Nanoscopic screening of exosomes from myeloma patients - exploring nature's own nanoparticles for potential diagnostic and therapeutic applications.
Prosjekt
- Prosjektnummer
- 46056651
- Ansvarlig person
- Katarzyna M. Psonka-Antonczyk
- Institusjon
- NTNU, NT Fak.
- Prosjektkategori
- Forskerstipend 2012
- Helsekategori
- Cancer
- Forskningsaktivitet
- 4. Detection and Diagnosis
Rapporter
Exosomes are lipid membrane vesicles secreted by cells that ferry proteins and RNAs. They are found in bodily fluids and ascites. Due to their signaling role they are considered as potential source of biomarkers for cancer diagnostics. Our objective is to characterize cancer-derived exosomes, to determine their possible role in cancer biology.The main goal of this project is to study exosomes, nanovesicles isolated from various multiple myeloma cell lines as well as from the samples collected from patients suffering from multiple myeloma to unravel their role in cancer biology. Single-molecule techniques such as atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) are employed to provide detailed physical and biochemical characterization of isolated vesicles. Identification of exosomes' subpopulations according to transmembrane protein’s repertoire and genetic content are expected to make progress in understanding their biological roles in cancer development and spreading. It may also result in identifying relevant biomarkers to be searched for in patients' blood and bone marrow samples in myeloma diagnostics.
This project is a multidisciplinary collaboration between biopolymer group at Dept of Physics, NTNU and the department of cancer research and molecular medicine at DMF, NTNU and St Olav Hospital. Exosomes isolates studied in this project are prepared from the various cell cultures (relevant healthy and malignant cell lines), healthy donors (blood samples) and hospitalized patients (blood and bone marrow samples donated by local bio-bank).
Exosomes are secreted by all cells and tissues and ferry cargo that is cell-of-origin specific Thus, when isolated from body fluids like blood or urine, exosomes represent a heterogeneous population in terms of their source. A series of tests was performed to establish an optimized protocol for exosomes isolation resulting in sample quality that is adequate for down-stream analysis of their content and for their characterization with AFM. Cell lines producing fluorescent vesicles (multiple myeloma and stroma cells) were examined by TIRFM to visualize the dynamics of exosomes and the process of their secretion. Co-culturing experiments showed a transfer of exosomes between different multiple myeloma cell lines as well as between the latter and the mesenchymal and stroma cells. Transfected cells to express green fluorescent proteins in the exosomes membrane were used for these studies. The obtained exosome trafficking result provides experimental evidence to the hypothesis of a possible function of exosomes as signalling entities in a novel communication route between distant and different tissues and organs in human body. Exosomes’ transfer was observed between malignant and healthy cultured cells indicating a possible role of exosomes in disease spreading. Multiple myeloma cells secrete vesicles that carry HGF on their surface. A signalling route between HGF+ cells and osteoblasts was investigated to show a possible role of multiple myeloma derived vesicles in bone marrow in bone remodelling. A series of stimulation experiments was performed to research the influence of pro- and anti-inflammatory cytokines on the secretion of vesicles in cancer, stroma and stem cells.
The results of co-culturing and stimulation experiments were presented as an oral presentation at the Nordic Physics Days meeting (Trondheim, 9-12 June 2015). The bone remodelling experiments are being prepared to be published.
Exosomes, vesicles secreted by healthy and malignant cells are loaded with a specific to cell-of-origin cargo and thus considered to be an attractive biomarker candidate for novel and early cancer diagnosis. In the project, we focus on investigation of exosomes using single-molecule techniques to address the vesicles’ role in disease progress.The main goal of this project is to investigate exosomes, nanovesicles isolated from various multiple myeloma cell lines as well as from the samples collected from patients suffering from multiple myeloma. Single-molecule techniques such as atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM) are employed to provide detailed physical and biochemical characterization. Identification of exosomes' subpopulations according to transmembrane protein’s repertoire and genetic content are expected to make progress in understanding their biological roles in cancer biology. It may also result in identifying relevant biomarkers to be searched for in patients' blood and bone marrow samples in myeloma diagnostics.
