Oncolytic adenoviruses are a promising treatment alternative for many advanced cancers, including colorectal cancer. However, clinical trials have demonstrated that single-agent therapy in advanced tumor masses is rarely curative. Poor spreading of the virus through tumor tissue is one of the major issues limiting efficacy. As oncolytic viruses kill preferentially cancer cells, high extracellular matrix (ECM) content constitutes potential barriers for viral penetration within tumors. In this study, the ECM-degrading proteases relaxin, hyaluronidase, elastase and macrophage metalloelastase (MME) were tested for their antitumor efficacy alone and in combination with oncolytic adenovirus. MME improved the overall antitumor efficacy of oncolytic adenovirus in subcutaneous HCT116 xenografts. In a liver metastatic colorectal cancer model, intra-tumoral treatment of primary tumors from HT29 cells with MME monotherapy or with oncolytic adenovirus inhibited tumor growth. Combination therapy showed no increased mortality in comparison with either monotherapy alone. Contradictory results of effects of MME on tumorigenesis and metastasis formation have been reported in the literature. This study demonstrates for the first time in a metastatic animal model that MME, as a monotherapy or in combination with oncolytic virus, does not increase tumor invasiveness. Co-administration of MME and oncolytic adenovirus may be a suitable approach for further optimization aiming at clinical applications for metastatic colorectal cancer. © 2012 Nature America, Inc. All rights reserved.