A multimodal imaging study to highlight elastin-derived peptide pro-tumoral effect in a pancreatic xenograft model.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is highly malignant with a very poor prognosis due to its silent development and metastatic profile with a 5-year survival rate below 10%. PDAC is characterised by an abundant desmoplastic stroma modulation that influences cancer development by extracellular matrix/cell interactions. Elastin is a key element of the extracellular matrix. Elastin degradation products (EDPs) regulate numerous biological processes such as cell proliferation, migration and invasion. The aim of the present study was to characterise for the first time the effect of two EDPs with consensus sequences "GxxPG" and "GxPGxGxG" (VG-6 and AG-9) on PDAC development. The ribosomal protein SA (RPSA) has been discovered recently, acting as a new receptor of EDPs on the surface of tumour cells, contributing to poor prognosis. METHODS: Six week-old female Swiss nude nu/nu (Nu(Ico)-Foxn1nu) mice were subcutaneously injected with human PDAC MIA PaCa-2/eGFP-FLuc+ cells, transduced with a purpose-made lentiviral vector, encoding green fluorescent protein (GFP) and Photinus pyralis (firefly) luciferase (FLuc). Animals were treated three times per week with AG-9 (n = 4), VG-6 (n = 5) or PBS (n = 5). The influence of EDP on PDAC was examined by multimodal imaging (bioluminescence imaging (BLI), fluorescence imaging (FLI) and magnetic resonance imaging (MRI). Tumour volumes were also measured using a caliper. Finally, immunohistology was performed at the end of the in vivo study. RESULTS: After in vitro validation of MIA PaCa-2 cells by optical imaging, we demonstrated that EDPs exacerbate tumour growth in the PDAC mouse model. While VG-6 stimulated tumour growth to some extent, AG-9 had greater impact on tumour growth. We showed that the expression of the RPSA correlates with a possible effect of EDPs in the PDAC model. Multimodal imaging allowed for longitudinal in vivo follow-up of tumour development. In all groups, we showed mature vessels ending in close vicinity of the tumour, except for the AG-9 group where mature vessels are penetrating the tumour reflecting an increase of vascularisation. CONCLUSIONS: Our results suggest that AG-9 strongly increases PDAC progression through an increase in tumour vascularisation.

Simultaneous in vivo PET/MRI using fluorine-18 labeled Fe3O4@Al(OH)3 nanoparticles: comparison of nanoparticle and nanoparticle-labeled stem cell distribution.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown potential for treatment of different diseases. However, their working mechanism is still unknown. To elucidate this, the non-invasive and longitudinal tracking of MSCs would be beneficial. Both iron oxide-based nanoparticles (Fe3O4 NPs) for magnetic resonance imaging (MRI) and radiotracers for positron emission tomography (PET) have shown potential as in vivo cell imaging agents. However, they are limited by their negative contrast and lack of spatial information as well as short half-life, respectively. In this proof-of-principle study, we evaluated the potential of Fe3O4@Al(OH)3 NPs as dual PET/MRI contrast agents, as they allow stable binding of [18F]F- ions to the NPs and thus, NP visualization and quantification with both imaging modalities. RESULTS: 18F-labeled Fe3O4@Al(OH)3 NPs (radiolabeled NPs) or mouse MSCs (mMSCs) labeled with these radiolabeled NPs were intravenously injected in healthy C57Bl/6 mice, and their biodistribution was studied using simultaneous PET/MRI acquisition. While liver uptake of radiolabeled NPs was seen with both PET and MRI, mMSCs uptake in the lungs could only be observed with PET. Even some initial loss of fluoride label did not impair NPs/mMSCs visualization. Furthermore, no negative effects on blood cell populations were seen after injection of either the NPs or mMSCs, indicating good biocompatibility. CONCLUSION: We present the application of novel 18F-labeled Fe3O4@Al(OH)3 NPs as safe cell tracking agents for simultaneous PET/MRI. Combining both modalities allows fast and easy NP and mMSC localization and quantification using PET at early time points, while MRI provides high-resolution, anatomic background information and long-term NP follow-up, hereby overcoming limitations of the individual imaging modalities.

