Focused Ultrasound-Enabled Blood-Based Liquid Biopsy (Sonobiopsy) for Brain Disease Diagnosis

Brain cancer severely threatens human health due to its disruption of neurological function, poor prognosis, and substantial reduction in quality of life. Glioblastoma (GBM) is the most devastating brain cancer; not only is it the most common malignant primary tumor in adults, but also it has a median survival of 14 months with a 5-year survival rate of less than 5%. Despite advances in multidisciplinary treatment that includes surgical resection, radiation therapy, and chemotherapy, almost all patients experience tumor progression and nearly universal mortality within 2 years. However, advances in patient care have suggested that the accurate diagnosis of molecular subtypes is critical for individualized targeted treatment and improving survival outcome for brain cancer patients.Conventional diagnostic evaluation begins with neuroimaging and continues with surgical tissue biopsy to confirm the diagnosis and acquire the molecular profile of the tumor. Though tissue biopsy is the gold standard for molecular characterization, there are significant risks for patients because the procedure is invasive. Liquid biopsy is a minimally invasive approach that enables genetic profiling by detecting circulating tumor-derived biomarkers that were shed by tumors into the blood circulation. However, blood-based liquid biopsy is inherently limited by the blood-brain barrier (BBB) that hinders the release of molecular biomarkers, leading to a low detection sensitivity for GBM. The combination of focused ultrasound (FUS) with microbubbles is an established technique to disrupt the BBB noninvasively and transiently with high precision (on the order of millimeter). Though this has conventionally been used to deliver drugs from the bloodstream to the brain tissue of interest, it is hypothesized that this FUS-induced BBB disruption enables molecules to be released from the tissue into the blood circulation. Under this “two-way trafficking” hypothesis, FUS-enabled blood-based liquid biopsy (sonobiopsy) can release brain tumor-derived biomarkers into the blood circulation to improve the sensitivity for noninvasive molecular characterization of GBM. In this work, we evaluated the feasibility, safety, and efficacy of sonobiopsy in small and large animal models to provide a minimally invasive, spatiotemporally-controlled, and sensitive molecular characterization of brain diseases. First, we evaluated the impact of different sonobiopsy parameters on the extent of biomarker release and tissue damage in a mouse GBM model. The blood collection time after FUS sonication was an important factor to minimize the effect of clearance and maximize the level of biomarkers detected in the plasma. Importantly, careful optimization of key sonobiopsy parameters, e.g., FUS pressure, microbubble dose, and sonication volume, was necessary to increase the release of circulating biomarkers while minimizing the potential for tissue damage. With the optimized parameters, sonobiopsy significantly increased the plasma level of GBM-derived biomarkers and improved the detection sensitivity for two clinically relevant mutations. Second, sonobiopsy was performed in a non-tumor pig model to demonstrate the potential for clinical translation. A customized sonobiopsy device was developed to target a specific brain area and release brain-specific biomarkers into the blood circulation. Importantly, sonobiopsy significantly increased the plasma level of these biomarkers without causing detectable tissue damage. This large animal study demonstrated that sonobiopsy has the potential to be safely translated to humans. To further underscore the potential for clinical translation of sonobiopsy, a pig GBM model was developed to assess the feasibility of sonobiopsy to release GBM-derived biomarkers and improve the detection sensitivity for two clinically relevant mutations. We achieved localized BBB disruption and the plasma level of GBM biomarkers significantly increased shortly after FUS sonication in the large animal tumor model. Importantly, sonobiopsy improved the detection sensitivity for two mutations without causing off-target damage. This addressed the fundamental limitation—obtaining specimens with a sufficient abundance of circulating tumor biomarkers—for the minimally invasive, sensitive molecular characterization of GBM. Lastly, we evaluated the impact of sonobiopsy as a platform technology to aid in the diagnosis of other brain diseases. After performing sonobiopsy in a transgenic mouse model of tauopathy, there was a significant increase in the plasma levels of pathologic proteins and a key marker for neurodegeneration. This demonstrated the potential to use sonobiopsy for the noninvasive diagnosis of neurodegenerative disorders. In summary, this work provided evidence that supports the clinical translation of sonobiopsy as a minimally invasive, spatiotemporally-controlled, and sensitive molecular characterization of brain diseases. This enhanced capability could have an important impact throughout the continuum of patient care from brain disease diagnosis and treatment monitoring to recurrence detection. In addition, sonobiopsy could support the investigation of disease-specific molecular mechanisms and accelerate the development of targeted therapy

