Comparative analyses of the biodistribution of 89Zr-labeled human serum albumin in the mouse and HET-CAM model

Introduction: Evaluation of specific accumulation and biodistribution of novel radioligands is mostly performed in xenograft mouse models. Also, accumulation of compounds based on the EPR effect (Enhanced Permeability and Retention) is analyzed in these models. In accordance with the 3Rs principles (Replacement, Reduction, Refinement), this study focuses on the xenograft HET-CAM (Hen’s Egg Test - Chorioallantoic Membrane) model as a possible alternative for the analysis of intratumoral accumulation and retention in blood. For this purpose, biodistribution ⁸⁹Zr-labeled human serum albumin is studied in the SCID mouse model as well as in the HET-CAM model using PET (Positron Emission Tomography) and MRI (Magnetic Resonance Imaging)

Methods: The radioligand [⁸⁹Zr]Zr-DFO-HSA was injected intravenously into SCID mice (n=5) and chicken embryos (n=4). Applied tumor types (TZM-bl/PC-3 or C4-2/PC-3) where either grafted to the subscapular region of the mice or in silicon rings onto the CAM. Dynamic PET scans and anatomic MR imaging were used to analyze distribution kinetics. Mice were analyzed over a period of 24h, while chick embryos were analyzed over a period of 1h. Accumulation in target tissues was quantified in ex vivo biodistribution analyses and possible intratumoral biodistribution of the ligand was verified by histological staining and autoradiography. PET data were analyzed in PMOD, and statistical analysis was performed using SPSS 28 and GraphPad Prism 9.3.1.

Results: Based on PET evaluations, an activity concentration of (31.8 ± 4.4) %IA/ml was detected in the blood of SCID mice and of (22.0 ± 7.4) %IA/ml in the blood of chick embryos after 1 hour of measurement. Simultaneously, an increase to (3.1 ± 0.9) %IA/ml in mice and to (2.8 ± 3.5) %IA/ml in the HET-CAM model was observed in PC-3 tumors within 60 minutes.

Similar kinetics were observed in mice and the HET-CAM model during the measurement period. Statistical analysis by comparing the kinetics of radioligands from mouse and HET-CAM model using linear regression revealed a strong significant correlation of the courses in blood (r=0.965, p<0.0001), but no significant correlation for PC-3 tumor (r=0.573; p=0.056).

When comparing the slopes of simple linear regression analyses (equivalent to accumulation over time) of the HET-CAM and mouse measurements, no statistically significant differences were detected for either blood or tumor kinetics.

Conclusions: Accumulation in tumor tissue and corresponding blood retention due to the EPR effect were demonstrated in both models. The strong significant correlation of the observed blood pool kinetics in both mice and HET-CAM xenograft systems suggest similar distribution and resorption characteristics of the radiolabeled HSA. Thus, the HET-CAM model may represents a potential alternative to the mouse model, not only regarding aspects of simpler handling and lower maintenance costs, but also in terms of the 3Rs principles.

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