Processes such as cell proliferation, angiogenesis, apoptosis, or invasion are strongly influenced by the surrounding microenvironment of the tumor. Therefore, the ability to change these surroundings represents an important property through which tumor cells are able to acquire specific functions necessary for tumor growth and dissemination. Matrix metalloproteinases (MMPs) constitute key players in this process, allowing tumor cells to modify the extracellular matrix (ECM) and release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression. Remodeling of the ECM by collagenolytic enzymes such as MMP1, MMP8, MMP13, or the membrane-bound MT1-MMP as well as by other membrane-anchored proteases is required for invasion and recruitment of novel blood vessels. However, the multiple roles of the MMPs do not all fit into a simple pattern. Despite the pro-tumorigenic function of certain metalloproteinases, recent studies have shown that other members of these families, such as MMP8 or MMP11, have a protective role against tumor growth and metastasis in animal models. These studies have been further expanded by large-scale genomic analysis, revealing that the genes encoding metalloproteinases, such as MMP8, MMP27, ADAM7, and ADAM29, are recurrently mutated in specific tumors, while several ADAMTSs are epigenetically silenced in different cancers. The importance of these proteases in modifying the tumor microenvironment highlights the need for a deeper understanding of how stroma cells and the ECM can modulate tumor progression.

Aim: Activation of sodium reabsorption by urinary proteases has been implicated in sodium retention associated with nephrotic syndrome. The study was designed to test the hypothesis that nephrotic proteinuria in mice after conditional deletion of podocin leads to urokinase-dependent, amiloride-sensitive plasmin-mediated sodium and water retention. Methods: Ten days after podocin knockout, urine and faeces were collected for 10 days in metabolic cages and analysed for electrolytes, plasminogen, protease activity and ability to activate γENaC by patch clamp and western blot. Mice were treated with amiloride (2.5 mg kg −1 for 2 days and 10 mg kg −1 for 2 days) or an antiurokinase-type plasminogen activator (uPA) targeting antibody (120 mg kg −1 /24 h) and compared to controls. Results: Twelve days after deletion, podocin-deficient mice developed significant protein and albuminuria associated with increased body wt, ascites, sodium accumulation and suppressed plasma renin. This was associated with increased urinary excretion of plasmin and plasminogen that correlated with albumin excretion, urine protease activity co-migrating with active plasmin, and the ability of urine to induce an amiloride-sensitive inward current in M1 cells in vitro. Amiloride treatment in podocin-deficient mice resulted in weight loss, increased sodium excretion, normalization of sodium balance and prevention of the activation of plasminogen to plasmin in

Degradation of proteins in the extracellular matrix is crucial for the multistep process of cancer invasion and metastasis. Compelling evidence has demonstrated the urokinase receptor (uPAR) and its cognate ligand, the urokinase plasminogen activator (uPA), to play critical roles in the concerted action of several proteolytic systems in generation of a high proteolytic potential required for tissue remodeling processes. uPAR is additionally cleaved by uPA on the cell surface, liberating domain I, resulting in abrogated pericellular proteolysis. The expression of both uPAR and uPA is significantly up-regulated during cancer progression and is primarily confined to the tumor-associated stromal compartment. Furthermore, both uPAR and uPA have proven to be prognostic markers in several types of cancer; high levels indicating poor survival. The cleaved forms of uPAR are also prognostic markers, and a potential diagnostic and predictive impact of the different uPAR forms has been reported. Hence, pericellular proteolysis seems to be a suitable target for anti-cancer therapy and numerous approaches have been pursued. Targeting of this process may be achieved by preventing the binding of uPA to uPAR on the cell surface and/or by direct inhibition of the catalytic activity of uPA. Both strategies have been pursued and inhibition of these functions has shown effect in xenogenic cancer models. Pericellular proteolysis has also been inhibited in vivo in mouse models of wound healing and hepatic fibrinolysis using mouse monoclonal antibodies (mAbs) against mouse uPA or uPAR. These reagents will target uPA and uPAR in both stromal cells and cancer cells, and their therapeutic potential can now be assessed in syngenic mouse cancer models.

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using genetargeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPAdependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.