Characterization of a preclinical PET insert in a 7 tesla MRI scanner: beyond NEMA testing.

This study evaluates the performance of the Bruker positron emission tomograph (PET) insert combined with a BioSpec 70/30 USR magnetic resonance imaging (MRI) scanner using the manufacturer acceptance protocol and the NEMA NU 4-2008 for small animal PET. The PET insert is made of 3 rings of 8 monolithic LYSO crystals (50 × 50 × 10 mm3) coupled to silicon photomultipliers (SiPM) arrays, conferring an axial and transaxial FOV of 15 cm and 8 cm. The MRI performance was evaluated with and without the insert for the following radiofrequency noise, magnetic field homogeneity and image quality. For the PET performance, we extended the NEMA protocol featuring system sensitivity, count rates, spatial resolution and image quality to homogeneity and accuracy for quantification using several MRI sequences (RARE, FLASH, EPI and UTE). The PET insert does not show any adverse effect on the MRI performances. The MR field homogeneity is well preserved (Diameter Spherical Volume, for 20 mm of 1.98 ± 4.78 without and -0.96 ± 5.16 Hz with the PET insert). The PET insert has no major effect on the radiofrequency field. The signal-to-noise ratio measurements also do not show major differences. Image ghosting is well within the manufacturer specifications (<2.5%) and no RF noise is visible. Maximum sensitivity of the PET insert is 11.0% at the center of the FOV even with simultaneous acquisition of EPI and RARE. PET MLEM resolution is 0.87 mm (FWHM) at 5 mm off-center of the FOV and 0.97 mm at 25 mm radial offset. The peaks for true/noise equivalent count rates are 410/240 and 628/486 kcps for the rat and mouse phantoms, and are reached at 30.34/22.85 and 27.94/22.58 MBq. PET image quality is minimally altered by the different MRI sequences. The Bruker PET insert shows no adverse effect on the MRI performance and demonstrated a high sensitivity, sub-millimeter resolution and good image quality even during simultaneous MRI acquisition.

Development of Superparamagnetic Nanoparticles Coated with Polyacrylic Acid and Aluminum Hydroxide as an Efficient Contrast Agent for Multimodal Imaging.

Early diagnosis of disease and follow-up of therapy is of vital importance for appropriate patient management since it allows rapid treatment, thereby reducing mortality and improving health and quality of life with lower expenditure for health care systems. New approaches include nanomedicine-based diagnosis combined with therapy. Nanoparticles (NPs), as contrast agents for in vivo diagnosis, have the advantage of combining several imaging agents that are visible using different modalities, thereby achieving high spatial resolution, high sensitivity, high specificity, morphological, and functional information. In this work, we present the development of aluminum hydroxide nanostructures embedded with polyacrylic acid (PAA) coated iron oxide superparamagnetic nanoparticles, Fe3O4@Al(OH)3, synthesized by a two-step co-precipitation and forced hydrolysis method, their physicochemical characterization and first biomedical studies as dual magnetic resonance imaging (MRI)/positron emission tomography (PET) contrast agents for cell imaging. The so-prepared NPs are size-controlled, with diameters below 250 nm, completely and homogeneously coated with an Al(OH)3 phase over the magnetite cores, superparamagnetic with high saturation magnetization value (Ms = 63 emu/g-Fe3O4), and porous at the surface with a chemical affinity for fluoride ion adsorption. The suitability as MRI and PET contrast agents was tested showing high transversal relaxivity (r2) (83.6 mM-1 s-1) and rapid uptake of 18F-labeled fluoride ions as a PET tracer. The loading stability with 18F-fluoride was tested in longitudinal experiments using water, buffer, and cell culture media. Even though the stability of the 18F-label varied, it remained stable under all conditions. A first in vivo experiment indicates the suitability of Fe3O4@Al(OH)3 nanoparticles as a dual contrast agent for sensitive short-term (PET) and high-resolution long-term imaging (MRI).

Targeting tumor cells and neovascularization using RGD-functionalized magnetoliposomes.

