.1016/j.biomaterials.2019.119228DOI Listing
September 2019

Ultrasound Microbubbles for Diagnosis and Treatment of Cardiovascular Diseases.

Semin Thromb Hemost 2020 Jul 16;46(5):545-552. Epub 2019 May 16.

Institute for Experimental Molecular Imaging (ExMI), RWTH Aachen University, Aachen, Germany.

Ultrasound (US) imaging of heart and major arteries and veins is among the most frequently used diagnostic techniques applied in humans. Conventional cardiovascular US sessions include anatomical B-mode and functional M-, pulsed-wave- and Doppler mode, which have their limitations in both precise cardiac chambers' delineation and small vessel imaging. The introduction of contrast-enhanced US, employing microbubble suspensions as contrast agent, has enabled a better delineation of heart chambers, the visualization of myocardial microvasculature, and the atherosclerotic plaque neovascularization. Moreover, specific disease-related molecular tracers have been developed by modifying the microbubbles with targeting ligands directed to biological markers exposed to the luminal side of the blood vessels. Microbubble functionalization has enabled in vivo molecular US imaging of various stages of atherosclerosis, from plaque initiation to plaque vulnerability, and neointima formation following revascularization procedures. Furthermore, oscillating microbubbles have been used to mechanically dissolve thrombus material and may act as carriers of drugs and nucleic acids that are released locally by US pulses. This review article summarizes recent advances in functional and molecular US images and discusses therapeutic applications of microbubbles. The addressed topics include an overview on microbubble formats, microbubble detection methods, molecular targets of cardiovascular diseases, and the use of microbubbles for thrombolysis and drug delivery.
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http://dx.doi.org/10.1055/s-0039-1688492DOI Listing
July 2020

A water-soluble PEGylated RGD-functionalized bisbithiophenyl diketopyrrolopyrrole as a photoacoustic sonophore.

Photochem Photobiol Sci 2018 May;17(5):617-621

DWI - Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50, 52076 Aachen, Germany.

Photoacoustic imaging presents an innocuous imaging modality with good penetration depth and resolution. To use this modality for detection and imaging of pathological sites, new imaging probes need to be developed to enhance the contrast over endogenous sonophores. These contrast agents should specifically bind to the site of interest, be non-toxic and be cleared renally if applied intravenously. Small organic dyes with absorption in the near infrared spectrum often exhibit good photoacoustic response. However, such dyes are often not water soluble or they are cytotoxic. Here, we present a novel PEGylated sonophore based on diketopyrrolopyrrole (DPP), which overcomes these limitations and can be functionalized with desired biological recognition motifs using thiol-yne click chemistry. Proof of concept is demonstrated by functionalizing the DPP-based probe with an RGD peptide, resulting in specific binding to endothelial (HUVEC) cells and an efficient photoacoustic response.
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http://dx.doi.org/10.1039/c8pp00069gDOI Listing
May 2018

Motion model ultrasound localization microscopy for preclinical and clinical multiparametric tumor characterization.

Nat Commun 2018 04 18;9(1):1527. Epub 2018 Apr 18.

Institute for Experimental Molecular Imaging, University Clinic Aachen, RWTH Aachen University, CMBS, Forckenbeckstr. 55, 52074, Aachen, Germany.

Super-resolution imaging methods promote tissue characterization beyond the spatial resolution limits of the devices and bridge the gap between histopathological analysis and non-invasive imaging. Here, we introduce motion model ultrasound localization microscopy (mULM) as an easily applicable and robust new tool to morphologically and functionally characterize fine vascular networks in tumors at super-resolution. In tumor-bearing mice and for the first time in patients, we demonstrate that within less than 1 min scan time mULM can be realized using conventional preclinical and clinical ultrasound devices. In this context, next to highly detailed images of tumor microvascularization and the reliable quantification of relative blood volume and perfusion, mULM provides multiple new functional and morphological parameters that discriminate tumors with different vascular phenotypes. Furthermore, our initial patient data indicate that mULM can be applied in a clinical ultrasound setting opening avenues for the multiparametric characterization of tumors and the assessment of therapy response.
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http://dx.doi.org/10.1038/s41467-018-03973-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5906644PMC
April 2018

Advanced Ultrasound Technologies for Diagnosis and Therapy.

J Nucl Med 2018 05 1;59(5):740-746. Epub 2018 Mar 1.

