Safety and efficacy of histotripsy delivery through overlying gas-filled small bowel in an ex vivo swine model.

PURPOSE: To evaluate the safety and efficacy of performing histotripsy through overlying gas-filled bowel in an ex vivo swine model. METHODS: An ex vivo model was created to simulate histotripsy treatment of solid organs through gas-filled bowel. Spherical 2.5 cm histotripsy treatments were performed in agar phantoms for each of five treatment groups: 1) control with no overlying bowel (n = 6), 2) bowel 0 cm above phantom (n = 6), 3) bowel 1 cm above phantom (n = 6), 4) bowel 2 cm above phantom (n = 6), and 5) bowel 0 cm above the phantom with increased treatment amplitude (n = 6). Bowel was inspected for gross and microscopic damage, and treatment zones were measured. A ray-tracing simulation estimated the percentage of therapeutic beam path blockage by bowel in each scenario. RESULTS: All histotripsy treatments through partial blockage were successful (24/24). No visible or microscopic damage was observed to intervening bowel. Partial blockage resulted in a small increase in treatment volume compared to controls (p = 0.002 and p = 0.036 for groups with bowel 0 cm above the phantom, p > 0.3 for bowel 1 cm and 2 cm above the phantom). Gas-filled bowel was estimated to have blocked 49.6%, 35.0%, and 27.3% of the therapeutic beam at 0, 1, and 2 cm, respectively. CONCLUSION: Histotripsy has the potential to be applied through partial gas blockage of the therapeutic beam path, as shown by this ex vivo small bowel model. Further work in an in vivo survival model appears indicated.

Nanoparticle-Mediated Histotripsy Using Dual-Frequency Pulsing Methods.

OBJECTIVE: Nanoparticle-mediated histotripsy (NMH) is a novel ablation method that combines nanoparticles as artificial cavitation nuclei with focused ultrasound pulsing to achieve targeted, non-invasive, and cell-selective tumor ablation. The study described here examined the effect of dual-frequency histotripsy pulsing on the cavitation threshold, bubble cloud characteristics, and ablative efficiency in NMH. High-speed optical imaging was used to analyze bubble cloud characteristics and to measure ablation efficiency for NMH inside agarose tissue phantoms containing perfluorohexane-filled nanocone clusters, which were previously developed to reduce the histotripsy cavitation threshold for NMH. METHODS: Dual-frequency histotripsy pulsing was applied at a 1:1 pressure ratio using a modular 500 kHz and 3 MHz dual-frequency array transducer. Optical imaging results revealed predictable, well-defined bubble clouds generated for all tested cases with similar reductions in the cavitation thresholds observed for single-frequency and dual-frequency pulsing. RESULTS: Dual-frequency pulsing was seen to nucleate small, dense clouds in agarose phantoms, intermediate in size of their component frequencies but closer in area to that of the higher component frequency. Red blood cell experiments revealed complete ablations were generated by dual-frequency NMH in all phantoms in <1500 pulses. This result was a significant increase in ablation efficiency compared with the ∼4000 pulses required in prior single-frequency NMH studies. CONCLUSION: Overall, this study indicates the potential for using dual-frequency histotripsy methods to increase the ablation efficacy of NMH.

Ultrasound-Guided Mechanical High-Intensity Focused Ultrasound (Histotripsy) Through an Acoustically Permeable Polyolefin-Based Cranioplasty Device.

INTRODUCTION: Histotripsy is a non-thermal focused ultrasound therapy in development for the non-invasive ablation of cancerous tumors. Intracranial histotripsy has been limited by significant pressure attenuation through the skull, requiring large, complex array transducers to overcome this effect. OBJECTIVE: Recently, a biocompatible, polyolefin-based cranioplasty device was developed to allow ultrasound (US) transmission into the intracranial space with minimal distortion. In this study, we investigated the in vitro feasibility of applying US-guided histotripsy procedures across the prosthesis. METHODS: Pressure waveforms and beam profiles were collected for singleand multi-element histotripsy transducers. Then, high-speed optical images of the bubble cloud with and without the prosthesis were collected in water and tissue-mimicking agarose gel phantoms. Finally, red blood cell (RBC) tissue phantom and excised brain tissue experiments were completed to test the ablative efficacy across the prosthesis. RESULTS: Single element tests revealed increased pressure loss with increasing transducer frequency and increasing transducer-to-prosthesis angle. Array transducer measurements at 1 MHz showed average pressure losses of >50% across the prosthesis. Aberration correction recovered up to 18% of the pressure lost, and high-speed optical imaging in water, agarose gels, and RBC phantoms demonstrated that histotripsy bubble clouds could be generated across the prosthesis at pulse repetition frequencies of 50-500 Hz. Histologic analysis revealed a complete breakdown of brain tissue treated across the prosthesis. Conclusion & Significance: Overall, the results of this study demonstrate that the cranial prosthesis may be used as an acoustic window through which intracranial histotripsy can be applied under US guidance without the need for large transcranial array transducers.

