Imaging angiogenesis in an intracerebrally induced model of brain macrometastasis using αv ß3 -targeted iron oxide microparticles.

Brain metastasis is responsible for a large proportion of cancer mortality, and there are currently no effective treatments. Moreover, the impact of treatments, particularly antiangiogenic 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 Arg-Gly-Asp peptide (RGD) targeting integrin αv ß3 . 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. T2 *-weighted imaging was used to visualise MPIO-induced hypointense voxels in vivo, and Prussian blue staining was used to visualise MPIO and endogenous iron histologically ex vivo. The RGD-MPIO showed target-specific binding in vivo, but the sensitivity of the agent for visualising 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. While pre-MPIO imaging will circumvent the issues surrounding pre-existing hypointensities and enable detection of specific contrast, our preliminary 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.

Prion protein "gamma-cleavage": characterizing a novel endoproteolytic processing event.

The cellular prion protein (PrP(C)) is a ubiquitously expressed protein of currently unresolved but potentially diverse function. Of putative relevance to normal biological activity, PrP(C) is recognized to undergo both α- and ß-endoproteolysis, producing the cleavage fragment pairs N1/C1 and N2/C2, respectively. Experimental evidence suggests the likelihood that these processing events serve differing cellular needs. Through the engineering of a C-terminal c-myc tag onto murine PrP(C), as well as the selective use of a far-C-terminal anti-PrP antibody, we have identified a new PrP(C) fragment, nominally 'C3', and elaborating existing nomenclature, 'γ-cleavage' as the responsible proteolysis. Our studies indicate that this novel γ-cleavage event can occur during transit through the secretory pathway after exiting the endoplasmic reticulum, and after PrP(C) has reached the cell surface, by a matrix metalloprotease. We found that C3 is GPI-anchored like other C-terminal and full length PrP(C) species, though it does not localize primarily at the cell surface, and is preferentially cleaved from an unglycosylated substrate. Importantly, we observed that C3 exists in diverse cell types as well as mouse and human brain tissue, and of possible pathogenic significance, γ-cleavage may increase in human prion diseases. Given the likely relevance of PrP(C) processing to both its normal function, and susceptibility to prion disease, the potential importance of this previously underappreciated and overlooked cleavage event warrants further consideration.

Glycosaminoglycan sulfation determines the biochemical properties of prion protein aggregates.

Prion diseases are transmissible neurodegenerative disorders associated with the conversion of the cellular prion protein, PrP(C), to a misfolded isoform called PrP(Sc). Although PrP(Sc) is a necessary component of the infectious prion, additional factors, or cofactors, have been shown to contribute to the efficient formation of transmissible PrP(Sc). Glycosaminoglycans (GAGs) are attractive cofactor candidates as they can be found associated with PrP(Sc) deposits, have been shown to enhance PrP misfolding in vitro, are found in the same cellular compartments as PrP(C) and have been shown to be disease modifying in vivo. Here we investigated the effects of the sulfated GAGs, heparin and heparan sulfate (HS), on disease associated misfolding of full-length recombinant PrP. More specifically, the degree of sulfation of these molecules was investigated for its role in modulating the disease-associated characteristics of PrP. Both heparin and HS induced a ß-sheet conformation in recombinant PrP that was associated with the formation of aggregated species; however, the biochemical properties of the aggregates formed in the presence of heparin or HS varied in solubility and protease resistance. Furthermore, these properties could be modified by changes in GAG sulfation, indicating that subtle changes in the properties of prion disease cofactors could initiate disease associated misfolding.

C-terminal peptides modelling constitutive PrPC processing demonstrate ameliorated toxicity predisposition consequent to α-cleavage.

Misfolding of PrPC (cellular prion protein) to ß-strand-rich conformations constitutes a key event in prion disease pathogenesis. PrPC can undergo either of two constitutive endoproteolytic events known as α- and ß-cleavage, yielding C-terminal fragments known as C1 and C2 respectively. It is unclear whether C-terminal fragments generated through α- and ß-cleavage, especially C2, influence pathogenesis directly. Consequently, we compared the biophysical properties and neurotoxicity of recombinant human PrP fragments recapitulating α- and ß-cleavage, namely huPrP-(112-231) (equating to C1) and huPrP-(90-231) (equating to C2). Under conditions we employed, huPrP-(112-231) could not be induced to fold into a ß-stranded isoform and neurotoxicity was not a feature for monomeric or multimeric assemblies. In contrast, huPrP-(90-231) easily adopted a ß-strand conformation, demonstrated considerable thermostability and was toxic to neurons. Synthetic PrP peptides modelled on α- and ß-cleavage of the unique Y145STOP (Tyr145→stop) mutant prion protein corroborated the differential toxicity observed for recombinant huPrP-(112-231) and huPrP-(90-231) and suggested that the persistence of soluble oligomeric ß-strand-rich conformers was required for significant neurotoxicity. Our results additionally indicate that α- and ß-cleavage of PrPC generate biophysically and biologically non-equivalent C-terminal fragments and that C1 generated through α-cleavage appears to be pathogenesis-averse.

