Exosomes in Tumor Angiogenesis
Exosomes are small vesicles ranging in size between 30 and 150 nm, derived from the luminal membranes of the endosome and are constitutively released by fusion with the cell membrane. Several studies have revealed that exosomes play crucial roles in mediating local and systemic cell communication through the horizontal transfer of information in the form of nucleic material and proteins. This is particularly relevant in the context of the tumor-microenvironment cross talk. Here we describe the method of isolating exosomes and their role in modifying the tumor environment and more specifically in enabling metastasis and promoting angiogenesis.
Key wordsExosome Angiogenesis Cancer Tumor Microenvironment Pre-metastatic niche
Exosomes have recently come to be recognized as critical components of intercellular communication capable of producing local and systemic changes. They are defined as small vesicles ranging in size between 30 and 150 nm and are released by all cells and particularly tumor cells. These vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. In fact, the functional impact of exosomes is imparted by the molecular components they carry—including protein and nucleic acids such as micro RNAs. At present, exosome-mediated processes have been implicated in the pathobiology of both solid tumors and hematologic malignancies. These microvesicles play an important role in angiogenesis  and in promoting tumorigenesis in many cancer types, in particular through the transfer of miRNAs, mRNA, and proteins [2, 3, 4]. The function of exosomes in tumor progression can be explained in part by the capacity of tumor-derived exosomes to modulate the host microenvironment—in a distant site—and produce conditions favorable for the proliferation and spread of neoplastic cells [5, 6, 7, 8]. These findings have raised the concept of the pre-metastatic niche, in which exosome-induced neoangiogenesis plays a central role.
In spite of the advances in elucidating the role of exosomes in cancer, there is an experimental challenge in studying these nanovesicles because of their small size and the difficulty in obtaining high yields of these vesicles from biological fluids such as plasma. Cell culture media and physiologic fluids are known to contain several types of shed membrane fragments and vesicles. For this reason, it is imperative to ensure that the purified vesicles are indeed exosomes and not other contaminating material before performing any functional analysis.
Here we will develop the different methods to isolate exosomes and describe their role in inducing tumor progression and metastasis by inducing neoangiogenesis.
2 Methods of Exosome Isolation
One of the challenges involved in exosome research is a lack of standard methodologies evaluating the usability, vesicle purity, and yield from cell-culture-conditioned media and biological fluids. Currently, the gold standard in the purification of exosomes is a process of differential centrifugation, consisting of low speed centrifugation to remove cells and debris followed by high speed centrifugation to pellet the exosomes The main disadvantage of this process is that it can be cumbersome when dealing with large volumes and can also negatively impact the yield and quality of isolated exosomes. Alternatives to ultracentrifugation are process involving ultrafiltration, or exosome isolation reagents.
Characterizing and assessing the purity of the isolated exosomes is also imperative before performing any functional analysis to ensure that they are not contaminated with other vesicles.
For exosomes from cell culture supernatants, FBS used in cell culture should be precleared of exosomes by ultracentrifugation at 100,000 × g for 3 h at 4 °C to remove any preexisting bovine-derived exosomes.
The ultracentrifugation process consists of several differential centrifugations. The first few centrifugation steps are designed to remove dead cells and large debris. At each of these centrifugation steps, the pellet is discarded and the supernatant is spun down again. The final supernatant is then ultracentrifuged at 100,000 × g to pellet the exosomes. The pellet is then washed in a large volume of PBS to remove any contaminating proteins or other vesicles and centrifuged one last time at the same high speed .
An alternative method is the ultrafiltration process in which supernatant fractions of cell-cultured media are filtered on 0.2 μM pore filters to remove contaminating apoptotic bodies, microvesicles, and cell debris. The resulting filtrate is then ultracentrifuged at 20,000 × g for 20 min. Exosomes are then harvested by ultracentrifugation at 100,000 × g for 70 min.
