Multi-Modal PET and MR Imaging in the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Model for Initial In Vivo Testing of Target-Specific Radioligands

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.

Tumor grafted – chick chorioallantoic membrane as an alternative model for biological cancer research and conventional/nanomaterial-based theranostics evaluation

“…Moreover, the addition of CT or MRI scans to PET could provide relatively accurate tumor volume estimation, as in ovo/vivo CT and ex ovo/vivo CT scans of the tumor were found to be comparable. In a similar manner, this was also the case for PET/MRI scans, where target-specificity was evaluated in ovo for prostate-specific membrane antigen (PSMA) positive and negative prostate carcinoma xenografts (Figure 3(b)) [99]. Previous mouse xenograft studies using the same PSMA-specific tracer demonstrated comparable accumulation values for both mice and CAM models.…”

“…To obtain high-resolution images, motion artifacts due to sudden movements of the chick embryo should be prevented. Anesthetics have been delivered to the chick embryo through fumigation or topical administration [98][99][100]. Alternatively, eggs could be pre-cooled at 4°C for at least 1 hour prior to imaging [83].…”

Fluorescent Nanodiamonds Encapsulated by Cowpea Chlorotic Mottle Virus (CCMV) Proteins for Intracellular 3D-Trajectory Analysis

“…It is worth noting that the HET-CAM method represents a potential alternative to animal experiments to assess toxicity and is widely applied to replace animal experiments. 42 In our HET-CAM assay, we observed hemorrhage from blood vessels that started within 2-4 seconds after applying 1% sodium dodecyl sulfate (SDS) as positive control. Lysis occurred after 27 seconds, and we observed coagulation within 24 hours.…”

“…These remained in the optical and spin active state (NV − ), which is essential for applications of ND-CP in bioimaging, tracking, and nanoscale sensing. The biocompatibility of ND-CP was investigated using HeLa cell line as well as in a chorioallantoic membrane (HET-CAM) ex vivo model 42 . As depicted in Figure 3B, the ND-CP showed low cytotoxicity upon treating cells at high concentrations of up to 200 µg/mL.…”

Patient-Derived Xenograft Models in Cancer Research

“…Finally, another alternative PDX platform is provided in the contribution by Winter et al [ 11 ] using the Hen’s egg test–chorioallantoic membrane (HET–CAM) PDX model. Like the ZF embryo, the chicken embryo lacks a functional immune system and is considered a non-life animal, making these PDX models an attractive platform for (“non-animal”) drug testing.…”