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(Cluj-Napoca) Role of exosomes in drug resistance of hematological malignancies
Romania (FASMR) - University of Medicine and Pharmacy "Iuliu Hatieganu", Cluj-Napoca
Research Center for Functional Genomics, Biomedicine and Translational Medicine, Cluj-Napoca, Romania
Professor Doctor Ioana Berindan-Neagoe
Professor Doctor Ioana Berindan-Neagoe, Doctor Jurj Anca
Type of Research Project
- Basic science
What is the background of the project?
Exosomes are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Increasing studies have shown that exosomes play an important role in tumour initiation, growth, progression, metastasis, drug resistance and immune escape. The project is focused on the exosomes exerted by cancer and normal cells in a malignant tumor specifically for hematological malignancies.The main interest is to establish why and how these exosomes have features to transport to normal cells their content that will generate drug resistance and high toxicity to normal cells.The project will look to the mechanisms for the biogenesis, release, uptake, structure and contents of exosomes; role of exosomes in normal cells and cancer cells; therapeutic potential of exosomes in cancer and future of exosomes; function of exosomes biogenesis and uptake inhibitors and exosomes removal from the circulations for cancer therapy. We will elaborate the specific mechanism by which exosomes affect the communication between tumours and the microenvironment. Finally, we report that exosomes may provide promising biomarkers for cancer diagnosis and represent new targets for cancer therapy.
What is the aim of the project?
The aim of the project is to use nanoparticles produced by cells called exosomes in order to identify a specific profiling for hematological malignancies related to drug resistance.
What techniques and methods are used?
The experiments will be performed using a chronic B cell lymphocytic leukemia cell line, Hs505.T. Cell viability will be evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells will be cultured at sub confluence before being washed three times with phosphate-buffered saline (PBS 1X, Gibco). After trypsinization, Hs505.T cells will be resuspended in culture media with 10% FBS and counted with Countess Automated Cell Counter (Invitrogen). The proliferation activity of the cells will be evaluated after 24 and 48 hours from treatment using MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma-Aldrich, Germany) test. Cells will be incubated for 2 hours with 1 mg/ml MTT solution and the metabolized formazan salt was resolubilised, afterwards we will read the absorbance at 570 nm with a multi-plate spectrophotometer BioTek Synergy. Exosomes-derived from chronic B-cell lymphocytic leukemia cell line, Hs505.T, will be isolated through ultracentrifugation. Hs505.T cells will be cultured in complete culture media supplemented with 10% fetal bovine serum heat inactivated. Conditioned media will be collected 48 and 96 hours after media exchange. Conditioned media will be centrifuged at 4000 x rpm(rotations per minute) for 10 minutes at room temperature to remove cells and cellular debris. Microvesicles will be isolated by media filtration with 0.22μm filter. The supernatant will be carefully removed without disturbing the pellet. This step will be followed by another ultra-centrifugation for 2 hours. After ultracentrifugation, the exosomes pellet was recovered and retained at -80˚C until further investigations. Exosomes size will be evaluated using NanoSight NS300 system with the camera level set at 14 for all recordings. According to Brownian motion, was determined the particle-size distribution of each particle. Total RNA extraction from Hs505.T cells will be performed using Tri Reagent (Invitrogen) according to manufacturer’s instruction. Reverse transcription was performed following protocol from Applied Biosystems. Gene expression evaluation was conducted using SYBR Select Master Mix (Applied Biosystems) and RT-qPCR will performed in a 10 µl volume using a 384-well plate.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
The students will work daily under the supervision of the tutor and coordinator of the center. The students will perform basic protocols in cell cultures like tests to check the viability of the cells, change media for the cell cultures, treat them with Fetal Bovine Serum in order to enhance their proliferation and treat them with different cytostatics in order to analyse the drug resistance. Assays using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide dye (MTT assay) will be performed in order to find out the right concentration of the drugs to be used during the experiments and the scratch assay to see how cell proliferation is altered with different treatments. Microscopy techniques will be performed in order to analyse the structural changes of the cells and the genetic technique Real Time Polymerase Chain Reaction (RT-PCR) will be used in order to evaluate the gene expression. The exosomes will be isolated from the cell cultures through ultracentrifugation, and their size will be characterised with the help of the NanoSight technique. Eventually, the exosomes will be internalized in other lines of cells and the changes in their morphology and function will be analysed through microscopy. The students will work daily under the supervision of the tutor and coordinator of the center.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Theoretical teaching will be provided. The students will receive some lectures for a better understanding of the project.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a presentation - The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Discipline, interest to work daily on a research project, interest to read and write papers are required from the students. Subjects passed: Cellular and Molecular Biology, Genetics
Are there any legal limitations in the student’s involvement
Type of students accepted
This project accepts: - Medical students - Pre-Medical students from the American-British system
- Niamh McNamee; Lorraine O'Driscoll.EXTRACELLULAR VESICLES AND ANTI-CANCER DRUG RESISTANCE. Biochim Biophys Acta Rev Cancer. 2018 Dec.
- Angelica Ortiz; Jun Gui; Farima Zahedi; Constantinos Koumenis; J. Alan Diehl; Serge Y. Fuchs. AN INTERFERON-DRIVEN OXYSTEROL-BASED DEFENSE AGAINST TUMOUR-DERIVED EXTRACELLULAR VESICLES. Cancer Cell. 2019 Jan 14
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