Mechanical decellularization of tissue volumes using boiling histotripsy

Boiling histotripsy (BH) is a new high intensity focused ultrasound (HIFU) ablation technique to mechanically fragmentize soft tissue into submicrometer fragments. So far, ultrasound has been used for BH treatment guidance and evaluation. The in vivo histopathological effects of this treatment are largely unknown. Here, we report on an MR guided BH method to treat subcutaneous tumors in a mouse model. The treatment effects of BH were evaluated one hour and four days later with MRI and histopathology, and compared with the effects of thermal HIFU (T-HIFU). The lesions caused by BH were easily detected with T2 w imaging as a hyper-intense signal area with a hypo-intense rim. Histopathological evaluation showed that the targeted tissue was completely disintegrated and that a narrow transition zone (<200 µm) containing many apoptotic cells was present between disintegrated and vital tumor tissue. A high level of agreement was found between T2 w imaging and H&E stained sections, making T2 w imaging a suitable method for treatment evaluation during or directly after BH. After T-HIFU, contrast enhanced imaging was required for adequate detection of the ablation zone. On histopathology, an ablation zone with concentric layers was seen after T-HIFU. In line with histopathology, contrast enhanced MRI revealed that after BH or T-HIFU perfusion within the lesion was absent, while after BH in the transition zone some micro-hemorrhaging appeared. Four days after BH, the transition zone with apoptotic cells was histologically no longer detectable, corresponding to the absence of a hypo-intense rim around the lesion in T2 w images. This study demonstrates the first results of in vivo BH on mouse tumor using MRI for treatment guidance and evaluation and opens the way for more detailed investigation of the in vivo effects of BH. Copyright © 2016 John Wiley & Sons, Ltd.

Boiling histotripsy is a promising non-invasive High-Intensity Focused Ultrasound (HIFU) technique that employs HIFU mechanical effects to fractionate solid tumours without causing any significant thermal damage. It has been suggested that boiling histotripsy may induce a strong immune response due to the absence of denatured antigenic protein at the HIFU focus. The underlying immunological mechanisms of this technique are, however, poorly understood. In this study, we demonstrated the feasibility of using boiling histotripsy to mechanically fractionate human breast adenocarcinoma cells (MDA-MB-231) and the potential immunological effects induced by boiling histotripsy, for the first time. Our results showed that mechanical stresses produced by boiling histotripsy promote immunogenic cell death of cancer cells via TNF-induced necrosis signaling pathway. This immunogenic cell death significantly increases secretions of damage-associated molecular patterns (CRT, HSP70, HMGB-1), pro-inflammatory cytokines (IFN-γ, IL-1α, IL-1β, IL-18) and chemokines (IL-8) which are related to M1 macrophage activation. Furthermore, the levels of these signaling proteins increase with the degree of mechanical damage induced by boiling histotripsy. Together, the results presented can suggest that boiling histotripsy could be a potential therapeutic approach for not only mechanically destroying solid tumours (e.g., breast cancer) but also promoting immunogenic cell death via TNF-induced necrosis to trigger antitumour immunity.

Background High intensity focused ultrasound (HIFU) is a non-ionizing and non-invasive application for benign and malign disease. Ultrasonic waves are concentrated to achieve high level of energy and therefore necrosis due to coagulation. Aims We will describe one pediatric case where an osteoidosteoma war successfully treated by HIFU. Methods A seven-year-old boy presented in our department for hand surgery due to indolent swelling of the distal phalanx of the right ring finger. Neither recent trauma, nor positive family history was present. Clinical examination showed significant enlargement of the distal phalanx with minimal pain on palpation and unlimited mobility. Conventional radiography showed discrete fuzzy sclerosis of the metaphyseal part of the growth plate. MRI was not conclusive. Bone biopsy showed reactive change with no signs for acute inflammatory event. Periodic checks were performed, with no change in clinical and radiographic aspect. Nine months after the first appointment, the patient presented with pain after minor trauma and increase of the swelling. Radiography showed a nidus in the distal phalanx leading to a suspected diagnosis of an osteoidosteoma. Therefore, MRI-guided HIFU (MR-HIFU) was recommended one year after the first appointment. Results MR-HIFU was performed to destroy the osteoidosteoma. Six months after the intervention the patient presented with no further exponential growth and the difference to the other phalanges was minimized. Furthermore, radiography showed no retention of the nidus with a plane distal phalanx. Conclusion MR-HIFU is a promising approach in the non-invasive treatment of tumorous neoplasm. Because of its non-ionizing ultrasonic waves, there is no upper limit in dosing. The deposition of heat is in general limited by the surrounding tissue. The main factors preventing MR-HIFU treatment are thick bone around the nidus, a superficial position with risk for skin burn and the vicinity to neural structures. Osteoidosteomas are generally rather small and therefore well suited for MR-HIFU.

