Juan D. Rojas

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.

Enhancing Nanoparticle Accumulation and Retention in Desmoplastic Tumors via Vascular Disruption for Internal Radiation Therapy.

Aggressive, desmoplastic tumors are notoriously difficult to treat because of their extensive stroma, high interstitial pressure, and resistant tumor microenvironment. We have developed a combination therapy that can significantly slow the growth of large, stroma-rich tumors by causing massive apoptosis in the tumor center while simultaneously increasing nanoparticle uptake through a treatment-induced increase in the accumulation and retention of nanoparticles in the tumor. The vascular disrupting agent Combretastatin A-4 Phosphate (CA4P) is able to increase the accumulation of radiation-containing nanoparticles for internal radiation therapy, and the retention of these delivered radioisotopes is maintained over several days. We use ultrasound to measure the effect of CA4P in live tumor-bearing mice, and we encapsulate the radio-theranostic isotope 177Lutetium as a therapeutic agent as well as a means to measure nanoparticle accumulation and retention in the tumor. This combination therapy induces prolonged apoptosis in the tumor, decreasing both the fibroblast and total cell density and allowing further tumor growth inhibition using a cisplatin-containing nanoparticle.

Optimizing Acoustic Activation of Phase Change Contrast Agents With the Activation Pressure Matching Method: A Review

Submicrometer phase-change contrast agents (PCCAs) consist of a liquid perfluorocarbon (PFC) core that can be vaporized by ultrasound (acoustic droplet vaporization) to generate contrast with excellent spatial and temporal control. When these agents, commonly referred to as nanodroplets, are formulated with cores of low boiling-point PFCs such as decafluorobutane and octafluoropropane, they can be activated with low-mechanical-index (MI) imaging pulses for diagnostic applications. Since the utilization of minimum MI is often desirable to avoid unnecessary biological effects, enabling consistent activation of these agents in an acoustic field is a challenge because the energy that must be delivered to achieve the vaporization threshold increases with depth due to attenuation. A novel vaporization approach called activation pressure matching (APM) has been developed to deliver the same pressure throughout a field of view in order to produce uniform nanodroplet vaporization and to limit the amount of energy that is delivered. In this paper, we discuss the application of this method with a Verasonics V1 Research Ultrasound System to modulate the output pressure from an ATL L11-5 transducer. Vaporization-pulse spacing optimization can be used in addition to matching the activation pressure through depth, and we demonstrate the feasibility of this approach both in vivo and in vitro. The use of optimized vaporization parameters increases the amount of time a single bolus of nanodroplets can generate useful contrast and provides consistent image enhancement in vivo. Therefore, APM is a useful technique for maximizing the efficacy of PCCA while minimizing delivered acoustic energy.

Super resolution contrast ultrasound imaging: Analysis of imaging resolution and application to imaging tumor angiogenesis

Tumor associated microvascular angiogenesis is an important biomarker for cancer diagnostics. However, ultrasound is challenged in the detection of these microvascular changes due to resolution and/or sensitivity limits. Recently proposed ultrasound localization microscopy technique can achieve a ten-fold resolution improvement compared with traditional ultrasound imaging and therefore, can achieve super-resolution vascular imaging. This study investigates the feasibility of this revolutionary technique to examine tumor associated microvascular angiogenesis. In vitro experimental results acquired from microtubes and in vivo 2D images from rat subcutaneous fibrosarcoma tumors show this technique might be a valuable tool for imaging tumor microvasculature.

In Vivo Assessment of the Potential for Renal Bio-Effects from the Vaporization of Perfluorocarbon Phase-Change Contrast Agents

Low-boiling-point perfluorocarbon phase-change contrast agents (PCCAs) provide an alternative to microbubble contrast agents. Although parameter ranges related to in vivo bio-effects of microbubbles are fairly well characterized, few studies have been done to evaluate the potential of bio-effects related to PCCAs. To bridge this gap, we present an assessment of biological effects (e.g., hemorrhage) related to acoustically excited PCCAs in the rodent kidney. The presence or absence of bio-effects was observed after sonication with various perfluorocarbon core PCCAs (decafluorobutane, octafluoropropane or a 1:1 mixture) and as a function of activation pulse mechanical index (MI; minimum activation threshold, which was a moderate MI of 0.81-1.35 vs. a clinical maximum of 1.9). Bio-effects on renal tissue were assessed through hematology and histology including measurement of blood creatinine levels and the quantity of red blood cell (RBC) casts present in hematoxylin and eosin-stained kidney tissue sections after sonication. Short-term (24 h) and long-term (2 and 4 wk) analyses were performed after treatment. Results indicated that bio-effects from PCCA vaporization were not observed at lower mechanical indices. At higher mechanical indices, bio-effects were observed at 24 h, although these were not observable 2 wk after treatment.

