Low-Intensity Pulsed Ultrasound

Ultrasound exposure within and just beyond diagnostic acoustic output levels have been investigated for therapeutic benefit (1–3 MHz, 0.02–1 W/cm²).³⁵ Low-intensity pulsed ultrasound (LIPUS) has been categorized as insufficient to generate significant thermal effects, but can cause tissue displacement or fluid streaming due to radiation force.³⁶ Given the low acoustic output of LIPUS systems, cavitation effects are unlikely without the addition of exogenous agents.³¹

Mild Hyperthermia

Hyperthermia refers to the heating of tissue to temperatures in the range of 41–45°C for durations of ~30–60 minutes. These effects are achieved with acoustic powers of approximately 10–100 W.^68-70 Mild heating has been shown to combine synergistically with other cancer therapies.^71-73 Hyperthermia has been shown to enhance tissue damage from radiation therapy,^{74, 75} and including in clinical trials for multiple tumor types.⁷³ Other applications of hyperthermia include enhanced drug delivery using thermosensitive liposomes^76-78 and enhanced immune effects for CAR-t cell immunotherapy.⁷⁹ The systems used to produce acoustic fields for hyperthermia include single or multi-element sources, phased arrays, or intracavity devices.^{72, 80} Volumetric heating can be accomplished through mechanical scanning, and/or phased arrays that offer electronic beam forming and steering. Recent investigations demonstrated the feasibility to design holographic lens transducers that produce uniform thermal dose profiles over arbitrary tumor volumes.⁸¹

Thermal Ablation

The application of focused ultrasound to cause thermal ablation has been extensively investigated for multiple targets. Focused ultrasound was applied to more than 98 k patients worldwide in 2022, the majority of which were thermal ablation cases.⁸² The typical fundamental frequency for thermal ablation ranges from 0.5 and 10 MHz, predominantly at the lower end of this spectrum.⁸³ Focused pulses have spatial-peak, time-average intensities (I_SPTA) from 0.001 to more than 1 kW/cm². The resultant effect is the generation of in situ temperatures ranging from 48°C to more than 70°C that causes cell death in minutes to seconds, respectively.⁸⁴ Therapeutic volumes larger than the focal region are achieved by targeting multiple locations sequentially or in an interleaved fashion.⁸⁵ For all but the most superficial targets, image guidance is employed with ultrasound or magnetic resonance imaging (MRI).

Many applications of thermal ablation have transitioned into clinical use since 2012. The Insightec Exablate Neuro received U.S. FDA approval for unilateral thalamotomy to treat essential tremor in 2016. The indications for use were expanded to include tremor-dominant Parkinson's disease in 2018.^{86, 87} A thermal lesion generated within the ventral intermediate nucleus ablates misfiring neurons responsible for symptoms.⁸⁸ This system uses a hemispherical array of 1024 elements to deliver transcranial ultrasound corrected for phase aberrations caused by the skull. The corrections are determined through simulations based on computed tomography scans of the skull. In situ corrections are also applied based on MRI thermometry to track mild heating from sub-therapeutic test pulses. Thalamotomy to treat tremor syndromes targets volumes of 100–200 mm³ at maximum temperatures around 56°C.⁸⁹ A large clinical trial demonstrated significant improvement in hand tremor scores with persistence of 36 months.⁹⁰ The Exablate Neuro is in clinical trials underway for targeting pediatric tumors (NCT03028246) and gliomas (NCT03100474). A trial targeting other brain tumors with the Exablate Neuro system was recently completed (NCT01698437).

The Sonalleve system was designed to target uterine fibroids, and has been adapted to treat primary and secondary bone cancers.^{91, 92} Thermal ablation has also been approved for palliative care of bone metastases with the Sonalleve.⁹³ Focused ultrasound is applied to disrupt nerves endings on the periosteum of the diseased bone to alleviate pain, not the lesion itself.⁹⁴ Thermal ablation has been cleared for other benign musculoskeletal tumors, such as osteoid osteomas.⁹⁵ For patients with hypertension, thermal ablation is being applied to tissue surrounding the main renal arterial supply.^{96, 97} The goal of this trial is to decrease the over-activity of nerves and reduce blood pressure (NCT02649426). Multiple focused and unfocused systems have been developed and approved for prostate ablation.^98-102 A healthcare cost analysis indicated focused ultrasound had lower expenditures than radical prostatectomy and external beam radiotherapy as primary treatment options.¹⁰³ There is also ongoing work to treat liver,⁸⁵ kidney,⁸⁵ breast cancers,¹⁰⁴ thyroid nodules,¹⁰⁵ desmoid tumors,¹⁰⁶ and nerves¹⁰⁷ with thermal ablation.

Catheter-Based Ultrasound

Intravascular catheter devices are used commonly by physicians to perform minimally invasive procedures. Clinical studies have demonstrated the safety and efficacy of the EKOS system, an FDA-cleared ultrasound-assisted, catheter-directed thrombolysis device to treat pulmonary embolism.^108-110 The device emits cylindrically symmetric ultrasound pulses within the thrombus between 2.0 and 2.25 MHz fundamental frequency, 1.5 MPa peak negative pressure, and 15% duty cycle.¹¹¹ Thrombolytic therapy is administered through infusion ports in the catheter during insonation. A clinical study regarding the benefit of the EKOS system was inconclusive, potentially due to insufficient statistical power in the experimental design.¹¹² An ongoing, multinational/multicenter/randomized/controlled clinical trial is underway designed to provide sufficient data to compare EKOS with an anticoagulant with anticoagulant alone.¹¹³ In addition to pulmonary embolism, pre-clinical studies have indicated that the EKOS system provides effective drug delivery for the treatment of peri-stent restenosis.^{114, 115}.

Other catheter-based ultrasound systems have been developed to treat vascular calcifications. Severe vascular calcification can inhibit balloon expansion during angioplasty and stent placement in a stenotic vessel.¹¹⁶ An FDA-approved device has been developed for intravascular lithotripsy to disrupt calcifications mechanically.^117-121 An electric spark discharge produces ultrasound waves that interact with calcifications in an analogous fashion to lithotripsy for renal calcifications (see Kidney Stone Management section).¹²² The system has been applied to peripheral and coronary targets, each of which requires a different number of pulses for effective treatment.

