We have shown that optical imaging with an affibody can be used to non-invasively monitor changes in Her2 expression in vivo
as a response to treatment with a specific Hsp90 inhibitor, whereas effects of the therapy on tumor volume were limited during the study period and statistically non-significant. Results of this study are very promising for the use of optical imaging as a molecular imaging tool for treatment monitoring in a clinical setting. This is the first work we know of to show the feasibility of optical imaging in the visualization of the response to Hsp90 therapy at a molecular level in living subjects. Affibody molecules have been used successfully in optical imaging studies before, for instance by Lee et al. targeting Her2 in a mouse model (26
), but the feasibility of treatment monitoring with the affibody in optical imaging studies has not been addressed yet. Other research groups have demonstrated the potential of target-specific radiotracers in PET imaging to measure the treatment effects on molecular targets (27
). Smith-Jones et al. monitored Her2 changes after treatment with a Hsp90 inhibitor (17-AAG) using PET imaging with 68
Ga-labelled trastuzumab F(ab
). They found a reduction in tumor uptake of 70% in BT474 breast tumor xenografts. The reduced uptake lasted until 5 days after treatment. Her2 levels were not determined ex vivo
, only imaging studies were performed. Oude Munnink et al. used PET imaging with full length 89
Zr-labelled trastuzumab to measure Her2 down-regulation after treatment with a Hsp90 inhibitor (NVP-AUY922) in SKOV-3 ovarian tumor xenografts (28
). They reported a reduction in tumor uptake of 41%. Immunohistochemistry confirmed the decrease in Her2 expression ex vivo
in a qualitative way only. Kramer-Marek et al. measured changes in Her2 expression after 17-DMAG treatment using the same affibody as we used in our study, but instead of using optical imaging they labeled the affibody with 18
F for PET imaging and only performed a single pre- and post-treatment scan (29
). They reported a reduction of 33% in a MCF7 cell line transfected with Her2 (Clone 18) and of 71% in BT474 breast tumor xenografts. Her2 downregulation was confirmed ex vivo
by western blot and ELISA.
Both Oude Munnink et al. and Kamer-Marek et al. compared a single post-treatment measurement with pre-treatment. Her2 expression was not monitored over a longer period of time. The strength of our study is that we followed each mouse over 10 days, which enabled us to see the Her2 levels decrease after treatment and recover after the treatment was stopped (which is in line with data from Smith-Jones et al. (27
)). This indicates that we can monitor the molecular changes non-invasively over time with our optical imaging strategy, whereas we did not observe significant changes in tumor volume during the study. Our in vivo
results of 22.5% signal reduction are consistent with the previous reports, considering that different cell lines were used for the tumor xenografts and that the imaging technique used was also different. In addition, correlating in vivo
optical imaging signal with ex vivo
Her2 levels by western blotting further supported our results. Although tumor volume did not change significantly after 17-DMAG treatment, in 2 of the 17-DMAG treated mice the Clone B tumors shrunk to very small volumes at day 9. To confirm that the changes in optical imaging signal were due to a decrease in Her2 expression levels and not caused by other non-specific anti-tumor effects of the drug, we correlated the in vivo
optical imaging signal with the ex vivo
Her2 expression levels not only at day 9, but also at day 3 in a subgroup of 8 mice. In that group we also closely monitored the tumor volume by ultrasound measurements up to day 3, and confirmed that there was no decrease in tumor volume after treatment (supplementary information
). Results indicate that the measured changes in optical imaging signal reflect the changes in Her2 expression after drug treatment.
An important advantage of optical imaging in comparison with PET imaging is that it does not use radioactive components or ionizing radiation and can thus be used more frequently. An additional advantage is that optical imaging agents are easier to generate and much cheaper than PET tracers. In contrast to radionuclide imaging approaches where over time the imaging signal disappears as a result of natural decay in addition to clearance from the subject, in optical imaging the clearance of the imaging signal is predominantly dependent on clearance of the imaging probe from the body. For this reason, small molecules with quick clearance, such as affibody molecules, may be preferable over large molecules in optical imaging. In addition, pre-injection optical signal can be measured and subtracted from subsequent imaging exams in optical imaging to adjust for the residual signal as done in this work.
Limitations of optical imaging include its limited spatial resolution and the complexity of its image reconstruction/quantitation. These aspects make it difficult to draw ROIs precisely around the tumor border. Chances are that different observers would draw ROIs differently. To evaluate if this would influence the results of our study, two different sizes of ROIs were also drawn besides the medium ROI we used for the primary results (an ROI drawn as accurately as possible around the apparent tumor border): a very small ROI in the center of the tumor, and a very large ROI around the entire tumor including some surrounding normal tissue. Average counts of all of these ROIs were calculated at all imaging time points. We found comparable differences in optical imaging signal post- and pre-treatment for all ROI sizes (see supplementary information
). This implies that the interpretation of the signal changes was not importantly influenced by the manner in which the ROI was drawn. Another limitation of the relatively low spatial resolution is that partial volume effects can lead to inaccurate optical imaging signals in very small lesions compared to the system’s spatial resolution.
Quantification of optical imaging signal is more complicated as compared to PET imaging in which percentage injected dose per gram of tissue can be calculated. Due to the fairly large background signal (noise) in vivo
, the correlation between in vivo
and in vitro
results is relatively limited. However, our results support that relative signal quantification with the right optical imaging set up is achievable and in the range of Oude Munnink et al. and Kamer-Marek et al. (28
), and that it is thus feasible to semi-quantitatively measure molecular changes over time using optical imaging.
In ongoing studies we are evaluating other molecular imaging agents, such as engineered antibodies and peptides, in the same xenograft model to make better comparisons between the different imaging agents. We aim to translate (one or more of) these molecular imaging agents to clinical studies. For clinical applications, deeper light penetration is required and therefore it will be advantageous to conjugate the targeted agents to a near-infrared dye with excitation wavelengths above 700–750 nm, for example IRdye 800CW (LI-COR Biosciences, Lincoln, NE), which has already been registered with the European regulatory authorities and the United States Food and Drug Administration in anticipation of clinical trials. Future preclinical studies will also include administering 17-DMAG more than once to repeatedly monitor the transient effect on Her2 expression over time, and investigating whether repeated probe injection within hours yields reproducible imaging results after pre-injection background subtraction to adjust for residual probe levels. If possible, we will be able to show the reproducibility of the entire optical imaging procedure, and not only from probe injection onwards which we showed to be highly reproducible (average COV 5.9%). This will give a better understanding of the magnitude of effects that can be measured with this optical imaging assay.
In conclusion, optical imaging with an affibody can be used for non-invasive in vivo imaging of Her2 expression and for monitoring the changes in Her2 expression as a response to treatment. This makes optical imaging a promising molecular imaging tool for treatment monitoring in preclinical models and potentially in patients.