Critical Advances for Democratizing Ultrasound Diagnostics in Human and Veterinary Medicine.

The democratization of ultrasound imaging refers to the process of making ultrasound technology more accessible. Traditionally, ultrasound imaging has been predominately used in specialized medical facilities by trained professionals. Advancements in technology and changes in the health-care landscape have inspired efforts to broaden the availability of ultrasound imaging to various settings such as remote and resource-limited areas. In this review, we highlight several key factors that have contributed to the ongoing democratization of ultrasound imaging, including portable and handheld devices, recent advancements in technology, and training and education. Examples of diagnostic point-of-care ultrasound (POCUS) imaging used in emergency and critical care, gastroenterology, musculoskeletal applications, and other practices are provided for both human and veterinary medicine. Open challenges and the future of POCUS imaging are presented, including the emerging role of artificial intelligence in technology development.

Super-Resolution Ultrasound Imaging for Monitoring the Therapeutic Efficacy of a Vascular Disrupting Agent in an Animal Model of Breast Cancer.

OBJECTIVE: Evaluate the use of super-resolution ultrasound (SRUS) imaging for the early detection of tumor response to treatment using a vascular-disrupting agent (VDA). METHODS: A population of 28 female nude athymic mice (Charles River Laboratories) were implanted with human breast cancer cells (MDA-MB-231, ATCC) in the mammary fat pad and allowed to grow. Ultrasound imaging was performed using a Vevo 3100 scanner (FUJIFILM VisualSonics Inc) equipped with the MX250 linear array transducer immediately before and after receiving bolus injections of a microbubble (MB) contrast agent (Definity, Lantheus Medical Imaging) via the tail vein. Following baseline ultrasound imaging, VDA drug (combretastatin A4 phosphate, CA4P, Sigma Aldrich) or control saline was injected via the placed catheter. After 4 or 24 hours, repeat ultrasound imaging along the same tumor cross-section occurred. Direct intratumoral pressure measurements were obtained using a calibrated sensor. All raw ultrasound data were saved for offline processing and SRUS image reconstruction using custom MATLAB software (MathWorks Inc). From a region encompassing the tumor space and the entire postprocessed ultrasound image sequence, time MB count (TMC) curves were generated in addition to traditional SRUS maps reflecting MB enumeration at each pixel location. Peak enhancement (PE) and wash-in rate (WIR) were extracted from these TMC curves. At termination, intratumoral microvessel density (MVD) was quantified using tomato lectin labeling of patent blood vessels. RESULTS: SRUS images exhibited a clear difference between control and treated tumors. While there was no difference in any group parameters at baseline (0 hour, P > .09), both SRUS-derived PE and WIR measurements in tumors treated with VDA exhibited significant decreases by 4 (P = .03 and P = .05, respectively) and 24 hours (P = .02 and P = .01, respectively), but not in control group tumors (P > .22). Similarly, SRUS derived microvascular maps were not different at baseline (P = .81), but measures of vessel density were lower in treated tumors at both 4 and 24 hours (P < .04). An inverse relationship between intratumoral pressure and both PE and WIR parameters were found in control tumors (R2 > .09, P < .03). CONCLUSION: SRUS imaging is a new modality for assessing tumor response to treatment using a VDA.

H-Scan Ultrasound Monitoring of Breast Cancer Response to Chemotherapy and Validation With Diffusion-Weighted Magnetic Resonance Imaging.

