In Vivo FRET Imaging of Tumor Endothelial Cells Highlights a Role of Low PKA Activity in Vascular Hyperpermeability.

Vascular hyperpermeability is a pathological hallmark of cancer. Previous in vitro studies have elucidated roles of various signaling molecules in vascular hyperpermeability; however, the activities of such signaling molecules have not been examined in live tumor tissues for technical reasons. Here, by in vivo two-photon excitation microscopy with transgenic mice expressing biosensors based on Förster resonance energy transfer, we examined the activity of protein kinase A (PKA), which maintains endothelial barrier function. The level of PKA activity was significantly lower in the intratumoral endothelial cells than the subcutaneous endothelial cells. PKA activation with a cAMP analogue alleviated the tumor vascular hyperpermeability, suggesting that the low PKA activity in the endothelial cells may be responsible for the tumor-tissue hyperpermeability. Because the vascular endothelial growth factor (VEGF) receptor is a canonical inducer of vascular hyperpermeability and a molecular target of anticancer drugs, we examined the causality between VEGF receptor activity and the PKA activity. Motesanib, a kinase inhibitor for VEGF receptor, activated tumor endothelial PKA and reduced the vascular permeability in the tumor. Conversely, subcutaneous injection of VEGF decreased endothelial PKA activity and induced hyperpermeability of subcutaneous blood vessels. Notably, in cultured human umbilical vascular endothelial cells, VEGF activated PKA rather than decreasing its activity, highlighting the remarkable difference between its actions in vitro and in vivo These data suggested that the VEGF receptor signaling pathway increases vascular permeability, at least in part, by reducing endothelial PKA activity in the live tumor tissue. Cancer Res; 76(18); 5266-76. ©2016 AACR.

Feasibility of poly(ethylene glycol) derivatives as diagnostic drug carriers for tumor imaging.

Poly(ethylene glycol) (PEG) is an artificial but biocompatible hydrophilic polymer that has been widely used in clinical products. To evaluate the feasibility of using PEG derivative itself as a tumor imaging carrier via an enhanced permeability and retention (EPR) effect, we prepared indium-111-labeled PEG ((111)In-DTPA-PEG) and indocyanine green (ICG)-labeled PEG (ICG-PEG) with PEG molecular weights of 5-40kDa and investigated their in vivo biodistribution in colon26 tumor-bearing mice. Thereafter, single-photon emission computed tomography (SPECT) and photoacoustic (PA) imaging studies were performed. The in vivo biodistribution studies demonstrated increased tumor uptake and a prolongation of circulation half-life as the molecular weight of PEG increased. Although the observed differences in in vivo biodistribution were dependent on the labeling method ((111)In or ICG), the tumor-to-normal tissue ratios were comparable. Because PEG-based probes with a molecular weight of 20kDa (PEG20) showed a preferable biodistribution (highest accumulation among tissues excised and relatively high tumor-to-blood ratios), an imaging study using (111)In-DTPA-PEG20 and ICG-PEG20 was performed. Colon26 tumors inoculated in the right shoulder were clearly visualized by SPECT 24h after administration. Furthermore, PA imaging using ICG-PEG20 also detected tumor regions, and the detected PA signals increased in proportion with the injected dose. These results suggest that PEG derivatives (20kDa) serve as robust diagnostic drug carriers for tumor imaging.

Development of anti-HER2 fragment antibody conjugated to iron oxide nanoparticles for in vivo HER2-targeted photoacoustic tumor imaging.

Photoacoustic (PA) imaging is a promising imaging modality that provides biomedical information with high sensitivity and resolution. Iron oxide nanoparticles (IONPs) have been regarded as remarkable PA contrast agents because of their low toxicity and biodegradable properties. However, IONP delivery is restricted by its modest leakage and retention in tumors. In this study, we designed IONPs (20nm, 50nm, and 100nm) conjugated with anti-HER2 moieties [whole IgG, single-chain fragment variable (scFv), and peptide] for HER2-targeted PA tumor imaging. The binding affinity, cellular uptake, and in vivo biodistribution were examined. We propose 20-nm anti-HER2 scFv-conjugated IONPs (SNP20) as a novel PA contrast agent. SNP20 demonstrated high affinity and specific binding to HER2-expressing cells; it selectively visualized HER2-positive tumors in PA imaging studies. These data indicate that SNP20 is a potential PA contrast agent for imaging of HER2-expressing tumors. FROM THE CLINICAL EDITOR: Iron oxide nanoparticles have been demonstrated to be good contrast agents for tumor imaging. They may also be useful in photoacoustic (PA) imaging, which can provide high sensitivity data and image resolution. The authors here coupled iron oxide nanoparticles with anti-HER2 antibody fragment and showed significant retention of these nanoparticles in tumors. This combination may provide another option for enhanced imaging of tumors.

Near-infrared dye-conjugated amphiphilic hyaluronic acid derivatives as a dual contrast agent for in vivo optical and photoacoustic tumor imaging.

Amphiphilic hyaluronic acid (HA) derivatives bearing hydrophobic indocyanine green dye derivatives and hydrophilic poly(ethylene glycol) were synthesized through the use of condensation and copper-catalyzed click cyclization reactions. The amphiphilic HA derivatives dissolved in water and formed self-assemblies in which the near-infrared dyes were tightly packed and arranged to form dimers or H-aggregates. By irradiating an aqueous solution of HA derivatives with near-infrared light, photoacoustic signals were detected along with fluorescence emission. Self-assemblies consisting of HA derivatives could smoothly accumulate in tumor tissues by passive tumor targeting. By utilizing HA derivatives as a contrast agent, tumor sites were clearly visualized by optical imaging as well as by photoacoustic tomography.