Development of a contrast-enhanced ultrasound guided high intensity focused ultrasound system for coagulation of liver parenchyma.

BACKGROUND: The liver is the most common organ injured in blunt abdominal trauma and makes up roughly 5% of all trauma admissions. Current treatments are invasive and resource-intensive, which may delay care. We aim to develop and validate a contrast-enhanced ultrasound (CEUS)guided noninvasive tool to treat liver lacerations at the bedside. METHODS: Two 1.8 MHz high-intensity focused ultrasound (HIFU) elements were coupled to a C1-6 diagnostic ultrasound probe and a Logiq E10 scanner (GE HealthCare) utilizing a custom enclosure for co-registered imaging and ablation. A phantom was created from polyacrylamide gel combined with thermochromic ink whose color changes above biological ablative temperatures (60 °C). The HIFU wave was focused approximately 0.5 cm below the surface utilizing a 50% duty cycle generating 11.9 MPa for 20, 30, 40, 50, and 60s. Experiments were repeated on ex vivo chicken livers in a water bath. Finally, the livers of 4 live swine underwent up to 6 CEUS-guided treatments using parameters optimized from in vitro work. RESULTS: Treatment of the phantom between 20-60s, produced ablation sizes from 0.016 to 0.4 cm 3 . The relationship between time and size was exponential (R 2 = 0.992). Ablation areas were also well visualized on with ultrasound imaging. The ex vivo liver ablation size at 20s was 0.37 cm 3 , at 30s was 0.66 cm 3 , and at 100 s was 5.0 cm 3 . For the in-vivo swine experiments, the average ablation area measured 2.0x0.75 cm with a maximum of 3.5x1.5 cm. CEUS was utilized with the contrast agent Definity (Lantheus) for identification of lacerations as well as immediate post operative evaluation of therapy. CONCLUSION: These experiments demonstrate the feasibility of CEUS guided transdermal HIFU ablation and the time-dependent size of ablation. This work warrants future investigations into using ultrasound to detect active bleeding and HIFU to coagulate grade III and IV liver laceration. STUDY TYPE: Therapeutic/care management.

Cavitation-induced pressure saturation: a mechanism governing bubble nucleation density in histotripsy.

Objective.Histotripsy is a noninvasive focused ultrasound therapy that mechanically disintegrates tissue by acoustic cavitation clouds. In this study, we investigate a mechanism limiting the density of bubbles that can nucleate during a histotripsy pulse. In this mechanism, the pressure generated by the initial bubble expansion effectively negates the incident pressure in the vicinity of the bubble. From this effect, the immediately adjacent tissue is prevented from experiencing the transient tension to nucleate bubbles. Approach.A Keller-Miksis-type single-bubble model was employed to evaluate the dependency of this effect on ultrasound pressure amplitude and frequency, viscoelastic medium properties, bubble nucleus size, and transducer geometric focusing. This model was further combined with a spatial propagation model to predict the peak negative pressure field as a function of position from a cavitating bubble.Main results. The single-bubble model showed the peak negative pressure near the bubble surface is limited to the inertial cavitation threshold. The predicted bubble density increased with increasing frequency, tissue viscosity, and transducer focusing angle. The simulated results were consistent with the trends observed experimentally in prior studies, including changes in density with ultrasound frequency and transducerF-number.Significance.The efficacy of the therapy is dependent on several factors, including the density of bubbles nucleated within the cavitation cloud formed at the focus. These results provide insight into controlling the density of nucleated bubbles during histotripsy and the therapeutic efficacy.

