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Project Title: „Molecular design of new luminescent compounds for diagnostic purposes”

Funding: European Regional Development Fund (ERDF), Measure “Support for applied research”

Project No.:

Period: 1st March 2017 – 29st February 2020

Project costs: 580 000,00 EUR

BMC Principle Investigator: Dr. biol. D. Pjanova

Project leading partner: Daugavpils Universitāte

In the framework of the project „Molecular design of new luminescent compounds for diagnostic purposes” the interdisciplinary fundamental research will be carried out, which will ensure the synthesis of new luminescent substances for different diagnostic tools. The project is aimed to the synthesis of new luminescent substances for the development of diagnostics methods in medicine and veterinary medicine. Application of newly prepared dyes opens up new opportunities for progress of innovative non-invasive, early, and express diagnostics at the global level. The project is divided in two stages; the first stage includes elaboration of optimal synthetic approaches for new luminophores, its spectral characterization and binding with biological objects (blood cells, animal, and parasite tissues etc.). The second part involves development of protocols. The project implementation will be based on the collaboration of scientific institutions of several established working groups in a complexity of chemical, parasitological, histological, physical, genomics, and clinical genomics methods. Moreover, during the project will be created several work groups, which include important diagnostic fields in medicine, veterinary medicine and biology. The results of the research will be concentrated on the development of new product prototype and protocols of new methods (e.g. histology, karyotyping, parasitology etc.). The synthesis of new sensored luminophores, developed innovative methods, and protocols will be important in Latvia and Europe after the end of the project as well. Through the realisation of this project will be solved significant questions in different fields of diagnostics and research.

Information published: 01.03.2017.

Progress of the project:

June 1, 2017 – August 31, 2017

During this reporting period, several cell freezing protocols have been tested and freezing has been found to affect the properties of cell membranes.  The search for the best cell refrigeration protocol continues. In addition, differences in spectral parameters between normal cells in culture (fibroblasts) and melanoma cells in culture were confirmed. Pilot experiments have been initiated to see if colour spectral parameters correlate with tumour cell migration e.g. potential to form metastases.

Information published: 31.08.2017.

Progress of the project:

September 1, 2017 –  November 31, 2017

In order to analyze whether the spectral parameters of sunthesized dyes correlate with tumor cell migration, an in vitro melanoma cell model is under the development. For that, different melanoma cell cultures and various cytokines, as cell migration initiators, have been tested and model optimization continues. Similarly, work on the analysis of spectral parameters changes for sunthesized dyes in plazma samples from cancer patients and controls continues.

Information published: 30.11.2017.

Progress of the project:

December 1, 2017 – February 28, 2018

The best results in migration tests were obtained using cytokine TNF-alpha and cell starvation (cell growing in serum-free medium). Clear differences in cell migration were obtained between fibroblast and melanoma cells in culture. Comparison of differences between melanoma cell lines continues. The analysis of spectral changes of fluorescent dyes at the single cell level using confocal scanning microscopy are initiated as well.

Information published: 28.02.2018.

Progress of the project:

March 1, 2018 – May 31, 2018

During this reporting period, mainly spectral measurements of newly synthesised fluorescent probes were performed using laser-scanning confocal microscopy. Measurements were made at the level of cell populations, individual cells and individual cell fragments, evaluating the properties of fluorescence probes and looking at the specificity of probe’s cellular localization. One of the analysed probes, benzatron derivative, showed a bright fluorescence without significant toxicity to the cells and was highly lipophilic. At the cellular level, this fluorochrome was bright in both the plasma membrane and the internal cellular membranes. In addition, the difference was found in the spectral properties of the probe between the tumour (melanoma) and the normal (fibroblast) cell’s cytoplasmic membranes, but in the case of intracellular membranes there was no difference. Research in this direction continues.

Information published: 31.05.2018.

Progress of the project:

June 1, 2018 – August 31, 2018

During the reporting period, different spectral measurements of newly synthesised fluorescent probes were continued using laser scanning confocal microscopy and similar to the previous period spectral measurements were performed at different levels of the cell (cell populations, individual cells, and individual cellular fragments). Parallel to measurements in human melanoma and fibroblast cell cultures, measurements were also made in peripheral blood mononuclear cells of human and mouse origin. Analyses have been made for several newly synthesised substances. In parallel, spectral measurements have also been obtained in plasma samples of additional melanoma patients and control persons. Data analysis is currently underway.

