In Situ Generated Novel 1H MRI Reporter for ß-Galactosidase Activity Detection and Visualization in Living Tumor Cells.

For wide applications of the lacZ gene in cellular/molecular biology, small animal investigations, and clinical assessments, the improvement of noninvasive imaging approaches to precisely assay gene expression has garnered much attention. In this study, we investigate a novel molecular platform in which alizarin 2-O-ß-d-galactopyranoside AZ-1 acts as a lacZ gene/ß-gal responsive 1H-MRI probe to induce significant 1H-MRI contrast changes in relaxation times T 1 and T 2 in situ as a concerted effect for the discovery of ß-gal activity with the exposure of Fe3+. We also demonstrate the capability of this strategy for detecting ß-gal activity with lacZ-transfected human MCF7 breast and PC3 prostate cancer cells by reaction-enhanced 1H-MRI T 1 and T 2 relaxation mapping.

Correlation of Tumor Hypoxia Metrics Derived from 18F-Fluoromisonidazole Positron Emission Tomography and Pimonidazole Fluorescence Images of Optically Cleared Brain Tissue.

18F-fluoromisonidazole (FMISO) positron emission tomography (PET) is a widely used noninvasive imaging modality for assessing hypoxia. We describe the first spatial comparison of FMISO PET with an ex vivo reference standard for hypoxia across whole tumor volumes. Eighteen rats were orthotopically implanted with C6 or 9L brain tumors and made to undergo FMISO PET scanning. Whole brains were excised, sliced into 1-mm-thick sections, optically cleared, and fluorescently imaged for pimonidazole using an in vivo imaging system. FMISO maximum tumor uptake, maximum tumor-to-cerebellar uptake (TCmax), and hypoxic fraction (extracted 110 minutes after FMISO injection) were correlated with analogous metrics derived from pimonidazole fluorescence images. FMISO SUVmax was not significantly different between C6 and 9L brain tumors (P = .70), whereas FMISO TCmax and hypoxic fraction were significantly greater for C6 tumors (P < .01). FMISO TCmax was significantly correlated with the maximum tumor pimonidazole intensity (ρ = 0.76, P < .01), whereas FMISO SUVmax was not. FMISO tumor hypoxic fraction was significantly correlated with the pimonidazole-derived hypoxic fraction (ρ = 0.78, P < .01). Given that FMISO TCmax and tumor hypoxic fraction had strong correlations with the pimonidazole reference standard, these metrics may offer more reliable measures of tumor hypoxia than conventional PET uptake metrics (SUVmax). The voxel-wise correlation between FMISO uptake and pimonidazole intensity for a given tumor was strongly dependent on the tumor's TCmax (ρ = 0.81, P < .01) and hypoxic fraction (ρ = 0.85, P < .01), indicating PET measurements within individual voxels showed greater correlation with pimonidazole reference standard in tumors with greater hypoxia.

A practical method for multimodal registration and assessment of whole-brain disease burden using PET, MRI, and optical imaging.

Many neurological diseases present with substantial genetic and phenotypic heterogeneity, making assessment of these diseases challenging. This has led to ineffective treatments, significant morbidity, and high mortality rates for patients with neurological diseases, including brain cancers and neurodegenerative disorders. Improved understanding of this heterogeneity is necessary if more effective treatments are to be developed. We describe a new method to measure phenotypic heterogeneity across the whole rodent brain at multiple spatial scales. The method involves co-registration and localized comparison of in vivo radiologic images (e.g. MRI, PET) with ex vivo optical reporter images (e.g. labeled cells, molecular targets, microvasculature) of optically cleared tissue slices. Ex vivo fluorescent images of optically cleared pathology slices are acquired with a preclinical in vivo optical imaging system across the entire rodent brain in under five minutes, making this methodology practical and feasible for most preclinical imaging labs. The methodology is applied in various examples demonstrating how it might be used to cross-validate and compare in vivo radiologic imaging with ex vivo optical imaging techniques for assessing hypoxia, microvasculature, and tumor growth.

2-D magnetic resonance spectroscopic imaging of the pediatric brain using compressed sensing.

