In Vivo PET Detection of Lung Micrometastasis in Mice by Targeting Endothelial VCAM-1 Using a Dual-Contrast PET/MRI Probe.

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.

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.

Biomimetic screening of class-B G protein-coupled receptors.

The 41-amino acid peptide corticotropin releasing factor (CRF) is a major modulator of the mammalian stress response. Upon stressful stimuli, it binds to the corticotropin releasing factor receptor 1 (CRF(1)R), a typical member of the class-B G-protein-coupled receptors (GPCRs) and a prime target in the treatment of mood disorders. To chemically probe the molecular interaction of CRF with the transmembrane domain of its cognate receptor, we developed a high-throughput conjugation approach that mimics the natural activation mechanism of class-B GPCRs. An acetylene-tagged peptide library was synthesized and conjugated to an azide-modified high-affinity carrier peptide derived from the CRF C-terminus using copper-catalyzed dipolar cycloaddition. The resulting conjugates reconstituted potent agonists and were tested in situ for activation of the CRF(1) receptor in a cell-based assay. By use of this approach we (i) defined the minimal sequence motif that is required for full receptor activation, (ii) identified the critical functional groups and structure-activity relationships, (iii) developed an optimized, highly modified peptide probe with high potency (EC(50) = 4 nM) that is specific for the activation domain of the receptor, and (iv) probed the behavioral role of CRF receptors in living mice. The membrane recruitment by a high-affinity carrier enhanced the potency of the tethered peptides by >4 orders of magnitude and thus allowed the testing of very weak initial fragments that otherwise would have been inactive on their own. As no chromatography purification of the test peptides was necessary, a substantial increase in screening throughput was achieved. Importantly, the peptide conjugates can be used to probe the endogenous receptor in its native environment in vivo.

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.

Vinyl sulfone: a versatile function for simple bioconjugation and immobilization.

The easy functionalization of tags and solid supports with the vinyl sulfone function is a valuable tool in omic sciences that allows their coupling with the amine and thiol groups present in the proteogenic residues of proteins, in mild and green conditions compatible with their biological function.

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.

Extracellular vesicle integrins act as a nexus for platelet adhesion in cerebral microvessels.

Circulating extracellular vesicles (EVs) regulate signaling pathways via receptor-ligand interactions and content delivery, after attachment or internalization by endothelial cells. However, they originate from diverse cell populations and are heterogeneous in composition. To determine the effects of specific surface molecules, the use of synthetic EV mimetics permits the study of specific EV receptor-ligand interactions. Here, we used endogenous EVs derived from the circulation of rats, as well as ligand-decorated synthetic microparticles (MPs) to examine the role of integrin αvß3 in platelet adhesion under flow in structurally intact cerebral arteries. At an intraluminal pressure of 50 mmHg and flow rate of 10 µl/min, platelets were delivered to the artery lumen and imaged with whole-field fluorescent microscopy. Under basal conditions very few platelets bound to the endothelium. However, adhesion events were markedly increased following the introduction of arginine-glycine-aspartate (RGD)-labelled synthetic MPs or endogenously-derived EVs from experimental stroke animals carrying excess RGD proteins, including vitronectin, CD40-ligand and thrombospondin-1. These data, which were generated in a dynamic and physiologically relevant system, demonstrate the importance of vesicle-carried RGD ligands in platelet adherence to the cerebrovascular endothelium and highlight the ability of synthetic EVs to isolate and identify key components of the molecular handshake between EVs and their targets.

VCAM-1-targeted MRI Enables Detection of Brain Micrometastases from Different Primary Tumors.

PURPOSE: A major issue for the effective treatment of brain metastasis is the late stage of diagnosis with existing clinical tools. The aim of this study was to evaluate the potential of vascular cell adhesion molecule 1 (VCAM-1)-targeted MRI for early detection of brain micrometastases in mouse models across multiple primary tumor types.Experimental Design: Xenograft models of brain micrometastasis for human breast carcinoma (MDA231Br-GFP), lung adenocarcinoma (SEBTA-001), and melanoma (H1_DL2) were established via intracardiac injection in mice. Animals (n = 5-6/group) were injected intravenously with VCAM-1-targeted microparticles of iron oxide (VCAM-MPIO) and, subsequently, underwent T 2*-weighted MRI. Control groups of naïve mice injected with VCAM-MPIO and tumor-bearing mice injected with nontargeting IgG-MPIO were included. RESULTS: All models showed disseminated micrometastases in the brain, together with endothelial VCAM-1 upregulation across the time course. T 2*-weighted MRI of all tumor-bearing mice injected with VCAM-MPIO showed significantly more signal hypointensities (P < 0.001; two-sided) than control cohorts, despite a lack of blood-brain barrier (BBB) impairment. Specific MPIO binding to VCAM-1-positive tumor-associated vessels was confirmed histologically. VCAM-1 expression was demonstrated in human brain metastasis samples, across all three primary tumor types. CONCLUSIONS: VCAM-1-targeted MRI enables the detection of brain micrometastases from the three primary tumor types known to cause the majority of clinical cases. These findings represent an important step forward in the development of a broadly applicable and clinically relevant imaging technique for early diagnosis of brain metastasis, with significant implications for improved patient survival.

