Xiaowen Hu

Ultrasound Molecular Imaging of Tumor Angiogenesis with an Integrin Targeted Microbubble Contrast Agent

Rationale and Objectives:Ultrasound molecular imaging is an emerging technique for sensitive detection of intravascular targets. Molecular imaging of angiogenesis has strong potential for both clinical use and as a research tool in tumor biology and the development of antiangiogenic therapies. Our objectives are to develop a robust ultrasound contrast agent platform using microbubbles (MB) to which targeting ligands can be conjugated by biocompatible, covalent conjugation chemistry, and to develop a pure low mechanical index (MI) imaging processing method and corresponding quantification method. The MB and the imaging methods were evaluated in a mouse model of breast cancer in vivo. Materials and Methods:We used a cyclic arginine-glycine-aspartic acid (cRGD) pentapeptide containing a terminal cysteine group conjugated to the surface of MB bearing pyridyldithio-propionate (PDP) for targeting &agr;v&bgr;3 integrins. As negative controls, MB without a ligand or MB bearing a scrambled sequence (cRAD) were prepared. To enable characterization of peptides bound to MB surfaces, the cRGD peptide was labeled with FITC and detected by plate fluorometry, flow cytometry, and fluorescence microscopy. Targeted adhesion of cRGD-MB was demonstrated in an in vitro flow adhesion assay against recombinant murine &agr;v&bgr;3 integrin protein and &agr;v&bgr;3 integrin-expressing endothelial cells (bEnd.3). The specificity of cRGD-MB for &agr;v&bgr;3 integrin was demonstrated by treating bEnd.3 EC with a blocking antibody. A murine model of mammary carcinoma was used to assess targeted adhesion and ultrasound molecular imaging in vivo. The targeted MB were visualized using a low MI contrast imaging pulse sequence, and quantified by intensity normalization and 2-dimensional Fourier transform analysis. Results:The cRGD ligand concentration on the MB surface was ∼8.2 × 106 molecules per MB. At a wall shear stress of 1.0 dynes/cm2, cRGD-MB exhibited 5-fold higher adhesion to immobilized recombinant &agr;v&bgr;3 integrin relative to nontargeted MB and cRAD-MB controls. Similarly, cRGD-MB showed significantly greater adhesion to bEnd.3 EC compared with nontargeted MB and cRAD-MB. In addition, cRGD-MB, but not nontargeted MB or cRAD-MB, showed significantly enhanced contrast signals with a high tumor-to-background ratio. The adhesion of cRGD-MB to bEnd.3 was reduced by 80% after using anti-&agr;v monoclonal antibody to treat bEnd.3. The normalized image intensity amplitude was ∼0.8, 7 minutes after the administration of cRGD-MB relative to the intensity amplitude at the time of injection, while the spatial variance in image intensity improved the detection of bound agents. The accumulation of cRGD-MB was blocked by preadministration with an anti-&agr;v blocking antibody. Conclusions:The results demonstrate the functionality of a novel MB contrast agent covalently coupled to an RGD peptide for ultrasound molecular imaging of &agr;v&bgr;3 integrin and the feasibility of quantitative molecular ultrasound imaging with a low MI.

Epoxy metabolites of docosahexaenoic acid (DHA) inhibit angiogenesis, tumor growth, and metastasis

Epidemiological and preclinical evidence supports that omega-3 dietary fatty acids (fish oil) reduce the risks of macular degeneration and cancers, but the mechanisms by which these omega-3 lipids inhibit angiogenesis and tumorigenesis are poorly understood. Here we show that epoxydocosapentaenoic acids (EDPs), which are lipid mediators produced by cytochrome P450 epoxygenases from omega-3 fatty acid docosahexaenoic acid, inhibit VEGF- and fibroblast growth factor 2-induced angiogenesis in vivo, and suppress endothelial cell migration and protease production in vitro via a VEGF receptor 2-dependent mechanism. When EDPs (0.05 mg⋅kg−1⋅d−1) are coadministered with a low-dose soluble epoxide hydrolase inhibitor, EDPs are stabilized in circulation, causing ∼70% inhibition of primary tumor growth and metastasis. Contrary to the effects of EDPs, the corresponding metabolites derived from omega-6 arachidonic acid, epoxyeicosatrienoic acids, increase angiogenesis and tumor progression. These results designate epoxyeicosatrienoic acids and EDPs as unique endogenous mediators of an angiogenic switch to regulate tumorigenesis and implicate a unique mechanistic linkage between omega-3 and omega-6 fatty acids and cancers.