Focused ultrasound for safe and effective release of brain tumor biomarkers into the peripheral circulation

The development of noninvasive approaches for brain tumor diagnosis and monitoring continues to be a major medical challenge. Although blood-based liquid biopsy has received considerable attention in various cancers, limited progress has been made for brain tumors, at least partly due to the hindrance of tumor biomarker release into the peripheral circulation by the blood-brain barrier. Focused ultrasound (FUS) combined with microbubbles induced BBB disruption has been established as a promising technique for noninvasive and localized brain drug delivery. Building on this established technique, we propose to develop FUS-enabled liquid biopsy technique (FUS-LBx) to enhance the release of brain tumor biomarkers (e.g., DNA, RNA, and proteins) into the circulation. The objective of this study was to demonstrate that FUS-LBx could sufficiently increase plasma levels of brain tumor biomarkers without causing hemorrhage in the brain. Mice with orthotopic implantation of enhanced green fluorescent protein (eGFP)-transfected murine glioma cells were treated using magnetic resonance (MR)-guided FUS system in the presence of systemically injected microbubbles at three peak negative pressure levels (0.59, 1.29, and 1.58 MPa). Plasma eGFP mRNA levels were quantified with the quantitative polymerase chain reaction (qPCR). Contrast-enhanced MR images were acquired before and after the FUS sonication. FUS at 0.59 MPa resulted in an increased plasma eGFP mRNA level, comparable to those at higher acoustic pressures (1.29 MPa and 1.58 MPa). Microhemorrhage density associated with FUS at 0.59 MPa was significantly lower than that at higher acoustic pressures and not significantly different from the control group. MRI analysis revealed that post-sonication intratumoral and peritumoral hyperenhancement had strong correlations with the level of FUS-induced biomarker release and the extent of hemorrhage. This study suggests that FUS-LBx could be a safe and effective brain-tumor biomarker release technique, and MRI could be used to develop image-guided FUS-LBx

Blood-brain barrier opening in a large animal model using closed-loop microbubble cavitation-based feedback control of focused ultrasound sonication

Focused ultrasound (FUS) in combination with microbubbles has been established as a promising technique for noninvasive and localized Blood-brain barrier (BBB) opening. Real-time passive cavitation detection (PCD)-based feedback control of the FUS sonication is critical to ensure effective BBB opening without causing hemorrhage. This study evaluated the performance of a closed-loop feedback controller in a porcine model. Calibration of the baseline cavitation level was performed for each targeted brain location by a FUS sonication in the presence of intravenously injected microbubbles at a low acoustic pressure without inducing BBB opening. The target cavitation level (TCL) was defined for each target based on the baseline cavitation level. FUS treatment was then performed under real-time PCD-based feedback controller to maintain the cavitation level at the TCL. After FUS treatment, contrast-enhanced MRI and ex vivo histological staining were performed to evaluate the BBB permeability and safety. Safe and effective BBB opening was achieved with the BBB opening volume increased from 3.8 ± 0.7 to 53.6 ± 23.3 m

The Relationship between Saccades and Locomotion

Human locomotion involves a complex interplay among multiple brain regions and depends on constant feedback from the visual system. We summarize here the current understanding of the relationship among fixations, saccades, and gait as observed in studies sampling eye movements during locomotion, through a review of the literature and a synthesis of the relevant knowledge on the topic. A significant overlap in locomotor and saccadic neural circuitry exists that may support this relationship. Several animal studies have identified potential integration nodes between these overlapping circuitries. Behavioral studies that explored the relationship of saccadic and gait-related impairments in normal conditions and in various disease states are also discussed. Eye movements and locomotion share many underlying neural circuits, and further studies can leverage this interplay for diagnostic and therapeutic purposes