Histotripsy

Mechanical bioeffects can also be used to ablate tissue. Histotripsy (histo: cells; tripsy: breaking) applies pulses 1 μs to 10 ms in duration with much higher spatial-peak, pulse-average intensities than used in thermal ablation to generate inertial cavitation (> 20 kW/cm² for histotripsy compared to 0.001 kW/cm² for thermal ablation).^{123, 124} Bubbles formed in the focal zone fractionate cells without heating the target.¹²⁵ There are multiple types of histotripsy, each of which use different mechanisms to generate bubbles.^{126, 127} Intrinsic-threshold histotripsy applies pulses of 1 cycle with a peak negative pressure that exceeds ~25 MPa.³¹ Bubbles are generated due to the tension of the ultrasound pulse. Shock-scattering histotripsy uses highly nonlinear pulses 3 to 20 cycles in duration, with peak negative pressures of 15 MPa or greater. A cloud of bubbles is formed due to interactions between the incident pulse, and the shock wave of the pulse that scatters from bubbles formed in the focal zone.¹²⁸ Boiling histotripsy relies on pulses 1–10 ms in duration that cause rapid shock wave-induced heating.¹²⁹ The increased temperature lowers the peak negative pressure required to cause bubble nucleation.¹³⁰ To date (2024), clinical trials are underway or have been completed to test the safety and technical success of histotripsy technology for the treatment of prostate tissue (NCT01896973), liver (NCT04572633), kidney (NCT05820087), pancreatic adenocarcinoma (NCT06282809), and calcified aortic stenosis (NCT03779620). Further, the FDA granted a de novo request for this technology in the treatment of lesions in the liver.

Pre-clinical studies have demonstrated that histotripsy sensitizes pathologies to other therapeutic approaches. Bubble activity decellularizes tissue, but it is not as effective for breaking down stiff extracellular structures.¹³¹ This property of histotripsy can be advantageous for targets that encompass major vessels with extensive collagen and fibrin.¹³² Extracellular structure can be problematic for applications like venous thrombosis, where fibrin will be undertreated and may serve as a nidus for re-thrombosis.¹³³ Combining histotripsy with a fibrinolytic drug has been shown to treat the cellular and extracellular clot components synergistically.¹³⁴ Histotripsy has been also shown to enhance the delivery of doxorubicin in a murine model of pancreatic cancer.¹³⁵

Systemic bioeffects have been observed when histotripsy is applied to free-flowing blood or venous thrombosis without sufficient anticoagulation. The mortality rate of swine in these studies was 45–50% compared to 0% when heparin was administered during insonation.^{136, 137} The precise cause of mortality in these studies is unknown. There were no cases of vascular perforation or pulmonary embolism. Histotripsy causes significant hemolysis,¹³⁴ which is a pathway for platelet activation and intravascular thrombosis.¹³⁸ Microclotting was observed in the heart and lung of pigs that expired during treatment,¹³⁷ consistent with platelet-induced intravascular thrombosis. Spontaneous thrombus formation may be beneficial when targeting certain lesions. Histotripsy has been applied successfully across the capsule of the liver and kidney without extraneous bleeding issues. This was accomplished when therapeutic and supratherapeutic dose of the antithrombotic warfarin were administered.¹³⁹ The lack of excessive bleeding was attributed in part due to thromboses in vessels that coincided with the treatment zone. The thrombus resolved over time, as evidenced by patent vessels in follow-up contrast-enhanced imaging.¹⁴⁰ Note that the antithrombotic warfarin is not an anti-platelet agent.¹³⁸

Kidney Stone Management

Physical Therapy

Ultrasound can be used to alleviate injuries that limit mobility or the performance of daily functions, such as osteoarthritis, soft tissue injuries, and myofascial pain. Unfocused ultrasound sources with low spatial-average, temporal-average intensities (0.02–2.5 W/cm²), heat-injured tissue to increase blood flow, and oxygen delivery to accelerate healing.^{166, 167} However, there is debate in the physical therapy community regarding the benefits of heating for these injuries.¹⁶⁸

Extracorporeal shock waves have been used to address musculoskeletal disorders such as plantar fasciitis and lateral epicondylitis. Success rates for shock wave therapy range from 30 to 90% in muscular applications (eg, plantar fasciitis, lateral epicondylitis, calcified tendinitis of the shoulder, and Achilles tendinopathy) and 50–85% in bone disorders (eg, non-union and delayed union of long bone fracture).¹⁶⁹ The therapeutic mechanisms of shock waves for benefit in physical therapy are not fully understood. The primary hypothesis is that shock waves cause interstitial and extracellular responses that promote tissue regeneration.¹⁷⁰ In addition to these direct effects of the shock wave, cavitation generated via boiling histotripsy has been investigated to address Achilles tendinopathy as an alternative to dry needling.¹⁷¹ Purely mechanical disruption was achieved with pulse durations of 0.1–1 ms in the form of fiber separation and fraying. A combination of thermal and mechanical damage was observed for longer pulse duration, indicating that a range of exposure parameters can be identified that achieve tendon disruption.