OBJECTIVES: H-scan ultrasound (US) imaging is a novel tissue characterization technique to detect apoptosis-induced changes in cancer cells after the initiation of effective drug treatment. The objective of the proposed research was to assess the sensitivity of 3-dimensional (3D) H-scan US technique for monitoring the response of breast cancer-bearing animals to neoadjuvant chemotherapy and correlate results to diffusion-weighted magnetic resonance imaging (DW-MRI) measurements of programmed cancer cell death. METHODS: Experimental studies used female mice (N = 18) implanted with human breast cancer cells. Animals underwent H-scan US and DW-MRI imaging on days 0, 1, 3, 7, and 10. After imaging at day 0, breast tumor-bearing nude mice were treated biweekly with an apoptosis-inducing drug. Texture analysis of H-scan US images explored spatial relationships between local US scattering. At day 10, H-scan measurements were compared with DW-MRI-derived apparent diffusion coefficient (ADC) values and histological findings. RESULTS: H-scan US imaging of low and high dose cisplatin-treated cancer-bearing animals revealed changes in image intensity suggesting a progressive decrease in aggregate US scatterer size that was not observed in control animals. Longitudinal trends discovered in H-scan US result matched with texture analysis and DW-MRI (P < .01). Further, analysis of the H-scan US image intensity and corresponding DW-MRI-derived ADC values revealed a strong linear correlation (R2  = .93, P < .001). These changes were due to cancer cell apoptotic activity and consider as early detectable biomarker during treatment. CONCLUSIONS: The 3D H-scan technique holds promise for assisting clinicians in monitoring the early response of breast cancer tumor to neoadjuvant chemotherapy and adding value to traditional diagnostic ultrasound examinations.

Intercellular Arc Signaling Regulates Vasodilation.

Injury responses require communication between different cell types in the skin. Sensory neurons contribute to inflammation and can secrete signaling molecules that affect non-neuronal cells. Despite the pervasive role of translational regulation in nociception, the contribution of activity-dependent protein synthesis to inflammation is not well understood. To address this problem, we examined the landscape of nascent translation in murine dorsal root ganglion (DRG) neurons treated with inflammatory mediators using ribosome profiling. We identified the activity-dependent gene, Arc, as a target of translation in vitro and in vivo Inflammatory cues promote local translation of Arc in the skin. Arc-deficient male mice display exaggerated paw temperatures and vasodilation in response to an inflammatory challenge. Since Arc has recently been shown to be released from neurons in extracellular vesicles (EVs), we hypothesized that intercellular Arc signaling regulates the inflammatory response in skin. We found that the excessive thermal responses and vasodilation observed in Arc defective mice are rescued by injection of Arc-containing EVs into the skin. Our findings suggest that activity-dependent production of Arc in afferent fibers regulates neurogenic inflammation potentially through intercellular signaling.SIGNIFICANCE STATEMENT Nociceptors play prominent roles in pain and inflammation. We examined rapid changes in the landscape of nascent translation in cultured dorsal root ganglia (DRGs) treated with a combination of inflammatory mediators using ribosome profiling. We identified several hundred transcripts subject to rapid preferential translation. Among them is the immediate early gene (IEG) Arc. We provide evidence that Arc is translated in afferent fibers in the skin. Arc-deficient mice display several signs of exaggerated inflammation which is normalized on injection of Arc containing extracellular vesicles (EVs). Our work suggests that noxious cues can trigger Arc production by nociceptors which in turn constrains neurogenic inflammation in the skin.

Formulation and Characterization of Chemically Cross-linked Microbubble Clusters.

The purpose of this study is to introduce a new concept of chemically cross-linked microbubble clusters (CCMCs), which are individual microbubble ultrasound contrast agents (UCAs) physically tethered together. We demonstrate a facile means of their production, characterize their size and stability, and describe how they can potentially be used in biomedical applications. By tethering UCAs together into CCMCs, we propose that novel methods of ultrasound mediated imaging and therapy can be developed through unique interbubble interactions in an ultrasound field. One of the major challenges in generating CCMCs is controlling aggregate sizes and maintaining stability against Ostwald ripening and coalescence. In this study, we demonstrate that chemically cross-linked microbubble clusters can produce small (<10 µm) quasi-stable complexes that slowly fuse into bubbles with individual gas cores. Furthermore, we demonstrate that this process can be driven with low-intensity ultrasound pulses, enabling a rapid fusion of clusters which could potentially be used to develop novel ultrasound contrast imaging and drug delivery strategies in future studies. The development of novel microbubble clusters presents a simple yet robust process for generating novel UCAs with a design that could allow for more versatility in contrast-enhanced ultrasound (CEUS), molecular imaging, and drug delivery applications. Additionally, microbubble clustering is a unique way to control size, shell, and gas compositions that can be used to study bubble ripening and coalescence in a highly controlled environment or study the behavior of mixed-microbubble populations.

