Development of a medical imaging-based technology for cancer treatment

The Electrical Impedance Mammography (EIM) device is an imaging system developed at the University of Sussex for the detection of breast lesions in vivo using quadrature detection of impedance. The work describes a novel technique to integrate Ultrasound-guided Focused Ultrasound Surgery (USgFUS) with the existing EIM system. The benefits that such a system could provide include the possibility of non-invasive detection, diagnosis and treatment of breast cancer all within a single device and involving no radiation. Furthermore the timescales involved would allow the process to be considered an outpatient procedure such that a patient can be diagnosed and treated on the same day using the same device. Various geometries of transducer were investigated for physical compatibility as well as the ability to target the entire specified volume, based on the dimensions of the existing system. Simulations were performed using a custom written code based on Huygen’s principle, allowing minimum surface area and power requirements to be determined and feasibility of designs to be evaluated. The use of phase differences in the excitation signals applied to individual elements was also investigated, thus the effect of steering the simulated focus could be observed, an important factor to consider when attempting to incorporate a transducer into a device with restricted dimensions. Resulting simulated pressure fields were used to obtain acoustic intensity fields, which could then be used as inputs in the Pennes Bio-Heat Transfer Equation (BHTE) allowing temperature distributions to be observed. Preliminary studies proved the feasibility of using the suggested transducer design in conjunction with the existing EIM system. Pressure fields and heating patterns were all within acceptable limits, confirming the ability of the device to effectively ablate cancerous tissue. Additionally the capability to steer the resultant focal point was validated, and a thermal dose model was implemented allowing different heating patterns to be quantitatively compared

How sonoporation disrupts cellular structural integrity: morphological and cytoskeletal observations

Posters: no. 1Control ID: 1672429OBJECTIVES: In considering sonoporation for drug delivery applications, it is essential to understand how living cells respond to this puncturing force. Here we seek to investigate the effects of sonoporation on cellular structural integrity. We hypothesize that the membrane morphology and cytoskeletal behavior of sonoporated cells under recovery would inherently differ from that of normal viable cells. METHODS: A customized and calibrated exposure platform was developed for this work, and the ZR-75-30 breast carcinoma cells were used as the cell model. The cells were exposed to either single or multiple pulses of 1 MHz ultrasound (pulse length: 30 or 100 cycles; PRF: 1kHz; duration: up to 60s) with 0.45 MPa spatial-averaged peak negative pressure and in the presence of lipid-shelled microbubbles. Confocal microscopy was used to examine insitu the structural integrity of sonoporated cells (identified as ones with exogenous fluorescent marker internalization). For investigations on membrane morphology, FM 4-64 was used as the membrane dye (red), and calcein was used as the sonoporation marker (green); for studies on cytoskeletal behavior, CellLight (green) and propidium iodide (red) were used to respectively label actin filaments and sonoporated cells. Observation started from before exposure to up to 2 h after exposure, and confocal images were acquired at real-time frame rates. Cellular structural features and their temporal kinetics were quantitatively analyzed to assess the consistency of trends amongst a group of cells. RESULTS: Sonoporated cells exhibited membrane shrinkage (decreased by 61% in a cell’s cross-sectional area) and intracellular lipid accumulation (381% increase compared to control) over a 2 h period. The morphological repression of sonoporated cells was also found to correspond with post-sonoporation cytoskeletal processes: actin depolymerization was observed as soon as pores were induced on the membrane. These results show that cellular structural integrity is indeed disrupted over the course of sonoporation. CONCLUSIONS: Our investigation shows that the biophysical impact of sonoporation is by no means limited to the induction of membrane pores: e.g. structural integrity is concomitantly affected in the process. This prompts the need for further fundamental studies to unravel the complex sequence of biological events involved in sonoporation.postprin

Developmental delays and subcellular stress as downstream effects of sonoporation

