Visualisierung und Analysen von Therapieansätzen durch fluoreszenzbasierte molekulare Bildgebungsverfahren

Preclinical in vivo analyses of therapeutic efficacy are an important prerequisite for the evaluation of new therapeutics. Optical molecular imaging in the far red or NIR range is a favourable method, since it allows measurements at different time points in one single animal. This not only reduces the amount of animals required per experiment, but in addition increases statistical significance. A431 cells have a very high expression of the epidermal growth factor receptor (EGFR). This receptor is also strongly over-expressed by a variety of tumors and thus qualifies as a target for therapeutic intervention. However, a well-known problem in EGFR-specific therapy is the down-regulation of the receptor. The aim of this work was the visualisation of therapeutic effects by the use of optical molecular imaging. For that, mainly 3 different aspects were tested. On one hand, tumor cells should be used, that allow the monitoring of cell growth during therapy in vivo on the basis of their inherent fluorescence. Furthermore, the induced apoptosis should be detectable using fluorescently labeled Annexin A5. Finally, after therapy receptor expression should be verified using a receptor-specific construct to exclude the possibility of development of therapy resistance due to down-regulation of the receptor. For the in vivo experiments, A431 tumor cells, that have been passaged in mice before, were transfected with the far red fluorescent protein Katushka (Kat 2). One in-house developed immunotoxin, consisting of a scFv targeted against the EGFR, fused to a deletion mutant of Pseudomonas aeruginosa Exotoxin A, showed specific binding and high toxicity to A431 cells. Fluorescent A431scM3-Kat2 cells were injected subcutaneously in vivo. After tumor development, treatment was executed with the immunotoxin or with the chemotherapeutic Doxorubicin. Tumor sizes were determined on basis of the cell-inherent fluorescence at different time points. Furthermore, tumor-specific EGFR-expression was assessed through binding of the same 425(scFv) coupled to a NIR dye. Treatment with the immunotoxin 425(scFv)-ETA’ led to a decelerated tumor growth without having an impact on the overall health of the mice, this unlike the chemotherapeutic Doxorubicin. The here described method allows the simultaneous assessment of several parameters in one single animal, like tumor growth and receptor expression. This is particularly important for the in vivo evaluation of new therapeutics and can be employed to monitor treatment of different types of tumors