Combination of Focused Ultrasound, Immunotherapy, and Chemotherapy: New Perspectives in Breast Cancer Therapy.

Focused ultrasound is a treatment modality increasingly used for diverse therapeutic applications, and currently approved for several indications, including prostate cancers and uterine fibroids. But what about breast cancer? Breast cancer is the most common and deadliest cancer in women worldwide. While there are different treatment strategies available, there is a need for development of more effective and personalized modalities, with fewer side effects. Therapeutic ultrasound is such an option, and this review summarizes the state of the art in their use for the treatment of breast cancer and evaluate potentials of novel treatment approaches combining therapeutic ultrasound, immuno- and chemo-therapies.

A simulation study on the sensitivity of transcranial ray-tracing ultrasound modeling to skull properties.

In transcranial focused ultrasound therapies, such as treating essential tremor via thermal ablation in the thalamus, acoustic energy is focused through the skull using a phased-array transducer. Ray tracing is a computationally efficient method that can correct skull-induced phase aberrations via per-element phase delay calculations using patient-specific computed tomography (CT) data. However, recent studies show that variations in CT-derived Hounsfield unit may account for only 50% of the speed of sound variability in human skull specimens, potentially limiting clinical transcranial ultrasound applications. Therefore, understanding the sensitivity of treatment planning methods to material parameter variations is essential. The present work uses a ray-tracing simulation model to explore how imprecision in model inputs, arising from clinically significant uncertainties in skull properties or considerations of acoustic phenomena, affects acoustic focusing quality through the skull. We propose and validate new methods to optimize ray-tracing skull simulations for clinical treatment planning, relevant for predicting intracranial target's thermal rise, using experimental data from ex-vivo human skulls.

Sonodynamic therapy for gliomas.

INTRODUCTION: Glioma remains incurable and a life limiting disease with an urgent need for effective therapies. Sonodynamic therapy (SDT) involves systemic delivery of non-toxic chemical agents (sonosensitizers) that accumulate in tumor cells or environment and are subsequently activated by exposure to low-frequency ultrasound to become cytotoxic agents. Herein, we discuss proposed mechanisms of action of SDT and provide recommendation for future research and clinical applications of SDT for gliomas. METHODS: Review of literature of SDT in glioma cell cultures and animal models published in Pubmed/MEDLINE before January, 2021. RESULTS: Different porphyrin and xanthene derivatives have proven to be effective sonosensitizers. Generation of reactive oxygen species and free radicals from water pyrolysis or sonosensitizers, or physical destabilization of cell membrane, have been identified as mechanisms of SDT leading to cell death. Numerous studies across glioma cell lines using various sonosensitizers and ultrasound parameters have documented tumoricidal effects of SDT. Studies in small animal glioma xenograft models have also consistently documented that SDT is associated with improved tumor control and longer survival of animals treated with SDT while avoiding damage of surrounding brain. There are no clinical trials completed to date regarding safety and efficacy of SDT in patients harboring gliomas, but some are beginning. CONCLUSIONS: Pre-clinical studies cell cultures and animal models indicate that SDT is a promising treatment approach for gliomas. Further studies should define optimal sonication parameters and sonosensitizers for gliomas. Clinical trials of SDT in patients harboring gliomas and other malignant brain tumors are currently underway.

Preparation of perfluorocarbon emulsions by premix membrane emulsification for Acoustic Droplet Vaporization (ADV) in biomedical applications.

