Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with dismal prognosis (1, 2). Despite extensive research and the discovery of several promising drug candidates, little progress in PDAC treatment has been reported in the last years (3, 4). Two facts can be behind these disappointing results. In one hand, although PDAC is still clinically treated as a single disease, three distinct molecular subtypes (classical, quasi-mesenchymal and exocrine) were recently described based on expression profiling of microdissected epithelial tumor cells of PDAC samples (5). Interestingly, in vitro studies revealed differences in drug response of two of the proposed subtypes for which cell lines were available. These results highlight the importance of considering PDAC as a heterogeneous disease and point to the stratification of patients as a possible way to improve PDAC treatment response. On another hand, an additional reason behind the limited efficacy of PDAC treatment might be the tumor microenvironment. PDAC is the solid tumor with the highest stromal content, which can account for up to 90% of the total tumor mass. The PDAC microenvironment is known to actively affect tumorigenesis (6) and to impair drug delivery (7). Thus, rendering the PDAC microenvironment as an appealing therapeutic target to improve PDAC care (8). We have developed a novel workflow to efficiently generate patient-derived orthotopic xenografts (PDX) and serum-free cell cultures from primary resected PDAC tumors. The established primary cell lines comprise for first time all three described PDAC subtypes. Additionally, when re-injected into immunodeficient mice, these cells generate xenografts with high pathological similarity to the original patient tumor, including a prominent stromal presence. To explore the differences in the microenvironment associated to the individual PDAC subtypes we have now generated gene expression profiles for the stroma of a number of xenografts from our PDX model representing all three subtypes. Besides, RNA sequencing from different sub-populations isolated from fresh primary human PDAC tumors (as separated by fluorescent activated cell sorting according to surface markers) may reveal interesting interactions between the different tumor compartments. We have developed a set of immunohistochemical markers to identify the PDAC-subtypes that can be used in patient paraffin sections. Hence, the RNAseq data of the different tumor populations can be also easily studied in the context of the different subtypes. We believe that these approaches will shed some light on how different stromal expression patterns are interconnected with different epithelial expression profiles and vice versa, and how this information can be ultimately exploited for patient stratification and therapy. 1. Hidalgo M. Pancreatic cancer. N Engl J Med 2010 2. Vincent A et al. Pancreatic cancer. Lancet, 2011 3. Werner J et al. Advanced-stage pancreatic cancer: therapy options. Nature Reviews Clinical Oncology, 2013 4. Hidalgo M et al. Translational therapeutic opportunities in ductal adenocarcinoma of the pancreas. Clin Cancer Res, 2012 5. Collisson EA et al. Subtypes of pancreatic ductal adenocarcinoma and their differing responses to therapy. Nat Med, 2011 6. Feig C et al. Targeting CXCL12 from FAP-expressing carcinoma-associated fibroblastssynergizes with anti-PD-L1 immunotherapy in pancreatic cancer. Proc Natl Acad Sci USA, 2013 7. Provenzano PP et al. Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma. Cancer Cell, 2012 8. Neesse A et al. Emerging concepts in pancreatic cancer medicine: targeting the tumor stroma. Onco Targets Ther, 2013 Citation Format: Elisa Espinet Espinet, Christian Eisen, Elisa M. Noll, Vanessa Vogel, Corinna Klein, Zuguang Gu, Matthias Schlesner, Tobias Bauer, Nathalia Giese, Roland Eils, Jens Werner, Wilko Weichert, Martin R. Sprick, Andreas Trumpp. Exploring the PDAC-subtype-associated microenvironment in PDX models and patients. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A61.