gms | German Medical Science

Objective: The chick embryo chorioallantoic membrane model (CAM) assays has been widely used to study angiogenesis and invasion of brain tumors. It constitutes an easy model including tumor angiogenesis that is optical accessible during tumor growth. Furthermore, investigation of tumorbiology in the CAM model helps to reduce animal experiments. However, tumor observation is only possible for a limited period. Our aim was to use the CAM assay for the investigation of tumor growth of human glioma cells lines with different genotyp and observe the biological characteristics.

Methods: We used different genetically modified U87 cell lines: U87 EGFP, U87 IDH-1wildtype, U87 IDH-1mutation. Cells were either cultured as adherent cells or as speroids and implanted on embryonic day 7, 8 or 10 on the CAM (4x10⁶ cells / egg, n=10 for each cell line). Tumor growth of U87-EGFP cell lines was monitored by fluorescence microscopy Tumors were allowed to grow until day E15 ( embryonic day E15), removed from the egg and fixed in formalin. Images of the wholemount preparation were obtained. The tissue was cryoprotected using sucrose, embedded in cryomedia and cryosections of 10 µm thickness were prepared. Sections were analyzed using haematoxylin and eosin stain or immunohistochemistry for Ki67, CD38 and a-SMA.

Results: Our results demonstrate that both, adherent U87 cells and U87 spheroids can be used for tumor induction in the CAM model and form glioma within one week. The tumors exhibited angiogenetic effects, high proliferation rates and the morphological characteristics of glioma. Utilization of adherent U87 cell lines resulted in higher survival rate of chicken embryo and higher rates of tumor formation than that with U87 spheroids (p<0.05). However, CAM with U87 spheroid cell lines model could be more favorable to study the glioma angiogenesis and antiangiogenesis. Comparison of tumor fromationra rate and the viablity of the chicken embry regarding the timepoint of tumor implantation showed that E10 is the most suitable day for implantion of the cell lines on the CAM. U87 IDH1 mutation and U87 IDH1 wildtype cell lines likewise formed tumors on the CAM retaining their initial genotype. Initial tests confirmed, that monitoring tumor formation using optical methods like fluorescence microscopy, Raman spectroscopy or label-free mulitphoton microscopy is feasible.

Conclusion: Our experiments provide a simple, fast-growing and effective chick embryo chorioallantoic membrane model of glioma. It opens the possiblility to study the mechanisms of tumor formation using advanced optical techniques beyond in vitro settings without the need to perform animal experiments.