Perfluorocarbon (PFC) droplets are used in acoustic droplet vaporization (ADV), a phenomenon where droplets vaporize into gas microbubbles under exposure to ultrasound. The size and the size distribution of a phase change contrast agent is an important factor in determining the ADV threshold and the biodistribution. Thus, high throughout manufacturing of uniform-sized droplets, required to maintain spatial control of the vaporization process, remains challenging. This work describes a parametric evaluation of a novel process using premix membrane emulsification (PME) to produce homogeneous PFC emulsions at high rate with moderate pressure using Shirasu Porous Glass (SPG) membranes. In this study, we investigated the effect of several process parameters on the resulting pressure and droplet size: membrane pore size, flow rate, and dispersed phase type. The functionality of the manufactured emulsions for ADV was also demonstrated. Vaporization of the PFC emulsions was obtained using an imaging ultrasound transducer at 7.813 MHz, and the ADV thresholds were determined. Here, the pressure threshold for ADV was determined to be 1.49 MPa for uniform-sized perfluorohexane (PFHex) droplets with a mean size of 1.51 µm and a sharp distribution (CV and span respectively of 20% and 0.6). Thus, a uniform-sized droplet showed a more homogeneous vaporization with a uniform response in the focal region of the transducer. Indeed, polydispersed droplets had a more diffuse response outside the focal region due to the presence of large droplets that vaporize at lower energies. The ADV threshold of uniform-sized PFC droplets was found to decrease with the droplet diameter and the bulk fluid temperature, and to increase with the boiling temperature of PFC and the presence of an oil layer surrounding the PFC core.

Investigation of the tumoricidal effects of sonodynamic therapy in malignant glioblastoma brain tumors.

OBJECTIVE: Glioblastoma is the most common primary brain tumor; survival is typically 12-18 months after diagnosis. We sought to study the effects of sonodynamic therapy (SDT) using 5-Aminolevulinic acid hydrochloride (5-ALA) and high frequency focused ultrasound (FUS) on 2 glioblastoma cell lines. PROCEDURE: Rat C6 and human U87 glioblastoma cells were studied under the following conditions: 1 mM 5-ALA (5-ALA); focused ultrasound (FUS); 5-ALA and focused ultrasound (SDT); control. Studied responses included cell viability using an MTT assay, microscopic changes using phase contract microscopy, apoptotic induction through a caspase-3 assay, and apoptosis staining to quantify cell death. RESULTS: SDT led to a marked decrease in cell extension and reduction in cell size. For C6, the MTT assay showed reductions in cell viability for 5-ALA, FUS, and SDT groups of 5%, 16%, and 47%, respectively compared to control (p < 0.05). Caspase 3 induction in C6 cells relative to control showed increases of 109%, 110%, and 278% for 5-ALA, FUS, and SDT groups, respectively (p < 0.05). For the C6 cells, caspase 3 staining positivity was 2.1%, 6.7%, 11.2%, and 39.8% for control, 5-ALA, FUS, and SDT groups, respectively. C6 Parp-1 staining positivity was 1.9%, 6.5%, 9.0%, and 37.8% for control, 5-ALA, FUS, and SDT groups, respectively. U87 cells showed similar responses to the treatments. CONCLUSIONS: Sonodynamic therapy resulted in appreciable glioblastoma cell death as compared to 5-ALA or FUS alone. The approach couples two already FDA approved techniques in a novel way to treat the most aggressive and malignant of brain tumors. Further study of this promising technique is planned.

Stimulation of Bone Repair with Ultrasound.

This chapter reviews the different options available for the use of ultrasound in the enhancement of fracture healing or in the reactivation of a failed healing process: LIPUS, shock waves and ultrasound-mediated delivery of bioactive molecules, such as growth factors or plasmids. The main emphasis is on LIPUS, or Low Intensity Pulsed Ultrasound, the most widespread and studied technique. LIPUS has pronounced bioeffects on tissue regeneration, while employing intensities within a diagnostic range. The biological response to LIPUS is complex as the response of numerous cell types to this stimulus involves several pathways. Known to-date mechanotransduction pathways involved in cell responses include MAPK and other kinases signaling pathways, gap-junctional intercellular communication, up-regulation and clustering of integrins, involvement of the COX-2/PGE2 and iNOS/NO pathways, and activation of the ATI mechanoreceptor. Mechanisms at the origin of LIPUS biological effects remain intriguing, and analysis is hampered by the diversity of experimental systems used in-vitro. Data point to clear evidence that bioeffects can be modulated by direct and indirect mechanical effects, like acoustic radiation force, acoustic streaming, propagation of surface waves, heat, fluid-flow induced circulation and redistribution of nutrients, oxygen and signaling molecules. One of the future engineering challenge is therefore the design of dedicated experimental set-ups allowing control of these different mechanical phenomena, and to relate them to biological responses. Then, the derivation of an 'acoustic dose' and the cross-calibration of the different experimental systems will be possible. Despite this imperfect knowledge of LIPUS biophysics, the clinical evidence, although most often of low quality, speaks in favor of the clinical use of LIPUS, when the economics of nonunion and the absence of toxicity of this ultrasound technology are taken into account.