Cosmetic Ultrasound

Esthetic medicine focuses on improving the appearance of patients. Therapeutic claims described for ultrasound in cosmetic medicine include fat reduction,¹⁷² wrinkle smoothing,¹⁷³ tightening of loose skin areas,¹⁷⁴ stretch mark removal,¹⁷⁵ cellulite/orange-peel skin treatment,¹⁷⁶ spider veins, telangiectasis,¹⁷⁷ abnormal pigmentations, acne, and scar reduction.¹⁷⁸ Reported studies testing ultrasound for cosmetic purposes have often been industry sponsored. There have been no randomized controlled trials, although a few clinical studies have compared treated and untreated sites within subjects.¹⁷⁹ Most reports have been based on single-center studies ranging in size from 6 to 152 subjects. Use of analgesia with paracetamol, or of anesthesia, has been sporadically reported. Reported adverse effects range from mild transient pain, erythema and edema of skin lasting a few days,¹⁸⁰ to more prevalent and wide-ranging effects,¹⁸¹ including pain after treatment (75%), edema (75%), bruising (66%), pain during treatment (66%), tingling (60%), erythema (45%), and skin burns (2/152 = 1.3%) that can occur at the second-degree level (1/152 = 0.65%). Pain scores were higher when higher energy levels were used.¹⁸² In several studies where blood lipids and other liver function tests were examined, no anomalies were detected after treatment.^{183, 184} Some studies have reported the appearance of hard subcutaneous nodules, a burning sensation, mild blisters, and 1 case of purpuric lesions.^{183, 185, 186} In general, patients describe most adverse effects resolved spontaneously within 4–12 weeks.

Immnuno-oncology

The immunostimulatory effects induced by ultrasound also hold promise in cancer treatment.^187-190 There are 2 distinct translational strategies. The first strategy targets diseases with a poor response to existing immunotherapies (eg, breast, ovary, prostate, pancreas, and primary brain tumors). The goal of this approach is to initiate or modulate an anti-tumor immune response. The second strategy seeks to boost treatment efficacy or delivery in cancers responsive to immunotherapies (eg, melanoma, kidney, and liver).

Histotripsy, thermal ablation, and hyperthermia have been explored for their immunological impacts.⁷⁹ Approaches that use exogenous microbubble nucleation agents in combination with ultrasound have also been shown to promote an immune response.¹⁹¹ Mechanical ablation with histotripsy can initiate damage-associated molecular patterns (DAMP) release, modulate cytokine and chemokine profiles, and reduce pro-tumor immune cells.¹⁹² Increased CD8+ infiltration was found in murine subcutaneous melanoma tumor (B16GP33 cell line) following histotripsy relative to radiation therapy or radiofrequency ablation.¹⁹³ These outcomes were hypothesized to result from preservation of tumor-presenting antigens under histotripsy, in contrast to thermal ablation which can denature protein structures.¹⁹⁴ Changes in tumor oxygenation due to histotripsy may also contribute to the observed immune response. Assessment of neuroblastoma tumor following histotripsy exposure in a murine orthotopic model indicated mitigation of tumor hypoxia, a primary prognostic of disease resistance.¹⁹⁵ These shifts were attributed to the proliferation of vasculature surrounding the treatment zone, providing means for blood flow and reoxygenation in the targeted tumor.¹⁹⁶ Thermal treatments alter vascular permeability/perfusion, induce heat shock proteins and pro-inflammatory cytokines/chemokines, boost immune cell infiltration and the cytotoxic activity of natural killer and CD8+ T cells.⁷⁹ Partial ablation strategies during thermal ablation might further enhance immune cell infiltration.¹⁹⁷ These effects can vary with tumor type. For instance, fibrous tumors like pancreatic cancer may benefit from mechanical disruption of the extracellular matrix to facilitate immune cell penetration infiltration.¹⁹⁸ Focused ultrasound and microbubbles have been used for blood–brain barrier (BBB) opening, which induce inflammation to neurogenesis stimulation, and modulation of microglial structure.^199-202 Clinical studies have documented immuno-modulation of cancers, including liver,²⁰³ breast,^{204, 205} thyroid,²⁰⁶ and others.²⁰⁷ Notably, thermal ablation of breast cancer results in an increase of activated dendritic cells, macrophages, and B lymphocytes.^{205, 208} Combining sonodynamic therapy (see Sonodynamic Therapy section) with anti-PD1/PD-L1 therapies initiates a favorable adaptive immune response in multiple tumor models.²⁰⁹ More immuno-competent tumors with pre-existing anti-tumor immune cells may respond better to non-ablative, lower-power treatments that amplify cytokine signaling without harming infiltrative immune cells.²¹⁰ Some tumors with significant pro-tumorigenic infiltration might require more aggressive ablative methods.²⁰⁴

Control of local diseases and at distant sites (abscopal effect) has been reported in case reports for thermal ablation of pancreatic cancer²¹¹ and mechanical ablation of liver cancer.²¹² There is a lack of evidence that therapeutic ultrasound alone can have an immuno-stimulatory effect strong enough to be curative. Several combinatorial approaches have been tested in preclinical tumor models, including thermal ablation with toll-like receptor agonist CpG and anti-PD1 in breast tumor models.^{213, 214} Histotripsy has been tested in combination with anti-CTLA-4 and anti-PD-L1 for neuroblastoma,²¹⁵ with anti-PD1 and CAR T cells for brain tumors,²¹⁶ and anti-CTLA-4 or CD40 agonist for melanoma.^{193, 217} Clinical trials are ongoing to assess the potential of combination treatment, including thermal ablation with checkpoint inhibitors to treat breast cancer and solid tumors (NCT04116320), and ultrasound and microbubble BBB opening to deliver nivolumab in melanoma brain metastases (NCT04021420) or pembrolizumab for recurrent glioblastoma (NCT05879120). Partial thermal ablation of tumors is also being tested to promote immune responses for undifferentiated pleomorphic sarcoma (NCT04123535). Key unresolved issues include determining effective exposures to generate the intended bioeffect, the sequence for combined therapies, and methods to monitor the immunological impacts.

