Evaluation of binding specificity of target-specific radioligands in xenografts by multi-modal PET and MR imaging using the Hen’s egg test chorioallantoic membrane (HET-CAM) model

Introduction: Target specificity is one of the main goals when developing new specific agents for oncological diagnosis and therapy. Animal experiments with xenografts mostly in mice are usually performed to obtain information about target specific tumor accumulation after systemic application, with the necessity of an appropriate animal test authorization. The HET-CAM model, an already established model system for tests regarding mucosal tolerance to chemical compounds, serves as an excellent alternative regarding animal welfare in the respect of the 3R principle (replacement, reduction, refinement). Chicken embryos are not considered animals before day 17 and, based on current knowledge, are not able to sense pain before day 15 of the development [1]. Therefore, for the HET-CAM model a special authorization is not required. Due to an undeveloped immune response, vascularized tumor xenografts can be established on the membrane surface and the target-specific accumulation can be studied after systemic application of the novel agent. The well-characterized PSMA-specific PET radiotracer ⁶⁸Ga-PSMA-11 was used to demonstrate the principle of the HET-CAM model for evaluation of specific radioligand accumulation. Materials and Methods: After six days of incubation at 37.8 °C, silicone rings were placed on the membrane of the opened chicken eggs. Tumor cells of the PSMA-positive cell line LNCaP C4-2 (1 x 10⁶ cells) and the PSMA-negative control PC-3 (7.5 x 10⁵ cells) were applied with Matrigel (40%, v/v) into the rings on day seven. MR and PET imaging was performed starting on day 12. Anatomical information was provided by high-resolution imaging using a small animal MR (BioSpin 117/16, Bruker) based on the protocol of Zuo et al. [2, 3]. 150 µl of ⁶⁸Ga-PSMA-11 solution was prepared and injected into a chorioallantoic membrane vessel followed by a dynamic 60 min PET scan (Focus 120, Concorde Microsystems Inc.). Tumors and parts of the membrane were excised for quantitative evaluation by gamma counter (COBRA II, Perkin Elmer). MRI and PET data were superimposed by fiducial registration using the 3D slicer software [4].

Results: Tumor growth was clearly proven by MR imaging. Accumulation of ⁶⁸Ga-PSMA-11 was observed in the PSMA-positive tumor and chick embryo in the PET images. Compared to the PC-3 tumors a higher accumulation of ⁶⁸Ga-PSMA-11 was observed in the LNCaP C4-2 tumors: 0.26 ± 0.71 %ID (LNCaP C4-2); 0.07 ± 0.05 %ID (PC-3); (n=20) and a ratio of 4.2 ± 4.0 (PSMA-specific/ non-specific) was calculated. A successful injection has been achieved for 65% of the eggs. During the PET measurement, the LNCaP C4-2 activity increased by 8.5 ± 2.6 %.

Conclusions: The principle of using a HET-CAM model with xenografts for quantitative evaluation of target-specific radiotracer accumulation in PET was successfully demonstrated. Fusion of multi-modal PET and MR images combined high-resolution anatomical details and high sensitivity for detection of peptide accumulation in the tumor. Thus, according to the 3R-principle, this model could serve as an alternative for many mouse experiments for the evaluation of novel radiopharmaceuticals with respect to target-specific tracer accumulation. References: [1] Deutscher Bundestag, Umwelt, Naturschutz, Reaktorsicherheit, Bildung und Forschung 2017 , Aktenzeichen : WD 8 - 3000 -030/17 [2] Zuo, Z. et al. Scientific reports 2017, 7, 46690. [3] Zuo, Z. et al. NMR in biomedicine 2015, 28 (4), 440-7. [4] Fedorov, A. et al. Magn Reson Imaging. 2012;30(9):1323-41.