In this study, we evaluated the in vivo efficacy of a model system using Ad vector modulation of an intratumoral HA protein. Traditionally, targeted MB studies have used different cell lines to verify MB binding to overexpressed receptors found in tissue states of disease. While convenient, the use of different cell lines in animal implantation and experimentation introduces considerable variation in tumor yield, cell line proliferation rates and tumor growth and metastatic ability. The use of Ad vectors to control and modulate receptor expression offers the ability to conduct molecular US imaging studies using the same cell line so the above noted variations are minimized. Without any bias of varying receptor expression, the mechanisms behind molecular US imaging are more easily explored using this model system.
Previously, we investigated the use of Ad vectors for modulating receptor expression and targeted MB binding in an in vitro
study (Saini et al. 2011
). Results showed a linear response between increased Ad-HA-GFP vector dose and receptor expression in the 2LMP breast cancer cell line. Additionally, it was shown that HA-targeted MB attachment was proportional to receptor density. This study concluded that the Ad vector approach is an ideal model to verify and analyze in vitro
targeted MB performance.
For in vivo
evaluation, viral infection occurred intratumorally and targeted MB “flash” sequences were used for the US imaging technique. This sequence has been widely used to quantify targeted MB binding in vivo
because it allows for direct measurement of MB accumulation in the vessels. Adherent MBs are quantitatively assessed by subtracting post-flash intensities from the pre-flash intensities (Willmann et al. 2008
). By modulating the Ad-HA-hSSTr2-GFP vector dose, expression of the exogenous cellular receptor (HA protein) within the tumor at low and high concentrations was achieved (Zinn et al. 2002
). In vivo
results showed that the HA-targeted MB frame intensity difference was significantly different for day 2 (high Ad-HA-GFP vector dose) than for day 1 (low Ad-HA-GFP dose). For IgG isotype control MBs, signal enhancement attributable to MB accumulation relative to HA-targeted MB was considerably lower. Therefore, MB accumulation is proportional viral dose and expression levels of the induced receptors in vivo
. Additionally, fluorescence imaging confirmed that high Ad vector dosing increases GFP expression, hence, more target proteins induced, compared with the low and no Ad vector dosing. The spatial distribution of the MB contrast enhancement was seen mostly in the tumor periphery. Weller et al. and Korpanty et al. have similarly shown peripheral enhancement to correlate with viable tissue (Korpanty et al. 2007
; Weller et al. 2005
). Future work would incorporate histologic analysis to correlate hyperechoic regions with viable tissues for further quantitative analysis of targeted MBs.
Due to the nature of the fixed circular ROI for image analysis, extratumoral intensities could have been included in the analysis, therefore, skewing results. One limitation to this study was that due to slight variation in positioning of the US probe, tumor cross-sections of the animals were not known to be consistent from day 1 to day 2. Therefore, the radius of the circular ROI was slightly different from day 1 to day 2. However, ROI radius and time points after MB injections were held constant between each mouse for both targeted and control MB imaging sessions. Additionally, the order of the ad-injection (low dose followed by a high dose after the imaging session) might have brought bias during experimentation, however, the order of the Ad vector dose allowed for a longitudinal study of the induced receptors while having a consistent tumor size and population for targeted and control MB imaging between the 2 days. Also, the lack of control mice without adenoviral dose was not present for quantitative analysis.
If receptor density increases (or decreases) during disease progression, molecular US imaging of targeted MBs can be useful for characterizing the diseased tissue. The model system introduced can be used extensively in preclinical molecular US imaging studies where minimal biologic variation in tumor-bearing animals is desirable. The correlation established between MB accumulation and increased cellular receptors may be useful in applications like receptor profiling and/or therapeutic monitoring. Presently, clinical translation of targeted MBs with the streptavidin-biotin bridge to conjugate MBs and antibody is not possible because of a strong immunogenic response in human. However, some groups have recently fabricated a MB with an alternate binding chemistry to avoid immunogenic response and, thus, have potential for clinical application (Pysz et al. 2010b
; Willmann et al. 2010
; Bzyl et al. 2011
; Pochon et al. 2011
In conclusion, the results of this study verified a model system using Ad vector techniques to modulate receptor protein expression and quantify targeted MB accumulation in vivo using molecular US imaging.