Sarah E. Shelton

Quantification of Microvascular Tortuosity during Tumor Evolution Using Acoustic Angiography

The recent design of ultra-broadband, multifrequency ultrasound transducers has enabled high-sensitivity, high-resolution contrast imaging, with very efficient suppression of tissue background using a technique called acoustic angiography. Here we perform the first application of acoustic angiography to evolving tumors in mice predisposed to develop mammary carcinoma, with the intent of visualizing and quantifying angiogenesis progression associated with tumor growth. Metrics compared include vascular density and two measures of vessel tortuosity quantified from segmentations of vessels traversing and surrounding 24 tumors and abdominal vessels from control mice. Quantitative morphologic analysis of tumor vessels revealed significantly increased vascular tortuosity abnormalities associated with tumor growth, with the distance metric elevated approximately 14% and the sum of angles metric increased 60% in tumor vessels versus controls. Future applications of this imaging approach may provide clinicians with a new tool in tumor detection, differentiation or evaluation, though with limited depth of penetration using the current configuration.

Acoustic characterization of contrast-to-tissue ratio and axial resolution for dual-frequency contrast-specific acoustic angiography imaging

Recently, dual-frequency transducers have enabled high-spatial-resolution and high-contrast imaging of vasculature with minimal tissue artifacts by transmitting at a low frequency and receiving broadband superharmonic echoes scattered by microbubble contrast agents. In this work, we examine the imaging parameters for optimizing contrast-totissue ratio (CTR) for dual-frequency imaging and the relationship with spatial resolution. Confocal piston transducers are used in a water bath setup to measure the SNR, CTR, and axial resolution for ultrasound imaging of nonlinear scattering of microbubble contrast agents when transmitting at a lower frequency (1.5 to 8 MHz) and receiving at a higher frequency (7.5 to 25 MHz). Parameters varied include the frequency and peak negative pressure of transmitted waves, center frequency of the receiving transducer, microbubble concentration, and microbubble size. CTR is maximized at the lowest transmission frequencies but would be acceptable for imaging in the 1.5 to 3.5 MHz range. At these frequencies, CTR is optimized when a receiving transducer with a center frequency of 10 MHz is used, with the maximum CTR of 25.5 dB occurring when transmitting at 1.5 MHz with a peak negative pressure of 1600 kPa and receiving with a center frequency of 10 MHz. Axial resolution is influenced more heavily by the receiving center frequency, with a weak decrease in measured pulse lengths associated with increasing transmit frequency. A microbubble population containing predominately 4-μm-diameter bubbles yielded the greatest CTR, followed by 1- and then 2-μm bubbles. Varying concentration showed little effect over the tested parameters. CTR dependence on transmit frequency and peak pressure were confirmed through in vivo imaging in two rodents. These findings may lead to improved imaging of vascular remodeling in superficial or luminal cancers such as those of the breast, prostate, and colon.

Vascular channels formed by subpopulations of PECAM1+ melanoma cells

Targeting the vasculature remains a promising approach for treating solid tumors; however, the mechanisms of tumor neovascularization are diverse and complex. Here we uncover a new subpopulation of melanoma cells that express the vascular cell adhesion molecule PECAM1, but not VEGFR-2, and participate in a PECAM1-dependent form of vasculogenic mimicry (VM). Clonally-derived PECAM1+ tumor cells coalesce to form PECAM1-dependent networks in vitro and they generate well-perfused, VEGF-independent channels in mice. The neural crest specifier AP-2α is diminished in PECAM1+ melanoma cells and is a transcriptional repressor of PECAM1. Reintroduction of AP-2α into PECAM1+ tumor cells represses PECAM1 and abolishes tube-forming ability whereas AP-2α knockdown in PECAM1− tumor cells up-regulates PECAM1 expression and promotes tube formation. Thus, VM-competent subpopulations, rather than all cells within a tumor, may instigate VM, supplant host-derived endothelium, and form PECAM1-dependent conduits that are not diminished by neutralizing VEGF.

