Amanda M. Hamilton

Nanoparticles coated with the tumor-penetrating peptide iRGD reduce experimental breast cancer metastasis in the brain

Metastasis is the main killer in cancer; consequently, there is great interest in novel approaches to prevent and treat metastatic disease. Brain metastases are particularly deadly, as the protection of the blood-brain barrier obstructs the passage of common anticancer drugs. This study used magnetic resonance imaging (MRI) to investigate the therapeutic effects of nanoparticles coated with a tumor-penetrating peptide (iRGD) against a preclinical model of breast cancer brain metastasis. Single doses of iRGD nanoparticle were administered intravenously, and the effect on tumor growth was observed over time. iRGD nanoparticles, when applied in the early stages of metastasis development, strongly inhibited tumor progression. Overall, this study demonstrated for the first time that a single dose of iRGD nanoparticle can have a significant effect on metastatic tumor progression and nonproliferative cancer cell retention when applied early in course of tumor development. These data suggest that iRGD nanoparticles may be useful in preventatively reducing metastasis after a cancer diagnosis has been established.Key messagesbSSFP MRI can be used to track nonproliferative iron-labeled cells and tumor development over time.iRGD-NW, when applied early, has a significant effect on metastatic tumor progression.Retained signal voids represent a subpopulation of nonproliferating tumor cells.Reduced cell retention and tumor burden show a role for iRGD-NW in metastasis prevention.iRGD target is universally expressed; thus, iRGD-NW should be clinically translatable.

Enzyme-Sensitive Magnetic Resonance Imaging Targeting Myeloperoxidase Identifies Active Inflammation in Experimental Rabbit Atherosclerotic Plaques

Background— Inflammation undermines the stability of atherosclerotic plaques, rendering them susceptible to acute rupture, the cataclysmic event that underlies clinical expression of this disease. Myeloperoxidase is a central inflammatory enzyme secreted by activated macrophages and is involved in multiple stages of plaque destabilization and patient outcome. We report here that a unique functional in vivo magnetic resonance agent can visualize myeloperoxidase activity in atherosclerotic plaques in a rabbit model. Methods and Results— We performed magnetic resonance imaging of the thoracic aorta of New Zealand White rabbits fed a cholesterol (n=14) or normal (n=4) diet up to 2 hours after injection of the myeloperoxidase sensor bis-5HT-DTPA(Gd) [MPO(Gd)], the conventional agent DTPA(Gd), or an MPO(Gd) analog, bis-tyr-DTPA(Gd), as controls. Delayed MPO(Gd) images (2 hours after injection) showed focal areas of increased contrast (>2-fold) in diseased wall but not in normal wall (P=0.84) compared with both DTPA(Gd) (n=11; P<0.001) and bis-tyr-DTPA(Gd) (n=3; P<0.05). Biochemical assays confirmed that diseased wall possessed 3-fold elevated myeloperoxidase activity compared with normal wall (P<0.01). Areas detected by MPO(Gd) imaging colocalized and correlated with myeloperoxidase-rich areas infiltrated by macrophages on histopathological evaluations (r=0.91, P<0.0001). Although macrophages were the main source of myeloperoxidase, not all macrophages secreted myeloperoxidase, which suggests that distinct subpopulations contribute differently to atherogenesis and supports our functional approach. Conclusions— The present study represents a unique approach in the detection of inflammation in atherosclerotic plaques by examining macrophage function and the activity of an effector enzyme to noninvasively provide both anatomic and functional information in vivo.

Comparison of Gadofluorine-M and Gd-DTPA for Noninvasive Staging of Atherosclerotic Plaque Stability Using MRI

Background—Inflammation and neovascularization play critical roles in the stability of atherosclerotic plaques. Whole-body quantitative assessment of these plaque features may improve patient risk-stratification for life-threatening thromboembolic events and direct appropriate intervention. In this report, we determined the utility of the MR contrast agent gadofluorine-M (GdF) for staging plaque stability and compared this to the conventional agent Gd-DTPA. Methods and Results—Five control and 7 atherosclerotic rabbits were sequentially imaged after administration of Gd-DTPA (0.2 mmol/kg) and GdF (0.1 mmol/kg) using a T1-weighted pulse sequence on a 3-T MRI scanner. Diseased aortic wall could be distinguished from normal wall based on wall-to-muscle contrast-to-noise values after GdF administration. RAM-11 (macrophages) and CD-31 (endothelial cells) immunostaining of MR-matched histological sections revealed that GdF accumulation was related to the degree of inflammation at the surface of plaques and the extent of core neovascularization. Importantly, an MR measure of GdF accumulation at both 1 and 24 hours after injection but not Gd-DTPA at peak enhancement was shown to correlate with a quantitative histological morphology index related to these 2 plaque features. Conclusions—GdF-enhanced MRI of atherosclerotic plaques allows noninvasive quantitative information about plaque composition to be acquired at multiple time points after injection (within 1 and up to 24 hours after injection). This dramatically widens the imaging window for assessing plaque stability that is currently attainable with clinically approved MR agents, therefore opening the possibility of whole-body (including coronary) detection of unstable plaques in the future and potentially improved mitigation of cataclysmic cardiovascular events.

Synthesis, characterization and in vitro cell compatibility study of a poly(amic acid) graft/cross-linked poly(vinyl alcohol) hydrogel.

Although physically cross-linked poly(vinyl alcohol) (PVA) hydrogels have tunable mechanical properties to match that of soft tissues, such as vascular tissue, their hydrophilic nature is not conducive to cell adhesion and spreading. For applications such as small diameter vascular grafts for coronary bypass both mechanical matching and hemocompatibility are important. Poly(amic acid) (PAA), derived from ethylene diamine tetraacetic dianhydride, is a cell-compatible polymer. It was grafted/cross-linked onto physically cross-linked PVA to provide cell compatibility. Functionalization was achieved via a one-step esterification reaction using 1,3-dicyclohexylcarbodiimide as the coupling agent and 4-dimethylaminopyridine as the catalyst. The success of the grafting reaction was verified using Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance spectroscopy and X-ray photoelectron spectroscopy. The mechanical properties of the starting PVA hydrogel were largely preserved after the grafting reaction within the physiological strain range of vascular tissue. In vitro cell culture studies using primary porcine endothelial cells confirmed cell compatibility of the PAA graft PVA hydrogel, making it an attractive candidate for small diameter vascular graft development.

Tracking and evaluation of dendritic cell migration by cellular magnetic resonance imaging

Cellular magnetic resonance imaging (MRI) is a means by which cells labeled ex vivo with a contrast agent can be detected and tracked over time in vivo. This technology provides a noninvasive method with which to assess cell-based therapies in vivo. Dendritic cell (DC)-based vaccines are a promising cancer immunotherapy, but its success is highly dependent on the injected DC migrating to a secondary lymphoid organ such as a nearby lymph node. There the DC can interact with T cells to elicit a tumor-specific immune response. It is important to verify DC migration in vivo using a noninvasive imaging modality, such as cellular MRI, so that important information regarding the anatomical location and persistence of the injected DC in a targeted lymph node can be provided. An understanding of DC biology is critical in ascertaining how to label DC with sufficient contrast agent to render them detectable by MRI. While iron oxide nanoparticles provide the best sensitivity for detection of DC in vivo, a clinical grade iron oxide agent is not currently available. A clinical grade (19) Fluorine-based perfluorcarbon nanoemulsion is available but is less sensitive, and its utility to detect DC migration in humans remains to be demonstrated using clinical scanners presently available. The ability to quantitatively track DC migration in vivo can provide important information as to whether different DC maturation and activation protocols result in improved DC migration efficiency which will determine the vaccines immunogenicity and ultimately the tumor immunotherapys outcome in humans.

Understanding Heterogeneity and Permeability of Brain Metastases in Murine Models of HER2-Positive Breast Cancer Through Magnetic Resonance Imaging: Implications for Detection and Therapy

