A white-light image and fluorescence uptake images of the targeted and untargeted imaging reporters at 60 min after injection are presented in , respectively, for a subcutaneous U251 tumor. The white-light image displays the typical morphology of the subcutaneously grown tumors, exposed and ready for imaging. The false colored images in , c demonstrate the quality of the typical fluorescence maps acquired at 700 and 800 nm wavelengths, respectively, in the Odyssey system at each imaging time point. Typical fluorescence uptake curves of targeted and untargeted reporter in a tumor region of interest, and the dual-reporter model fits, are depicted for all tumor lines in . There were clear differences between the uptake curves in different tumor lines, with a proportionately greater separation between targeted and untargeted reporter uptake observed for tumor lines expected to express more EGFR.
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 (more ...)
Additional fluorescence images of the uptake of untargeted and targeted reporter at 60 min post-reporter injection in all tumor lines are presented in the first two columns of , respectively. The average fluorescence contrast-to-background ratios (CBR) for the targeted reporter of the various tumors were generally quite low: 0.33±0.44, 3.35± 4.32, 1.60±1.95, 1.03±1.28, and 0.50±0.65 for the A431, AsPC-1, U251, 9L-GFP, and blocked U251 groups, respectively (this can be visualized in ). The low contrast observed suggests that much of the fluorescence uptake at early time points is non-specific-uptake related and not binding related (this is reflected in the similarities in the uptake maps of the targeted and untargeted reporters), and the large variance in CBR values within groups was likely related to inter-subject variability in tumor blood supply. Despite the low CBR observed for all tumor lines, the trend of fluorescence did match the expected EGFR expression levels of the various tumors except for the A431 tumor, which demonstrated very low fluorescence uptake. It is possible that the uptake was so low because of a high compressibility of the tumors, which may have caused a constricted blood flow to these tumors, since the animals were imaged tumor side down. However, Aerts et al.
also observed a low uptake of targeted reporter in the A431 line in an imaging setup that did not have the potential to restrict blood supply [23
], suggesting that the tumor has an inherently poor blood supply.
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 (more ...)
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 (more ...)
Corresponding in vivo
binding potential maps are presented in the third column of . In these maps, the Logan-based dual-reporter model was employed on a pixel-by-pixel basis to demonstrate the differences in binding potential throughout the imaging field. The more accurate, yet less robust-to-noise, Lammertsma-based dual-reporter model was used to calculate the average non-displaceable binding potential (BPND
)—a product of target affinity and receptor expression [16
]—from the uptake curves in all tumors ( presents typical fits of the model to the data). The average BPND
in each tumor group were significantly greater than that of the control leg tissue (p
<0.05). Furthermore, average BPND
measures between tumor groups were statistically significant from each other (p
<0.05), with the exception of A431 and AsPC-1. The trend in BPND
matched the predicted trend in EGFR expression, where A431 demonstrated the highest BPND
9L-GFP the lowest, and AsPC-1 and U251 in between. Furthermore, the lowest BPND
was observed in the blocked U251 tumors and the leg muscles (tissue with no expected appreciable levels of EGFR expression). Moreover, the CBR of the BPND
s was considerably larger than the fluorescence uptake: 47.2±14.7, 39.5±6.2, 23.3±4.6, 7.5±3.5, and 3.8±0.8 for the A431, AsPC-1, U251, 9L-GFP, and blocked U251, respectively. The non-zero CBR of the blocked U251 suggests either that the blocking was incomplete or that there are slight differences in either non-specific binding or the plasma input curve between the two reporters. However, the excellent correlation between in vivo
and in vitro
binding potentials suggests that whatever the reason, it does not seem to affect the quantitative potential of the approach. Further studies are on going using fluorescently labeled, targeted, and untargeted affibodies to employ reporters that are more chemically similar.
demonstrates the validation of the dual-reporter BPND measurements against ex vivo measures of binding potential from tissue slices. A very strong linear correlation was observed (r=0.99, p<0.01) with a slope of 1.80±0.48 and an intercept of −0.58±0.84. Some mismatch between the in vivo and ex vivo binding potentials were expected since the ex vivo measurement was based on the assumption that the blood concentration was negligible (leading to a possible underestimation of the ex vivo BPND). Furthermore, it was not possible to remove the effect of autofluorescence in the ex vivo images, which was more pronounced for the untargeted images (leading to the possible overestimation in the slope).