BACKGROUND: Magnetic resonance spectroscopic imaging helps to determine abnormal brain tissue conditions by evaluating metabolite concentrations. Although a powerful technique, it is underutilized in routine clinical studies because of its long scan times. OBJECTIVE: In this study, we evaluated the feasibility of scan time reduction in metabolic imaging using compressed-sensing-based MR spectroscopic imaging in pediatric patients undergoing routine brain exams. MATERIALS AND METHODS: We retrospectively evaluated compressed-sensing reconstructions in MR spectroscopic imaging datasets from 20 pediatric patients (11 males, 9 females; average age: 5.4±4.5 years; age range: 3 days to 16 years). We performed retrospective under-sampling of the MR spectroscopic imaging datasets to simulate accelerations of 2-, 3-, 4-, 5-, 7- and 10-fold, with subsequent reconstructions in MATLAB. Metabolite maps of N-acetylaspartate, creatine, choline and lactate (where applicable) were quantitatively evaluated in terms of the root-mean-square error (RMSE), peak amplitudes and total scan time. We used the two-tailed paired t-test along with linear regression analysis to statistically compare the compressed-sensing reconstructions at each acceleration with the fully sampled reference dataset. RESULTS: High fidelity was maintained in the compressed-sensing MR spectroscopic imaging reconstructions from 50% to 80% under-sampling, with the RMSE not exceeding 3% in any dataset. Metabolite intensities and ratios evaluated on a voxel-by-voxel basis showed no statistically significant differences and mean metabolite intensities showed high correlation compared to the fully sampled reference dataset up to an acceleration factor of 5. CONCLUSION: Compressed-sensing MR spectroscopic imaging has the potential to reduce MR spectroscopic imaging scan times for pediatric patients, with negligible information loss.

A faster PISTOL for 1 H MR-based quantitative tissue oximetry.

Quantitative mapping of oxygen tension (pO2 ), noninvasively, could potentially be beneficial in cancer and stroke therapy for monitoring therapy and predicting response to certain therapies. Intracellular pO2 measurements may also prove useful in tracking the health of labeled cells and understanding the dynamics of cell therapy in vivo. Proton Imaging of Siloxanes to map Tissue Oxygenation Levels (PISTOL) is a relatively new oximetry technique that measures the T1 of administered siloxanes such as hexamethyldisiloxane (HMDSO), to map the tissue pO2 at various locations with a temporal resolution of 3.5 minutes. We have now developed a siloxane-selective Look-Locker imaging sequence equipped with an echo planar imaging (EPI) readout to accelerate PISTOL acquisitions. The new tissue oximetry sequence, referred to as PISTOL-LL, enables the mapping of HMDSO T1 , and hence tissue pO2 in under one minute. PISTOL-LL was tested and compared with PISTOL in vitro and in vivo. Both sequences were used to record dynamic changes in pO2 of the rat thigh muscle (healthy Fischer rats, n = 6), and showed similar results (P > 0.05) as the other, with each sequence reporting a significant increase in pO2 (P < 0.05) under hyperoxia compared with steady state normoxia. This study demonstrates the ability of the new sequence in rapidly and accurately mapping the pO2 changes and accelerating quantitative 1 H MR tissue oximetry by approximately 4-fold. The faster PISTOL-LL technique could enable dynamic 1 H oximetry with higher temporal resolution for assesing tissue oxygentation and tracking the health of transplanted cells labeled with siloxane-based probes. With minor modifications, this sequence can be useful for 19 F applications as well.

A role for dynamic contrast-enhanced magnetic resonance imaging in predicting tumour radiation response.

BACKGROUND: Dynamic contrast-enhanced (DCE) MRI may provide prognostic insights into tumour radiation response. This study examined quantitative DCE MRI parameters in rat tumours, as potential biomarkers of tumour growth delay following single high-dose irradiation. METHODS: Dunning R3327-AT1 prostate tumours were evaluated by DCE MRI following intravenous injection of Gd-DTPA. The next day tumours were irradiated (single dose of 30 Gy), while animals breathed air (n=4) or oxygen (n=4); two animals were non-irradiated controls. Growth was followed and tumour volume-quadrupling time (T4) was compared with pre-irradiation DCE assessments. RESULTS: Irradiation caused significant tumour growth delay (T4 ranged from 28 to 48 days for air-breathing rats, and 40 to 75 days for oxygen-breathing rats) compared with the controls (T4=7 to 9 days). A strong correlation was observed between T4 and extravascular-extracellular volume fraction (ve) irrespective of the gas inhaled during irradiation. There was also a correlation between T4 and volume transfer constant (K(trans)) for the air-breathing group alone. CONCLUSIONS: The data provide rationale for expanded studies of other tumour sites, types and progressively patients, and are potentially significant, as many patients undergo contrast-enhanced MRI as part of treatment planning.

