Species-Specific Urokinase Receptor Ligands Reduce Glioma Growth and Increase Survival Primarily by an Antiangiogenesis Mechanism
Total Page：16
File Type：pdf, Size：1020Kb
Laboratory Investigation (2004) 84, 667–678 & 2004 USCAP, Inc All rights reserved 0023-6837/04 $25.00 www.laboratoryinvestigation.org Research Articles Species-specific urokinase receptor ligands reduce glioma growth and increase survival primarily by an antiangiogenesis mechanism Xingyao Bu1, Vazgen Khankaldyyan1, Ignacio Gonzales-Gomez1, Susan Groshen2, Wei Ye2, Shaoqiu Zhuo3,*, Jaume Pons3,w, Jennifer R Stratton3,w, Steven Rosenberg3,z and Walter E Laug1 1Department of Pediatrics; 2Department of Preventive Medicine, Childrens Center for Cancer and Blood Diseases, Childrens Hospital Los Angeles, Los Angeles, CA, USA and 3USC Keck School of Medicine and Chiron Corporation, Emeryville, CA, USA Species-specific urokinase receptor (uPAR) ligands with improved pharmacokinetics were generated by site- specific mutagenesis and amino-terminal pegylation. These molecules were used to probe the role of uPAR in brain tumor progression and angiogenesis. The ligands blocked endothelial cell tube formation in Matrigel in a species-specific manner and reduced both baseline and uPA amino-terminal fragment-stimulated cell migration on vitronectin gradients. Treatment of U87MG gliomas implanted orthotopically in mice with single species- specific or combination uPAR ligands resulted in significant decreases in tumor size, which translated to increases in survival time, and which were most significant when the murine-specific ligand was included. Further analysis of tumors showed that the reduced sizes were correlated with a decrease in tumor cell proliferation and mean vessel density and an increase in tumor cell apoptosis. In addition, a large increase in collagen deposition was observed in the treated groups. Statistical analysis showed that the combination therapy demonstrated a clear synergy as compared to the individual agent treatments. These results suggest that the major role of the uPAR system in brain tumor progression is in the stromal compartment and particularly in neovascularization, a hallmark of invasive brain tumors. Laboratory Investigation (2004) 84, 667–678, advance online publication, 19 April 2004; doi:10.1038/labinvest.3700089 Keywords: urokinase receptor; brain tumor; angiogenesis; proteolysis; adhesion The urokinase-type plasminogen activator (uPA) uPAR with catalytically inactive uPA fragments lead system has been implicated in cancer progression, to reductions in tumor growth, tumor angiogenesis, invasion, and angiogenesis as well as tumor prog- and an increase in tumor dormancy in a variety of nosis.1–5 This system is central to cell-associated tumor types.3,8–11 In addition, studies of human proteolytic activity as well as cell migration and tumors have shown that different components of the adhesion, through interactions between the uroki- uPA system are expressed by both transformed nase receptor (uPAR) and integrins, as well as epithelial cells and stromal cells.12,13 Finally, studies extracellular matrix components, especially vitro- using uPA knock-out mice have shown a direct role nectin.6,7 Several in vivo studies have indicated that for stromal uPA in tumor progression in some modulation of the expression of uPAR or blockade of systems,14,15 although uPAR blockade in the absence of uPA has also shown in vivo effects.16 Brain tumors, and specifically gliomas, have been shown to overexpress components of the uPA Correspondence: W Laug, MD, Childrens Hospital Los Angeles, system, and tumor grade has been shown to 4650 Sunset Blvd, MS #57, Los Angeles, CA 90027, USA. correlate with expression levels.17 Reduction in E-mail: [email protected] *Current address: Diadexus, Inc., 343 Oyster Point Blvd, S. San levels of uPAR expression using antisense techno- Francisco, CA 94080, USA. logy has been shown to decrease tumor growth in an wCurrent address: Rinat Neuroscience, 3155 Porter Drive, Palo in vivo model.18 In addition, gliomas are reported to Alto, CA 94304, USA. be one of the most highly vascularized tumors, zCurrent address: XDx, Inc., 750 Gateway Blvd, Suite H, S. San Francisco, CA 94080, USA. suggesting that antiangiogenic approaches to these Received 21 November 2003; revised and accepted 21 January aggressive tumors might be useful. In this study, we 2004; published online 19 April 2004 have taken advantage of the species specificity of the uPA receptor ligands inhibit glioma growth XBuet al 668 uPA:uPAR interaction19 to probe the role of both nectin was purchased from Promega (Madison, MI, human tumor and murine stromal uPAR in tumor USA) and Matrigel from BD Biosciences (San Diego, growth and angiogenesis in an orthotopic human CA, USA). Human ATF was purchased from Amer- xenograft model of glioma in immunodeficient mice. ican Diagnostica. The results suggest that stromal uPAR, especially on endothelial cells, plays a predominant role in glioma tumor growth, and blockade of murine Cell Line stromal uPAR in this setting, increases survival, decreases tumor growth and tumor neovasculari- The human