Investigation of Optimum Production Conditions and the Stability of ß-Cyclodextrin-Perfluorocarbon Nanocone Clusters for Histotripsy Applications.

Nanocone clusters (NCCs) have been developed as clusters with inclusion complexes of FDA-approved ß-cyclodextrin (ßCD) and perfluorocarbons (PFC) (i.e., perfluoropentane (PFP) and perfluorohexane (PFH)) and have shown promise in nanoparticle-mediated histotripsy (NMH) applications owing to their lowered cavitation threshold, ease of production, and fluorocarbon quantification. However, there is still a lack of information on the best conditions of the synthesis of NCCs as a product that can have a maximum determinable fluorocarbon content and maintain the stability of the NCC during synthesis and when used as histotripsy agents or exposed to physiological conditions. These concerns about the stability of the clusters and the best possible formulation are investigated in the current work. The cluster formation potential was tested taking into consideration the nature of both PFCs and ßCD by employing different synthesis conditions in terms of solution and environmental parameters such as concentration of solvent, stoichiometry between ßCD and PFCs, temperature, pH, solvent type, etc. The best route of synthesis was then translated into various batch sizes and investigated in terms of the PFC loading and yield. These studies revealed that preparing NCCs in double-distilled water in an ice bath at the optimized solution concentration gave the highest yields and optimal PFC loading, as determined from gas chromatography. Furthermore, the stability of the clusters with different stoichiometries was scrutinized in varying concentrations, mechanical disruption times, pH levels, and temperature conditions, showing effects on each cluster's particle size in dynamic light scattering, visualized in transmission electron microscopy, and cavitation behavior in agarose gel tissue phantoms. These studies revealed stable clusters for all formulations, with PFH-containing NCCs emerging to be the most stable in terms of their cluster size and bubble formation potential in histotripsy. Finally, the shelf life of these clusters was investigated using DLS, which revealed a stable cluster. In conclusion, NCCs have shown high stability in terms of both synthesis, which can be replicated in gram-level production, and the cluster itself, which can be exposed to harsher conditions and still form stable bubbles in histotripsy.

Effects of pulse repetition frequency on bubble cloud characteristics and ablation in single-cycle histotripsy.

Objective. Histotripsy is a cavitation-based ultrasound ablation method in development for multiple clinical applications. This work investigates the effects of pulse repetition frequency (PRF) on bubble cloud characteristics and ablative capabilities for histotripsy using single-cycle pulsing methods.Approach.Bubble clouds produced by a 500 kHz histotripsy system at PRFs from 0.1 to 1000 Hz were visualized using high-speed optical imaging in 1% agarose tissue phantoms at peak negative pressures,p-, of 2-36 MPa.Main results.Results showed a decrease in the cavitation cloud threshold with increasing PRF, ranging from 26.7 ± 0.5 MPa at 0.1 Hz to 15.0 ± 1.9 MPa at 1000 Hz. Bubble cloud analysis showed cavitation clouds generated at low PRFs (0.1-1 Hz) were characterized by consistently dense bubble clouds (41.7 ± 2.8 bubbles mm-2at 0.1 Hz), that closely matched regions of the focus above the histotripsy intrinsic threshold. Bubble clouds formed at higher PRFs measured lower cloud densities (23.1 ± 4.0 bubbles mm-2at 1000 Hz), with the lowest density measured for 10 Hz (8.8 ± 4.1 bubbles mm-2). Furthermore, higher PRFs showed increased pulse-to-pulse correlation, characteristic of cavitation memory effects; however, bubble clouds still filled the entire volume of the focus due to their initial density and enhanced bubble expansion from the restimulation of residual nuclei at the higher PRFs. Histotripsy ablation assessed through lesion analysis in red blood cell (RBC) phantoms showed higher PRFs generated lesions with lower adherence to the initial focal region compared to low PRF ablations; however, no trend of decreasing ablation efficiency with PRF was observed, with similar efficiencies observed for all the PRFs tested in this study.Significance.Notably, this result is different than what has previously been shown for shock-scattering histotripsy, which has shown decreased ablation efficiencies at higher PRFs. Overall, this study demonstrates the essential effects of PRF on single-cycle histotripsy procedures that should be considered to help guide future histotripsy pulsing strategies.