Magnetic resonance imaging-guided high-intensity focused ultrasound (MRIgFUS) is a novel technique that may have the potential for precise image-guided thermocoagulation of intracranial lesions. The system delivers small volumetric sonications from an ultrasound phased array transmitter that focuses energy selectively to destroy the target with verification by magnetic resonance imaging-generated thermal maps. A Phase I clinical study was initiated to treat patients with recurrent glioma with MRIgFUS. To date, three patients with histologically verified recurrent glioblastoma multiforme have been treated with MRIgFUS. All patients underwent craniectomy 7 to 10 days before therapy to create a bony window for the ultrasound treatment. Sonications were applied to induce thermocoagulation of the enhancing tumor mass. Long-term radiological follow-up and post-treatment tissue specimens were available for all patients. MRIgFUS treatment resulted in immediate changes in contrast-enhanced T1-, T2-, and diffusion-weighted magnetic resonance imaging scans in the treated regions with subsequent histological evidence of thermocoagulation. In one patient, heating of brain tissue in the sonication path resulted in a secondary focus outside the target causing neurological deficit. New software modifications were developed to address this problem. In this first clinical report, MRIgFUS was demonstrated to be a potentially effective means of destroying tumor tissue by thermocoagulation, although with an associated morbidity and the inherent invasive nature of the procedure requiring creation of a bone window. A modified technology to allow MRIgFUS treatment through a closed cranium is being developed.

ObjectiveIf bowels and other structures are in the pathway of high-intensity focused ultrasound (HIFU) beam during magnetic resonance image-guided HIFU (MRgFUS) therapy, filling to the bladder and the rectum and then emptying the bladder (i.e., the BRB technique) is used to avoid them. A modified BRB technique might be useful method to using a uterine elevator method or by inducing uterus downward traction to lower the position of the uterus.MethodsA total of 156 patients who had undergone MRgFUS surgery treatment for uterine fibroids from March 2015 to February 2016 were included in this retrospective study. Of the 156 patients, 40 were treated using a uterine elevator while 29 were treated using downward traction of uterus. HIFU was performed using Philips Achieva 1.5 Tesla MR and Sonalleve HIFU system.ResultsMRgFUS surgery was feasible with modified BRB technique in 69 cases. Using uterine elevator method, the intensity of HIFU for group with antefletxio uteri was significantly lower than that for the group without antefletxio uteri (105.37±17.62 vs. 118.71±26.88 W). The group with downward traction of uterus induced was found to have significantly lower intensity of HIFU compared to the group without downward traction of uterus induced (110.26±22.60 vs. 130.51±27.81 W).ConclusionModified BRB technique was useful in avoiding bowels and other structures located in HIFU beam pathway during MRgFUS treatment to ablate uterine fibroids.

Histological and Biochemical Analysis of Mechanical and Thermal Bioeffects in Boiling Histotripsy Lesions Induced by High Intensity Focused Ultrasound

Effect of pulse repetition frequency of high-intensity focused ultrasound on in vitro thrombolysis

Enhancing boiling histotripsy efficacy with a tandem pulse sequence: Immediate hypoechoic Sonograhy and expanded lesion size.

High intensity focused ultrasound (HIFU) has been introduced for treatment of cardiac arrhythmias because it offers the ability to create rapid tissue modification in confined volumes without directly contacting the myocardium. In spite of the benefits of HIFU, a number of limitations have been reported, which hindered its clinical adoption. In this study, we used a multimodal approach to evaluate thermal and nonthermal effects of HIFU in cardiac ablation. We designed a computer controlled system capable of simultaneous fluorescence mapping and HIFU ablation. Using this system, linear lesions were created in isolated rabbit atria (n=6), and point lesions were created in the ventricles of whole-heart (n=6) preparations by applying HIFU at clinical doses (4-16 W). Additionally, we evaluate the gap size in ablation lines necessary for conduction in atrial preparations (n=4). The voltage sensitive dye di-4-ANEPPS was used to assess functional damage produced by HIFU. Optical coherence tomography and general histology were used to evaluate lesion extent. Conduction block was achieved in 1 (17%) of 6 atrial preparations with a single ablation line. Following 10 minutes of rest, 0 (0%) of 6 atrial preparations demonstrated sustained conduction block from a single ablation line. Tissue displacement of 1 to 3 mm was observed during HIFU application due to acoustic radiation force along the lesion line. Additionally, excessive acoustic pressure and high temperature from HIFU generated cavitation, causing macroscopic tissue damage. A minimum gap size of 1.5 mm was found to conduct electric activity. This study identified 3 potential mechanisms responsible for the failure of HIFU ablation in cardiac tissues. Both acoustic radiation force and acoustic cavitation, in conjunction with inconsistent thermal deposition, can increase the risk of lesion discontinuity and result in gap sizes that promote ablation failure.