Institute for Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany

Ultrasound is among the most rapidly advancing imaging techniques. Functional methods such as elastography have been clinically introduced, and tissue characterization is improved by contrast-enhanced scans. Here, novel superresolution techniques provide unique morphologic and functional insights into tissue vascularization. Functional analyses are complemented by molecular ultrasound imaging, to visualize markers of inflammation and angiogenesis. The full potential of diagnostic ultrasound may become apparent by integrating these multiple imaging features in radiomics approaches. Emerging interest in ultrasound also results from its therapeutic potential. Various applications of tumor ablation with high-intensity focused ultrasound are being clinically evaluated, and its performance strongly benefits from the integration into MRI. Additionally, oscillating microbubbles mediate sonoporation to open biologic barriers, thus improving the delivery of drugs or nucleic acids that are coadministered or coformulated with microbubbles. This article provides an overview of recent developments in diagnostic and therapeutic ultrasound, highlighting multiple innovation tracks and their translational potential.
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http://dx.doi.org/10.2967/jnumed.117.200030DOI Listing
May 2018

Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial Dysregulation.

Arterioscler Thromb Vasc Biol 2018 01 30;38(1):40-48. Epub 2017 Nov 30.

From the Institute for Molecular Cardiovascular Research (IMCAR) (A.C., Z.W., M.S., S.A.-R., M.v.Z., E.A.L.), and Institute for Experimental Molecular Imaging (ExMI) (A.C., Z.W., A.R., T.L., F.K.), University Hospital Aachen, RWTH Aachen, Germany; Victor Babes National Institute of Pathology, Bucharest, Romania (A.C.); AYOXXA Biosystems GmbH, Cologne, Germany (O.G.); Department of Targeted Therapeutics, University of Twente, Enschede, The Netherlands (T.L.); Department of Genetics and Molecular Cell Biology, School for Oncology and Developmental Biology (GROW), Maastricht University, The Netherlands (M.v.Z., R.R.K.); Department of Biochemistry, School for Cardiovascular Diseases (CARIM), Maastricht University, The Netherlands (M.v.Z., C.W.); German Centre for Cardiovascular Research, partner site Munich Heart Alliance (DZHK), Germany (C.W.); Institute for Cardiovascular Prevention, Ludwig-Maximilians-Universität München, Munich, Germany (C.W.); and Human Genetic Laboratory, University for Medicine and Pharmacy, Craiova, Romania (E.A.L.).

Objective: The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations.

Approach And Results: Flow-dependent endothelial dysfunction was induced in apolipoprotein E-deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A-targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [<0.001] and ≈5× [<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A-targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side.

Conclusions: Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A-targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.
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http://dx.doi.org/10.1161/ATVBAHA.117.309503DOI Listing
January 2018

Nilotinib Enhances Tumor Angiogenesis and Counteracts VEGFR2 Blockade in an Orthotopic Breast Cancer Xenograft Model with Desmoplastic Response.

Neoplasia 2017 Nov 20;19(11):896-907. Epub 2017 Sep 20.

Institute for Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, Aachen, Germany. Electronic address:

Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR)-targeted therapies predominantly affect nascent, immature tumor vessels. Since platelet-derived growth factor receptor (PDGFR) blockade inhibits vessel maturation and thus increases the amount of immature tumor vessels, we evaluated whether the combined PDGFR inhibition by nilotinib and VEGFR2 blockade by DC101 has synergistic therapy effects in a desmoplastic breast cancer xenograft model. In this context, besides immunohistological evaluation, molecular ultrasound imaging with BR55, the clinically used VEGFR2-targeted microbubbles, was applied to monitor VEGFR2-positive vessels noninvasively and to assess the therapy effects on tumor angiogenesis. DC101 treatment alone inhibited tumor angiogenesis, resulting in lower tumor growth and in significantly lower vessel density than in the control group after 14 days of therapy. In contrast, nilotinib inhibited vessel maturation but enhanced VEGFR2 expression, leading to markedly increased tumor volumes and a significantly higher vessel density. The combination of both drugs led to an almost similar tumor growth as in the DC101 treatment group, but VEGFR2 expression and microvessel density were higher and comparable to the controls. Further analyses revealed significantly higher levels of tumor cell-derived VEGF in nilotinib-treated tumors. In line with this, nilotinib, especially in low doses, induced an upregulation of VEGF and IL-6 mRNA in the tumor cells in vitro, thus providing an explanation for the enhanced angiogenesis observed in nilotinib-treated tumors in vivo. These findings suggest that nilotinib inhibits vessel maturation but counteracts the effects of antiangiogenic co-therapy by enhancing VEGF expression by the tumor cells and stimulating tumor angiogenesis.
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http://dx.doi.org/10.1016/j.neo.2017.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5608503PMC
November 2017

Bio-degradable highly fluorescent conjugated polymer nanoparticles for bio-medical imaging applications.

