For a blood-borne cancer therapeutic agent to be effective, it must cross the blood vessel wall to reach cancer cells in adequate quantities, and it must overcome the resistance conferred by the local microenvironment around cancer cells. The brain microenvironment can thwart the effectiveness of drugs against primary brain tumours as well as brain metastases. In this Review, we highlight the cellular and molecular components of the blood–brain barrier (BBB), a specialized neurovascular unit evolved to maintain brain homeostasis. Tumours are known to compromise the integrity of the BBB, resulting in a vasculature known as the blood–tumour barrier (BTB), which is highly heterogeneous and characterized by numerous distinct features, including non-uniform permeability and active efflux of molecules. We discuss the challenges posed by the BBB and BTB for drug delivery, how multiple cell types dictate BBB function and the role of the BTB in disease progression and treatment. Finally, we highlight emerging molecular, cellular and physical strategies to improve drug delivery across the BBB and BTB and discuss their impact on improving conventional as well as emerging treatments, such as immune checkpoint inhibitors and engineered T cells. A deeper understanding of the BBB and BTB through the application of single-cell sequencing and imaging techniques, and the development of biomarkers of BBB integrity along with systems biology approaches, should enable new personalized treatment strategies for primary brain malignancies and brain metastases. This Review discusses the structure and function of the blood–brain barrier (BBB) and how brain tumours and brain metastases compromise BBB integrity. It also discusses the challenges the BBB poses for cancer therapy and how these might be overcome.

The blood–brain barrier and blood–tumour barrier in brain tumours and metastases

Although the use of ultrasonic plane-wave transmissions rather than line-per-line focused beam transmissions has been long studied in research, clinical application of this technology was only recently made possible through developments in graphical processing unit (GPU)-based platforms Far beyond a technological breakthrough, the use of plane or diverging wave transmissions enables attainment of ultrafast frame rates (typically faster than 1000 frames per second) over a large field of view This concept has also inspired the emergence of completely novel imaging modes which are valuable for ultrasound-based screening, diagnosis, and therapeutic monitoring In this review article, we present the basic principles and implementation of ultrafast imaging In particular, present and future applications of ultrafast imaging in biomedical ultrasound are illustrated and discussed

Ultrafast imaging in biomedical ultrasound

Amyloid-β (Aβ) peptide has been implicated in the pathogenesis of Alzheimer's disease (AD). We present a nonpharmacological approach for removing Aβ and restoring memory function in a mouse model of AD in which Aβ is deposited in the brain. We used repeated scanning ultrasound (SUS) treatments of the mouse brain to remove Aβ, without the need for any additional therapeutic agent such as anti-Aβ antibody. Spinning disk confocal microscopy and high-resolution three-dimensional reconstruction revealed extensive internalization of Aβ into the lysosomes of activated microglia in mouse brains subjected to SUS, with no concomitant increase observed in the number of microglia. Plaque burden was reduced in SUS-treated AD mice compared to sham-treated animals, and cleared plaques were observed in 75% of SUS-treated mice. Treated AD mice also displayed improved performance on three memory tasks: the Y-maze, the novel object recognition test, and the active place avoidance task. Our findings suggest that repeated SUS is useful for removing Aβ in the mouse brain without causing overt damage, and should be explored further as a noninvasive method with therapeutic potential in AD.

Scanning ultrasound removes amyloid-β and restores memory in an Alzheimer’s disease mouse model

The superparamagnetic properties of magnetic nanoparticles (MNPs) allow them to be guided by an externally positioned magnet and also provide contrast for MRI. However, their therapeutic use in treating CNS pathologies in vivo is limited by insufficient local accumulation and retention resulting from their inability to traverse biological barriers. The combined use of focused ultrasound and magnetic targeting synergistically delivers therapeutic MNPs across the blood–brain barrier to enter the brain both passively and actively. Therapeutic MNPs were characterized and evaluated both in vitro and in vivo, and MRI was used to monitor and quantify their distribution in vivo. The technique could be used in normal brains or in those with tumors, and significantly increased the deposition of therapeutic MNPs in brains with intact or compromised blood–brain barriers. Synergistic targeting and image monitoring are powerful techniques for the delivery of macromolecular chemotherapeutic agents into the CNS under the guidance of MRI.

https://www.pnas.org/content/pnas/107/34/15205.full.pdf

Magnetic resonance monitoring of focused ultrasound/magnetic nanoparticle targeting delivery of therapeutic agents to the brain

High-precision motion estimation has become essential in ultrasound-based techniques such as time-domain Doppler and elastography. Normalized cross-correlation (NCC) has been shown as one of the best motion estimators. However, a significant drawback is its associated computational cost, especially when RF signals are used. In this paper, a method based on sum tables developed elsewhere is adapted for fast NCC calculation in ultrasound-based motion estimation, and is tested with respect to the speed enhancement of the specific application of ultrasound-based motion estimation. Both the numerator and denominator in the NCC definition are obtained through pre-calculated sum tables to eliminate redundancy of repeated NCC calculations. Unlike a previously reported method, a search region following the principle of motion estimation is applied in the construction of sum tables. Because an exhaustive search and high window overlap are typically used for highest quality imaging, the computational cost of the proposed method is significantly lower than that of the direct method using the NCC definition, without increasing bias and variance characteristics of the motion estimation or sacrificing the spatial resolution. Therefore, high quality, high spatial resolution, and high calculation speed can be all simultaneously obtained using the proposed methodology. The high efficiency of this method was verified using RF signals from a human abdominal aorta in vivo. For the parameters typically used, a real-time, very high frame rate of 310 frames/s was achieved for the motion estimation. The proposed method was also extended to 2-D NCC motion estimation and motion estimation with other algorithms. The technique could thus prove very useful and flexible for real-time motion estimation as well as in other fields such as optical flow and image registration.