This project is a multidisciplinary collaboration between biopolymer group at Dept of Physics, NTNU and the department of cancer research and molecular medicine at DMF, NTNU and St Olav Hospital. Exosomes samples studied in this project are prepared from the various cell cultures (healthy and malignant cells), healthy donors (blood samples) and hospitalized patients (blood and bone marrow samples donated by local bio-bank).
Exosomes isolated from body fluids are believed to be rather heterogeneous population in terms of their cell-of-origin. Development of substrates for selective binding of exosomes based on the individual receptors on the exosomes membranes provides a platform to investigate vesicles at the level of individual subpopulations. The protocols for the manufacturing of chemically- and biochemically-modified surfaces for exosomes binding based on specific interactions between membranous receptors and antibodies are under development and testing. In parallel, a series of tests was performed to establish an optimized protocol for exosomes isolation resulting in sample quality that is adequate for single-molecule techniques. Fluorescently labelled vesicles isolated from cultured cell lines were visualized with TIRFM. Cell lines producing fluorescent vesicles were examined by TIRFM. Co-culturing experiments showed a transfer of fluorescently labelled exosomes between different multiple myeloma cell lines as well as between the latter and the mesenchymal and stroma cells. Such a result provides experimental evidence to the hypothesis of a possible function of exosomes as a novel communication route between distant and different tissues and organs in human body. Exosomes’ transfer was observed between malignant and healthy cultured cells indicating a possible role of exosomes in disease spreading. Test AFM experiments are being performed to establish a strategy to access the elasticity of individual vesicles by nanoindentation. Size- and concentration- profiling of vesicles secreted by cells stimulated with various pro-inflammatory factors was performed to study if and how such stimulation can alter the production and secretion of vesicles.
Exosomes, vesicles secreted by healthy and malignant human cells to the body fluids are carriers of a variety of a specific to cell-of-origin cargo (RNAs, proteins). These nanovesicles are considered to be a potential source of biomarkers exploited in innovative cancer diagnosis. Characterization of exosomes harvested from healthy and diseased state might give information about their role in the cancer biology.The main aim of the project is to characterize exosomes derived from multiple myeloma patients with single-molecule techniques, atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM), to provide detailed physical and biochemical description. Identification of exosomes’ cargo components and deciphering their genetic content will shed light to the role of exosomes in cancer biology. Linking the components to their origin and functions might result in identification of potential biomarkers that can be exploited in multiple myeloma diagnostics by screening potential patients’ samples like blood and bone marrow.
This project emerged as collaboration between NTNU, Dept. of Physics, biopolymer group and St. Olav Hospital, Dept. of Cancer Research bringing together expertise in single molecule techniques like AFM, TIRFM and NTA (nanoparticle tracking analysis), culturing cells and isolating exosomes from diverse sources. Exosomes that are investigated within presented project are harvested from various cell cultures (healthy and malignant cells), healthy donors (blood samples) and hospitalized patients (blood and bone marrow samples donated by local bio-bank).
As exosomes are secreted by various cells and tissues in the human body and released to the body fluids, it is believed that they are rather heterogeneous population of vesicles in terms of their composition and cargo. Moreover, based on their characteristics exosomes can be linked back to their cell-of-origin. Therefore, it is believed that sorting exosomes into subpopulations based on their individual receptors at the membrane will provide information about the diversity and sources of a given exosomes’ sample. In this project it is aimed to develop set biochemically modified substrates for selective binding of exosomes based on the individual receptors on the exosomes membrane providing a platform to investigate vesicles at the level of individual subpopulations. The protocols for the manufacturing of chemically- and biochemically-modified surfaces for exosomes binding based on specific interactions between membranous receptors and antibodies are currently being developed and optimized.
Various cell lines were used to mimic different microenvironment of the human body i.e. individual components of healthy bone marrow as well as malignant cells therein. Isolated exosomes were fluorescently labelled and visualized with TIRFM. Cell lines producing fluorescent vesicles were used to investigate a possibility of exosomes’ transfer between different cell lines by co-culturing them providing experimental evidence to exosomes as a novel communication route between distant and different tissues and organs in human body. Exosomes’ transfer was observed between malignant and healthy cultured cells indicating a possible role of exosomes in disease spreading. Exosomes isolated from cells stimulated with various pro-inflammatory factors were characterized in terms of their size and concentration to study if and how various stress stimuli can alter the production and secretion of vesicles.
