Abstract/Video opens by clicking at the talk title.
Abstract/Video opens by clicking at the talk title.
|12:00 p.m.||PS 18-01||
Introductory Lecture (#676)
1 Memorial Sloan Kettering Cancer Center, New York, United States of America
|12:18 p.m.||PS 18-02||
The Role of DUOX in Gut Inflammation: MR, CT and PET Imaging of the Alternative Animal Model Manduca sexta (#320)
Anton G. Müller1, Heinz H. F. Müller2, Michael Hentschel3, Christoph-Rüdiger von Bredow4, Yvette von Bredow5, Martin Hardt6, Tina Trenczek7
1 Justus-Liebig-University of Giessen, General Zoology and Developmental Biology, Giessen, Germany
Bacteria heavily colonize animal epithelia. The dual oxidase (DUOX) is part of the first line defense in gut epithelia and attacks pathogenic bacteria via synthesis of HOCl. In an unbalanced microbiome, bacteria derived uracil activates epithelial DUOX. In humans, DUOX plays an essential part in the etiology of inflammatory bowel disease. Because human DUOX and its immunological context are similar to MsDUOX, we established M. sexta as a model to understand the role of DUOX in gut inflammation. In this study, we tested if the activation of DUOX can cause gut inflammation in M. sexta.
We established contrast-enhanced CT, MR and FDG-PET imaging in M. sexta in Müller et. al 2019. Production of DUOX associated HOCl after uracil feeding was proven by conversion of R19s into its fluorescence state in the midgut of M. sexta. Additionally, intestinal transcription levels of DUOX were documented via RT-PCR. Then, larvae fed with uracil were compared to uracil negative control larvae and larvae fed with uracil and DPI (diphenyleneiodonium), a DUOX specific inhibitor or fed with uracil and NAC (N-acetylcysteine), a ROS-specific scavenger, using CT, MR and PET imaging. The ultrastructure of the gut epithelium after uracil treatment was investigated using scanning electron microscopy.
The occurrence of HOCl in the gut of uracil fed but not in control larvae was demonstrated. HOCl production in larvae fed with uracil and DPI sowed a significant reduction of HOCL production compared to uracil only fed larvae. Furthermore, survival kinetics of larvae fed with uracil showed a significantly lower survival (p < 0.0001) and a significantly lower weight at L2d4 (p = 0.0208). We detected a significant higher CT and MR contrast-enhanced gut wall thickness (p < 0.0001, p = 0.0146), CT and MR gut wall signal enhancement (p < 0.0001, p = 0.004) and FDG uptake (p = 0.039) in larvae fed with uracil compared to the uracil-negative control group. Inflammation was further confirmed using scanning electron microscopy. Larvae fed with uracil and DPI, or uracil and NAC, showed significantly reduced production of HOCl (DPI) and a significant difference in CT, MR and PET imaging compared to animals fed with uracil only.
We can present strong indications that M. sexta DUOX (MsDUOX) is activated via uracil, and catalyzes the production of ROS, which causes gut inflammation. This new aspect should be considered and may lead to new strategies understanding inflammatory bowel diseases
 A. G. Müller, F. H. H. Müller, M. Hentschel, M. Kampschulte, F. H. Leinberger and T. E. Trenczek, Insect Larvae in Medical Imaging: New Screening System for Gut Inflammatory Compounds Using CT, MR, and PET, 105th Scientific Assembly and Annual Meeting of the Radiological Society of North America (RSNA) 2019
Keywords: inflammation, alternativ animal model, CT, MRI, PET
|12:30 p.m.||PS 18-03||
Copper-free click conjugation of P-selectin antibody to lipid-shelled microbubbles for imaging inflammation in a murine model of inflammatory bowel disease (#321)
Una Goncin1, Wendy Bernhard2, Ronald Geyer2, Steven Machtaler1
1 University of Saskatchewan, Department of Medical Imaging, College of Medicine,, Saskatoon, Canada
Inflammatory bowel disease (IBD) is a chronic disease with no cure. Patients require disease monitoring over their entire lives, where use of expensive imaging modalities is not financially sustainable. We lack a low-cost molecular imaging approach to monitor regions of active IBD over time. Microbubbles (MBs) and ultrasound (US) are an ideal contrast agent/modality combination for repeated molecular imaging. Our goal is use Cu-free click chemistry for adding a P-selectin antibody to the surface of lipid-shelled MBs, and use it to image inflammation in a murine model of acute IBD.
