Figure 4. Modified microbubble contrast agents were not retained at significant levels in nonmalignant vasculature.

B-mode images (black and white) are shown overlaid with CPS-mode images (green). CPS-mode is sensitive to ultrasound signal typically produced by microbubbles oscillating within an ultrasound field. (A) In the absence of ultrasound contrast agent (no contrast) there was no CPS-mode signal within the region of interest (dotted rectangle) outside of the tumor margins. Tissue artifacts generated the CPS-mode signal observed in the absence of contrast agent. Contrast agent freely flowing through both tumor and non-tumor vasculature generated CPS-mode signal throughout the field of view (panel A, perfusion, middle frames) with either streptavidin-coated (control, upper frames) or SFRP2- targeted ultrasound contrast agent (lower frames). No signal remained within the region of interest drawn outside of the tumor margins after allowing all freely flowing contrast agent to be cleared from the vasculature, while SFRP2 specific signal was retained within the tumor margins. (B) Modified microbubble contrast agents were not retained within kidney vasculature. Freely flowing streptavidin-loaded microbubbles (panel B, control, upper frames) or SFRP2 - targeted microbubbles (panel B, lower frames) were allowed to clear from the vasculature prior to three-dimensional molecular imaging. Single frames are shown from two different animals (animal 1 or animal 2). The dotted oval region of interest represents the location of the kidney (K) and there was no significant difference in average pixel intensity after injection of either streptavidin-loaded or SFRP2 -targeted microbubbles. In contrast, the liver (L) retained both modified microbubble contrast agents to a high degree. White scale bars in panels A and B represent 5 mm.