PURPOSE: Magnetoliposomes (MLs) have shown great potential as magnetic resonance imaging contrast agents and as delivery vehicles for cancer therapy. Targeting the MLs towards the tumor cells or neovascularization could ensure delivery of drugs at the tumor site. In this study, we evaluated the potential of MLs targeting the αvß3 integrin overexpressed on tumor neovascularization and different tumor cell types, including glioma and ovarian cancer. METHODS: MLs functionalized with a Texas Red fluorophore (anionic MLs), and with the fluorophore and the cyclic Arginine-Glycine-Aspartate (cRGD; cRGD-MLs) targeting the αvß3 integrin, were produced in-house. Swiss nude mice were subcutaneously injected with 107 human ovarian cancer SKOV-3 cells. Tumors were allowed to grow for 3 weeks before injection of anionic or cRGD-MLs. Biodistribution of MLs was followed up with a 7T preclinical magnetic resonance imaging (MRI) scanner and fluorescence imaging (FLI) right after injection, 2h, 4h, 24h and 48h post injection. Ex vivo intratumoral ML uptake was confirmed using FLI, electron paramagnetic resonance spectroscopy (EPR) and histology at different time points post injection. RESULTS: In vivo, we visualized a higher uptake of cRGD-MLs in SKOV-3 xenografts compared to control, anionic MLs with both MRI and FLI. Highest ML uptake was seen after 4h using MRI, but only after 24h using FLI indicating the lower sensitivity of this technique. Furthermore, ex vivo EPR and FLI confirmed the highest tumoral ML uptake at 4 h. Last, a Perl's stain supported the presence of our iron-based particles in SKOV-3 xenografts. CONCLUSION: Uptake of cRGD-MLs can be visualized using both MRI and FLI, even though the latter was less sensitive due to lower depth penetration. Furthermore, our results indicate that cRGD-MLs can be used to target SKOV-3 xenograft in Swiss nude mice. Therefore, the further development of this particles into theranostics would be of interest.

CT-2A neurospheres-derived high-grade glioma in mice: a new model to address tumor stem cells and immunosuppression.

Recently, several promising treatments for high-grade gliomas (HGGs) failed to provide significant benefit when translated from the preclinical setting to patients. Improving the animal models is fundamental to overcoming this translational gap. To address this need, we developed and comprehensively characterized a new in vivo model based on the orthotopic implantation of CT-2A cells cultured in neurospheres (NS/CT-2A). Murine CT-2A methylcholanthrene-induced HGG cells (C57BL/6 background) were cultured in monolayers (ML) or NS and orthotopically inoculated in syngeneic animals. ML/CT-2A and NS/CT-2A tumors' characterization included the analysis of tumor growth, immune microenvironment, glioma stem cells (GSCs), vascularization and metabolites. The immuno-modulating properties of NS/CT-2A and ML/CT-2A cells on splenocytes were tested in vitro Mice harboring NS/CT-2A tumors had a shorter survival than those harboring ML/CT-2A tumors (P=0.0033). Compared to standard ML/CT-2A tumors, NS/CT-2A tumors showed more abundant GSCs (P=0.0002 and 0.0770 for Nestin and CD133, respectively) and regulatory T cells (Tregs, P=0.0074), and a strong tendency towards an increased vascularization (P=0.0503). There were no significant differences in metabolites' composition between NS/ and ML/CT-2A tumors. In vitro, NS were able to drive splenocytes towards a more immunosuppressive status by reducing CD8+ T cells (P=0.0354) and by promoting Tregs (P=0.0082), macrophages (MF, P=0.0019) and their M2 subset (P=0.0536). Compared to standard ML/CT-2A tumors, NS/CT-2A tumors show a more aggressive phenotype with increased immunosuppression and GSCs proliferation. Because of these specific features, the NS/CT-2A model represents a clinically relevant platform in the search for new HGG treatments aimed at reducing immunosuppression and eliminating GSCs.

