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

项目总结 在美国，肝细胞癌是癌症相关死亡增长最快的原因， 这是导致肝硬变患者死亡的主要原因之一--任何慢性肝损伤的最终结果。肝细胞癌是 治疗困难，结果在过去30年里几乎没有改善，5年存活率低于20%和 发病率/死亡率比接近1。肝癌的检测、预防和治疗的创新战略是 它的发展将在很大程度上依赖于肝癌的小鼠模型。海流 模型是不充分的，因为它们没有考虑起源细胞，这可能会有显著的 对肿瘤启动、维持和治疗脆弱性的影响。虽然肝脏被认为是 作为中心代谢中心，由于其惊人的再生能力，肝脏中的细胞异质性主要是 未被开发的。沿着肝小叶内的门静脉到中轴，基因表达存在着深刻的差异， 观察新陈代谢、低氧和倍性。无论这些差异是否反映了肿瘤的差异 它们是否会影响代谢性疾病或癌症的发生，目前尚不清楚。近年来， 关于是否存在肝脏干细胞/祖细胞一直存在激烈的争议。将这些混合在一起 争论中，用于推动肝细胞癌发展的损伤分析通常不能模拟常见的病因，如非 酒精性脂肪性肝炎(NASH)，它正在成为美国最常见的肝硬变原因，我们假设 建立肝癌模型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 在临床相关伤害的情况下。我们认为，确定特定的细胞亚型会导致 对于我们更好地建立肝细胞癌模型，能够从基因上干扰这些细胞是至关重要的。不幸的是， 该领域没有老鼠试剂来操纵肝脏中的大部分异质性。为了解决这个问题， 我的实验室已经优化了CRISPR基因组编辑方法，以快速生成血统追踪小鼠。九个新的 已经产生了标记不同区带特定和祖先种群的Creer敲入模型， 有效地使我们领域中可用的Creer线路的数量翻了两番。我们将使用这些工具来追踪和 以系统的方式对细胞类型进行基因操作，以确定最重要的再生细胞 人群(目标1)和起源的肝癌细胞(S)(目标2)在临床相关化学和 营养损伤。然后我们会问，对常见的肝癌驱动基因进行细胞类型特异性基因操作是否会有帮助 揭示肝细胞亚群之间不同的转化能力(目标3)。在这方面取得了成功 项目将为社区提供大量重要的Creer工具，让现场专注于 更有可能转化并揭示控制肿瘤发展的途径的重要亚群 在这些牢房里。

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.