Nat Commun 2017 09 7;8(1):470. Epub 2017 Sep 7.

DWI-Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50, 52076, Aachen, Germany.

Conjugated polymer nanoparticles exhibit strong fluorescence and have been applied for biological fluorescence imaging in cell culture and in small animals. However, conjugated polymer particles are hydrophobic and often chemically inert materials with diameters ranging from below 50 nm to several microns. As such, conjugated polymer nanoparticles cannot be excreted through the renal system. This drawback has prevented their application for clinical bio-medical imaging. Here, we present fully conjugated polymer nanoparticles based on imidazole units. These nanoparticles can be bio-degraded by activated macrophages. Reactive oxygen species induce scission of the conjugated polymer backbone at the imidazole unit, leading to complete decomposition of the particles into soluble low molecular weight fragments. Furthermore, the nanoparticles can be surface functionalized for directed targeting. The approach opens a wide range of opportunities for conjugated polymer particles in the fields of medical imaging, drug-delivery, and theranostics.Conjugated polymer nanoparticles have been applied for biological fluorescence imaging in cell culture and in small animals, but cannot readily be excreted through the renal system. Here the authors show fully conjugated polymer nanoparticles based on imidazole units that can be bio-degraded by activated macrophages.
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http://dx.doi.org/10.1038/s41467-017-00545-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5589938PMC
September 2017

Balancing Passive and Active Targeting to Different Tumor Compartments Using Riboflavin-Functionalized Polymeric Nanocarriers.

Nano Lett 2017 08 27;17(8):4665-4674. Epub 2017 Jul 27.

Institute for Experimental Molecular Imaging, University Hospital and Helmholtz Institute for Biomedical Engineering, RWTH Aachen , Pauwelsstrasse 30, 52074 Aachen, Germany.

Riboflavin transporters (RFTs) and the riboflavin carrier protein (RCP) are highly upregulated in many tumor cells, tumor stem cells, and tumor neovasculature, which makes them attractive targets for nanomedicines. Addressing cells in different tumor compartments requires drug carriers, which are not only able to accumulate via the EPR effect but also to extravasate, target specific cell populations, and get internalized by cells. Reasoning that antibodies are among the most efficient targeting systems developed by nature, we consider their size (∼10-15 nm) to be ideal for balancing passive and active tumor targeting. Therefore, small, short-circulating (10 kDa, ∼7 nm, t ∼ 1 h) and larger, longer-circulating (40 kDa, ∼13 nm, t ∼ 13 h) riboflavin-targeted branched PEG polymers were synthesized, and their biodistribution and target site accumulation were evaluated in mice bearing angiogenic squamous cell carcinoma (A431) and desmoplastic prostate cancer (PC3) xenografts. The tumor accumulation of the 10 kDa PEG was characterized by rapid intercompartmental exchange and significantly improved upon active targeting with riboflavin (RF). The 40 kDa PEG accumulated in tumors four times more efficiently than the small polymer, but its accumulation did not profit from active RF-targeting. However, RF-targeting enhanced the cellular internalization in both tumor models and for both polymer sizes. Interestingly, the nanocarriers' cell-uptake in tumors was not directly correlated with the extent of accumulation. For example, in both tumor models the small RF-PEG accumulated much less strongly than the large passively targeted PEG but showed significantly higher intracellular amounts 24 h after iv administration. Additionally, the size of the polymer determined its preferential uptake by different tumor cell compartments: the 10 kDa RF-PEGs most efficiently targeted cancer cells, whereas the highest uptake of the 40 kDa RF-PEGs was observed in tumor-associated macrophages. These findings imply that drug carriers with sizes in the range of therapeutic antibodies show balanced properties with respect to passive accumulation, tissue penetration, and active targeting. Besides highlighting the potential of RF-mediated (cancer) cell targeting, we show that strong tumor accumulation does not automatically mean high cellular uptake and that the nanocarriers' size plays a critical role in cell- and compartment-specific drug targeting.
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http://dx.doi.org/10.1021/acs.nanolett.7b01171DOI Listing
August 2017

Molecular Ultrasound Imaging of αvβ3-Integrin Expression in Carotid Arteries of Pigs After Vessel Injury.

Invest Radiol 2016 12;51(12):767-775

From the *Institute for Experimental Molecular Imaging, University Clinic and Helmholtz Center for Biomedical Engineering, and †Department of Diagnostic and Interventional Neuroradiology, RWTH Aachen University, Aachen, Germany.