αP-selectin was conjugated to DBCO-PEG4-NHS for 1 hr at RT. MBs were produced by sonicating a perfluorobutane-sparged solution containing solubilized phospholipids including DSPE-PEG2000-Azide (Fig. 1A). MBs were incubated with αP-selectin-DBCO for up to 2 hrs. MB labeling was validated with confocal microscopy after incubation with a fluorescent αRat-IgG. Acute IBD was induced using 4% dextran sulfate sodium (DSS) over 4 - 8 days in 5 of 8 female FVB mice until weight loss was observed. Mouse bowels were imaged using non-linear contrast mode following i.v. bolus of 5x107 MBs. MBs circulated for 4 min before the molecular imaging signal was collected. Each mouse received a bolus of both targeted and non-targeted MBs (unlabelled), allowing for clearance between, and analyzed using VEVOCQ.
MB-antibody labelling using DBCO-Azide click reaction was confirmed using confocal microscopy, where a positive fluorescent signal was detected (Fig. 1B). αP-selectin-targeted MBs were prepared and used to image control (no inflammation) and treated mice (with acute bowel inflammation). In control mice, a low differential targeted enhancement (dTE) signal was detected using P-selectin targeted (0.05 ± 0.08 a.u; n=3) and non-targeted MBs (0.01 a.u; n=3) in control mice (Fig. 1C, top). There was a significant increase in dTE signal in mice with acute bowel inflammation using P-selectin targeted MBs (2.02 ± 0.44 a.u; n=5; P < 0.05) in comparison to non-targeted MBs (0.65 ± 0.66 a.u; n=4; (Fig. 1C, bottom).
Using an Azide-DBCO Cu-free click reaction, we were able to construct a targeted MB to the vascular inflammatory marker P-selectin, which generated a detectable US molecular imaging signal. These data show potential of this approach for quick, cost-efficient labelling of pre-formed lipid-shelled MBs that may provide clinicians with an alternative tool for identifying and monitoring disease progression in IBD patients.
Fig. 1. Cu-free antibody labeling of microbubbles (MBs) for ultrasound imaging of acute murine IBD.
A) Schematic of a perfluorobutane-filled, lipid-shelled MB containing a DSPE-PEG2000-Azide lipid conjugated to anti-P-selectin-DBCO. B) Anti-P-selectin (red) labelling on MB surface. C) US-molecular imaging with non-targeted (top), and P-selectin targeted MBs (bottom) in mice with and without acute IBD (B-mode: left; ultrasound molecular imaging signal: right; green ROI = bowel). D) Differential targeted enhancement (d.T.E; a.u) signal using non-targeted and P-selectin targeted MBs (mean ± standard deviations in control (n=3) and acute IBD mice (n=4)).
Keywords: click-chemistry, p-selectin, contrast agent, inflammation, ultrasound molecular imaging
|12:42 p.m.||PS 18-04||
Non invasive in vivo detection of infiltrating CD8+ T cells in non alcoholic steatohepatitis (NASH) (#495)
Vera Jörke1, Stefania Pezzana1, Dominik Sonanini1, 7, Dominik Seyfried1, Andreas Maurer1, 8, Andreas Schmid1, Alessandro Mascioni2, Irene Gonzales-Mendez3, 8, Leticia Quintanilla-Fend3, 8, Mathias Heikenwälder4, Bernd J. Pichler1, 8, Manfred Kneilling1, 5, 8, Johannes Schwenck1, 6, 8
1 Eberhard Karls University Tübingen, Werner Siemens Imaging Center, Tübingen, Germany
Liver steatosis induced by high caloric diet frequently leads to non-alcoholic steatohepatitis (NASH) and is a major cause of hepatocellular carcinoma (HCC). The progression to NASH and HCC is driven by the inflammatory infiltrate including CD8+ T cells although the exact mechanisms still unknown. Currently, invasive biopsies and subsequent histopathological analysis are the gold standard in the diagnostic of NASH. Here, we investigated the feasibility of non invasive positron emission tomography (PET) using a radiolabeled anti CD8 minibody to characterize the inflammatory infiltrate in NASH.