Monitoring Early Breast Cancer Response to Neoadjuvant Therapy Using H-Scan Ultrasound Imaging: Preliminary Preclinical Results.

OBJECTIVE: H-scan imaging is a new ultrasound technique used to visualize the relative size of acoustic scatterers. The purpose of this study was to evaluate the use of H-scan ultrasound imaging for monitoring early tumor response to neoadjuvant treatment using a preclinical breast cancer animal model. METHODS: Real-time H-scan ultrasound imaging was implemented on a programmable ultrasound scanner (Vantage 256; Verasonics Inc., Kirkland, WA) equipped with an L11-4v transducer. Bioluminescence and H-scan ultrasound was used to image luciferase-positive breast cancer-bearing mice at baseline and at 24, 48, and 168 hours after administration of a single dose of neoadjuvant (paclitaxel) or sham treatment. Animals were euthanized at 48 or 168 hours, and tumors underwent histologic processing to identify cancer cell proliferation and apoptosis. RESULTS: Baseline H-scan ultrasound images of control and therapy group tumors were comparable, but the latter exhibited significant changes over the 7-day study (P < .05). At termination, there was a marked difference between the H-scan ultrasound images of control and treated tumors (P < .05). Specifically, H-scan ultrasound images of treated tumors were more blue in hue than images obtained from control tumors. There was a significant linear correlation between the predominance of the blue hue found in the H-scan ultrasound images and intratumoral apoptotic activity (R2 > 0.40, P < .04). CONCLUSION: Preliminary preclinical results suggest that H-scan ultrasound imaging is a new and promising tissue characterization modality. H-scan ultrasound imaging may provide prognostic value when monitoring early tumor response to neoadjuvant treatment.

Super-Resolution Ultrasound Imaging of Skeletal Muscle Microvascular Dysfunction in an Animal Model of Type 2 Diabetes.

OBJECTIVES: To evaluate the use of super-resolution ultrasound (SR-US) imaging for quantifying microvascular changes in skeletal muscle using a mouse model of type 2 diabetes. METHODS: Study groups were young, standard chow-fed male C57BL/6J mice (lean group) and high fat diet-fed older mice (obese group). After an overnight fast, dynamic contrast-enhanced US imaging was performed on the proximal hind limb adductor muscle group for 10 minutes at baseline and again at 1 and 2 hours during administration of a hyperinsulinemic-euglycemic clamp. Dynamic contrast-enhanced US images were collected on a clinical US scanner (Acuson Sequoia 512; Siemens Healthcare, Mountain View, CA) equipped with a 15L8 linear array transducer. Dynamic contrast-enhanced US images were processed with a spatiotemporal filter to remove tissue clutter. Individual microbubbles were localized and counted to create an SR-US image. A frame-by-frame analysis of the microbubble count was generated (ie, time-microbubble count curve [TMC]) to estimate tissue perfusion and microvascular blood flow. The conventional time-intensity curve (TIC) was also generated for comparison. RESULTS: In vivo SR-US imaging could delineate microvascular structures in the mouse hind limb. Compared with lean animals, insulin-induced microvascular recruitment was attenuated in the obese group. The SR-US-based TMC analysis revealed differences between lean and obese animal data for select microvascular parameters (P < .04), which was not true for TIC-based measurements. Whereas the TMC and TIC microvascular parameters yielded similar temporal trends, there was less variance associated with the TMC-derived values. CONCLUSIONS: Super-resolution US imaging is a new modality for measuring the microvascular properties of skeletal muscle and dysfunction from type 2 diabetes.

Contrast-enhanced ultrasound imaging using pulse inversion spectral deconvolution.

