Reading the exosome

Once thought of as simple cellular waste disposal units, exosomes are now known to be messengers closely intertwined with a number of cellular processes. As such they can hold a wealth of information – but how can we take advantage of these vesicles to improve health? Nina Koliha and Stefan Wild from Miltenyi Biotec GmbH explain…

Communication is important – particularly between cells in multicellular organisms. During development as well as for adaption to their surroundings, cells communicate within their direct environment via cell-to-cell contact or with distant cells via messengers. Complex, new messengers – exosomes – were discovered in 1983 simultaneously by the research groups of Rose Johnstone¹ and Philip D. Stahl².

Exosomes are small vesicles about 100 times smaller than a cell, and – like a cell – are formed by a lipid membrane. They are generated and loaded inside the cell and are mainly composed of lipids, proteins, and nucleic acids. Loaded exosomes can be secreted by cells and move through the body, for example, in the blood stream to reach their target cells. Proteins on the surface of exosomes can be recognised by cellular receptors on distant cells within the body, inducing a signal similar to cell-to-cell communication. Other proteins such as enzymes or transcription factors can be taken up by the cell and act within the target cells. Likewise, genetic material encoded by nucleic acids can be transferred and can influence the cellular machinery. Compared to signalling molecules like hormones or cytokines, exosomes are complex structures and can transport a variety of information affecting different functions of the target cell. As exosomes can be secreted and taken up by virtually every cell type, one can imagine the powerful and complex communication network they enable. In addition to diverse potential functions and transported information, it will be key to know the ‘addresser’ – the exosome-secreting cell – and the addressed target cells.

Researchers have now developed a new analysis tool to simplify the analysis of proteins on the surface of exosomes³. The surface protein composition could give scientists information about the secreting cell and potentially the intended target cells. The new tool uses colour-coded beads which can be distinguished by flow cytometry instruments. Each bead type has been coupled to an antibody that recognises one specific surface protein. Exosomes carrying the respective surface protein will be bound by the respective bead type and the bound exosomes can be detected by fluorescent labelling. In the tool, 37 different antibodies are included to detect distinctive expression patterns on exosomes derived from various cell types. Most antibodies were selected to recognise surface markers assigned to specific cell types. Exosomes carrying the same cell type–specific surface marker likely derive from the respective cell type. A positive signal for one bead type indicates binding of exosomes due to the specific surface marker and provides information about the secreting cell type. In a complex mixture like blood, different exosomes will bind to different beads according to the respective surface protein. Thereby, positive signals give clues about the composition of the cells secreting exosomes into the blood stream.

[caption id="attachment_55913" align="alignnone" width="620"] Exosomes are small vesicles about 100 times smaller than a cell[/caption]

Immune cell–derived exosomes are of particular interest because they could reflect immune responses. In addition, they are known to assist and enhance the functions of their originating immune cells. Dendritic cells play an important role in presenting antigens from pathogens to activate specific T cells. Activated T cells proliferate, recognise the pathogen by this antigen, and destroy the pathogen. Besides the mature dendritic cells themselves, exosomes secreted by mature dendritic cells can activate T cells. Exosomes can also induce other antigen-presenting cells to activate T cells additionally?. In contrast, exosomes from immature dendritic cells have a contrary effect and promote tolerance. Therefore, depending on the maturation status of the dendritic cell, exosomes can indicate an immunologic challenge or induce tolerance within the body. Immunosuppressive effects were also observed for exosomes from so-called regulatory T cells. They can induce programmed cell death (apoptosis) of target immune cells and thereby attenuate expansion of activated immune cells. In mice, regulatory T cell–derived exosomes were observed to inhibit the secretion of immune-activating cytokines and the growth of other immune cells necessary to activate immune responses?.

Compared to signalling molecules like hormones or cytokines, exosomes are complex structures

Tumour cells also use the exosomal communication system to support their survival within the body. One aspect of tumour survival is the escape from anti-tumour immune responses. For this purpose, tumour cells extensively secrete exosomes that are capable of inducing apoptosis of immune cells, inhibiting the maturation of immune cells into functional members of the immune system, and down-regulating proteins on immune cells important for efficient immune responses?. Even immune cell–derived exosomes were found to be reprogrammed in favour of the tumour. In tumour-bearing mouse models, immune cell–derived exosomes promote tumour migration and accelerate tumour growth?. Other immune cell–derived exosomes prepare blood vessels for the tumour’s increased need for oxygen and nutrients?.

Tumour exosomes also serve as shuttles to spread tumour growth – promoting proteins to normal cells. Surrounding cells are thereby turned into tumour cells. Hector Peinado’s research group revealed that melanoma-derived exosomes can manipulate bone marrow progenitor cells via proteins to promote invasion, migration, and metastasis of primary tumour cells?. In the course of drug-based tumour therapy, some tumour cells develop resistance against the drug resulting in the production of drug-resistance proteins. Exosomes are able to transfer such proteins to turn drug-sensitive tumour cells into tumour cells that are resistant against the drug and survive therapy¹?. The composition of exosomes in blood could give indications about tumour progression and potential immune responses.

For diagnostics, exosomes provide important advantages. Samples can, for example, be obtained in a non-invasive fashion by simply drawing blood or taking saliva or urine samples instead of tissue biopsies. This comes along with the option to take multiple samples for monitoring over time. In addition, exosome samples from body fluids are a mixture of different exosome populations from various cell types. This is particularly advantageous in tumour diagnosis because tumours are mostly a collection of differentially mutated cells. In contrast to tissue biopsies, exosome samples more likely reflect the landscape of the heterogenous tumour and its microenvironment and therefore allow a more detailed analysis. In diagnosing prostate cancer, exosomes were superior compared to blood samples with a higher specificity leading to more reliable results. Indicative protein cargo and/or increased exosome numbers were observed for many cancer types, including melanoma, gastric, ovarian, breast, prostate and lung cancer.

Tumour exosomes also serve as shuttles to spread tumour growth – promoting proteins to normal cells

Besides the diagnostic potential of exosomes, researchers are aiming to use exosomes for therapeutic approaches. The knowledge about the versatile functions of exosomes might be the starting point to design favourable exosomes for therapy. For example, exosomes from regulatory T cells might be useful in inducing transplant tolerance. In rats, the transfer of regulatory T cell–derived exosomes prolonged the survival and the function of kidney transplants5. For the development of a tumour therapy, Laurence Zitvogel’s research group has already taken a first step: They showed exosomes from dendritic cells loaded with antigenic peptides were able to induce potent immune responses that led to the regression of established tumours in mice¹¹. Exosomes are an important tool for intercellular communication playing roles in complex processes like immune response or tumorigenesis. With improved methods to investigate the composition of exosomes researchers can shed light on the diverse addressor cells secreting exosomes. Furthermore, the exosomal cargo can give insight about potential functions in target cells. In the future, we might be able to read, even manipulate, the exosomal messages and use exosomes for diagnosis and therapies.

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Nina Koliha is a Research Assistant and PhD student at Miltenyi Biotec GmbH in Bergisch Gladbach, Germany.

Stefan Wild is a Senior Scientist in Research and Development at Miltenyi Biotec GmbH in Bergisch Gladbach, Germany.