Acceleration of ultrasound thermal therapy by patterned acoustic droplet vaporization.

One application of acoustic droplet vaporization (ADV), a method of converting biocompatible microdroplets into microbubbles, is to enhance locally high intensity focused ultrasound (HIFU) therapy. Two objectives are pursued here: (1) the controlled creation of a bubble trench prior to HIFU using ADV and (2) use of the trench for increasing ablation volumes, lowering acoustic powers, and decreasing therapy duration. Thermally responsive phantoms were made with perfluorocarbon emulsion. Compound lesions were formed in a laboratory setting and a clinical magnetic resonance imaging (MRI)-guided HIFU system. Linear and spiral patterned compound lesions were generated in trenches. A larger fraction of the HIFU beam is contained to increase the generation of heat. Using the laboratory system, a 90 mm linear length spiral trench was formed in 30 s with mechanical beam steering. Comparatively, the clinical HIFU system formed a 19.9 mm linear length spiral trench in approximately 1 s with electronic beam steering. Lesions were imaged optically and with MRI. A uniform thermal ablation volume of 3.25 mL was achieved in 55.4 s (4-times faster than standard clinical HIFU and 14-times larger volume versus sum of individual lesions). Single lesions showed a 400% volume increase.

Radiosensitization of Allogenic Subcutaneous C6 Glioma Model with Focused Ultrasound-Induced Mild Hyperthermia.

The radiosensitization potential of focused ultrasound (FUS)-induced mild hyperthermia was assessed in an allogenic subcutaneous C6 glioma tumor model in rats. Mild hyperthermia at 42 °C was induced in tumors using a single-element 350 kHz FUS transducer. Radiation was delivered with a small animal radiation research platform using a single-beam irradiation technique. The combined treatment involved 20 min of FUS hyperthermia immediately before radiation. Tumor growth changes were observed one week post-treatment. A radiation dose of 2 Gy alone showed limited tumor control (30% reduction). However, when combined with FUS hyperthermia, there was a significant reduction in tumor growth compared to other treatments (tumor volumes: control-1174 ± 554 mm3, FUS-HT-1483 ± 702 mm3, 2 Gy-609 ± 300 mm3, FUS-HT + 2 Gy-259 ± 186 mm3; ANOVA p < 0.00001). Immunohistological analysis suggested increased DNA damage as a short-term mechanism for tumor control in the combined treatment. In conclusion, FUS-induced mild hyperthermia can enhance the effectiveness of radiation in a glioma tumor model, potentially improving the outcome of standard radiation treatments for better tumor control.

Breast mass characterization using 3-dimensional automated ultrasound as an adjunct to digital breast tomosynthesis: a pilot study.