Targeted opening of the blood-brain barrier using VCAM-1 functionalised microbubbles and "whole brain" ultrasound.

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.

Inhibition of Anti-Inflammatory Macrophage Phenotype Reduces Tumour Growth in Mouse Models of Brain Metastasis.

Breast cancer brain metastasis is a significant clinical problem and carries a poor prognosis. Although it is well-established that macrophages are a primary component of the brain metastasis microenvironment, the role of blood-derived macrophages (BDM) and brain-resident microglia in the progression of brain metastases remains uncertain. The aim of this study, therefore, was to determine the role, specifically, of pro- and anti-inflammatory BDM and microglial phenotypes on metastasis progression. Initial in vitro studies demonstrated decreased migration of EO771 metastatic breast cancer cells in the presence of pro-inflammatory, but not anti-inflammatory, stimulated RAW 264.7 macrophages. In vivo, suppression of the anti-inflammatory BDM phenotype, specifically, via myeloid knock out of Krüppel-like Factor 4 (KLF4) significantly reduced EO771 tumour growth in the brains of C57BL/6 mice. Further, pharmacological inhibition of the anti-inflammatory BDM and/or microglial phenotypes, via either Colony Stimulating Factor 1 Receptor (CSF-1R) or STAT6 pathways, significantly decreased tumour burden in two different syngeneic mouse models of breast cancer brain metastasis. These findings suggest that switching BDM and microglia towards a more pro-inflammatory phenotype may be an effective therapeutic strategy in brain metastasis.

Metabolomic Biomarkers in Blood Samples Identify Cancers in a Mixed Population of Patients with Nonspecific Symptoms.

PURPOSE: Early diagnosis of cancer is critical for improving patient outcomes, but cancers may be hard to diagnose if patients present with nonspecific signs and symptoms. We have previously shown that nuclear magnetic resonance (NMR) metabolomics analysis can detect cancer in animal models and distinguish between differing metastatic disease burdens. Here, we hypothesized that biomarkers within the blood metabolome could identify cancers within a mixed population of patients referred from primary care with nonspecific symptoms, the so-called "low-risk, but not no-risk" patient group, as well as distinguishing between those with and without metastatic disease. EXPERIMENTAL DESIGN: Patients (n = 304 comprising modeling, n = 192, and test, n = 92) were recruited from 2017 to 2018 from the Oxfordshire Suspected CANcer (SCAN) pathway, a multidisciplinary diagnostic center (MDC) referral pathway for patients with nonspecific signs and symptoms. Blood was collected and analyzed by NMR metabolomics. Orthogonal partial least squares discriminatory analysis (OPLS-DA) models separated patients, based upon diagnoses received from the MDC assessment, within 62 days of initial appointment. RESULTS: Area under the ROC curve for identifying patients with solid tumors in the independent test set was 0.83 [95% confidence interval (CI): 0.72-0.95]. Maximum sensitivity and specificity were 94% (95% CI: 73-99) and 82% (95% CI: 75-87), respectively. We could also identify patients with metastatic disease in the cohort of patients with cancer with sensitivity and specificity of 94% (95% CI: 72-99) and 88% (95% CI: 53-98), respectively. CONCLUSIONS: For a mixed group of patients referred from primary care with nonspecific signs and symptoms, NMR-based metabolomics can assist their diagnosis, and may differentiate both those with malignancies and those with and without metastatic disease. See related commentary by Van Tine and Lyssiotis, p. 1477.

Selective blood-brain barrier permeabilization of brain metastases by a type 1 receptor-selective tumor necrosis factor mutein.

