Licensed in 5/17/2013,  Solid Tumor Research Center (STRC), is currently located at Urmia University of Medical Sciences, Shafa St., Ershad Blv, Urmia, West Azarbaijan, Iran. STRC, as a scientific research centre, is planned to develop research on solid tumors and applied research which could result in understanding cancer-related issues including risk factors,  diagnosis, improvement, control, treatment and also enhancement of the quality of cancer patients life. In line with Iran's 20-year vision plan and macroeconomic goals, and also according to the ministry and university goals, STRC seeks to become an active and supportive research centre with several cancer-related research groups.

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Scentific comments:

     I: Extracellular Vesicles:

            In the past decade, EVs are known to mediate intercellular communication in various cell types [5]. EVs represent heterogeneous populations of very small, bi-phospholipid membranous particles of cells with ability to carry bioactive molecules such as protein, lipid, mRNA, miRNA. Even, it was proved that small DNA sequences are also released by almost eukaryotic cells [4, 20]. Three major subpopulations of EVs are generally identified based on the origin, way of generation and size: (i) exosomes, (ii) MVs, and (iii) apoptotic bodies (ABs) [20, 21] . Exosomes belong to a large family of membrane vesicles referred to as extracellular vesicles. First, exosomes were discovered in 1983 by two independent work groups directed by Harding and Pan [22, 23].

In 1983, Johnstone et al. termed these vesicles as “exosomes” [24]. Exosomes, nano-sized (30-120 nm) membranous vesicles, are directly originated via intercellular regulated process [25]. These small particles, generated from endosomal compartment, multivesicular body (MVB), are routinely located in the cytoplasm. It is clear that the invagination of MVB membrane results in the formation of intraluminal vesicles (ILVs) in MVBs lumen. MVBs could selectively fuse with a lysosome to degrade their contents. Alternatively, MVBs may fuse with the plasma membrane and release ILVs contents to extracellular space as exosome for the purposes of intercellular transport and signaling [26, 27].

Scientists believe that MVB membrane composition determines the fate of MVBs. For abscission of MVs, Rab-GTPase family mediates intracellular trafficking and docking of MVB to plasma membrane [28]. Most experimental evidence suggests that many molecules contribute to exosome biogenesis and cargo sorting. For instance,  endosomal sorting complex required for transport (ESCRT) machine is complexes of diverse proteins that located on the outer side of the MVB membrane are involved in the sorting of ubiquitinated proteins into exosomes [29]. In the ESCRT independent mechanism, other tetraspanins such as CD63, CD81, and CD82, abundantly founded on exosome membrane, mediate exosome biogenesis. Observations confirmed the involvement of sphingomyelinase, protein kinase D family, trinucleotide repeat-containing gene 6A protein (TNRC6A), and Argonaute-2 (AGO2) in exosome biogenesis and cargo packaging [30, 31]. The common molecular markers are tetraspanins, CD9, CD63, CD81, CD82, CD151, intercellular adhesion molecule-1 (ICAM1), CD51, CD61, Alix, milk fat globule-E8/lactoferrin, CD80, CD86, CD96, Rab5b, and major histocompatibility complex (MHC) class, I and II [32, 33]. Various cells including non-stem/stem cells have capability to release exosomes into the surrounding milieu.

These nanovesicles have both favorable physiological rules and even harmful pathological roles in biological processes. Using transmission electron microscopy analysis, exosomes are represented as cup-shaped vesicles with a diameter between ∼30–120 nm, but the spherical appearance of exosomes will be achieved when cryoEM technique is used, showing the real-state exosomes morphology [34]. MVs have a broad size range of 100-1000 nm in diameter, a heterogeneous subpopulation of EVs, generating through outward budding and abscission of membrane vesicles from the cell plasma membrane. This mechanism is similar to the abscission step in cytokinesis [5]. MV biogenesis also shares resemblances with the mechanism of virus outward protrusion from cells. Many cell types, especially ECs, platelets, and erythrocytes release MVs.

It is believed that MVs appear to be formed and released in response to stimuli, but exosomes could be produced constitutively in each condition [35, 36]. A great deal of research on MVs show some of these irregular bags actively bind to annexin V, suggesting an enrichment of MVs membrane with phosphatidylserine.By monitoring phosphatidylethanolamine-specific peptide (i.e. duramycin), Larson and colleagues declared that some MVs have not capacity to bind to Annexin V or lactadherin and some MVs filled with this phospholipid resident (ref). Growing evidence show MVs along with exosomes participate in various cells signaling process. These particles transfer a variety of bioactive molecules including proteins, lipids, and acids nucleic through regulated mechanism [37].

MVs composition was specifically altered in response to certain stimuli for example, in prothrombotic conditions, platelets produce large-sized MVs transfer such factors that stimulate the endothelial barrier function. After the development of thrombus, platelet-derived MVs primarily were enriched with factors in the favor of elimination of thrombogenesis [38]. These observations support the idea that MVs represent a heterogeneous population of vesicles in size and content. Identification and isolation of MVs certain subpopulation are of great importance for studies on EVs biology and function [5, 38]. Apoptotic bodies (ABs), the largest EVs, are released through outward blebbing and fragmentations of the plasma membrane of cells undergoing apoptosis. Rho-associated kinase 1 (ROCK1) is actively recruited to the formation of ABs in which fragments ranging between 1 and 5 µM are released as heterogeneous particles.

Studies on ABs formation mechanisms support the potential role of  caspase-3 in activating of ROCK1 that, in turn, phosphorylates myosin light chain (MLC) and further promotes membrane blebbing [39]. It has previously been shown that phosphorylation of MLC and following ATPase activity of this chain result in the actin-myosin cytoskeletal interaction that interrupts nuclear integrity. This action, in turn, causes chromosomal DNA fragmentation and packaging of DNA fragments into ABs blebs [40]. These particles may harbor whole organelles and nuclear fragments including fragmented DNA and histones [41].

As ABs encompass proteins, DNA fragments, and microRNAs, however, they could facilitate intercellular communication via transporting of these bioactive agents and may contribute to the progress of several diseases[42, 43]. For example, a work by Berda-Haddad and coworkers in 2011 declared that endothelial cell-derived ABs enriched with the cytokine IL-1α can actively promote chemokine release and cause sterile inflammation [43].Moreover, it has been well-established that ABs plays a key role in stimulating phagocytosis through the presentation of signals; therefore inhibit induction of secondary necrosis [44].

Exsosome biogenesis through ESCRT-dependent and ESCRT independent mechanism; Key roles of Rab proteins  

Routes of EVs Uptake

                  Once EVs released into the ECM, they can potentially deliver their cargo to target cells. Involvement of possible mechanisms in EVs uptake route was early described in the scientific literature [45, 46]. Due to heterogeneity in EVs population and target cells properties, three possible mechanisms may be exploited by the EVs; (i) internalization; (ii) direct fusion; (iii)  receptor-ligand interaction [45, 47]. In the internalization pathway, EVs were taken up by cells via a variety of endocytic routes, including clathrin-dependent endocytosis, caveolin-mediated uptake, macropinocytosis, phagocytosis, and lipid raft-mediated internalization [48].

              Another promising entry mechanism is direct fusion, in which lipid bilayers actively merge and then EVs cargo was released into the cytoplasmic part of target cells [49]. Similarly to most membrane fusion process, in this pathway, membrane fusion is controlled by a variety of proteins such as Rab GTPase proteins, SNAREs, and Sec1/Munc-18 related proteins (SM-proteins) [50]. Finally, by using ligand-receptor interaction, EVs could activate intracellular signaling without internalization. In this route, various ligands and adhesion molecules participate directly or indirectly in the binding of EVs to target cells. For example, mature DCs derived EVs bearing on their surface the intercellular adhesion molecule 1 (ICAM1) that bind to the lymphocyte function-associated antigen 1 receptor(LFA1-R) localized on the PM of antigen presenting cells (APCs)[51] or stimulated T-cells [52]. As a matter of fact, understanding of mechanisms involved in EVs uptake is a pivotal key in design EVs related delivery systems and discovery of intercellular signaling.  

                                                    Exosome biogenesis, trafficking  and uptake.  J.Rezaie et al., 2018

Bio-application of exosomes

             Exosomes are starting to gather attention in cancer therapeutics and diagnostics, with several applications in drug delivery, tumor immunotherapy, and diagnostic biomarkers. Their unique strengths include enhanced passive targeting due to small size, indigenous nature, and the ability to cross biological barriers. However, the cumbersome nature of the methods required for isolation/purification, inability to distinguish between different cancer stages, and incomplete understanding of their impact on the immune system are some of the current limitations with this technology. It is anticipated that sophisticated engineering and detailed clinical studies that address these limitations will lead to the translation of exosome‐based.

By: Dr.J.Rezaie et al 2018 .   11/28/2018,      12:31:42

Ref: Rezaie, J., et al., Exosomes and their application in biomedical field: difficulties and advantages. Molecular neurobiology, 2018. 55(4): p. 3372-3393.

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Ref: doi:  [10.1016/j.biocel.2012.08.022] .

Provided by Dr. J.Rezaie