Magneto-acoustic protein nanostructures for non-invasive imaging of tissue mechanics in vivo.

Measuring cellular and tissue mechanics inside intact living organisms is essential for interrogating the roles of force in physiological and disease processes. Current agents for studying the mechanobiology of intact, living organisms are limited by poor light penetration and material stability. Magnetomotive ultrasound is an emerging modality for real-time in vivo imaging of tissue mechanics. Nonetheless, it has poor sensitivity and spatiotemporal resolution. Here we describe magneto-gas vesicles (MGVs), protein nanostructures based on gas vesicles and magnetic nanoparticles that produce differential ultrasound signals in response to varying mechanical properties of surrounding tissues. These hybrid nanomaterials significantly improve signal strength and detection sensitivity. Furthermore, MGVs enable non-invasive, long-term and quantitative measurements of mechanical properties within three-dimensional tissues and in vivo fibrosis models. Using MGVs as novel contrast agents, we demonstrate their potential for non-invasive imaging of tissue elasticity, offering insights into mechanobiology and its application to disease diagnosis and treatment.

Remote control of mechanochemical reactions under physiological conditions using biocompatible focused ultrasound.

External control of chemical reactions in biological settings with spatial and temporal precision is a grand challenge for noninvasive diagnostic and therapeutic applications. While light is a conventional stimulus for remote chemical activation, its penetration is severely attenuated in tissues, which limits biological applicability. On the other hand, ultrasound is a biocompatible remote energy source that is highly penetrant and offers a wide range of functional tunability. Coupling ultrasound to the activation of specific chemical reactions under physiological conditions, however, remains a challenge. Here, we describe a synergistic platform that couples the selective mechanochemical activation of mechanophore-functionalized polymers with biocompatible focused ultrasound (FUS) by leveraging pressure-sensitive gas vesicles (GVs) as acousto-mechanical transducers. The power of this approach is illustrated through the mechanically triggered release of covalently bound fluorogenic and therapeutic cargo molecules from polymers containing a masked 2-furylcarbinol mechanophore. Molecular release occurs selectively in the presence of GVs upon exposure to FUS under physiological conditions. These results showcase the viability of this system for enabling remote control of specific mechanochemical reactions with spatiotemporal precision in biologically relevant settings and demonstrate the translational potential of polymer mechanochemistry.

Ultrasound-controllable engineered bacteria for cancer immunotherapy.

Rapid advances in synthetic biology are driving the development of genetically engineered microbes as therapeutic agents for a multitude of human diseases, including cancer. The immunosuppressive microenvironment of solid tumors, in particular, creates a favorable niche for systemically administered bacteria to engraft and release therapeutic payloads. However, such payloads can be harmful if released outside the tumor in healthy tissues where the bacteria also engraft in smaller numbers. To address this limitation, we engineer therapeutic bacteria to be controlled by focused ultrasound, a form of energy that can be applied noninvasively to specific anatomical sites such as solid tumors. This control is provided by a temperature-actuated genetic state switch that produces lasting therapeutic output in response to briefly applied focused ultrasound hyperthermia. Using a combination of rational design and high-throughput screening we optimize the switching circuits of engineered cells and connect their activity to the release of immune checkpoint inhibitors. In a clinically relevant cancer model, ultrasound-activated therapeutic microbes successfully turn on in situ and induce a marked suppression of tumor growth. This technology provides a critical tool for the spatiotemporal targeting of potent bacterial therapeutics in a variety of biological and clinical scenarios.

Acoustically triggered mechanotherapy using genetically encoded gas vesicles.

Recent advances in molecular engineering and synthetic biology provide biomolecular and cell-based therapies with a high degree of molecular specificity, but limited spatiotemporal control. Here we show that biomolecules and cells can be engineered to deliver potent mechanical effects at specific locations inside the body through ultrasound-induced inertial cavitation. This capability is enabled by gas vesicles, a unique class of genetically encodable air-filled protein nanostructures. We show that low-frequency ultrasound can convert these biomolecules into micrometre-scale cavitating bubbles, unleashing strong local mechanical effects. This enables engineered gas vesicles to serve as remotely actuated cell-killing and tissue-disrupting agents, and allows genetically engineered cells to lyse, release molecular payloads and produce local mechanical damage on command. We demonstrate the capabilities of biomolecular inertial cavitation in vitro, in cellulo and in vivo, including in a mouse model of tumour-homing probiotic therapy.

Ultrasensitive ultrasound imaging of gene expression with signal unmixing.

