Molecular ultrasound assessment of colorectal tumor angiogenesis with endoglin-targeted contrast microbubbles

Colorectal cancer (CRC) remains the third most popular cancer in US. CRC biopsy usually shows substantially increased angiogenesis, which is promoted by a number of pro-angiogenic growth factors. Among all these factors, the expression of endoglin has been remarkably up-regulated on actively proliferating endothelial cells of neo-vasculature. Endoglin has been an independent clinical pathological target for the tumor aggressiveness assessment. In this study, we developed endoglin-targeted microbubbles (MBs), and employed targeted microbubbles enhanced ultrasound (US) imaging to assess the endoglin expression levels in neo-vasculature for non-invasive assessment of colorectal tumor angiogenesis. Endoglin-targeted microbubbles and control microbubbles (MBs) were prepared according to standard protocol. A parallel-plate flow chamber was employed, in which endoglin-targeted MBs and untargeted control MBs were tested across mouse SVR angiosarcoma cells (positive endoglin expression) and mouse 4T1 cells (negative endoglin expression) with the adhesion quantified. In vivo contrast enhanced US imaging (Vevo 2100; VisualSonics) was conducted using these two types of MBs at different progression stages in a subcutaneous COLO 201 xenograft model in nude mice (n=16). Finally, statistical analysis of endoglin expression levels was performed between in vivo molecular US signals and ex vivo endoglin expression levels from immunohistochemical test. Cell attachment of endoglin-targeted MBs was significantly higher than untargeted control MBs. Endoglin-positive SVR cells bound significantly more endoglin-targeted MBs than negative control 4T1 cells, and MBs attachment significantly correlated with endoglin expression levels on cells. There was a good correlation between in vivo molecular US signals using endoglin-targeted MBs and ex vivo expression levels of endoglin from immunoblotting result. The results indicate that the molecular US is much potential for non-invasive assessment of the expression levels of endoglin in colorectal tumor angiogenesis.