brain tumor cell line U87MG (glioblas- zation, and increases tumor cell apoptosis. These toma) was purchased from ATCC, VA, USA and results show the importance of the stromal cell uPA grown in RPMI medium with 10% fetal bovine 1 system contribution to brain tumor progression. serum in 5% CO2 at 37 C. Primary human and mouse brain capillary endothelial cells (BCEC) were isolated, characterized, and grown as described22 Materials and methods and used between passages 5 and 12. Materials Human uPA1-48 (h1-48) was expressed in recombi- Pharmacokinetics 20 nant yeast as described previously. The quadruple The in vivo half-lives of uPA1-48 and PEGh1-48 mutant of h1-48 with high affinity for the murine were determined after intravenous (i.v.) injection in uPAR (hm1-48, huPA1-48, N22Y, N27R, H29R, 8 mice. Proteins were injected at 10 mg/kg and blood W30R) was expressed in the same system. Peptides samples were collected at various time points and were purified from yeast cell-conditioned media by the amount of human uPAR inhibitory activity was S-Fractogel ion exchange chromatography. Specific measured using a uPAR binding assay as previously amino-terminal pegylation of these peptides was 21 described, but in the presence of a constant amount performed by the method of Gaertner and Offord. (50%) of mouse plasma.23 The half-life of uPA1-48 Specifically, purified huPA1-48 and the mutant was determined to be less than 10 min whereas protein were concentrated to 7–10 mg/ml by diafil- PEGh1-48 had a half-life of 130 min. Subsequent tration, the pH was adjusted to 6.8, and three molar experiments demonstrated that PEGh1-48 was very equivalents of freshly prepared sodium metaperio- efficiently absorbed after subcutaneous injection, date were added. The reaction was incubated for as well. 30 min at room temperature with stirring and protected from light and was then quenched by the addition of sodium acetate to a final pH of 4.5. After Brain Tumors dialysis against 30 mM sodium acetate, pH 4.5, the pegylation reaction was performed using a 3–5-fold Details of the xenotransplant model in nu/nu mice molar excess of methoxy-PEG-hydrazide 20 000 MW have been described previously.24 In brief, 106 tumor (Shearwater Polymers, Huntsville, AL, USA) at 371C cells suspended in 10 ml serum-free RPMI medium for 30 h with gentle stirring and protected from light. were injected over 10 min into the forebrain at The pegylated proteins were purified by Fractogel 1.5 mm lateral and 0.5 mm anterior to the Bregma, cation exchange chromatography, diafiltered against at a depth of 2.5 mm. The burr hole was closed with a 10 000 MW Amicon filter, and any contaminating bone wax. Mice were kept under general anesthesia endotoxin removed by passage through a TMAE during the procedure with administration of keta- Fractogel column equilibrated with phosphate- mine (100 mg/kg) and xylazine (10 mg/kg). Subcuta- buffered saline (PBS). The resulting purified pegy- neous administration of mouse or human pegylated lated peptides were analyzed by matrix-assisted uPA1-48 was twice weekly at 300 mg/100 ml when laser desorption mass spectroscopy, SDS-PAGE, given alone or at 100 mg/100 ml each when given human and murine uPAR radio-receptor-binding combined. The weight of animals was monitored assays, surface plasmon resonance (BIACore), and twice weekly and mice were killed when showing amino-acid analysis. In all cases, the resulting signs of tumor progression such as cachexia, hunch- proteins showed a molecular mass centered around back presentation, or any signs of paralysis. The 27 kDa, endotoxin of o0.5 EU/mg (LAL assay), and remaining mice were killed when drug supply 495% purity by SDS-PAGE. became limiting. Tumor size is given in millimeters Antibodies used for FACS analysis and immuno- and represents the largest diameter on serial sec- histochemistry were monoclonal mouse anti-human tions of the tumor-containing brain area. uPAR (CD 87) antibody 3936 (American Diagnostica, For histological studies, brains of killed animals Greenwich, CT, USA), polyclonal rabbit anti-mouse were immediately removed and either snap frozen uPAR antibody (Chiron), rat anti-mouse CD31 in liquid nitrogen for immunofluorescence studies monoclonal antibody (MEC 13.3, BD Biosciences, or fixed in 10% buffered formalin and embedded in San Diego, CA, USA), and monoclonal mouse anti- paraffin for hematoxylin and eosin (H&E) and human Ki-67 (DAKO, Carpinteria, CA, USA). Vitro- Masson’s Trichrome staining and immunohisto- Laboratory Investigation (2004) 84, 667–678 uPA receptor ligands inhibit glioma growth XBuet al 669 chemistry. Tumor size was determined. Animal phoresis and underlayed with a regular fibrin– studies were carried out according to the NIH agarose indicator gel.25 Clear zones of lysis, indicat- guidelines and approved by the local animal care ing presence of active uPA, were identified after 6 h committee. of incubation at 371C. Adhesion Assay Endothelial Tube Formation Assay Untreated tissue culture plates (48 wells/plate) were covered with vitronectin at 10 mg/ml and incubated Matrigel (Becton Dickinson) was added (150 ml) to overnight at 41C.