CATHEPSIN B AND GLIOMA INVASION LISA L. DEMCHIK,$,% MANSOUREH SAMENI,$ KEVIN NELSON,} TOM MIKKELSEN} and BONNIE F. SLOANE$,%* $Department of Pharmacology, Wayne State University, Detroit, MI 48201, USA; %Barbara Ann Karmanos Cancer Institute, Wayne State University, Detroit, MI 48201, USA; }Henry Ford Midwest Neuro-Oncology Center and Department of Neurology, Henry Ford Hospital, Detroit, MI 48202, USA AbstractÐIncreased expression of cathepsin B has been reported in a number of human and animal tumors. This has also been observed in human gliomas where increases in cathepsin B mRNA, protein, activity and secretion parallel malignant progression. In the present study, we showed that cathepsin B was directly involved in glioma cell invasion. Activity of cathepsin B was an order of magnitude higher in glioma tissue than in matched normal brain. Inhibitors of cysteine proteases reduced invasion of glioma cells in two in vitro models: invasion through Matrigel and in®ltration of a glioma spheroid into a normal brain aggregate. Glioma spheroids expressed higher levels of cathepsin B than did monolayers and the ability of subclones diering in cathepsin B activity to in®ltrate normal brain aggregates paral- leled their cathepsin B activity. We con®rmed that intracellular staining for cathepsin B occurs at the cell periphery and in cell processes and observed extracellular staining on the cell surface. In addition, we demonstrated that intracellular cathepsin B located at the cell periphery and in processes was active. The cell surface cathepsin B colocalized with areas of degradation of an extracellular matrix com- ponent. We hypothesize that the increased expression of active cathepsin B in gliomas leads to increases in invasion in vitro and in vivo and have developed a xenotransplant model in which this hypothesis can be tested. # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. INTRODUCTION Cathepsin B is one of many proteases involved in protein degradation within the lysosome. The cysteine protease cathepsin B functions as both an endopeptidase and an exopeptidase against a broad variety of substrates 6 . Cathepsin B has been implicated in a number of disease processes such as Alzheimer's 3 , arthritis 13 and tumor progression 1 . Localized degradation of the extracellular matrix (ECM) by proteases such as cathepsin B is a necessary step in tumor invasion 9 and other pathological processes. In many cancers, increases in cathepsin B message, protein and activity as well as alterations in tracking, localization and secretion of the enzyme have been observed 20 . Staining for cathepsin B message and protein has been shown to be most intense at the invasive, leading edge of tumors 8 suggesting that cathepsin B may enhance invasion. One of the features of primary malignant brain tumors is their ability to invade locally into the normal brain tissue. Recent studies in our laboratory have shown that cathepsin B is involved in glioma progression and invasion. Overexpression of cathepsin B mRNA transcripts has been identi®ed in both human glioma cell lines and biopsy samples. Transcript abundance increases in parallel with malignant progression of gliomas from astrocytomas to glioblastoma multiforme 15,19 . Increased expression of cathepsin B protein as determined by immunoblot analysis and by immunohistochemistry correlates with increased expression of cathepsin B transcripts. Increased secretion of procathepsin B by human gliomas in vitro has also been observed 10 . Immuno¯uorescent staining for cathepsin B in glioma cell lines with increased cathepsin B activity is distributed throughout the cells in both cytoplasmic processes and perinuclear regions. In contrast, staining for cathepsin B in normal cells, such as human Int. J. Devl Neuroscience, Vol. 17, Nos. 5±6, pp. 483±494, 1999 # 1999 ISDN. Published by Elsevier Science Ltd All rights reserved. Printed in Great Britain 0736-5748/99 $20.00 + 0.00 PII: S0736-5748(99)00011-8 www.elsevier.com/locate/ijdevneu *To whom correspondence should be addressed. Abbreviations: DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylinocarbocyanine perchlorate, DiO, 3,3'-dioctadecyloxacarbo- cyanine perchlorate, ECM, extracellular matrix, DMEM, Dulbecco's modi®ed Eagle's medium, FITC, ¯uorescein, MEM, minimal essential medium, PBS, phosphate buered saline. 483