ARTICLE ABSTRACTAggressive metastasis is the chief cause of the high morbidity and mortality associated with pancreatic cancer, yet the basis for its aggressive behavior remains elusive. Extracellular DNA (exDNA) is a recently discovered component of inflammatory tissue states. Here, we report that exDNA is present on the surface of pancreatic cancer cells where it is critical for driving metastatic behavior. exDNA was abundant on the surface and vicinity of cultured pancreatic cancer cells but absent from normal pancreas cells. Strikingly, treatment of cancer cell cultures with DNase I to degrade DNA nonspecifically reduced metastatic characters associated with matrix attachment, migration, and invasion. We further assessed the role of exDNA in pancreatic cancer metastasis in vivo using an orthotopic xenograft model established by implantation of pancreatic cancer cells expressing firefly luciferase. Noninvasive bioluminescent imaging confirmed that DNase I treatment was sufficient to suppress tumor metastasis. Mechanistic investigations suggested the existence of a positive feedback loop in which exDNA promotes expression of the inflammatory chemokine CXCL8, which leads to higher production of exDNA by pancreatic cancer cells, with a significant reduction in CXCL8 levels achieved by DNase I treatment. Taken together, our results strongly suggest that exDNA contributes to the highly invasive and metastatic character of pancreatic cancer. Cancer Res; 73(14); 4256–66. ©2013 AACR.