Other options include employing exosome isolation reagents such as ExoQuick (System Bioscience), Exosome Isolation Reagent (Life Technologies) or Exo-spin (Cell Guidance System). In this case, the biofluid is collected and centrifuged at 3000 × g for 15 min to remove cell debris. The supernatant is then transferred to a new sterile tube and the exosome isolation reagent is added to precipitate exosomes. At this point, the exosomes should appear as a beige/white pellet at the bottom of the tube. The use of exosome isolation reagent is more controversial as the potential for contamination by other vesicles is likely increased.
Once the isolation of exosomes is complete, the next step consists of the identification of isolated vesicles as exosomes and requires morphological analysis. Due to their small size, the most effective means of characterizing and visualizing exosomes is by electron microscopy combined with immunogold labeling of exosome-specific markers such as CD81 and CD63. Other methods to identify exosome-specific markers such as flow cytometry (requiring bead-conjugated antibodies) and western blot can be used. Characterizing the size of the vesicles by NanoSight is also helpful as it indicates the potential contamination by other vesicles.
3 Exosomes Are Involved in the Metastatic Process
Tumorigenicity and metastatic spread is instigated by cancer initiating cells that require a niche at the distant target site referred to as the pre-metastatic niche. Evidence suggests that these cancer-initiating cells prepare their homing bed before arrival, implying the involvement of soluble mediators in this process. Today, a large variety of contributing factors have been identified as being involved in preparing the pre-metastatic niche with tumor-derived exosomes being heralded as the driving force behind this process. Exosomes carry mRNA, miRNA, and proteins that are function-competent. These microvesicles spread throughout the body but bind to and get taken up by only select target cells. The process of binding and uptake can severely affect the target cells. It initiates activation of signal transduction and transferred RNA and proteins then account for gene silencing as well as over-expression of specific mRNA or proteins. Rapid progress in understanding the early role of exosome in metastasis has led to new hopes that we may be able to prevent metastatic spread by interfering with the pre-metastatic niche.
Mounting evidence suggests the central role of tumor-derived exosomes in modulating the distant tumor microenvironment. Melanoma-derived exosomes injected in naïve mice have been shown to induce neoangiogenesis at pre-metastatic niche sites before the presence of any tumor cells . In this process, melanoma tumor cells with high expression of the RAB family members were more prone to metastasize. The RAB proteins are regulators of membrane trafficking and exosome formation and their expression correlates with the secretion of exosomes. In this study, RAB27A RNA interference decreased exosome production, preventing bone marrow pre-metastatic education and reducing tumor growth and metastasis, suggesting a central role for exosomes in the pre-metastatic process.
In the context of multiple myeloma, it has been recently reported that the protein content of exosomes isolated from myeloma bone marrow stromal cells was enriched for oncogenic proteins, cytokines, and kinases, including IL-6, CCL2, and fibronectin . This supports the notion that bone marrow stromal cells support tumorigenesis not only through paracrine mechanisms like growth factors [10, 11] but also through direct transfer of microRNAs and proteins from exosomes thus creating a favorable niche for the expansion of the malignant clone.
A recent study by Hoshino et al. demonstrated that various different types of tumor-derived exosomes fuse preferentially with resident cells at their predicted destination . This concept of metastatic organotropism argues that exosomes taken up by organ-specific cells prepare and prime the pre-metastatic niche. Proteomic analysis of exosomes revealed distinct integrin expression patterns perhaps indicating the predilection for exosomes to target specific cells types or organs. Integrins α6β4 and α6β1 were associated with lung metastasis while exosomal integrins αvβ5 was linked to liver metastasis. Targeting these integrins in turn decreased exosome uptake as well as lung and liver metastasis.