Optimization of molecularly targeted MRI in the brain: empirical comparison of sequences and particles.

BACKGROUND: Molecular MRI is an evolving field of research with strong translational potential. Selection of the appropriate MRI sequence, field strength and contrast agent depend largely on the application. The primary aims of the current study were to: 1) assess the sensitivity of different MRI sequences for detection of iron oxide particles in mouse brain; 2) determine the effect of magnetic field strength on detection of iron oxide particles in vivo; and 3) compare the sensitivity of targeted microparticles of iron oxide (MPIO) or ultra-small superparamagnetic iron oxide (USPIO) for detection of vascular cell adhesion molecule-1 (VCAM-1) in vivo. METHODS: Mice were injected intrastriatally with interleukin 1ß to induce VCAM-1 expression on the cerebral vasculature. Subsequently, animals were injected intravenously with either VCAM-MPIO or VCAM-USPIO and imaged 1 or 13 hours post-injection, respectively. MRI was performed at 4.7, 7.0, or 9.4 T, using three different T2*-weighted sequences: single gradient echo 3D (GE3D), multi-gradient echo 3D (MGE3D) and balanced steady-state free precession 3D (bSSFP3D). RESULTS: MGE3D yielded the highest signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) for the detection of iron oxide particles. All sequences showed a significant increase in SNR and CNR from 4.7 to 7.0 T, but no further improvement at 9.4 T. However, whilst targeted MPIO enabled sensitive detection of VCAM-1 expression on the cerebral vasculature, the long half-life (16.5 h vs 1.2 min) and lower relaxivity per particle (1.29×10-14 vs 1.18×10-9 Hz L/particle) of USPIO vs. MPIO rendered them impractical for molecular MRI. CONCLUSION: These findings demonstrate clear advantages of MPIO compared to USPIO for molecularly-targeted MRI, and indicate that the MGE3D sequence is optimal for MPIO detection. Moreover, higher field strengths (7.0/9.4 T) showed enhanced sensitivity over lower field strengths (4.7 T). With the development of biodegradable MPIO, these agents hold promise for clinical translation.

Covalent assembly of nanoparticles as a peptidase-degradable platform for molecular MRI.

Ligand-conjugated microparticles of iron oxide (MPIO) have the potential to provide high sensitivity contrast for molecular magnetic resonance imaging (MRI). However, the accumulation and persistence of non-biodegradable micron-sized particles in liver and spleen precludes their clinical use and limits the translational potential of MPIO-based contrast agents. Here we show that ligand-targeted MPIO derived from multiple iron oxide nanoparticles may be coupled covalently through peptide linkers that are designed to be cleaved by intracellular macrophage proteases. The synthesized particles possess potential characteristics for targeted MRI contrast agents, including high relaxivity, unappreciable sedimentation, clearance from circulation and no overt toxicity. Importantly, we demonstrate that these particles are rapidly degraded both in vitro and in vivo, and that the targeted probes can be used for detection of inflammation in vivo using MRI. This approach provides a platform for molecular MRI contrast agents that is potentially more suitable for translation to humans.

Molecular magnetic resonance imaging of angiogenesis in vivo using polyvalent cyclic RGD-iron oxide microparticle conjugates.

Angiogenesis is an essential component of tumour growth and, consequently, an important target both therapeutically and diagnostically. The cell adhesion molecule α(v)ß(3) integrin is a specific marker of angiogenic vessels and the most prevalent vascular integrin that binds the amino acid sequence arginine-glycine-aspartic acid (RGD). Previous studies using RGD-targeted nanoparticles (20-50 nm diameter) of iron oxide (NPIO) for magnetic resonance imaging (MRI) of tumour angiogenesis, have identified a number of limitations, including non-specific extravasation, long blood half-life (reducing specific contrast) and low targeting valency. The aim of this study, therefore, was to determine whether conjugation of a cyclic RGD variant [c(RGDyK)], with enhanced affinity for α(v)ß(3), to microparticles of iron oxide (MPIO) would provide a more sensitive contrast agent for imaging of angiogenic tumour vessels. Cyclic RGD [c(RGDyK)] and RAD [c(RADyK)] based peptides were coupled to 2.8 µm MPIO, and binding efficacy tested both in vitro and in vivo. Significantly greater specific binding of c(RGDyK)-MPIO to S-nitroso-n-acetylpenicillamine (SNAP)-stimulated human umbilical vein endothelial cells in vitro than PBS-treated cells was demonstrated under both static (14-fold increase; P < 0.001) and flow (44-fold increase; P < 0.001) conditions. Subsequently, mice bearing subcutaneous colorectal (MC38) or melanoma (B16F10) derived tumours underwent in vivo MRI pre- and post-intravenous administration of c(RGDyK)-MPIO or c(RADyK)-MPIO. A significantly greater volume of MPIO-induced hypointensities were found in c(RGDyK)-MPIO injected compared to c(RADyK)-MPIO injected mice, in both tumour models (P < 0.05). Similarly, administration of c(RGDyK)-MPIO induced a greater reduction in mean tumour T(2)* relaxation times than the control agent in both tumour models (melanoma P < 0.001; colorectal P < 0.0001). Correspondingly, MPIO density per tumour volume assessed immunohistochemically was significantly greater for c(RGDyK)-MPIO than c(RADyK)-MPIO injected animals, in both melanoma (P < 0.05) and colorectal (P < 0.0005) tumours. In both cases, binding of c(RGDyK)-MPIO co-localised with α(v)ß(3) expression. Comparison of RGD-targeted and dynamic contrast enhanced (DCE) MRI assessment of tumour perfusion indicated sensitivity to different vascular features. This study demonstrates specific binding of c(RGDyK)-MPIO to α(v)ß(3) expressing neo-vessels, with marked and quantifiable contrast and rapid clearance of unbound particles from the blood circulation compared to NPIO. Combination of this molecular MRI approach with conventional DCE MRI will enable integrated molecular, anatomical and perfusion tumour imaging.