OBJECTIVES: The purpose of this study was to retrospectively evaluate the effect of 3-dimensional automated ultrasound (3D-AUS) as an adjunct to digital breast tomosynthesis (DBT) on radiologists' performance and confidence in discriminating malignant and benign breast masses. METHODS: Two-view DBT (craniocaudal and mediolateral oblique or lateral) and single-view 3D-AUS images were acquired from 51 patients with subsequently biopsy-proven masses (13 malignant and 38 benign). Six experienced radiologists rated, on a 13-point scale, the likelihood of malignancy of an identified mass, first by reading the DBT images alone, followed immediately by reading the DBT images with automatically coregistered 3D-AUS images. The diagnostic performance of each method was measured using receiver operating characteristic (ROC) curve analysis and changes in sensitivity and specificity with the McNemar test. After each reading, radiologists took a survey to rate their confidence level in using DBT alone versus combined DBT/3D-AUS as potential screening modalities. RESULTS: The 6 radiologists had an average area under the ROC curve of 0.92 for both modalities (range, 0.89-0.97 for DBT and 0.90-0.94 for DBT/3D-AUS). With a Breast Imaging Reporting and Data System rating of 4 as the threshold for biopsy recommendation, the average sensitivity of the radiologists increased from 96% to 100% (P > .08) with 3D-AUS, whereas the specificity decreased from 33% to 25% (P > .28). Survey responses indicated increased confidence in potentially using DBT for screening when 3D-AUS was added (P < .05 for each reader). CONCLUSIONS: In this initial reader study, no significant difference in ROC performance was found with the addition of 3D-AUS to DBT. However, a trend to improved discrimination of malignancy was observed when adding 3D-AUS. Radiologists' confidence also improved with DBT/3DAUS compared to DBT alone.

Theranostics in the vasculature: bioeffects of ultrasound and microbubbles to induce vascular shutdown.

Ultrasound-triggered microbubbles destruction leading to vascular shutdown have resulted in preclinical studies in tumor growth delay or inhibition, lesion formation, radio-sensitization and modulation of the immune micro-environment. Antivascular ultrasound aims to be developed as a focal, targeted, non-invasive, mechanical and non-thermal treatment, alone or in combination with other treatments, and this review positions these treatments among the wider therapeutic ultrasound domain. Antivascular effects have been reported for a wide range of ultrasound exposure conditions, and evidence points to a prominent role of cavitation as the main mechanism. At relatively low peak negative acoustic pressure, predominantly non-inertial cavitation is most likely induced, while higher peak negative pressures lead to inertial cavitation and bubbles collapse. Resulting bioeffects start with inflammation and/or loose opening of the endothelial lining of the vessel. The latter causes vascular access of tissue factor, leading to platelet aggregation, and consequent clotting. Alternatively, endothelium damage exposes subendothelial collagen layer, leading to rapid adhesion and aggregation of platelets and clotting. In a pilot clinical trial, a prevalence of tumor response was observed in patients receiving ultrasound-triggered microbubble destruction along with transarterial radioembolization. Two ongoing clinical trials are assessing the effectiveness of ultrasound-stimulated microbubble treatment to enhance radiation effects in cancer patients. Clinical translation of antivascular ultrasound/microbubble approach may thus be forthcoming.

Low-Cost Thermochromic Quality Assurance Phantom for Therapeutic Ultrasound Devices: A Proof of Concept.

High-intensity focused ultrasound (HIFU) transducer acoustic output can vary over time as a result of an inconsistent power supply, damage to the transducer or deterioration over time. Therefore, easy implementation of a daily quality assurance (DQA) method is of great importance for pre-clinical research and clinical applications. We present here a thermochromic material-based phantom validated by thermal simulations and found to provide repeatable visual power output assessments in fewer than 15 s that are accurate to within 10%. Whereas current available methods such as radiation force balance measurements provide an estimate of the total acoustic power, we explain here that the thermochromic phantom is sensitive to the shape of the acoustic field at focus by changing the aperture of a multi-element transducer with a fixed acoustic power. The proposed phantom allows the end user to visually assess the transducer's functionality without resorting to expensive, time-consuming hydrophone measurements or image analysis.

Interrogating the CD27:CD70 axis in αCD40-dependent control of pancreatic adenocarcinoma.

