Functional imaging of pericellular proteolysis in cancer cell invasion Katarina Wolf *, Peter Friedl Department of Dermatology, Rudolf-Virchow Center, DFG Center for Experimental Biomedicine, University of Wuerzburg, Josef-Schneider-Strasse 2, 97080 Wuerzburg, Germany Received 2 August 2004; accepted 10 October 2004 Available online 10 December 2004 Abstract Proteolytic interactions between cells and extracellular matrix (ECM) are involved in many physiological and pathological processes, such as embryogenesis, wound healing, immune response, and cancer. The visualization of cell-mediated proteolysis towards ECM is thus required to understand basic mechanisms of tissue formation and repair, such as the breakdown and structural remodelling of ECM, inflammatory changes of tissue integrity, and the formation of proteolytic trails by moving cells. A panel of synergistic techniques for the visualization of pericellular proteolysis in live and fixed samples allow monitoring the of proteolytic tumor cell invasion in three-dimensional (3D) fibrillar collagen matrices in vitro. These include the quantification of collagenolysis by measuring the release of collagen fragments, the detection of protease expression and local activity by dequenching of fluorogenic substrate, and the staining of cleavage-associated neoepitopes together with changes in matrix structure. In combination, these approaches allow the high-resolution mapping of pericellular proteolysis towards ECM substrata including individual focal cleavage sites and the interplay between cell dynamics and alterations in the tissue architecture. © 2004 Elsevier SAS. All rights reserved. Keywords: Collagen type I; Collagenases; Protease activity; Collagen fragments; Quenched and dequenched FITC 1. Introduction The interaction of cells with extracellular matrix (ECM) scaffolds represents the basis for organ structure and func- tion [1]. In the healthy organism, the ECM not only forms a stable physical environment for cells but also undergoes a continuous slow and regulated molecular turnover leading to structural remodelling. Under pathologic conditions, such as wound healing, angiogenesis, inflammation and cancer, tis- sue structures become more rapidly modified by enhanced assembly and disassembly of ECM components, including de novo secretion and degradation of ECM compounds. Changes in tissue architecture and composition, in turn, can strongly impact the behaviour of embedded cells, thus the knowledge of structural and functional mechanisms of tissue remodelling is important for understanding physiological and pathological cells behaviour in vitro and in vivo. Tissue assembly is achieved by the secretion, cross- linking and -stabilization of ECM monomers, executed by cross-linking enzymes. On the other hand, tissue disassem- bly and remodelling requires the action of matrix-degrading enzymes produced and activated by resident or immigrated cells in a time- and space-coordinated manner. ECM degrad- ing enzymes comprise several protease classes, such as the matrix metalloproteinases (MMPs), serine, cysteine, and aspartate proteases. These enzymes possess a major function towards cleavage of matrix components, i.e., collagen and other substrates, and further play a role in cell growth, sur- vival, and migration [2]. In many tissues, collagen is the most abundant and structurally important component. Fibrillar collagens maintain tissue stability and further provide anchoring points for other molecules, such as fibronectin, pro- teoglycans, and growth factors, which can bind to ECM. Col- lagen is cleaved by collagenases (MMP-1, -2, -8, -13 or membrane-anchored membrane type 1 (MT)-MMP (MMP- 14)) and cathepsins (e.g. cathepsins B, K, L) [2]. These enzymes are either secreted or act in a cell membrane-bound form, thereby mediating their pericellular activity. Many pathological conditions, including cancer invasion result in the up-regulation and increased activity of matrix-degrading enzymes. Although extended biochemical studies have shed light into complex activation and inhibition cascades leading * Corresponding author. Tel.: +49-931-201-267-18; fax: +49-931-201-267-00. E-mail address: wolf_k@klinik.uni-wuerzburg.de (K. Wolf). Biochimie 87 (2005) 315–320 www.elsevier.com/locate/biochi 0300-9084/$ - see front matter © 2004 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2004.10.016