The urokinase protease system as a target for breast cancer prognosis and therapy: Technical considerations

The majority of cancer patients do not die from their primary tumor but from distant metastases. Understanding the biology underlying these processes is of essential importance to specifically target the cause of malignancy. Proteases degrade the extracellular matrix surrounding the primary tumor, thereby facilitating tumor invasion and metastasis. Cathepsins, matrix metalloproteinases, and serine proteases such as the urokinase plasminogen activator and plasmin, in concert with their respective inhibitors and receptors, initiate tissue degradation and remodeling. Furthermore, components of these proteolytic systems exert additional functions, not related to their proteolytic activity, effecting cell adhesion, migration, proliferation, and vessel growth. In breast cancer, elevated antigen content of urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 is an indicator of poor prognosis and therefore helps to identify the patients likely to experience disease recurrence (metastasis) and/or early death. Such high-risk patients do benefit from adjuvant systemic chemotherapy. uPA and PAI-1 antigen is assessed in primary tumor tissue extracts by ELISA. We evaluated common macro-methodologies for disintegration of the primary tumor tissue and preparation of the tumor tissue extract, starting from fresh tumor tissue blocks. In addition, we present a new micro-extraction procedure using cryostat sections as the source of tumor tissue. Such techniques allow rapid and reproducible quantitative determination of uPA and PAI-1, even in small tumor specimens.