In vivo quantification of tumor receptor binding potential with dual-reporter molecular imaging

Abstract

Purpose: Receptor availability represents a key component of current cancer management. However, no approaches have been adopted to do this clinically, and the current standard of care is invasive tissue biopsy. A dual-reporter methodology capable of quantifying available receptor binding potential of tumors in vivo within a clinically relevant time scale is presented.

Procedures: To test the methodology, a fluorescence imaging-based adaptation was validated against ex vivo and in vitro measures of epidermal growth factor receptor (EGFR) binding potential in four tumor lines in mice, each line expected to express a different level of EGFR.

Results: A strong correlation was observed between in vivo and ex vivo measures of binding potential for all tumor lines (r = 0.99, p < 0.01, slope = 1.80 ± 0.48, and intercept = -0.58 ± 0.84) and between in vivo and in vitro for the three lines expressing the least amount of EGFR (r = 0.99, p < 0.01, slope = 0.64 ± 0.32, and intercept = 0.47 ± 0.51).

Conclusions: By providing a fast and robust measure of receptor density in tumors, the presented methodology has powerful implications for improving choices in cancer intervention, evaluation, and monitoring, and can be scaled to the clinic with an imaging modality like SPECT.

Fig. 1

Compartment model for dual-reporter approach for determining receptor status in vivo. An example of some raw experimental results—specifically, the white-light image of a subcutaneous A431 tumor, the uptake of the untargeted reporter, and the uptake of the targeted reporter—is depicted in a–c, respectively. The units of fluorescence are arbitrary, and the scale is equivalent for both the targeted and untargeted fluorescence maps. An illustration of the presumed distribution of both targeted and untargeted reporter concentrations within the tissue is presented in d. Specifically, it is assumed that the untargeted reporter (green) is dispersed throughout the blood compartment and the extravascular, intercellular (“free”) space, and that the targeted reporter (red) is dispersed throughout the blood, free, and “bound” space (i.e., attached to the specific receptor). The compartment model associated with the distribution of the untargeted reporter (the green dashed box) and the compartment model associated with the distribution of the targeted reporter (the red dashed box) are presented in e. A picture of the mouse slide setup for imaging is presented in f.

Fig. 2

Targeted and untargeted reporter uptake. Typical fluorescence uptake curves of targeted (red circles) and untargeted (green “x's”) reporter over time (in minutes) in blocked U251, 9L-GFP, U251, AsPC-1, and A431 tumors are presented in a–e, respectively. These curves correspond to the specific tumors maps displayed in Fig. 3. The fluorescence in these curves is normalized to the maximum fluorescence in the targeted reporter within the 60-min window. The black dashed line in each subfigure displays the fit of the targeted reporter uptake curve using the dual-reporter model.

Fig. 3

Binding potential maps. The tumor lines (i.e., the rows of the table) were ordered to represent the expected levels of epidermal growth factor receptor availability, with availability increasing from the top to the bottom. The first column displays typical fluorescence uptake of the untargeted (Untar.) reporter in tissue including and surrounding each tumor line examined. The second column displays the uptake of the epidermal growth factor-tagged targeted (Tar.) reporter in the same tumors. These images in the first two columns were taken at 700 and 800 nm, respectively, using the LI-COR Odyssey scanner at 60 min post-reporter injection. The units of fluorescence are arbitrary, and the scale is equivalent for both the targeted and untargeted fluorescence maps. The third column presents the binding potential (BP) maps of the corresponding tumors in the first two columns. These were calculated using the Logan graphical adaptation of the dual-reporter model. Binding potential is a unitless value proportional to receptor expression.

Fig. 4

Binding potential vs. targeted reporter uptake. In a, a boxplot of the binding potentials (BP) calculated from the average targeted and untargeted reporter uptake curves in each tumor is presented against tumor line in descending order of expected epidermal growth factor receptor expression (*p<0.05, ^#p<0.05). As a comparison, b displays the corresponding box plot of targeted fluorescence reporter uptake in each tumor at 60 min post-reporter injection.

Fig. 5

Validation of in vivo binding potential. In a, the correlation of the average in vivo BP of the 9L-GFP (purple), U251 (green), AsPC-1 (blue), and A431 (red) tumor groups to the corresponding ex vivo measured ratio of targeted to untargeted fluorescence from 10-µm tissue slices compared (r=0.99, p<0.01, slope=1.80±0.48, intercept=−0.58±0.84). In b, the correlation between in vivo BP and in vitro BP (measured from flow cytometry and histology) is displayed. The tumor groups are colored as in c with a significant correlation being observed for the 9L-GFP, U251, and AsPC-1 groups (r=0.99, p<0.01, slope= 0.64±0.32, intercept=0.47±0.51).