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Reporting period: 2018-01-01 to 2018-12-31

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.
A list of key parts of the work performed from Jan 01, 2016 to the Dec 31, 2018:

CASPASES chemical tools:

1. The development of small-molecule fluorogenic substrates and activity based probes for the investigation of apoptotic caspases (-3, -7, -8, -9, and -10).
2. Evaluation of the specificity of obtained activity based probes toward a panel of apoptotic caspases in in vitro experiments.
3. Using selected substrates and activity based probes we studied apoptosis mechanisms (extra- and intra-cellular) in various cancer cell lines.
4. We have developed new selective chemical tools (substrates and inhibitors) for investigation of human caspase 2. The selectivity of these probes were confirmed by performing kinetic studies and in vitro competitive labeling toward six human apoptotic caspases. We also demonstrated that one of the caspase-2 inhibitor can selectively block reversine-mediated caspase-2 activation in HCT-116 cells.

Related publications:
A) Poreba M et al Scientific Reports, 2017, doi: 10.1038/srep43135
B) Poreba M et al. Nature Protocols 2017, doi: 10.1038/nprot.2017.091
C) Ramirez MLG, Poreba M, et al. JBC 2018 doi: 10.1074/jbc.RA117.001329
D) Poreba M et al CDD 2019 doi: 10.1038/s41418-018-0110-y
E) Poreba M, et al CDD 2019 doi: 10.1038/s41418-019-0329-2
F) Groborz K et al CDD doi: 10.1038/s41418-019-0364-z

CATHEPSIN/LEGUMAIN chemical tools:

1. Using our HyCoSuL technology, we developed new very selective fluorescent-labeled small molecule inhibitors for studying human cysteine cathepsins (cathepsin L and cathepsin B) and legumain in cancer cells..
2. We tested the specificity of obtained activity based probes toward cathepsin L, cathepsin B and legumain in in vitro experiments and used them to visualize these enzymes in living cells (fluorescence microscopy).

Related publications:
G) Poreba M et al CCB 2016 doi: 10.1016/j.chembiol.2016.05.020
H) Kasperkiewicz P et al FEBS Journal 2017 doi: 10.1111/febs.14001
I) Poreba M et al Chemical Science 2018 doi: 10.1039/c7sc04303a
J) Poreba M et al Chemical Science 2019 doi: 10.1039/c9sc00997c


1. We developed a first panel of metal-tagged, mass cytometry-suitable chemical probes for dissecting proteases activtiy. Next, we used these probes to visualize cathepsin L, cathepsin B, legumain, and caspase-3 in cancer cells and human Peripheral Blood Mononuclear Cells (PBMCs) using mass cytometry. This is the first time ever, when mass cytometry-suitable chemical probes are used to detect enzymes activity. Moreover, we developed an integrated platform where we used both metal-tagged probes and metal-tagged antibodies to dissect proteolytic landscape in various populations (T cells, B cells, NK cells) in human PBMCs.
2. We also used our metal-labeled probes for the visualization of proteases in mice-models (cancer cell lines and cells isolated from mouse organs - kidney, liver, brain, and spleen).

Related publication:
K) Poreba M et al 2019, under review

Important remark: Since our mass cytometry data were very promising in context of multiparametric enzymes analysis, the Rector of our University approved to purchase mass cytometer (Helios, 3rd generation) to support our further research projects. We received this instrument on October 2018, and we have set up the first mass cytometry facility in Poland. Currently, I am responsible for training of other researchers on this instrument. We also submitted several grant proposals in which mass cytometer is a key technology, making our research very competitive in our field.
In this project we significantly improved the selectivity of chemical probes for proteolytic enzymes, and establish a detailed procedure for the parallel imaging of protease activities in biological samples. We also developed a novel class of metal-labelled chemical probes, which we used for the parallel visualization of proteases in cancer cells.

As mentioned in the grant proposal the societal implications of the project is the better understanding of the role of proteolytic enzymes in cancer progression, that can be further used for more tailored and personalized anticancer therapies.