Single-cell magnetic imaging using a quantum diamond microscope.

We apply a quantum diamond microscope for detection and imaging of immunomagnetically labeled cells. This instrument uses nitrogen-vacancy (NV) centers in diamond for correlated magnetic and fluorescence imaging. Our device provides single-cell resolution and a field of view (∼1 mm(2)) two orders of magnitude larger than that of previous NV imaging technologies, enabling practical applications. To illustrate, we quantified cancer biomarkers expressed by rare tumor cells in a large population of healthy cells.

Epigenetic memory in induced pluripotent stem cells.

Somatic cell nuclear transfer and transcription-factor-based reprogramming revert adult cells to an embryonic state, and yield pluripotent stem cells that can generate all tissues. Through different mechanisms and kinetics, these two reprogramming methods reset genomic methylation, an epigenetic modification of DNA that influences gene expression, leading us to hypothesize that the resulting pluripotent stem cells might have different properties. Here we observe that low-passage induced pluripotent stem cells (iPSCs) derived by factor-based reprogramming of adult murine tissues harbour residual DNA methylation signatures characteristic of their somatic tissue of origin, which favours their differentiation along lineages related to the donor cell, while restricting alternative cell fates. Such an 'epigenetic memory' of the donor tissue could be reset by differentiation and serial reprogramming, or by treatment of iPSCs with chromatin-modifying drugs. In contrast, the differentiation and methylation of nuclear-transfer-derived pluripotent stem cells were more similar to classical embryonic stem cells than were iPSCs. Our data indicate that nuclear transfer is more effective at establishing the ground state of pluripotency than factor-based reprogramming, which can leave an epigenetic memory of the tissue of origin that may influence efforts at directed differentiation for applications in disease modelling or treatment.

In vivo molecular target assessment of matrix metalloproteinase inhibition.

A number of different matrix metalloproteinase (MMP) inhibitors have been developed as cytostatic and anti-angiogenic agents and are currently in clinical testing. One major hurdle in assessing the efficacy of such drugs has been the inability to sense or image anti-proteinase activity directly and non-invasively in vivo. We show here that novel, biocompatible near-infrared fluorogenic MMP substrates can be used as activatable reporter probes to sense MMP activity in intact tumors in nude mice. Moreover, we show for the first time that the effect of MMP inhibition can be directly imaged using this approach within hours after initiation of treatment using the potent MMP inhibitor, prinomastat (AG3340). The developed probes, together with novel near-infrared fluorescence imaging technology will enable the detailed analysis of a number of proteinases critical for advancing the therapeutic use of clinical proteinase inhibitors.

In vivo imaging of tumors with protease-activated near-infrared fluorescent probes.

We have developed a method to image tumor-associated lysosomal protease activity in a xenograft mouse model in vivo using autoquenched near-infrared fluorescence (NIRF) probes. NIRF probes were bound to a long circulating graft copolymer consisting of poly-L-lysine and methoxypolyethylene glycol succinate. Following intravenous injection, the NIRF probe carrier accumulated in solid tumors due to its long circulation time and leakage through tumor neovasculature. Intratumoral NIRF signal was generated by lysosomal proteases in tumor cells that cleave the macromolecule, thereby releasing previously quenched fluorochrome. In vivo imaging showed a 12-fold increase in NIRF signal, allowing the detection of tumors with submillimeter-sized diameters. This strategy can be used to detect such early stage tumors in vivo and to probe for specific enzyme activity.

Tat peptide-derivatized magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells.

The ability to track the distribution and differentiation of progenitor and stem cells by high-resolution in vivo imaging techniques would have significant clinical and research implications. We have developed a cell labeling approach using short HIV-Tat peptides to derivatize superparamagnetic nanoparticles. The particles are efficiently internalized into hematopoietic and neural progenitor cells in quantities up to 10-30 pg of superparamagnetic iron per cell. Iron incorporation did not affect cell viability, differentiation, or proliferation of CD34+ cells. Following intravenous injection into immunodeficient mice, 4% of magnetically CD34+ cells homed to bone marrow per gram of tissue, and single cells could be detected by magnetic resonance (MR) imaging in tissue samples. In addition, magnetically labeled cells that had homed to bone marrow could be recovered by magnetic separation columns. Localization and retrieval of cell populations in vivo enable detailed analysis of specific stem cell and organ interactions critical for advancing the therapeutic use of stem cells.