A sensitive TLRH targeted imaging technique for ultrasonic molecular imaging

The primary goals of ultrasound molecular imaging are the detection and imaging of ultrasound contrast agents (microbubbles), which are bound to specific vascular surface receptors. Imaging methods that can sensitively and selectively detect and distinguish bound microbubbles from freely circulating microbubbles (free microbubbles) and surrounding tissue are critically important for the practical application of ultrasound contrast molecular imaging. Microbubbles excited by low-frequency acoustic pulses emit wide-band echoes with a bandwidth extending beyond 20 MHz; we refer to this technique as transmission at a low frequency and reception at a high frequency (TLRH). Using this wideband, transient echo, we have developed and implemented a targeted imaging technique incorporating a multifrequency colinear array and the Siemens Antares imaging system. The multifrequency colinear array integrates a center 5.4-MHz array, used to receive echoes and produce radiation force, and 2 outer 1.5-MHz arrays used to transmit low-frequency incident pulses. The targeted imaging technique makes use of an acoustic radiation force subsequence to enhance accumulation and a TLRH imaging subsequence to detect bound microbubbles. The radiofrequency (RF) data obtained from the TLRH imaging subsequence are processed to separate echo signatures between tissue, free microbubbles, and bound microbubbles. By imaging biotin-coated microbubbles targeted to avidin-coated cellulose tubes, we demonstrate that the proposed method has a high contrast-to-tissue ratio (up to 34 dB) and a high sensitivity to bound microbubbles (with the ratio of echoes from bound microbubbles versus free microbubbles extending up to 23 dB). The effects of the imaging pulse acoustic pressure, the radiation force subsequence, and the use of various slow-time filters on the targeted imaging quality are studied. The TLRH targeted imaging method is demonstrated in this study to provide sensitive and selective detection of bound microbubbles for ultrasound molecularly targeted imaging.

Insonation of targeted microbubbles produces regions of reduced blood flow within tumor vasculature.

ObjectivesIn ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects. Materials and MethodsA total of 114 mice carrying either Met-1 or neu deletion mutant (NDL) tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5-MHz color-Doppler pulses, 4 or 2 MPa peak-negative pressure, 8.1-millisecond pulse repetition period, 6-cycle pulse length, and 900-millisecond insonation). Microbubbles conjugated with cyclic-arginine-glycine-aspartic acid (cRGD) or cyclic-aspartic-acid-glycine-tyrosine (3-NO2)-glycine-hydroxyproline-asparagine (LXY-3) peptides or control (no peptide) microbubbles were injected, and contrast pulse sequencing was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects. ResultsAfter the accumulation of targeted microbubbles and high-pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (P < 0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (P < 0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (P < 0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5/18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150 kDa fluorescein-isothiocyanate (FITC)-dextran were observed by histology and confocal microscopy. Preinjection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals. ConclusionHigh-pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within 2 syngeneic mouse tumor models for ∼30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.

Leveraging the power of ultrasound for therapeutic design and optimization

Contrast agent-enhanced ultrasound can facilitate personalized therapeutic strategies by providing the technology to measure local blood flow rate, to selectively image receptors on the vascular endothelium, and to enhance localized drug delivery. Ultrasound contrast agents are micron-diameter encapsulated bubbles that circulate within the vascular compartment and can be selectively imaged with ultrasound. Microbubble transport-based estimates of local blood flow can quantify changes resulting from anti-angiogenic therapies and facilitate differentiation of angiogenic mechanisms. Microbubbles that are conjugated with targeting ligands attach to endothelial surface receptors that are upregulated in disease, providing high signal-to-noise ratio images of pathological vasculature. In addition to imaging applications, microbubbles can be used to enhance localized gene and drug delivery, either by changing membrane and vascular permeability or by carrying and locally releasing cargo. Our goal in this review is to provide an overview of the use of contrast-enhanced ultrasound methodologies in the design and evaluation of therapeutic strategies with emphases on quantitative blood flow mapping, molecular imaging, and enhanced drug delivery.

