Validation of controlled receptor expression using adenoviral techniques and targeted ultrasound imaging

This paper details a model system for evaluating targeted ultrasound (US) contrast agents using adenoviral (Ad) vectors for controlled receptor expression. Breast cancer cell receptor density in vitro was modulated by varying the multiplicity of infection (MOI) from 0 to 100. Target receptors were induced using a GFP-positive Ad vector for gene transfer and expression of the human somatostatin receptor subtype 2a (hSSTr2) with a hemagglutinin (HA) tag. Subsequently, receptor expression and anti-HA antibody (Ab) binding was examined with flow cytometry. Targeted US contrast agents (MB) were created by conjugating either biotinylated anti-HA or isotype control Ab to the surface of biotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Ad infected cells with to test in vitro MB binding. An in vivo study was performed in tumor-bearing nude athymic mice with induced HA-hSSTr2-GFP receptor expression by intratumoral injection of the Ad vector (MOI of 100). Mice were sorted and injected via the tail vein with the two MB groups followed by US imaging. 24 hrs later mice groups were switched and the MB study repeated. Experimental in vitro results found GFP expression to be directly correlated with Ad MOI (R2 = 0.96). Increasing the Ad MOI produced a corresponding increase in binding and accumulation of anti-HA Ab on the cell surface (P < 0.01). However, no differences was found between Cy5.5-labeled anti-HA Ab exposed cell groups at an MOI of 0 (P > 0.29) or in the control Ab group (P > 0.44) indicating minimal nonspecific binding. No difference was found between cells groups incubated with control MBs (P > 0.42) regardless of receptor density. However, cells exposed to targeted MBs showed increased levels of cell binding proportional to receptor expression levels (P < 0.02). Images taken from in vivo experiments were analyzed by two blinded reviewers in consensus to compare intratumoral MB accumulation. It was concluded that 78% (7 of 9) of US images from the targeted MB group exhibited increased local intratumoral accumulation compared to the control group. Overall, this study demonstrates use of an Ad vector for selectively controlling cellular expression and modulation of the receptor density. Furthermore, results demonstrate targeted MB accumulation at the receptor site.