Skin Permeabilization

The skin presents a large and easily accessible pathway for the delivery of therapeutics to the body. Drugs that are hydrophilic or greater than 500 Daltons have limited penetration across the skin,^{218, 219} and require injection or surgical approaches for administration. Ultrasound has been shown to permeabilize the skin in both animal models and human skin,²²⁰ and for a wide variety of drugs ranging from molecules to microparticles.²²¹ Both thermal and cavitation effects have been indicated as a possible mechanism of action. Ultrasound frequencies from 100 kHz to greater than 10 MHz have been investigated, and may contribute to variability of cavitation from endogenous nuclei.^{222, 223} Exogenous microbubble nucleation agents are also employed to achieve the intended bioeffects (see Types of Exogenous Agents section).²²⁴ Ultrasound can be administered through either traditional piezoelectric transducers or wearable devices.²²⁵

Types of Exogenous Agents

Blood–Brain/Blood–Spinal Cord Barrier

First demonstrated in 2001,³⁰¹ focused ultrasound insonation of circulating microbubbles is capable of safely and reversibly increasing the permeability of the BBB and blood–spinal cord barrier (BSCB) using stable cavitation.^{302, 303} The BBB and BSCB exclude most drugs from extravasating into the brain or spinal cord, which complicates treatment for malignancies in these biological structures. Ultrasound-mediated BBB and BSCB disruption has enabled the delivery of multiple drugs, antibodies, genes, viruses, and even cells in pre-clinical animal models.^304-306

Ultrasound-mediated BBB disruption was first tested clinically to treat Alzheimer's disease in 2018.³⁰⁷ In this clinical trial, the primary goal was technical success of BBB disruption. The primary safety endpoints were met, which demonstrated the BBB could be reversibly disrupted without hemorrhage, swelling, or neurological deficits. No clinically significant change in patient cognition or function was observed. A pre-clinical study indicated BBB disruption alone is capable of reducing amyloid-β plaque burden in a mouse model of Alzheimer's disease.³⁰⁸ Reduction in amyloid-β has also been observed in patients for BBB opening only,^309-312 and when focused ultrasound was combined with aducanumab.³¹³ Imaging with 18F-florbetaben PET indicated the amyloid-β plaque burden was reduced in regions targeted with focused ultrasound relative to the control hemisphere after 26 weeks.

Several other clinical BBB opening studies are ongoing, including the delivery of chemotherapy for Her2-positive brain metastases (NCT03714243). Another trial is ongoing that seeks to deliver the synthetic enzyme Cerezyme to the brains of patients with Parkinson's disease (NCT04370665). A phase 1 trial is underway to test the feasibility of BBB opening to enhance the delivery of paclitaxel to the peritumoral brain of patients with recurrent glioblastoma with a device transplanted in the skull during surgical resection (NCT04528680). Pharmacokinetic analysis from the trial indicates that operation of the device to activate circulating microbubbles increased the concentration of albumin-bound paclitaxel in sonicated portions of the by a factor of 3 to 4, and carboplatin by a factor of 5 to 9.³¹⁴ These effects were achieved with no observed treatment-related neurological deficits, and closing of the BBB over the course of an hour following the procedure.

Clinical results of BBB opening reflect conservative exposure parameters (eg, pulsing parameters to minimize BBB disruption), with no reported serious adverse outcomes. Pre-clinical evidence encourages this caution. Disruption of the BBB can be accomplished across a wide range of exposure frequencies, peak negative pressures, pulse durations, and microbubble formulations. Off-target bioeffects have been observed at the upper range of peak negative pressures and microbubble concentrations investigated, including red blood cell extravasation, dilated blood vessels, astroglia scars, upregulation in the transcription of proinflammatory cytokines and chemokines, glial cell activation, macrophage infiltration, and modulation of microglial activities.²⁰² A mild and temporary inflammatory response was shown to promote the removal of cellular debris.³¹⁵ The relevance of this finding is uncertain given the use of an increased microbubble concentration of approximately 5- to 10-fold increased relative to other pre-clinical or clinical studies.^{313, 316}

Confirmation of safe exposure parameters can be achieved with MRI to determine the duration and spatial extent of BBB opening, and identify adverse events such as extravasation or bleeding.^{317, 318} To account for changes in the in situ acoustic field given the heterogeneity of the skull, patient-specific simulations can be performed based on pre-treatment computerized tomography (CT) data.³¹⁹ Real-time passive cavitation detection is also being investigated to gauge the efficacy and safety of BBB procedures.^{316, 320}

In addition to enhancing drug delivery, focused ultrasound-induced BBB disruption can be used for diagnostic purposes. Liquid biopsy has entered clinical practice to guide treatment for numerous tumor types,³²¹ but remains challenging for brain cancers due to the BBB.³²² The use of focused ultrasound to permeabilize the BBB is thought to enable “2-way trafficking,” both to enhance drug delivery as well as increase the prevalence of tumor-derived biomarkers in the bloodstream.³²³ Further, focused ultrasound provides spatiotemporal control to determine the location of suspicious lesions. This sonobiopsy method improves the detection of glioblastoma mutations in murine and porcine models^{324, 325} A first-in-human sonobiopsy trial with neuronavigation delivery to apply focused ultrasound indicated an enrichment in plasma circulating DNA in patients with high-grade glioma.³²⁶ Cavitation monitoring was used to mitigate any inertial activity,³²⁷ and there was no observable tissue damage in this trial.

Similar to the BBB, the BSCB prevents dissemination of therapeutics from the vasculature.³²⁸ This barrier is problematic for the 5% of patients with solid tumors that develop leptomeningeal metastases.³²⁹ Inspired by work on BBB disruption, investigators have developed methods to improve the delivery of therapeutics through BSCB via focused ultrasound-induced microbubble activity. Initial studies in a rodent model of leptomeningeal metastases demonstrated good suppression of tumor growth when trastuzumab was combined with focused ultrasound and microbubbles. Findings indicated that the tumor volume was reduced for the focused ultrasound group relative to trastuzumab alone.³³⁰ A particular challenge applying focused ultrasound to the BSCB is bone that surrounds the target, which cause standing waves that decrease targeting accuracy.³³¹ To overcome this challenge, specialized dual aperture sources that use short burst, phase keying exposures have been proposed to mitigate standing waves.³³² This approach has been shown to be successful for penetrating the BSCB in rodents and swine.^{302, 333}