Immune checkpoint blockade immunotherapy has radically changed patient outcomes in multiple cancer types. Pancreatic cancer is one of the notable exceptions, being protected from immunotherapy by a variety of mechanisms, including the presence of a dense stroma and immunosuppressive myeloid cells. Previous studies have demonstrated that CD40 stimulation can remodel the tumor microenvironment in a manner that promotes effector immune cell responses and can cooperate with immune checkpoint inhibition for durable tumor control mediated by T cells. Here we confirm the capability of this combination therapy to dramatically, and durably, control pancreatic cancer growth in an orthotopic model and that the immune memory to this cancer is primarily a function of CD4+ T cells. We extend this understanding by demonstrating that recruitment of recently primed T cells from the draining lymph nodes is not necessary for the observed control, suggesting that the pre-existing intra-tumoral cells respond to the combination therapy. Further, we find that the efficacy of CD40 stimulation is not dependent upon CD70, which is commonly induced on dendritic cells in response to CD40 agonism. Finally, we find that directly targeting the receptor for CD70, CD27, in combination with the TLR3 agonist polyIC, provides some protection despite failing to increase the frequency of interferon gamma-secreting T cells.

Guidelines for immunological analyses following focused ultrasound treatment.

Focused ultrasound (FUS) is a powerful emerging tool for non-invasive, non-ionizing targeted destruction of tumors. The last two decades have seen a growing body of preclinical and clinical literature supporting the capacity of FUS to increase nascent immune responses to tumors and to potentiate cancer immunotherapies (e.g. checkpoint inhibitors) through a variety of means, including immune modulation and drug delivery. With the rapid acceleration of this field and a multitude of FUS immunotherapy clinical trials having now been deployed worldwide, there is a need to streamline and standardize the methodology for immunological analyses field-wide. Recently, the Focused Ultrasound Foundation and Cancer Research Institute partnered to convene a group of over 85 leaders to discuss the nexus of FUS and immuno-oncology. The guidelines documented herein were assembled in response to recommendations that emerged from this discussion, emphasizing the urgent need for heightened accessibility of immune analysis methods and standardized protocols unique to the field. These guidelines are designated for existing stakeholders in the FUS immuno-oncology domain or those newly entering the field, to provide guidance on collection, storage, and immunological profiling of tissue or blood specimens in the context of FUS immunotherapy studies, and additionally offer templates for standardized deployment of these methods based on collective experience gained within the field to date. These guidelines are tumor-agnostic and provide evidence-based, consensus-based recommendations for both preclinical and clinical immune analysis of tissue and blood specimens.

Recommendations for Reporting Therapeutic Ultrasound Treatment Parameters.

These recommendations are intended to provide guidance and to encourage best practice in reporting therapeutic ultrasound treatment parameters. Detailed uniform reporting will allow testing of therapy ultrasound systems and protocols, cross-comparison of studies between different teams using different systems and validation of therapeutic bio-effects. These recommendations have been divided into two sets, one for clinical and one for preclinical studies, each with stratified reporting categories, to account for the disparities in expertise and access to equipment between sites. The recommendations are intended to be useful for clinicians and researchers, for ethical and funding review boards and for the editors and reviewers of scientific journals.

Low-Cost 3-D Hydrophone Scanning Tank with MATLAB GUI Control.

The Focused Ultrasound Foundation has developed a low-cost, validated, open-source hydrophone scanner for the spatial characterization of ultrasound transducers. Assembly instructions and a MATLAB control graphical user interface are provided such that the device can be easily replicated for less than $1000 in roughly 40 person-hours. The low-cost scanning tank's performance was compared with data collected with a commercial automated scanning tank. Pressure measurements of a focused transducer and a planar transducer had less than a 10% difference between the two scanning systems. Two-dimensional automated scans (20 × 20 mm at 0.25-mm resolution) took the low-cost scanning tank 45 min compared with the commercial system's 30 min. A reproducibility study found that the low-cost scanner made consistent peak negative pressure measurements as reflected by the low coefficient of variation for both focused (1.88%) and planar (0.98%) transducers. The low-cost scanner described here is a viable alternative for ultrasound laboratories needing efficient, accurate characterization of ultrasound transducers.

