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One of the challenges of transcutaneous high-intensity focused ultrasound (HIFU) therapies, especially ones relying heavily on shock formation, such as boiling histotripsy (BH), is the loss of focusing from aberration induced by the heterogeneities of the body wall. Here, a methodology to execute aberration correction in vivo is proposed. A custom BH system consisting of a 1.5-MHz phased array of 256 elements connected to a Verasonics V1 system is used in pulse/echo mode on a porcine model under general anesthesia. Estimation of the time shifts needed to correct for aberration in the liver and kidney is done by maximizing the value of the coherence factor on the acquired backscattered signals. As this process requires multiple pulse/echo sequences on a moving target to converge to a solution, tracking is also implemented to ensure that the same target is used between each iteration. The method was validated by comparing the acoustic power needed to generate a boiling bubble at one target with aberration correction and at another target within a 5-mm radius without aberration correction. Results show that the aberration correction effectively lowers the acoustic power required to reach boiling by up to 45%, confirming that it indeed restored formation of the nonlinear shock front at the focus.

Supplementary Materials and Methods. Description of additional methods and procedures used in the study.

Supplementary Figure Legends. Legends for Supplementary Figures S1-S2.

Supplementary Table S2. Estimated regression coefficients from equation 2.

Supplementary Figure S1. A multispectral image of an in vivo murine subcutaneous pancreatic tumor treated with pHIFU with varying levels of peak-rarefactional pressure.

Pancreatic cancer is characterized by extensive stromal desmoplasia, which decreases blood perfusion and impedes chemotherapy delivery. Breaking the stromal barrier could both increase perfusion and permeabilize the tumor, enhancing chemotherapy penetration. Mechanical disruption of the stroma can be achieved using ultrasound-induced bubble activity—cavitation. Cavitation is also known to result in microstreaming and could have the added benefit of actively enhancing diffusion into the tumors. Here, we report the ability to enhance chemotherapeutic drug doxorubicin penetration using ultrasound-induced cavitation in a genetically engineered mouse model (KPC mouse) of pancreatic ductal adenocarcinoma. To induce localized inertial cavitation in pancreatic tumors, pulsed high-intensity focused ultrasound (pHIFU) was used either during or before doxorubicin administration to elucidate the mechanisms of enhanced drug delivery (active vs. passive drug diffusion). For both types, the pHIFU exposures that were associated with high cavitation activity resulted in disruption of the highly fibrotic stromal matrix and enhanced the normalized doxorubicin concentration by up to 4.5-fold compared with controls. Furthermore, normalized doxorubicin concentration was associated with the cavitation metrics (P < 0.01), indicating that high and sustained cavitation results in increased chemotherapy penetration. No significant difference between the outcomes of the two types, that is, doxorubicin infusion during or after pHIFU treatment, was observed, suggesting that passive diffusion into previously permeabilized tissue is the major mechanism for the increase in drug concentration. Together, the data indicate that pHIFU treatment of pancreatic tumors when resulting in high and sustained cavitation can efficiently enhance chemotherapy delivery to pancreatic tumors. Cancer Res; 75(18); 3738–46. ©2015 AACR.

Background: Upper urinary tract stones are increasingly prevalent in pet cats and are difficult to manage. Surgical procedures to address obstructing ureteroliths have short- and long-term complications, and medical therapies (e.g. fluid diuresis and smooth muscle relaxants) are infrequently effective. Burst wave lithotripsy is a non-invasive, ultrasound-guided, handheld focused ultrasound technology to disintegrate urinary stones, which is now undergoing human clinical trials in awake unanesthetized subjects. Results: In this study, we designed and performed in vitro testing of a modified burst wave lithotripsy system to noninvasively fragment stones in cats. The design accounted for differences in anatomic scale, acoustic window, skin-to-stone depth, and stone size. Prototypes were fabricated and tested in a benchtop model using 35 natural calcium oxalate monohydrate stones from cats. In an initial experiment, burst wave lithotripsy was performed using peak ultrasound pressures of 7.3 (n = 10), 8.0 (n = 5), or 8.9 MPa (n =10) for up to 30 minutes. Fourteen of 25 stones fragmented to in vivo exposure of kidneys and ureters in a porcine animal model. Conclusions: These data support further evaluation of burst wave lithotripsy as a noninvasive intervention for obstructing ureteroliths in cats.