Sonothrombolysis

Ultrasound has been explored as a means to expedite dissolution of a thrombus for the treatment of stroke, pulmonary embolism, myocardial infarction, and venous thrombosis.³³⁴ Depending on the exposure conditions, ultrasound can act either as a standalone therapy, or as adjuvant to thrombolytic drugs. Acoustic cavitation is a primary mechanism of action to enhance outcomes for sonothrombolysis therapies.³³⁵ Stable cavitation can act as a micro-pump to enhance the penetration of thrombolytic drugs into the thrombus while removing fibrin degradation products.³³⁶ Minimal changes have been observed in the clot burden for stable cavitation alone, demonstrating that thrombolytics are a necessary component for this treatment strategy.³³⁷ The ability of stable cavitation to enhance a thrombolytic drug depends on the clot subtype.³³⁸ Stable cavitation effectively enhances the action of thrombolytics for unretracted, porous clots. These outcomes enable a dose reduction of thrombolytic drugs,³³⁹ a key requirement to mitigate bleeding complications associated with thrombolytic therapy. Thrombolytic therapy is not improved via stable cavitation in clots characteristic of subacute or chronic disease. These aged thrombus subtypes are more resistant to thrombolytic therapy³⁴⁰ but may be prone to inertial cavitation.³⁴¹ Indeed, sonothrombolysis has been demonstrated to be effective in acute thrombus for histotripsy or histotripsy-like exposures and retracted thrombus.^{134, 342-345}

Multiple devices have been explored for sonothrombolysis, ranging from focused transducers to imaging probes with extended acoustic outputs.^346-348 Transcranial systems developed to treat stroke include unfocused, low-frequency systems for rapid treatment based on landmark-based targeting,^349-351 and MRI-guided focused transducers.³⁵² Venous thromboemboli have been treated with intravascular and extracorporeal systems. The EKOS endovascular system is used for pulmonary embolisms that are surrounded by bone and air-filled tissue.³⁵³ The system was cleared by the U.S. FDA in 2005, though this clearance did not include coadministration of a cavitation nucleation agent. Newer intravascular systems have been developed with an intention to co-deliver nanometer-sized droplets as bubble nucleation agents.^{354, 355} Extracorporeal systems are primarily developed for targeting deep vein thrombosis in the iliofemoral vasculature. Both spherically and cylindrically focused transducers have been produced for rapid targeting through electronic steering along the length of the vessel.^{347, 348}

Outcomes for clinical testing of sonothrombolysis techniques have been mixed. The “CLOTBUST-ER” trial (NCT01098981) found no difference at 90 days for stroke patients treated with standard-of-care thrombolytic alone with or without 2-MHz transcranial ultrasound exposure.³⁵⁶ The trial ended early because outcomes were not improved relative to the administration of thrombolytic alone. There were no issues in terms of patient safety, consistent with metanalysis of prior studies that used ultrasound to enhance thrombolytic drugs.³⁵⁷ It was hypothesized that the hands-free device designed for this study provided inadequate coverage of the thrombus. Similar outcomes were observed in the Norwegian Sonothrombolysis in Acute Stroke Study (NOR-SASS),³⁵⁸ which included the addition of a microbubble agent. High mechanical index diagnostic ultrasound pulses (5-μs duration, MI = 1.1–1.5, 1.8 MHz) in combination with microbubbles have been shown to restore epicardial and microvascular flow in acute ST-segment elevation myocardial infarction (NCT02410330).³⁵⁹ Patients who received ultrasound showed an improvement in terms of resolution of the ST-segment and infarct volume relative to percutaneous coronary intervention alone. These results suggest there is a role for sonothrombolysis, though technological developments are needed to ensure effective and safe microbubble activation.

Sonodynamic Therapy

The known capacity of inertial cavitation to produce rare chemical species has been investigated as a means to provide therapeutic benefit.³⁶⁰ Photosensitive agents that generate reactive oxygen species following exposure to light have been studied for over a century,³⁶¹ including to saturate tumors with cytotoxic chemicals (ie, photodynamic therapy). Sonodynamic therapy sought to provide an acoustic analog to photodynamic therapy,^{362, 363} capitalizing on the fact that several photosensitizing agents can also be activated by ultrasound.³⁶⁴ Unlike light, ultrasound can be applied to targets several centimeters deep, giving sonodynamic therapy a distinct advantage relative to photodynamic therapy.³⁶⁵ Common sonosensitizing agents include titanium dioxide,³⁶⁶ Rose Bengal,³⁶⁷ PpIX,³⁶⁸ and fluorescein.³⁶⁹ The inclusion of 5-aminolevulinic acid (5-ALA) increases the uptake of sonosensitizers like PpIX in proliferating cells such as glioma, thereby disrupting the heme pathway to promote apoptosis following ultrasound exposure.^370-372

The precise mechanism of activation for sonodynamic therapy remains under investigation,³⁷³ though light generation during inertial cavitation remains a common theme.³⁷⁴ To accentuate cavitation effects, several groups aim to develop moieties to co-deliver bubble nuclei with sonodynamic agents.^{287, 367} There are significant differences in gas content between microbubble contrast agents (eg, perfluorocarbon) and early studies into bubble-induced light emissions (eg, air with a need for noble gasses).³⁷⁵ Nevertheless, photons have been detected from ultrasound activation of microbubbles in vitro and in vivo.^{376, 377} Despite their lack of noble gasses, oxygen-loaded microbubbles have been shown to generate sufficient light to activate the sonodynamic agents.³⁷⁶ These findings demonstrate there is still a lack of understanding of the primary mechanisms of the process for light generation during cavitation. Given that a primary mechanism for sonodynamic therapy is the formation of reactive oxygen species, the therapy has been shown to have poor outcomes in hypoxic tumors that lack molecular oxygen.³⁷⁸ Hypoxia is a ubiquitous eventual outcome for all solid tumors.³⁷⁹ To ensure efficacy for sonodynamic therapy, oxygen-loaded microbubbles have been developed to titrate the targeted tissue to a normoxic state.³⁸⁰

There are several clinical trials that have tested sonodynamic therapy. A cohort of 3 patients with metastatic breast cancer were treated using a combination of sonodynamic and photodynamic therapy.³⁸¹ The targeted tumors displayed good regression, though patients experienced pain as the result of treatment. In a study with 115 patients, various cancer diagnoses were treated with sonodynamic and photodynamic therapy.³⁸² Outcomes with the combination approach were favorable, with an increase in median survival time and no adverse events. Positive outcomes were linked to an acute inflammatory response that activated the innate immune system. Additional trials are underway to test sonodynamic therapy for neuro-oncology applications (NCT06039709, NCT04845919, NCT04559685, NCT05362409, NCT05370508, and NCT05123534).

