Development of a Harmonic Motion Imaging guided Focused Ultrasound system for breast tumor characterization and treatment monitoring

Breast cancer is the most common cancer and the second leading cause of cancer death among women. About 1 in 8 U.S. women (about 12%) will develop invasive breast cancer over the course of their lifetime. Existing methods of early detection of breast cancer include mammography and palpation, either by patient self-examination or clinical breast exam. Palpation is the manual detection of differences in tissue stiffness between breast tumors and normal breast tissue. The success of palpation relies on the fact that the stiffness of breast tumors is often an order of magnitude greater than that of normal breast tissue, i.e., breast lesions feel ''hard'' or ''lumpy'' as compared to normal breast tissue. A mammogram is an x-ray that allows a qualified specialist to examine the breast tissue for any suspicious areas. Mammography is less likely to reveal breast tumors in women younger than 50 years with denser breast than in older women. When a suspicious site is detected in the breast through a breast self-exam or on a screening mammogram, the doctor may request an ultrasound of the breast tissue. A breast ultrasound can provide evidence about whether the lump is a solid mass, a cyst filled with fluid, or a combination of the two. An invasive needle biopsy is the only diagnostic procedure that can definitely determine if the suspicious area is cancerous. In the clinic, 80% of women who have a breast biopsy do not have breast cancer. Most women with breast cancer diagnosed will have some type of surgery to remove the tumor. Depending on the type of breast cancer and how advanced it is, the patient might need other types of treatment as well, such as chemotherapy and radiation therapy. Image-guided minimally-invasive treatment of localized breast tumor as an alternative to traditional breast surgery, such as high intensity focused ultrasound (HIFU) treatment, has become a subject of intensive research. HIFU applies extreme high temperatures to induce irreversible cell injury, tumor apoptosis and coagulative necrosis. Compared with conventional surgical procedures the main advantages of HIFU ablation lie in the fact that it is non-invasive, less scarring and less painful, allowing for shorter recovery time. HIFU can be guided by MRI (MRgFUS) or by conventional diagnostic ultrasound (USgFUS). Worldwide, thousands of patients with uterine fibroids, liver cancer, breast cancer, pancreatic cancer, bone tumors, and renal cancer have been treated by USgFUS. In this dissertation, the objective is to develop an integrated Harmonic Motion Imaging guided Focused Ultrasound (HMIgFUS) system as a clinical monitoring technique for breast HIFU with the added capability of detecting tumors for treatment planning, evaluation of tissue stiffness changes during HIFU ablation for treatment monitoring in real time, and assessment of thermal lesion sizes after treatment evaluation. A new HIFU treatment planning method was described that used oscillatory radiation force induced displacement amplitude variations to detect the HIFU focal spot before lesioning. Using this method, we were able to visualize the HMIgFUS focal region at variable depths. By comparing the estimated displacement profiles with lesion locations in pathology, we demonstrated the feasibility of using this HMI-based technique to localize the HIFU focal spot and predict lesion location during the planning phase. For HIFU monitoring, a HIFU lesion detection and ablation monitoring method was first developed using oscillatory radiation force induced displacement amplitude variations in real time. Using this method, the HMIgFUS focal region and lesion formation were visualized in real time at a feedback rate of 2.4 Hz. By comparing the estimated lesion size against gross pathology, the feasibility of using HMIgFUS to monitor treatment and lesion formation without interruption is demonstrated. In order to reduce the imaging time, it is shown in this dissertation that using the steered FUS beam, HMI can be used to image a 2.3 times larger ROI without requiring physical movement of the transducer. Using steering for HMI can be used to shorten the total imaging duration without requiring physical movement of the transducer. For the application of breast tumor, HMI and HMIgFUS were optimized and applied to ex vivo breast tissue. The results showed that HMI is experimentally capable of mapping and differentiating stiffness in normal and abnormal breast tissues. HMIgFUS can also successfully generate thermal lesions on normal and pathological breast tissues. HMI has also been applied to post-surgical breast mastectomy specimens to mimic the in vivo environment. In the end, the first HMI clinical system has been built with added capability of GUP-based parallel beamforming. A clinical trial has been approved at Columbia University to image breast tumor on patient. The HMI clinical system has shown to be able to map fibroadenoma mass on two patients with valid HMI displacement. The study in this dissertation may yield an early-detection technique for breast cancer without any age discrimination and thus, increase the survival rate