Male C57BL/6J mice were fed with high fat choline deficient diet (HFCD) to induce NASH over a time course of 12 months according to Wolf et al. (Cancer Cell 2014) or with standard chow as control. To examine the presence of CD8+ T cells in the liver in vivo we labeled the anti CD8 minibody df-IAB42 with the PET isotope 89Zr. Static PET scans combined with sequential magnetic resonance imaging (MRI) from mice with HFCD and standard diet were performed 24h after intravenous 89Zr-df-IAB42 injection. Attenuation correction was performed using the transmission data from the integrated 57Co source. We harvested liver tissue and conducted flow cytometry as well as histopathology (H&E) and immunohistochemistry (IHC) focusing on CD8+ T cells. All data is presented as mean ± SEM.
The body weight of mice with HFCD diet increased significantly compared to the control group with standard diet (55±5 vs. 33±5 g). PET imaging 24h p.i. revealed an intense 89Zr-df-IAB42 uptake in lymphatic tissue like the spleen and the lymph nodes in both experimental groups. In the livers of HFCD mice the 89Zr-df-IAB42 uptake was significantly enhanced compared to the livers of mice with standard diet (15.1±0.9 vs. 9.8±0.5 %injected dose). Flow cytometry confirmed a significant increase of CD8+ T cells in the liver tissue of HFCD mice compared to controls (26.1±1.7% vs. 15.6±1.9%), while only slight insignificant differences in CD4+ T cells where observed. H&E staining of liver tissue from mice fed with HFCD revealed typical histological features of NASH like steatosis, fibrosis and hepatocyte ballooning. IHC indicated an elevation in CD8+ T cells in the liver tissue of HFCD mice compared to the standard diet group (16.2 ± 3.0 vs. 6.6 ± 1.7 cells per field of view).
Here we demonstrated the feasibility of non-invasive in vivo monitoring of the CD8+ T cell infiltrate in NASH representing a novel tool to study the role of CD8+ T cells during disease progression from steatosis to NASH and HCC. Furthermore, translation of our preclinical results into the clinic may help to stratify NASH patients for a more individualized patient-centered care.
Keywords: NASH, HCC, PET, CD8
|12:54 p.m.||PS 18-05||
Imaging of the myeloid cell activation with 4-[18F]fluoronaphthol (#35)
Federica Pisaneschi1, Seth T. Gammon1, Vincenzo Paolillo2, Sarah A. Qureshy1, David Piwnica-Worms1
1 UT MD Anderson Cancer Center, Cancer Systems Imaging, Houston, Texas, United States of America
Dysregulation of the innate immune system contributes to the pathophysiology of many diseases, including obesity, rheumatoid arthritis and diabetes. In oncology, the role of innate immunity is complex: neutrophils can suppress tumors but depending on context, neutrophils and myeloid-derived suppressor cells can promote tumor growth. Reactive oxygen species (ROS), generated by NADPH oxidase 2 (Nox2) and Myeloperoxidase (MPO), are key reactive molecules in many inflammatory states. Nox2- and MPO-derived ROS are known to oxidize naphthol, the latter shown to bind to neutrophils in cell culture.1
We propose 4-[18F]Fluoronaphthol ([18F]4FN), naphthol fluorinated bioisoster, as a novel radiopharmaceutical to detect active inflammation by PET. [18F]4FN was synthesized by copper-mediated radiofluorination2 on an automated synthetic module (GE Tracerlab). [18F]4FN was tested in vitro for its ability to be retained by "neutrophil-like" human cells upon activation with phorbol-12-myristate-13-acetate (PMA) and in vivo on three models of acute inflammation: PMA-induced ear mild contact dermatitis, lipopolysaccharide (LPS)-induced ankle arthritis and LPS-induced whole body toxic shock. [18F]4FN ability to detect inflammation by PET was compared to clinically used [18F]FDG and 68Ga-citrate. MPO and Nox2 involvement in the trapping of [18F]4FN was proven using inhibitors and knock-out mice.
