Investigation of pancreatic tumorigenesis by derivation of organoids, their immortalization, and genetic modification in vitro

通过类器官的衍生、永生化和体外基因修饰研究胰腺肿瘤的发生

• 批准号: 445331015
• 负责人: Dr. Michael Johannes Pflüger
• 金额: --
• 依托单位: Department of Pathology
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/445331015?language=en
• 关键词: Investigation; pancreatic; tumorigenesis; derivation; organoids

Pancreatic ductal adenocarcinoma (PDAC) is one of the most dreadful diseases of the human body with dismal prognosis. This is ultimately based on two aspects: late detection and poorly effective therapies. The recent two decades yielded great knowledge in the molecular profile and pathogenesis of PDAC. Pancreatic tumorigenesis is regarded to be a stepwise process. Alterations accumulating in driver genes (KRAS, CDKN2A, TP53, SMAD4) aside with other genetic disturbances lead to invasive ductal epithelium. In vitro models of pancreatic precursor lesions are scarce, which prevents new insights into the complex molecular and pathogenic processes inflicted by these specific alterations. Modern genome engineering (CRISPR/Cas9) facilitates accurate genetic modification of cells. Therefore, we attempt to introduce all four driver gene alterations sequentially into normal, unmodified pancreatic epithelial duct cells. This will derive novel pancreatic cancer precursor cell lines harboring specific genetic modifications. In addition, we will modify pancreatic duct epithelium with the immortalization vector hTERT to establish a new cell line of normal duct epithelium. Both approaches will be conducted utilizing three-dimensional culture conditions (organoids). Resulting cells will be further examined and thoroughly characterized. Those cell lines are desperately needed to investigate the effects of genetic alterations in the malignant phenotype of invasive duct epithelium and its precursors in vitro. Better fundamental knowledge of pathway perturbations affected by these and other genetic changes may reveal better targets for early detection and therapy.

胰腺导管腺癌（PDAC）是人类最可怕的疾病之一，预后极差。这最终是基于两个方面：发现晚和治疗效果差。近二十年来，人们对PDAC的分子特征和发病机制有了深入的了解。胰腺肿瘤的发生被认为是一个逐步的过程。驱动基因（KRAS、CDKN 2A、TP 53、SMAD 4）中积累的改变以及其他遗传干扰导致浸润性导管上皮。胰腺前体病变的体外模型很少，这阻碍了对这些特定改变所造成的复杂分子和致病过程的新见解。现代基因组工程（CRISPR/Cas9）有助于细胞的精确遗传修饰。因此，我们尝试将所有四种驱动基因改变依次引入正常的未修饰的胰腺上皮导管细胞。这将衍生出具有特定遗传修饰的新型胰腺癌前体细胞系。此外，我们还将用永生化载体hTERT修饰胰腺导管上皮细胞，建立一种新的正常导管上皮细胞系。这两种方法都将利用三维培养条件（类器官）进行。将对所得细胞进行进一步检查和彻底表征。这些细胞系是迫切需要的，以研究遗传变异的影响，在恶性表型的浸润性导管上皮及其前体细胞在体外。更好的基础知识的影响，这些和其他遗传变化的途径扰动可能揭示更好的目标，早期检测和治疗。