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

胰腺导管腺癌是人类最可怕的疾病之一，预后极差。这归根结底是基于两个方面：发现晚和治疗效果不佳。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循序渐进的过程。驱动基因(KRAS、CDKN2A、TP53、Smad4)的改变与其他遗传障碍一起导致导管上皮的侵袭。胰腺前体病变的体外模型很少，这阻碍了对这些特定变化引起的复杂分子和致病过程的新见解。现代基因组工程(CRISPR/CAS9)促进了细胞的精确遗传修饰。因此，我们试图将所有四种驱动基因的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基因修饰的新型胰腺癌前驱体细胞系。此外，我们还将用永生化载体hTERT修饰胰管上皮细胞，建立一种新的正常胰管上皮细胞系。这两种方法都将利用三维培养条件(有机物质)进行。由此产生的细胞将得到进一步的检查和彻底的表征。这些细胞系迫切需要用来研究基因改变对体外侵袭性导管上皮及其前体细胞恶性表型的影响。更好地了解受这些和其他基因变化影响的途径扰动的基础知识，可能会揭示早期发现和治疗的更好靶点。

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.