Acoustic Droplet Initiated Radiosensitivity of Hepatocellular Carcinoma - Project Summary Hepatocellular carcinoma (HCC) is the third leading cause of cancer mortality worldwide and the fastest growing malignancy in the United States. The use of radioembolization or external beam radiation are frequently used for HCC treatment. However, both these techniques demonstrate a treatment response of only 25-89% depending on the evaluation criteria used and progression free survival of roughly 13 months. Consequently, additional strategies for HCC radiosensitization are greatly needed. Contrast-enhanced ultrasound is a well- established technique for HCC imaging and employs ultrasound-sensitive microbubbles to better visualize tumor vascularity. Ultrasound-triggered microbubble destruction (UTMD) and nanobubble destruction has been shown to sensitize tumors to radiotherapy by inducing vascular endothelial cell apoptosis and increasing ceramide production. Our group has demonstrated the feasibility, safety, and preliminary efficacy of using UTMD for sensitizing HCC to radiotherapy in animal models and, more recently, in a randomized clinical trial. However, we believe the magnitude of these bioeffects is limited by the size of UCAs (1-8 µm in diameter), which restricts their extravasation into the tumor tissue and limits the shear forces generated during cavitation. One attractive alternative to UCAs is acoustic droplets, which are composed of liquid perfluorocarbons encapsulated above their boiling point. The smaller size of these agents (< 200 nm) and stability relative to nanobubbles permits greater penetration into the tumor interstitium compared to ultrasound-sensitive bubbles. Acoustic droplets can be locally vaporized and destroyed using diagnostic ultrasound equipment, thereby generating larger local forces on the surrounding tissue than can be achieved with UTMD. Consequently, this proposal will investigate the use of acoustic droplets to improve HCC radiosensitivity relative to UCA. Two specific droplet formulations will be used, prioritizing either efficient clinical translation or droplet stability in vivo. In aim 1 we will characterize acoustic droplets made via condensation of a commercially approved UCA and by benchtop fabrication of phospholipid- coated perfluorobutane droplets, and evaluate their ability to initiate ceramide production, cellular apoptosis, and radiosensitivity relative to micro- and nanobubbles following ultrasound-triggering in vitro. Aim 2 will evaluate the ability of the two acoustic droplet formulations to generate ceramide production and vascular disruption relative to micro- and nanobubbles in a subcutaneous syngeneic model of HCC as well as explore the effects on tumor immune response. Finally, aim 3 will assess the potential improvement of the two acoustic droplet formulations relative to ultrasound-sensitive gas bubbles to act as a radiosensitizer in an orthotopic model of human HCC. At the conclusion of this work, we will have evaluated the mechanisms and potential therapeutic advantages of using acoustic droplet vaporization for HCC radiosensitization. These approaches are clinically translatable and may ultimately improve HCC patient survival by improving radiotherapy response.