Boiling histotripsy (BH) is a focused ultrasound technology that uses millisecond-long pulses with shock fronts to induce mechanical tissue ablation. The pulsing scheme and mechanisms of BH differ from those of cavitation cloud histotripsy, which was previously developed for benign prostatic hyperplasia. The goal of the work described here was to evaluate the feasibility of using BH to ablate fresh ex vivo human prostate tissue as a proof of principle for developing BH for prostate applications. Fresh human prostate samples (N = 24) were obtained via rapid autopsy (24 h after death, institutional review board exempt). Samples were analyzed using shear wave elastography to ensure that mechanical properties of autopsy tissue were clinically representative. Samples were exposed to BH using 10- or 1-ms pulses with 1% duty cycle under real-time B-mode and Doppler imaging. Volumetric lesions were created by sonicating 1-4 rectangular planes spaced 1 mm apart, containing a grid of foci spaced 1-2 mm apart. Tissue then was evaluated grossly and histologically, and the lesion content was analyzed using transmission electron microscopy and scanning electron microscopy. Observed shear wave elastography characterization of ex vivo prostate tissue (37.9 ± 22.2 kPa) was within the typical range observed clinically. During BH, hyperechoic regions were visualized at the focus on B-mode, and BH-induced bubbles were also detected using power Doppler. As treatment progressed, hypoechoic regions of tissue appeared, suggesting successful tissue fractionation. BH treatment was twofold faster using shorter pulses (1 ms vs. 10 ms). Histological analysis revealed lesions containing completely homogenized cell debris, consistent with histotripsy-induced mechanical ablation. It was therefore determined that BH is feasible in fresh ex vivo human prostate tissue producing desired mechanical ablation. The study supports further work aimed at translating BH technology as a clinical option for prostate ablation.

Despite the growing incidence of urinary stones, the fundamental interventions for urinary stones have remained the same for several decades. However, technical innovations in ultrasound have enabled new strategies for stone management. Our team aims to employ these methods to establish a new paradigm to treat stones in an office or clinic rather than a surgical suite. Technologies include new imaging methods to detect and characterize stones, burst wave lithotripsy to fragment stones using focused ultrasound rather than shock waves, and ultrasonic propulsion to promote clearance of fragments, all using a single ultrasound platform. This presentation will describe the principles and development of these technologies, as well as the fruitful partnerships between teams of basic scientists, engineers, physicians, and industry that have enabled rapid development of these concepts to successful clinical trials. [Work supported by NIDDK P01 DK043881 and the Focused Ultrasound Foundation.]

Pulsed focused ultrasound (pFUS) exposures utilizing short, nonlinearly distorted pulses at low duty cycle have been shown to enhance drug and gene delivery to targeted tissue through inertial cavitation activity. Passive cavitation detection (PCD) and mapping of broadband emissions are current conventional methods to monitor and quantify cavitation but provide limited spatial resolution. Here, plane-wave Doppler imaging was used with PCD to quantify pFUS-induced cavitation in ex vivo bovine tissues and in vivo surgically exposed porcine liver, kidney, and pancreas. A 1.5 MHz FUS transducer (aperture 75 mm, F-number 0.75) was used to deliver 60 pulses (duration 1 ms, 0.1% duty cycle, focal pressure p+ = 70i–110 MPa, p− = 13–20 MPa). A coaxially mounted ATL P7-4 ultrasound imaging probe was used for PCD during the FUS pulse, and Doppler and B-mode sequences. Disrupted tissue areas were collected for histology and compared to Doppler power images. Maximum Doppler power was found to correlate to broadband noise level for each FUS pulse. The Doppler power map integrated over the exposure was observed to correlate spatially with tissue disruption area from histology, which thus represents a promising real-time metric for quantifying cavitation activity induced by pFUS exposures. [Work supported by NIH R01CA154451, R01EB025187, and R01EB23910.]

