Exosome studies

Dose verification using 3D printed phantoms
Nanodosimetry – particle track structure measurements at the DNA level
The lung and prostate cancers are the most common men's cancers, which are treated in clinical practice with different methods, such as: surgery, radiotherapy, chemotherapy, immunotherapy. However, new methods of cancer treatment are still being investigated therefore the interest in exosomes studies is increasing in the last few years. The exosomes which are present in human body fluids can be isolated easily from cell culture medium and studied in in vitro experiments. Since exosome characteristics (miRNAs, mRNAs and proteins with the potential to control signaling pathways, lipid bilayer membrane of exosomes protecting their content) they can be used as a good delivery vector in therapy. The main goal of my project is to review the exosomes’ effects on prostate cancer progression, especially to the tumorigenesis and metastasis. Characteristics of exosomes Exosomes are spherical membrane nanovesicles ranging from 50 to 150 nm in diameter and their density varies between 1.1 and 1.9 g/cm3. They have a double membrane structure, which contain miRNAs, mRNAs, proteins lipids and viral particles. They are released by most eukaryotic cells, both healthy and affected, and are involved in cell-to-cell communication. They are classically considered as extracellular vesicles, which originate from intracellular budding from multivesicular bodies (MVBs). As a result of this intracellular budding and subsequent internalization in the endosome lumen, intraluminal vesicles (ILVs) are formed, i.e. precursors of exosomes. Endosomes that accumulate ILVs in their interior are referred to as MVBs. MVBs migrate towards the cell surface, and finally their membrane fuses with the membrane of the parental cell, and the contents are released into the extracellular space. This results in the release of vesicles called exosomes into the extracellular environment. Cell lines There are two types of human prostate cancer cell lines: PC-3, DU-145, and one normal RWPE-1 used in this project. PC-3 is the line, which was derived from a metastatic site: bone, and characterised by high invasiveness (grade IV). DU-145 is the line, which was derived from a metastatic site: brain, and had relatively low invasiveness compared to PC-3 cells (grade II). RWPE-1 was established from epithelial cells derived from the peripheral zone of a histologically normal adult human prostate and transfected with a single copy of the human papillomavirus 18 (HPV-18).
Exosome isolation and NTA The supernatant containing exosomes produced by irradiated cells will be collected according to a few steps protocoles and it will be used for exosomes isolation. The exosomes isolation will be performed in a few stages within our scientific cooperation with the Medical University of Warsaw (WUM). The number and size of isolated exosomes will be investigated using Nanoparticle Tracking Analysis (NTA) available at WUM. NTA is a technique used to visualize and measure the size and concentration of exosomes based on the analysis of Brownian motion. Particles in the sample are visualized by the illumination with a laser beam. The scattered light of the particles is recorded with a light sensitive CCD or CMOS camera. Molecular diffusion is determined by tracking changes in a single particle set and then converted to a hydrodynamic diameter (using the Stokes-Einstein equation). After the NTA analysis, the samples will submit to gene expression tests.
  • Beata Brzozowska
  • Józef Ginter
  • Wioletta Olejarz
  • Tomasz Lorenc
  • Katarzyna Życieńska
  • Adrianna Tartas
  • Mateusz Filipek
  • Beata Pszczółkowska
Partial financing: funds for cooperation between WUM-UW (Polish title: Badanie wpływu promieniowania jonizującego na profil egzosomów pochodzących z komórek raka prostaty o różnej promieniowrażliwości; dr G. Kubiak-Tomaszewska, mgr A. Tartas)