Objectives: Interventions such as balloon angioplasty can cause vascular injury leading to platelet activation, thrombus formation, and inflammatory response. This induces vascular smooth muscle cell activation and subsequent re-endothelialization with expression of αvβ3-integrin by endothelial cells and vascular smooth muscle cell. Thus, poly-N-butylcyanoacrylate microbubbles (MBs) targeted to αvβ3-integrin were evaluated for monitoring vascular healing after vessel injury in pigs using molecular ultrasound imaging.

Materials And Methods: Approval for animal experiments was obtained. The binding specificity of αvβ3-integrin-targeted MB to human umbilical vein endothelial cells was tested with fluorescence microscopy. In vivo imaging was performed using a clinical ultrasound system and an 8-MHz probe. Six mini pigs were examined after vessel injury in the left carotid artery. The right carotid served as control. Uncoated MB, cDRG-coated MB, and αvβ3-integrin-specific cRGD-coated MB were injected sequentially. Bound MBs were assessed 8 minutes after injection using ultrasound replenishment analysis. Measurements were performed 2 hours, 1 and 5 weeks, and 3 and 6 months after injury. In vivo data were validated by immunohistochemistry.

Results: Significantly stronger binding of cRGD-MB than MB and cDRG-MB to human umbilical vein endothelial cells was found (P < 0.01). As vessel injury leads to upregulation of αvβ3-integrin, cRGD-MBs bound significantly stronger (P < 0.05) in injured carotid arteries than at the counter side 1 week after vessel injury and significant differences could also be observed after 5 weeks. After 3 months, αvβ3-integrin expression decreased to baseline and binding of cRGD-MB was comparable in both vessels. Values remained at baseline also after 6 months.

Conclusions: Ultrasound imaging with RGD-MB is promising for monitoring vascular healing after vessel injury. This may open new perspectives to assess vascular damage after radiological interventions.
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http://dx.doi.org/10.1097/RLI.0000000000000282DOI Listing
December 2016

Low-Dose Molecular Ultrasound Imaging with E-Selectin-Targeted PBCA Microbubbles.

Mol Imaging Biol 2016 Apr;18(2):180-90

Department of Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Purpose: Our objective was to determine the lowest diagnostically effective dose for E-selectin-targeted poly n-butyl cyanoacrylate (PBCA)-shelled microbubbles and to apply it to monitor antiangiogenic therapy effects.

Procedures: PBCA-shelled microbubbles (MBs) coupled to an E-selectin-specific peptide were applied in mice carrying MLS or A431 carcinoma xenografts scaling down the MB dosage to the lowest level where binding could be examined with a 18-MHz small animal ultrasound transducer. Differences in E-selectin expression in the two carcinoma xenografts were confirmed by enzyme-linked immunosorbent assay (ELISA). In addition, MLS tumor-bearing mice under antiangiogenic therapy were monitored using E-selectin-targeted MBs at the lowest applicable dose. Therapy effects on tumor vascularization were verified by immunohistological analyses.

Results: The minimally required dosage was 7 × 10(7) MBs/kg body weight. This dosage was sufficient to enable E-selectin detection in high E-selectin-expressing MLS tumors, while low E-selectin-expressing A431 tumors required almost 2.5-fold higher doses. At the dose of 7 × 10(7) MBs/kg body weight, a decrease in E-selectin MB binding under antiangiogenic therapy could be assessed (being significant after 3 days of treatment; p < 0.0001), which was in line with the significant drop in E-selectin-positive area fractions that was found histologically (p < 0.05).

Conclusions: Molecular ultrasound imaging with our E-selectin-targeted MB and therapy monitoring was possible down to a dose of 7 × 10(7) MBs/kg body weight (equates to 66 μg PBCA/kg and 4.6 mg PBCA/70 kg). Improvements in choice of targets, MB composition, and other MB detection methods may improve sensitivity and lead to reliable detection results of clinically transferrable MBs at even lower dosage levels.
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http://dx.doi.org/10.1007/s11307-015-0894-9DOI Listing
April 2016

Squamous Cell Carcinoma Xenografts: Use of VEGFR2-targeted Microbubbles for Combined Functional and Molecular US to Monitor Antiangiogenic Therapy Effects.

Radiology 2016 Feb 27;278(2):430-40. Epub 2015 Aug 27.

From the Department of Experimental Molecular Imaging, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany (S.C.B., A.R., T.L., F.K., W.L.); Bracco Suisse SA, Geneva, Switzerland (F.T.); and Merck Serono, Darmstadt, Germany (R.S.).

