Elsevier

Journal of Proteomics

Volume 198, 30 April 2019, Pages 87-97
Journal of Proteomics

Exosome beads array for multiplexed phenotyping in cancer

https://doi.org/10.1016/j.jprot.2018.12.023Get rights and content

Abstract

Exosomes are small extracellular vesicles (EV) released from all cells that differ from others EV in their cellular origin, abundance and biogenesis. These different types of extracellular vesicles are recognized as potential markers of human diseases, including cancer and, in recent years, there has been an important advance in the molecular characterization of exosomes from different types of cancer. In particular, due to their presence and stability in most body fluids and the similarity of their content with tumor cells, exosomes have great potential as non-invasive biomarkers for liquid biopsy. Nevertheless, the use of exosomes for diagnostic purposes has been limited by the lack of reproducible methods. Flow cytometry is a technique well adapted for a reproducible analysis of clinical samples. However, conventional flow cytometers do not allow the detection of particles <300 nm based on forward scattered light (FSC), and therefore do not allow the direct detection of exosomes. To overcome this limitation, the use of microsphere bead-based flow cytometry assays is proposed, which, together with an adequate selection of markers, would contribute to making liquid biopsy based on exosomes a reality.

Significance

  • Exosome play a crucial role in cell-to-cell communication.

  • Tumor exosomes are abundant in cancer patient’s peripheral blood, also in the central nervous fluid systems which contribute to spread their function from proximal to distance tissues.

  • Exosomes are an emerging field that could be exploited such as non-invasive biomarker and delivery vehicles in cancer therapy.

  • There is an urgent need for technological advancements on exosome isolation, detection and characterization.

Introduction

New genomic and proteomic technologies make possible to establish molecular patterns that may be correlated with the etiology, type or invasive nature of a tumor, and even with response to treatment. For this reason, the individualized molecular characterization of each tumor is becoming more frequent in the clinics, facilitating prognosis and guiding treatment decisions, contributing to improve the patient's quality of life and clinical behavior. In order to perform the molecular characterization of a tumor, a biopsy from a lesion is usually necessary, which in most cases involve an invasive, frequently painful, relatively expensive procedures and causing problems in the evolution of the tumor tissue. In addition, tumors show high cellular heterogeneity and might evolve exponentially over time. Since cancer is a dynamic pathology, clinical decisions based on historical biopsy data are not useful in many cases for diagnosis and prognosis purposes. However, an alternative to overcome these difficulties relies on the analysis of molecules, vesicles and cells released by tumors to bodily fluids which could reveal much more information than a tissue biopsies information provided by tissue biopsies as an. This approach is named liquid biopsy and consists on a rapid test that is performed on a sample of blood or other body fluid, with the aim of analyzing different tumor materials such as DNA, RNA, proteins, extracellular vesicles or whole cells. Liquid biopsies have the potential to help doctors to detect and classify diseases, monitor the response to treatment and to analyze tumor mechanisms of drug resistance. When talking about liquid biopsy three specific approaches are usually referred to: circulating tumor cells (CTC), cell-free DNA (cDNA) and exosomes [1]. In this review, we will focus mainly on the EVs exosomes.

Section snippets

Exosome biogenesis

Exosomes are small (~40–150 nm) extracellular vesicles (EV) released from all cells and found in body fluids and cell culture supernatant that differ from the rest of EV regarding cellular origin, abundance and biogenesis. Exosomes are generated by fusion of a specialized endosome, known as the multivesicular body (MVB) with a plasma membrane [2]. Specifically, exosome generation takes place through inward budding of the plasma membrane to form intracellular endosomes. Additional invagination's

Exosome composition

In addition to their particular biogenesis, exosomes are unique in their molecular composition at the protein, lipid and nucleic acid levels, and said molecular content (qualitative/quantitative) being a reflection of the state of the precursor tissue or cell at the time exosome generation,

From protein point of view, exosomes include cytosolic, nuclear and plasma soluble proteins. Although the direct comparison of the proteins presented in the exosomes with the cell surface proteins from the

The role of exosomes in cancer

At first glance, exosomes was described as a garbage bag of cellular waste, disposing excess and/or non-functional cellular components, such as poorly degraded molecules by lysosomal system; nowadays, it seems proven that exosomes also function as cellular shuttles that play a crucial role in cell-to-cell communication. Then, the main function consist in the transport of bioactive molecules between cells, including proteins, metabolites, RNA (mRNA, miRNA, long non-coding RNA), DNA (mtDNA,

Exosomes in cancer liquid biopsy

One of the biggest challenges in oncology, it is the early diagnosis, for which new sensitive, specific, stable and easily sampled biomarkers are required; in addition it is also desired to avoid side effects of invasive biopsies and contribute to reduce excess treatment, psychological stress of the patients.

Bearing in mind all the aspects, the exosomes have a great potential to serve as a liquid biopsy tool in these diseases. In particular, tumoral exosomes most likely are useful as biomarkers

Cytometry beads assays for exosome analysis and characterization

The starting volume of sample has a considerable effect on the tehcniques used for isolation and detection, nowadays most researchers (87%) process between 1 and 50 samples per month while only a 4% process >100 samples per month. On the other hand, 71% of these same researchers process between 5 and 100 ml of starting sample volume, while the other 29% work with sample volumes <5 ml. In fact, 100% of researchers who work with biological fluids process sample volumes lower than 1 ml, except for

Bead arrays for exosome phenotyping

In a typical discovery-oriented experiment, a biological sample is analyzed by separating, quantifying and identifying as many exosomes as possible, often with emphasis on those exosomes with altered abundance relative to a reference sample. Although microarray technology has undoubtedly an important role in discovery-oriented proteomics, it seems evident that it will also be particularly suitable for EV research.

In particular, the phenotype of exosomes is particularly important in the

Conclusions and perspectives

In summary, there has been an important advance in the molecular characterization of circulating tumor-derived exosomes which have been recognized as a promising source of biomarkers for cancer diagnosis by less invasive procedures such as liquid biopsy.

However, the clinical use of exosomes has been limited by the lack of adequate methods for sensitive detection of exosomes on body fluids and by the absence of specific markers of the different subpopulations of exosomes present in patient

Acknowledgments

We gratefully acknowledge financial support from the Spanish Health Institute Carlos III (ISCIII) for the grants: FIS PI17/01930 and CB16/12/00400. Fundación Solórzano FS/38-2017. The Proteomics Unit belongs to ProteoRed, PRB3-ISCIII, supported by grant PT17/0019/0023, of the PE I + D + I 2017-2020, funded by ISCIII and FEDER.

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