[18F]4FN (A) was synthesized in 90 minutes, with an activity yield of 6.8±2.5% (n=22), >99% radiochemical purity and up to 140 GBq/µmol molar activity. The tracer has at least a 4 h shelf-stability and was stable in mouse plasma for at least 1 h. When incubated with “neutrophil-like” human cells (all-trans-retinoic acid-differentiated HL-60 ALL cells, B) for 30 min, [18F]4FN showed a 4 fold increase in retention in cells treated with PMA. MPO-inhibitor 4-ABAH could block the signal. In vivo, upon PMA-inflammation of one earlobe, [18F]4FN imaging yielded good contrast-to-noise ratios in two independent strains of adult female mice (Balb/c and C57Bl6), both by IV and IP injection (C). [18F]4FN yielded superior contrast to [18F]FDG (D). In an LPS model of ankle arthritis, [18F]4FN detected the inflamed site and correlated well with L-012 (E).3 In the LPS whole body toxic shock model, [18F]4FN retention was significantly higher when compared to 68Ga-citrate.
[18F]4FN could be readily synthesized with high molar activity and good yields, it was stable in both buffer and mouse plasma and appeared to be a suitable PET agent for monitoring ROS produced by activation of the innate immune system in deep tissues. Mechanistically, [18F]4FN seems to be linked to MPO and Nox2 expression, via enzyme-mediated ROS production. Superiority to [18F]4FDG and 68Ga-citrate warrants clinical translation of [18F]4FN.
We thank the Nuclear Magnetic Resonance Facility and Small Animal Imaging Facility (SAIF), supported by the MD Anderson Cancer Center Support Grant CA016672.
 Eastmond, D. A.; French, R. C.; Ross, D.; Smith, M. T. Chem.-Biol. Interact. 1987, 63, 47.
 Tredwell, M.; Preshlock, S. M.; Taylor, N. J.; Gruber, S.; Huiban, M.; Passchier, J.; Mercier, J.; Génicot, C.; Gouverneur, V. Angewandte Chem. Int. Edit. 2014, 53, 7751
 Gross, S.; Gammon, S.; Moss, B. L.; Rauch, D.; Harding, J.; Heinecke, J. W.; Ratner, L.; Piwnica-Worms, D. Nat. Med. 2009, 15, 455.
[18F]-4-Fluoronaphthol PET to detect activation of the innate immune system
A) Structure of [18F]4FN; B) PMA-induced HL-60 cell retention of [18F]4FN compared to vehicle, and inhibition of MPO with 4-ABAH; C) Retention in vivo of [18F]4FN in PMA-induced ear inflammation model compared to non-inflamed ear, with both IP and IV delivery of [18F]4FN; D) Comparison of [18F]4FN and [18F]FDG; E) [18F]4FN PET/CT scan of LPS-induced ankle articular inflammation model and L-012 BLI image of the same mouse confirm [18F]4FN and L-012 correlation.
Keywords: innate immunity, PET imaging, 18F-fluoronaphthol, MPO, ROS
|1:06 p.m.||PS 18-06||
Targeted photodynamic therapy of activated synovial fibroblast in rheumatoid arthritis: a study in murine and human synovium (#132)
Daphne N. Dorst1, Sanne A. M. van Lith1, Mark Rijpkema1, Mijke Buitinga2, Peter Laverman1, Marti Boss1, Maarten Brom1, Desirée Bos1, Anne Freimoser-Grundschober3, Christian Klein3, Birgitte Walgreen1, Peter van der Kraan1, Marije Koenders1, Martin Gotthardt1
1 Radboudumc, Nijmegen, Netherlands
Activated synovial fibroblasts (ASF) play an important role in the pathogenesis of rheumatoid arthritis (RA), contributing to the pro-inflammatory environment in the joint and degradation of cartilage and bone. Depleting ASF, e.g. by targeted therapy, could ameliorate both these hallmarks of RA. ASF are characterized by the expression of fibroblast activation protein (FAP). Here, we investigated the potential of FAP-targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 conjugated with the photosensitizer IRDye700DX (28H1-700DX) to selectively kill ASF.
The ability of FAP-tPDT to induce cell death in ASF was investigated in FAP-expressing 3T3 fibroblasts, primary synovial fibroblasts and synovial biopsies from RA patients. The feasibility to treat arthritis was evaluated in the murine collagen-induced arthritis (CIA) model. Mice with established CIA in their hind legs received 28H1-700DX or PBS control i.v. and were exposed to 690 nm light. Development of arthritis was scored over time. Biopsies and cultured cells were incubated with 28H1-700DX for 4h and subsequently exposed to light. Controls were either incubated in plain buffer or not exposed to light. For the cell culture, viability was measured using CellTiter-Glo. Biopsies were stained for caspase-3, γH2AX, TUNEL staining and FAP expression.
