PROVIST · PROTEASES VISUALIZATION DURING TUMOR PROGRESSION

PROTEASES VISUALIZATION DURING TUMOR PROGRESSION

The main goal of this project was to develop a new chemical approach to investigate proteases participating in cancer progression. To do this we designed and synthesized small molecule inhibitors labeled with stable metal isotopes, which make the suitable for mass cytometry analysis. We selected several cysteine proteases as a proof of concept for our studies (caspases -3, -7, -8, and -9, cathepsins L, and B, and legumain). Caspases are known to play a central role in apoptosis – a process of programmed cell death by which unwanted/non-functional cells are removed from organism. On the other hand tumor progression can be correlated with elevated levels of some proteases like cysteine cathepsins or legumain. It has been demonstrated that increased level of these enzymes in solid tumors or in some extracellular fluids can be a useful markers for multiple cancers, however their precisely role have yet to be carefully dissected. During this project I have been investigating the activity of these proteases in multiple biological systems (cancer cells, mice models, human cancer samples) using the metal-labelled chemical markers, I developed. Such an approach enabled us to develop first in class chemical toolbox for the parallel visualization of proteolytic activity, making this project very unique and interdisciplinary. The main problem being addressed in this project is the parallel analysis of multiple proteases participating in cancer progression. The application of fluorescent-labeled probes allows only for the analysis of several proteases activity, due to overlapping of fluorescence spectra of fluorochromes. Mass cytometry is a unique technique, which uses metal stable isotopes as tags. Each metal isotope gives a single peaks on m/z analysis, and these peaks do not overlap across the metals. Thus it is possible to analyze multiple various parameters in a single experiment. The second problem that we aimed to solve is the overlapping substrate specificity across caspases and cathepsins. Most commonly used activity based probes lack the specificity, because they contain in their structure only 20 natural amino acids. However our group has recently demonstrated that the use of unnatural amino acids in fluorescent, combinatorial peptide libraries can significantly improve the selectivity of substrates and probes towards multiple proteases. The scope of this project is important for the society, as the understanding of cancer biology, especially in context of caspases and cathepsins, may help to develop more efficient and more personalized therapies to combat this disease. The toolbox of selective probes we developed may also help other research groups in their cancer-related studies. Final conclusions of the action: 1. We reached our research goal that was stated in the project proposal - we developed the first panel of mass cytometry-suitable chemical probes for proteases visualization in various samples (cancer cells in vitro, human cancer cells ex vivo, mouse cells ex vivo, human PBMCs ex vivo) and we proved that the parallel viualization of multiple proteases with metal-tagged probes is possible. 2. We have recently the first mass cytometry facility in Poland, as we want to use the unique technology we developed in our future research projects.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз