Epigenetic MRI: Noninvasive imaging of DNA methylation in the brain.

Both neuronal and genetic mechanisms regulate brain function. While there are excellent methods to study neuronal activity in vivo, there are no nondestructive methods to measure global gene expression in living brains. Here, we present a method, epigenetic MRI (eMRI), that overcomes this limitation via direct imaging of DNA methylation, a major gene-expression regulator. eMRI exploits the methionine metabolic pathways for DNA methylation to label genomic DNA through 13C-enriched diets. A 13C magnetic resonance spectroscopic imaging method then maps the spatial distribution of labeled DNA. We validated eMRI using pigs, whose brains have stronger similarity to humans in volume and anatomy than rodents, and confirmed efficient 13C-labeling of brain DNA. We also discovered strong regional differences in global DNA methylation. Just as functional MRI measurements of regional neuronal activity have had a transformational effect on neuroscience, we expect that the eMRI signal, both as a measure of regional epigenetic activity and as a possible surrogate for regional gene expression, will enable many new investigations of human brain function, behavior, and disease.

Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound.

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn2+-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn2+-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

Ultrasound controlled mechanophore activation in hydrogels for cancer therapy.

Mechanophores are molecular motifs that respond to mechanical perturbance with targeted chemical reactions toward desirable changes in material properties. A large variety of mechanophores have been investigated, with applications focusing on functional materials, such as strain/stress sensors, nanolithography, and self-healing polymers, among others. The responses of engineered mechanophores, such as light emittance, change in fluorescence, and generation of free radicals (FRs), have potential for bioimaging and therapy. However, the biomedical applications of mechanophores are not well explored. Herein, we report an in vitro demonstration of an FR-generating mechanophore embedded in biocompatible hydrogels for noninvasive cancer therapy. Controlled by high-intensity focused ultrasound (HIFU), a clinically proven therapeutic technique, mechanophores were activated with spatiotemporal precision to generate FRs that converted to reactive oxygen species (ROS) to effectively kill tumor cells. The mechanophore hydrogels exhibited no cytotoxicity under physiological conditions. Upon activation with HIFU sonication, the therapeutic efficacies in killing in vitro murine melanoma and breast cancer tumor cells were comparable with lethal doses of H2O2 This process demonstrated the potential for mechanophore-integrated HIFU combination as a noninvasive cancer treatment platform, named "mechanochemical dynamic therapy" (MDT). MDT has two distinct advantages over other noninvasive cancer treatments, such as photodynamic therapy (PDT) and sonodynamic therapy (SDT). 1) MDT is ultrasound based, with larger penetration depth than PDT. 2) MDT does not rely on sonosensitizers or the acoustic cavitation effect, both of which are necessary for SDT. Taking advantage of the strengths of mechanophores and HIFU, MDT can provide noninvasive treatments for diverse cancer types.

Targeted Imaging Agent to HSP70 Induced In Vivo.

Heat shock protein expression can be induced by heat shock making it possible to artificially modulate their levels noninvasively in vivo in a spatially and temporally controlled manner. Here, we report the use of the major heat shock protein 70 (HSP70) as an inducible target by using the small molecule deoxyspergualin (DSG) conjugated to the near-infrared fluorophore (Cy5.5). We demonstrate that heat induction in the form of localized hyperthermia of normal tissue in living mice results in sufficient HSP70 overexpression for detection with DSG-Cy5.5 conjugate. This effect is dependent on total energy delivered and reaches maximum fluorescence signal in 6 to 8 hours post heat induction and declines over a period of up to 24 hours. These results suggest that DSG-Cy5.5 agent accumulates in tissue with elevated HSP70 by heat.

High-intensity focused ultrasound-induced mechanochemical transduction in synthetic elastomers.

While study in the field of polymer mechanochemistry has yielded mechanophores that perform various chemical reactions in response to mechanical stimuli, there is not yet a triggering method compatible with biological systems. Applications such as using mechanoluminescence to generate localized photon flux in vivo for optogenetics would greatly benefit from such an approach. Here we introduce a method of triggering mechanophores by using high-intensity focused ultrasound (HIFU) as a remote energy source to drive the spatially and temporally resolved mechanical-to-chemical transduction of mechanoresponsive polymers. A HIFU setup capable of controlling the excitation pressure, spatial location, and duration of exposure is employed to activate mechanochemical reactions in a cross-linked elastomeric polymer in a noninvasive fashion. One reaction is the chromogenic isomerization of a naphthopyran mechanophore embedded in a polydimethylsiloxane (PDMS) network. Under HIFU irradiation evidence of the mechanochemical transduction is the observation of a reversible color change as expected for the isomerization. The elastomer exhibits this distinguishable color change at the focal spot, depending on ultrasonic exposure conditions. A second reaction is the demonstration that HIFU irradiation successfully triggers a luminescent dioxetane, resulting in localized generation of visible blue light at the focal spot. In contrast to conventional stimuli such as UV light, heat, and uniaxial compression/tension testing, HIFU irradiation provides spatiotemporal control of the mechanochemical activation through targeted but noninvasive ultrasonic energy deposition. Targeted, remote light generation is potentially useful in biomedical applications such as optogenetics where a light source is used to trigger a cellular response.

Alpha Particle Enhanced Blood Brain/Tumor Barrier Permeabilization in Glioblastomas Using Integrin Alpha-v Beta-3-Targeted Liposomes.

Glioblastoma (GBM) is the most common primary malignant astrocytoma characterized by extensive invasion, angiogenesis, hypoxia, and micrometastasis. Despite the relatively leaky nature of GBM blood vessels, effective delivery of antitumor therapeutics has been a major challenge due to the complications caused by the blood-brain barrier (BBB) and the highly torturous nature of newly formed tumor vasculature (blood tumor barrier-BTB). External beam radiotherapy was previously shown to be an effective means of permeabilizing central nervous system (CNS) barriers. By using targeted short-ranged radionuclides, we show for the first time that our targeted actinium-225-labeled αvß3-specific liposomes (225Ac-IA-TLs) caused catastrophic double stranded DNA breaks and significantly enhanced the permeability of BBB and BTB in mice bearing orthotopic GBMs. Histologic studies revealed characteristic α-particle induced double strand breaks within tumors but was not significantly present in normal brain regions away from the tumor where BBB permeability was observed. These findings indicate that the enhanced vascular permeability in these distal regions did not result from direct α-particle-induced DNA damage. On the basis of these results, in addition to their direct antitumor effects, 225Ac-IA-TLs can potentially be used to enhance the permeability of BBB and BTB for effective delivery of systemically administered antitumor therapeutics. Mol Cancer Ther; 16(10); 2191-200. ©2017 AACR.

Phosphatidylserine-Targeted Nanotheranostics for Brain Tumor Imaging and Therapeutic Potential.

Phosphatidylserine (PS), the most abundant anionic phospholipid in cell membrane, is strictly confined to the inner leaflet in normal cells. However, this PS asymmetry is found disruptive in many tumor vascular endothelial cells. We discuss the underlying mechanisms for PS asymmetry maintenance in normal cells and its loss in tumor cells. The specificity of PS exposure in tumor vasculature but not normal blood vessels may establish it a useful biomarker for cancer molecular imaging. Indeed, utilizing PS-targeting antibodies, multiple imaging probes have been developed and multimodal imaging data have shown their high tumor-selective targeting in various cancers. There is a critical need for improved diagnosis and therapy for brain tumors. We have recently established PS-targeted nanoplatforms, aiming to enhance delivery of imaging contrast agents across the blood-brain barrier to facilitate imaging of brain tumors. Advantages of using the nanodelivery system, in particular, lipid-based nanocarriers, are discussed here. We also describe our recent research interest in developing PS-targeted nanotheranostics for potential image-guided drug delivery to treat brain tumors.

Enhancing tissue permeability with MRI guided preclinical focused ultrasound system in rabbit muscle: From normal tissue to VX2 tumor.

High Intensity Focused Ultrasound (HIFU) is an emerging noninvasive, nonionizing physical energy based modality to ablate solid tumors with high power, or increase local permeability in tissues/tumors in pulsed mode with relatively low power. Compared with traditional ablative HIFU, nondestructive pulsed HIFU (pHIFU) is present in the majority of novel applications recently developed for enhancing the delivery of drugs and genes. Previous studies have demonstrated the capability of pHIFU to change tissue local permeability for enhanced drug delivery in both mouse tumors and mouse muscle. Further study based on bulk tissues in large animals and clinical HIFU system revealed correlation between therapeutic effect and thermal parameters, which was absent in the previous mouse studies. In this study, we further investigated the relation between the therapeutic effect of pHIFU and thermal parameters in bulky normal muscle tissues based on a rabbit model and a preclinical HIFU system. Correlation between therapeutic effect and thermal parameters was confirmed in our study on the same bulk tissues although different HIFU systems were used. Following the study in bulky normal muscle tissues, we further created bulky tumor model with VX2 tumors implanted on both hind limbs of rabbits and investigated the feasibility to enhance tumor permeability in bulky VX2 tumors in a rabbit model using pHIFU technique. A radiolabeled peptidomimetic integrin antagonist, 111In-DOTA-IA, was used following pHIFU treatment in our study to target VX2 tumor and serve as the radiotracer for follow-up single-photon emission computed tomography (SPECT) scanning. The results have shown significantly elevated uptake of 111In-DOTA-IA in the area of VX2 tumors pretreated by pHIFU compared with the control VX2 tumors not being pretreated by pHIFU, and statistical analysis revealed averaged 34.5% enhancement 24h after systematic delivery of 111In-DOTA-IA in VX2 tumors pretreated by pHIFU compared with the control VX2 tumors.

Preliminary Therapy Evaluation of (225)Ac-DOTA-c(RGDyK) Demonstrates that Cerenkov Radiation Derived from (225)Ac Daughter Decay Can Be Detected by Optical Imaging for In Vivo Tumor Visualization.

The theranostic potential of (225)Ac-based radiopharmaceuticals continues to increase as researchers seek innovative ways to harness the nuclear decay of this radioisotope for therapeutic and imaging applications. This communication describes the evaluation of (225)Ac-DOTA-c(RGDyK) in both biodistribution and Cerenkov luminescence imaging (CLI) studies. Initially, La-DOTA-c(RGDyK) was prepared as a non-radioactive surrogate to evaluate methodologies that would contribute to an optimized radiochemical synthetic strategy and estimate the radioactive conjugate's affinity for αvß3, using surface plasmon resonance spectroscopy. Surface plasmon resonance spectroscopy studies revealed the IC50 and Ki of La-DOTA-c(RGDyK) to be 33 ± 13 nM and 26 ± 11 nM, respectively, and suggest that the complexation of the La(3+) ion to the conjugate did not significantly alter integrin binding. Furthermore, use of this surrogate allowed optimization of radiochemical synthesis strategies to prepare (225)Ac-DOTA-c(RGDyK) with high radiochemical purity and specific activity similar to other (225)Ac-based radiopharmaceuticals. This radiopharmaceutical was highly stable in vitro. In vivo biodistribution studies confirmed the radiotracer's ability to target αvß3 integrin with specificity; specificity was detected in tumor-bearing animals using Cerenkov luminescence imaging. Furthermore, tumor growth control was achieved using non-toxic doses of the radiopharmaceutical in U87mg tumor-bearing nude mice. To our knowledge, this is the first report to describe the CLI of αvß3 (+) tumors in live animals using the daughter products derived from (225)Ac decay in situ. This concept holds promise to further enhance development of targeted alpha particle therapy.

Remote spatiotemporally controlled and biologically selective permeabilization of blood-brain barrier.

The blood-brain barrier (BBB), comprised of brain endothelial cells with tight junctions (TJ) between them, regulates the extravasation of molecules and cells into and out of the central nervous system (CNS). Overcoming the difficulty of delivering therapeutic agents to specific regions of the brain presents a major challenge to treatment of a broad range of brain disorders. Current strategies for BBB opening are invasive, not specific, and lack precise control over the site and timing of BBB opening, which may limit their clinical translation. In the present report, we describe a novel approach based on a combination of stem cell delivery, heat-inducible gene expression and mild heating with high-intensity focused ultrasound (HIFU) under MRI guidance to remotely permeabilize BBB. The permeabilization of the BBB will be controlled with, and limited to where selected pro-inflammatory factors will be secreted secondary to HIFU activation, which is in the vicinity of the engineered stem cells and consequently both the primary and secondary disease foci. This therapeutic platform thus represents a non-invasive way for BBB opening with unprecedented spatiotemporal precision, and if properly and specifically modified, can be clinically translated to facilitate delivery of different diagnostic and therapeutic agents which can have great impact in treatment of various disease processes in the central nervous system.

Systems biology approach to studying proliferation-dependent prognostic subnetworks in breast cancer.

Tumor proliferative capacity is a major biological correlate of breast tumor metastatic potential. In this paper, we developed a systems approach to investigate associations among gene expression patterns, representative protein-protein interactions, and the potential for clinical metastases, to uncover novel survival-related subnetwork signatures as a function of tumor proliferative potential. Based on the statistical associations between gene expression patterns and patient outcomes, we identified three groups of survival prognostic subnetwork signatures (SPNs) corresponding to three proliferation levels. We discovered 8 SPNs in the high proliferation group, 8 SPNs in the intermediate proliferation group, and 6 SPNs in the low proliferation group. We observed little overlap of SPNs between the three proliferation groups. The enrichment analysis revealed that most SPNs were enriched in distinct signaling pathways and biological processes. The SPNs were validated on other cohorts of patients, and delivered high accuracy in the classification of metastatic vs non-metastatic breast tumors. Our findings indicate that certain biological networks underlying breast cancer metastasis differ in a proliferation-dependent manner. These networks, in combination, may form the basis of highly accurate prognostic classification models and may have clinical utility in guiding therapeutic options for patients.

Multi-scale modeling for the transmission of influenza and the evaluation of interventions toward it.

Mathematical modeling of influenza epidemic is important for analyzing the main cause of the epidemic and finding effective interventions towards it. The epidemic is a dynamic process. In this process, daily infections are caused by people's contacts, and the frequency of contacts can be mainly influenced by their cognition to the disease. The cognition is in turn influenced by daily illness attack rate, climate, and other environment factors. Few existing methods considered the dynamic process in their models. Therefore, their prediction results can hardly be explained by the mechanisms of epidemic spreading. In this paper, we developed a heterogeneous graph modeling approach (HGM) to describe the dynamic process of influenza virus transmission by taking advantage of our unique clinical data. We built social network of studied region and embedded an Agent-Based Model (ABM) in the HGM to describe the dynamic change of an epidemic. Our simulations have a good agreement with clinical data. Parameter sensitivity analysis showed that temperature influences the dynamic of epidemic significantly and system behavior analysis showed social network degree is a critical factor determining the size of an epidemic. Finally, multiple scenarios for vaccination and school closure strategies were simulated and their performance was analyzed.

Tumor lysing genetically engineered T cells loaded with multi-modal imaging agents.

Genetically-modified T cells expressing chimeric antigen receptors (CAR) exert anti-tumor effect by identifying tumor-associated antigen (TAA), independent of major histocompatibility complex. For maximal efficacy and safety of adoptively transferred cells, imaging their biodistribution is critical. This will determine if cells home to the tumor and assist in moderating cell dose. Here, T cells are modified to express CAR. An efficient, non-toxic process with potential for cGMP compliance is developed for loading high cell number with multi-modal (PET-MRI) contrast agents (Super Paramagnetic Iron Oxide Nanoparticles - Copper-64; SPION-(64)Cu). This can now be potentially used for (64)Cu-based whole-body PET to detect T cell accumulation region with high-sensitivity, followed by SPION-based MRI of these regions for high-resolution anatomically correlated images of T cells. CD19-specific-CAR(+)SPION(pos) T cells effectively target in vitro CD19(+) lymphoma.

Computational modeling of 3D tumor growth and angiogenesis for chemotherapy evaluation.

Solid tumors develop abnormally at spatial and temporal scales, giving rise to biophysical barriers that impact anti-tumor chemotherapy. This may increase the expenditure and time for conventional drug pharmacokinetic and pharmacodynamic studies. In order to facilitate drug discovery, we propose a mathematical model that couples three-dimensional tumor growth and angiogenesis to simulate tumor progression for chemotherapy evaluation. This application-oriented model incorporates complex dynamical processes including cell- and vascular-mediated interstitial pressure, mass transport, angiogenesis, cell proliferation, and vessel maturation to model tumor progression through multiple stages including tumor initiation, avascular growth, and transition from avascular to vascular growth. Compared to pure mechanistic models, the proposed empirical methods are not only easy to conduct but can provide realistic predictions and calculations. A series of computational simulations were conducted to demonstrate the advantages of the proposed comprehensive model. The computational simulation results suggest that solid tumor geometry is related to the interstitial pressure, such that tumors with high interstitial pressure are more likely to develop dendritic structures than those with low interstitial pressure.

In vivo MRI detection of carotid atherosclerotic lesions and kidney inflammation in ApoE-deficient mice by using LOX-1 targeted iron nanoparticles.

Lectin-like Oxidized Low-Density Lipoprotein Receptor 1 (LOX-1) plays a key role in atherosclerotic plaque initiation, formation and rupture, as well as in hyperlipidemia-induced glomerular disease. Here we report a sensitive, specific and biocompatible LOX-1-targeted-USPIO for the noninvasive MR imaging of LOX-1 within carotid atherosclerotic lesions and glomerular disease in apoE-deficient mice. In vitro analysis showed the highest uptake of targeted USPIOs in only activated RAW264.7 macrophages, and in vivo MRI studies showed signal loss in carotid atherosclerotic lesions after administration of targeted USPIOs at 8h and 24h. These areas of signal loss were correlated with the presence of nanoparticles in the atherosclerotic lesions, and immunohistochemistry and Perl's staining confirmed the co-localization of the LOX-1/macrophages/MMP-9 and targeted nanoparticles. Finally, additional studies suggest that this targeted probe may have potential to noninvasively image early glomerular disease. This finding may provide important methods for characterizing vulnerable atherosclerotic plaques and hyperlipidemia-induced glomerular diseases. FROM THE CLINICAL EDITOR: A functionalized USPIO-based negative contrast material was used in this study, demonstrating feasibility of sensitive MRI-based detection of atherosclerotic plaque formation in the carotid arteries and in the renal cortex, paving the way to potential future clinical applications.

Near-infrared fluorescence imaging of murine atherosclerosis using an oxidized low density lipoprotein-targeted fluorochrome.

The aim of this study was to explore the feasibility of detecting plaques using an NIR797 fluorochrome-labeled, anti-oxLDL antibody (anti-oxLDL-NIR797) and near-infrared fluorescence (NIRF) imaging in a murine model of atherosclerosis. Anti-mouse oxLDL polyclonal antibodies were conjugated to NIR797 dyes to synthesis oxLDL-targeted NIRF probe. In situ and ex vivo NIRF imaging of the high-cholesterol diet-induced atherosclerotic lesions of apoE-/- mice (baseline) as well as ex vivo NIRF imaging in the progression and regression group (without or with atorvastatin treatment for another 8 weeks) were performed 24 h after an intravenous injection of 1 mg/kg of anti-oxLDL-NIR797, while phosphate-buffered saline (PBS) was used for the controls. The plaque areas were investigated using Oil Red O (ORO) staining. Aortas isolated from the apoE-/- mice 24 h post-injection exhibited a selective, strong, heterogeneous NIRF signal enhancement in the aortic root, arch, and bifurcation, whereas the PBS and competitive inhibition groups had limited NIRF signal changes (p < 0.05). There was a significant correlation between ORO staining and NIRF in the atherosclerotic aortas that received anti-oxLDL-NIR797. Immunofluorescence studies confirmed the colocalization of the oxLDL/macrophages and NIR797 fluorochromes. Furthermore, the atherosclerotic lesions of atorvastatin-treated mice showed reduced anti-oxLDL-NIR797 uptake and oxLDL expression. These results indicate that NIRF plaque imaging is feasible with an oxLDL-targeted NIRF probe. Thus, oxLDL-based molecular imaging of atherosclerotic plaques is feasible and may provide important methods for characterizing vulnerable plaques and monitoring the response to therapeutic interventions for atherosclerosis.

Digitization of medicine: how radiology can take advantage of the digital revolution.

In the era of medical cost containment, radiologists must continually maintain their actual and perceived value to patients, payers, and referring providers. Exploitation of current and future digital technologies may be the key to defining and promoting radiology's "brand" and assure our continued relevance in providing predictive, preventive, personalized, and participatory medicine. The Association of University of Radiologists Radiology Research Alliance Digitization of Medicine Task Force was formed to explore the opportunities and challenges of the digitization of medicine that are relevant to radiologists, which include the reporting paradigm, computational biology, and imaging informatics. In addition to discussing these opportunities and challenges, we consider how change occurs in medicine, and how change may be effected in medical imaging community. This review article is a summary of the research of the task force and hopefully can be used as a stimulus for further discussions and development of action plans by radiology leaders.

Multivalency of non-peptide integrin αVß3 antagonist slows tumor growth.

Multivalency is a powerful strategy for achieving high-affinity molecular binding of compounds to increase their therapeutic potency or imaging potential. In our study, multivalent non-peptide integrin αvß3 antagonists (IA) were designed for antitumor therapy. Docking and molecular dynamics were employed to explore the binding modes of IA monomer, dimer, and trimer. In silico, one IA unit binds tightly in the active site with similar pose to native ligand RGD and other parts of dimer and trimer contribute extra binding affinities by interacting with residues in vicinity of the original site. In vitro studies demonstrated that increasing valency results in increasing antiproliferative and antiorganizational effects against endothelial cells (HUVECs), and a much weaker effect on melanoma B16F10 cells. The antitumor efficacies of the IA multivalent compounds were evaluated in subcutaneous B16F10 melanoma tumor-bearing mice. At 30 mg/kg dose, the mean masses of tumors harvested 18 days after inoculation were significantly reduced (p<10(-7)) by 36±9%, 49±8%, and 71±7% for the IA monomer, dimer, and trimer groups, relative to control. The importance of multivalency was demonstrated to be highly significant beyond the additive effect of the extra pharmacological sites (p=0.00011). These results suggest that the major target of these anti-αvß3 compounds is the neovasculature rather than the cancer cells, and the success of a multivalent strategy depends on the details of the components and linker. This is the first integrin αvß3 multivalent ligand showing clear enhancement in antitumor effectiveness.

MRI-based prediction of pulsed high-intensity focused ultrasound effect on tissue transport in rabbit muscle.

PURPOSE: To design an algorithm for optimizing pulsed high intensity focused ultrasound (p-HIFU) treatment parameters to maximize tissue transport while minimizing thermal necrosis based on MR image guidance. MATERIALS AND METHODS: P-HIFU power, duty cycle, and treatment duration were varied to generate different levels of thermal and mechanical deposition in rabbit muscle. Changes in T2-weighted and T1 contrast-enhanced (CE) signal were assessed immediately following treatment and at 24 h. Transport parameters were extracted by means of T1-weighted dynamic contrast-enhanced MRI (DCE-MRI) technique at 0 and 24-h time points. RESULTS: Successful p-HIFU treatment was indicated by focal hyperintensity on the T2-weighted image immediately post-treatment, suggesting increased fluid (edema), with little intensity change in CE image. After 24 h, the affected region expanded along the muscle fiber accompanied by clear hyperintensity in CE image (contrast uptake). Quantitative DCE-MRI analysis revealed statistically significant increases in both leakage rate and extracellular space, accompanied by a decrease in clearance rate. CONCLUSION: Successful p-HIFU treatment was mainly correlated to tissue heating. Tissue transport properties following treatment success would result in improved contact between drug and targets in both time and space. MRI is the key to controlling treatment by means of thermometry and also monitoring efficacy by means of T2-weighted imaging.