Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential

Background/Objectives: Hepatoblastoma (HB), the most common pediatric liver cancer, often bears mutations in and/or otherwise deregulates the oncogenic transcription factors β-catenin (B), YAP (Y) and NRF2 (N). HB research is hampered by a paucity of established cell lines, particularly those possessing these molecular drivers. All combinations of B, Y and N (BY, BN, YN and BYN) are tumorigenic when overexpressed in murine livers, but it has not been possible to establish cell lines from primary tumors. Recently, we found that concurrent, in vivo Crispr-mediated targeting of the Cdkn2a tumor suppressor locus allows for immortalized cell lines to be efficiently generated. Methods: We derived and characterized five immortalized cell lines from Cdkn2a-targeted BN and YN HBs. Results: Four of the above five cell lines retained their ability to grow as subcutaneous or "pseudo-metastatic" pulmonary tumors in the immunocompetent mice from which they originated. Most notably, when maintained under hypoxic conditions for as little as 2 days, BN cells transiently upregulated the expression of numerous endothelial cell (EC)-specific genes and acquired EC-like properties that benefited tumor growth. These lines and those from previously derived BY and BYN HBs also possessed similar sensitivities to four commonly employed chemotherapeutic drugs. Conclusions: The above-described approach is currently the only means to generate HB cell lines with pre-selected and clinically relevant oncogenic drivers. Its generic nature should also allow bespoke HB cell lines with other oncogenic drivers to be readily produced. A collection of such cell lines will be useful for studying tumor cell-to-EC trans-differentiation, interactions with the immune environment and drug sensitivities.