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 theCdkn2atumor suppressor locus allows for immortalized cell lines to be efficiently generated.Methods: We derived and characterized five immortalized cell lines fromCdkn2a-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.
背景/目的：肝母细胞瘤（HB）作为最常见的儿童肝脏恶性肿瘤，常伴有致癌转录因子β-连环蛋白（B）、YAP（Y）及NRF2（N）的突变和/或功能失调。当前HB研究受限于已建立的细胞系数量稀少，尤其是携带这些分子驱动因子的细胞模型。虽然在小鼠肝脏中过表达B、Y、N的所有组合（BY、BN、YN及BYN）均可诱发肿瘤，但始终无法从原发肿瘤中成功建立细胞系。近期我们发现，通过CRISPR技术同时靶向Cdkn2a抑癌基因位点，可在体内高效获得永生化细胞系。 方法：本研究从靶向Cdkn2a的BN型与YN型HB肿瘤中成功构建并鉴定了五种永生化细胞系。 结果：其中四种细胞系在具有免疫活性的源代小鼠体内，仍保持皮下或"假转移性"肺肿瘤的生长能力。尤为重要的是，BN细胞在低氧条件下培养仅需2天，即可瞬时上调大量内皮细胞特异性基因表达，并获得促进肿瘤生长的内皮细胞样特性。这些细胞系与先前建立的BY、BYN型HB细胞系，对四种常用化疗药物表现出相似的敏感性。 结论：本研究所建立的方法是目前唯一能够生成具有预设且临床相关致癌驱动因子的HB细胞系的技术路径。该方法的通用性使其可便捷地构建携带其他致癌驱动因子的定制化HB细胞系。此类细胞系集合将为研究肿瘤细胞向内皮细胞的转分化、免疫微环境相互作用及药物敏感性提供重要工具。
Derivation of Genetically Defined Murine Hepatoblastoma Cell Lines with Angiogenic Potential
肿瘤(癌症)患者之家