Validation of controlled receptor expression using adenoviral techniquesand targeted ultrasound imaging

This paper details a model system for evaluating targeted ultrasound (US)contrast agents using adenoviral (Ad) vectors for controlled receptorexpression. Breast cancer cell receptor density in vitro was modulated byvarying the multiplicity of infection (MOI) from 0 to 100. Target receptors wereinduced using a GFP-positive Ad vector for gene transfer and expression of thehuman somatostatin receptor subtype 2a (hSSTr2) with a hemagglutinin (HA) tag.Subsequently, receptor expression and anti-HA antibody (Ab) binding was examinedwith flow cytometry. Targeted US contrast agents (MB) were created byconjugating either biotinylated anti-HA or isotype control Ab to the surface ofbiotin coated MBs via a streptavidin bridge. Targeted MBs were incubated with Adinfected cells with to test in vitro MB binding. An in vivo study was performedin tumor-bearing nude athymic mice with induced HA-hSSTr2-GFP receptorexpression by intratumoral injection of the Ad vector (MOI of 100). Mice weresorted and injected via the tail vein with the two MB groups followed by USimaging. 24 hrs later mice groups were switched and the MB study repeated.Experimental in vitro results found GFP expression to be directly correlatedwith Ad MOI (R² 0.96). Increasing the Ad MOI produced a correspondingincrease 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 Abexposed 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 cellsgroups incubated with control MBs (P=0.42) regardless of receptor density.However, cells exposed to targeted MBs showed increased levels of cell bindingproportional to receptor expression levels (P=0.02). Images taken from in vivoexperiments were analyzed by two blinded reviewers in consensus to compareintratumoral MB accumulation. It was concluded that 78% (7 of 9) of US imagesfrom the targeted MB group exhibited increased local intratumoral accumulationcompared to the control group. Overall, this study demonstrates use of an Advector for selectively controlling cellular expression and modulation of thereceptor density. Furthermore, results demonstrate targeted MB accumulation atthe receptor site.