In vivo imaging of proteolytic enzyme activity using a novel molecular reporter.

The single biggest challenge facing in vivo imaging techniques is to develop biocompatible molecular beacons that are capable of specifically and accurately measuring in vivo targets at the protein, RNA, or DNA level. Our efforts have focused on developing activatable imaging probes to measure specific enzyme activities in vivo. Using cathepsin D as a model target protease, we synthesized a long-circulating, synthetic graft copolymer bearing near-infrared (NIR) fluorochromes positioned on cleavable substrate sequences. In its native state, the reporter probe was essentially nonfluorescent at 700 nm due to energy resonance transfer among the bound fluorochromes (quenching) but became brightly fluorescent when the latter were released by cathepsin D. NIR fluorescence signal activation was linear over at least 4 orders of magnitude and specific when compared with scrambled nonsense substrates. Using matched rodent tumor models implanted into nude mice expressing or lacking the targeted protease, it could be shown that the former generated sufficient NIR signal to be directly detectable and that the signal was significantly different compared with negative control tumors. The developed probes should find widespread applications for real-time in vivo imaging of a variety of clinically relevant proteases, for example, to detect endogenous protease activity in disease, to monitor the efficacy of protease inhibitors, or to image transgene expression.

Tyrosinase mutants are capable of prodrug activation in transfected nonmelanotic cells.

Tyrosinase has been suggested as a prodrug-converting enzyme for the treatment of melanoma. We hypothesized that tyrosinase expression in transfected nonmelanotic cells can be used in a gene therapy paradigm of prodrug activation. To verify our hypothesis, we used the following tyrosinase variants: (a) a full-length human tyrosinase clone (T); (b) a mutant lacking the COOH-terminal cytoplasmic domain (TdeltaC); (c) a mutant lacking the COOH-terminal transmembrane and cytoplasmic domains (TdeltaTC); and (d) a fusion with the eight COOH-terminal amino acids of lysosome-associated membrane protein-1 (TL). Expression of mutant and wild-type tyrosinases was induced by transfection in nontumorigenic human cells of epithelial origin (293HEK, MCF-10A adenoma, and NHDF-Ad human dermal fibroblasts) as well as in tumor cells (9L gliosarcoma, MCF7 adenocarcinoma, and HT-1080 fibrosarcoma). When compared with the wild-type tyrosinase transfectants, truncated mutant expression resulted in higher mRNA levels that paralleled higher enzyme activity of the truncated mutants. Two model tyrosinase prodrugs, hydroxyphenyl-propanol (HPP) and N-acetyl-4-S-cysteaminylphenol (NAcSCAP) inhibited proliferation and caused cell death of transfected cells in a dose-dependent manner. Effects of prodrug treatment were compared for tumorigenic cells and their nontumorigenic counterparts. Two truncated mutants (TdeltaC and TdeltaTC) showed low endogenous cytotoxicity and efficiently suppressed proliferation and induced cytotoxicity in transfected tumor cells in the presence of NAcSCAP. Overall, these results indicate that the developed tyrosinase mutants hold promise as prodrug activation systems for tumoral gene therapy.

Real-time high dynamic range laser scanning microscopy.

In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Design of metal-binding green fluorescent protein variants.

