Abstract Hyaluronan (HA), a ubiquitous high-molar-mass linear glycosaminoglycan (GAG) plays a critical role in cell adhesion, motility, growth and differentiation. HA accumulation is associated with many human cancers. Human tumor cells can form HA rich pericellular matrix which might favor tumor growth by preventing the access of host immune cells to malignant cells. Monoclonal antibody (MAb) based therapeutics are emerging as an important component of therapy for human malignancies. Despite the very high specificity of monoclonal antibodies, resistance to MAb therapy is common. Antibody Dependent Cell-mediated Cytotoxicity (ADCC) is an important effector for the clinical efficacy of MAbs and relies on effective interaction between the Fc domain of antigen bound antibody and FcγRIII positive immune cells. We hypothesize that pericellular HA coating may contribute to resistance of HA-rich malignant cells to MAb mediated therapy by preventing efficient access of effector cells to their tumor targets. Enzymatic depletion of HA with PEGPH20 hyaluronidase could potentially sensitize such tumors to MAb mediated therapy. remove ell surface and ated therapy by preventing tumor cells contact EGFR positive breast carcinoma cell line MDA-MB-231luc and genetically engineered HAS2 over expressing MDA-MB-231HAS2 were used to investigate the role of HA-rich pericellular matrix in inhibition of ADCC. ADCC assay was performed by incubating Calcein AM labeled monolayer cells with monoclonal antibody (1h at 37°C) followed by co-incubation of recombinant human IL-2 (rhIL-2) activated human peripheral blood mononuclear cells (PBMC, 5h at 37°C, 5% CO2) with or without PEGPH20. Live cells from each treatment were assessed by fluorescence readings at Ex/Em 488nm/537nm (SpectraMax M2e plate reader) at the end of the assay. Percentages of cell killing were calculated against control. The student's t test was used to compare the difference between treatments. Particle exclusion assay demonstrated that HAS2 over expressing MDA-MB-231HAS2 cells formed a pericellular coat that inhibited access of the rhIL-2 activated PBMC to tumor cells. Incubation with PEGPH20 removed the HA-rich pericellular matrix and promoted contact of PBMC to tumor cells. Incubation of Erbitux with rhIL-2 activated PBMC induced a dose dependent killing of both MDA-MB-231luc and MDA-MB-231HAS2 cells. PEGPH20 increased ADCC by 10% on MDA-MB-231luc (HAlow) and 25% on MDA-MB-231HAS2 (HAhigh) tumor cells. Our results show that the HA-rich pericellular matrix on adherent tumor cells inhibits ADCC and PEGPH20 depletion of this coat sensitizes the tumor cells to ADCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3665. doi:10.1158/1538-7445.AM2011-3665