Investigating cell death responses associated with histotripsy ablation of canine osteosarcoma.

BACKGROUND: Osteosarcoma (OS) is the most frequently occurring primary bone tumor in dogs and people and innovative treatment options are profoundly needed. Histotripsy is an emerging tumor ablation modality, and it is essential for the clinical translation of histotripsy to gain knowledge about the outcome of nonablated tumor cells that could remain postablation. The objective of this study was to characterize the cell death genetic signature and proliferation response of canine OS cells post a near complete histotripsy ablation (96% ± 1.5) and to evaluate genetic cell death signatures associated with histotripsy ablation and OS in vivo. METHODS: In the current study, we ablated three canine OS cell lines with a histotripsy dose that resulted in near complete ablation to allow for a viable tumor cell population for downstream analyses. To assess the in vivo cell death genetic signature, we characterized cell death genetic signature in histotripsy-ablated canine OS tumors collected 24-h postablation. RESULTS: Differential gene expression changes observed in the 4% viable D17 and D418 cells, and histotripsy-ablated OS tumor samples, but not in Abrams cells, were associated with immunogenic cell death (ICD). The 4% viable OS cells demonstrated significantly reduced proliferation, compared to control OS cells, in vitro. CONCLUSION: Histotripsy ablation of OS cell lines leads to direct and potentially indirect cell death as evident by, reduced proliferation in remaining viable OS cells and cell death genetic signatures suggestive of ICD both in vitro and in vivo.

A review of the development of histotripsy for extremity tumor ablation with a canine comparative oncology model to inform human treatments.

Cancer is a devasting disease resulting in millions of deaths worldwide in both humans and companion animals, including dogs. Treatment of cancer is complex and challenging and therefore often multifaceted, as in the case of osteosarcoma (OS) and soft tissue sarcoma (STS). OS predominantly involves the appendicular skeleton and STS commonly develops in the extremities, resulting in treatment challenges due to the need to balance wide-margin resections to achieve local oncological control against the functional outcomes for the patient. To achieve wide tumor resection, invasive limb salvage surgery is often required, and the patient is at risk for numerous complications which can ultimately lead to impaired limb function and mobility. The advent of tumor ablation techniques offers the exciting potential of developing noninvasive or minimally invasive treatment options for extremity tumors. One promising innovative tumor ablation technique with strong potential to serve as a noninvasive limb salvage treatment for extremity tumor patients is histotripsy. Histotripsy is a novel, noninvasive, non-thermal, and non-ionizing focused ultrasound technique which uses controlled acoustic cavitation to mechanically disintegrate tissue with high precision. In this review, we present the ongoing development of histotripsy as a non-surgical alternative for extremity tumors and highlight the value of spontaneously occurring OS and STS in the pet dog as a comparative oncology research model to advance this field of histotripsy research.

Bubble cloud characteristics and ablation efficiency in dual-frequency intrinsic threshold histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ∼25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single-frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.

Clinical translation of abdominal histotripsy: a review of preclinical studies in large animal models.

Histotripsy is an emerging noninvasive, non-thermal, and non-ionizing focused ultrasound (US) therapy that can be used to destroy targeted tissue. Histotripsy has evolved from early laboratory prototypes to clinical systems which have been comprehensively evaluated in the preclinical environment to ensure safe translation to human use. This review summarizes the observations and results from preclinical histotripsy studies in the liver, kidney, and pancreas. Key findings from these studies include the ability to make a clinically relevant treatment zone in each organ with maintained collagenous architecture, potentially allowing treatments in areas not currently amenable to thermal ablation. Treatments across organ capsules have proven safe, including in anticoagulated models which may expand patients eligible for treatment or eliminate the risk associated with taking patients off anti-coagulation. Treatment zones are well-defined with imaging and rapidly resorb, which may allow improved evaluation of treatment zones for residual or recurrent tumor. Understanding the effects of histotripsy in animal models will help inform physicians adopting histotripsy for human clinical use.

Ablative and Immunostimulatory Effects of Histotripsy Ablation in a Murine Osteosarcoma Model.

