Monica Siepmann

Targeted Ultrasound Imaging of Cancer: An Emerging Technology on its Way to Clinics

Ultrasound is one of the workhorses in clinical cancer diagnosis. In particular, it is routinely used to characterize lesions in liver, urogenital tract, head and neck and soft tissues. During the last years image quality steadily improved, which, among others, can be attributed to the development of harmonic image analysis. Microbubbles were introduced as intravascular contrast agents and can be detected with superb sensitivity and specificity using contrast specific imaging modes. By aid of these unspecific contrast agents tissues can be characterised regarding their vascularity. Antibodies, peptides and other targeting moieties were bound to microbubbles to target sites of angiogenesis and inflammation intending to get more disease-specific information. Indeed, many preclinical studies proved the high potential of targeted ultrasound imaging to better characterize tumors and to more sensitively monitor therapy response. Recently, first targeted microbubbles had been developed that meet the pharmacological demands of a clinical contrast agent. This review articles gives an overview on the history and current status of targeted ultrasound imaging of cancer. Different imaging concepts and contrast agent designs are introduced ranging from the use of experimental nanodroplets to agents undergoing clinical evaluation. Although it is clear that targeted ultrasound imaging works reliably, its broad acceptance is hindered by the user dependency of ultrasound imaging in general. Automated 3D-scanning techniques-like being used for breast diagnosis - and novel 3D transducers will help to make this fascinating method clinical reality.

Advanced characterization and refinement of poly N-butyl cyanoacrylate microbubbles for ultrasound imaging.

We aimed to develop and characterize poly n-butylcyanoacrylate (PBCA) microbubbles (MBs) with a narrow size distribution. MBs were synthesized by established emulsion polymerization techniques, size-isolated by centrifugation and functionalized for molecular imaging by coating their surface with streptavidin. The physical and acoustic properties of the parent solution, different-size isolated populations and functionalized MBs were measured and compared. As expected from negative zeta potentials at pH 7, cryo scanning electron microscopy showed no aggregates. In phantoms MBs were destructible at high mechanical indices and showed a frequency-dependent attenuation and backscattering. The MBs were stable in solution for more than 14 weeks and could be lyophilized without major damage. However, for injection, small needle diameters and high injection rates are shown to be critical because both lead to MB destruction. In summary, when being handled correctly, size-isolated PBCA MBs are promising candidates for preclinical functional and molecular ultrasound imaging.

Synergistic effects of sonoporation and taurolidin/TRAIL on apoptosis in human fibrosarcoma.

Sonodynamic therapy, in combination with ultrasound contrast agents, proved to enhance the uptake of chemotherapeutics in malignant cells. HT1080 fibrosarcoma cells were treated in vitro with a combination of ultrasound SonoVue™-microbubbles and taurolidine (TRD) plus tumor necrosis factor related apoptosis inducing ligand (TRAIL). Apoptosis was measured by TdT-mediated dUTP-biotin nick end labelling (TUNEL) assay and fluorescence activated cell sorting (FACS) analysis. Gene expression was analysed by RNA-microarray. The apoptotic effects of TRD and TRAIL on human fibrosarcoma are enhanced by sonodynamic therapy and additional application of contrast agents, such as SonoVue™ by 25%. A broad change in the expression of genes related to apoptotic pathways is observed when ultrasound and microbubbles act synchronously in combination with the chemotherapeutics (e.g. BIRC3, NFKBIA and TNFAIP3). Some of these genes have already been proven to play a role in programmed cell death in human fibrosarcoma (HSPA1A/HSPA1B, APAF1, PAWR, SOCS2) or were associated with sonication induced apoptosis (CD44). Further studies are needed to explore the options of sonodynamic therapy on soft tissue sarcoma and its molecular mechanisms.