Array-based functional screening for genes that regulate vascular endothelial differentiation of Flk1-positive progenitors derived from embryonic stem cells.

Functional genomics is a central topic of current biological research, where a reverse genetic approach is one of the most promising strategies to discover functions of novel genes. Such an approach requires high-throughput methodologies to assess biological functions for a huge number of genes. We have developed a transfection array that permits parallel introduction of multiple plasmids separately into living cells. The feasibility of this array was examined in an assay system. Eleven genes were over-expressed alone, or in combination in vascular progenitors derived from embryonic stem cells. Endothelial differentiation of the cells was monitored through a stably transformed EGFP reporter construct that is expressed only in endothelial cells. Transcriptional activators that promote endothelial differentiation, such as Ets1 and Sox7, were identified. In addition, the assays also revealed an inhibitory effect on endothelial differentiation by several of the factors. These results demonstrate the feasibility of the transfection array for use in cell-based, high-throughput functional assays.

Layer-by-layer assembly of cationic lipid and plasmid DNA onto gold surface for stent-assisted gene transfer.

Intravascular stent-assisted gene transfer is an advanced approach for the therapy of vascular diseases such as atherosclerosis and stenosis. This approach requires a stent that allows local and efficient administration of therapeutic genes to the target cells at the vascular wall. To create such a stent, a method was developed for loading plasmid DNA onto the metal surface. The method involves the formation of self-assembled monolayer on the noble metal surface followed by electrostatic layer-by-layer (LBL) assembly of a cationic lipid/plasmid DNA complex and free plasmid DNA. In this in vitro feasibility study, the thin plainer film and the wire of gold were used as a substrate. The LBL assembly process was characterized by surface plasmon resonance spectroscopy and static contact angle measurement. Plasmid DNA loaded in the multilayer exhibited improved resistance against nuclease digestion. When cultured directly on the DNA-loaded surface, cells were transfected to express exogenous gene in the DNA loading-dependent manner. Plasmid DNA could also be transferred to endothelial cells from its apical side by placing the DNA-loaded gold wire onto the cell layer.

Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer.

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode made of indium-tin oxide. The surface with adsorbed poly(ethyleneimine) and DNA was characterized by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and contact angle measurements. These analyses revealed that the alternate adsorption process generated a multilayered assembly of cationic poly(ethyleneimine) and anionic DNA. For the spatially and temporally specific gene transfer, cells were cultured on the plasmid-loaded electrode and then a short electric pulse was applied to the cell-electrode system. It was shown that, upon electric pulsing, the plasmid was released from the electrode and transferred into the cells, resulting in efficient gene expression even in primary cultured cells. Transfection could be effected for hippocampal neurons after 3-day culture on the plasmid-loaded electrode, which indicated the feasibility of selecting the time of transfection. Our results also showed that electroporation could be performed in a spatially specific manner by using a plasmid-arrayed electrode, demonstrating the feasibility of the method for the fabrication of transfected cell microarrays.

Spatially and temporally controlled gene transfer by electroporation into adherent cells on plasmid DNA-loaded electrodes.

Functional characterization of human genes is one of the most challenging tasks in current genomics. Owing to a large number of newly discovered genes, high-throughput methodologies are greatly needed to express in parallel each gene in living cells. To develop a method that allows efficient transfection of plasmids into adherent cells in spatial- and temporal-specific manners, we studied electric pulse-triggered gene transfer using a plasmid-loaded electrode. A plasmid was loaded on a gold electrode surface having an adsorbed layer of poly(ethyleneimine), and cells were then plated directly onto this modified surface. The plasmid was detached from the electrode by applying a short electric pulse and introduced into the cells cultured on the electrode, resulting in efficient gene expression, even in primary cultured cells. The location of transfected cells could be restricted within a small area on a micropatterned electrode, showing the versatility of the method for spatially controlled transfection. Plasmid transfection could also be performed in a temporally controlled manner without a marked loss of the efficiency when an electric pulse was applied within 3 days after cell plating. The method described here will provide an efficient means to transfer multiple genes, in parallel, into cultured mammalian cells for high-throughput reverse genetics research.

Micropatterned, self-assembled monolayers for fabrication of transfected cell microarrays.

The aim of this study was to develop the cell microarray that allows efficient transfer of multiple genes into mammalian cells cultured on the microarray in a high-throughput fashion. A microarray was fabricated using a gold-coated glass plate having a micropatterned, self-assembled monolayer of alkanethiols carrying ionic and nonionic terminal groups. Plasmid DNA and a cationic lipid were loaded by alternate electrostatic adsorption to the microspots to obtain a plasmid DNA microarray. The loading and the release of lipid-DNA complex were studied by, respectively, the fluorescence staining of DNA and the imaging of the microarray with a surface plasmon resonance (SPR) apparatus. The transfection efficiency was evaluated by directly plating and culturing human embryonic kidney cells onto the microarray. The results demonstrated that cells which adhered to the DNA-loaded spots were transfected to express the encoded model proteins for several days. The chemistry of the monolayers and the number of alternate adsorption cycles had large effects on the efficiency of transfection. This may be explained from the availability of the lipid-DNA complex to the cells directly contacted. We conclude that the micropatterned, self-assembled monolayers greatly facilitate regionally defined loading of DNAs and expression of the encoded protein in mammalian cells.