In Vivo Feasibility of Real-Time Monitoring of Focused Ultrasound Surgery (FUS) Using Harmonic Motion Imaging (HMI)

Abstract-In this study, the Harmonic Motion Imaging for Focused Ultrasound (HMIFU) technique is applied to monitor changes in mechanical properties of tissues during thermal therapy in a transgenic breast cancer mouse model in vivo. An HMIFU system, composed of a 4.5-MHz focused ultrasound (FUS) and a 3.3-MHz phased-array imaging transducer, was mechanically moved to image and ablate the entire tumor. The FUS transducer was driven by an amplitude-modulated (AM) signal at 15 Hz. The acoustic intensity (I sp ta ) was equal to 1050 W/cm 2 at the focus. A digital low-pass filter was used to filter out the spectrum of the FUS beam and its harmonics prior to displacement estimation. The resulting axial displacement was estimated using 1-D crosscorrelation on the acquired RF signals. Results from two mice with eight lesions formed in each mouse (16 lesions total) showed that the average peak-to-peak displacement amplitude before and after lesion formation was respectively equal to 17.34 ± 1.34 µm and 10.98 ± 1.82 µm (p < 0.001). Cell death was also confirmed by hematoxylin and eosin histology. HMI displacement can be used to monitor the relative tissue stiffness changes in real time during heating so that the treatment procedure can be performed in a timeefficient manner. The HMIFU system may, therefore, constitute a cost-efficient and reliable alternative for real-time monitoring of thermal ablation. Index Terms-Acoustic radiation force, breast cancer, focused ultrasound surgery (FUS), harmonic motion imaging, highintensity focused ultrasound (HIFU), in vivo, monitoring, noninvasive estimation, tissue ablation, ultrasound

Harmonic Motion Imaging for Abdominal Tumor Detection and High-Intensity Focused Ultrasound Ablation Monitoring: An In Vivo Feasibility Study in a Transgenic Mouse Model of Pancreatic Cancer

Abstract-Harmonic motion imaging (HMI) is a radiationforce-based elasticity imaging technique that tracks oscillatory tissue displacements induced by sinusoidal ultrasonic radiation force to assess the resulting oscillatory displacement denoting the underlying tissue stiffness. The objective of this study was to evaluate the feasibility of HMI in pancreatic tumor detection and high-intensity focused ultrasound (HIFU) treatment monitoring. The HMI system consisted of a focused ultrasound transducer, which generated sinusoidal radiation force to induce oscillatory tissue motion at 50 Hz, and a diagnostic ultrasound transducer, which detected the axial tissue displacements based on acquired radio-frequency signals using a 1-D cross-correlation algorithm. For pancreatic tumor detection, HMI images were generated for pancreatic tumors in transgenic mice and normal pancreases in wild-type mice. The obtained HMI images showed a high contrast between normal and malignant pancreases with an average peak-to-peak HMI displacement ratio of 3.2. Histological analysis showed that no tissue damage was associated with HMI when it was used for the sole purpose of elasticity imaging. For pancreatic tumor ablation monitoring, the focused ultrasound transducer was operated at a higher acoustic power and longer pulse length than that used in tumor detection to simultaneously induce HIFU thermal ablation and oscillatory tissue displacements, allowing HMI monitoring without interrupting tumor ablation. HMI monitoring of HIFU ablation found significant decreases in the peak-to-peak HMI displacements before and after HIFU ablation with a reduction rate ranging from 15.8% to 57.0%. The formation of thermal lesions after HIFU exposure was confirmed by histological analysis. This study demonstrated the feasibility of HMI in abdominal tumor detection and HIFU ablation monitoring

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

Biomechanical Assessment and Monitoring of Thermal Ablation Using Harmonic Motion Imaging for Focused Ultrasound (HMIFU)