Siloxane Nanoprobes for Labeling and Dual Modality Functional Imaging of Neural Stem Cells.

Cell therapy represents a promising therapeutic for a myriad of medical conditions, including cancer, traumatic brain injury, and cardiovascular disease among others. A thorough understanding of the efficacy and cellular dynamics of these therapies necessitates the ability to non-invasively track cells in vivo. Magnetic resonance imaging (MRI) provides a platform to track cells as a non-invasive modality with superior resolution and soft tissue contrast. We recently reported a new nanoprobe platform for cell labeling and imaging using fluorophore doped siloxane core nanoemulsions as dual modality ((1)H MRI/Fluorescence), dual-functional (oximetry/detection) nanoprobes. Here, we successfully demonstrate the labeling, dual-modality imaging, and oximetry of neural progenitor/stem cells (NPSCs) in vitro using this platform. Labeling at a concentration of 10 µL/10(4) cells with a 40%v/v polydimethylsiloxane core nanoemulsion, doped with rhodamine, had minimal effect on viability, no effect on migration, proliferation and differentiation of NPSCs and allowed for unambiguous visualization of labeled NPSCs by (1)H MR and fluorescence and local pO2 reporting by labeled NPSCs. This new approach for cell labeling with a positive contrast (1)H MR probe has the potential to improve mechanistic knowledge of current therapies, and guide the design of future cell therapies due to its clinical translatability.

GdDO3NI, a nitroimidazole-based T1 MRI contrast agent for imaging tumor hypoxia in vivo.

Tumor hypoxia is known to affect sensitivity to radiotherapy and promote development of metastases; therefore, the ability to image tumor hypoxia in vivo could provide useful prognostic information and help tailor therapy. We previously demonstrated in vitro evidence for selective accumulation of a gadolinium tetraazacyclododecanetetraacetic acid monoamide conjugate of 2-nitroimidazole (GdDO3NI), a magnetic resonance imaging T1-shortening agent, in hypoxic cells grown in tissue culture. We now report evidence for accumulation of GdDO3NI in hypoxic tumor tissue in vivo. Our data show that GdDO3NI accumulated significantly (p < 0.05) in the central, poorly perfused regions of rat prostate adenocarcinoma AT1 tumors (threefold higher concentration than for the control agent) and showed better clearance from well-perfused regions and complete clearance from the surrounding muscle tissue. Inductively coupled plasma mass spectroscopy confirmed that more GdDO3NI than control agent was retained in the central region and that more GdDO3NI was retained in the central region than at the periphery. These results show the utility of GdDO3NI to image tumor hypoxia and highlight the potential of GdDO3NI for application to image-guided interventions for radiation therapy or hypoxia-activated chemotherapy.

Acceleration of conventional data acquisition in dynamic contrast enhancement: comparing keyhole approaches with compressive sensing.

Dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI) has become a valuable clinical tool for cancer diagnosis and prognosis. DCE MRI provides pharmacokinetic parameters dependent on the extravasation of small molecular contrast agents, and thus high temporal resolution and/or spatial resolution is required for accurate estimation of parameters. In this article we investigate the efficacy of 2 undersampling approaches to speed up DCE MRI: a conventional keyhole approach and compressed sensing-based imaging. Data reconstructed from variants of these methods has been compared with the full k-space reconstruction with respect to data quality and pharmacokinetic parameters Ktrans and ve. Overall, compressive sensing provides better data quality and reproducible parametric maps than key-hole methods with higher acceleration factors. In particular, an undersampling mask based on a priori precontrast data showed high fidelity of reconstructed data and parametric maps up to 5× acceleration.

Generation of polypeptide-templated gold nanoparticles using ionizing radiation.