Enhanced Stable Cavitation and Nonlinear Acoustic Properties of Poly(butyl cyanoacrylate) Polymeric Microbubbles after Bioconjugation.

Microbubbles (MB) are used as ultrasound (US) contrast agents in clinical settings because of their ability to oscillate upon exposure to acoustic pulses and generate nonlinear responses with a stable cavitation profile. Polymeric MB have recently attracted increasing attention as molecular imaging probes and drug delivery agents based on their tailorable acoustic responses, high drug loading capacity, and surface functionalization capabilities. While many of these applications require MB to be functionalized with biological ligands, the impact of bioconjugation on polymeric MB cavitation and acoustic properties remains poorly understood. Hence, we here evaluated the effects of MB shell hydrolysis and subsequent streptavidin conjugation on the acoustic behavior of poly(butyl cyanoacrylate) (PBCA) MB. We show that upon biofunctionalization, MB display higher acoustic stability, stronger stable cavitation, and enhanced second harmonic generation. Furthermore, functionalized MB preserve the binding capabilities of streptavidin conjugated on their surface. These findings provide insights into the effects of bioconjugation chemistry on polymeric MB acoustic properties, and they contribute to improving the performance of polymer-based US imaging and theranostic agents.

Evaluation of Pseudorandom Sonications for Reducing Cavitation With a Clinical Neurosurgery HIFU Device.

Transcranial high-intensity focused ultrasound is used in clinics for treating essential tremor (ET) and proposed for many other brain disorders. This promising treatment modality requires high energy resulting eventually in undesired cavitation and potential side effects. The goals of the present work were: 1) to evaluate the potential increase of the cavitation threshold using pseudorandom gated sonications and 2) to assess the heating capabilities with such sonications. The experiments were performed with the transcranial magnetic resonance (MR)-compatible ExAblate Neuro system (InSightec, Haifa, Israel) operating at a frequency of 670 kHz, either in continuous wave (CW) or with pseudorandom gated sonications of 50% duty cycle. Cavitation activity with the two types of sonications was compared using chemical dosimetry of hydroxyl radical production at the focus of the transducer, after propagation in water or through a human skull. Heating trials were performed in a hydrogel tissue-mimicking material embedded in a human skull to mimic a clinical situation. The temperature was measured by MR-thermometry when focusing at the geometrical focus and steering off focus up to 15 mm. Compared with CW sonications, the use of gated sonication did not affect the efficiency (60%) nor the steering abilities of the transducer. After propagation through a human skull, gated sonication required a higher pressure level (10 MPa) to initiate cavitation as compared with CW (5.8 MPa). Moreover, at equivalent acoustic power above the cavitation threshold, the level of cavitation activity initiated with gated sonications was much lower with gated sonication than with continuous sonications, almost half after propagation through water and one-third after propagation through a skull. This lowered cavitation activity may be attributed to a breaking of the dynamic of the bubbles moving from monochromatic to more broadband sonications and to the removal of residual cavitation nuclei between pulses with gated sonications. The heating capability was not affected by the gated sonications, and similar temperature increases were reached at focus with both types of sonications when sonicating at equivalent acoustic power, both in water or after propagation through a human skull (+15 °C at 325 W for 10 s). These data, acquired with a clinical system, suggest that gated sonication could be an alternative to continuous sonications when cavitation onset is an issue.

Quantitative analysis of in-vivo microbubble distribution in the human brain.