Motion corrected cadence CPS ultrasound for quantifying response to vasoactive drugs in a rat kidney model

OBJECTIVE To establish the ability of contrast-enhanced motion corrected cadence pulse sequencing (CPS) to detect changes in renal blood flow induced by vasoactive substances in rats. METHODS Ultrasound contrast media was administered as a constant rate infusion into a phantom at a known rate and CPS data acquired. Rats were anesthetized and predrug CPS estimates of replenishment rate were made for the right kidney. Real-time motion correction was applied, and parametric images were generated from the CPS data. Group 1 rats (n = 7) were administered a vasodilator and group 2 rats (n = 3) were administered a vasoconstrictor. The CPS imaging of the kidney was repeated after ample time for drug effects to occur. RESULTS Contrast CPS accurately estimated flow velocity in the phantom model. In addition, CPS defined statistically significant differences between pre- and postdrug blood flow in the renal medulla (vasodilator, P < .01; vasoconstrictor, P < .0001) and cortex (vasoconstrictor, P < .0001). CONCLUSIONS We conclude that motion-corrected CPS ultrasound provides real-time quantification of renal blood flow alterations and may prove useful for the assessment of blood flow in transplanted kidneys.

Novel Ultrasound and DCE-MRI Analyses after Antiangiogenic Treatment with a Selective VEGF Receptor Inhibitor

We report a comparison between tumor perfusion estimates acquired using contrast-enhanced MRI and motion-corrected contrast-enhanced ultrasound before and after treatment with AG-028262, a potent vascular endothelial growth factor receptor tyrosine kinase inhibitor. Antiangiogenic activity was determined by assessing weekly ultrasound and MRI images of rats with bilateral hind flank mammary adenocarcinomas before and after treatment with AG-028262. Images were acquired with a spoiled gradient, 1.5 T magnetic resonance sequence and a destruction-replenishment ultrasound protocol. For ultrasound, a time to 80% contrast replenishment was calculated for each tumor voxel; for MR imaging, a measure of local flow rate was estimated from a linear fit of minimum to maximum intensities. AG-028262 significantly decreased tumor growth and increased the time required to replenish tumor voxels with an ultrasound contrast agent from 2.66 to 4.54 s and to fill with an MR contrast agent from 29.5 to 50.8 s. Measures of flow rate derived from MRI and ultrasound demonstrated a positive linear correlation of r2 = 0.86.

Reduced blood flow in murine tumors after the destruction of bound, targeted microbubbles

The insonation of circulating microbubbles (MBs) by low frequency (1 MHz) ultrasound (US) pulses has previously been associated with changes in vascular permeability and local changes in blood flow. Here, using a clinical scanner, 5 MHz insonation of bound, targeted MBs is demonstrated to locally alter blood flow in murine tumors. Peptide-targeted MBs were administrated into murine Met-1 and NDL tumor models via tail vein injection (5×107 MBs). Thirty frames of CPS contrast images (Siemens Sequoia 512, 0.09 MI, 10 Hz frame rate) were recorded to assess tumor blood flow. Seven minutes after injection, freely-circulating MBs had cleared from the blood stream leaving bound MBs that had accumulated in the tumor vasculature. At this time, 5 MHz pulses with a peak negative pressure (PNP) of 2 or 4 MPa, a pulse length of 5 cycles and a pulse repetition period of 8.1 ms were transmitted for 0.9 second. Five minutes after the high-pressure pulse sequence, a second dose of MBs was injected and 30 frames of CPS images were acquired. Optical images of systemically-injected FITC-dextran (MW=150,000), pre-administration of an anti-CD41 antibody, and histology were used to investigate the possible mechanism for the vascular changes. After the insonation of bound MBs with a 4 MPa PNP, additional regions of reduced blood flow were observed in 71% of Met-1 tumors (n=28) and 40% of NDL tumors (n = 10). In Met-1 tumors insonified with 4 MPa pulses, the area over which reduced blood flow was observed increased from 22±13% to 63±17% (pμ0.01) of the tumor region of interest. Decreasing the PNP to 2 MPa decreased the percentage of Met-1 tumors with additional regions of reduced blood flow from 71% to 28%. Histological analysis of Met-1 tumors after 4 MPa insonation demonstrated that the mean microvascular diameter in insonified tumors was approximately 17±8 μm, compared to 7±4 μm in control tumors (pμ0.01). Extravasation of FITC-dextran was observed in 4 MPa insonified, but not control, Met-1 tumors. The results suggest that high-pressure insonation of targeted MBs which had accumulated at high concentration, may result in changes in blood flow.