Sonoporation

Ultrasound has been shown to accelerate drug and gene transfection for a number of years.³⁸³ Compared with other transfection methods, sonoporation provides the best potential for clinical adoption due to its high specificity, deep tissue penetration, and low cost.³⁸⁴ The generation of transient pores in cells via cavitation is hypothesized to be the primary mechanism of action for sonoporation.³⁸⁵ Hence, microbubble contrast agents are a critical component for any sonoporation strategy. The precise mechanisms of pore formation are under investigation. Stable cavitation generates microstreaming that increases shear stresses near the cell wall,³⁸⁶ which has been associated with membrane pore generation.³⁸⁵ Inertial cavitation effects may also contribute, including shock waves or jetting from an asymmetric collapse.^387-389 The observed duration of permeabilization ranges from a few seconds up to 24 hours.³⁹⁰ In some cases, the pore may be permanent. For example, 1-MHz pulses of 10 cycles and 0.85-MPa peak negative pressure were applied to microbubbles affixed to MRC-5 fetal fibroblasts.³⁹¹ Openings less than 3 μm in diameter resealed the cell membrane, but pores larger than ~6 μm diameter persisted indefinitely. Beyond the physical mechanisms responsible for pore generation, the resulting increased penetration of molecules into the cell cytoplasm is also not fully understood. Hypotheses range from non-selective poration of the cell membrane, endocytosis stimulation, and large cell membrane wounds.³⁹⁰

There have been few updates to clinical adoption of sonoporation since 2012, though several new applications have emerged. Multiple studies have investigated sonoporation for mRNA and DNA vaccines delivery.^{392, 393} To facilitate vaccine delivery, designer microbubbles were developed with mRNA incorporated into the lipid shell.³⁹⁴ Ultrasound-induced microbubbles activity enhanced transfection by more than a factor of 40 relative to intramuscular injection, resulting in modification of genetic expression for more than 400 days.³⁹³ Sonoporation methods have been developed to load the cryoprotectant trehalose into erythrocytes, enabling storage of the formed blood elements at room temperature. The resulting product is a “freeze-dried blood” for transfusions. More than 95% of erythrocytes can be recovered following sonoporation, and ~90% after rehydration.³⁹⁵ Microfluidics devices are under development to perform sonoporation to accentuate trehalose transfection.³⁹⁶

Cavitation-Enabled Therapy for Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy is a life-threatening condition associated with progressive heart malfunction, which can lead to sudden death in otherwise healthy young athletes.³⁹⁷ Available treatment methods are invasive, including intra-cardiac surgery and alcohol ablation.^{398, 399} Inertial cavitation can reduce excessive heart muscle to address hypertrophic cardiomyopathy. Proof-of-concept for this approach was demonstrated in a rodent model using ultrasound pulses of 1.5 MHz fundamental frequency and 4 MPa peak negative pressure. These exposure conditions resulted in good cavitation, as visualized on B-mode grayscale with a 10 MHz imaging probe.^{400, 401} Therapeutic efficacy was indicated by premature ventricular contractions induced by the cardiomyocyte injury, and measurements of troponin.⁴⁰² Ultrasound pulses were ECG-gated to coincide with the end of systole, which resulted in the best production of premature complexes with minimal heart function disruption.⁴⁰³ The resulting effect was tissue reduction indicated on vital staining.⁴⁰⁴ The lesions healed within ~6 weeks with minor fibrin formation but effective heart function.⁴⁰⁵ Application of this approach on a genetic rat model of hypertrophic cardiomyopathy resulted in a 16% reduction in endocardial wall thickness, which was considered a clinically significant outcome.⁴⁰⁶

An undesirable consequence of the treatment was swelling in heavily treated volumes which led to infarction-like injury. Swelling associated with these off-target effects was substantially reduced by steroids, and cardiac fibrosis limited by losartan. The prevalence of excessive injury was reduced when the fractionating therapy occurred over the course of 3 treatment sessions separated by 1 week.⁴⁰⁷ These results demonstrate the promise of cavitation-enabled tissue reduction for hypertrophic cardiomyopathy, though additional tests are required prior to the initiation of a large-scale clinical study.

Radiosensitization

Therapeutic ultrasound in combination with microbubbles has been shown to increase the sensitivity of tumors to radiation therapy. Disruption of the BBB with ultrasound combined with systemic microbubbles increases regional blood flow and oxygenation.⁴⁰⁸ These conditions are necessary for the formation of free radical by radiation, and the resultant DNA damage.³⁷⁹ Radiotherapy combined with ultrasound-induced BBB opening was found to enhance tumor control and prolonged survival compared to radiotherapy alone in a murine model of glioblastoma.⁴⁰⁹ These results led to a prospective pilot study for patients with recurrent high-grade glioma that included reirradiation for disease control (NCT04988750). A BBB opening procedure was performed 2 hours prior to fractioned stereotactic radiosurgery or conventional radiotherapy. Analysis of the initial 6 patients indicated there were no BBB opening-related adverse events. One patient had a partial response after the combined procedure or an objective response rate of 16.7%.

Inertial cavitation also increases tumor sensitization to radiation therapy,⁴¹⁰ potentially via vascular damage.^411-417 Ultrasound-activated microbubbles damage endothelial cells and induce thrombosis, similar to outcomes of high-dose radiation. Acidic sphingomyelinase (ASMase) activation within the endothelium results in ceramide-induced cell death and rapid apoptosis.^{413, 417-420} This process enhances low (less than 6 Gy) and high (more than 8 Gy) radiation doses to provide a synergistic approach to cancer treatment.^{412, 413, 416, 417, 419, 421, 422} These results indicate endothelial cell death due to vascular shutdown and direct canonical DNA damage are primary contributors to ultrasound-enhanced radiotherapy.

