Establishment of xenografts from human pancreatic tumors for genetic screening of molecular targets with therapeutic potential

Author(s)

Luís Bruno da Cruz e Alves de Moraes

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2018-12-14

Examining board members

Eduardo Moraes Reis; Maria Mitzi Brentani; Miriam Galvonas Jasiulionis Leon; Deborah Schechtman; Mari Cleide Sogayar

• Advisor: Eduardo Moraes Reis

Abstract

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 ofthis 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 control of the silencing library, production of lentiviral vectors and adjustment of the experimental conditions for transduction and selection of the target cells. Only three of the cell lines evaluated (AsPC-1, MIA PaCa-2 and PANC-1) were permissible for transduction by the lentiviral vectors, and were accordingly used in the screening of epigenetic targets. The analysis of data obtained in this trial is ongoing and the results will be used for definition of potential candidate targets. In conclusion, valuable resources to support research on pancreatic cancer have been developed. The established collection of PDXs as well as the newly derived cell line constitutes a permanent and stable source of PDAC cells for molecular analyzes and functional studies seeking to elucidate aspects of this disease that are still poorly understood. Additionally, both the reagents generated and the expertise gained from the RNAi assay against epigenetic targets will have inordinate usefulness in future investigations to identify genes with major functions in maintaining the malignant phenotype, and consequently with the potential to be exploited therapeutically. (AU)

• FAPESP's process: 14/03943-6 - Development of human pancreatic tumor xenografts for genetic screening of molecular targets with therapeutic potential
• Grantee: Luís Bruno da Cruz e Alves de Moraes
• Support Opportunities: Scholarships in Brazil - Doctorate