Acoustic reporter genes (ARGs) that encode air-filled gas vesicles enable ultrasound-based imaging of gene expression in genetically modified bacteria and mammalian cells, facilitating the study of cellular function in deep tissues. Despite the promise of this technology for biological research and potential clinical applications, the sensitivity with which ARG-expressing cells can be visualized is currently limited. Here we present burst ultrasound reconstructed with signal templates (BURST)-an ARG imaging paradigm that improves the cellular detection limit by more than 1,000-fold compared to conventional methods. BURST takes advantage of the unique temporal signal pattern produced by gas vesicles as they collapse under acoustic pressure above a threshold defined by the ARG. By extracting the unique pattern of this signal from total scattering, BURST boosts the sensitivity of ultrasound to image ARG-expressing cells, as demonstrated in vitro and in vivo in the mouse gastrointestinal tract and liver. Furthermore, in dilute cell suspensions, BURST imaging enables the detection of gene expression in individual bacteria and mammalian cells. The resulting abilities of BURST expand the potential use of ultrasound for non-invasive imaging of cellular functions.

Achieving Spatial and Molecular Specificity with Ultrasound-Targeted Biomolecular Nanotherapeutics.

The precise targeting of cells in deep tissues is one of the primary goals of nanomedicine. However, targeting a specific cellular population within an entire organism is challenging due to off-target effects and the need for deep tissue delivery. Focused ultrasound can reduce off-targeted effects by spatially restricting the delivery or action of molecular constructs to specific anatomical sites. Ultrasound can also increase the efficiency of nanotherapeutic delivery into deep tissues by enhancing the permeability of tissue boundaries, promoting convection, or depositing energy to actuate cellular activity. In this review we focus on the interface between biomolecular engineering and focused ultrasound and describe the applications of this intersection in neuroscience, oncology, and synthetic biology. Ultrasound can be used to trigger the transport of therapeutic payloads into a range of tissues, including specific regions of the brain, where it can be targeted with millimeter precision through intact skull. Locally delivered molecular constructs can then control specific cells and molecular pathways within the targeted region. When combined with viral vectors and engineered neural receptors, this technique enables noninvasive control of specific circuits and behaviors. The penetrant energy of ultrasound can also be used to more directly actuate micro- and nanotherapeutic constructs, including microbubbles, vaporizable nanodroplets, and polymeric nanocups, which nucleate cavitation upon ultrasound exposure, leading to local mechanical effects. In addition, it was recently discovered that a unique class of acoustic biomolecules-genetically encodable nanoscale protein structures called gas vesicles-can be acoustically "detonated" as sources of inertial cavitation. This enables the targeted disruption of selected cells within the area of insonation by gas vesicles that are engineered to bind cell surface receptors. It also facilitates ultrasound-triggered release of molecular payloads from engineered therapeutic cells heterologously expressing intracellular gas vesicles. Finally, focused ultrasound energy can be used to locally elevate tissue temperature and activate temperature-sensitive proteins and pathways. The elevation of temperature allows noninvasive control of gene expression in vivo in cells engineered to express thermal bioswitches. Overall, the intersection of biomolecular engineering, nanomaterials and focused ultrasound can provide unparalleled specificity in controlling, modulating, and treating physiological processes in deep tissues.

SUSHI: Sparsity-Based Ultrasound Super-Resolution Hemodynamic Imaging.

Identifying and visualizing vasculature within organs and tumors has major implications in managing cardiovascular diseases and cancer. Contrast-enhanced ultrasound scans detect slow-flowing blood, facilitating noninvasive perfusion measurements. However, their limited spatial resolution prevents the depiction of microvascular structures. Recently, super-localization ultrasonography techniques have surpassed this limit. However, they require long acquisition times of several minutes, preventing the detection of hemodynamic changes. We present a fast super-resolution method that exploits sparsity in the underlying vasculature and statistical independence within the measured signals. Similar to super-localization techniques, this approach improves the spatial resolution by up to an order of magnitude compared to standard scans. Unlike super-localization methods, it requires acquisition times of only tens of milliseconds. We demonstrate a temporal resolution of ~25 Hz, which may enable functional super-resolution imaging deep within the tissue, surpassing the temporal resolution limitations of current super-resolution methods, e.g., in neural imaging. The subsecond acquisitions make our approach robust to motion artifacts, simplifying in vivo use of super-resolution ultrasound.

Biomolecular Ultrasound and Sonogenetics.

Visualizing and modulating molecular and cellular processes occurring deep within living organisms is fundamental to our study of basic biology and disease. Currently, the most sophisticated tools available to dynamically monitor and control cellular events rely on light-responsive proteins, which are difficult to use outside of optically transparent model systems, cultured cells, or surgically accessed regions owing to strong scattering of light by biological tissue. In contrast, ultrasound is a widely used medical imaging and therapeutic modality that enables the observation and perturbation of internal anatomy and physiology but has historically had limited ability to monitor and control specific cellular processes. Recent advances are beginning to address this limitation through the development of biomolecular tools that allow ultrasound to connect directly to cellular functions such as gene expression. Driven by the discovery and engineering of new contrast agents, reporter genes, and bioswitches, the nascent field of biomolecular ultrasound carries a wave of exciting opportunities.

