Chorioallantoic membrane assay: Vascularized 3-dimensional cell culture system for human prostate cancer cells as an animal substitute model

Purpose: Chorioallantoic membranes have been used as a reliable biomedical assay system for many years. Chicken eggs in the early phase of breeding ar e between in vitro and in vivo systems but may provide an immunodeficient, vascularized test environment. We tested this model as an in vivo system fo r prostate cancer research. Materials and Methods: Single cell suspensions of LNCaP, PC-3 and Tsu-Prl h uman prostatic cancer cell lines as well as 2 immortalized normal human pro state epithelial cell lines were inoculated on the chorioallantoic membrane of fertilized chicken eggs on day 5 or 6 of breeding. Tumor growth and via bility of the embryo was evaluated by stereo microscopy. At day 10 the memb ranes were removed and embedded in paraffin. Cell morphology was assessed a fter hematoxylin and eosin staining. Cellular expression of cytokeratin, pr ostate specific antigen and androgen receptor as well as apoptosis inductio n was confirmed by immunohistochemistry. Results: Three days after tumor cell inoculation on the extraembryonic vasc ular system of the chorioallantoic membrane cell growth and formation of 3- dimensional tumors became apparent in 100% of inoculated membranes. Strong neo-angiogenesis was detected next to the established tumors and tumor cell s invading the stroma of the chorioallantoic membrane. Cytokeratin expressi on as well as prostate specific antigen and androgen receptor in LNCaP cell s confirmed the human prostate tumor origin. Assessment of quantitative in vivo apoptosis induction in LNCaP cells after intravenous injection of the phorbol ester 12-0-tetradecanoylphorbol-13-acetate confirmed the model as a versatile in vivo system. Conclusions: The well vascularized chorioallantoic membrane of bred chicken eggs is a suitable system for early in vivo cancer research. Reliable grow th of prostate cancer cell lines is feasible and allows the evaluation of p roliferation and apoptosis induction after intravascular or topic applicati on of anticancer drugs. Exploitation of this assay enables a substantial re duction in or substitution for subsequent animal experiments.