Targeting microbubbles with Shiga-Toxin B-subunit

The targeting moiety (B-subunit) of the Shiga toxin (STxB) is a very potent ligand for the glycolipid Gb3, which is expressed in ovarian, colorectal and breast carcinomas. It is also present on endothelial cells of tumor neovascularization. This study demonstrates the use of STxB for targeting microbubbles onto cancerous cells. STxB-functionalized microbubbles and biotinylated controls were incubated with colorectal carcinoma HT29 in opticell plates. The culture plates were observed by fluorescence microscopy. The plates were then installed in a water-bath under a 8 MHz transducer array. STxB and control microbubbles were also injected in nude mice with subcutaneous breast tumors, before ultrasound imaging. FACS analysis demonstrated that STxB was stably associated with the microbubbles. Fluorescence microscopy showed that STxB-functionalized microbubbles adhered favorably to the Gb3 expressing cells, as compared to cells in which Gb3 expression was inhibited. Disruption ultrasonography of the culture plates showed a 12 dB difference in average backscatter intensity of the surface of Gb3 expressing cells, compared to Gb3-negative cells. An intensity difference of 18 dB was also observed between cells that were incubated with STxB-functionalized-microbubbles, as compared to unspecific microbubbles. The experiment in mice showed a significant increase in microbubble-signal intensity within gb3-positive tumors after being injected with STxBmicrobubbles. These in vitro and in vivo experiments showed that STxBfunctionalized microbubbles bind specifically to cells expressing the Gb3 glycolipid. The targeting moieties of toxins are a new group of ligands for microbubbles and have several advantages compared to antibodies and small peptides.