Background: Osteosarcoma (OS) is the most frequently occurring malignant bone tumor in humans, primarily affecting children and adolescents. Significant advancements in treatment options for OS have not occurred in the last several decades, and the prognosis remains grim with only a 70% rate of 5-year survival. The objective of this study was to investigate the focused ultrasound technique of histotripsy as a novel, noninvasive treatment option for OS. Methods: We utilized a heterotopic OS murine model to establish the feasibility of ablating OS tumors with histotripsy in a preclinical setting. We investigated the local immune response within the tumor microenvironment (TME) via immune cell phenotyping and gene expression analysis. Findings: We established the feasibility of ablating heterotopic OS tumors with ablation characterized microscopically by loss of cellular architecture in targeted regions of tumors. We observed greater populations of macrophages and dendritic cells within treated tumors and the upregulation of immune activating genes 72 h after histotripsy ablation. Interpretation: This study was the first to investigate histotripsy ablation for OS in a preclinical murine model, with results suggesting local immunomodulation within the TME. Our results support the continued investigation of histotripsy as a novel noninvasive treatment option for OS patients to improve clinical outcomes and patient prognosis.

Probability of Cavitation in a Custom Iron-Based Coupling Medium for Transcranial Magnetic Resonance-Guided Focused Ultrasound Procedures.

OBJECTIVE: A coupling bath of circulating, chilled, degassed water is essential to safe and precise acoustic transmittance during transcranial magnetic resonance-guided focused ultrasound (tMRgFUS) procedures, but the circulating water impairs the critical real-time magnetic resonance imaging (MRI). An iron-based coupling medium (IBCM) using iron oxide nanoparticles previously developed by our group increased the relaxivity of the coupling bath such that it appears to be invisible on MRI compared with degassed water. However, the nanoparticles also reduced the pressure threshold for cavitation. To address this concern for prefocal cavitation, our group recently developed an IBCM of electrosterically stabilized and aggregation-resistant poly(methacrylic acid)-coated iron oxide nanoparticles (PMAA-FeOX) with a similar capability to reduce the MR signal of degassed water. This study examines the effect of the PMAA-FeOX IBCM on the cavitation threshold. METHODS: Increasing concentrations of PMAA-FeOX nanoparticles in degassed, deionized water were placed at the focus of two different transducers to assess low and high duty-cycle pulsing parameters which are representative of two modes of focused ultrasound being investigated for tMRgFUS. Passive cavitation detection and high-speed optical imaging were used to measure cavitation threshold pressures. RESULTS: The mean cavitation threshold was determined in both cases to be indistinguishable from the degassed water control, between 6-8 MPa for high duty-cycle pulsing (CW) and between 25.5-26.5 MPa for very low duty-cycle pulsing. CONCLUSION: The findings of this study indicate that an IBCM of PMAA-FeOX nanoparticles is a possible solution to reducing MRI interference from the coupling bath without increasing the risk of prefocal cavitation.

Ultrasound-guided noninvasive pancreas ablation using histotripsy: feasibility study in an in vivo porcine model.

Pancreatic cancer is a malignant disease associated with poor survival and nearly 80% present with unresectable tumors. Treatments such as chemotherapy and radiation therapy have shown overall improved survival benefits, albeit limited. Histotripsy is a noninvasive, non-ionizing, and non-thermal focused ultrasound ablation modality that has shown efficacy in treating hepatic tumors and other malignancies. In this novel study, we investigate histotripsy for noninvasive pancreas ablation in a pig model. In two studies, histotripsy was applied to the healthy pancreas in 11 pigs using a custom 32-element, 500 kHz histotripsy transducer attached to a clinical histotripsy system, with treatments guided by real-time ultrasound imaging. A pilot study was conducted in 3 fasted pigs with histotripsy applied at a pulse repetition frequency (PRF) of 500 Hz. Results showed no pancreas visualization on coaxial ultrasound imaging due to overlying intestinal gas, resulting in off-target injury and no pancreas damage. To minimize gas, a second group of pigs (n = 8) were fed a custard diet containing simethicone and bisacodyl. Pigs were euthanized immediately (n = 4) or survived for 1 week (n = 4) post-treatment. Damage to the pancreas and surrounding tissue was characterized using gross morphology, histological analysis, and CT imaging. Results showed histotripsy bubble clouds were generated inside pancreases that were visually maintained on coaxial ultrasound (n = 4), with 2 pigs exhibiting off-target damage. For chronic animals, results showed the treatments were well-tolerated with no complication signs or changes in blood markers. This study provides initial evidence suggesting histotripsy's potential for noninvasive pancreas ablation and warrants further evaluation in more comprehensive studies.