Distinct Ventral Pallidal Neural Populations Mediate Separate Symptoms of Depression

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression

Sonothermogenetics for noninvasive and cell-type specific deep brain neuromodulation

Background: Critical advances in the investigation of brain functions and treatment of brain disorders are hindered by our inability to selectively target neurons in a noninvasive manner in the deep brain. Objective: This study aimed to develop sonothermogenetics for noninvasive, deep-penetrating, and cell-type-specific neuromodulation by combining a thermosensitive ion channel TRPV1 with focused ultrasound (FUS)-induced brief, non-noxious thermal effect. Methods: The sensitivity of TRPV1 to FUS sonication was evaluated in vitro. It was followed by in vivo assessment of sonothermogenetics in the activation of genetically defined neurons in the mouse brain by two-photon calcium imaging. Behavioral response evoked by sonothermogenetic stimulation at a deep brain target was recorded in freely moving mice. Immunohistochemistry staining of ex vivo brain slices was performed to evaluate the safety of FUS sonication. Results: TRPV1 was found to be an ultrasound-sensitive ion channel. FUS sonication at the mouse brain in vivo selectively activated neurons that were genetically modified to express TRPV1. Temporally precise activation of TRPV1-expressing neurons was achieved with its success rate linearly correlated with the peak temperature within the FUS-targeted brain region as measured by in vivo magnetic resonance thermometry. FUS stimulation of TRPV1-expressing neurons at the striatum repeatedly evoked locomotor behavior in freely moving mice. FUS sonication was confirmed to be safe based on inspection of neuronal integrity, inflammation, and apoptosis markers. Conclusions: This noninvasive and cell-type-specific neuromodulation approach with the capability to stimulate deep brain has the promise to advance the study of the intact nervous system and uncover new ways to treat neurological disorders

Characterization of magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU)-induced large-volume hyperthermia in deep and superficial targets in a porcine model

Purpose To characterize temperature fields and tissue damage profiles of large-volume hyperthermia (HT) induced by magnetic resonance-guided high-intensity focused ultrasound (MRgHIFU) in deep and superficial targets in vivo in a porcine model. Methods Nineteen HT sessions were performed in vivo with a commercial MRgHIFU system (Sonalleve® V2, Profound Medical Inc., Mississauga, ON, Canada) in hind leg muscles of eight pigs with temperature fields of cross-sectional diameter of 58-mm. Temperature statistics evaluated in the target region-of-interest (tROI) included accuracy, temporal variation, and uniformity. The impact of the number and location of imaging planes for feedback-based temperature control were investigated. Temperature fields were characterized by time-in-range (TIR, the duration each voxel stays within 40–45 °C) maps. Tissue damage was characterized by contrast-enhanced MRI, and macroscopic and histopathological analysis. The performance of the Sonalleve® system was benchmarked against a commercial phantom. Results Across all HT sessions, the mean difference between the average temperature (Tavg) and the desired temperature was −0.4 ± 0.5 °C; the standard deviation of temperature 1.2 ± 0.2 °C; the temporal variation of Tavg for 30-min HT was 0.6 ± 0.2 °C, and the temperature uniformity was 1.5 ± 0.2 °C. A difference of 2.2-cm (in pig) and 1.5-cm (in phantom) in TIR dimensions was observed when applying feedback-based plane(s) at different locations. Histopathology showed 62.5% of examined HT sessions presenting myofiber degeneration/necrosis within the target volume. Conclusion Large-volume MRgHIFU-mediated HT was successfully implemented and characterized in a porcine model in deep and superficial targets in vivo with heating distributions modifiable by user-definable parameters