Nanoscale imaging and quantification of local proteolytic activity

Area of Science:

• Biophysics
• Cancer Biology
• Materials Science

Background:

• Proteolytic cleavage of the extracellular matrix (ECM) is crucial for tumor invasion and impacts cancer cell behavior.
• Quantifying local proteolytic activity and ECM alterations near tumor cells is challenging.
• Tumor cells secrete proteases as inactive proenzymes activated by cleavage, with activity concentrated near the cells.

Purpose of the Study:

• To develop a novel method for measuring proteolytic activity in the cellular microenvironment.
• To enable high-resolution imaging and functional investigation of ECM alterations.
• To precisely quantify local ECM proteolysis at the nanoscale.

Main Methods:

• Utilized atomic force microscopy (AFM) combined with fluorescence microscopy (FM).
• Seeded amelanotic melanoma cells (A7-clone) on fluorescent gelatin and collagen-IV coatings.
• Measured nanoscale volume, 3D protein structure, and fluorescence reduction due to proteolysis.

Main Results:

• AFM enabled nanoscale volume measurement and 3D reconstruction of proteins, showing ECM cleavage restricted to the tumor cell microenvironment.
• Detected significant decreases in molecular weight (-76.6%), matrix volume (-46.6%), and height (-38.1%) in proteolyzed gelatin.
• AFM revealed stronger degradation of gelatin than collagen-IV by A7 cells and demonstrated similar parameter changes without FM.

Conclusions:

• AFM provides specific quantification and imaging of local proteolytic processes at the nanometer level.
• This method offers a unique approach for evaluating tumor cell invasiveness and metastatic potential.
• The technique allows functional assessment of proteolytic activity in small-scale samples.