Multivalent neoglycoconjugates by regiospecific cycloaddition of alkynes and azides using organic-soluble copper catalysts.

[reaction: see text] The construction of multivalent neoglycoconjugates is efficiently achieved by the regiospecific catalytic cycloaddition of alkynes and azides using the organic-soluble copper complexes (Ph(3)P)(3).CuBr and (EtO)(3)P.CuI. The simultaneous use of microwave irradiation shortened notably the reaction times.

Molecular MRI approaches to the detection of CNS inflammation.

Inflammation is a key component of many neurological diseases, yet our understanding of the contribution of these processes to tissue damage remains poor. For many such diseases, magnetic resonance imaging (MRI) has become the method of choice for clinical diagnosis. However, many of the MRI parameters that enable disease detection, such as passive contrast enhancement across a compromised blood-brain barrier, are weighted towards late-stage disease. Moreover, whilst these methods may report on disease severity, they are not able to provide information on either disease activity or the underlying molecular processes. There is a need, therefore, to develop methods that enable earlier disease detection, potentially long before clinical symptoms become apparent, together with identification of specific molecular processes that may guide specific therapy. This chapter describes the methodology for the synthesis and validation of two novel, functional MRI-detectable probes, based on microparticles of iron oxide (MPIO), which target endothelial adhesion molecules. These contrast agents enable the detection of acute brain inflammation in vivo, at a time when pathology is undetectable by conventional MRI. Such molecular MRI methods are opening new vistas for the acute diagnosis of CNS disease, together with the possibility for individually tailored therapy and earlier, more sensitive assessment of the efficacy of novel therapies.

Molecular imaging with optical coherence tomography using ligand-conjugated microparticles that detect activated endothelial cells: rational design through target quantification.

OBJECTIVES: Optical coherence tomography (OCT) is a high resolution imaging technique used to assess superficial atherosclerotic plaque morphology. Utility of OCT may be enhanced by contrast agents targeting molecular mediators of inflammation. METHODS AND RESULTS: Microparticles of iron oxide (MPIO; 1 and 4.5 µm diameter) in suspension were visualized and accurately quantified using a clinical optical coherence tomography system. Bound to PECAM-1 on a plane of cultured endothelial cells under static conditions, 1 µm MPIO were also readily detected by OCT. To design a molecular contrast probe that would bind activated endothelium under conditions of shear stress, we quantified the expression (basal vs. TNF-activated; molecules µm(-2)) of VCAM-1 (not detected vs. 16 ± 1); PECAM-1 (132 ± 6 vs. 198 ± 10) and E-selectin (not detected vs. 46 ± 0.6) using quantitative flow cytometry. We then compared the retention of antibody-conjugated MPIO targeting each of these molecules plus a combined VCAM-1 and E-selectin (E+V) probe across a range of physiologically relevant shear stresses. E+V MPIO were consistently retained with highest efficiency (P < 0.001) and at a density that provided conspicuous contrast effects on OCT pullback. CONCLUSION: Microparticles of iron oxide were detectable using a clinical OCT system. Assessment of binding under flow conditions recommended an approach that targeted both E-selectin and VCAM-1. Bound to HUVEC under conditions of flow, targeted 1 µm E+V MPIO were readily identified on OCT pullback. Molecular imaging with OCT may be feasible in vivo using antibody targeted MPIO.

Click multivalent neoglycoconjugates as synthetic activators in cell adhesion and stimulation of monocyte/machrophage cell lines.

The efficient synthesis of fluorescent and non-fluorescent multivalent neoglycoconjugates is described by means of the Cu(i) catalyzed azide-alkyne 1,3-dipolar cycloaddition ("click-chemistry"). A well-defined glycopolymer, glycocyclodextrin or glycocluster architecture displaying galactose or lactose epitopes has been chosen. Cellular assays using U-937 and RAW 264.7 monocyte/macrophage cells showed that these glycocompounds have the capability to act as synthetic activators mimicking the lipopolysaccharide (LPS) effects. Thus, the click compounds promote cell adhesion and stimulation of monocytes, measured as an increase in the amount of TNFalpha, facilitating their differentiation to macrophages.