Improving hepatocellular carcinoma mouse modeling by understanding the malignant potential and biology of liver cell subpopulations

项目摘要 肝细胞癌（HCC）是美国癌症相关死亡增长最快的原因， 肝硬化是任何慢性肝损伤的最终结果，是导致肝硬化患者死亡的主要原因之一。HCC是 在过去的30年里，难以治疗和结局几乎没有改善，5年生存率低于20%， 发病率与死亡率之比接近1检测、预防和治疗HCC的创新策略是 这是迫切需要的，其发展将在很大程度上取决于肝癌的小鼠模型。当前 模型是不够的，因为它们没有考虑到细胞的起源，这将可能有显着的 对肿瘤发生、维持和治疗脆弱性的影响。虽然肝脏被认为是 作为一个中心代谢中心，由于其惊人的再生能力，肝脏中的细胞异质性主要是 未开发的沿着肝小叶内的中心轴，基因表达存在显著差异， 观察代谢、缺氧和倍性。这些差异是否反映了肿瘤性 潜在的，以及它们是否影响代谢疾病或致癌作用，尚不清楚。近年来， 关于是否存在肝干/祖细胞一直存在激烈的争议。为了复合这些 然而，用于驱动HCC发展的损伤测定通常不能模拟常见的病因学，例如非- 酒精性脂肪性肝炎（NASH）是美国肝硬化最常见的原因。 HCC建模的一个关键问题是缺乏对不同细胞类型如何导致癌症的理解， 在临床相关损伤的情况下。我们相信，识别出引起癌症的特定细胞亚型 然后能够遗传干扰这些细胞对我们更好地模拟HCC至关重要。不幸的是， 该领域没有小鼠试剂来操纵肝脏中的大部分异质性。为了解决这一问题， 我的实验室已经优化了CRISPR基因组编辑方法，以快速生成谱系追踪小鼠。九个新 已经产生了标记不同区域特异性和祖细胞群体的CreER敲入模型， 有效地将我们领域可用的CreER线的数量增加了四倍。我们将使用这些工具来跟踪和 以系统的方式遗传操纵细胞类型，以识别最重要的再生细胞 在临床相关的化学和生物学背景下， 营养伤害。然后，我们将询问，对常见HCC驱动基因进行细胞类型特异性基因操作是否会有所帮助 以揭示肝细胞亚群之间的不同转化能力（目的3）。胜任这 该项目将为社区提供一个重要的CreER工具的大型面板，让该领域专注于 重要的亚群，更有可能转化，并揭示控制肿瘤发展的途径 在这些细胞中。

PROJECT SUMMARY Hepatocellular carcinoma (HCC) is the fastest growing cause of cancer-related death in the United States and one of the leading causes of death in patients with cirrhosis – the end result of any chronic liver injury. HCC is difficult to treat and outcomes have barely improved over the last 30 years, with 5-year survival under 20% and an incidence-to-mortality ratio near 1. Innovative strategies for detection, prevention, and treatment for HCC are desperately needed, the development of which will depend heavily on mouse models of liver cancer. The current models are inadequate because they do not take into account the cells of origin, which will likely have a significant impact on tumor initiation, maintenance, and therapeutic vulnerabilities. While the liver is appreciated for being a central metabolic hub and for its astounding regenerative capacity, cellular heterogeneity in the liver is mostly unexplored. Along the portal to central axis within the hepatic lobule, profound differences in gene expression, metabolism, hypoxia, and ploidy are observed. Whether or not these differences reflect differences in neoplastic potential, and whether or not they influence metabolic disease or carcinogenesis, is unclear. In recent years, there has been intense controversy about whether or not there is a liver stem/progenitor cell. To compound these debates, the injury assays used to drive HCC development often do not model common etiologies such as non- alcoholic steatohepatitis (NASH), which is emerging as the most common cause of cirrhosis in the U.S. We posit that a critical problem for HCC modeling is a lack of understanding of how different cell types contribute to cancer in the context of clinically relevant injuries. We believe that identifying the specific cellular subtypes that give rise to HCC and then being able to genetically perturb these cells is critical for us to better model HCC. Unfortunately, the field does not have mouse reagents to manipulate much of the heterogeneity in the liver. To address this, my lab has optimized CRISPR genome-editing methods to rapidly generate lineage tracing mice. Nine new CreER knock-in models that label different zone-specific and progenitor populations have been produced, effectively quadrupling the number of CreER lines available to our field. We will use these tools to trace and genetically manipulate cell types in a systematic fashion in order to identify the most important regenerative cell populations (Aim 1) and the HCC cell(s) of origin (Aim 2) in the context of clinically-relevant chemical and nutritional injuries. We will then ask if cell type specific gene manipulation of common HCC driver genes will help to uncover different transformation competencies between hepatocyte subpopulations (Aim 3). Success in this project will provide the community with a large panel of important CreER tools, allow the field to focus on important subpopulations that are more likely to transform, and reveal pathways that control tumor development in these cells.