I seek three years' funding to acquire the training needed to develop an independent research program examining cellular pathways that drive pancreatic ductal adenocarcinoma (PDA), with the ultimate aim of identifying new targets that could be used to develop treatments for this highly aggressive malignancy. I hypothesize that a subset of the gene expression differences between neoplastic and untransformed pancreatic cells represent cellular factors that function to promote PDA tumorigenesis, and the goal of this proposal is to define such factors. While previous studies have attempted to identify gene expression changes associated with PDA tumorigenesis, these studies suffered from a number of limitations, including the lack of a pure population of untransformed pancreatic ductal cells as a control and the presence of abundant stromal cells in pancreatic tumor specimens, making any gene expression changes identified in these samples difficult to attribute to neoplastic cells. In addition, the functional relevance of many genes identified through such studies to the development of pancreatic cancer has not been determined. Here, I propose to use an innovative three-dimensional organoid culture system that allows for the propagation of both normal and neoplastic pancreatic cells to define the cellular pathways driving PDA tumorigenesis. Specifically, I will first advance my training in bioinformatic approaches, which will be used to compare the gene expression profiles of normal, premalignant, and malignant pancreatic cells grown as organoids. Expression data will be integrated with other available datasets to define genes upregulated in neoplastic cells that are likely to promote tumorigenesis. In a preliminary study, I have identified a number of promising candidate genes upregulated in premalignant and malignant murine organoids, including S100a7a, which encodes a Ca+2-signaling protein previously found upregulated in human pancreatic lesions. I now plan to extend this analysis to identify genes upregulated in cancer cells derived from human pancreatic tumors and propagated as organoids. Second, I will learn flow cytometry and single cell analysis approaches, which will allow me to isolate normal and neoplastic pancreatic cells from primary tissue. Sorted cells will be used to confirm expression changes identified using the organoid system and to explore the cellular heterogeneity of those changes in vivo. Finally, I will develop the training to functionally assay tumorigenesis pathways, which will be used to determine the effect of promising candidate genes on tumorigenesis. I will genetically deplete or overexpress candidate genes of interest to look for changes in ductal organoid proliferation and survival. I will also determine whether candidate knockdown impairs tumor formation following orthotopic transplantation. Through the development of core competencies in bioinformatic, single cell, and cancer biology approaches, this fellowship will help me to elucidate new pathways that drive pancreatic tumorigenesis, and will place me in an excellent position to start my own independent research laboratory further dissecting these pathways.