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 (more ...)
The BPND measurements also correlated well with the in vitro BP measurements (from flow cytometry and histology) for the 9L-GFP, U251, and AsPC-1 tumor groups (), but not for the A431, which displayed a very high expression of EGFR in vitro. For comparison purposes, the average binding potential measured in vivo, ex vivo, and in vitro for all tumor lines, as well as the average flow cytometry measures of receptor density per cell and the histological measures of cell density, are displayed in .
The average in vivo, ex vivo, and in vitro binding potentials for each tumor line, in addition to the average number of receptors per cell measured by flow cytometry and average in vivo cell density measured by histology.
The discrepancy between in vivo
and in vitro
measures of EGFR expression in the A431 tumor line was also observed by Aerts et al.
], and McLarty et al.
demonstrated that in vitro
measures of receptor density in tumor lines that express abnormally high levels of HER2 receptor severely overestimate the in vivo
measured binding potential [24
]. These results highlight a potential pitfall in using in vitro
measures to predict in vivo
receptor expression characteristics. That is, in vitro
measures determine the total number of receptors, and in vivo
measures presumably determine the total amount of receptors available for binding
]; therefore, if the in vivo
structure or biology of a tumor inhibit a significant fraction of binding to receptors, in vitro
measures can grossly overestimate the in vivo
receptor availability. A number of factors can influence the total number of receptors available for binding in vivo, e.g.
, the binding site barrier effect [25
] and high inter-tumoral pressure [26
], which impair the ability of a reporter to diffuse far from blood vessels.
While the results of this study highlight the potential of the dual-reporter approach to quantify receptor expression in vivo
, there are some potential limitations that require further study. First, the model requires that other than the ability of the targeted reporter to bind to a specific receptor, the untargeted and targeted reporters have identical pharmacokinetics. In this study, a somewhat arbitrary untargeted reporter was used that was found to have a similar plasma uptake curve to the targeted reporter [15
], to display a similar uptake curve to the targeted reporter in the blocking study (). However, in the future, it may be preferable to use an enantiomer of the targeted reporter to minimize any differences between the reporters. For example, for EGFR targeting, it would be possible to use an anti-EGFR Affibody® imaging agent for the targeted reporter and a negative control Affibody® imaging agent for the untargeted reporter [27
]. Second, even if the targeted and untargeted reporters are otherwise identical, the model also relies on the assumption that the system has reached a steady state and that the bound and free states of the targeted reporter are in an instantaneous equilibrium. The validity of these assumptions was superficially tested by a full kinetic model using approximate kinetic parameter inputs: less than a 4% error in the dual-reporter model-measured binding potential was found (results not shown). However, further simulation studies are ongoing to fully characterize the sensitivity of the dual-reporter to all model assumptions: e.g.
, the affect of discrepancies between the two reporters in terms of plasma kinetics and non-specific binding, the affect of cellular internalization, and the affect of reporter affinity (i.e.
, how reversible or irreversible the targeted reporter binding is to its receptor). Finally, in this study, the fluorescence of the untargeted and targeted reporters was measured at considerably different wavelengths (700 and 800 nm, respectively). Therefore, due to potential differences in tissue light absorption and scattering properties at these two wavelengths, it is possible that the detection sensitivity volume profiles of the two reporters could differ. Further investigation is necessary to fully elucidate the potential magnitude of this effect and to determine the affect this discrepancy could have on the modeling; however, it is known that the optical properties of tissue at these two wavelengths are relatively similar [28
], and the epi-illumination geometry of the imaging system used makes the images at both wavelengths particularly surface weighted, exemplified by the spatial resolution in , c in which small blood vessels can be discriminated.
In conclusion, this is a report and validation of a novel, quick, and robust in vivo methodology for measuring receptor expression in tumors. There are several significant and widespread implications of this approach. For preclinical work, the approach could help guide drug discovery and development by providing an in vivo estimate of receptor expression before and after treatment to better analyze the efficacy of proposed therapeutics. The approach can also be applied clinically for cancer staging to stratify patients for treatment or to monitor therapy using an optical fiber probe in conjunction with tissue biopsy or using non-invasive imaging modality like single photon emission tomography that is capable of dual-reporter imaging on a human scale.