Insights from preclinical studies on ultrasound vascular ablation have led to several clinical trials. A pilot clinical trial (NCT03199274) explored ultrasound-triggered microbubble destruction to enhance the treatment response in hepatocellular carcinoma patients undergoing transarterial radioembolization.⁴²³ Ultrasound pulses with a 1.5 MHz fundamental frequency, 2.3 μs duration, and mechanical index of 1.13 were applied at a rate of 100 Hz for several minutes prior to radioembolization. A higher tumor response rate was noted in participants who received microbubble destruction compared to those who received radioembolization alone. No significant complications were reported in this study. A first-in-human, phase 1 prospective interventional trial (NCT04431674) of 8 breast cancer patients assessed fractionated radiotherapy combined with focused ultrasound-microbubble treatment (800 kHz fundamental frequency, 570 kPa peak negative pressure, and 16-cycle tone burst over a 50 ms period repeated for 5–10 minutes). The ultrasound microbubble treatment was applied an hour prior to the first and fifth radiotherapy fractions.⁴²⁴ Seven patients experienced a complete response at the treated site over 3 or more months of follow-up. There were no late effects related to radiotherapy or toxicity from the ultrasound-microbubble treatment, indicating a significant potential for radiosensitization. These results have prompted another clinical trial underway to test ultrasound microbubble destruction to radiosensitize head and neck cancers (NCT04431648).

Mechanical Ablation

Histotripsy relies on peak negative pressures in excess of 25 MPa to generate spontaneous bubbles for tissue ablation (see Histotripsy section).¹²⁶ Such pressures can be difficult to achieve in situ due to factors such as aberration and complex acoustic paths.^425-427 To mitigate these issues, nanodroplets have been explored to lower the threshold for bubble nucleation. Formulations of nanodroplets to target micro-metastases were developed and tested in vitro.⁴²⁸ Initial studies found a significant decrease in the threshold for bubble cloud formation with ~200 nm diameter nanodroplets (~10 MPa) compared to without the droplets (~28 MPa) for 500-kHz pulses of 2 cycle duration.²²⁸ The droplet transition threshold was found to increase with frequency for single-cycle pulses,⁴²⁹ in contrast to measurements with non-histotripsy insonations.²⁴² These results suggest homogenous nucleation of the perfluorocarbon liquid under histotripsy,⁴²⁹ whereas heating via superharmonic focusing is a mechanism for non-histotripsy droplet transition.^{257, 430} A larger number of pulses was required for complete treatment with nanodroplets relative to insonation without droplets, indicating exogenous nuclei lower the efficiency of histotripsy relative to intrinsic nuclei. Other nuclei for histotripsy have also been investigated, including nanocones and microbubbles.^{226, 431, 432} Nanobubbles combined with low frequency, low amplitude ultrasound (~80 kHz fundamental frequency, ~250 kPa peak negative pressure) have been shown to produce mechanical ablation in vitro and in vivo.^{282, 433, 434}

Ultrasound Imaging

Magnetic Resonance Imaging

Computed Tomography

Hounsfield units are a relative quantitative measurement of radio density.⁵¹⁷ There is temperature dependence for Hounsfield units, which make CT a potential method to monitor thermal therapies.⁵¹⁸ Real-time monitoring with CT is not common due to the associated ionizing radiation. The primary use of CT is to assess ablation outcomes. Similar to MRI, regions of ablation appear as hypointense on contrast CT acquired with a 3-phase sequence.^{436, 519} There can be a rim of enhancement surrounding the primary ablation zone indicative of benign physiologic response to thermal injury. The enhancing rim is usually resolved over the course of 1 month,⁵²⁰ and the hypointense ablation zone involutes as tissue recovers from the treatment.⁵¹⁹

A further use of CT is for treatment planning of transcranial or spinal procedures with multi-element phased array sources. The approximate mapping between Hounsfield units and acoustic properties are included in calculations to estimate the in situ acoustic field.⁵²¹ The calculation is used to develop appropriate targeting strategies to account for aberration through appropriate phasing of each element of the therapeutic source.⁵²² Information about aberration collected with CT can also be used to correct passive imaging for treatment monitoring, and hence better localization of cavitation.⁴⁵⁹

There can be limitations with identifying lesions with the imaging probe coaxial to the therapy source, particularly for deep-seated targets. To address this limitation, methods that use computed tomography have been developed in the pre-treatment planning phase for histotripsy procedures.⁵²³ This approach uses a well-characterized phantom to determine the transfer functions between a cone-beam CT system and robotic arm use for placement of the histotripsy source. Once registered, the robotic positioner for the histotripsy system places the transducers within to target regions under cone-beam CT with 1.4 ± 0.5 mm precision.

Tissue-Mimicking Materials

Therapeutic ultrasound systems are often calibrated or evaluated with tissue-mimicking materials (TMMs). Compared to animal and human studies, experiments with TMMs are relatively inexpensive, easier to conduct, more reproducible, and have fewer regulatory hurdles. For most applications, it is important that TMMs model the attenuation coefficient and speed of sound to approximate the propagation of ultrasound correctly. The nonlinearity coefficient should be considered for TMMs used to test high Gol'dberg number conditions,⁵²⁴ including thermal ablation,⁵²⁵ histotripsy,⁵²⁶ and lithotripsy.¹⁴³ Quantitative acoustic parameters (eg, backscatter coefficient) should be properly addressed to evaluate an ultrasound-guided system.⁴⁷⁰ For MRI-guided therapies, the longitudinal relaxation time (T1) and effective spin–spin-relaxation time (T2*) should be replicated.^{527, 528}