Purpose: To assess the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted and nontargeted ultrasonography (US) to depict antiangiogenic therapy effects and to investigate whether first-pass kinetics obtained with VEGFR2-targeted microbubbles provide independent data about tumor vascularization.

Materials And Methods: Governmental approval was obtained for animal experiments. Vascularization in response to anti-vascular endothelial growth factor receptor or vehicle-control treatment (10 per group) in HaCaT-ras A-5RT3 xenografts was longitudinally assessed in mice by means of first-pass kinetics of nontargeted microbubbles (BR1, BR38; Bracco, Geneva, Switzerland) and VEGFR2-targeted microbubbles (BR55, Bracco) before and 4, 7, and 14 days after therapy. VEGFR2 expression was determined 8 minutes after BR55 injection with destruction-replenishment analysis. US data were validated with immunohistochemistry. Significant differences were evaluated with the Mann-Whitney test.

Results: First-pass analysis with BR1, BR38, and BR55 showed similar tendencies toward decreasing vascularization, with a stronger decrease in tumors treated with anti-VEGF antibody. The median signal intensity (in arbitrary units [au]) of anti-VEGF antibody-treated versus control tumors at day 14 was as follows: BR1, 5.2 au (interquartile range [IQR], 3.2 au) vs 11.3 au (IQR, 10.0 au), respectively; BR38, 6.2 au (IQR, 3.5) vs 10.0 au (IQR, 7.8); and BR55, 9.5 au (IQR, 6.0 au) vs 13.8 au (IQR, 9.8) (P = .0230). VEGFR2 assessment with BR55 demonstrated significant differences between both groups throughout the therapy period (median signal intensity of anti-VEGF antibody-treated vs control tumors: 0.04 au [IQR, 0.1 au] vs 0.14 au [IQR, 0.08 au], respectively, at day 4, P = .0058; 0.04 au [IQR, 0.06 au] vs 0.13 au [IQR, 0.09 au] at day 7, P = .0058; and 0.06 au [IQR, 0.11 au] vs 0.16 au [IQR, 0.15 au] at day 14, P = .0247). Immunohistochemistry confirmed the lower microvessel density and VEGFR2-positive area fraction in tumors treated with anti-VEGF antibody.

Conclusion: Antiangiogenic therapy effects were detected earlier and more distinctly with VEGFR2-targeted US than with functional US. First-pass analyses with BR55, BR38, and BR1 revealed similar results, with a decrease in vascularization during therapy. Functional data showed that BR55 is not strongly affected by early binding of the microbubbles to VEGFR2. Thus, functional and molecular imaging of angiogenesis can be performed with BR55 within one examination.
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http://dx.doi.org/10.1148/radiol.2015142899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5410993PMC
February 2016

Erythropoietin improves the accumulation and therapeutic effects of carboplatin by enhancing tumor vascularization and perfusion.

Theranostics 2015 1;5(8):905-18. Epub 2015 May 1.

1. Experimental Molecular Imaging, Medical Faculty, RWTH Aachen University, Aachen, Germany.

Recombinant human erythropoietin (rhuEpo) is currently under debate for the treatment of chemotherapy-induced anemia due to clinical trials showing adverse effects in Epo-treated patients and the discovery of the erythropoietin-receptor (EpoR) in tumor and endothelial cells. Here, using Epo-Cy5.5 as theranostic near-infrared fluorescent probe we analyzed the effects of rhuEpo as co-medication to carboplatin in non-small-cell-lung-cancer (NSCLC)-xenografts with different tumor cell EpoR-expression (H838 ~8-fold higher than A549). Nude mice bearing subcutaneous A549 and H838 NSCLC-xenografts received either only carboplatin or carboplatin and co-medication of rhuEpo in two different doses. Tumor sizes and relative blood volumes (rBV) were longitudinally measured by 3D-contrast-enhanced ultrasound (3D-US). Tumoral EpoR-levels were determined by combined fluorescence molecular tomography (FMT)/ micro computed tomography (µCT) hybrid imaging. We found that rhuEpo predominantly acted on the tumor endothelium. In both xenografts, rhuEpo co-medication significantly increased vessel densities, diameters and the amount of perfused vessels. Accordingly, rhuEpo induced EpoR-phoshorylation and stimulated proliferation of endothelial cells. However, compared with solely carboplatin-treated tumors, tumor growth was significantly slower in the groups co-medicated with rhuEpo. This is explained by the Epo-mediated vascular remodeling leading to improved drug delivery as obvious by a more than 2-fold higher carboplatin accumulation and significantly enhanced tumor apoptosis. In addition, co-medication of rhuEpo reduced tumor hypoxia and diminished intratumoral EpoR-levels which continuously increased during carboplatin (Cp) -treatment. These findings suggest that co-medication of rhuEpo in well balanced doses can be used to improve the accumulation of anticancer drugs. Doses and indications may be personalized and refined using theranostic EpoR-probes.
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http://dx.doi.org/10.7150/thno.11304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4440446PMC
February 2016

RGD-decorated conjugated polymer particles as fluorescent biomedical probes prepared by Sonogashira dispersion polymerization.

