Angiogenesis has been known as a hallmark of solid tumor cancers for decades, yet ultrasound has been limited in its ability to detect the microvascular changes associated with malignancy. Here, we demonstrate the potential of 'ultrasound localization microscopy' applied volumetrically in combination with quantitative analysis of microvascular morphology, as an approach to overcome this limitation. This pilot study demonstrates our ability to image complex microvascular patterns associated with tumor angiogenesis in-vivo at a resolution of tens of microns -substantially better than the diffraction limit of traditional clinical ultrasound, yet using an 8 MHz clinical ultrasound probe. Furthermore, it is observed that data from healthy and tumor-bearing tissue exhibit significant differences in microvascular pattern and density. Results suggests that with continued development of these novel technologies, ultrasound has the potential to detect biomarkers of cancer based on the microvascular 'fingerprint' of malignant angiogenesis rather than through imaging of blood flow dynamics or the tumor mass itself.

Many studies have explored phase-change contrast agents (PCCAs) that can be vaporized by an ultrasonic pulse to form microbubbles for ultrasound imaging and therapy. However, few investigations have been published demonstrating the utility and characteristics of PCCAs as contrast agents in vivo. In this study, we examine the properties of low boiling point nanoscale PCCAs evaluated in vivo, and compare data to conventional microbubbles with respect to contrast generation and circulation properties. In order to do this, we develop a custom pulse sequence to vaporize and image PCCAs using the Verasonics research platform and a clinical array transducer. Results show that droplets can produce similar contrast enhancement to microbubbles (7.29 to 18.24 dB over baseline, depending on formulation), and can be designed to circulate for as much as 3.3 times longer than microbubbles. This study also demonstrates for the first time the ability to capture contrast wash-out kinetics of the target organ as a measure of vascular perfusion.

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-to-tissue 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.

Acoustic angiography imaging of microbubble contrast agents utilizes the superharmonic energy produced from excited microbubbles, and enables high-contrast, high-resolution imaging. However, the exact mechanism by which broadband harmonic energy is produced is not fully understood. In order to elucidate the role of microbubble shell fragmentation in superharmonic signal production, simultaneous optical and acoustic measurements were performed on individual microbubbles at transmit frequencies from 1.75 to 3.75 MHz and pressures near the shell fragmentation threshold for microbubbles of varying diameter. High-amplitude, broadband superharmonic signals were produced with shell fragmentation, while weaker signals (approximately 25% of peak amplitude) were observed in the presence of shrinking bubbles. Furthermore, when imaging populations of stationary microbubbles with a dual-frequency ultrasound imaging system, a sharper decline in image intensity with respect to frame number was observed for 1 μm bubbles than for 4 μm bubbles. Finally, in a study of two rodents, increasing frame rate from 4 to 7 Hz resulted in a decrease in mean steady-state image intensity of 27% at 1000 kPa and 29% at 1300 kPa. While the existence of superharmonic signals when bubbles shrink has the potential to prolong the imaging efficacy of microbubbles, parameters such as frame rate and peak pressure must be balanced with expected re-perfusion rate in order to maintain adequate contrast during in vivo imaging.

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.