OBJECTIVES: Brain metastases due to breast cancer are increasing, and the prognosis is poor. Lack of effective therapy is attributed to heterogeneity of breast cancers and their resulting metastases, as well as impermeability of the blood–brain barrier (BBB), which hinders delivery of therapeutics to the brain. This work investigates three experimental models of HER2 + breast cancer brain metastasis to better understand the inherent heterogeneity of the disease. We use magnetic resonance imaging (MRI) to quantify brain metastatic growth and explore its relationship with BBB permeability. DESIGN: Brain metastases due to breast cancer cells (SUM190-BR3, JIMT-1-BR3, or MDA-MB-231-BR-HER2) were imaged at 3 T using balanced steady-state free precession and contrast-enhanced T1-weighted spin echo sequences. The histology and immunohistochemistry corresponding to MRI were also analyzed. RESULTS: There were differences in metastatic tumor appearance by MRI, histology, and immunohistochemistry (Ki67, CD31, CD105) across the three models. The mean volume of an MDA-MB-231-BR-HER2 tumor was significantly larger compared to other models (F2,12 = 5.845, P < .05); interestingly, this model also had a significantly higher proportion of Gd-impermeable tumors (F2,12 = 22.18, P < .0001). Ki67 staining indicated that Gd-impermeable tumors had significantly more proliferative nuclei compared to Gd-permeable tumors (t[24] = 2.389, P < .05) in the MDA-MB-231-BR-HER2 model. CD31 and CD105 staining suggested no difference in new vasculature patterns between permeable and impermeable tumors in any model. CONCLUSION: Significant heterogeneity is present in these models of brain metastases from HER2 + breast cancer. Understanding this heterogeneity, especially as it relates to BBB permeability, is important for improvement in brain metastasis detection and treatment delivery.

Statin treatment of hypercholesterolemic-induced aortic valve sclerosis

BACKGROUND Aortic valve sclerosis (AVS) is a common inflammatory heart valve disease prevalent in the population over the age of 65 years. Several published clinical and animal studies have examined the ability of statin treatment to modify disease progression. Clinical trials yielded conflicting results, and animal studies examined the effects of statins prior to the onset of disease. Our study assessed the effect of dietary modification and/or statin treatment on established aortic valve disease in a rabbit model of AVS to examine the tissue response to therapy. METHODS Aortic valve sclerosis was induced in male New Zealand White rabbits by dietary cholesterol supplementation. Rabbits were followed over 2.5 years, with the introduction of statins and/or dietary changes for the second half of the study. At end point, valve function was examined by magnetic resonance imaging. Excised aortic valve cusp tissue was surveyed for thickness, lipid accumulation, protein deposition, calcification, and cellular infiltration. RESULTS By 15 months, cholesterol-fed valves exhibited thickening due to significant lipid content, macrophage infiltration, and osteopontin expression. By 30 months, the untreated disease had progressed to include elevated collagen deposition, lymphocyte invasion, and calcification. With treatment, however, the valve cusps exhibited significant pathological changes including diminished immune cell infiltration and osteopontin expression. Unfortunately, lipid was retained and calcification persisted in all treated valves. CONCLUSIONS In established AVS, the cellular response to statin therapy does not result in full regression of the sclerotic process.

Exploring cell compatibility of a fibronectin-functionalized physically crosslinked poly(vinyl alcohol) hydrogel.

Physically crosslinked poly(vinyl alcohol) (PVA) hydrogels prepared using a low-temperature thermally cycled process have tunable mechanical properties that fall within the range of soft tissues, including cardiovascular tissue. An approach to render it hemocompatible is by endothelization, but its hydrophilic nature is not conducive to cell adhesion and spreading. We investigated the functionalization reaction of this class of PVA hydrogel with fibronectin (FN) for adhesion and spreading of primary porcine radial artery cells and vascular endothelial cells. These are cells relevant to small-diameter vascular graft development. FN functionalization was achieved using a multistep reaction, but the activation step involving carbonyl diimidazole normally required for chemically crosslinked PVA was found to be unnecessary. The reaction resulted in an increase in the elastic modulus of the PVA hydrogel but is still well within the range of cardiovascular tissue. Confocal microscopy confirmed the adhesion and spreading of both cell types on the PVA-FN surfaces, whereas cells failed to adhere to the PVA control. This is a first step toward an alternative for the realization of a synthetic replacement small-diameter vascular graft.

Early Identification of Aortic Valve Sclerosis Using Iron Oxide Enhanced MRI

To test the ability of MION‐47 enhanced MRI to identify tissue macrophage infiltration in a rabbit model of aortic valve sclerosis (AVS).

Application of dual 19F and iron cellular MRI agents to track the infiltration of immune cells to the site of a rejected stem cell transplant

Cellular MRI) was used to detect implanted human mesenchymal stem cells (hMSCs) and the resulting macrophage infiltration that occurs in response to xenotransplantation.