Chem Commun (Camb) 2015 Jun;51(45):9358-61

DWI - Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50, 52074 Aachen, Germany.

Here, we present a facile one-step Sonogashira dispersion polymerization affording monodisperse conjugated polymer particles bearing accessible acetylene moieties on the surface. These acetylene groups are easily functionalized with biological recognition motifs using thiol-yne click chemistry. The resulting functional particles are applied as fluorescent probes for imaging of activated endothelial cells, which take up the particles via receptor mediated endocytosis.
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http://dx.doi.org/10.1039/c4cc10092aDOI Listing
June 2015

In-vivo detection of the erythropoietin receptor in tumours using positron emission tomography.

Eur Radiol 2015 Feb 9;25(2):472-9. Epub 2014 Sep 9.

Department for Experimental Molecular Imaging (ExMI), Medical Faculty, RWTH Aachen University, Pauwelsstraße 20, 52074, Aachen, Germany.

Objective: Recombinant human erythropoietin (rhuEpo) is used clinically to treat anaemia. However, rhuEpo-treated cancer patients show decreased survival rates and erythropoietin receptor (EpoR) expression has been found in patient tumour tissue. Thus, rhuEpo application might promote EpoR(+) tumour progression. We therefore developed the positron emission tomography (PET)-probe (68)Ga-DOTA-rhuEpo and evaluated its performance in EpoR(+) A549 non-small-cell lung cancer (NSCLC) xenografts.

Methods: (68)Ga-DOTA-rhuEpo was generated by coupling DOTA-hydrazide to carbohydrate side-chains of rhuEpo. Biodistribution was determined in tumour-bearing mice 0.5, 3, 6, and 9 h after probe injection. Competition experiments were performed by co-injecting (68)Ga-DOTA-rhuEpo and rhuEpo in five-fold excess. Probe specificity was further evaluated histologically using Epo-Cy5.5 stainings.

Results: The blood half-life of (68)Ga-DOTA-rhuEpo was 2.6 h and the unbound fraction was cleared by the liver and kidney. After 6 h, the highest tumour to muscle ratio was reached. The highest (68)Ga-DOTA-rhuEpo accumulation was found in liver (10.06 ± 6.26%ID/ml), followed by bone marrow (1.87 ± 0.53%ID/ml), kidney (1.58 ± 0.39%ID/ml), and tumour (0.99 ± 0.16%ID/ml). EpoR presence in these organs was histologically confirmed. Competition experiments showed significantly (p < 0.05) lower PET-signals in tumour and bone marrow at 3 and 6 h.

Conclusion: (68)Ga-DOTA-rhuEpo shows favourable pharmacokinetic properties and detects EpoR specifically. Therefore, it might become a valuable radiotracer to monitor EpoR status in tumours and support decision-making in anaemia therapy.

Key Points: • PET-probe (68) Ga-DOTA-rhuEpo was administered to assess the EpoR status in vivo • (68) Ga-DOTA-rhuEpo binds specifically to EpoR positive organs in vivo • Tumour EpoR status determination might enable decision-making in anaemia therapy with rhuEpo.
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http://dx.doi.org/10.1007/s00330-014-3413-5DOI Listing
February 2015

Influence of repetitive contrast agent injections on functional and molecular ultrasound measurements.

Ultrasound Med Biol 2014 Oct 9;40(10):2468-75. Epub 2014 Jul 9.

Department of Experimental Molecular Imaging, RWTH Aachen University, Aachen, Germany.

Quantitative contrast-enhanced ultrasound plays an important role in tumor characterization and treatment assessment. Besides established functional ultrasound techniques, ultrasound molecular imaging using microbubbles targeted to disease-associated markers is increasingly being applied in pre-clinical studies. Often, repeated injections of non-targeted or targeted microbubbles during the same imaging session are administered. However, the influence of repeated injections on the accuracy of the quantitative data is unclear. Therefore, in tumor-bearing mice, we investigated the influence of multiple injections of non-targeted microbubbles (SonoVue) on time to peak and peak enhancement in liver and tumor tissue and of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted contrast agents (MicroMarker) on specific tumor accumulation. We found significantly decreasing values for time to peak and a tendency for increased values for peak enhancement after multiple injections. Repeated injections of VEGFR2-targeted microbubbles led to significantly increased tumor accumulation, which may result from the exposure of additional binding sites at endothelial surfaces caused by mechanical forces from destroyed microbubbles.
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http://dx.doi.org/10.1016/j.ultrasmedbio.2014.04.010DOI Listing
October 2014

A low molecular weight zinc2+-dipicolylamine-based probe detects apoptosis during tumour treatment better than an annexin V-based probe.