BACKGROUND: Metastasis to the brain is a major challenge with poor prognosis. The blood-brain barrier (BBB) is a significant impediment to effective treatment, being intact during the early stages of tumor development and heterogeneously permeable at later stages. Intravenous injection of tumor necrosis factor (TNF) selectively induces BBB permeabilization at sites of brain micrometastasis, in a TNF type 1 receptor (TNFR1)-dependent manner. Here, to enable clinical translation, we have developed a TNFR1-selective agonist variant of human TNF that induces BBB permeabilization, while minimizing potential toxicity. METHODS: A library of human TNF muteins (mutTNF) was generated and assessed for binding specificity to mouse and human TNFR1/2, endothelial permeabilizing activity in vitro, potential immunogenicity, and circulatory half-life. The permeabilizing ability of the most promising variant was assessed in vivo in a model of brain metastasis. RESULTS: The primary mutTNF variant showed similar affinity for human TNFR1 than wild-type human TNF, similar affinity for mouse TNFR1 as wild-type mouse TNF, undetectable binding to human/mouse TNFR2, low potential immunogenicity, and permeabilization of an endothelial monolayer. Circulatory half-life was similar to mouse/human TNF and BBB permeabilization was induced selectively at sites of micrometastases in vivo, with a time window of ≥24 hours and enabling delivery of agents within a therapeutically relevant range (0.5-150 kDa), including the clinically approved therapy, trastuzumab. CONCLUSIONS: We have developed a clinically translatable mutTNF that selectively opens the BBB at micrometastatic sites, while leaving the rest of the cerebrovasculature intact. This approach will open a window for brain metastasis treatment that currently does not exist.

Microbubbles Containing Lysolipid Enhance Ultrasound-Mediated Blood-Brain Barrier Breakdown In Vivo.

Ultrasound and microbubbles (MBs) offer a noninvasive method of temporarily enhancing blood-brain barrier (BBB) permeability to therapeutics. To reduce off-target effects, it is desirable to minimize the ultrasound pressures required. It has been shown that a new formulation of MBs containing lysolipids (Lyso-MBs) can increase the cellular uptake of a model drug in vitro. The aim of this study is to investigate whether Lyso-MBs can also enhance BBB permeability in vivo. Female BALB/c mice are injected with either Lyso-MBs or control MBs and gadolinium-DTPA (Gd-DTPA) and exposed to ultrasound (500 kHz, 1 Hz pulse repetition frequency, 1 ms pulse length, peak-negative pressures 160-480 kPa) for 2 min. BBB permeabilization is measured via magnetic resonance imaging (7.0 T) of Gd-DTPA extravasation and subsequent histological examination of brain tissue to assess serum immunoglobulin G (IgG) extravasation (n = 8 per group). An approximately twofold enhancement in BBB permeability is produced by the Lyso-MBs at the highest ultrasound pressure compared with the control. These findings indicate that modifying the composition of phospholipid-shelled MBs has the potential to improve the efficiency of BBB opening, without increasing the ultrasound pressure amplitude required. This is particularly relevant for delivery of therapeutics deep within the brain.

A novel molecular magnetic resonance imaging agent targeting activated leukocyte cell adhesion molecule as demonstrated in mouse brain metastasis models.

Molecular magnetic resonance imaging (MRI) allows visualization of biological processes at the molecular level. Upregulation of endothelial ALCAM (activated leukocyte cell adhesion molecule) is a key element for leukocyte recruitment in neurological disease. The aim of this study, therefore, was to develop a novel molecular MRI contrast agent, by conjugating anti-ALCAM antibodies to microparticles of iron oxide (MPIO), for detection of endothelial ALCAM expression in vivo. Binding specificity of ALCAM-MPIO was demonstrated in vitro under static and flow conditions. Subsequently, in a proof-of-concept study, mouse models of brain metastasis were induced by intracardial injection of brain-tropic human breast carcinoma, lung adenocarcinoma or melanoma cells to upregulate endothelial ALCAM. At selected time-points, mice were injected intravenously with ALCAM-MPIO, and ALCAM-MPIO induced hypointensities were observed on T2*-weighted images in all three models. Post-gadolinium MRI confirmed an intact blood-brain barrier, indicating endoluminal binding. Correlation between endothelial ALCAM expression and ALCAM-MPIO binding was confirmed histologically. Statistical analysis indicated high sensitivity (80-90%) and specificity (79-83%) for detection of endothelial ALCAM in vivo with ALCAM-MPIO. Given reports of endothelial ALCAM upregulation in numerous neurological diseases, this advance in our ability to image ALCAM in vivo may yield substantial improvements for both diagnosis and targeted therapy.

