Abstract
Proteolytic cleavage of extracellular matrix (ECM) is a critical feature of tumor cell invasion, and affects cancer cell growth, differentiation, apoptosis, and migration. Malignant cells secrete most proteases as inactive proenzymes that undergo proteolytic cleavage for activation, and proteolytic activity is elevated in close proximity to these cells. Therefore, local activity rather than protease concentration determines ECM proteolysis. Precise quantification of local proteolytic activity, functional investigation, and high resolution imaging of morphological ECM alterations have proven difficult. In this study, we present a novel approach for measuring proteolytic activity in the microenvironment of cells by using atomic force microscopy (AFM). Amelanotic melanoma cells (A7โclone) were seeded on fluorescent gelatin or collagenโIV coatings. Proteolysis reduced fluorescence of these coatings. Fluorescence microscopy (FM) in combination with AFM was used to maneuver the AFMโtip to tumor cell induced proteolytic spots. AFM enabled nanoscale volume measurement, threeโdimensional reconstruction of single proteins and demonstrated that ECM cleavage is restricted to the proteolytic microenvironment of cancer cells. This method detected significant decreases in molecular weight of protein clusters (โ76.6%), matrix volume (โ46.6%), and height (โ38.1%) between intact and proteolyzed gelatin. Similar parameter changes were demonstrated without FM, by AFMโscanning gelatin in close proximity to invasive cells. Furthermore, AFM depicted significantly stronger local degradation of gelatin than collagenโIV by A7โcells. Taken together, AFM allows specific quantification and imaging of local proteolytic processes at a nanometer level, thus providing a unique method for the functional evaluation of invasiveness and metastatic potential of tumor cells in small scale samples. ยฉ 2005 WileyโLiss, Inc.