A contrast-enhanced ultrasound (CEUS) imaging approach, termed pulse inversion spectral deconvolution (PISD), is introduced. The approach uses two Gaussian-weighted Hermite polynomials to form two inverted pulse sequences. The two inversed pulses are then used to filter ultrasound (US) backscattered data and discrimination of the linear and nonlinear signal components. A research US scanner equipped with a linear array transducer was used for data acquisition. The receive data from all channels are shaped using plane wave imaging beamforming with angular compounding (from one to nine angles). In vitro data was collected with a tissue mimicking flow phantom perfused with an US contrast agent using PISD and traditional nonlinear (NLI) US imaging as comparison. The role of imaging frequency (between 4.5 and 6.25 MHz) and mechanical index (from 0.1 to 0.3) were evaluated. Preliminary in vivo data was collected in the hindlimb of three healthy mice. Preliminary experimental findings indicate that the PISD contrast-to-tissue ratio was improved nearly ten times compared to the NLI US imaging approach. Also, the spatial resolution was improved due to the effect of deconvolution and spatial angular compounding. Overall, PISD is a promising postprocessing technique for real-time CEUS imaging.

Impact of hydrostatic pressure on phase-change contrast agent activation by pulsed ultrasound.

A phase-change contrast agent (PCCA) can be activated from a liquid (nanodroplet) state using pulsed ultrasound (US) energy to form a larger highly echogenic microbubble (MB). PCCA activation is dependent on the ambient pressure of the surrounding media, so any increase in hydrostatic pressure demands higher US energies to phase transition. In this paper, the authors explore this basic relationship as a potential direction for noninvasive pressure measurement and foundation of a unique technology the authors are developing termed tumor interstitial pressure estimation using ultrasound (TIPE-US). TIPE-US was developed using a programmable US research scanner. A custom scan sequence interleaved pulsed US transmissions for both PCCA activation and detection. An automated US pressure sweep was applied, and US images were acquired at each increment. Various hydrostatic pressures were applied to PCCA samples. Pressurized samples were imaged using the TIPE-US system. The activation threshold required to convert PCCA from the liquid to gaseous state was recorded for various US and PCCA conditions. Given the relationship between the hydrostatic pressure applied to the PCCA and US energy needed for activation, phase transition can be used as a surrogate of hydrostatic pressure. Consistent with theoretical predictions, the PCCA activation threshold was lowered with increasing sample temperature and by decreasing the frequency of US exposure, but it was not impacted by PCCA concentration.

New Ultrasound Techniques Promise Further Advances in AKI and CKD.

AKI and CKD are important clinical problems because they affect many patients and the associated diagnostic and treatment paradigms are imperfect. Ultrasound is a cost-effective, noninvasive, and simple imaging modality that offers a multitude of means to improve the diagnosis, monitoring, and treatment of both AKI and CKD, especially considering recent advances in this technique. Ultrasound alone can attenuate AKI and prevent CKD by stimulating the splenic cholinergic anti-inflammatory pathway. Additionally, microbubble contrast agents are improving the sensitivity and specificity of ultrasound for diagnosing kidney disease, especially when these agents are conjugated to ligand-specific mAbs or peptides, which make the dynamic assessment of disease progression and response to treatment possible. More recently, drug-loaded microbubbles have been developed and the load release by ultrasound exposure has been shown to be a highly specific treatment modality, making the potential applications of ultrasound even more promising. This review focuses on the multiple strategies for using ultrasound with and without microbubble technology for enhancing our understanding of the pathophysiology of AKI and CKD.

Molecular Hyperthermia: Spatiotemporal Protein Unfolding and Inactivation by Nanosecond Plasmonic Heating.

Spatiotemporal control of protein structure and activity in biological systems has important and broad implications in biomedical sciences as evidenced by recent advances in optogenetic approaches. Here, this study demonstrates that nanosecond pulsed laser heating of gold nanoparticles (GNP) leads to an ultrahigh and ultrashort temperature increase, coined as "molecular hyperthermia", which causes selective unfolding and inactivation of proteins adjacent to the GNP. Protein inactivation is highly dependent on both laser pulse energy and GNP size, and has a well-defined impact zone in the nanometer scale. It is anticipated that the fine control over protein structure and function enabled by this discovery will be highly enabling within a number of arenas, from probing the biophysics of protein folding/unfolding to the nanoscopic manipulation of biological systems via an optical trigger, to developing novel therapeutics for disease treatment without genetic modification.