Cancer remains, one of the major public health problems in the United States as well as many other countries worldwide. According to the World Health Organization, cancer is currently the leading cause of death worldwide, accounting for 7.6 million deaths annually, and 25% of the annual death was due to Cancer during the year of 2011. In the long history of the cancer treatment field, many treatment options have been established up to date. Traditional procedures include surgical procedures as well as systemic therapies such as biologic therapy, chemotherapy, hormone therapy, and radiation therapy. Nevertheless, side-effects are often associated with such procedures due to the systemic delivery across the entire body. Recently technologies have been focused on localized therapy under minimally or noninvasive procedure with imaging-guidance, such as cryoablation, laser ablation, radio‐frequency (RF) ablation, and High Intensity F-ocused Ultrasound (HIFU). HIFU is a non-invasive procedure aims to coagulate tissue thermally at a localized focal zone created with noninvasively emitting a set of focused ultrasound beams while the surrounding healthy tissues remain relatively untreated. Harmonic Motion Imaging for Focused Ultrasound (HMIFU) is a dynamic, radiation-force-based imaging technique, which utilizes a single HIFU transducer by emitting an Amplitude-modulated (AM) beam to both thermally ablate the tumor while inducing a stable oscillatory tissue displacement at its focal zone. The oscillatory response is then estimated by a cross-correlation based motion tracking technique on the signal collected by a confocally-aligned diagnostic transducer. HMIFU addresses the most critical aspect and one of the major unmet needs of HIFU treatment, which is the ability to perform real-time monitoring and mapping of tissue property change during the HIFU treatment. In this dissertation, both the assessment and monitoring aspects of HMIFU have been investigated fundamentally and experimentally through development of both a 1-D and 2-D based system. The performance assessment of HMIFU technique in depicting the lesion size increase as well as the lesion-to-background displacement contrast was first demonstrated using a 3D, FE-based interdisciplinary simulation framework. Through the development of 1-D HMIFU system, a multi-parametric monitoring approach was presented where presented where the focal HMI displacement, phase shift (Δφ), and correlation coefficients were monitored along with thermocouple and PCD under the HIFU treatment sequence with boiling and slow denaturation. For HIFU treatments with slow denaturation, consistent displacement increase-then-decrease trend was observed, indicating tissue softening-then-stiffening and phase shift increased with treatment time in agreement with mechanical testing outcomes. The correlation coefficient remained high throughout the entire treatment time under a minimized broadband energy and boiling mechanism. Contrarily, both displacement and phase shift changes lacked consistency under HIFU treatment sequences with boiling due to the presence of strong boiling mechanism confirmed by both PCD and thermocouple monitoring. In order to facilitate its clinical translation, a fully-integrated, clinically 2D real-time HMIFU system was also developed, which is capable of providing 2D real-time streaming during HIFU treatment up to 15 Hz without interruption. Reproducibility studies of the system showed consistent displacement estimation on tissue-mimicking phantoms as well as monitoring of tissue-softening-then-stiffening phase change across 16 out of 19 liver specimens (Increasing rate in phase shift (Δφ): 0.73±0.69 %/s, Decreasing rate in phase shift (Δφ): 0.60±0.19 %/s) along with thermocouple monitoring (Increasing: 0.84±1.15 %/ °C, Decreasing: 2.03± 0.93%/ °C) and validation of tissue stiffening using mechanical testing. In addition, the 2-D HMIFU system feasibility on preclinical pancreatic tumor mice model was also demonstrated in vivo, where HMI displacement decreases were observed across three of five treatment locations on the kP(f)c model at 20.8±6.84, 18.6±1.46, and 24.0±5.43%, as well as across four of the seven treatment locations on the KPC model at 39.5±2.98%, 34.5±21.5%, 16.0±3.05%, and 35.0±3.12% along with H and E histological confirmation. In order to improve the quantitative monitoring aspect of HMIFU, a novel, model-independent method for the estimating Young's modulus based on strain profile was also implemented, where 1-D HMIFU system showed feasibilities on polyacrylamide phantom (EHMI/E ≈ 2.3) and liver specimen (EHMI/E ≈ 8.1), and 2-D HMIFU system showed feasibilities on copolymer phantom(EHMI/E ≈ 30.4), liver specimen(EHMI/E ≈ 211.3), as well as HIFU treated liver specimen (EHMI,end/EHMI,beginning ≈ 5.96). In conclusion, the outcomes from the aforementioned studies successfully showed the feasibility of both HMIFU systems in multi-parametric monitoring of HIFU treatment with slow denaturation and boiling, which prepares its stage towards clinical translation