A Multimodal Imaging Approach Enables In Vivo Assessment of Antifungal Treatment in a Mouse Model of Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus causes life-threatening lung infections in immunocompromised patients. Mouse models are extensively used in research to assess the in vivo efficacies of antifungals. In recent years, there has been an increasing interest in the use of noninvasive imaging techniques to evaluate experimental infections. However, single imaging modalities have limitations concerning the type of information they can provide. In this study, magnetic resonance imaging and bioluminescence imaging were combined to obtain longitudinal information on the extent of developing lesions and fungal load in a leukopenic mouse model of invasive pulmonary aspergillosis (IPA). This multimodal imaging approach was used to assess changes occurring within lungs of infected mice receiving voriconazole treatment starting at different time points after infection. The results showed that IPA development depends on the inoculum size used to infect animals and that disease can be successfully prevented or treated by initiating intervention during early stages of infection. Furthermore, we demonstrated that a reduction in fungal load is not necessarily associated with the disappearance of lesions on anatomical lung images, especially when antifungal treatment coincides with immune recovery. In conclusion, multimodal imaging allows an investigation of different aspects of disease progression or recovery by providing complementary information on dynamic processes, which are highly useful for assessing the efficacy of (novel) therapeutic compounds in a time- and labor-efficient manner.

Bronchoscopic fibered confocal fluorescence microscopy for longitudinal in vivo assessment of pulmonary fungal infections in free-breathing mice.

Respiratory diseases, such as pulmonary infections, are an important cause of morbidity and mortality worldwide. Preclinical studies often require invasive techniques to evaluate the extent of infection. Fibered confocal fluorescence microscopy (FCFM) is an emerging optical imaging technique that allows for real-time detection of fluorescently labeled cells within live animals, thereby bridging the gap between in vivo whole-body imaging methods and traditional histological examinations. Previously, the use of FCFM in preclinical lung research was limited to endpoint observations due to the invasive procedures required to access lungs. Here, we introduce a bronchoscopic FCFM approach that enabled in vivo visualization and morphological characterisation of fungal cells within lungs of mice suffering from pulmonary Aspergillus or Cryptococcus infections. The minimally invasive character of this approach allowed longitudinal monitoring of infection in free-breathing animals, thereby providing both visual and quantitative information on infection progression. Both the sensitivity and specificity of this technique were high during advanced stages of infection, allowing clear distinction between infected and non-infected animals. In conclusion, our study demonstrates the potential of this novel bronchoscopic FCFM approach to study pulmonary diseases, which can lead to novel insights in disease pathogenesis by allowing longitudinal in vivo microscopic examinations of the lungs.

Pretargeted PET Imaging Using a Bioorthogonal 18F-Labeled trans-Cyclooctene in an Ovarian Carcinoma Model.

In cancer research, pretargeted positron emission tomography (PET) imaging has emerged as an effective two-step approach that combines the excellent target affinity and selectivity of antibodies with the advantages of using short-lived radionuclides such as fluorine-18. One possible approach is based on the bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) reaction between tetrazines and trans-cyclooctene (TCO) derivatives. Here, we report the first successful use of an 18F-labeled small TCO compound, [18F]1 recently developed in our laboratory, to perform pretargeted immuno-PET imaging. The study was performed in an ovarian carcinoma mouse model, using a trastuzumab-tetrazine conjugate.

Sodium Iodide Symporter PET and BLI Noninvasively Reveal Mesoangioblast Survival in Dystrophic Mice.

Muscular dystrophies are a heterogeneous group of myopathies, characterized by muscle weakness and degeneration, without curative treatment. Mesoangioblasts (MABs) have been proposed as a potential regenerative therapy. To improve our understanding of the in vivo behavior of MABs and the effect of different immunosuppressive therapies, like cyclosporine A or co-stimulation-adhesion blockade therapy, on cell survival noninvasive cell monitoring is required. Therefore, cells were transduced with a lentiviral vector encoding firefly luciferase (Fluc) and the human sodium iodide transporter (hNIS) to allow cell monitoring via bioluminescence imaging (BLI) and small-animal positron emission tomography (PET). Non-H2 matched mMABs were injected in the femoral artery of dystrophic mice and were clearly visible via small-animal PET and BLI. Based on noninvasive imaging data, we were able to show that co-stim was clearly superior to CsA in reducing cell rejection and this was mediated via a reduction in cytotoxic T cells and upregulation of regulatory T cells.