Calreticulin Regulates Neointima Formation and Collagen Deposition following Carotid Artery Ligation.

BACKGROUND/AIMS: The endoplasmic reticulum (ER) stress protein, calreticulin (CRT), is required for the production of TGF-ß-stimulated extracellular matrix (ECM) by fibroblasts. Since TGF-ß regulates vascular fibroproliferative responses and collagen deposition, we investigated the effects of CRT knockdown on vascular smooth-muscle cell (VSMC) fibroproliferative responses and collagen deposition. METHODS: Using a carotid artery ligation model of vascular injury, Cre-recombinase-IRES-GFP plasmid was delivered with microbubbles (MB) to CRT-floxed mice using ultrasound (US) to specifically reduce CRT expression in the carotid artery. RESULTS: In vitro, Cre-recombinase-mediated CRT knockdown in isolated, floxed VSMCs decreased the CRT transcript and protein, and attenuated the induction of collagen I protein in response to TGF-ß. TGF-ß stimulation of collagen I was partly blocked by the NFAT inhibitor 11R-VIVIT. Following carotid artery ligation, CRT staining was upregulated with enhanced expression in the neointima 14-21 days after injury. Furthermore, Cre-recombinase-IRES-GFP plasmid delivered by targeted US reduced CRT expression in the neointima of CRT-floxed mice and led to a significant reduction in neointima formation and collagen deposition. The neointimal cell number was also reduced in mice, with a local, tissue-specific knockdown of CRT. CONCLUSIONS: This work establishes a novel role for CRT in mediating VSMC responses to injury through the regulation of collagen deposition and neointima formation.

Volumetric contrast-enhanced ultrasound imaging of renal perfusion.

OBJECTIVES: To determine whether volumetric contrast-enhanced ultrasound (US) imaging has the potential to monitor changes in renal perfusion after vascular injury. METHODS: Volumetric contrast-enhanced US uses a series of planar image acquisitions, capturing the nonlinear second harmonic signal from microbubble contrast agents flowing in the vasculature. Tissue perfusion parameters (peak intensity [IPK], time to peak intensity [TPK], wash-in rate [WIR], and area under the curve [AUC]) were derived from time-intensity curve data collected during in vitro flow phantom studies and in vivo animal studies of healthy and injured kidneys. For the flow phantom studies, either the contrast agent concentration was held constant (10 µL/L) with varying volumetric flow rates (10, 20, and 30 mL/min), or the flow rate was held constant (30 mL/min) with varying contrast agent concentrations (5, 10, and 20 µL/L). Animal studies used healthy rats or those that underwent renal ischemia-reperfusion injury. Renal studies were performed with healthy rats while the transducer angle was varied for each volumetric contrast-enhanced US image acquisition (reference or 0°, 45°, and 90°) to determine whether repeated renal perfusion measures were isotropic and independent of transducer position. Blood serum biomarkers and immunohistology were used to confirm acute kidney injury. RESULTS: Flow phantom results revealed a linear relationship between microbubble concentrations injected into the flow system and the IPK, WIR, and AUC (R(2) > 0.56; P < .005). Furthermore, there was a linear relationship between volume flow rate changes and the TPK, WIR, and AUC (R(2) > 0.77; P < .005). No significant difference was found between the transducer angle during data acquisition and any of the perfusion measures (P > .60). After induction of renal ischemia-reperfusion injury in the rat animal model (n = 4), volumetric contrast-enhanced US imaging of the injured kidney revealed an initial reduction in renal perfusion compared to control animals, followed by progressive recovery of vascular function. CONCLUSIONS: Volumetric contrast-enhanced US-based renal perfusion imaging may prove clinically feasible for detecting and monitoring acute kidney injury.

Multiparametric ultrasound imaging for early-stage steatosis: Comparison with magnetic resonance imaging-based proton density fat fraction.