Microbubbles (MB) are widely used as contrast agents to perform contrast-enhanced ultrasound (CEUS) imaging and as acoustic amplifiers of mechanical bioeffects incited by therapeutic-level ultrasound. The distribution of MBs in the brain is not yet fully understood, thereby limiting intra-operative CEUS guidance or MB-based FUS treatments. In this paper we describe a robust platform for quantification of MB distribution in the human brain, allowing to quantitatively discriminate between tumoral and normal brain tissues and we provide new information regarding real-time cerebral MBs distribution. Intraoperative CEUS imaging was performed during surgical tumor resection using an ultrasound machine (MyLab Twice, Esaote, Italy) equipped with a multifrequency (3-11 MHz) linear array probe (LA332) and a specific low mechanical index (MI < 0.4) CEUS algorithm (CnTi, Esaote, Italy; section thickness, 0.245 cm) for non-destructive continuous MBs imaging. CEUS acquisition is started by enabling the CnTI PEN-M algorithm automatically setting the MI at 0.4 with a center frequency of 2.94 MHz-10 Hz frame rate at 80 mm-allowing for continuous non-destructive MBs imaging. 19 ultrasound image sets of adequate length were selected and retrospectively analyzed using a custom image processing software for quantitative analysis of echo power. Regions of interest (ROIs) were drawn on key structures (artery-tumor-white matter) by a blinded neurosurgeon, following which peak enhancement and time intensity curves (TICs) were quantified. CEUS images revealed clear qualitative differences in MB distribution: arteries showed the earliest and highest enhancement among all structures, followed by tumor and white matter regions, respectively. The custom software built for quantitative analysis effectively captured these differences. Quantified peak intensities showed regions containing artery, tumor or white matter structures having an average MB intensity of 0.584, 0.436 and 0.175 units, respectively. Moreover, the normalized area under TICs revealed the time of flight for MB to be significantly lower in brain tissue as compared with tumor tissue. Significant heterogeneities in TICs were also observed within different regions of the same brain lesion. In this study, we provide the most comprehensive strategy for accurate quantitative analysis of MBs distribution in the human brain by means of CEUS intraoperative imaging. Furthermore our results demonstrate that CEUS imaging quantitative analysis enables discernment between different types of brain tumors as well as regions and structures within the brain. Similar considerations will be important for the planning and implementation of MB-based imaging or treatments in the future.

Ultrasound Molecular Imaging for the Guidance of Ultrasound-Triggered Release of Liposomal Doxorubicin and Its Treatment Monitoring in an Orthotopic Prostatic Tumor Model in Rat.

Liposome encapsulation of drugs is an interesting approach in cancer therapy to specifically release the encapsulated drug at the desired treatment site. In addition to thermo-, pH-, light-, enzyme- or redox-responsive liposomes, which have had promising results in (pre-) clinical studies, ultrasound-triggered sonosensitive liposomes represent an exciting alternative to locally trigger the release from these cargos. Localized drug release requires precise tumor visualization to produce a targeted and ultrasound stimulus. We used ultrasound molecular imaging (USMI) with BR55, a vascular endothelial growth factor receptor 2 (VEGFR2)-targeted ultrasound contrast agent, to guide ultrasound-triggered release of sonosensitive liposomes encapsulating doxorubicin (L-DXR) in an orthotopic prostatic rodent tumor model. Forty-eight hours after L-DXR injection, local release of doxorubicin was triggered with a confocal ultrasound device with two focused transducers, 1.1-MHz center frequency, and peak positive and negative pressures of 20.5 and 13 MPa at focus. Tumor size decreased by 20% in 2 wk with L-DXR alone (n = 9) and by 70% after treatment with L-DXR and confocal ultrasound (n = 7) (p < 0.01). The effect of doxorubicin on perfusion/vascularity and VEGFR2 expression was evaluated by USMI and immunohistochemistry of CD31 and VEGFR2 and did not reveal differences in perfusion or VEGFR2 expression in the absence or after the triggered release of liposomes. USMI can provide precise guidance for ultrasound-triggered release of liposomal doxorubicin mediated by a confocal ultrasound device; moreover, the combination of B-mode imaging and USMI can help to follow the response of the tumor to the therapy.