Pancreatic cancer is a leading cause of cancer-related death. Desmoplastic pancreatic tumors exhibit excessive extracellular matrix (ECM) and are thus highly resistant to anticancer therapeutics, since the ECM restricts drug penetration and dispersion. Here, we designed and generated two hypoxia-responsive and cancer-specific hybrid promoters, H(mT)E and H(E)mT. Transgene expression driven by each hybrid promoter was markedly higher under hypoxic conditions than normoxic conditions. Moreover, H(E)mT-driven transgene expression was highly cancer-specific and was superior to that of H(mT)E-driven expression. A decorin-expressing oncolytic adenovirus (Ad; oH(E)mT-DCN) replicating under the control of the H(E)mT promoter induced more potent and highly cancer-specific cell death compared with its cognate control oncolytic Ad, which harbored the endogenous Ad E1A promoter. Moreover, oH(E)mT-DCN exhibited enhanced antitumor efficacy compared with both the clinically approved oncolytic Ad ONYX-015 and its cognate control oncolytic Ad lacking DCN. oH(E)mT-DCN treatment also attenuated the expression of major ECM components, such as collagen I/III, elastin and fibronectin and induced tumor cell apoptosis, leading to extensive viral dispersion within orthotopic pancreatic tumors and pancreatic cancer patient-derived tumor spheroids. Collectively, these findings demonstrate that oH(E)mT-DCN exhibits potent antitumor efficacy by degrading the ECM and inducing apoptosis in a multifunctional process. This process facilitates the dispersion and replication of oncolytic Ad, making it an attractive candidate for the treatment of aggressive and desmoplastic pancreatic cancer.Pancreatic cancer is the fourth leading cause of cancerrelated death in Europe and the United States.1 Only 10-20% of all patients with pancreatic cancer are resectable at presentation. The US National Cancer Institute recently reported that the overall 5-year relative survival rate for 2002-2008 was 5.8%, and nearly 90% of all patients were dead 1 year after diagnosis, with a median survival of <6 months. 2,3 One experimental treatment option of particular interest for pancreatic cancer is oncolytic virotherapy, which uses viral vectors to preferentially replicate in tumor cells and induce cancer cell death. Among the various viral vectors that have been tested, adenovirus (Ad) has been most extensively used in gene therapy applications, and sufficient data from randomized clinical trials support its safety in patients. 4-6Although oncolytic Ad-mediated cancer gene therapy is highly promising, significant therapeutic efficacy of oncolytic Ad for the treatment of localized tumors has not yet been achieved in clinical trials; thus, further improvements are needed. 7,8 Hypoperfusion and desmoplasia are two prominent features of pancreatic cancer that attenuate the therapeutic efficacy of cancer therapeutics.9 Aberrant extracellular matrix (ECM) functions as a protective layer that prevents drug penetration and diffusion into the pancreatic tumors, contributing t...