BACKGROUND: The prevalence of liver diseases, especially steatosis, requires a more convenient and noninvasive tool for liver diagnosis, which can be a surrogate for the gold standard biopsy. Magnetic resonance (MR) measurement offers potential, however ultrasound (US) has better accessibility than MR. PURPOSE: This study aims to suggest a multiparametric US approach which demonstrates better quantification and imaging performance than MR imaging-based proton density fat fraction (MRI-PDFF) for hepatic steatosis assessment. METHODS: We investigated early-stage steatosis to evaluate our approach. An in vivo (within the living) animal study was performed. Fat inclusions were accumulated in the animal livers by feeding a methionine and choline deficient (MCD) diet for 2 weeks. The animals (n = 19) underwent US and MR imaging, and then their livers were excised for histological staining. From the US, MR, and histology images, fat accumulation levels were measured and compared: multiple US parameters; MRI-PDFF; histology fat percentages. Seven individual US parameters were extracted using B-mode measurement, Burr distribution estimation, attenuation estimation, H-scan analysis, and shear wave elastography. Feature selection was performed, and the selected US features were combined, providing quantification of fat accumulation. The combined parameter was used for visualizing the localized probability of fat accumulation level in the liver; This procedure is known as disease-specific imaging (DSI). RESULTS: The combined US parameter can sensitively assess fat accumulation levels, which is highly correlated with histology fat percentage (R = 0.93, p-value < 0.05) and outperforms the correlation between MRI-PDFF and histology (R = 0.89, p-value < 0.05). Although the seven individual US parameters showed lower correlation with histology compared to MRI-PDFF, the multiparametric analysis enabled US to outperform MR. Furthermore, this approach allowed DSI to detect and display gradual increases in fat accumulation. From the imaging output, we measured the color-highlighted area representing fatty tissues, and the fat fraction obtained from DSI and histology showed strong agreement (R = 0.93, p-value < 0.05). CONCLUSIONS: We demonstrated that fat quantification utilizing a combination of multiple US parameters achieved higher performance than MRI-PDFF; therefore, our multiparametric analysis successfully combined selected features for hepatic steatosis characterization. We anticipate clinical use of our proposed multiparametric US analysis, which could be beneficial in assessing steatosis in humans.

Generalized mathematical framework for contrast-enhanced ultrasound imaging with pulse inversion spectral deconvolution.

A generalized mathematical framework for performing contrast-enhanced ultrasound (CEUS) imaging is introduced. Termed pulse inversion spectral deconvolution (PISD), this CEUS approach is founded on Gaussian derivative functions (GDFs). PISD pulses are used to form two inverted pulse sequences, which are then used to filter backscattered ultrasound (US) data for isolation of the nonlinear (NL) microbubble (MB) signal component. An US scanner equipped with a linear array transducer was used for data acquisition. With a vascular flow phantom perfused with MBs, data was collected using PISD and NL-based CEUS imaging. The role of wide-beam transmit aperture size (32 or 64 elements) was also evaluated using an US pulse frequency of 6.25 MHz. Image enhancement was quantified by a contrast-to-noise ratio (CNR). Preliminary in vivo data was collected in the hindlimb and kidney of healthy rats. Overall, in vitro wide-beam CEUS imaging using an aperture size of 64 elements yielded improved CNR values. Specifically, PISD-based CEUS imaging produced CNR values of 37.3 dB. For comparison, CNR values obtained using B-mode US or NL approaches were 2.1 and 12.1 dB, respectively. In vivo results demonstrated that PISD-based CEUS imaging improved vascular visualization compared to the NL imaging strategy.

High-Resolution Ultrasound Characterization of Local Scattering in Cancer Tissue.