Quality Assurance

Clinical-focused ultrasound systems are tested on a regular basis to identify possible malfunctions prior to patient treatment. Several publications document quality assurance (QA) protocols for single-institution studies.^548-550 A report by the American Association of Physicists in Medicine (AAPM) Task Group 241 includes recommendations for periodic testing of MRI-guided focused ultrasound systems.^{551, 552} The recommendations are divided into categories according to the frequency of inspection. Tests that are conducted daily or before each patient treatment include transducer focusing capability, transducer steering, imaging signal-to-noise ratio, safety interlock evaluation, visual check of equipment for damage, and inspection of the coupling membrane. Tests to be conducted every 6 months or every twenty patients include motor system evaluation, table positioning and homing capability, the accuracy of MRI thermometry, planning/delivery software function evaluation, cavitation detection, and degassing system. Tests that should be conducted annually or every 100 patients include acoustic output power with a radiation force balance and ultrasound beam characterization with a hydrophone.⁵⁵¹

The AAPM Task Group 333 was formed in 2019 to develop a more detailed MRI-guided focused ultrasound systems QA protocol that should be executed before each patient treatment. Further, the protocol was tested in an interlaboratory comparison study. The protocol included specifications for a new phantom that was custom-designed for MRI-guided focused ultrasound systems. Detailed methods for measurements of peak temperature rise, targeting error, signal-to-noise ratio, and assessing the thermal ablation volume were included as part of data acquisition required for the protocol. Five institutes were included in the interlaboratory comparison study, each with a variety of commercial and custom therapy systems. The study was completed in 2023, and processing of collected data was underway at the time this manuscript was written (June 2024).

Reporting Therapeutic Ultrasound Treatment Parameters

Recommendations have been developed to document therapeutic ultrasound studies to promote experimental reproducibility.⁵⁵³ There are separate sets of recommendations for clinical and preclinical studies to provide flexibility for differences in expertise and equipment among institutions. The reporting recommendations include descriptions of driving electronics, transducer, hydrophone-based acoustic output characterization, numerical simulations, monitoring methods, and the intended bioeffect.

Measurements of the acoustic field with a hydrophone remain a mainstay parameter needed to ensure the reproducibility of therapeutic ultrasound studies. The effective size of a hydrophone element can be much larger than its geometric size, particularly at low frequencies.⁵⁵⁴ Methods have been developed to compensate for spatial averaging across the hydrophone sensitive element and for hydrophone frequency-dependent sensitivity,⁵⁵⁵ including for broadband therapeutic ultrasound pressure pulses.^555-559 The broadband nature of therapeutic ultrasound pulses is due to nonlinear propagation, resulting in energy at the fundamental frequency of the excitation (f₀) and harmonic frequencies (nf₀, where n = 1,2,3, …n_max, where n_max can exceed 100).^{130, 560} The −3 dB beamwidths of the harmonic beam components of the therapeutic ultrasound acoustic field decrease with n. Critical values of n can exist for an excitation with harmonic beamwidths smaller than the sensitive element of the hydrophone (Figure 5). The resulting effect is equivalent to the application of a low pass filter to pressure waveforms recorded by the hydrophone. To address the need for spatial averaging correction, an inverse-filter method has been shown to improve the accuracy for hydrophone measurements of focused ultrasound pressure fields.⁵⁶¹ This method was adopted into an International Electrotechnical Commission (IEC) standard.⁵⁶² To facilitate implementation of the spatial averaging correction, a simplified method was developed that is equivalent to the full model.^{563, 564}

Numerical Simulations

The acoustic output for operational driving conditions of therapeutic sources can be sufficient to either damage hydrophones or cause inaccurate measurements due to cavitation or excessive heating. Numerical simulations have been employed to address the gap in field measurements. Modeling may provide a better estimate of the field than measurements for some conditions due to limitations in the hydrophone bandwidth or spatial averaging effects.¹³⁰ The IEC has released technical specification TS62556 to provide guidelines in the use of numerical calculations to report a subset of field parameters for therapeutic ultrasound devices.^565-568

There are multiple open-source codes available to calculate the linear,⁵⁶⁹ nonlinear,^{570, 571} and shocked^{560, 572} acoustic fields and pressure waveforms of ultrasound sources (Table 2). Some codes also calculate Pennes bioheat transfer equation in parallel.^{566, 573, 574} These codes can often be run with a high-level computing language (eg, MATLAB) to facilitate ease of use. The assumptions integrated into calculations for each model should be considered when analyzing data.⁵⁷⁵ For example, sources that are highly focused (eg, f-number less than 1.2) require additional considerations to address diffraction correctly.⁵⁷² Increased computational power may be required in the form of workstations or clusters for highly nonlinear waveforms that include shock waves.⁵⁷⁶ Differences among models to predict acoustic fields are unknown. The development of benchmarks for numerical computations is a topic being reviewed by IEC Technical Committee 87.

Patient Safety

Precautions for MRI-guided focused ultrasound procedures have been reviewed previously at length.⁵⁵¹ Briefly, therapeutic ultrasound transducers can potentially apply excessive heat to superficial tissues between the source and the intended target.^{551, 577, 578} This problem can be ameliorated by cycling the transducer power to allow for adequate cooling between exposures,⁵⁷⁹ circulating cooling water,⁵⁸⁰ or using cooling balloons.^{581, 582}

Sub-therapeutic exposures can be performed to verify the accuracy of targeting. Extra care should be taken to avoid collateral exposure to nearby tissues that are thermally sensitive, such as nerves, bones, and bowel. Cavitation detection should be used to minimize the risk of undesired mechanical bioeffects.⁵⁵¹

The standard to promote patient safety for therapeutic ultrasound procedures (IEC 60601-2-62) does not allow: 1) display of incorrect numerical values associated with the therapy; 2) production of unwanted ultrasound output; 3) production of excessive ultrasound output; 4) reflection of excessive ultrasonic power at the transducer/patient interface due to inadequate coupling; 5) unwanted targeting of tissue regions away from the intended target region; and 6) production of unwanted thermal or mechanical tissue damage in or distal to the region of interest.