Fate-mapping temporally marked precursor populations to hepatocellular carcinoma

肝细胞癌的时间标记前体群体的命运图谱

• 批准号: 8303586
• 负责人: Katherine S Koch
• 金额: $ 20.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303586
• 关键词: AFP gene; Address; Adenovirus Vector; Adult; Biological; Biological Assay; Carcinogen exposure; Carcinogens; Cell Culture Techniques; Cell Lineage; Cells; Characteristics; Chemicals; Choline; Choline Deficiency; Clinical; Coupled; Diet; Diethylnitrosamine; Differentiation and Growth; Elements; Environmental Exposure; Ethionine; Etiology; Exclusion; Exposure to; Fetoprotein; Genes; Genetically Engineered Mouse; Goals; Growth; Hepatic; Hepatocarcinogenesis; Hepatocyte; Hepatotoxicity; Histology; Human; Immune; Immunofluorescence Immunologic; Immunohistochemistry; In Vitro; Infection; Investigation; Kinetics; Knowledge; Lead; Liver; Liver Stem Cell; Malignant - descriptor; Malignant Neoplasms; Malignant neoplasm of liver; Maps; Mediating; Methods; Minority; Mission; Modeling; Molecular Biology; Monitor; Mus; National Institute of Allergy and Infectious Disease; Outcome; Pathology; Population; Population Study; Premalignant; Primary carcinoma of the liver cells; Property; Regimen; Regulatory Element; Reporter; Reporter Genes; Reporting; Research; Rodent; Rodent Model; Role; SCID Mice; Serum; Staining method; Stains; Stem cells; Suspension substance; Suspensions; System; Testing; Time; Tissues; Transplantation; base; cancer stem cell; cell type; chemical carcinogen; environmental chemical; feeding; in vivo; intrahepatic; oval cell; progenitor; promoter; recombinase; research study; stem; tissue culture; treatment strategy; tumorigenic; vector; vector control

DESCRIPTION (provided by applicant): The long-term objectives of this application are to identify the cellular origins of hepatocellular carcinoma ('HCC'). Many intrahepatic candidates might serve as HCC-progenitor cells, including mature hepatocytes, tissue-specific bipotential stem cells called 'oval' cells, multipotential liver stem cells, and postulated malignant HCC cells with spheroid-forming and stem cell-like properties ('liver cancer stem cells' [LCSCs]). Thus far, rodent models of chemical hepatocarcinogenesis support HCC-progenitor roles only for hepatocytes, although the validity of their roles to the exclusion of other candidates has not been established rigorously, and appear to depend on the carcinogen and its regimen of administration. That most markers for intrahepatic HCC progenitors are not specific to a single cell type or HCC has confounded simple fate-mapping. Therefore, to define a population for study, a unifying hypothesis is proposed that hepatic oval cells, determined in part here by characteristic tiny size and combination of markers, are, in fact, premalignant and HCC-spheroid, HCC, and LCSC progenitors. Accordingly, {a standard hepatocarcinogenic regimen} known to induce oval cells will be utilized in combination with mice capable of being expression-specifically, heritably targeted. The carcinogen- induced ?1-fetoprotein expressing ('AFP+') oval cells, a minority population in both rodent and human adult liver and a controversial, but longstanding HCC-progenitor candidate, will then be followed with a new two-step fate mapping approach. Experiments will be guided by three specific Aims: in Aim 1, {ROSA26 ZsGreen1 ('ZSG1') mice will be fed an hepatocarcinogenic diet (choline-deficiency + ethionine)} to monitor the induction of AFP+ oval cells and their putative descendants (HCCs; HCC-derived spheroids; postulated LCSCs), as evaluated by standard biological methods of histology, immunohistochemistry, immunofluorescence, {epifluorescence} and cell culture; in Aim 2, a control ('Ad-E'), and an experimental adenoviral vector expressing Cre recombinase governed by mouse 5'Afp regulatory elements ('Ad-Afp-Cre'), will be constructed and validated by methods of molecular biology, 1o culture and immunostaining. {Potential vector liver toxicity will also be assessed}; in Aim 3, carcinogen-induced AFP+ oval cells will be 'marked' specifically in ROSA mice after carcinogen feeding, by precisely timed, and transient Ad-Afp-Cre infection, to activate heritable ROSA {ZSG1} reporter genes solely in AFP+ oval cells from this narrow time window, and their subsequent descendants. This approach, plus ROSA and liver cell marker co-staining will be used to follow, and characterize the carcinogen-induced oval cell fates. In vitro growth and in vivo transplantation studies will be performed with ROSA {ZSG1+} HCCs, and HCC- derived spheroids to determine their stem cell-like and tumorigenic properties. {The WHO reports that 696,000 people died from HCC in 2008; these numbers are increasing, and environmental chemicals are among major causes.} It is expected that knowledge of the basic cellular etiologies of hepatocarcinogenesis will lead to targeting strategies for treatment of HCC. This research addresses missions of the NCI, NIDDKD and NIAID. PUBLIC HEALTH RELEVANCE: More than 690,000 people throughout the world die yearly from liver cancer (hepatocellular carcinoma ['HCC']), an insidious, aggressive cancer which is caused by many factors, including environmental exposure to chemical carcinogens. The intrahepatic cells that are attacked by chemical carcinogens, and eventually produce malignant and metastatic HCCs, have not been identified unequivocally, and although hepatocytes are implicated as one such progenitor cell type, controversy, speculation and limited approaches have continued to confound this conclusion, as well as {further studies} and isolation of the other candidates. Investigations in this proposal will be based on a new hypothesis and new fate-mapping approach, broadly applicable to other cell populations and systems, to mark heritably, identify, and follow HCC-progenitor cells (including recently proposed 'liver cancer stem cells'), thus contributing to knowledge of the cellular etiology of HCC, and enabling its potential clinical targeting.