Molecular Acoustic Angiography: A New Technique for High-resolution Superharmonic Ultrasound Molecular Imaging

Ultrasound molecular imaging utilizes targeted microbubbles to bind to vascular targets such as integrins, selectins and other extracellular binding domains. After binding, these microbubbles are typically imaged using low pressures and multi-pulse imaging sequences. In this article, we present an alternative approach for molecular imaging using ultrasound that relies on superharmonic signals produced by microbubble contrast agents. Bound bubbles were insonified near resonance using a low frequency (4 MHz) element and superharmonic echoes were received at high frequencies (25-30 MHz). Although this approach was observed to produce declining image intensity during repeated imaging in both in vitro and in vivo experiments because of bubble destruction, the feasibility of superharmonic molecular imaging was demonstrated for transmit pressures, which are sufficiently high to induce shell disruption in bound microbubbles. This approach was validated using microbubbles targeted to the αvβ3 integrin in a rat fibrosarcoma model (n = 5) and combined with superharmonic images of free microbubbles to produce high-contrast, high-resolution 3-D volumes of both microvascular anatomy and molecular targeting. Image intensity over repeated scans and the effect of microbubble diameter were also assessed in vivo, indicating that larger microbubbles yield increased persistence in image intensity. Using ultrasound-based acoustic angiography images rather than conventional B-mode ultrasound to provide the underlying anatomic information facilitates anatomic localization of molecular markers. Quantitative analysis of relationships between microvasculature and targeting information indicated that most targeting occurred within 50 μm of a resolvable vessel (>100 μm diameter). The combined information provided by these scans may present new opportunities for analyzing relationships between microvascular anatomy and vascular targets, subject only to limitations of the current mechanically scanned system and microbubble persistence to repeated imaging at moderate mechanical indices.

Optimization of Contrast-to-Tissue Ratio Through Pulse Windowing in Dual-Frequency “Acoustic Angiography” Imaging

Early-stage tumors in many cancers are characterized by vascular remodeling, indicative of transformations in cell function. We have previously presented a high-resolution ultrasound imaging approach to detecting these changes that is based on microbubble contrast agents. In this technique, images are formed from only the higher harmonics of microbubble contrast agents, producing images of vasculature alone with 100- to 200-μm resolution. In this study, shaped transmit pulses were used to image the higher broadband harmonic echoes of microbubble contrast agents, and the effects of varying pulse window and phasing on microbubble and tissue harmonic echoes were evaluated using a dual-frequency transducer in vitro and in vivo. An increase in the contrast-to-tissue ratio of 6.8 ± 2.3 dB was observed in vitro using an inverted pulse with a cosine window relative to a non-inverted pulse with a rectangular window. The increase in mean image intensity resulting from contrast enhancement in vivo in five rodents was 13.9 ± 3.0 dB greater for an inverted cosine-windowed pulse and 17.8 ± 3.6 dB greater for a non-inverted Gaussian-windowed pulse relative to a non-inverted pulse with a rectangular window. Implications for pre-clinical and diagnostic imaging are discussed.

The “Fingerprint” of Cancer Extends Beyond Solid Tumor Boundaries: Assessment With a Novel Ultrasound Imaging Approach

Goal: Abnormalities of microvascular morphology have been associated with tumor angiogenesis for more than a decade, and are believed to be intimately related to both tumor malignancy and response to treatment. However, the study of these vascular changes in-vivo has been challenged due to the lack of imaging approaches which can assess the microvasculature in 3-D volumes noninvasively. Here, we use contrast-enhanced “acoustic angiography” ultrasound imaging to observe and quantify heterogeneity in vascular morphology around solid tumors. Methods: Acoustic angiography, a recent advance in contrast-enhanced ultrasound imaging, generates high-resolution microvascular images unlike anything possible with standard ultrasound imaging techniques. Acoustic angiography images of a genetically engineered mouse breast cancer model were acquired to develop an image acquisition and processing routine that isolated radially expanding regions of a 3-D image from the tumor boundary to the edge of the imaging field for assessment of vascular morphology of tumor and surrounding vessels. Results: Quantitative analysis of vessel tortuosity for the tissue surrounding tumors 3 to 7 mm in diameter revealed that tortuosity decreased in a region 6 to 10 mm from the tumor boundary, but was still significantly elevated when compared to control vasculature. Conclusion: Our analysis of angiogenesis-induced changes in the vasculature outside the tumor margin reveals that the extent of abnormal tortuosity extends significantly beyond the primary tumor mass. Significance: Visualization of abnormal vascular tortuosity may make acoustic angiography an invaluable tool for early tumor detection based on quantifying the vascular footprint of small tumors and a sensitive method for understanding changes in the vascular microenvironment during tumor progression.

