Materials and Methods
The Vevo 2100 High Resolution Ultrasound Imaging System was used to acquire all of the images in this study. Images were acquired from athymic nude mice implanted with hepatocarcinoma cells subdermally in the hind limb four to five weeks before imaging.
Nontargeted MicroMarker contrast agents were used to enhance the visualization of blood flow down to the capillary level. The contrast agents were injected intravenously, typically through the tail vein, and circulated freely through functional blood vessels. The microbubbles were 2–3 µm in size and were made up of a phospholipid shell containing a polyethylene glycol outer shell, along with a perfluorobutane/nitrogen gas core.
A bolus injection of the nontargeted MicroMarker contrast agents provides a wash-in curve for analysis (Figure 1). Here, two regions of interest were drawn on the tumor—the purple outlining the entire tumor and the pink outlining an area with increased resistance to perfusion. Software analysis tools allow for quantification of the peak enhancement, which is a measure of relative blood volume, and for quantification of the time to peak, which is a measure of relative blood velocity.
In Figure 1, the highly resistive area shows a much longer time to peak, therefore, blood is moving into that area at a much lower velocity than the whole tumor, the peak enhancement is also much lower, indicating a lower blood volume when compared to the whole tumor.
Target-ready MicroMarker contrast agents are similar in structure to the non-targeted MicroMarker contrast agents, however, there is a streptavidin molecule attached to the polyethylene glycol (PEG) molecule that makes up the outermost layer of the shell. This molecule allows for the conjugation of any biotinylated molecule to the microbubble, such as a primary antibody for vascular endothelial growth factor receptor 2 (VEGFR2). The conjugated antibody, for example, can then bind to its ligand on the surface of endothelial cells.
A negative control, in the form of an isotype control antibody, is utilized to allow for the quantification of any non-specific binding of the contrast agent.
A destructive pulse must be applied in order to perform the quantification; a destruction curve can then be plotted for both the isotype control and VEGFR2 antibodies (Figure 2). Quantification involves calculating the change in contrast enhancement following the destruction pulse. The results from this study show some nonspecific binding, however, there is substantial specific VEGFR2 binding in this tumor model.