Posters: no. 2Control ID: 1672434OBJECTIVES: The biological impact of sonoporation has often been overlooked. Here we seek to obtain insight into the cytotoxic impact of sonoporation by gaining new perspectives on anti-proliferative characteristics that may emerge within sonoporated cells. We particularly focused on investigating the cell-cycle progression kinetics of sonoporated cells and identifying organelles that may be stressed in the recovery process. METHODS: In line with recommendations on exposure hardware design, an immersion-based ultrasound platform has been developed. It delivers 1 MHz ultrasound pulses (100 cycles; 1 kHz PRF; 60 s total duration) with 0.45 MPa peak negative pressure to a cell chamber that housed HL-60 leukemia cells and lipid-shelled microbubbles at a 10:1 cell-tobubble ratio (for 1e6/ml cell density). Calcein was used to facilitate tracking of sonoporated cells with enhanced uptake of exogenous molecules. The developmental trend of sonoporated cells was quantitatively analyzed using BrdU/DNA flow cytometry that monitors the cell population’s DNA synthesis kinetics. This allowed us to measure the temporal progression of DNA synthesis of sonoporated cells. To investigate whether sonoporation would upset subcellular homeostasis, post-exposure cell samples were also assayed for various proteins using Western blot analysis. Analysis focus was placed on the endoplasmic reticulum (ER): an important organelle with multi-faceted role in cellular functioning. The post-exposure observation time spanned between 0-24 h. RESULTS: Despite maintaining viability, sonoporated cells were found to exhibit delays in cell-cycle progression. Specifically, their DNA synthesis time was lengthened substantially (for HL-60 cells: 8.7 h for control vs 13.4 h for the sonoporated group). This indicates that sonoporated cells were under stress: a phenomenon that is supported by our Western blot assays showing upregulation of ER-resident enzymes (PDI, Ero1), ER stress sensors (PERK, IRE1), and ER-triggered pro-apoptotic signals (CHOP, JNK). CONCLUSIONS: Sonoporation, whilst being able to facilitate internalization of exogenous molecules, may inadvertently elicit a cellular stress response. These findings seem to echo recent calls for reconsideration of efficiency issues in sonoporation-mediated drug delivery. Further efforts would be necessary to improve the efficiency of sonoporation-based biomedical applications where cell death is not desirable.postprin

A study on the change in plasma membrane potential during sonoporation

Posters: no. 4Control ID: 1680329OBJECTIVES: There has been validated that the correlation of sonoporation with calcium transients is generated by ultrasound-mediated microbubbles activity. Besides calcium, other ionic flows are likely involved in sonoporation. Our hypothesis is the cell electrophysiological properties are related to the intracellular delivery by ultrasound and microbubbles. In this study, a real-time live cell imaging platform is used to determine whether plasma membrane potential change is related to the sonoporation process at the cellular level. METHODS: Hela cells were cultured in DMEM supplemented with 10% FBS in Opticell Chamber at 37 °C and 5% CO2, and reached 80% confluency before experiments. The Calcein Blue-AM, DiBAC4(3) loaded cells in the Opticell chamber filled with PI solution and Sonovue microbubbles were immerged in a water tank on a inverted fluorescence microscope. Pulsed ultrasound (1MHz freq., 20 cycles, 20Hz PRF, 0.2-0.5MPa PNP) was irradiated at the angle of 45° to the region of interest for 1s.The real-time fluorescence imaging for different probes was acquired by a cooled CCD camera every 20s for 10min. The time-lapse fluorescence images were quantitatively analyzed to evaluate the correlation of cell viability, intracellular delivery with plasma membrane potential change. RESULTS: Our preliminary data showed that the PI fluorescence, which indicated intracellular delivery, was immediately accumulated in cells adjacent to microbubbles after exposure, suggesting that their membranes were damaged by ultrasound-activated microbubbles. However, the fluorescence reached its highest level within 4 to 6 minutes and was unchanged thereafter, indicating the membrane was gradually repaired within this period. Furthermore, using DIBAC4(3), which detected the change in the cell membrane potential, we found that the loss of membrane potential might be associated with intracellular delivery, because the PI fluorescence accumulation was usually accompanied with the change in DIBAC4 (3) fluorescence. CONCLUSIONS: Our study suggests that there may be a linkage between the cell membrane potential change and intracellular delivery mediated by ultrasound and microbubbles. We also suggest that other ionic flows or ion channels may be involved in the cell membrane potential change in sonoporation. Further efforts to explore the cellular mechanism of this phenomenon will improve our understanding of sonoporation.postprin

