Immunotherapies have significantly improved the prognosis of patients with advanced hepatocellular carcinoma (HCC), although more than 70% of patients still do not respond to this first-line treatment. Many new combination strategies are currently being explored, which drastically increases the need for preclinical models that would allow large-scale testing of new immunotherapies and their combinations. We developed several in ovo (in the egg) human liver cancer models, based on human tumor xenografts of different liver cancer cell lines on the chicken embryo's chorioallantoic membrane. We characterized the angiogenesis, as well as the collagen accumulation and tumor immune microenvironment, and tested atezolizumab (anti-PD-L1) plus bevacizumab (anti-VEGF) treatment. Our results show the involvement of chicken immune cells in tumor growth, reproducing a classical non-inflamed "cold" as well as inflamed "hot" tumor status, depending on the in ovo liver cancer model. The treatment by atezolizumab and bevacizumab was highly efficient in the "hot" tumor model PLC/PRF/5 in ovo with the reduction of tumor size by 76% (p ≤ .0001) compared with the control, whereas the efficacy was limited in the "cold" Hep3B in ovo tumor. The contribution of the anti-PD-L1 blockade to the anti-tumoral effect in the PLC/PRF/5 in ovo model was demonstrated by the efficacy of atezolizumab monotherapy (p = .0080, compared with the control). To conclude, our study provides a detailed characterization and rational arguments that could help to partially replace conventional laboratory animals with a more ethical model, suited to the current needs of preclinical research of new immunotherapies for liver cancer.
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