First-in-Human Study of Acoustic Angiography in the Breast and Peripheral Vasculature

Screening with mammography has been found to increase breast cancer survival rates by about 20%. However, the current system in which mammography is used to direct patients toward biopsy or surgical excision also results in relatively high rates of unnecessary biopsy, as 66.8% of biopsies are benign. A non-ionizing radiation imaging approach with increased specificity might reduce the rate of unnecessary biopsies. Quantifying the vascular characteristics within and surrounding lesions represents one potential target for assessing likelihood of malignancy via imaging. In this clinical note, we describe the translation of a contrast-enhanced ultrasound technique, acoustic angiography, to human imaging. We illustrate the feasibility of this technique with initial studies in imaging the hand, wrist and breast using Definity microbubble contrast agent and a mechanically steered prototype dual-frequency transducer in healthy volunteers. Finally, this approach was used to image pre-biopsy Breast Imaging Reporting and Data System (BI-RADS) 4 and 5 lesions <2 cm in depth in 11 patients. Results indicate that sensitivity and spatial resolution are sufficient to image vessels as small as 0.2 mm in diameter at depths of ~15 mm in the human breast. Challenges observed include motion artifacts, as well as limited depth of field and sensitivity, which could be improved by correction algorithms and improved transducer technologies.

Optimization of contrast-to-tissue ratio and role of bubble destruction in dual-frequency contrast-specific “acoustic angiography” imaging

Recently, dual-frequency transducers have enabled high-spatial resolution, high-contrast imaging of microvasculature by transmitting at a low frequency and receiving broadband superharmonic echoes from microbubble contrast agents at a higher frequency. In this work, we examine the imaging parameters for optimizing contrast-to-tissue ratio (CTR) for dual-frequency imaging and the relationship between bubble destruction and broadband harmonic signal production. CTR was assessed in vitro by acquiring scattered echoes by bubbles and beef muscle for transmit pressures up to 2 MPa, transmit frequencies from 1.5-8 MHz, and receive frequencies from 7.5 to 25 MHz. Optimum CTR (25.5 dB) was found to occur at the lowest transmit frequencies, though a broad peak exists within the 1.5-3.5 MHz range. At these frequencies, CTR is optimized when receiving at a center frequency of 10 - 15 MHz. A 4 μm-diameter microbubble population yielded ~5 dB higher CTR than a 1 μm population. Single bubble behavior was assessed with simultaneous acoustic and optical recordings. For n=250 single bubbles subjected to five consecutive single-cycle pulses (100-500 kPa), three primary categories of bubble behavior were observed optically: 1) no change in bubble diameter, 2) bubble shrinking (deflation), and 3) immediate bubble destruction (fragmentation). Matched acoustic data indicate that superharmonic signals having the broadest bandwidth and highest energy are associated with shell fragmentation. In the deflation case, a weaker superharmonic signal is produced with an amplitude approximately 25% of the signal in the shell fragmentation case. Similar regimes were observed in vivo, suggesting that bubble diameter, transmit frequency, peak negative pressure, and frame rate must be selected in light of the intended application, accounting for attenuation and local perfusion rate in the region of interest.

Which properties of the vascular architecture are reflected by dynamic contrast-enhanced ultrasound imaging of dispersion and wash-in rate? A comparison with acoustic angiography

Tumor growth requires formation of new angiogenic vessels, which differ in their morphology from those of healthy tissue. These vascular abnormalities result in altered blood flow dynamics, which can be assessed by dynamic contrast-enhanced ultrasound (DCE-US). Two distinct approaches are typically employed in DCE-US following an intravenous injection of ultrasound contrast agent: assessment of perfusion or dispersion parameters from the measured time intensity curves [1]. In this paper, we compare maps of dispersion and perfusion with those of acoustic angiography (AA), a 3D high-resolution technique capable of capturing the vascular morphology [2]. We aim at determining those properties of the vascular architecture that are reflected by perfusion and dispersion parameters, as well as their evolution over time as tumor grows. To this end, a longitudinal study has been performed with tumor models in 3 rats.

In-vivo characterization of angiogenesis in tumor-bearing rats using multiple scattering of ultrasound

There are significant differences in microvasculature density, tortuosity, and anisotropy of cancerous and benign tissue, which makes non-invasive methods to quantify these microvascular architectural properties highly relevant for applications like monitoring the response to anti-angiogenic treatments. We present a method to quantify these properties using ultrasound multiple scattering from contrast agent microbubbles injected into the vasculature.