Magic bubbles: utilizing histotripsy to modulate the tumor microenvironment and improve systemic anti-tumor immune responses.

Focused Ultrasound (FUS) is emerging as a promising primary and adjunct therapy for the treatment of cancer. This includes histotripsy, which is a noninvasive, non-ionizing, non-thermal ultrasound guided ablation modality. As histotripsy has progressed from bench-to-bedside, it has become evident that this therapy has benefits beyond local tumor ablation. Specifically, histotripsy has the potential to shift the local tumor microenvironment from immunologically 'cold' to 'hot'. This is associated with the production of damage associated molecular patterns, the release of a selection of proinflammatory mediators, and the induction of inflammatory forms of cell death in cells just outside of the treatment zone. In addition to the induction of this innate immune response, histotripsy can also improve engagement of the adaptive immune system and promote systemic anti-tumor immunity targeting distal tumors and metastatic lesions. These tantalizing observations suggest that, in settings of widely metastatic disease burden, selective histotripsy of a limited number of accessible tumors could be a means of maximizing responsiveness to systemic immunotherapy. More work is certainly needed to optimize treatment strategies that best synergize histotripsy parameters with innate and adaptive immune responses. Likewise, rigorous clinical studies are still necessary to verify the presence and repeatability of these phenomena in human patients. As this technology nears regulatory approval for clinical use, it is our expectation that the insights and immunomodulatory mechanisms summarized in this review will serve as directional guides for rational clinical studies to validate and optimize the potential immunotherapeutic role of histotripsy tumor ablation.

Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

OBJECTIVE: New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. METHODS: Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. RESULTS: Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. CONCLUSION: This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Bubble Cloud Characteristics and Ablation Efficiency in Dual-Frequency Intrinsic Threshold Histotripsy.

Histotripsy is a non-thermal focused ultrasound ablation method that destroys tissue through the generation and activity of acoustic cavitation bubble clouds. Intrinsic threshold histotripsy uses single-cycle pulses to generate bubble clouds when the dominant negative pressure phase exceeds an intrinsic threshold of ~25-30 MPa. The ablation efficiency is dependent upon the size and density of bubbles within the bubble cloud. This work investigates the effects of dual-frequency pulsing schemes on the bubble cloud behavior and ablation efficiency in intrinsic threshold histotripsy. A modular 500 kHz:3 MHz histotripsy transducer treated agarose phantoms using dual-frequency histotripsy pulses with a 1:1 pressure ratio from 500 kHz and 3 MHz frequency elements and varying arrival times for the 3 MHz pulse relative to the arrival of the 500 kHz pulse (-100 ns, 0 ns, and +100 ns). High-speed optical imaging captured cavitation effects to characterize bubble cloud and individual bubble dynamics. The effects of dual-frequency pulsing on lesion formation and ablation efficiency were also investigated in red blood cell (RBC) phantoms. Results showed that the single bubble and bubble cloud size for dual-frequency cases were intermediate to published results for the component single frequencies of 500 kHz and 3 MHz. Additionally, bubble cloud size and dynamics were shown to be altered by the arrival time of the 3 MHz pulse with respect to the 500 kHz pulse, with more uniform cloud expansion and collapse observed for early (-100 ns) arrival. Finally, RBC phantom experiments showed that dual-frequency exposures were capable of generating precise lesions with smaller areas and higher ablation efficiencies than previously published results for 500 kHz or 3 MHz. Overall, results demonstrate dual-frequency histotripsy's ability to modulate bubble cloud size and dynamics can be leveraged to produce precise lesions at higher ablation efficiencies than previously observed for single-frequency pulsing.

Catheter-Based Medical Device Biofilm Ablation Using Histotripsy: A Parameter Study.

