Metastatic tumours in the brain now represent one of the leading causes of death from cancer. Current treatments are largely ineffective owing to the combination of late diagnosis and poor delivery of therapies across the blood-brain barrier (BBB). Conjugating magnetic resonance imaging (MRI) contrast agents with a monoclonal antibody for VCAM-1 (anti-VCAM1) has been shown to enable detection of micrometastases, two to three orders of magnitude smaller in volume than those currently detectable clinically. The aim of this study was to exploit this targeting approach to enable localised and temporary BBB opening at the site of early-stage metastases using functionalised microbubbles and ultrasound. Methods: Microbubbles functionalised with anti-VCAM1 were synthesised and shown to bind to VCAM-1-expressing cells in vitro. Experiments were then conducted in vivo in a unilateral breast cancer brain metastasis mouse model using Gadolinium-DTPA (Gd-DTPA) enhanced MRI to detect BBB opening. Following injection of Gd-DTPA and targeted microbubbles, the whole brain volume was simultaneously exposed to ultrasound (0.5 MHz, 10% duty cycle, 0.7 MPa peak negative pressure, 2 min treatment time). T1-weighted MRI was then performed to identify BBB opening, followed by histological confirmation via immunoglobulin G (IgG) immunohistochemistry. Results: In mice treated with targeted microbubbles and ultrasound, statistically significantly greater extravasation of Gd-DTPA and IgG was observed in the left tumour-bearing hemisphere compared to the right hemisphere 5 min after treatment. No acute adverse effects were observed. There was no investigation of longer term bioeffects owing to the nature of the study. Conclusion: The results demonstrate the feasibility of using targeted microbubbles in combination with low intensity ultrasound to localise opening of the BBB to metastatic sites in the brain. This approach has potential application in the treatment of metastatic tumours whose location cannot be established a priori with conventional imaging methods.

Current clinical diagnostic imaging methods for lung metastases are sensitive only to large tumours (1–2 mm cross-sectional diameter), and early detection can dramatically improve treatment. We have previously demonstrated that an antibody-targeted MRI contrast agent based on microparticles of iron oxide (MPIO; 1 μm diameter) enables the imaging of endothelial vascular cell adhesion molecule-1 (VCAM-1). Using a mouse model of lung metastasis, upregulation of endothelial VCAM-1 expression was demonstrated in micrometastasis-associated vessels but not in normal lung tissue, and binding of VCAM-MPIO to these vessels was evident histologically. Owing to the lack of proton MRI signals in the lungs, we modified the VCAM-MPIO to include zirconium-89 (89Zr, t1/2 = 78.4 h) in order to allow the in vivo detection of lung metastases by positron emission tomography (PET). Using this new agent (89Zr-DFO-VCAM-MPIO), it was possible to detect the presence of micrometastases within the lung in vivo from ca. 140 μm in diameter. Histological analysis combined with autoradiography confirmed the specific binding of the agent to the VCAM-1 expressing vasculature at the sites of pulmonary micrometastases. By retaining the original VCAM-MPIO as the basis for this new molecular contrast agent, we have created a dual-modality (PET/MRI) agent for the concurrent detection of lung and brain micrometastases.

AimsAccording to Braak's hypothesis, it is plausible that Parkinson's disease (PD) originates in the enteric nervous system (ENS) and spreads to the brain through the vagus nerve. In this work, we studied whether inflammatory bowel diseases (IBDs) in humans can progress with the emergence of pathogenic alpha-synuclein (alpha-syn) in the gastrointestinal tract and midbrain dopaminergic neurons.MethodsWe have analysed the gut and the ventral midbrain from subjects previously diagnosed with IBD and form a DSS-based rat model of gut inflammation in terms of alpha-syn pathology.ResultsOur data support the existence of pathogenic alpha-syn in both the gut and the brain, thus reinforcing the potential role of the ENS as a contributing factor in PD aetiology. Additionally, we have analysed the effect of a DSS-based rat model of gut inflammation to demonstrate (i) the appearance of P-alpha-syn inclusions in both Auerbach's and Meissner's plexuses (gut), (ii) an increase in alpha-syn expression in the ventral mesencephalon (brain) and (iii) the degeneration of nigral dopaminergic neurons, which all are considered classical hallmarks in PD.ConclusionThese results strongly support the plausibility of Braak's hypothesis and emphasise the significance of peripheral inflammation and the gut-brain axis in initiating alpha-syn aggregation and transport to the substantia nigra, resulting in neurodegeneration. In this article, we include data demonstrating pathogenic insoluble phosphorylated alpha-synuclein (P-alpha-syn) aggregates in the gastrointestinal tract from patients earlier diagnosed with inflammatory bowel disease (IBD). Using thioflavin-S staining along with P-alpha-syn in the human midbrain of IBD cases, we have identified a group of highly depigmented neurons showing insoluble alpha-syn aggregates. Using a rat model of IBD, we show that long-lasting inflammation of the gastrointestinal tract leads to striking neuropathological features typically associated with Parkinson's disease. image