Tissue-mimicking gels provide a cost-effective medium to optimize histotripsy treatment parameters with immediate feedback. Agarose and polyacrylamide gels are often used to evaluate treatment outcomes as they mimic the acoustic properties and stiffness of a variety of soft tissues, but they do not exhibit high toughness, a characteristic of fibrous connective tissue. To mimic pathologic fibrous tissue found in benign prostate hyperplasia (BPH) and other diseases that are potentially treatable with histotripsy, an optically transparent hydrogel with high toughness was developed that is a hybrid of polyacrylamide and alginate. The stiffness was established using shear wave elastography (SWE) and indentometry techniques and was found to be representative of human BPH ex vivo prostate tissue. Different phantom compositions and excised ex vivo BPH tissue samples were treated with a 700-kHz histotripsy transducer at different pulse repetition frequencies. Post-treatment, the hybrid gels and the tissue samples exhibited differential reduction in stiffness as measured by SWE. On B-mode ultrasound, partially treated areas were present as hyperechoic zones and fully liquified areas as hypoechoic zones. Phase contrast microscopy of the gel samples revealed liquefaction in regions consistent with the target lesion dimensions and correlated to findings identified in tissue samples via histology. The dose required to achieve liquefaction in the hybrid gel was similar to what has been observed in ex vivo tissue and greater than that of agarose of comparable or higher Young's modulus by a factor10. These results indicate that the developed hydrogels closely mimic elasticities found in BPH prostate ex vivo tissue and have a similar response to histotripsy treatment, thus making them a useful cost-effective alternative for developing and evaluating different treatment protocols.

Inertial cavitation induced by pulses of nonlinearly distorted focused ultrasound (FUS) at moderate intensity can result in mild mechanical disruption of tissue, short of its complete mechanical disintegration—histotripsy. This effect can be used to enhance diffusion of subsequently systemically administered drugs or biologics. Previously, single-element passive cavitation detection (PCD) of broadband noise emissions was successfully used to quantify tissue disruption and enhancement in drug concentration. This metric, however, has limitations: minimal spatial resolution and challenges with calibration when used at large depths in acoustically variable tissue. To address this, we developed a combination of passive and active Doppler-based methods that relied on relative, rather than absolute signal metrics. Specifically, destructive cavitation behaviors were previously linked to substantial motion of the bubbles during the FUS pulse due to acoustic radiation force and shock scattering. This was reflected in the backscattered FUS harmonics as Doppler shift and measurable from PCD. The bubbles were observed to dissolve within milliseconds following each FUS pulse, thus their distribution could be visualized as an area of rapid change via fast plane wave Doppler ensemble following the FUS pulse. The Doppler power distribution was spatially correlated with the area of tissue disruption in the in vivo experiments. [Work supported by NIH R01CA154451, R01EB025187, and R01EB23910.]

Abscesses are infected walled-off collections of pus and bacteria. They can affect any part of the body. Current treatment is typically limited to antibiotics, catheter drainage and hospitalization, or surgical wash-out. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. Histotripsy generates localized cavitation without heating and represents a potential new noninvasive treatment modality. Successful histotripsy treatments were demonstrated with gram-negative E-coli in both in vitro and in vivo studies [Brayman et al., UMB 44, 1996–2008 (2018); Matula et al., UMB 47, 603–619 (2020)]. Interestingly, preliminary data showed that boiling histotripsy was less effective than cavitation histotripsy in killing bacteria. In this talk, we will describe more in-depth studies that compare cavitation histotripsy (low number of cycles, high-pulse repetition rate) with boiling histotripsy (high number of cycles, low-pulse repetition rate) on inactivation rates of microbes. [Work funded in part by NIH NIBIB No. R01EB019365.]

Pancreatic ductal adenocarcinomas are characterized by a complex and robust tumor microenvironment (TME) consisting of fibrotic tissue, excessive levels of hyaluronan (HA), and immune cells. We utilized quantitative multi-parametric magnetic resonance imaging (mp-MRI) methods at 14 Tesla in a genetically engineered KPC (KrasLSL-G12D/+, Trp53LSL-R172H/+, Cre) mouse model to assess the complex TME in advanced stages of tumor development. The whole tumor, excluding cystic areas, was selected as the region of interest for data analysis and subsequent statistical analysis. Pearson correlation was used for statistical inference. There was a significant correlation between tumor volume and T2 (r = −0.66), magnetization transfer ratio (MTR) (r = 0.60), apparent diffusion coefficient (ADC) (r = 0.48), and Glycosaminoglycan-chemical exchange saturation transfer (GagCEST) (r = 0.51). A subset of mice was randomly selected for histological analysis. There were positive correlations between tumor volume and fibrosis (0.92), and HA (r = 0.76); GagCEST and HA (r = 0.81); and MTR and CD31 (r = 0.48). We found a negative correlation between ADC low-b (perfusion) and Ki67 (r = −0.82). Strong correlations between mp-MRI and histology results suggest that mp-MRI can be used as a non-invasive tool to monitor the tumor microenvironment.