Exosome beads array for multiplexed phenotyping in cancer
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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