Eur Radiol 2014 Feb;24(2):363-70

Objectives: Molecular imaging of apoptosis is frequently discussed for monitoring cancer therapies. Here, we compare the low molecular weight phosphatidylserine-targeting ligand zinc2+-dipicolylamine (Zn2+-DPA) with the established but reasonably larger protein annexin V.

Methods: Molecular apoptosis imaging with the fluorescently labelled probes annexin V (750 nm, 36 kDa) and Zn2+-DPA (794 nm, 1.84 kDa) was performed in tumour-bearing mice (A431). Three animal groups were investigated: untreated controls and treated tumours after 1 or 4 days of anti-angiogenic therapy (SU11248). Additionally, μPET with 18 F-FDG was performed. Imaging data were displayed as tumour-to-muscle ratio (TMR) and validated by quantitative immunohistochemistry.

Results: Compared with untreated control tumours, TUNEL staining indicated significant apoptosis after 1 day (P < 0.05) and 4 days (P < 0.01) of treatment. Concordantly, Zn2+-DPA uptake increased significantly after 1 day (P < 0.05) and 4 days (P < 0.01). Surprisingly, annexin V failed to detect significant differences between control and treated animals. Contrary to the increasing uptake of Zn2+-DPA, 18 F-FDG tumour uptake decreased significantly at days 1 (P < 0.05) and 4 (P < 0.01).

Conclusions: Increase in apoptosis during anti-angiogenic therapy was detected significantly better with the low molecular weight probe Zn2+-DPA than with the annexin V-based probe. Additionally, significant treatment effects were detectable as early using Zn2+-DPA as with measurements of the glucose metabolism using 18 F-FDG.

Key Points: • The detection of apoptosis by non-invasive imaging is important in oncology. • A new low molecular weight probe Zn2+-DPA shows promise in depicting anti-angiogenic effects. • The small Zn2+-DPA ligand appears well suited for monitoring therapy. • Treatment effects are detectable just as early with Zn2+-DPA as with 18F-FDG.
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http://dx.doi.org/10.1007/s00330-013-3014-8DOI Listing
February 2014

Ultrasound molecular imaging of E-selectin in tumor vessels using poly n-butyl cyanoacrylate microbubbles covalently coupled to a short targeting peptide.

Invest Radiol 2013 Dec;48(12):843-50

From the *Department of Experimental Molecular Imaging, University Clinic and Helmholtz Center for Biomedical Engineering, RWTH Aachen University, Aachen, Germany; †Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal; ‡Institute for Molecular Cardiovascular Research, University Clinic, RWTH Aachen University, Aachen; and §Academic Radiology Baden-Baden, Diagnostic and Interventional Radiology, University Medical Center Heidelberg, Baden-Baden, Germany.

Objectives: The purposes of this study were the development and preclinical evaluation of clinically translatable E-selectin-specific ultrasound contrast agents based on a peptide ligand with the recognition sequence IELLQAR.

Materials And Methods: The E-selectin-specific peptide was synthesized through solid phase peptide synthesis and covalently attached to poly n-butylcyanoacrylate-stabilized microbubbles with an air core. Quantification of the microbubble surface coverage with peptides was performed through flow cytometry. Targeted adhesion of peptide-coated microbubbles was investigated in vitro using parallel plate flow chamber assays on tumor necrosis factor-α-stimulated human umbilical vein endothelial cells. In vivo imaging was performed in nude mice bearing human ovarian carcinoma xenografts (MLS), followed by ex vivo immunohistochemistry validation of E-selectin expression.

Results: Success of peptide synthesis was validated through preparative reverse phase high-pressure liquid chromatography and electronspray ionization-mass spectrometry. Results of the flow cytometry revealed approximately 4000 E-selectin-specific peptides/microbubble surface. Results of the in vitro experiments demonstrated the specificity of peptide-coated microbubbles to E-selectin (1.10 ± 0.48 vs 0.19 ± 0.09 bound microbubbles per cell, before and after competition respectively; P < 0.01). The in vivo imaging enabled specific assessment of E-selectin expression in MLS carcinoma xenografts (5.21 ± 3.41 vs 1.37 ± 0.67 contrast intensity before and after competition, respectively; P < 0.05).