Ultrasound Molecular Imaging of VEGFR-2 in Clear-Cell Renal Cell Carcinoma Tracks Disease Response to Antiangiogenic and Notch-Inhibition Therapy.

Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. Methods A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. Results The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. Conclusion This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.

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.

In Vivo Molecular Imaging Using Low-Boiling-Point Phase-Change Contrast Agents: A Proof of Concept Study

Sub-micron phase-change contrast agents (PCCAs) have been proposed as a tool for ultrasound molecular imaging based on their potential to extravasate and target extravascular markers and also because of the potential to image these contrast agents with a high contrast-to-tissue ratio. We compare in vivo ultrasound molecular imaging with targeted low-boiling-point PCCAs and targeted microbubble contrast agents. Both agents were targeted to the intravascular (endothelial) integrin αvß3via a cyclic RGD peptide (cyclo-Arg-Gly-Asp-D-Tyr-Cys) mechanism and imaged in vivo in a rodent fibrosarcoma model, which exhibits angiogenic microvasculature. Signal intensity was measured using two different techniques, conventional contrast-specific imaging (amplitude/phase modulation) and a droplet vaporization imaging sequence, which detects the unique signature of vaporizing PCCAs. Data indicate that PCCA-specific imaging is more sensitive to small numbers of bound agents than conventional contrast imaging. However, data also revealed that contrast from targeted microbubbles was greater than that provided by PCCAs. Both control and targeted PCCAs were observed to be retained in tissue post-vaporization, which was expected for targeted agents but not expected for control agents. The exact mechanism underlying this observation remains unknown.

Vaporization Detection Imaging: A Technique for Imaging Low-Boiling-Point Phase-Change Contrast Agents with a High Depth of Penetration and Contrast-to-Tissue Ratio

Phase-change contrast agents (PCCAs) possess advantages over microbubble contrast agents, such as the ability to extravasate and circulate longer in the vasculature that could enhance the diagnostic capabilities of contrast-enhanced ultrasound. PCCAs typically have a liquid perfluorocarbon (PFC) core that can be vaporized into echogenic microbubbles. Vaporization of submicron agents filled with liquid PFCs at body temperature usually requires therapeutic pressures higher than typically used for diagnostic imaging, but low-boiling-point PCCAs using decafluorobutane or octafluoropropane can be vaporized using pressures in the diagnostic imaging regime. Low-boiling-point PCCAs produce a unique acoustic signature that can be separated from tissue and bubble signals to make images with high contrast-to-tissue ratios. In this work, we explore the effect of pulse length and concentration on the vaporization signal of PCCAs and a new technique to capture and use the signals to make high contrast-to-tissue ratio images in vivo. The results indicate that using a short pulse may be ideal for imaging because it does not interact with created bubbles but still produces strong signals for making images. Furthermore, it was found that capturing PCCA vaporization signals produced higher contrast-to-tissue ratio values and better depth of penetration than imaging the bubbles generated by droplet activation using conventional contrast imaging techniques. The resolution of the vaporization signal images is poor because of the low frequency of the signals, but their high sensitivity may be used for applications such as molecular imaging, where the detection of small numbers of contrast agents is important.

A new preclinical ultrasound platform for widefield 3D imaging of rodents

Noninvasive in vivo imaging technologies enable researchers and clinicians to detect the presence of disease and longitudinally study its progression. By revealing anatomical, functional, or molecular changes, imaging tools can provide a near real-time assessment of important biological events. At the preclinical research level, imaging plays an important role by allowing disease mechanisms and potential therapies to be evaluated noninvasively. Because functional and molecular changes often precede gross anatomical changes, there has been a significant amount of research exploring the ability of different imaging modalities to track these aspects of various diseases. Herein, we present a novel robotic preclinical contrast-enhanced ultrasound system and demonstrate its use in evaluating tumors in a rodent model. By leveraging recent advances in ultrasound, this system favorably compares with other modalities, as it can perform anatomical, functional, and molecular imaging and is cost-effective, portable, and high throughput, without using ionizing radiation. Furthermore, this system circumvents many of the limitations of conventional preclinical ultrasound systems, including a limited field-of-view, low throughput, and large user variability.