描述（由申请人提供）：本申请的长期目标是确定肝细胞癌的细胞起源（“ HCC”）。许多肝内候选者可能充当HCC - 促进剂细胞，包括成熟的肝细胞，组织特异性的双重电势干细胞，称为“椭圆形”细胞，多能肝干细胞和假定的恶性HCC细胞 具有球体形成和干细胞样性质（“肝癌干细胞” [LCSC]）。到目前为止，尽管尚未严格确定其角色对排除其他候选者的作用的有效性，但啮齿动物肝癌发生的啮齿动物模型仅支持肝细胞的HCC - 促进剂角色，并且似乎取决于致癌物及其管理方案。肝内HCC祖细胞的大多数标记并非特定于单个细胞类型或HCC都使简单的命运映射混淆。因此，为了定义研究人群，提出了一个统一的假设，即在此部分由特征性的小尺寸和标记物组合确定的肝椭圆形细胞实际上是前态度和HCC-SPHEROID，HCC和LCSC祖细胞。因此，{已知诱导椭圆形细胞的标准肝癌方案}将与能够特定于表达的小鼠结合使用，可遗传。致癌物诱导的？1-五蛋白蛋白表达（'AFP+'）椭圆形细胞，啮齿动物和人类成人肝脏中的少数群体以及有争议的，但长期存在的HCC - 促进剂候选者，然后将采用新的两步性命运方法。实验将由三个具体目的指导：在AIM 1，{Rosa26 Zsgreen1（'ZSG1'）小鼠将喂食肝癌饮食（胆碱缺乏 +乙氨酸）}，以监测AFP +卵形细胞及其假定的遗传者（HCCSSSSSSS; HCCCS; hccss serperiend oferideSsssperiveSsspersepsssspects;组织学，免疫组织化学，免疫荧光，{epifluorescence}和细胞培养的生物学方法；在AIM 2中，将通过分子生物学，1O培养和免疫染色的方法来构建和验证AIM 2，对照（'AD-E'）和由小鼠5'AFP调节元件（“ AD-AFP-CRE”）所控制的CRE重组酶（“ AD-AFP-CRE”）的实验性腺病毒载体。 {也将评估潜在的肝毒性}； in Aim 3, carcinogen-induced AFP+ oval cells will be 'marked' specifically in ROSA mice after carcinogen feeding, by precisely timed, and transient Ad-Afp-Cre infection, to activate heritable ROSA {ZSG1} reporter genes solely in AFP+ oval cells from this narrow time window, and their subsequent descendants.这种方法以及ROSA和肝细胞标记物将使用共同染色，并表征致癌物诱导的椭圆形细胞命运。将使用ROSA {ZSG1+} HCC和HCC衍生的球体进行体外生长和体内移植研究，以确定其干细胞样和肿瘤性特性。 {WHO报告说，2008年有696,000人死于HCC；这些数字正在增加，环境化学物质是主要原因之一。}预计肝癌发生的基本细胞病因的知识将导致针对治疗HCC的策略。 这项研究涉及NCI，NIDDKD和NIAID的任务。 公共卫生相关性：全世界有690,000多人每年死于肝癌（肝细胞癌['HCC']），这是一种阴险，侵略性的癌症，是由许多因素引起的，包括环境暴露于化学癌。尚未明确鉴定出受到化学致癌物并最终产生恶性和转移性HCC攻击并最终产生恶性和转移性HCC的肝内细胞，尽管肝细胞被认为是一种祖细胞类型，争议，推测和有限的方法，但继续进行了这一结论，并进一步研究了其他研究，并进一步研究了其他研究。该提案中的研究将基于一种新的假设和新的命运映射方法，该方法广泛地适用于其他细胞群体和系统，以标记可遗传的，识别和遵循HCC - 促进性细胞（包括最近提出的“肝癌干细胞”），从而有助于对HCC的细胞病因的了解，并实现其潜在的临床靶标。