Protease assays for cancer diagnostics

Abstract

Numerous proteases are known to be necessary for cancer development and progression including Matrix Metalloproteinases (MMPs), Tissue Serine Proteases, and Cathepsins. The goal of this research is to develop a Fe/Fe₃O₄ nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Our nanoparticle-based “light switches” for measuring protease activity, consist of fluorescent cyanine dyes which are directly attached to Fe/Fe₃O₄ nanoparticles and porphyrins that are attached to Fe/Fe₃O₄ nanoparticles via consensus sequences. The consensus (cleavage) sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe₃O₄ nanoparticle resulting in highly sensitive (down to 1 x 10⁻¹⁶ mol L⁻¹ for 12 proteases), selective, and fast nanoplatforms (required time: 60 min.). Upon escape, the emission intensity of the organic dye will significantly increase, which can be detected using fluorescence spectroscopy. In order to demonstrate the potential of this new technology of early recognition of various cancers several analysis types have been used. Blood and urine samples from human cancer patients and healthy volunteers, tissue and blood serum samples from human cancer patients, and canine urine and blood serum samples are some of those types. Blood samples from human cancer patients and healthy volunteers were used to demonstrate the potential of this new technology for the early recognition of breast and lung cancers. We were able to establish several proteases with diagnostic potential for breast cancer and non-small cell lung cancer. It is very likely that different cancers will feature different “protease signatures”, meaning that different proteases will be activated, depending on the origin of cancer. This permits the diagnosis of various solid tumors at different stages. Tissue samples were collected from normal tissues, from the boundary of the tumor and from the tumor of the same person. Performed fluorescence experiments clearly indicate that tissue samples from the tumor show the highest fluorescence indicating the highest concentration of the protease. Results can be used excellently in a diagnostic system for breast cancer. Based on our results measuring protease signatures offers an inexpensive and fast approach towards early cancer diagnostics.