Exosomes are nano-sized (30–150 nm) extracellular vesicles (EVs) formed by a lipid bilayer surrounding an organelle-deprived cytosol with several biomolecules, such as proteins, glycans, lipids, RNA and DNA [8,9]. These vesicles are naturally found in various body fluids, such as blood plasma and urine, and they are produced by most (if not all) cells [10]. Exosomes’ biogenesis starts with the invagination of the endosomal limiting membranes resulting in intraluminal vesicles (ILVs) inside the endosome, forming the so-called multivesicular body (MVB) [9,11]. This MVB will then fuse with the cell membrane, releasing the ILVs which are now referred to as exosomes [9]. Therefore, contrary to other types of EVs that result from direct plasma membrane budding, exosomes come from the endocytic pathway [9]. In addition to the site of the biogenesis, another feature that distinguishes exosomes from other types of EVs is the size. EVs produced by the cell from direct budding of the plasma membrane are considered to be larger than exosomes (100–1000 nm) [11,12]. Exosomes’ production is a complex process which involves several different protein machineries, namely: (1) endosomal sorting complexes required for transport (ESCRT) to regulate ILV formation; (2) tetraspanins, transmembrane proteins which enable vesicle formation by promoting membrane curvature; (3) Rab GTPase proteins for endosomal trafficking control and (4) several enzymes, such as sphingomyelinase, to produce ceramides and promote vesicle formation [9,11,12]. Interestingly, some of these protein networks and complexes are also involved in the formation of other types of EVs and, therefore, despite having distinctive sites for biogenesis and sizes, exosomes and other EVs share common protein machinery [9,12]. Importantly, the need for such a complex protein machinery for exosome formation implies that this is a heavily regulated process involving the secretion of specific substrates, which suggest a critical function for these vesicles, as discussed in the next section. Another piece of evidence of the specificity and selectivity of exosomes’ biogenesis is the heterogeneity of the exosomes’ population with different subpopulations exhibiting different molecular compositions and organ distribution, and suggesting different roles [13]. Recent studies suggest the classification of the subpopulations of exosomes as follows: Exo-Large (90–120 nm), Exo-Small (60–80 nm) and membrane-less exomeres (<50 nm) [13].

Exosomes’ lipidic composition shares similarities to that of membrane lipid rafts, and it includes ceramides, sphingolipids, cholesterol and glycerophospholipids [10,14]. The protein content of exosomes is very broad as it can contain cytosolic or nuclear proteins, transport-involved or adhesion-related and also membrane-bound proteins [10]. Some examples of this wide variety of proteins include heat shock protein (HSP70, HSP90), tetraspanins (CD9, CD63, CD81 and CD82), epithelial cell adhesion molecule (EpCAM) and proteins involved in exosomes biogenesis (ESCRT complex, ALIX, TSG101) [15]. For more specific details on exosomes composition, a manually curated web database containing information about exosomal proteins, RNAs and lipids is available at www.exocarta.org (accessed on 04 October 2021) [16].

Exosomes were initially thought to be a mechanism through which cells could eliminate unnecessary proteins [17,18]. However, the past decades were marked by a notable development in the study of exosomes and it is now demonstrated that these EVs play a critical role in intercellular communication, which is of paramount importance in the context of tumor progression and metastases [8,9,19,20,21].

Even though the mechanisms by which exosomes are taken up by recipient cells is not fully understood, several studies bring evidence of a non-random process which is dependent on transmembrane proteins [22,23]. For example, a study by Kuroda et al. recently identified possible receptors for the uptake of exosomes derived from SK-Mel-28 melanoma cells in human brain capillary endothelial cells (hCMEC/D3) [22]. This study revealed that the uptake of SK-Mel-28-derived exosomes by hCMEC/D3 cells occurs via macropinocytosis and receptor-mediated pathways, with major contribution of the presence of CD46 in hCMEC/D3 [22]. This selective uptake by recipient cells together with the heavily regulated loading process briefly described in the previous section, supports the specific and critical function of these vesicles in intercellular communication.

Recent reports have demonstrated that the successful development of brain metastases rely on a complex intercellular communication occurring between metastatic cancer cells and brain stroma cells, which involves secreted proteins or small vesicles, namely exosomes [24,25]. The existence of various mechanisms by which exosomes are capable of positively or negatively influencing brain colonization by cancer cells is evidence of an immense complex intercellular communication network, which can be considered as a target for new therapeutic approaches or as an inspiration for new drug delivery strategies. In the next sections, we will review some roles of exosomes in tumor progression with focus on intercellular communication within the tumor microenvironment (TME) and in pre-metastatic niche (PMN).