Ultrasound (US) has afforded an approach to tissue characterization for more than a decade. The challenge is to reveal hidden patterns in the US data that describe tissue function and pathology that cannot be seen in conventional US images. Our group has developed a high-resolution analysis technique for tissue characterization termed H-scan US, an imaging method used to interpret the relative size of acoustic scatterers. In the present study, the objective was to compare local H-scan US image intensity with registered histologic measurements made directly at the cellular level. Human breast cancer cells (MDA-MB 231, American Type Culture Collection, Manassas, VA, USA) were orthotopically implanted into female mice (N = 5). Tumors were allowed to grow for approximately 4 wk before the study started. In vivo imaging of tumor tissue was performed using a US system (Vantage 256, Verasonics Inc., Kirkland, WA, USA) equipped with a broadband capacitive micromachined ultrasonic linear array transducer (Kolo Medical, San Jose, CA, USA). A 15-MHz center frequency was used for plane wave imaging with five angles for spatial compounding. H-scan US image reconstruction involved use of parallel convolution filters to measure the relative strength of backscattered US signals. Color codes were applied to filter outputs to form the final H-scan US image display. For histologic processing, US imaging cross-sections were carefully marked on the tumor surface, and tumors were excised and sliced along the same plane. By use of optical microscopy, whole tumor tissue sections were scanned and digitized after nuclear staining. US images were interpolated to have the same number of pixels as the histology images and then spatially aligned. Each nucleus from the histologic sections was automatically segmented using custom MATLAB software (The MathWorks Inc., Natick, MA, USA). Nuclear size and spacing from the histology images were then compared with local H-scan US image features. Overall, local H-scan US image intensity exhibited a significant correlation with both cancer cell nuclear size (R2 > 0.27, p < 0.001) and the inverse relationship with nuclear spacing (R2 > 0.17, p < 0.001).

Simultaneous imaging of ultrasonic relative backscatter and attenuation coefficients for quantitative liver steatosis assessment.

Prevalence of liver disease is continuously increasing and nonalcoholic fatty liver disease (NAFLD) is the most common etiology. We present an approach to detect the progression of liver steatosis based on quantitative ultrasound (QUS) imaging. This study was performed on a group of 55 rats that were subjected to a control or methionine and choline deficient (MCD) diet known to induce NAFLD. Ultrasound (US) measurements were performed at 2 and 6 weeks. Thereafter, animals were humanely euthanized and livers excised for histological analysis. Relative backscatter and attenuation coefficients were simultaneously estimated from the US data and envelope signal-to-noise ratio was calculated to train a regression model for: (1) fat fraction percentage estimation and (2) performing classification according to Brunt's criteria in grades (0 <5%; 1, 5-33%; 2, >33-66%; 3, >66%) of liver steatosis. The trained regression model achieved an [Formula: see text] of 0.97 (p-value < 0.01) and a RMSE of 3.64. Moreover, the classification task reached an accuracy of 94.55%. Our results suggest that in vivo QUS is a promising noninvasive imaging modality for the early assessment of NAFLD.

Image-guided focused ultrasound-mediated molecular delivery to breast cancer in an animal model.

Tumors become inoperable due to their size or location, making neoadjuvant chemotherapy the primary treatment. However, target tissue accumulation of anticancer agents is limited by the physical barriers of the tumor microenvironment. Low-intensity focused ultrasound (FUS) in combination with microbubble (MB) contrast agents can increase microvascular permeability and improve drug delivery to the target tissue after systemic administration. The goal of this research was to investigate image-guided FUS-mediated molecular delivery in volume space. Three-dimensional (3-D) FUS therapy functionality was implemented on a programmable ultrasound scanner (Vantage 256, Verasonics Inc.) equipped with a linear array for image guidance and a 128-element therapy transducer (HIFUPlex-06, Sonic Concepts). FUS treatment was performed on breast cancer-bearing female mice (N= 25). Animals were randomly divided into three groups, namely, 3-D FUS therapy, two-dimensional (2-D) FUS therapy, or sham (control) therapy. Immediately prior to the application of FUS therapy, animals received a slow bolus injection of MBs (Definity, Lantheus Medical Imaging Inc.) and near-infrared dye (IR-780, surrogate drug) for optical reporting and quantification of molecular delivery. Dye accumulation was monitored viain vivooptical imaging at 0, 1, 24, and 48 h (Pearl Trilogy, LI-COR). Following the 48 h time point, animals were humanely euthanized and tumors excised forex vivoanalyzes. Optical imaging results revealed that 3-D FUS therapy improved delivery of the IR-780 dye by 66.4% and 168.1% at 48 h compared to 2-D FUS (p= 0.18) and sham (p= 0.047) therapeutic strategies, respectively.Ex vivoanalysis revealed similar trends. Overall, 3-D FUS therapy can improve accumulation of a surrogate drug throughout the entire target tumor burden after systemic administration.