A fast ultrasound molecular imaging method and its 3D visualization in vivo

Using targeted microbubbles (MBs), ultrasound molecular imaging can be used to selectively and specifically visualize upregulated vascular receptors. In order to acquire bound MB echoes, a delay of ~7-15 minutes is commonly required for the clearance of freely circulating MBs. Here, we test whether echoes from MBs can be distinguished from the surrounding tissue, based on the transmission of pulses at low (1.5 MHz) and reception at high (5.5 MHz) frequencies (TLRH), without the requirement for destructive pulses. Pulses with a peak negative pressure of 230 kPa were transmitted (10 fps) and a 7th order IIR pulse-to-pulse filter was applied to the TLRH radiofrequency (RF) data to distinguish the signature of bound MBs from that of flowing MBs. 3D images of the accumulation of intravenously-administrated integrin-targeted MBs in a Met-1 mouse tumor model were acquired. An in vitro study demonstrated that the T2R15 contrast imaging technique has a ~2-fold resolution improvement over 2MHz contrast pulse sequencing (CPS) imaging. By applying the 7th order IIR filter to the TLRH RF data acquired at 2 minutes, echoes from flowing MBs in the surrounding tissue region were suppressed by 26±2 dB, while the signal intensity within the tumor was suppressed by 4±1 dB. The targeted images correctly represented the distribution of bound MBs. After the filter, the signal intensity resulting from cyclic RGD bearing MBs was 25±2 dB higher than that after the injection of non-targeted MBs.

Spatial fourier transform processing of cRGD microbubble echoes in mouse tumors

The development of targeted ultrasound contrast agents has brought ultrasound imaging into the arena of molecular imaging. In particular, targeted microbubbles are now utilized to detect angiogenesis in the early phase of tumor development. The detection of bound agents in a low mechanical index imaging scheme is desirable for clinical application. Here, we present a novel quantitative ultrasound contrast imaging method using the 2D Fourier transform for such targeted contrast agents. In this study, microbubbles coated with RGD peptides were used to target αvβ3-bearing endothelial cells. The quantitative imaging method was based on the low mechanical-index contrast pulse sequencing (CPS) technology (Siemens Medical Solutions) and employed time-domain averaging to suppress echoes from freely circulating microbubbles, thus enhancing echoes from bound microbubbles. Intensity normalization over the ROI was employed to compensate for variations encountered during in vivo studies, such as differences in injected microbubble dose and vascular morphology. The spatial Fourier transform of the time-averaged images was calculated to assess image smoothness. Then, we compared the spatial Fourier spectra of the images and calculated the −6 dB Fourier width with and without high amplitude microbubble destruction. The proposed imaging method was verified in vitro. Images were acquired from Met-1 syngeneic tumors in mice after injection, 7 minutes later, and after a destructive sequence. The normalized intensity of bound microbubbles in the tumor region was 0.8±0.1, compared with a normalized intensity of 0.02±0.02 in the same region for a control (non-targeted) microbubble and 0.1±0.1 for a scrambled peptide. The normalized intensity of bound microbubbles in the surrounding vessels and tissues was negligible. The −6 dB Fourier width was 2.7±0.2 cycles/mm for targeted and 0.8±0.1 cycles/mm for freely circulating control microbubbles. With the application of image averaging, subtraction of the microbubble-image intensity after a destructive pulse was not required for bound bubble discrimination. The quantitative strategy using the spatial Fourier Transform was successfully implemented and is independent of attenuation.