Ultra-Low-Dose Ultrasound Molecular Imaging for the Detection of Angiogenesis in a Mouse Murine Tumor Model How Little Can We See?

Objectives: The objective of this study was to evaluate the minimum microbubble dose for ultrasound molecular imaging to achieve statistically significant detection of angiogenesis in a mouse model. Materials and Methods: The preburst minus postburst method was implemented on a Verasonics ultrasound research scanner using a multiframe compounding pulse inversion imaging sequence. Biotinylated lipid (distearoyl phosphatidylcholine-based) microbubbles that were conjugated with antivascular endothelial growth factor 2 (VEGFR2) antibody (MBVEGFR2) or isotype control antibody (MBControl) were injected into mice carrying adenocarcinoma xeno-grafts. Different injection doses ranging from 5 x 10(4) to 1 x 10(7) microbubbles per mouse were evaluated to determine the minimum diagnostically effective dose. Results: The proposed imaging sequence was able to achieve statistically significant detection (P < 0.05, n = 5) of VEGFR2 in tumors with a minimum MBVEGFR2 injection dose of only 5 x 10(4) microbubbles per mouse (distearoyl phosphatidylcholine at 0.053 ng/g mouse body mass). Nonspecific adhesion of MBControl at the same injection dose was negligible. In addition, the targeted contrast ultrasound signal of MBVEGFR2 decreased with lower microbubble doses, whereas nonspecific adhesion of MBControl increased with higher microbubble doses. Conclusions: The dose of 5 x 10(4) microbubbles per animal is now the lowest injection dose on record for ultrasound molecular imaging to achieve statistically significant detection of molecular targets in vivo. Findings in this study provide us with further guidance for future developments of clinically translatable ultrasound molecular imaging applications using a lower dose of microbubbles.