Information published: 31.05.2018.

Progress of the project:

September 1, 2018 – November 30, 2018

Two of the previously analysed fluorescent probes have shown different spectral properties in normal and tumour (melanoma) cells. At present, work continues on the analysis of these probes in relation to the migration potential of tumour cells. For this purpose, a number of primary melanoma cell cultures have been established from human tumour material, all of which are melanocyte marker (HMB45) positive. In parallel, these cultures are analysed with additional molecular markers for melanoma. After these tests will be completed, the cancer cell migration assays will be performed. In addition, work on the analysis of human and mouse lymphocytes with probes still continues.

Information published: 30.11.2018.

Progress of the project:

1 December 2018 – 28 February 2019

During the reporting period, an in-depth molecular analysis of primary melanoma cell cultures was performed, showing that melanoma-specific markers are expressed in three out of the eight primary cultures. Next, the migration ability of cells in these three melanoma cell cultures was analysed. The obtained results were ambiguous and will be repeated. In addition, the establishment of primary melanoma cell cultures from human tumour material will be continued. Spectral analysis of dyes in melanoma patient’s plasma showed that changes in spectral properties of at least one probe correlated with the disease stage. We plan to validate this correlation in a larger sample set, including as many patients with late-stage melanomas as possible.

Information published: 28.02.2019.

Progress of the project:

1 March 2019 – 31 May 2019

During the reporting period, the number of analysed tumor cell cultures was increased. In addition to fibroblast and melanoma cell cultures, also breast, stomach, lung and pancreatic cancer cell cultures were analysed. This is done with the aim to show that the ability of benzatron derivatives to recognize changes in the plasma membrane is a universal phenomenon and is not limited to the ability to recognize melanoma cells only. The initial analysis shows that the localisation of probes in the cell is similar in all of the analysed cultures. In all analysed cell cultures spectral measurements were also made. Spectral analysis continues.

Information published 31.05.2019.

Progress of the project:

1 June 2019 – 31 August 2019

The spectral properties of fluorescent probes were analysed in additional different tumour cell lines. The spectral properties of probes differ between the cells of different tumours, indicating differences between these cells and biochemical composition of the cell membranes, probably mainly due to the different lipid composition. However, this is a hypothesis that should be further tested. In patients with tumours, spectral analysis of lymphocytes and correlation analysis with immunological parameters, such as CD38 lymphocyte activity marker, have been initiated in parallel with their plasma analysis.

Information published 30.08.2019.

Progress of the project:

1 September 2019 – 30 November 2019

Although the cells of different tumours showed differences in the spectral properties of probes, they were only significant in the case of melanoma compared to normal cells (fibroblasts). In the case of melanoma, the spectral features of probes between B16 (low metastatic potential) and B16-F10 (high metastatic potential) melanoma cells were also found. Differences were mainly observed at the plasma membrane level. This indicates, first, that the probes analysed can distinguish melanoma cells with increased metastatic potential from melanoma cells with lower metastatic potential and, secondly, that the metastatic potential of melanoma may depend on the structure of the plasma membrane of melanoma cells. Possible structural differences between melanoma cell membranes should be further explored. Analysis of plasma samples from melanoma patients showed no significant difference between measurements in patients and controls. Work is ongoing on the predominant cellular localization of probes and possible differences in membrane structures between metastatic and non-metastatic melanoma cells.

Information published 29.11.2019.

Progress of the project:

1 December 2020 – 29 February 2020

“All dyes selected for evaluation in human lymphocytes isolated from peripheral blood as well as for in depth analyses in different cell cultures showed a bright fluorescence without significant toxicity to the cells as shown by viability tests and both fluorescent and laser scanning confocal microscopy. At the cellular level, these dyes exhibit bright fluorescence in both the plasma membrane and the internal cellular membranes. In addition, the differences were found in the spectral properties of the dyes between the tumour (SK-MEL 28 melanoma) and the normal (HS-68 fibroblasts) cell’s cytoplasmic membranes.

Dyes under investigation have also been characterized in relation to plasma albumin conformational changes as measured by its spectral differences. Correlation between spectral characteristics of dyes and plasma albumin state in different pathologies including diabetes and cancer, e.g. melanoma, patients has been established. “

Information published 28.02.2020.