Diglycylcysteine motifs bind reduced oxo-compounds of technetium-99m, an important isotope in nuclear imaging. We suggested a system for detecting gene expression employing the effect of oxo[99mTc]technetate (Tc(V)O3+) transchelation and coordination with redox amino acid motifs. DNA fragments encoding diglycylcysteine (GGC) binding motifs were prepared by PCR and positioned downstream from the green fluorescent protein (GFP) cDNA insert. Using a Bluescript (+) vector with the fusion protein positioned under the control of a lac promoter, we obtained several E. coli clones expressing the following GFP fusion peptides: (1) GFP-P1 bearing a 'hydrophilic' C-terminal peptide (LEGGGCEGGC) containing two residues of glutamic acid and C-terminal cysteine (2) GFP-P2 carrying a 'hydrophobic' (LGGGGCGGGCGI) peptide (3) a control GFP fusion peptide with deleted C-terminal portion. Bacterial lysates obtained from the corresponding clones were tested for oxo[99mTc] technetate transchelation from a glucoheptonate complex. We found, using a solid phase assay, that radioactivity associated with protein lysates obtained from clones expressing GFP-P2 fusions were 3-4 fold higher than lysates prepared from a clone expressing a truncated GFP fusion protein lacking the C-terminal GGC motifs. High expression of GFP fusions (5-21% of total protein) was demonstrated by electrophoresis and verified by immunoblotting. Specific association of the isotope with GFP-P2 fusion proteins was detected upon incubation of gels in the presence of [99mTc]glucoheptonate, while no binding of oxo[99mTc]technetate to GFP-P1 was revealed. We demonstrated, by using semi-quantitative autoradiography, that there is a 10-fold higher binding of oxotechnetate to GFP-P2 than to a control GFP fusion protein. The implications of the study for in vivo gene expression imaging are discussed.

Trapping of dextran-coated colloids in liposomes by transient binding to aminophospholipid: preparation of ferrosomes.

A procedure is described that allows to increase the efficiency of the loading of liposomes with dextran-stabilized iron oxides (MION). The method produces a preparation of liposomes (REVs) with high iron oxide content as a result of transient binding of oxidized dextran with amino groups of aminophospholipids. Phosphatidylethanolamine (PE)-containing lipid mixtures (PC/DOPE/CH or SM/DOPE/CH, 9:2:9 molar ratio) in organic phase were combined with oxidized MION at pH 8. Liposomes then were obtained by reversed-phase evaporation. Liposomes, 263 +/- 89 nm in diameter, contained up to 11.8 mol Fe/mol phospholipid (encapsulation yield 49%). 10.2% of liposome-associated iron was dissociated from liposomes upon changing the pH to 4.5. When lipid compositions of extracts prepared from liposomes incubated at pH 4.5 and pH 8.0 were compared, an increase of relative PE-content in extracts of liposomes incubated at lowered pH was detected. This indicates a dissociation of imine bonds between aldehydes on the MION surface and PE. The accessibility of liposomal PE for acylation was demonstrated by modification with an activated ester of methoxy poly(ethylene glycol) succinate. Control liposomes, containing no aminophospholipid, or PE-containing liposomes obtained in the presence of non-oxidized MION, were 3.5-5-fold less effective for MION encapsulation and showed extensive aggregation.

Measuring transferrin receptor gene expression by NMR imaging.

The human transferrin receptor (hTfR) has been used as a model molecular target to direct therapeutic agents to tumor cells and to shuttle drugs across the blood-brain-barrier. We show in the current study that receptor expression and regulation can be visualized by NMR imaging, when the receptor is probed with a sterically protected iron containing magnetic hTfR probe. We were able to demonstrate that the novel receptor probe was an iron source that could enter the cells via the hTfR but did not play an immediate role in iron downregulation of hTfR within incubation times tested. Using genetically engineered rat 9L gliosarcoma cell lines with three different forms of the hTfR, we also demonstrated that receptor expression and regulation can be visualized by NMR imaging using the probe. This research provides proof of the principle that it is possible to image receptor gene expression and regulation and it demonstrates that it may be possible to image gene transfer in vivo.

Noninvasive in vivo measurement of beta-cell mass in mouse model of diabetes.

Pancreatic beta-cell mass (BCM) is a major determinant of the quantity of insulin that can be secreted. BCM is markedly reduced in type 1 diabetes because of selective autoimmune destruction of beta-cells. Accurate assessment of BCM in human diabetes is limited to autopsy studies, which usually suffer from inadequate clinical information; thus, the development of noninvasive means of BCM measurement could be important in intervention therapy. The goal of this study was to develop such noninvasive methods for measuring BCM featuring target-specific imaging probes and to investigate whether this technique is feasible, accurate, and predictive of BCM in normal and diabetic states. Using a beta-cell-specific monoclonal antibody IC2, modified with a radioisotope chelator for nuclear imaging, we showed that highly specific binding and accumulation to beta-cells occurs after intravenous administration of the probe, with virtually no binding to exocrine pancreas or stromal tissues. Furthermore, we observed a direct correlation between accumulation of the probe with BCM in diabetic and normal animals. Nuclear imaging of the animals that received an injection of the radioactive probe showed major difference in signal intensity between normal and diabetic pancreases. The results from this study set the route for further development of imaging probes for measuring BCM that would aid in diagnosis and treatment of diabetic patients in the clinic.