A fast normalized cross-correlation calculation method for motion estimation

The therapeutic efficacy of neurological agents is severely limited, because large compounds do not cross the blood-brain barrier (BBB). Focused ultrasound (FUS) sonication in the presence of microbubbles has been shown to temporarily open the BBB, allowing systemically administered agents into the brain. Until now, polydispersed microbubbles (1-10 ?m in diameter) were used, and, therefore, the bubble sizes better suited for inducing the opening remain unknown. Here, the FUS-induced BBB opening dependence on microbubble size is investigated. Bubbles at 1-2 and 4-5 ?m in diameter were separately size-isolated using differential centrifugation before being systemically administered in mice (n = 28). The BBB opening pressure threshold was identified by varying the peak-rarefactional pressure amplitude. BBB opening was determined by fluorescence enhancement due to systemically administered, fluorescent-tagged, 3-kDa dextran. The identified threshold fell between 0.30 and 0.46 MPa in the case of 1-2 ?m bubbles and between 0.15 and 0.30 MPa in the 4-5 ?m case. At every pressure studied, the fluorescence was greater with the 4-5 ?m than with the 1-2 ?m bubbles. At 0.61 MPa, in the 1-2 ?m bubble case, the fluorescence amount and area were greater in the thalamus than in the hippocampus. In conclusion, it was determined that the FUS-induced BBB opening was dependent on both the size distribution in the injected microbubble volume and the brain region targeted.

/pdf/microbubble-size-dependence-of-focused-ultrasound-induced-4s4vc0dwbs.pdf

Microbubble-Size Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening in Mice In Vivo

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agent's molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525MHz, peak-rarefactional pressure in situ: 570 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared with 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa into the brain parenchyma. This noninvasive and localized BBB opening technique could, thus, provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro but whose in vivo translation has been hampered by their associated BBB impermeability. (E-mail: ek2191@columbia.edu).

/pdf/molecules-of-various-pharmacologically-relevant-sizes-can-1xp23drjoo.pdf

Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo

High frame-rate ultrasound RF data acquisition has been proved to be critical for novel cardiovascular imaging techniques, such as high-precision myocardial elastography, pulse wave imaging (PWI), and electromechanical wave imaging (EWI). To overcome the frame-rate limitations on standard clinical ultrasound systems, we developed an automated method for multi-sector ultrasound imaging through retrospective electrocardiogram (ECG) gating on a clinically used open architecture system. The method achieved both high spatial (64 beam density) and high temporal resolution (frame rate of 481 Hz) at an imaging depth up to 11 cm and a 100% field of view in a single breath-hold duration. Full-view imaging of the left ventricle and the abdominal aorta of healthy human subjects was performed using the proposed technique in vivo. ECG and ultrasound RF signals were simultaneously acquired on a personal computer (PC). Composite, full-view frames both in RF- and B-mode were reconstructed through retrospective combination of seven small (20%) juxtaposed sectors using an ECG-gating technique. The axial displacement of the left ventricle, in both long-axis and short-axis views, and that of the abdominal aorta, in a long-axis view, were estimated using a RF-based speckle tracking technique. The electromechanical wave and the pulse wave propagation were imaged in a cineloop using the proposed imaging technique. Abnormal patterns of such wave propagation can serve as indicators of early cardiovascular disease. This clinical system could thus expand the range of applications in cardiovascular elasticity imaging for quantitative, noninvasive diagnosis of myocardial ischemia or infarction, arrhythmia, abdominal aortic aneurysms, and early-stage atherosclerosis.

A composite high-frame-rate system for clinical cardiovascular imaging

Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer's double transgenic mice using focused ultrasound.

Systems and methods for composite elastography and wave imaging are presented In exemplary embodiments of the present invention an imaging modality field of view, such as that of ultrasound, can be divided into N sectors, each having 1/Nth of a full field of view In exemplary embodiments of the present invention a temporal series of 2D or 3D ultrasound images for each of the N sectors can be acquired over one or more periods of a periodic signal A periodic signal can also be acquired simultaneously, wherein each of said series of 2D ultrasound images for each sector can be triggered or gated using said periodic signal The data from the various N sectors can be synchronized in time using the ECG signals, and the ultrasound signals from each of the N sectors combined to generate a series of composite ultrasound images at the frame rate of the Individual sectors.

Shougang Wang

Papers

Microbubble-Size Dependence of Focused Ultrasound-Induced Blood–Brain Barrier Opening in Mice In Vivo

Molecules of various pharmacologically-relevant sizes can cross the ultrasound-induced blood-brain barrier opening in vivo

A composite high-frame-rate system for clinical cardiovascular imaging

Noninvasive and transient blood-brain barrier opening in the hippocampus of Alzheimer's double transgenic mice using focused ultrasound.

Systems and methods for composite elastography and wave imaging