ObjectivePancreatic ductal adenocarcinoma (PDA) is characterised by stromal desmoplasia and vascular dysfunction, which critically impair drug delivery. This study examines the role of an abundant extracellular matrix component, the megadalton glycosaminoglycan hyaluronan (HA), as a novel therapeutic target in PDA.MethodsUsing a genetically engineered mouse model of PDA, the authors enzymatically depleted HA by a clinically formulated PEGylated human recombinant PH20 hyaluronidase (PEGPH20) and examined tumour perfusion, vascular permeability and drug delivery. The preclinical utility of PEGPH20 in combination with gemcitabine was assessed by short-term and survival studies.ResultsPEGPH20 rapidly and sustainably depleted HA, inducing the re-expansion of PDA blood vessels and increasing the intratumoral delivery of two chemotherapeutic agents, doxorubicin and gemcitabine. Moreover, PEGPH20 triggered fenestrations and interendothelial junctional gaps in PDA tumour endothelia and promoted a tumour-specific increase in macromolecular permeability. Finally, combination therapy with PEGPH20 and gemcitabine led to inhibition of PDA tumour growth and prolonged survival over gemcitabine monotherapy, suggesting immediate clinical utility.ConclusionsThe authors demonstrate that HA impedes the intratumoral vasculature in PDA and propose that its enzymatic depletion be explored as a means to improve drug delivery and response in patients with pancreatic cancer.

Hyaluronan (HA) is a glycosaminoglycan polymer that often accumulates in malignancy. Megadalton complexes of HA with proteoglycans create a hydrated connective tissue matrix, which may play an important role in tumor stroma formation. Through its colloid osmotic effects, HA complexes contribute to tumor interstitial fluid pressure, limiting the effect of therapeutic molecules on malignant cells. The therapeutic potential of enzymatic remodeling of the tumor microenvironment through HA depletion was initially investigated using a recombinant human HA-degrading enzyme, rHuPH20, which removed HA-dependent tumor cell extracellular matrices in vitro. However, rHuPH20 showed a short serum half-life (t 1/2 < 3 minutes), making depletion of tumor HA in vivo impractical. A pegylated variant of rHuPH20, PEGPH20, was therefore evaluated. Pegylation improved serum half-life (t 1/2 = 10.3 hours), making it feasible to probe the effects of sustained HA depletion on tumor physiology. In high-HA prostate PC3 tumors, i.v. administration of PEGPH20 depleted tumor HA, decreased tumor interstitial fluid pressure by 84%, decreased water content by 7%, decompressed tumor vessels, and increased tumor vascular area >3-fold. Following repeat PEGPH20 administration, tumor growth was significantly inhibited (tumor growth inhibition, 70%). Furthermore, PEGPH20 enhanced both docetaxel and liposomal doxorubicin activity in PC3 tumors (P < 0.05) but did not significantly improve the activity of docetaxel in low-HA prostate DU145 tumors. The ability of PEGPH20 to enhance chemotherapy efficacy is likely due to increased drug perfusion combined with other tumor structural changes. These results support enzymatic remodeling of the tumor stroma with PEGPH20 to treat tumors characterized by the accumulation of HA. Mol Cancer Ther; 9(11); 3052-64. ©2010 AACR.

Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet–induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet–induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance.

Elevated interstitial fluid pressure can present a substantial barrier to drug delivery in solid tumors. This is particularly true of pancreatic ductal adenocarcinoma, a highly lethal disease characterized by a robust fibroinflammatory response, widespread vascular collapse, and hypoperfusion that together serve as primary mechanisms of treatment resistance. Free-fluid pressures, however, are relatively low in pancreatic ductal adenocarcinoma and cannot account for the vascular collapse. Indeed, we have shown that the overexpression and deposition in the interstitium of high-molecular-weight hyaluronan (HA) is principally responsible for generating pressures that can reach 100 mmHg through the creation of a large gel-fluid phase. By interrogating a variety of tissues, tumor types, and experimental model systems, we show that an HA-dependent fluid phase contributes substantially to pressures in many solid tumors and has been largely unappreciated heretofore. We investigated the relative contributions of both freely mobile fluid and gel fluid to interstitial fluid pressure by performing simultaneous, real-time fluid-pressure measurements with both the classical wick-in-needle method (to estimate free-fluid pressure) and a piezoelectric pressure catheter transducer (which is capable of capturing pressures associated with either phase). We demonstrate further that systemic treatment with pegylated recombinant hyaluronidase (PEGPH20) depletes interstitial HA and eliminates the gel-fluid phase. This significantly reduces interstitial pressures and leaves primarily free fluid behind, relieving the barrier to drug delivery. These findings argue that quantifying the contributions of free- and gel-fluid phases to hydraulically transmitted pressures in a given cancer will be essential to designing the most appropriate and effective strategies to overcome this important and frequently underestimated resistance mechanism.

Extensive accumulation of the glycosaminoglycan hyaluronan is found in pancreatic cancer. The role of hyaluronan synthases 2 and 3 (HAS2, 3) was investigated in pancreatic cancer growth and the tumor microenvironment. Overexpression of HAS3 increased hyaluronan synthesis in BxPC-3 pancreatic cancer cells. In vivo, overexpression of HAS3 led to faster growing xenograft tumors with abundant extracellular hyaluronan accumulation. Treatment with pegylated human recombinant hyaluronidase (PEGPH20) removed extracellular hyaluronan and dramatically decreased the growth rate of BxPC-3 HAS3 tumors compared to parental tumors. PEGPH20 had a weaker effect on HAS2-overexpressing tumors which grew more slowly and contained both extracellular and intracellular hyaluronan. Accumulation of hyaluronan was associated with loss of plasma membrane E-cadherin and accumulation of cytoplasmic β-catenin, suggesting disruption of adherens junctions. PEGPH20 decreased the amount of nuclear hypoxia-related proteins and induced translocation of E-cadherin and β-catenin to the plasma membrane. Translocation of E-cadherin was also seen in tumors from a transgenic mouse model of pancreatic cancer and in a human non-small cell lung cancer sample from a patient treated with PEGPH20. In conclusion, hyaluronan accumulation by HAS3 favors pancreatic cancer growth, at least in part by decreasing epithelial cell adhesion, and PEGPH20 inhibits these changes and suppresses tumor growth.