The lethality of pancreatic ductal adenocarcinoma is universal, even in patients who present with small primary tumors and undergo surgery with curative intent. Therefore, determining whether there are specific molecular alterations contributing to the final transformation of pre- malignant cells, both for early detection and for patients with precursor lesions adjacent to their surgical resection site, may have clinical benefits. Since the investigation of pancreatic cancer in human and mouse tissues is challenging due to the low neoplastic cellularity and poorly defined tumor margins, we have developed a novel organoid cell culture system to expand primary mouse and human normal, pre-malignant and malignant pancreatic ductal cells. These organoids will be comprehensively analyzed for molecular alterations using transcriptomic, proteomic and other omics level approaches in order to identify candidates that correlate with the earliest stages of pancreatic cancer progression. Specific molecular alterations will be considered for potential therapeutic and/or diagnostic development in mouse and human tissues, using conventional and single cell analyses. Such findings will enable the identification of at risk patients, development of new therapeutic strategies, and provide new insights regarding how to prevent pancreatic cancer occurrence and recurrence. Significance The failure of disease management in pancreatic cancer patients following surgery is due to either local tumor regrowth or distant metastatic spread. In addition, if pre-malignant cells that are likely transform into a frank malignancy could be identified and treated before forming a tumor, many lives could be saved. In order to detect and treat pre-malignant cells that are poised to become tumorigenic in mice and patients, we will develop new organoid model systems and use these systems to better characterize and understand the transition from a pre- malignant disease state to invasive, metastatic adenocarcinoma.