Evaluation of high frequency ultrasound methods and contrast agents for characterising tumor response to anti-angiogenic treatment

PURPOSE To compare non-enhanced and contrast-enhanced high-frequency 3D Doppler ultrasound with contrast-enhanced 2D and 3D B-mode imaging for assessing tumor vascularity during antiangiogenic treatment using soft-shell and hard-shell microbubbles. MATERIALS AND METHODS Antiangiogenic therapy effects (SU11248) on vascularity of subcutaneous epidermoid-carcinoma xenografts (A431) in female CD1 nude mice were investigated longitudinally using non-enhanced and contrast-enhanced 3D Doppler at 25 MHz. Additionally, contrast-enhanced 2D and 3D B-mode scans were performed by injecting hard-shell (poly-butyl-cyanoacrylate-based) and soft-shell (phospholipid-based) microbubbles. Suitability of both contrast agents for high frequency imaging and the sensitivity of the different ultrasound methods to assess early antiangiogenic therapy effects were investigated. Ultrasound data were validated by immunohistology. RESULTS Hard-shell microbubbles induced higher signal intensity changes in tumors than soft-shell microbubbles in 2D B-mode measurements (424 ± 7 vs. 169 ± 8 A.U.; p<0.01). In 3D measurements, signals of soft-shell microbubbles were hardly above the background (5.48 ± 4.57 vs. 3.86 ± 2.92 A.U.), while signals from hard-shell microbubbles were sufficiently high (30.5 ± 8.06 A.U). Using hard-shell microbubbles 2D and 3D B-mode imaging depicted a significant decrease in tumor vascularity during antiangiogenic therapy from day 1 on. Using soft-shell microbubbles significant therapy effects were observed at day 4 after therapy in 2D B-mode imaging but could not be detected in the 3D mode. With non-enhanced and contrast-enhanced Doppler imaging significant differences between treated and untreated tumors were found from day 2 on. CONCLUSION Hard-shell microbubble-enhanced 2D and 3D B-mode ultrasound achieved highest sensitivity for assessing therapy effects on tumor vascularisation and were superior to B-mode ultrasound with soft-shell microbubbles and to Doppler imaging.

Influence of shell composition on the resonance frequency of microbubble contrast agents.

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050-0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10(-8) N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.

Imaging tumor vascularity by tracing single microbubbles

In high frequency B-Mode images the circulation of single microbubbles through tumor vessels can be observed. We propose the identification of these individual MBs to image tumor vascularity. In addition to the morphological information, a spatial map of the perfusion can be obtained with this technique. The method is tested on B-Mode images of a tumor xenograft in a nude mouse. Circulation of BR38 microbubbles (Bracco, Geneva, Switzerland) was imaged with a Vevo 2100 small animal imaging system at 40 MHz transmit frequency (Visualsonics, Toronto, Canada). Bubbles were identified by filtering subtraction images with a size selective Difference of Gaussians filter. An image of the vascularity is created by mapping the microbubble centroid positions. This map allows a quantitative evaluation of perfused area and the number of microbubbles passing through each pixel. Our first results show that the identification of single bubbles is feasible. The images show improved vessel resolution in comparison to standard maximum intensity persistence images.

A Statistical Model for the Quantification of Microbubbles in Destructive Imaging

Rationale and Objectives:Quantification of targeted ultrasound contrast agents allows for the monitoring of endothelial marker expressions on a molecular level. In this study, a statistical correction is provided, which allows for improved precision in estimating the concentration of microbubbles (MBs) from Doppler images. Doppler imaging can be used to display the destruction of single MBs. However, concentrations will generally be too high to distinguish the individual events resulting in an inaccurate microbubble (MB) quantification. Therefore, a mathematical description of destruction events in Doppler images is developed which yields a correction formula for the concentration estimate from the color pixel density. Methods:The mathematical model is experimentally verified in gelatin phantoms using a high resolution imaging system (Vevo 770) and experimental cyanoacrylate MBs. Sensitive Particle Acoustic Quantification (SPAQ) is used to quantify MB in a defined volume. The SPAQ step size is varied from 32 to 127 &mgr;m to demonstrate the validity of the model for high color pixel densities. Results:The corrected acoustic quantification shows the expected linear dependence on the step size and thus the amount of MBs in the images (R2 = 0.95). At SPAQ step sizes up to 127 &mgr;m, a MB concentration of 2.7 × 106 MBs/mL can be quantified. Conclusions:The results demonstrate the validity of the proposed correction. Quantification results of the SPAQ technique were considerably improved. The resulting formula is readily applied to SPAQ measurements at no additional expense.

Monitoring of Insonicated Microbubble Behavior and their Effect on Sonoporation Supported Chemotherapy of Fibrosarcoma Cells

Fibrosarcoma shows low response rates to cytotoxic agents. A method to improve chemotherapeutic effects could be microbubble (MB) enhanced sonoporation, which acts through the transient opening of cell membranes due to ultrasound. In this study the behavior of insonicated MB clouds is acoustically monitored and their effect on sonoporation supported chemotherapy of fibrosarcoma cells is analyzed.

Monitoring and modeling of microbubble behavior during ultrasound mediated transfection of cell monolayers

The large scale oscillation of insonified microbubbles (MBs) is considered to be the primary effect for sonoporation and thus enhances cell transfection in gene therapy. MB destruction on the other hand is suspected to lead to lower transfection rates. For future in vivo therapy, online acoustic monitoring could be used to identify these effects and to determine optimal pulse sequence parameters adaptively. As a first step, we monitor MB cloud behavior optically and acoustically during ultrasound mediated transfection of cell monolayers. Opticellreg containers are used to grow monolayers of 293T cells. The containers are filled with a medium containing GFP expressing plasmid DNA and SonoVuereg MBs. Each container is placed in water in the focus of a single element transducer emitting 5 cycles sine-bursts at 1.1 MHz repeated 150 times at 3 Hz. The peak negative pressure varies from 0.29 to 1.53 MPa. A second transducer (1 MHz center frequency) detects transmitted signals on the opposite side. The transducers horizontally scan the entire cell monolayer in a rectangular grid with a spacing of 6 mm. Transmitted and backscattered therapy signals are recorded. For optical MB monitoring, a microscope coupled to a high speed camera is used. The transfection rate is determined by flow cytometry after incubating the cells for 48 hours. The acoustical transmission results reveal MB destruction, which is confirmed by optical MB monitoring. Furthermore, an exponential model of MB destruction in suspensions can be fitted to the monolayer situation. A correlation of the point in time of the maximum of the backscattered signal with the point in time of maximum bubble expansion can be identified. Transfection efficiency, bubble extension and the maximum of the backscattered signals at MB resonance frequency rise with increasing peak negative pressure. In this study, the correlation of sonoporation efficiency and MB extension at cell layers is demonstrated by online monitoring. MB cloud dynamics are acoustically monitored and identified during sonoporation therapy for different excitation peak negative pressures. This is a first step towards adaptively optimizing transfection efficiency in sonoporation therapy by online acoustic monitoring.

Phase shift variance imaging for contrast agent detection

A novel ultrasound contrast agent detection method that selectively detects microbubble (MB) destruction is presented. The detection is based on the variance of the phase shift of consecutive echoes. The variance for stationary echoes or motion at constant speed is determined by the noise of the acquisition system and is theoretically predicted. We hypothesize that the phase shift variance of ultrasound echoes increases during MB destruction. It is expected that this PSV is higher than the PSV for stationary echoes or motion thus allowing the detection of the agent. The hypothesis is experimentally verified in a motion phantom. In a second experiment, MB in very low concentrations are imaged to demonstrate the sensitivity of the method. The results show good agreement with theory and exhibit a clear increase in PSV for MB vs. tissue (44.8 dB). Thus, the phase shift variance can be utilized for ultrasound contrast agent detection. Microbubbles in a concentration of 100 bubbles per ml are sufficient to cause detectable signals with a contrast to tissue ratio of up to 34.7 dB. The acquisition is implemented on a free programmable clinical device. No additions or hardware modifications are necessary making the method easily translatable to most clinical systems.