Real-time imaging of cellular dynamics during low-intensity pulsed ultrasound exposure

Control ID: 1671584Oral Session 5 - Bioeffects of therapeutic ultrasoundOBJECTIVE: Although the therapeutic potential of low-intensity pulsed ultrasound is unquestionable, the wave-matter interactions involved in the process remain to be vaguely characterized. Here we seek to undertake a series of in-situ cellular imaging studies that aim to analyze the mechanical impact of low-intensity pulsed ultrasound on attached fibroblasts from three different aspects: membrane, cytoskeleton, and nucleus. METHODS: Our experimental platform comprised an in-house ultrasound exposure hardware that was coupled to a confocal microscopy system. The waveguided ultrasound beam was geometrically aligned to the microscope’s fieldof-view that corresponds to the center of a polystyrene dish containing fibroblasts. Short ultrasound pulses (5 cycles; 2 kHz PRF) with 0.8 MPa peak acoustic pressure (0.21 W/cm2 SPTA intensity) were delivered over a 10 min period. Live imaging was performed on both membrane (CellMask) and cytoskeleton (actin-GFP, tubulin-RFP) over the entire observation period (up to 30 min after end of exposure). Also, pre- and post-exposure fixed-cell imaging was conducted on the nucleus (Hoechst 33342) and two cytoskeleton components related to stress fibers: F-actin (phalloidin-FITC) and vincullin (Alexa Fluor 647 conjugated). To study whether mechanotransduction was responsible in mediating ultrasound-cell interactions, some experiments were conducted with the addition of gadolinium that blocks stretch-sensitive ion channels. RESULTS: Cell shrinkage was evident over the course of low-intensity pulsed ultrasound exposure. This was accompanied with contraction of actin and tubulin. Also, an increase in central stress fibers was observed at the end of exposure, while the nucleus was found to have decreased in size. Interestingly, after the exposure, a significant rebound in cell volume was observed over a 30 min. period. These effects were not observed in cases with gadolinium blockage of mechanosensitive ion channels. CONCLUSIONS: Our results suggest that low-intensity pulsed ultrasound would transiently induce remodeling of a cell’s membrane and cytoskeleton, and it will lead to repression of nucleus. This indicates that ultrasound after all represents a mechanical stress on cellular membrane. The post-exposure outgrowth phenomenon is also of practical relevance as it may be linked to the stimulatory effects that have been already observed in low-intensity pulsed ultrasound treatments.postprin

The use of ultrasound to create tissue hyperthermia to support the treatment of cancer

The value of mild hyperthermia in improving the outcome of radiotherapy and chemotherapy treatments is well established. However, clinical applications are currently restricted to accessible tumours, with the application of controlled hyperthermia in solid tumours deep within the body presenting an unresolved problem. Ultrasound is an attractive heating technique because of its ability to create a focus at depth which can be steered around the tumour volume. However, despite considerable research no clinically usable transducers for deep tumour applications have resulted. In this thesis the underlying principles that govern the characteristics of phased array transducers have been examined. The concept of an idealised phased array has been introduced, and analysis of simulated fields from such arrays has enabled a new set of equations to be defined which relate the geometry of the field to the fundamental array design parameters (including the array diameter, radius of curvature and frequency of operation). Further simulations have examined the impact of secondary array design parameters (such as the individual element size, number density and layout geometry) which modify the field from that of the idealised case. Analysis of these has enabled an upper limit to be placed on the element size within any planar array in order to prevent undesirable changes in the characteristics of the focal region. A fifteen element phased array with a random element distribution has been constructed based on the design principles established in the simulation work. Measurements of the inter-element cross-coupling have been made, demonstrating that acoustic coupling dominated for inter-element pitches of less than 8 mm, while electrical coupling dominated at larger inter-element pitches. The field produced by the array in an acoustic tank has been characterised and compared against simulation predictions, showing good agreement in terms of the geometries of the focal region and the grating lobes. However, a number of differences have also been identified. In particular, the focal region was closer to the surface of the physical transducer in the measured fields compared to the simulation results, and there were numerous small high intensity regions between the surface of the transducer and the focus which were absent from the simulated fields. A sensitivity analysis, using a simulated factorial experiment, has been performed to identify the origin of these differences, with the results indicating that the presence of a secondary vibrational mode within the elements of the array was the principal causative factor. Finally, calculations have been performed which demonstrate the feasibility of manufacturing an array suitable for the application of mild hyperthermia in deep tumours based on the array design scheme presented in this thesis. Potential extensions of the array design have also been described which would improve the behaviour of the array under steering and provide further increase in the focal intensity

