Simultaneous enzyme overlay membrane (EOM)-based in situ zymography and immunofluorescence technique reveals cathepsin B-like activity in a subset of tumour vessels

Abstract.

The analysis of the invasion front of oral squamous cell carcinoma (OSCC) has revealed a fundamental invasion-associated remodelling of the extracellular matrix as the result of a complex regulated interplay of matrix synthesis and matrix degradation. Cysteine proteinases, in particular cathepsin B, are implicated in tumour invasion in vivo and in vitro and are thought to be important mediators of metastasis. An in situ zymographic assay based on enzyme overlay membranes (EOMs) was established to define the tissue localisation of cathepsin B activity in OSCC. Using confocal laser scanning microscopy we present a double-labelling method for the rapid and reproducible simultaneous detection of cathepsin B-like activity and cellular or extracellular antigens based on an EOM and immunofluorescence technique on frozen sections. Applying this method, cathepsin B-like activity was mainly found in vascular structures within the invasive front of OSCC. Therefore, the results suggest a particular pathogenic role of cathepsin B in tumour angioneogenesis. The method can simply be transferred to other enzymes and can be recommended for more extensive studies of proteolytic activity in human malignancies.