Improved Contrast-Enhanced Power Doppler Using a Coherence-Based Estimator.

While plane-wave imaging can improve the performance of power Doppler by enabling much longer ensembles than systems using focused beams, the long-ensemble averaging of the zero-lag autocorrelation R(0) estimates does not directly decrease the mean noise level, but only decreases its variance. Spatial variation of the noise due to the time-gain compensation and the received beamforming aperture ultimately limits sensitivity. In this paper, we demonstrate that the performance of power Doppler imaging can be improved by leveraging the higher lags of the autocorrelation [e.g., R(1), R(2),…] instead of the signal power (R(0)). As noise is completely uncorrelated from pulse-to-pulse while the flow signal remains correlated significantly longer, weak signals just above the noise floor can be made visible through the reduction of the noise floor. Finally, as coherence decreases proportionally with respect to velocity, we demonstrate how signal coherence can be targeted to separate flows of different velocities. For instance, we show how long-time-range coherence of microbubble contrast-enhanced flow specifically isolates slow capillary perfusion (as opposed to conduit flow).

Fast Vascular Ultrasound Imaging With Enhanced Spatial Resolution and Background Rejection.

Ultrasound super-localization microscopy techniques presented in the last few years enable non-invasive imaging of vascular structures at the capillary level by tracking the flow of ultrasound contrast agents (gas microbubbles). However, these techniques are currently limited by low temporal resolution and long acquisition times. Super-resolution optical fluctuation imaging (SOFI) is a fluorescence microscopy technique enabling sub-diffraction limit imaging with high temporal resolution by calculating high order statistics of the fluctuating optical signal. The aim of this work is to achieve fast acoustic imaging with enhanced resolution by applying the tools used in SOFI to contrast-enhance ultrasound (CEUS) plane-wave scans. The proposed method was tested using numerical simulations and evaluated using two in-vivo rabbit models: scans of healthy kidneys and VX-2 tumor xenografts. Improved spatial resolution was observed with a reduction of up to 50% in the full width half max of the point spread function. In addition, substantial reduction in the background level was achieved compared to standard mean amplitude persistence images, revealing small vascular structures within tumors. The scan duration of the proposed method is less than a second while current super-localization techniques require acquisition duration of several minutes. As a result, the proposed technique may be used to obtain scans with enhanced spatial resolution and high temporal resolution, facilitating flow-dynamics monitoring. Our method can also be applied during a breath-hold, reducing the sensitivity to motion artifacts.

Functional Flow Patterns and Static Blood Pooling in Tumors Revealed by Combined Contrast-Enhanced Ultrasound and Photoacoustic Imaging.

Alterations in tumor perfusion and microenvironment have been shown to be associated with aggressive cancer phenotypes, raising the need for noninvasive methods of tracking these changes. Dynamic contrast-enhanced ultrasound (DCEUS) and photoacoustic (PA) imaging serve as promising candidates-one has the ability to measure tissue perfusion, whereas the other can be used to monitor tissue oxygenation and hemoglobin concentration. In this study, we investigated the relationship between the different functional parameters measured with DCEUS and PA imaging, using two morphologically different hind-limb tumor models and drug-induced alterations in an orthotopic breast tumor model. Imaging results showed some correlation between perfusion and oxygen saturation maps and the ability to sensitively monitor antivascular treatment. In addition, DCEUS measurements revealed different vascular densities in the core of specific tumors compared with their rims. Noncorrelated perfusion and hemoglobin concentration measurements facilitated discrimination between blood lakes and necrotic areas. Taken together, our results illustrate the utility of a combined contrast-enhanced ultrasound method with photoacoustic imaging to visualize blood flow patterns in tumors. Cancer Res; 76(15); 4320-31. ©2016 AACR.

Co-option of Liver Vessels and Not Sprouting Angiogenesis Drives Acquired Sorafenib Resistance in Hepatocellular Carcinoma.