Improving Delineation of True Tumor Volume With Multimodal MRI in a Rat Model of Brain Metastasis.

PURPOSE: Brain metastases are almost universally lethal with short median survival times. Despite this, they are often potentially curable, with therapy failing only because of local relapse. One key reason relapse occurs is because treatment planning did not delineate metastasis margins sufficiently or accurately, allowing residual tumor to regrow. The aim of this study was to determine the extent to which multimodal magnetic resonance imaging (MRI), with a simple and automated analysis pipeline, could improve upon current clinical practice of single-modality, independent-observer tumor delineation. METHODS AND MATERIALS: We used a single rat model of brain metastasis (ENU1564 breast carcinoma cells in BD-IX rats), with and without radiation therapy. Multimodal MRI data were acquired using sequences either in current clinical use or in clinical trial and included postgadolinium T1-weighted images and maps of blood flow, blood volume, T1 and T2 relaxation times, and apparent diffusion coefficient. RESULTS: In all cases, independent observers underestimated the true size of metastases from single-modality gadolinium-enhanced MRI (85 ± 36 µL vs 131 ± 40 µL histologic measurement), although multimodal MRI more accurately delineated tumor volume (132 ± 41 µL). Multimodal MRI offered increased sensitivity compared with independent observer for detecting metastasis (0.82 vs 0.61, respectively), with only a slight decrease in specificity (0.86 vs 0.98). Blood flow maps conferred the greatest improvements in margin detection for late-stage metastases after radiation therapy. Gadolinium-enhanced T1-weighted images conferred the greatest increase in accuracy of detection for smaller metastases. CONCLUSIONS: These findings suggest that multimodal MRI of brain metastases could significantly improve the visualization of brain metastasis margins, beyond current clinical practice, with the potential to decrease relapse rates and increase patient survival. This finding now needs validation in additional tumor models or clinical cohorts.

Correction to: Amyloid Precursor Protein Mediates Neuronal Protection from Rotenone Toxicity.

The original version of this article unfortunately contained a mistake in the author name. The family name of Dr. Vanessa A. Johannsen should be written as "Johanssen."

Analysis of Prion Protein Conformation Using Circular Dichroism Spectroscopy.

According to the protein-only hypothesis of prion propagation, the pathogenesis of prion disease is due to the misfolding of cellular PrP (PrPC) which gives rise to disease-associated PrPSc. This misfolding results in the predominantly α-helix secondary structure of PrP becoming increasingly ß-sheet. Prion protein researchers often employ circular dichroism (CD) spectroscopy to rapidly analyze and identify the degree of α-helix and ß-sheet content in their recombinant protein and peptide samples. CD is a nondestructive method of determining protein secondary structure and can be used to monitor the protein structural changes in various environments, e.g., pH and temperature. CD can also be used to investigate kinetic and thermodynamic characteristics of proteins and peptides.

Unusual clinical and molecular-pathological profile of gerstmann-Sträussler-Scheinker disease associated with a novel PRNP mutation (V176G).

IMPORTANCE: Here we describe the unusual clinical and molecular-neuropathological profile of a case of Gerstmann-Sträussler-Scheinker disease associated with a novel prion protein (PRNP) gene mutation. OBSERVATIONS: This case report from the Australian National Creutzfeldt-Jakob Disease Registry concerns a 61-year-old British-born woman with no history of neurodegenerative disorder in first-degree relatives. Rapidly progressive dementia, altered behavior, and cerebellar ataxia dominated the clinical picture in the period immediately following minor elective surgery, with death 1 month later in an akinetic-mute state. Brain histopathological examination revealed neuronal loss, scant foci of spongiform change, and diffuse multicentric amyloid plaques, selectively immunoreactive for prion protein, within the cerebral and cerebellar cortices and deep gray matter. Tau immune-reactive neurofibrillary tangles and neuritic threads were present in the cerebral cortex. PRNP sequencing demonstrated a valine to glycine mutation at codon 176, with valine homozygosity at polymorphic codon 129. Western-blot analysis of frozen brain tissue displayed a nonclassic protease-resistant prion protein banding pattern, with a prominent approximately 8-kDa protease-resistant fragment. CONCLUSIONS AND RELEVANCE: Reported is a proband with a novel PRNP mutation associated with neuropathologically confirmed Gerstmann-Sträussler-Scheinker disease displaying a somewhat unusual constellation of clinicopathological features, which overall subserve to further broaden an already diverse phenotypic spectrum.