Abstract
The validation of novel target-specific radioligands requires animal experiments mostly using mice with xenografts. A pre-selection based on a simpler in vivo model would allow to reduce the number of animal experiments, in accordance with the 3Rs principles (reduction, replacement, refinement). In this respect, the chick embryo or hen’s egg test–chorioallantoic membrane (HET-CAM) model is of special interest, as it is not considered an animal until day 17. Thus, we evaluated the feasibility of quantitative analysis of target-specific radiotracer accumulation in xenografts using the HET-CAM model and combined positron emission tomography (PET) and magnetic resonance imaging (MRI). For proof-of-principle we used established prostate-specific membrane antigen (PSMA)-positive and PSMA-negative prostate cancer xenografts and the clinically widely used PSMA-specific PET-tracer [68Ga]Ga-PSMA-11. Tracer accumulation was quantified by PET and tumor volumes measured with MRI (n = 42). Moreover, gamma-counter analysis of radiotracer accumulation was done ex-vivo. A three- to five-fold higher ligand accumulation in the PSMA-positive tumors compared to the PSMA-negative tumors was demonstrated. This proof-of-principle study shows the general feasibility of the HET-CAM xenograft model for target-specific imaging with PET and MRI. The ultimate value for characterization of novel target-specific radioligands now has to be validated in comparison to mouse xenograft experiments.
Highlights
The development of target-specific radiolabeled probes like on the basis of small molecules, peptides, antibodies, or nanoparticles, always requires information on biodistribution and in particular on the level of specific binding of the novel probe to the target in the in vivo situation, which means specific radiotracer accumulation in the target-expressing region of interest [1,2,3,4,5]
Four more chick embryos had to be excluded detectable despite successful injection, because of extended paravasation of radiotracer adjacent to the tumor excluded despite successful injection, because of extended paravasation of radiotracer adjacent to the areas and consecutive spillover of activity which made a meaningful analysis of tracer accumulation in tumor areas and consecutive spillover of activity which made a meaningful analysis of tracer the tumors either by positron emission tomography (PET) or gamma-counter measurements impossible
(brown color), while a weak staining is is visible forfor. In this feasibility study we demonstrated successful imaging and analysis of target-specific radiotracer accumulation in a hen’s egg test–chorioallantoic membrane (HET-CAM) xenograft model by combined PET and magnetic resonance imaging (MRI), using the prostate-specific membrane antigen (PSMA)-specific PET tracer [68Ga]Ga-PSMA-11 and PSMA-positive and PSMA-negative tumor cell lines
Summary
The development of target-specific radiolabeled probes like on the basis of small molecules, peptides, antibodies, or nanoparticles, always requires information on biodistribution and in particular on the level of specific binding of the novel probe to the target in the in vivo situation, which means specific radiotracer accumulation in the target-expressing region of interest [1,2,3,4,5]. Alternative methods to these animal experiments are of high interest regarding animal welfare according to the 3Rs principles (reduction, replacement, refinement), and to potentially speed up the development of novel radioligands by simpler and effective screening tools In this respect, the hen’s egg test-chorioallantoic membrane (HET-CAM) model is a promising alternative for animal experiments, as in many countries, chick embryos are not considered live animals before embryo development day (EDD) 17 or hatching. Quick and inexpensive model, HET-CAM has already been used for studies on, e.g., angiogenesis [21], cancer progression [22], pharmacology [23], and radiotherapy [24] It is well suited for xenograft studies, as the chick embryo is not fully immunocompetent at early development stages. The immune system components further diversify until EDD18 when the embryo is fully immunocompetent [27]
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