Conclusions: Clinically translatable microbubbles that were covalently coupled to the short E-selectin-specific peptide (IELLQAR) enabled specific imaging of the E-selectin expression in tumor vessels in vivo.
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http://dx.doi.org/10.1097/RLI.0b013e31829d03ecDOI Listing
December 2013

Accuracy of a clinical PET/CT vs. a preclinical μPET system for monitoring treatment effects in tumour xenografts.

Eur J Radiol 2013 Aug 26;82(8):1318-24. Epub 2013 Feb 26.

Department of Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany.

Purpose: Small animal imaging is of growing importance for preclinical research and drug development. Tumour xenografts implanted in mice can be visualized with a clinical PET/CT (cPET); however, it is unclear whether early treatment effects can be monitored. Thus, we investigated the accuracy of a cPET versus a preclinical μPET using (18)F-FDG for assessing early treatment effects.

Materials And Methods: The spatial resolution and the quantitative accuracy of a clinical and preclinical PET were evaluated in phantom experiments. To investigate the sensitivity for assessing treatment response, A431 tumour xenografts were implanted in nude mice. Glucose metabolism was measured in untreated controls and in two therapy groups (either one or four days of antiangiogenic treatment). Data was validated by γ-counting of explanted tissues.

Results: In phantom experiments, cPET enabled reliable separation of boreholes≥5mm whereas μPET visualized boreholes≥2mm. In animal studies, μPET provided significantly higher tumour-to-muscle ratios for untreated control tumours than cPET (3.41±0.87 vs. 1.60±.0.28, respectively; p<0.01). During treatment, cPET detected significant therapy effects at day 4 (p<0.05) whereas μPET revealed highly significant therapy effects even at day one (p<0.01). Correspondingly, γ-counting of explanted tumours indicated significant therapy effects at day one and highly significant treatment response at day 4. Correlation with γ-counting was good for cPET (r=0.74; p<0.01) and excellent for μPET (r=0.85; p<0.01).

Conclusion: Clinical PET is suited to investigate tumour xenografts≥5mm at an advanced time-point of treatment. For imaging smaller tumours or for the sensitive assessment of very early therapy effects, μPET should be preferred.
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http://dx.doi.org/10.1016/j.ejrad.2013.01.028DOI Listing
August 2013

Liver dysplasia: US molecular imaging with targeted contrast agent enables early assessment.

Radiology 2013 May 29;267(2):487-95. Epub 2013 Jan 29.

Department of Experimental Molecular Imaging, Internal Medicine III, and Nuclear Medicine, RWTH-Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany.

Purpose: To investigate the ability of vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted ultrasonographic (US) microbubbles for the assessment of liver dysplasia in transgenic mice.

Materials And Methods: Animal experiments were approved by the governmental review committee. Nuclear factor-κB essential modulator knock-out mice with liver dysplasia and wild-type mice underwent liver imaging by using a clinical US system. Two types of contrast agents were investigated: nontargeted, commercially available, second-generation microbubbles (SonoVue) and clinically translatable PEGylated VEGFR2-targeted microbubbles (BR55). Microbubble kinetics was investigated over the course of 4 minutes. Targeted contrast material-enhanced US signal was quantified 5 minutes after injection. Competitive in vivo binding experiments with BR55 were performed in knock-out mice. Immunohistochemical and hematoxylin-eosin staining of liver sections was performed to validate the in vivo US results. Groups were compared by using the Mann-Whitney test.

Results: Peak enhancement after injection of SonoVue and BR55 did not differ in healthy and dysplastic livers (SonoVue, P = .46; BR55, P = .43). Accordingly, immunohistochemical findings revealed comparable vessel densities in both groups. The specificity of BR55 to VEGFR2 was proved by in vivo competition (P = .0262). While the SonoVue signal decreased similarly in healthy and dysplastic livers during the 4 minutes, there was an accumulation of BR55 in dysplastic livers compared with healthy ones. Furthermore, targeted contrast-enhanced US signal indicated a significantly higher site-specific binding of BR55 in dysplastic than healthy livers (P = .005). Quantitative immunohistologic findings confirmed significantly higher VEGFR2 levels in dysplastic livers (P = .02).

Conclusion: BR55 enables the distinction of early stages of liver dysplasia from normal liver.
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http://dx.doi.org/10.1148/radiol.13120220DOI Listing
May 2013
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