OBJECTIVE: Biofilm formation in medical catheters is a major source of hospital-acquired infections which can produce increased morbidity and mortality for patients. Histotripsy is a non-invasive, non-thermal focused ultrasound therapy and recently has been found to be effective at removal of biofilm from medical catheters. Previously established histotripsy methods for biofilm removal, however, would require several hours of use to effectively treat a full-length medical catheter. Here, we investigate the potential to increase the speed and efficiency with which biofilms can be ablated from catheters using histotripsy. METHODS: Pseudomonas aeruginosa (PA14) biofilms were cultured in in vitro Tygon catheter mimics and treated with histotripsy using a 1 MHz histotripsy transducer and a variety of histotripsy pulsing rates and scanning methods. The improved parameters identified in these studies were then used to explore the bactericidal effect of histotripsy on planktonic PA14 suspended in a catheter mimic. RESULTS: Histotripsy can be used to remove biofilm and kill bacteria at substantially increased speeds compared with previously established methods. Near-complete biofilm removal was achieved at treatment speeds up to 1 cm/s, while a 4.241 log reduction in planktonic bacteria was achieved with 2.4 cm/min treatment. CONCLUSION: These results represent a 500-fold increase in biofilm removal speeds and a 6.2-fold increase in bacterial killing speeds compared with previously published methods. These findings indicate that histotripsy shows promise for the treatment of catheter-associated biofilms and planktonic bacteria in a clinically relevant time frame.

Histotripsy ablation for the treatment of feline injection site sarcomas: a first-in-cat in vivo feasibility study.

PURPOSE: Feline soft tissue sarcoma (STS) and injection site sarcoma (fISS) are rapidly growing tumors with low metastatic potential, but locally aggressive behavior. Histotripsy is a non-invasive focused ultrasound therapy using controlled acoustic cavitation to mechanically disintegrate tissue. In this study, we investigated the in vivo safety and feasibility of histotripsy to treat fISS using a custom 1 MHz transducer. MATERIALS AND METHODS: Three cats with naturally-occurring STS were treated with histotripsy before surgical removal of the tumor 3 to 6 days later. Gross and histological analyses were used to characterize the ablation efficacy of the treatment, and routine immunohistochemistry and batched cytokine analysis were used to investigate the acute immunological effects of histotripsy. RESULTS: Results showed that histotripsy ablation was achievable and well-tolerated in all three cats. Precise cavitation bubble clouds were generated in all patients, and hematoxylin & eosin stained tissues revealed ablative damage in targeted regions. Immunohistochemical results identified an increase in IBA-1 positive cells in treated tissues, and no significant changes in cytokine concentrations were identified post-treatment. CONCLUSIONS: Overall, the results of this study demonstrate the safety and feasibility of histotripsy to target and ablate superficial feline STS and fISS tumors and guide the clinical development of histotripsy devices for this application.

Development of a Synthetic, Injectable Hydrogel to Capture Residual Glioblastoma and Glioblastoma Stem-Like Cells with CXCL12-Mediated Chemotaxis.

Glioblastoma (GBM), characterized by high infiltrative capacity, is the most common and deadly type of primary brain tumor in adults. GBM cells, including therapy-resistant glioblastoma stem-like cells (GSCs), invade the healthy brain parenchyma to form secondary tumors even after patients undergo surgical resection and chemoradiotherapy. New techniques are therefore urgently needed to eradicate these residual tumor cells. A thiol-Michael addition injectable hydrogel for compatibility with GBM therapy is previously characterized and optimized. This study aims to develop the hydrogel further to capture GBM/GSCs through CXCL12-mediated chemotaxis. The release kinetics of hydrogel payloads are investigated, migration and invasion assays in response to chemoattractants are performed, and the GBM-hydrogel interactions in vitro are studied. With a novel dual-layer hydrogel platform, it is demonstrated that CXCL12 released from the synthetic hydrogel can induce the migration of U251 GBM cells and GSCs from the extracellular matrix microenvironment and promote invasion into the synthetic hydrogel via amoeboid migration. The survival of GBM cells entrapped deep into the synthetic hydrogel is limited, while live cells near the surface reinforce the hydrogel through fibronectin deposition. This synthetic hydrogel, therefore, demonstrates a promising method to attract and capture migratory GBM cells and GSCs responsive to CXCL12 chemotaxis.