Assessment of Transarterial Chemoembolization Using Super-resolution Ultrasound Imaging and a Rat Model of Hepatocellular Carcinoma.

OBJECTIVE: Hepatocellular carcinoma (HCC) is a highly prevalent form of liver cancer diagnosed annually in 600,000 people worldwide. A common treatment is transarterial chemoembolization (TACE), which interrupts the blood supply of oxygen and nutrients to the tumor mass. The need for repeat TACE treatments may be assessed in the weeks after therapy with contrast-enhanced ultrasound (CEUS) imaging. Although the spatial resolution of traditional CEUS has been restricted by the diffraction limit of ultrasound (US), this physical barrier has been overcome by a recent innovation known as super-resolution US (SRUS) imaging. In short, SRUS enhances the visible details of smaller microvascular structures on the 10 to 100 µm scale, which unlocks a host of new clinical opportunities for US. METHODS: In this study, a rat model of orthotopic HCC is introduced and TACE treatment response (to a doxorubicin-lipiodol emulsion) is assessed using longitudinal SRUS and magnetic resonance imaging (MRI) performed at 0, 7 and 14 d. Animals were euthanized at 14 d for histological analysis of excised tumor tissue and determination of TACE response, that is, control, partial response or complete response. CEUS imaging was performed using a pre-clinical US system (Vevo 3100, FUJIFILM VisualSonics Inc.) equipped with an MX201 linear array transducer. After administration of a microbubble contrast agent (Definity, Lantheus Medical Imaging), a series of CEUS images were collected at each tissue cross-section as the transducer was mechanically stepped at 100 µm increments. SRUS images were formed at each spatial position, and a microvascular density metric was calculated. Microscale computed tomography (microCT, OI/CT, MILabs) was used to confirm TACE procedure success, and tumor size was monitored using a small animal MRI system (BioSpec 3T, Bruker Corp.). RESULTS: Although there were no differences at baseline (p > 0.15), both microvascular density levels and tumor size measures from the complete responder cases at 14 d were considerably lower and smaller, respectively, than those in the partial responder or control group animals. Histological analysis revealed tumor-to-necrosis levels of 8.4%, 51.1% and 100%, for the control, partial responder and complete responder groups, respectively (p < 0.005). CONCLUSION: SRUS imaging is a promising modality for assessing early changes in microvascular networks in response to tissue perfusion-altering interventions such as TACE treatment of HCC.

A single photodynamic priming protocol augments delivery of ⍺-PD-L1 mAbs and induces immunogenic cell death in head and neck tumors.

Photodynamic priming (PDP) leverages the photobiological effects of subtherapeutic photodynamic therapy (PDT) regimens to modulate the tumor vasculature and stroma. PDP also sensitizes tumors to secondary therapies, such as immunotherapy by inducing a cascade of molecular events, including immunogenic cell death (ICD). We and others have shown that PDP improves the delivery of antibodies, among other theranostic agents. However, it is not known whether a single PDP protocol is capable of both inducing ICD in vivo and augmenting the delivery of immune checkpoint inhibitors. In this rapid communication, we show for the first time that a single PDP protocol using liposomal benzoporphyrin derivative (Lipo-BPD, 0.25 mg/kg) with 690 nm light (75 J/cm2 , 100 mW/cm2 ) simultaneously doubles the delivery of ⍺-PD-L1 antibodies in murine AT-84 head and neck tumors and induces ICD in vivo. ICD was observed as a 3-11 fold increase in tumor cell exposure of damage-associated molecular patterns (Calreticulin, HMGB1, and HSP70). These findings suggest that this single, highly translatable PDP protocol using clinically relevant Lipo-BPD holds potential for improving immunotherapy outcomes in head and neck cancer. It can do so by simultaneously overcoming physical barriers to the delivery of immune checkpoint inhibitors, and biochemical barriers that contribute to immunosuppression.