Boiling histotripsy (BH) is a HIFU approach that uses millisecond-long ultrasound bursts with high-amplitude shocks to mechanically fractionate tissue. In this work, the performance of a BH pre-clinical system operating at 1.5 MHz was characterized by hydrophone measurements in water and lesion formation capabilities were tested in ex vivo tissue. The system comprises a 256 element array driven by Verasonics Ultrasound Engine with a 1.2 kW external power source enhanced by an additional capacitor bank. The maximum pulse average acoustic power of the system was 2.2 kW (as measured by radiation force balance), sustained for 10 ms with &amp;lt;15% drop in amplitude. At the focus, a fully developed shock of 100 MPa amplitude formed at 275 W acoustic power. The steering range defined at -3 dB in both linear and nonlinear beam was 19 mm laterally and 38 mm axially forming the region where 100 MPa shocks can be achieved with less than twofold increase in acoustic power. Within the axial steering range, shock formation characteristics varied substantially due to changes in the effective transducer F-number. The system was successfully used to produce volumetric BH lesions in ex vivo tissue using electronic steering only. [Work supported by NIH R01EB7643, R01GM122859, and R01EB025187.]

Effect of pulse parameters on ablation efficiency in dual-frequency HIFU therapy

Investigation of Thrombolysis and Safety of Stand-Alone High-Intensity Focused Ultrasound Therapy in Partially Occluded Ex-Vivo Animal Models.

Hyperecho as the Indicator of Tissue Necrosis During Microbubble-Assisted High Intensity Focused Ultrasound: Sensitivity, Specificity and Predictive Value

Boiling histotripsy is an experimental noninvasive focused ultrasound therapy that applies shocked ms-length pulses to achieve mechanical disintegration of a targeted tissue. Localized delivery of high-amplitude shocks causes rapid heating, resulting in boiling of the tissue. The interaction of incident shocks with the boiling bubble results in tissue disruption and liquefaction without significant thermal injury. Simulations are utilized to design and characterize therapy sources, predicting focal waveforms, shock amplitudes, and boiling times. Transducers have been developed to generate focal shock amplitudes &amp;gt;70 MPa and achieve rapid boiling at depth in tissue. Therapy systems including ultrasound-guided single-element sources and clinical MRI-guided phased arrays have been successfully used to create ex vivo and in vivo lesions at ultrasound frequencies in the 1–3 MHz range. Histological and biochemical analyses show mechanical disruption of tissue architecture with minimal thermal effect, similar to cavitation-based histotripsy. Atomization as observed with acoustic fountains has been proposed as an underlying mechanism of tissue disintegration. This promising technology is being explored for several applications in tissue ablation, as well as new areas such as tissue engineering and biomarker detection. [Work supported by NIH 2T32DK007779-11A1, R01EB007643-05, 1K01EB015745, and NSBRI through NASA NCC 9-58.]

A wide variety of treatment protocols have been employed in high intensity focused ultrasound (HIFU) treatments, and the resulting bioeffects observed include both mechanical as well as thermal effects. In recent studies, there has been significant interest in generating purely mechanical damage using protocols with short, microsecond pulses. Tissue erosion effects have been attained by operating HIFU sources using short pulses of 10–20 cycles, low duty cycles (<1%), and pulse average intensities of greater than 20 kW/cm2. The goal of this work was to use a modified pulsing protocol, consisting of longer, millisecond‐long pulses of ultrasound and to demonstrate that heating and rapid millisecond boiling from shock wave formation can be harnessed to induce controlled mechanical destruction of soft tissue. Experiments were performed in excised bovine liver and heart tissue using a 2‐MHz transducer. Boiling activity was monitored during exposures using a high voltage probe in parallel with the HIFU source. In situ acoustic fields and heating rates were determined for exposures using a novel derating approach for nonlinear HIFU fields. Several different exposure protocols were used and included varying the duty cycle, pulse length, and power to the source. After exposures, the tissue was sectioned, and the gross lesion morphology was observed. Different types of lesions were induced in experiments that ranged from purely thermal to purely mechanical depending on the pulsing protocol used. Therefore, shock wave heating and millisecond boiling may be an effective method for reliably generating significant tissue erosion effects.