The Chicken Embryo as a relevant model for Hepatocellular Carcinoma
GARCIA P. 1,2,3, WANG Y. 3, VIALLET J. 3, DECAENS T. 1,2,4, MONTAUT E. 1,2,5, EMADALI A. 1,2,5, MACEK JILKOVA Z. 1,2,4
1 Universite Grenoble Alpes (UGA), Grenoble, France; 2 Institute for Advanced Biosciences, CNRS UMR 5309-INSERM U1209, Grenoble, France; 3 INOVOTION, La Tronche, France; 4 Service díHepato-gastroenterologie, Pole Digidune, CHU Grenoble Alpes, Grenoble, France; 5 Pole Recherche, CHU Grenoble Alpes, Grenoble, France
Primary liver cancer is the 3rd leading cause of cancer-related death, with hepatocellular carcinoma (HCC) accounting for more than 80% of cases. Currently, 70% of patients don't respond to immunotherapies. Therefore, a reliable immunocompetent model is needed to test new treatments. We have developed four in ovo (in egg) liver cancer models, based on grafting human tumor cells onto the chicken embryo's chorioallantoic membrane (CAM).
4 relevant liver cancer cell lines (HepG2, Hep3B, HuH7, and PLC/PRF/5) were grafted in ovo on embryonic development day (EDD) 9, before being collected at EDD18. Egg survival and tumor weight have been measured to determine the optimal grafting conditions. Tumors were then collected, fixed in formalin, and embedded in paraffin for further histology studies: Hematoxylin/Eosin and Sirius Red staining, and anti-Ki-67 and anti-CD3 immunohistochemistry. Finally, RNA sequencing studies have been performed on the human transcriptome, allowing both an in vitro/in ovo comparison and a deep model description.
The morphology of the in ovo tumor growth depended on the cell line. Collagen accumulation was homogeneous between each cell line, except for HepG2. We observed the highest cell proliferation in the PLC/PRF/5 in ovo model. Both the tumor weight and the T cell infiltration were the lowest in the HuH7 in ovo model. RNA sequencing data unveiled the homogeneity of in ovo models and striking differences in the enrichment of gene sets associated with human HCC and inflammation.
We have developed four different models of human liver cancer cell lines in ovo, all bearing their specific characteristics, allowing for a wide and varied panel of studies. Overall, this project provides an in-depth characterization of in ovo HCC models and rational arguments that could lead to the replacement of conventional animal models in preclinical HCC research.