Análise dos efeitos do transplante de células mononucleares da medula óssea em camundongos submetidos à lesão eletrolítica do hipocampo dorsalDissertação apresentada ao Programa de

estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e estável de células de PDAC para análises moleculares e estudos funcionais que busquem elucidar aspectos da doença ainda pouco compreendidos.Adicionalmente, os reagentes gerados e a expertise adquirida com os ensaios realizados com a biblioteca de shRNAs contra alvos epigenéticos serão de grande utilidade em futuras investigações para identificar genes com funções importantes na manutenção do fenótipo tumoral, e consequentemente com potencial para serem explorados terapeuticamente. Palavras-chave: Câncer de pâncreas. Xenoenxerto. Linhagem celular tumoral. Epigenética. RNA de interferência. shRNA ABSTRACT Moraes L.B.C.A. Establishment of xenografts from human pancreatic tumors for genetic screening of molecular targets with therapeutic potential. 2018. 174 p. PhD Thesis -Graduate Program in Biochemistry. Instituto de Química, Universidade de São Paulo, São Paulo. Pancreatic ductal adenocarcinoma (PDAC), the most prevalent type of pancreatic cancer, is a highly aggressive and lethal neoplasm. There is a pressing need to identify vulnerabilities in PDAC suited to be exploited as therapeutic targets, and the use of preclinical models recapitulating the biological complexity and clinical heterogeneity of the disease is central to this task. Patient-derived tumor tissue xenografts (PDX), established in immunodeficient mice, replicate with great similarity the main characteristics of the original tumor and thus constitute a valuable tool for drug testing and functional studies. In this work, 17 surgical samples of human PDAC were implanted subcutaneously in athymic nude mice. Seven tumors (41%) were successfully grafted and have been maintained through successive generations of recipient animals. Histological examination of six of these xenografts identified morphological characteristics compatible with the recognized patterns of human PDAC, as well as a consistent similarity of their histological differentiation status in relation to the profiles verified in the original tumors. In vitro culture of cells derived from one of these xenografts resulted in a new pancreatic cancer cell line, with morphology and growth kinetics comparable to those of other pancreatic tumor cells.The tumorigenic potential of this freshly derived cell line was validated in vivo, with a consistent tumor formation following inoculation into nude mice. To take advantage of this resource to investigate potential therapeutic targets in PDAC, a screening of molecular vulnerabilities was performed through large-scale gene silencing with RNA interference (RNAi). A lentiviral library containing 4492 short hairpin RNAs (shRNAs), targeting about 350 genes involved in epigenetic regulation, was employed for the search of susceptibility genes in the PDX-derived cells and in other five pancreatic tumor cell lines (AsPC-1, BxPC -3, Capan-1, MIA PaCa-2 and PANC-1). Initially, a series of preliminary experiments were carried out aiming at the amplification and quality co...