Abstract Pancreatic cancer is one of most deadly cancers with a 5-year survival rate of 6%. Accumulation of hyaluronan (HA) is found in about 87% of human pancreatic adenocarcinomas, and removal of HA suppresses tumor growth in HA-rich preclinical models. In a transgenic pancreatic cancer mouse model (LSL-KrasG12D/+;LSLTrp53R172H/+;Pdx-1-Cre, KPC), removal of HA by pegylated human recombinant PH20 hyaluronidase (PEGPH20) inhibits tumor growth and increases survival in combination with gemcitabine compared to gemcitabine monotherapy. In this study, we explored the role of HA synthesizing (HAS) enzymes HAS2 and HAS3 and HA accumulation in pancreatic cancer tumor growth and remodeling of tumor microenvironment. HAS2 and HAS3 were overexpressed in BxPC3 human pancreatic cancer cells using lentiviral vectors. Stable HAS2 and HAS3 overexpressing pancreatic cancer cell lines secreted more HA to culture medium and produced larger pericellular HA matrices than parental BxPC3 cells. In vivo, overexpression of HAS2 or HAS3 led to an increase in BxPC3 xenograft tumor growth (peritibial i.m. tumor model) compared to parental cells. Interestingly, overexpression of HAS3 was more effective to enhance tumor growth than overexpression of HAS2. In addition, massive accumulation of extracellular HA was found in HAS3 overexpressing tumors while HAS2 overexpressing tumors contained both extracellular and intracellular HA. Treatment with PEGPH20 removed the majority of extracellular HA and induced a 87% reduction of tumor volume in BxPC3 HAS3 model (p<0.001) but had weaker effect on BxPC3 HAS2 (33%, p<0.001) and BxPC3 tumors (36%, p<0.01). Accumulation of extracellular HA was associated with enriched tumor stroma, loss of membranous E-cadherin and accumulation of cytoplasmic β-catenin in pancreatic cancer cells, suggesting HA-induced epithelial-mesenchymal transition (EMT). Removal of HA by PEGPH20 reversed the remodeling of the tumor stroma and induced translocation of E-cadherin and β-catenin to the plasma membrane.Translocation of E-cadherin was also observed in the KPC pancreatic tumors after PEGPH20 treatment. In conclusion, accumulation of extracellular HA by HAS3 overexpression favors tumor growth and leads to a strong response to PEGPH20 in a pancreatic cancer xenograft model. Deposition of extracellular HA is associated with optimization of the tumor microenvironment and EMT. Depletion of HA by PEGPH20 reverses changes in the tumor stroma and induces translocation of epithelial markers to the plasma membrane. Citation Format: Anne Kultti, Chunmei Zhao, Susan Zimmerman, Ryan J. Osgood, Yanling Chen, Rebecca Symons, Ping Jiang, Curtis B. Thompson, David A. Tuveson, Gregory I. Frost, H Michael Shepard, Zhongdong Huang. Extracellular hyaluronan accumulation by hyaluronan synthase 3 promotes pancreatic cancer growth and modulates tumor microenvironment via epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4844. doi:10.1158/1538-7445.AM2014-4844