Ultrasound contrast agents (UCAs) have the ability to go throughout the body and reach anywhere there is active vascularity. As clinicians and scientists, we use this to our advantage for diagnostic and therapeutic purposes. A unique characteristic of a UCA is its ability to generate nonlinear responses at sufficient pressure. These nonlinear responses produce harmonics in an acoustic field. UCAs undergo natural oscillations, and, at higher pressures, these UCAs can produce bioeffects. Inertial cavitation is the transient destruction of UCAs from increased pressure, while stable cavitation is a constant oscillation. Both of these cavitation states can produce shear stress on vessel walls, particularly endothelial cells.

Endothelial cells are the cell layer that lines all blood vessels in the body and are responsible for many functions but primarily control the passage of nutrients into tissues.¹ In normal tissue, endothelial cells are uniform and form an organized monolayer network with tight junction connections.¹ However, in tumor endothelial cells (TECs), it is much more of a chaotic process. In TECs, long, fragile cytoplasmic projections extend into the vessel lumen, creating small openings and gaps in the vessel wall.¹ In addition to smaller gaps, larger openings (up to 1.5 µm) have also been identified, which make cell closures more difficult. These small and large gaps in TECs can be harnessed to produce endothelial cell apoptosis by destroying UCAs via inertial cavitation. Utilizing inertial cavitation has been shown to produce endothelial cell apoptosis (cell death) by generating bioeffects (ie, shock waves, microjets, micro streams, and thermal effects) that mechanically perturb TEC membranes.

Moreover, there are many components to a cell membrane, which goes beyond the scope of this blog post; however, sphingolipids are an essential enzyme to a cell membrane.² Sphingolipids help maintain cellular homeostasis and are closely associated with cellular biologic functions, such as proliferation, apoptosis, or oxidative stress on endothelial cells.² Some sphingolipids, such as ceramide, are important second signaling molecules that determine cell proliferation or death.

Traditionally, radiation therapy has been thought to act by damaging the DNA of cells via double and single DNA strand breaks, resulting in apoptosis. However, more recent studies have suggested that blood vessels are the determining factor of tumor response to radiation therapy at high doses (>8–19 Gy).³ Endothelial cells exposed to high doses of radiation upregulate the acid-sphingomyelinase pathway (ASMASE), which hydrolyzes sphingomyelin (dominant sphingolipid) into apoptosis, with ceramide acting as the second messenger. El Kaffas et al suggest that a primary factor why endothelial cells respond differently than other cell types is because endothelial cells have a 20x enrichment of a nonlyosomal secretory form of the ASMASE enzyme.^4,5 This enzyme is associated with membrane remodeling, restructuring, responses to shear stress, and activation of ceramide from cell stressors.^6,7 Additionally, endothelial cells are more sensitive to shear stress and mechanical forces.⁴ Radiation therapy and inertial cavitation treatments separately have been shown to produce tumor cell death. Combining these two treatments results in an increased accumulation of ceramide production and apoptosis, leading to improved tumor radiosensitivity.

Over the past decade, primarily in pre-clinical research, the benefits of combining microbubbles and radiation have been shown. From one of the original pre-clinical studies validating this concept, Czarnota et al showed that inertial cavitation and radiation therapy in a human prostate xenograft model in mice led to a 10-fold increase in ceramide-related endothelial cell death, confirming the benefits of the combined relationship.⁷ In addition, the study demonstrated a significant effect with a decreased radiotherapy dose (2 Gy versus 8 Gy) when combined with inertial cavitation compared to stand-alone treatment regimens. This mechanism has mainly been validated with in vivo studies. However, my group at Thomas Jefferson University has a first-ever pilot clinical trial incorporating ultrasound-triggered microbubble destruction (UTMD) to sensitize hepatocellular carcinoma in patients that receive the locoregional therapy, trans-arterial radioembolization (TARE).⁸ In an interim analysis, there was a greater prevalence of tumor response in the patients receiving TARE plus UTMD as opposed to in those who received TARE alone. Additionally, lab values and liver function tests demonstrated no significant differences between study groups, indicating that adding microbubble cavitation does not affect patient safety or liver functions.

This study is an ongoing clinical trial and will complete enrollment within the next 6 months, and this concept has been incorporated in non-HCC tumors in an ongoing pilot clinical trial at Thomas Jefferson University (NCT# 03199274). As a result of our research to date, we have learned that incorporating UTMD with radiation therapy may help sensitize tumors for improved survival and treatment outcomes. Additional clinical research is needed in this field because the combination treatment regimen may be easily incorporated into non-liver tumors.

References

1. Dudley AC. Tumor endothelial cells. Cold Spring Harb Perspect Med 2012; 2:a006536. doi: 10.1101/cshperspect.a006536.
2. Lai Y, Tian Y, You X, Du J, Huang J. Effects of sphingolipid metabolism disorders on endothelial cells. Lipids Health Dis 2022: 21(1):101. doi: 10.1186/s12944-022-01701-2.
3. Paris F et al. Endothelial apoptosis as the primary lesion initiating intestinal radiation damage in mice. Science 2001; 293(5528):293–297. doi: 10.1126/science.1060191.
4. El Kaffas A and Czarnota GJ. Biomechanical effects of microbubbles: from radiosensitization to cell death. Future Oncol 2015; 11(7):1093–1108. doi: 10.2217/fon.15.19.
5. Tabas I. Secretory sphingomyelinase. Chem Phys Lipids 1999; 102(1):123–130. doi: 10.1016/S0009-3084(99)00080-8.
6. El Kaffas A, Al-Mahrouki A, Hashim A, Law N, Giles A, Czarnota GJ. Role of acid sphingomyelinase and ceramide in mechano-acoustic enhancement of tumor radiation responses. J Natl Cancer Inst 2018; 110(9):1009–1018. doi: 10.1093/jnci/djy011.
7. Czarnota GJ et al. Tumor radiation response enhancement by acoustical stimulation of the vasculature. Proc Natl Acad Sci U S A  2012; 109(30): E2033-E2041. doi: 10.1073/pnas.1200053109.
8. Eisenbrey JR et al. US-triggered microbubble destruction for augmenting hepatocellular carcinoma response to transarterial radioembolization: a randomized pilot clinical trial. Radiology 2021; 298:450–457. doi: 10.1148/radiol.2020202321.

Corinne Wessner, MS, MBA, RDMS, RVT, is a research sonographer at Thomas Jefferson University and a PhD candidate at Drexel University in the School of Biomedical Engineering, Science and Health Systems in Philadelphia, PA. She is also Vice Chair of the American Institute of Ultrasound in Medicine’s (AIUM’s) High-Frequency Clinical and Preclinical Imaging Community (2023–2025).

Interested in reading more from Corinne Wessner? Check out these articles from the Journal of Ultrasound in Medicine (JUM). Members of AIUM can access them for free after logging in to the AIUM. Join the AIUM today!):

• Activation of Phase Change Contrast Agents Using Ionizing Radiation
• Improved Sensitivity of Ultrasound-Based Subharmonic Aided Pressure Estimation Using Monodisperse Microbubbles
• Contrast-Enhanced Ultrasound in Small Intestinal Ischemia: Proof of Concept