Table containing all brain metastatic cell lines in the BrMPanel

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly anti-angiogenic therapeutics, is difficult to ascertain using current magnetic resonance imaging (MRI) methods. Imaging of the angiogenic vasculature has been successfully carried out in solid tumours using microparticles of iron oxide (MPIO) conjugated to a peptide (RGD) targeting the integrin α v β 3 . The aim of this study was to determine whether RGD-MPIO could be used to identify angiogenic blood vessels in brain metastases in vivo. A mouse model of intracerebrally implanted brain macrometastasis was established through intracerebral injection of 4T1-GFP cells. T 2 * weighted imaging was used to visualize MPIO induced hypointense voxels in vivo, and Prussian blue staining was used to visualize MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualizing angiogenic vessels per se was reduced by the presence of endogenous iron-laden macrophages in larger metastases, resulting in pre-existing hypointense areas within the tumour. Further, our data suggest that peptide-targeted MPIO, but not antibody-targeted MPIO, are taken up by perivascular macrophages within the macrometastatic microenvironment, resulting in additional nonspecific contrast. Whilst pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our findings suggest that the use of antibodies rather than peptides as the targeting ligand may represent a preferable route forward for new angiogenesis-targeted molecular MRI agents.

Supplementary Data from Metabolomic Biomarkers in Blood Samples Identify Cancers in a Mixed Population of Patients with Nonspecific Symptoms

1: JAG1 antibody characterization. 2: antibody inhibition of JAG1 mediated Notch signalling in tumor cell lines. 3: antibody inhibition of JAG1 signalling in vascular cells. 4: antibody inhibition of JAG1 signalling in HUVEC-HUVSMC co-culture. 5: effect of anti-Notch pathway treatment on MDA-MB231 2D growth and J1-65D titration and effect on 3D growth and gene expression. 6: J1-65D treatment of established OVCAR3 xenograft. 7: JAG1 antibodies cross-reactivity with rat Jag1. 8: anti-JAG1 treatment effect in a rat brain metastasis model.

<p>Additional info regarding COX-1 and iNOS</p>

<p>Additional info regarding colocalisation studies</p>

Additional info regarding the ENU model

<p>Additional info regarding COX-1 and iNOS</p>

<p>Additional info regarding the use of WP1066</p>

Understanding brain structure and function often requires combining data across different modalities and scales to link microscale cellular structures to macroscale features of whole brain organisation. Here we introduce the BigMac dataset, a resource combining in vivo MRI, extensive postmortem MRI and multi-contrast microscopy for multimodal characterisation of a single whole macaque brain. The data spans modalities (MRI and microscopy), tissue states (in vivo and postmortem), and four orders of spatial magnitude, from microscopy images with micrometre or sub-micrometre resolution, to MRI signals on the order of millimetres. Crucially, the MRI and microscopy images are carefully co-registered together to facilitate quantitative multimodal analyses. Here we detail the acquisition, curation, and first release of the data, that together make BigMac a unique, openly-disseminated resource available to researchers worldwide. Further, we demonstrate example analyses and opportunities afforded by the data, including improvement of connectivity estimates from ultra-high angular resolution diffusion MRI, neuroanatomical insight provided by polarised light imaging and myelin-stained histology, and the joint analysis of MRI and microscopy data for reconstruction of the microscopy-inspired connectome. All data and code are made openly available.

<p>Supplementary Methods, Figure Legends and Tables</p>

Abstract The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood–brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Magnetic resonance imaging is a powerful tool in preclinical research of diseases with associated neuroinflammation, as it allows high-resolution imaging without harmful ionizing radiation. The development of targetable iron oxide contrast agents, such as microparticles of iron oxide (MPIO), has greatly enhanced molecular MRI for superior detection of target molecules. Cell adhesion molecules (CAMs) are upregulated early in neuroinflammation on the luminal side of activated endothelial cells within the blood-brain barrier, making them a highly accessible target. Targeting MPIO with CAM antibodies has improved the early detection of neuropathologies in preclinical models, as well as providing further insight into disease progression and mechanisms. This chapter highlights the uses of molecular MRI, specifically CAM-targeted MPIO, for investigating multiple sclerosis, stroke, epilepsy, and brain cancer and how the advances made in these preclinical models have the potential to be transferred to other neuropathologies and tissues.

<p>Additional info regarding OX42</p>

Additional info regarding the Lv-CNTF model

Supplementary material and methods. Supplementary table 1: cell lines and growth conditions. Supplementary table 2: Notch ligands and control protein for plate coating. Supplementary table 3: qPCR primers. Supplementary table 4: flow cytometry antibodies. Supplementary table 5: ICC/IHC antibodies. Supplementary table 6: titration of Notch1-JAG1 binding inhibition by JAG1 antibodies. Supplementary table 7: amino acid sequence of JAG1 antibodies. Supplementary table 8: blood analysis on antibody-treated tumor-bearing rats.

Supplementary Material, including Supplementary Methods describing the Random Effects Model. Supplementary Results showing example quantitative T1/T2 maps from the rat tumour model (Supplementary Figure S1), the standard curve for making quantitative protein concentration measurements using Coomassie staining (Supplementary Figure S2), the result of a conventional MTRasym APT analysis approach in the rat tumour model (Supplementary Figure S3), and the histological validation that areas of high protein concentration correlate with areas of increased hypoxia, increased vessel area and higher cellularity in the rat tumour model (Supplementary Figure S4). Supplementary Table S1 shows the mean {plus minus} standard deviation of T1 and T2 relaxation times measured in different tissue ROIs from both the rat tumour and mouse tumour models.