Ionizing radiation, including γ rays and X-rays, are high-energy electromagnetic radiation with diverse applications in nuclear energy, astrophysics, and medicine. In this work, we describe the use of ionizing radiation and cysteine-containing elastin-like polypeptides (C(n)ELPs, where n = 2 or 12 cysteines in the polypeptide sequence) for the generation of gold nanoparticles. In the presence of C(n)ELPs, ionizing radiation doses higher than 175 Gy resulted in the formation of maroon-colored gold nanoparticle dispersions, with maximal absorbance at 520 nm, from colorless metal salts. Visible color changes were not observed in any of the control systems, indicating that ionizing radiation, gold salt solution, and C(n)ELPs were all required for nanoparticle formation. The hydrodynamic diameters of nanoparticles, determined using dynamic light scattering, were in the range of 80-150 nm, while TEM imaging indicated the formation of gold cores 10-20 nm in diameter. Interestingly, C2ELPs formed 1-2 nm diameter gold nanoparticles in the absence of radiation. Our results describe a facile method of nanoparticle formation in which nanoparticle size can be tailored based on radiation dose and C(n)ELP type. Further improvements in these polypeptide-based systems can lead to colorimetric detection of ionizing radiation in a variety of applications.

Novel S-Gal(®) analogs as (1)H MRI reporters for in vivo detection of ß-galactosidase.

The quantitative assessment of gene expression and related enzyme activity in vivo could be important for the characterization of gene altering diseases and therapy. The development of imaging techniques, based on specific reporter molecules may enable routine non-invasive assessment of enzyme activity and gene expression in vivo. We recently reported the use of commercially available S-Gal(®) as a ß-galactosidase reporter for (1)H MRI, and the synthesis of several S-Gal(®) analogs with enhanced response to ß-galactosidase activity. We have now compared these analogs in vitro and have identified the optimal analog, C3-GD, based on strong T1 and T2 response to enzyme presence (ΔR1 and ΔR2~1.8 times S-Gal(®)). Moreover, application is demonstrated in vivo in human breast tumor xenografts. MRI studies in MCF7-lacZ tumors implanted subcutaneously in athymic nude mice (n=6), showed significant reduction in T1 and T2 values (each~13%) 2h after intra-tumoral injection of C3-GD, whereas the MCF7 (wild type) tumors showed slight increase. Thus, C3-GD successfully detects ß-galactosidase activity in vivo and shows promise as a lacZ gene (1)H MR reporter molecule.

A Multi-Camera System for Bioluminescence Tomography in Preclinical Oncology Research.

Bioluminescent imaging (BLI) of cells expressing luciferase is a valuable noninvasive technique for investigating molecular events and tumor dynamics in the living animal. Current usage is often limited to planar imaging, but tomographic imaging can enhance the usefulness of this technique in quantitative biomedical studies by allowing accurate determination of tumor size and attribution of the emitted light to a specific organ or tissue. Bioluminescence tomography based on a single camera with source rotation or mirrors to provide additional views has previously been reported. We report here in vivo studies using a novel approach with multiple rotating cameras that, when combined with image reconstruction software, provides the desired representation of point source metastases and other small lesions. Comparison with MRI validated the ability to detect lung tumor colonization in mouse lung.

Synthesis and characterization of a hypoxia-sensitive MRI probe.

Tissue hypoxia occurs in pathologic conditions, such as cancer, ischemic heart disease and stroke when oxygen demand is greater than oxygen supply. An imaging method that can differentiate hypoxic versus normoxic tissue could have an immediate impact on therapy choices. In this work, the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) with a 2-nitroimidazole attached to one carboxyl group via an amide linkage was prepared, characterized and tested as a hypoxia-sensitive MRI agent. A control complex, Gd(DO3A-monobutylamide), was also prepared in order to test whether the nitroimidazole side-chain alters either the water proton T(1) relaxivity or the thermodynamic stability of the complex. The stabilities of these complexes were lower than that of Gd(DOTA)(-) as expected for mono-amide derivatives. The water proton T(1) relaxivity (r(1)), bound water residence lifetime (τ(M)) and rotational correlation time (τ(R)) of both complexes was determined by relaxivity measurements, variable temperature (17) O NMR spectroscopy and proton nuclear magnetic relaxation dispersion (NMRD) studies. The resulting parameters (r(1) =6.38 mM(-1) s(-1) at 20 MHz, τ(M) =0.71 µs, τ(R) =141 ps) determined for the nitroimidazole derivative closely parallel to those of other Gd(DO3A-monoamide) complexes of similar molecular size. In vitro MR imaging experiments with 9L rat glioma cells maintained under nitrogen (hypoxic) versus oxygen (normoxic) gas showed that both agents enter cells but only the nitroimidazole derivative was trapped in cells maintained under N(2) as evidenced by an approximately twofold decrease in T(1) measured for hypoxic cells versus normoxic cells exposed to this agent. These results suggest that the nitroimidazole derivative might serve as a molecular reporter for discriminating hypoxic versus normoxic tissues by MRI.