BACKGROUND: The anti-angiogenic Sorafenib is the only approved systemic therapy for advanced hepatocellular carcinoma (HCC). However, acquired resistance limits its efficacy. An emerging theory to explain intrinsic resistance to other anti-angiogenic drugs is 'vessel co-option,' ie, the ability of tumors to hijack the existing vasculature in organs such as the lungs or liver, thus limiting the need for sprouting angiogenesis. Vessel co-option has not been evaluated as a potential mechanism for acquired resistance to anti-angiogenic agents. METHODS: To study sorafenib resistance mechanisms, we used an orthotopic human HCC model (n = 4-11 per group), where tumor cells are tagged with a secreted protein biomarker to monitor disease burden and response to therapy. Histopathology, vessel perfusion assessed by contrast-enhanced ultrasound, and miRNA sequencing and quantitative real-time polymerase chain reaction were used to monitor changes in tumor biology. RESULTS: While sorafenib initially inhibited angiogenesis and stabilized tumor growth, no angiogenic 'rebound' effect was observed during development of resistance unless therapy was stopped. Instead, resistant tumors became more locally infiltrative, which facilitated extensive incorporation of liver parenchyma and the co-option of liver-associated vessels. Up to 75% (±10.9%) of total vessels were provided by vessel co-option in resistant tumors relative to 23.3% (±10.3%) in untreated controls. miRNA sequencing implicated pro-invasive signaling and epithelial-to-mesenchymal-like transition during resistance development while functional imaging further supported a shift from angiogenesis to vessel co-option. CONCLUSIONS: This is the first documentation of vessel co-option as a mechanism of acquired resistance to anti-angiogenic therapy and could have important implications including the potential therapeutic benefits of targeting vessel co-option in conjunction with vascular endothelial growth factor receptor signaling.

Denoising of Contrast-Enhanced Ultrasound Cine Sequences Based on a Multiplicative Model.

BACKGROUND: Speckle noise is an inherent characteristic of dynamic contrast-enhanced ultrasound (DCEUS) movies and ultrasound images in general. Speckle noise considerably reduces the quality of these images and limits their clinical use. Currently, temporal compounding and maximum intensity persistence (MIP) are among the most widely accepted processing methods enabling the visualization of vasculature using DCEUS. GOAL: A different approach has been used in this study, in order to improve the noise removal, while enabling the investigation of CEUS dynamics. METHODS: A multiplicative model for the formation of DCEUS speckled images is adopted and the log-transformed cines are processed. A preprocessing step was performed, locally removing low value outliers. Due to the fast-changing spatial distribution of microbubbles inside the vasculature, the noise in consecutive DCEUS frames is independent, facilitating its removal by temporal denoising. Noise reduction is efficiently achieved by wavelet denoising, in which the signal's wavelet coefficients are thresholded and small-value noise-related coefficients are discarded. The main advantage of using wavelet denoising in the present context is its ability to estimate ultrasound contrast agents' (UCA) concentration over time adaptively, without assuming a model or predefining the signal's degree of smoothness. The performance of wavelet denoising was compared against MIP, temporal compounding, and Log-normal model fitting. RESULTS: Phantom experiments showed improved SNR, using wavelet denoising over a wide range of UCA concentrations (MicroMarker, 0.001-1%). In the in vivo tests, improved noise removal was achieved, reflected by a significantly lower coefficient of variation in homogeneous vascular regions (p < 0.01).

Lysyl oxidase-like-2 promotes tumour angiogenesis and is a potential therapeutic target in angiogenic tumours.

Lysyl oxidase-like 2 (LOXL2), a secreted enzyme that catalyzes the cross-linking of collagen, plays an essential role in developmental angiogenesis. We found that administration of the LOXL2-neutralizing antibody AB0023 inhibited bFGF-induced angiogenesis in Matrigel plug assays and suppressed recruitment of angiogenesis promoting bone marrow cells. Small hairpin RNA-mediated inhibition of LOXL2 expression or inhibition of LOXL2 using AB0023 reduced the migration and network-forming ability of endothelial cells, suggesting that the inhibition of angiogenesis results from a direct effect on endothelial cells. To examine the effects of AB0023 on tumour angiogenesis, AB0023 was administered to mice bearing tumours derived from SKOV-3 ovarian carcinoma or Lewis lung carcinoma (LLC) cells. AB0023 treatment significantly reduced the microvascular density in these tumours but did not inhibit tumour growth. However, treatment of mice bearing SKOV-3-derived tumours with AB0023 also promoted increased coverage of tumour vessels with pericytes and reduced tumour hypoxia, providing evidence that anti-LOXL2 therapy results in the normalization of tumour blood vessels. In agreement with these data, treatment of mice bearing LLC-derived tumours with AB0023 improved the perfusion of the tumour-associated vessels as determined by ultrasonography. Improved perfusion and normalization of tumour vessels after treatment with anti-angiogenic agents were previously found to improve the delivery of chemotherapeutic agents into tumours and to result in an enhancement of chemotherapeutic efficiency. Indeed, treatment with AB0023 significantly enhanced the anti-tumourigenic effects of taxol. Our results suggest that inhibition of LOXL2 may prove beneficial for the treatment of angiogenic tumours.