Fluorescence molecular imaging of small animal tumor models.

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.

In vivo imaging of gene and cell therapies.

Molecular imaging can be broadly defined as the in vivo characterization and measurement of biological processes at the cellular and molecular level. In contrast to commonly used clinical imaging, it sets forth to probe the molecular abnormalities that are the basis of disease, rather than imaging the end effects of these molecular alterations. Development of new imaging technologies requires a multidisciplinary collaboration between biologists, chemists, physicists, and imaging scientists to create novel agents, signal amplification strategies, and imaging techniques that successfully address these questions. In this article we attempt to present some of the recent developments and show how molecular imaging can be used, at least experimentally, to assess specific molecular targets for gene- and cell-based therapies. In particular, we place emphasis on the development and use of experimental small-animal models, which are particularly inclined toward this approach, primarily in combination with magnetic resonance (MR), radionuclide, and optical imaging. In the future, specific imaging of disease targets will allow earlier detection and characterization of disease, as well as earlier and direct molecular assessment of treatment efficacy.

The development of in vivo imaging systems to study gene expression.

Imaging techniques are currently being developed to map the topography and level of gene expression following gene therapy. To date, two different imaging strategies have been investigated--using marker genes encoding either intracellular enzymes or cell-surface receptors. The first approach employs the ability of certain enzymes to modify imaging prodrugs, so that tissue accumulation of such drugs reflects the expression. The second approach utilises cell-surface expression of a ligand-binding receptor that can be detected using imaging tracers. In this review, we discuss nuclear-and magnetic-resonance-image techniques that have been developed to detect gene expression.

Mapping the in vivo distribution of herpes simplex virions.

We describe a method for labeling enveloped viral particles with a radiotracer, indium-111, allowing labeled viruses to be traced in vivo by nuclear imaging. After initial optimization experiments, a labeling efficiency of 83% (incorporation yield) was achieved for herpes simplex virus (HSV), resulting in a specific activity of 30 microCi/10(9) PFU. The labeling procedure did not significantly reduce the infectivity of the labeled virus and the virus did not release any significant amounts of the radionuclide within 12 hr after labeling. Sequential imaging of animals after intravenous administration of the labeled virus showed fast accumulation in the liver and redistribution from the blood pool (immediately after injection) to liver and spleen (12-24 hr after injection). At 12 hr after injection 7% of the virus-associated (111)In had been eliminated from the body and the remaining organ distribution of the virus was as follows: spleen 2.87 +/- 0.54% ID/g; liver, 2.60 +/- 0.51% ID/g; kidney, 0.98 +/- 0.31% ID/g; lung, 0.57 +/- 0.10% ID/g; [corrected] and lower amounts in other organs. Our results indicate that the described method allows qualitative and quantitative assessment of viral biodistribution in vivo by nuclear imaging.

Selective uptake of viral and monocrystalline particles delivered intra-arterially to experimental brain neoplasms.

