Development of a Targeted Ultrasound Microbubble for Early Detection of Ovarian Cancer
Authors: Flannery Meghan M. , Dorayappan Kalpana Deepa Priya , Calo Corinne A. , Kalmar Eileen L. , Tweedle Michael F. , Suarez Adrian A. , Cohn David E. , Karuppaiyah Selvendiran 
Published: April 2020

Background and Objective

Ovarian cancer is the deadliest gynecologic malignancy. Although curable in up to 90% of patients diagnosed during early‐stage disease, most patients are not diagnosed until late‐stage, at which point, the overall 5‐year survival rate is less than 25%. This disparity clearly shows that early detection is a key factor for improving patient survival. However, there is no accurate screening tool available, and currently utilized diagnostic modalities lack specificity for early disease. Transvaginal ultrasound, the imaging gold standard for routine examination of ovarian masses, has many advantages that make it an ideal diagnostic tool, such as its low cost, safety, and sensitivity. Despite its advantages, ultrasound is lacking in specificity, as it is limited in its ability to distinguish between benign and malignant ovarian masses. This study seeks to address this deficiency via a novel technique known as targeted ultrasound. Targeted ultrasound involves the use of micron‐sized (~1–4μm) gas‐filled bubbles stabilized by an outer shell to which targeting molecules, such as antibodies, are conjugated. Microbubbles are injected intravenously, where they travel systemically to bind to their target. Upon confrontation with ultrasound waves, they create a unique ultrasound backscatter, indicating the presence of the target. Due to their micron‐scale size, microbubbles are too large to exit the vascular space and enter surrounding tissues; therefore, targets must be expressed on endothelial cells lining the luminal surface of blood vessels. Previous studies have shown tissue factor (TF) to be a unique, tumor‐specific marker expressed in a number of different tumor types, including ovarian cancer. Although not expressed on normal, healthy vascular endothelial cells (VECs), TF expression has been shown on tumor‐associated VECs. Additionally, TF is associated with tumor angiogenesis ‐ an early event in tumorigenesis. This unique expression profile makes TF an attractive target for early cancer detection via targeted ultrasound. The objective of this study was to develop a TF‐targeted ultrasound microbubble for early detection of ovarian cancer.

Methods

TF expression on murine and human ovarian cancer tissues was examined using western blotting and immunohistochemistry. Targeted microbubbles were made by conjugating an anti‐TF antibody to the outer shell of microbubbles via streptavidin‐biotin chemistry. Their binding efficacy and specificity was validated using in vitro cell binding assays.

Results

TF expression was shown on ovarian tumor‐associated VECs in patient and murine tissues. TF‐targeted microbubbles, with the ability to detect and bind to TF‐expressing cells in vitro, were developed.

Conclusions

Based upon these findings, we anticipate that in vivo translation of TF‐targeted microbubbles will show effective detection of tumor VECs in a murine model of ovarian cancer. Development of a targeted microbubble against a unique tumor marker, such as TF, would have significant implications towards increasing the number of early‐stage ovarian cancer diagnoses and ultimately, help save the lives of more women affected by this disease.