**3.1. Biological roles of EVs**

agglutinating agent as well as two centrifugation steps. Exoquick has been claimed to produce the highest concentration of EVs when compared to differential centrifugation or salting out

**Figure 2.** Summary of procedures for isolation of EVs from biological fluids or cell cultures.

Currently, there is no standard isolation protocol for clearly discriminating the different classes of EVs whether by size, density, morphology of the particles or molecular markers [2]. Various procedures have been described in an attempt to separate them. Among the different groups of EVs, the isolation of exosomes is the one most frequently reported in the literature [13]. Separation of exosomes from MVs usually involves a combination of low-speed differential centrifugation steps followed by sucrose gradient ultracentrifugation [21]. Apoptotic bodies can be collected at low-speed centrifugation of approximately 2,000 g. Microvesicles need a higher speed ranging from 10,000 to 20,000 g. Exosomes are pelleted by ultracentrifugation above 100,000 g for 1 hour or more [12]. Alternatively, immune selection of MVs can be performed instead of the differential centrifugation step. This involves the adherence of MVs to magnetic beads bearing antibodies against tumour-associated markers found on the surface of MVs. Ultracentrifugation would still be needed following this immunoselection in order to recover the exosomes in the eluate from the magnetic beads. Apoptotic bodies, on the other hand, can be separated from exosomes by flotation on a continuous sucrose gradient. Separa‐

methods [14].

88 Tumor Metastasis

EVs that are derived from healthy cells transfer signals to other cells, which are needed to maintain their physiological homeostasis and biological functions such as growth, differen‐ tiation and apoptotic death. They exert their effects through multiple pathways, directly activating cell surface receptors through bioactive lipid ligands and proteins, integrating their membrane contents into the recipient cell plasma membrane and delivering effectors, such as transcription factors, oncogenes, small and large non-coding regulatory RNAs, mRNAs and infectious particles into recipient cells. Consequently, EVs contribute to the maintenance of normal physiology [12].

The following are some examples of the role of EVs in maintaining a wide range of cellular and biological functions:

I. Regulation of immune responses.

EVs might trigger adaptive immune responses or suppress inflammation in a tolerogenic manner [13]. They have been shown to implement immune suppression by several mecha‐ nisms, such as enhancing the function of regulatory T cells, suppressing natural killer (NK cells, and inhibiting monocyte differentiation [12].

II. The nervous system.

The secretion of EVs can contribute to a range of neurobiological functions. For example, increased release of EVs containing neurotransmitter receptors from cortical neurons follow‐ ing enhanced glutamatergic activity [14].