Development of arthritis in the CIA model was initially mitigated in the treated group (AUC change in arthritis score 0.82±0.27 vs 0.46±0.17 for control and treated groups, respectively, p<0.05). FAP-tPDT performed on cultured fibroblasts showed a light-dose-dependent increase in cell death. Cell viability was not affected in the controls. Radiant exposures of 52.8, 105.6 and 158.4 J/cm2 decreased cell viability significantly compared to control (38.6±6.9%, 67.5±10.9% and 80.6±5.8% respectively, all p<0.001). After FAP-tPDT, expression of γH2AX, caspase-3 and TUNEL was increased in FAP positive areas compared to pre-treatment biopsies (0.94±0.58, 0.47±0.41, 0.87±0.47 for γH2AX, caspase-3 and TUNEL, respectively), indicating successful ASF targeting.
After successful targeting of FAP-positive cells in vitro and during collagen-induced arthritis, we demonstrate that FAP-tPDT induces cell death of FAP-positive activated fibroblasts in synovial tissue from RA patients. This is a first indication that FAP-targeted PDT can be a feasible new treatment strategy in patients with RA.
Keywords: Photodynamic therapy, Fibroblast, Rheumatoid arthritis, FAP
|1:18 p.m.||PS 18-07||
Unraveling the multifaceted in vivo fate of active-targeting nanomedicines (#536)
Alexandros Marios Sofias1, 2, 3, Carlos Pérez-Medina3, 4, Geir Bjørkøy2, Twan Lammers1, 5, 6, Willem Mulder3, 7, Sjoerd Hak2, 8
1 RWTH Aachen University, Aachen, Germany
Active-targeting nanomedicines have been extensively evaluated as therapeutic and diagnostic agents in cancer and other diseases. Their potential to improve drug delivery relies on their ability to target receptors or other macromolecules that are overexpressed in diseased tissues. Published “proof” of their targeting potential is often based on in vitro or ex vivo data. In addition, the investigation of these nanomedicines is often limited to the identification of interactions only with the target cell, while their engagement with non-target cells or organs is being neglected.
We decorated liposomes and nanoemulsions with clinically trialed targeting ligands (cRGD). We used two established ligand decoration procedures to assess how these affect nanoparticle in vivo behavior (1). Additionally, labeling the nanoparticles with radioactive nuclides or fluorescent molecules, allowed us to obtain quantitative real-time insights at organism and organ level (PET/CT imaging) (2) as well as at tissue and cellular level (intravital microscopy) (3). To corroborate the real-time observations, we performed ex vivo gamma counting, flow cytometry, and immunohistochemistry. Lastly, in order to investigate whether our in vivo observations are disease-dependent, we studied nanoparticle in vivo behavior in both cancerous and inflammatory lesions.
By using this uniquely complementary experimental approach on approximately 350 mice, we identified that nanoparticle ligand-decoration procedures significantly affect nanoparticle interactions with immune cells and that ligand decoration can severely affect nanoparticle pharmacokinetic and biodistribution properties. We also observed that cRGD-nanoparticles are extensively taken up by circulating phagocytes, which were previously not considered target-cells. Majorly, we identified a unique targeting mechanism in which these phagocytosed nanoparticles actively home to tumor and inflammation sites in an immune-cell mediated fashion.
The realization that myeloid immune cells are involved in nanoparticle delivery processes establishes cell-mediated targeting as an independent mechanism that should be studied for therapeutic and diagnostic purposes.
AcknowledgmentHMN (AMS: 90062100, 90284100; SH: 90262100); AHA (CPM: 16SDG31390007), NIH (WJMM: R01 CA220234); NFR (SH: 230788/F20).
 Sofias et al., Mol. Pharmaceutics, 15, 12, 5754-5761 (2018)
 Pérez-Medina et al., Nat. Commun., 7, 11838 (2016)
 Sofias et al., Mol. Imaging Biol., 1-8 (2019)
Keywords: Nanomedicine, Immune-cell targeting, Tumor-homing phagocytes, Intravital Microscopy, PET/CT imaging