Principles and emerging applications of nanomagnetic materials in medicine.

The development of nanometer-scale magnetic materials for biomedical applications spans the interface between the physical sciences and biology. Applications of these materials are rapidly becoming important in medicine and enable targeted therapies and diagnostics. At the same time, specific applications add focus to the development of novel magnetic materials and facilitate a deeper understanding of the physical mechanisms behind their function. This review presents a broad, nontechnical overview of the basis of magnetism in materials at the nanometer scale and describes how these materials are created, characterized, and used. Specific emerging applications in medical diagnostics and therapies are discussed, including cancer cell targeting for thermal ablation, tissue engineering, and three-dimensional noninvasive molecular imaging. Challenges in these fields are discussed, including the toxicity and delivery of magnetic nanomaterials and the sensitivity of imaging and therapeutic techniques. The development of novel nanomagnetic nanomaterials should continue to accelerate as new applications are identified and researchers uncover new mechanisms to increase and modulate magnetism at the nanometer scale.

Dual 19F/1H MR gene reporter molecules for in vivo detection of ß-galactosidase.

Increased emphasis on personalized medicine and novel therapies requires the development of noninvasive strategies for assessing biochemistry in vivo. The detection of enzyme activity and gene expression in vivo is potentially important for the characterization of diseases and gene therapy. Magnetic resonance imaging (MRI) is a particularly promising tool, since it is noninvasive and has no associated radioactivity, yet penetrates deep tissue. We now demonstrate a novel class of dual (1)H/(19)F nuclear magnetic resonance (NMR) lacZ gene reporter molecule to specifically reveal enzyme activity in human tumor xenografts growing in mice. We report the design, synthesis, and characterization of six novel molecules and evaluation of the most effective reporter in mice in vivo. Substrates show a single (19)F NMR signal and exposure to ß-galactosidase induces a large (19)F NMR chemical shift response. In the presence of ferric ions, the liberated aglycone generates intense proton MRI T(2) contrast. The dual modality approach allows both the detection of substrate and the imaging of product enhancing the confidence in enzyme detection.

Compressive sensing could accelerate 1H MR metabolic imaging in the clinic.

PURPOSE: To retrospectively evaluate the fidelity of magnetic resonance (MR) spectroscopic imaging data preservation at a range of accelerations by using compressed sensing. MATERIALS AND METHODS: The protocols were approved by the institutional review board of the university, and written informed consent to acquire and analyze MR spectroscopic imaging data was obtained from the subjects prior to the acquisitions. This study was HIPAA compliant. Retrospective application of compressed sensing was performed on 10 clinical MR spectroscopic imaging data sets, yielding 600 voxels from six normal brain data sets, 163 voxels from two brain tumor data sets, and 36 voxels from two prostate cancer data sets for analysis. The reconstructions were performed at acceleration factors of two, three, four, five, and 10 and were evaluated by using the root mean square error (RMSE) metric, metabolite maps (choline, creatine, N-acetylaspartate [NAA], and/or citrate), and statistical analysis involving a voxelwise paired t test and one-way analysis of variance for metabolite maps and ratios for comparison of the accelerated reconstruction with the original case. RESULTS: The reconstructions showed high fidelity for accelerations up to 10 as determined by the low RMSE (< 0.05). Similar means of the metabolite intensities and hot-spot localization on metabolite maps were observed up to a factor of five, with lack of statistically significant differences compared with the original data. The metabolite ratios of choline to NAA and choline plus creatine to citrate did not show significant differences from the original data for up to an acceleration factor of five in all cases and up to that of 10 for some cases. CONCLUSION: A reduction of acquisition time by up to 80%, with negligible loss of information as evaluated with clinically relevant metrics, has been successfully demonstrated for hydrogen 1 MR spectroscopic imaging.

