Abstract Hyaluronan (HA), a nonsulfated glycosaminoglycan, is a significant component of the extracellular matrix of many solid tumors. HA accumulation has been correlated with local invasion, the presence of distal metastasis, higher tumor grade, and poorer overall survival. We have previously demonstrated that sustained HA removal, accomplished with a novel pegylated human recombinant hyaluronidase PH20 (PEGPH20), inhibited tumor growth and enhanced chemotherapeutic activity in HA-rich xenografts (Thompson et al, 2010). As endogenous lysosomal hyaluronidase expression has been implicated as a tumor promoter (Kovar et al, 2006), and exogenous hyaluronidase has been shown to act as a tumor suppressor (Shuster et al, 2002), we aimed to determine whether exogenous PEGPH20 administration alters the metastatic behavior of malignant cells in solid tumors. In a first set of experimental metastasis studies, HA-rich prostate PC3 (2e5) or breast MDA-MB-231 (1e5) cells were intracardially injected (IC) into nude mice (NCR nu/nu). Mice were subsequently treated (>20 min) with a single dose of PEGPH20 (4.5 mg/kg, IV) or vehicle and tumor seeding into organs tracked via bioluminescent imaging over time (n=8/group). In a second set of spontaneous metastasis experiments, prostate PC3 (2e6) or breast MDA-MB-231-Has2 (2e6) cells were injected into either the tibia periosteum or mammary fad pad of nude mice, respectively. MDA-MB-231-Has2 cells are a MDA-MB-231 Has 2 synthase overexpressing clone, shown to overexpress HA both in vitro and in vivo. When PC3 tumors reached ∼375 mm3 (n=11/group), and MDA-MB-231-Has2 reached ∼400 mm3 (n=6/group), mice received PEGPH20 (4.5 mg/kg, IV) or vehicle, q3d × 8 or q3d × 5, respectively. Mice were subsequently sacrificed and lymph node metastasis assessed histologically. Following the initial set of IC experiments, no significant difference in the number or frequency of metastatic nodules, as assessed by bioluminescence, was observed following PEGPH20 treatment versus control in either cell line (PC3, p=0.49; MDA-MB-231, p=0.89). In the second set of experiments, no difference in lymph node metastasis in either cell line was detected (PC3, p=0.68; MDA-MB-231, p=0.55). Finally, no histological evidence of increased metastases was observed in any organs examined (liver, kidney, heart, spleen or lung) following PEGPH20 administration in either study. Taken together, these results suggest exogenous PEGPH20 administration does not impact metastatic behavior in the PC3 prostate, MDA-MB-231 breast or MDA-MB-231-Has2 breast preclinical models. 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 442. doi:10.1158/1538-7445.AM2011-442