In this study we investigated the intra-arterial delivery of viral and nonviral particles to experimental brain tumors. A herpes simplex virus (HSV) vector and monocrystalline iron oxide nanoparticles (MION) were injected into the internal carotid artery of Fisher 344 rats harboring intracerebral 9L gliosarcomas, using bradykinin to disrupt the blood-tumor barrier. Brain and internal organs were stained both for virus-mediated gene expression and for iron. Quantitative comparisons of gene expression and MION uptake with and without blood-tumor barrier disruption were performed in the center and at the periphery of the tumor mass, as well as in normal brain. In addition, MION distribution was traced in vivo by MR imaging. Delivery of HSV into 9L gliosarcoma cells was greatly enhanced by intra-carotid bradykinin infusion. Virus-mediated expression of the HSV-thymidine kinase (TK) and beta-galactosidase gene products was highest at the tumor periphery as compared to the tumor center. Selective HSV infection of multiple tumor foci was achieved in both hemispheres without affecting normal brain. MION uptake was high at the tumor periphery even without blood-tumor barrier disruption. Bradykinin increased MION uptake predominantly in the center of the tumor with virtually no effect at the periphery. These findings show that selective blood-tumor barrier disruption by bradykinin can be used to enhance HSV-mediated gene delivery to tumor cells in the periphery of brain tumors. A crucial aspect in the treatment of malignant brain tumors is the eradication of tumor cells infiltrating the brain; bradykinin may facilitate access of vectors to these areas by selective disruption of their neovasculature.

Detection of spontaneous schwannomas by MRI in a transgenic murine model of neurofibromatosis type 2.

Spontaneous schwannomas were detected by magnetic resonance imaging (MRI) in a transgenic murine model of neurofibromatosis type 2 (NF2) expressing a dominant mutant form of merlin under the Schwann cell-specific P0 promoter. Approximately 85% of the investigated mice showed putative tumors by 24 months of age. Specifically, 21% of the mice showed tumors in the intercostal muscles, 14% in the limb muscles, 7% in the spinal cord and spinal ganglia, 7% in the external ear, 14% in the muscle of the abdominal region, and 7% in the intestine; 66% of the female mice had uterine tumors. Multiple tumors were detected by MRI in 21% of mice. The tumors were isointense with muscle by T1-weighted MRI, showed strong enhancement following administration of gadolinium-DTPA, and were markedly hyperintense by T2-weighted MRI, all hallmarks of the clinical manifestation. Hematoxylin and eosin staining and immunohistochemistry indicated that the tumors consisted of schwannomas and Schwann cell hyperplasias. The lesions stained positively for S-100 protein and a marker antigen for the mutated transgenic NF2 protein, confirming that the imaged tumors and areas of hyperplasia were of Schwann cell origin and expressed the mutated NF2 protein. Tumors were highly infectable with a recombinant herpes simplex virus type 1 vector, hrR3, which contains the reporter gene, lacZ. The ability to develop schwannoma growth with a noninvasive imaging technique will allow assessment of therapeutic interventions.

Treatment of experimental brain tumors with trombospondin-1 derived peptides: an in vivo imaging study.

Antiangiogenic and antiproliferative effects of synthetic D-reverse peptides derived from the type 1 repeats of thrombospondin (TSP1) were studied in rodent C6 glioma and 9L gliosarcomas. To directly measure tumor size and vascular parameters, we employed in vivo magnetic resonance (MR) imaging and corroborated results by traditional morphometric tissue analysis. Rats bearing either C6 or 9L tumors were treated with TSP1-derived peptide (D-reverse amKRFKQDGGWSHWSPWSSac, n=13) or a control peptide (D-reverse amKRAKQAGGASHASPASSac, n=12) at 10 mg/kg, administered either intravenously or through subcutaneous miniosmotic pumps starting 10 days after tumor implantation. Eleven days later, the effect of peptide treatment was evaluated. TSP1 peptide-treated 9L tumors (50.7+/-44.2 mm3, n=7) and C6 tumors (41.3+/-34.2 mm3, n=6) were significantly smaller than tumors treated with control peptide (9L: 215.7+/-67.8 mm3, n=6; C6: 184.2+/-105.2 mm3, n=6). In contrast, the in vivo vascular volume fraction, the mean vascular area (determined by microscopy), and the microvascular density of tumors were not significantly different in any of the experimental groups. In cell culture, TSP1, and the amKRFKQDGGWSHWSPWSSac peptide showed antiproliferative effects against C6 with an IC of 45 nM for TSP1. These results indicate that TSP1-derived peptides retard brain tumor growth presumably as a result of slower de novo blood vessel formation and synergistic direct antiproliferative effects on tumor cells. We also show that in vivo MR imaging can be used to assess treatment efficacy of novel antiangiogenic drugs non-invasively, which has obvious implications for clinical trials.