Dual-modality, dual-functional nanoprobes for cellular and molecular imaging.

An emerging need for evaluation of promising cellular therapies is a non-invasive method to image the movement and health of cells following transplantation. However, the use of a single modality to serve this purpose may not be advantageous as it may convey inaccurate or insufficient information. Multi-modal imaging strategies are becoming more popular for in vivo cellular and molecular imaging because of their improved sensitivity, higher resolution and structural/functional visualization. This study aims at formulating Nile Red doped hexamethyldisiloxane (HMDSO) nanoemulsions as dual modality (Magnetic Resonance Imaging/Fluorescence), dual-functional (oximetry/detection) nanoprobes for cellular and molecular imaging. HMDSO nanoprobes were prepared using a HS15-lecithin combination as surfactant and showed an average radius of 71±39 nm by dynamic light scattering and in vitro particle stability in human plasma over 24 hrs. They were found to readily localize in the cytosol of MCF7-GFP cells within 18 minutes of incubation. As proof of principle, these nanoprobes were successfully used for fluorescence imaging and for measuring pO(2) changes in cells by magnetic resonance imaging, in vitro, thus showing potential for in vivo applications.

Quantitative tissue oxygen measurement in multiple organs using 19F MRI in a rat model.

Measurement of individual organ tissue oxygen levels can provide information to help evaluate and optimize medical interventions in many areas including wound healing, resuscitation strategies, and cancer therapeutics. Echo planar (19) F MRI has previously focused on tumor oxygen measurement at low oxygen levels (pO(2)) <30 mmHg. It uses the linear relationship between spin-lattice relaxation rate (R(1)) of hexafluorobenzene (HFB) and pO(2). The feasibility of this technique for a wider range of pO(2) values and individual organ tissue pO(2) measurement was investigated in a rat model. Spin-lattice relaxation times (T(1) = 1/R(1)) of hexafluorobenzene were measured using (19) F saturation recovery echo planar imaging. Initial in vitro studies validated the linear relationship between R(1) and pO(2) from 0 to 760 mmHg oxygen partial pressure at 25, 37, and 41°C at 7 Tesla for hexafluorobenzene. In vivo experiments measured rat tissue oxygen (ptO2) levels of brain, kidney, liver, gut, muscle, and skin during inhalation of both 30 and 100% oxygen. All organ ptO(2) values significantly increased with hyperoxia (P < 0.001). This study demonstrates that (19) F MRI of hexafluorobenzene offers a feasible tool to measure regional ptO2 in vivo, and that hyperoxia significantly increases ptO2 of multiple organs in a rat model.

S-Gal, a novel 1H MRI reporter for beta-galactosidase.

Reporter genes and associated enzyme activity are becoming increasingly significant for research in vivo. The lacZ gene and beta-galactosidase (beta-gal) expression have long been exploited as reporters of biologic manipulation at the molecular level, and a noninvasive detection strategy based on proton MRI is particularly attractive. 3,4-Cyclohexenoesculetin beta-D-galactopyranoside (S-Gal) is a commercial histologic stain, which forms a black precipitate in the presence of beta-gal and ferric ions, suggesting potential detectability by MRI. Generation of the precipitate is now shown to cause strong T(2)* relaxation, revealing beta-gal activity. A series of tests with the enzyme in vitro and with tumor cells shows that this approach can be used as an assay for beta-gal activity. Proof of principle is shown in human breast tumor xenografts in mice. Upon direct injection of a mixture of 3,4-cyclohexenoesculetin beta-D-galactopyranoside and ferric ammonium citrate, intense contrast was observed immediately in MCF7-lacZ tumors, but not in wild-type tumors. 3,4-Cyclohexenoesculetin beta-D-galactopyranoside activation in combination with ferric ions introduces a novel approach for assaying enzyme activity by MRI in vivo.