Cancer is a leading cause of death worldwide. Exosomes, vesicles secreted by healthy and malignant cells are considered to be an attractive biomarker candidate for novel and early cancer diagnosis. In the project, we focus on investigation of exosomes using single-molecule technique as an tool to address the vesicles’ role in disease progress.The main goal of this project is to investigate exosomes, nanovesicles secreted by cells, derived from multiple myeloma patients with single-molecule techniques such as atomic force microscopy (AFM) and total internal reflection fluorescence microscopy (TIRFM), to provide detailed physical and biochemical characterization. Identification of exosomes' subpopulations according to transmembrane protein’s repertoire and genetic content are expected to make progress in understanding their biological roles in cancer biology. It may also result in identifying relevant biomarkers to be searched for in patients' blood and bone marrow samples in myeloma diagnostics.
This project is a close collaboration between biopolymer group at Dept of Physics, NTNU and the department of cancer research and molecular medicine at DMF, NTNU and St Olav Hospital. Exosomes samples studied in this project are prepared from the various cell cultures (healthy and malignant cells), healthy donors (blood samples) and hospitalized patients (blood and bone marrow samples donated by local bio-bank). Exosomes isolated from body fluids are believed to be rather heterogeneous mixtures of vesicles in terms of their cell-of-origin. Development of substrates for selective binding of exosomes based on the individual receptors on the exosomes membranes provides a platform to investigate vesicles at the level of individual subpopulations. The protocols for the manufacturing of chemically- and biochemically-modified surfaces for exosomes binding based on specific interactions between membranous receptors and antibodies are currently being developed and optimized. Fluorescently labelled vesicles isolated from cultured cell lines were visualized with TIRFM. Cell lines producing fluorescent vesicles were used in co-culturing studies to examine a possible transfer of exosomes between different cell lines. Such a result provides experimental evidence to the hypothesis of a possible function of exosomes as a novel communication route between distant and different tissues and organs in human body. Exosomes’ transfer was observed between malignant and healthy cultured cells indicating a possible role of exosomes in disease spreading. Preliminary experiments with TIRFM were performed to examine the possibility of observing the trafficking and secretion of exosomes in cultured cells in real time. Size- and concentration- profiling of vesicles secreted by cells stimulated with various pro-inflammatory factors was performed to study if and how such stimulation can alter the production and secretion of vesicles.
Preliminary results were presented as a poster at the annual meeting of International Society for Extracellular Vesicles. Exosomes activity was also presented and advertised with a poster at the Annual NTNU Nanolab Meeting.
Vitenskapelige artikler
Strømme O, Psonka-Antonczyk KM, Stokke BT, Sundan A, Brede G
Multiple myeloma cells secrete nanovesicles that stimulate IL-11 secretion in osteoblast-like recipient cells
J. Extracell. Vesicles 2012, 1: 18179 - http://dx.doi.org/10.3402/jev.v1i0.18179
Araldi E, Kramer-Albers EM, Hoen E, Peinado H, Psonka-Antonczyk KM, Rao P, van Niel G, Yanez-Mo M, Nazarenko I
International Society for Extracellular Vesicles: first annual meeting, April 17-21, 2012: ISEV-2012
J. Extracell. Vesicles 2012, 1: 19995 - http://dx.doi.org/10.3402/jev.v1i0.19995
Psonka-Antonczyk KM, Brede G, Stokke BT
Nanoparticle tracking analysis of extracellular vesicles – nature’s own nanoparticles
poster - 7th Annual NTNU Nanolab Meeting; 2012-12-12
Deltagere
- Bjørn Torger Stokke Prosjektleder
- Anders Waage Prosjektdeltaker
- Anders Sundan Prosjektdeltaker
- Gaute Brede Prosjektdeltaker
- Katarzyna Maria Psonka-Antonczyk Forsker
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