Cavitation methods in therapeutic ultrasound : techniques, mechanisms, and system design

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, February 2004.Includes bibliographical references (leaves 134-151).Focused ultrasound is currently being developed as a non-invasive thermal ablation technique for benign and cancerous tumors in several organ systems. Although these therapies are designed to ablate tissue purely by thermal means, cavitation, the formation and collapse of gas bubbles, can occur. These bubbles can be unpredictable in their timing and location and often interfere with thermal therapies. Therefore, focused ultrasound techniques have tried to avoid bubbles and their effects. However, gas bubbles in vivo have some potential useful features for therapy. They greatly enhance local ultrasound absorption, and can on their own induce mechanical damage to the tissue. In addition, bubble clouds can block ultrasound wave propagation, providing a means to protect vital tissues during ablation of nearby pathology. If induced and controlled properly, cavitation in focused ultrasound therapy could potentially be very beneficial. The first aim of this research is to design and test in vivo ultrasound exposures that induce cavitation at appropriate times and take advantage of their absorption enhancing properties. In addition, methods to monitor and control cavitation induction and the associated therapy will be investigated. Second, a theoretical bubble model and acoustic field simulations will be used to design optimal pressure fields which very tightly control the cavitation location. These models will also be used to investigate methods for reducing the acoustic powers needed to induce cavitation while preventing off focus cavitation. For the final phase of the research a multi-channel, multi-frequency ultrasound amplifier system capable of delivering optimal exposures via large scale phased array systems will be developed and tested. In(cont.) total, the thesis research will justify applications for cavitation in ultrasound therapy, and develop the technology and methodology to optimally use cavitation and monitor its effects in vivo.by Shunmugavelu D. Sokka.Ph.D

Spatial control of cavitation in therapeutic ultrasound

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2005.Includes bibliographical references (p. 60-65).Inertial cavitation has been implicated as the primary mechanism for a host of emerging applications. In all these applications, the main concern is to induce cavitation in perfectly controlled locations in the field; this means specifically to be able to achieve cavitation threshold at the geometrical focus of the transducer without stimulating its near field. In this study, we make use of dual-frequency methods to preferentially lower the cavitation threshold at the focus relative to the rest of the field. One family of dual-frequency driving waveforms is evaluated in a bubble model incorporating rectified diffusion. Theoretical predictions based on Sokka's work (Sokka 2003a) are confirmed in vitro using Optison[TM], a commercially available contrast agent. The performance of the rest of acoustic field in suppressing cavitation when cavitation is induced at the focus is investigated theoretically and checked experimentally. This first part shows that dual-frequency phased arrays could be used to precisely control cavitation. Cavitation threshold is proved to be 1.2 times higher in the near field than at the focus. One of the main limitations of the aforementioned protocol is that it is tightly controlled. As an example, Optison[TM] has a mean bubble size of 2 - 4.5 [micro]m, which means that the initial bubble radii will fall in this range. Since cavitation threshold has been proved to depend on this parameter, using ultrasound contrast agents allows for more predictable results. Therefore, in the second half of this study, we propose a focused ultrasound protocol that induces and monitors gas bubbles at the focus and allows for ex vivo validation of the aforementioned theoretical results. The experiments involve fresh rabbit tissue and a statistical analysis is performed over data collected from back muscle.(cont.) Moreover, the experimental apparatus is designed to be MRI-compatible to make future in vivo assessments feasible. This second half of the study demonstrates that the theoretical predictions made earlier can reliably be used to predict dual-frequency cavitation thresholds. It also suggests that clinical use of dual-frequency excitations might be a solution to the problem of spatial control of cavitation.by Thomas P. Gauthier.S.M