Studies investigating pancreatic ductal adenocarcinoma (PDAC) have also demonstrated that intercellular communication is critical for metastatic progression in this aggressive cancer . In fact, PDAC-derived exosomes were shown to induce liver pre-metastatic niche formation in mice and consequently increase liver metastatic burden. Uptake of these tumor-derived exosomes by the liver resulted in TGF-β secretion and upregulation of fibronectin production. This resulting fibrotic microenvironment then enhanced the recruitment of bone marrow derived macrophages. It was also noted that macrophage migration inhibitory factor (MIF) was highly expressed in tumor-derived exosomes and its blockade prevented the formation of this liver pre-metastatic niche and thus inhibited metastasis. Additionally, patients with pancreatic tumors that did not progress had markedly lower MIF in their exosomes than patients who later developed liver metastasis. These results suggest that exosomal MIF does indeed prime the liver in preparation for metastasis and may have potential prognostic and therapeutic implications in preventing the progression of this cancer.
In addition to the role of exosomes in promoting tumormetastasis, their function and mechanism of action has been shown to extend to enhancing angiogenesis in the microenvironment. In fact, recent evidence suggests that exosomes’ capability to induce angiogenesis in the pre-metastatic niche plays a central role in the metastatic process.
4 Angiogenesis in Cancer
Angiogenesis, the growth of new blood vessels from pre-existing ones, is an important process in the pathogenesis of malignant, infectious, fibro-proliferative, and inflammatory diseases . This process is controlled by multiple growth factors and signaling pathways, and critically depends upon the tight balance of pro-angiogenic and antiangiogenic factors .
It is widely accepted that acquiring angiogenic capacities represents a fundamental hallmark leading pre-cancerous cells to become cancerous. After neoplastic transformation the net balance between pro-angiogenic and anti-angiogenic molecules in the tumor tissue is tipped to favor angiogenesis, the so called ‘angiogenic switch’ . The vascular endothelial growth factor, VEGF, is thought to be the most important as VEGF acts pro-angiogenic by augmenting all steps of angiogenesis: vascular permeability, endothelial cell proliferation, endothelial cell migration or invasion into the surrounding tissue, and finally capillary-like tube formation . Various inhibitors interfering with VEGF/VEGFR have received FDA approval and are currently approved in clinical use . Many other growth factors and pathways have also been implicated in the formation of cancer associated vessels.
The family of fibroblast growth factors (FGF) is involved in neurogenesis, embryonic and post-natal organ development, branching morphogenesis, angiogenesis, and in the pathogenesis of cancer. FGF-2 by binding to its receptor FGFR-1, is thought to be the major family member mediating neongiogenesis . The platelet-derived growth factor (PDGF) is frequently upregulated in tumors and has been shown to cooperate with FGF-2 to promote tumor angiogenesis and metastasis development . Delta-like ligand 4 (Dll4) is a member of the Delta/Jagged family of transmembrane ligands binding to Notch receptor [15, 16]. The delta-Notch pathway regulates the artery–vein differentiation but it seems to be also importantly implicated in the stimulation of blood vessel formation. Its major role is to mediate cell–cell communication and controls of cell determination thus playing a pivotal role in vascular development. The role of Angiopoietin 2 in angiogenesis is generally considered as an antagonist for Ang1, thereby inhibiting Ang1-promoted Tie2 signaling, which is critical for blood vessel maturation and stabilization . However the role of Ang1/Ang2/Tie2 pathways in tumor-associated angiogenesis remain controversial. While some studies have shown that upregulation of Ang2 correlates with tumor growth and angiogenesis development of various types of cancers, other studies have reported that specific induction of Ang2 in gliomas, mammary carcinomas, and lung carcinomas inhibited tumor growth and metastasis .
Apart from these growth factors and signaling pathways many other subcellular systems also contribute to the process of neovessel formation.
Proteolytic degradation of matrix proteins is essential for transmigration of activated endothelial cells through the basal membrane into surrounding matrix . In this context, the plasminogen system, the matrix-metalloproteinase (MMP) system, as well as the heparanase and chymase families are thought to be important . For example, a large body of in vitro and in vivo data has shown an important role of the urokinase (uPA)/plasminogen system in angiogenesis and cancer. Moreover blood vessel formation critically depends on extracellular signaling as well as the connection with the extracellular matrix (ECM) proteins, such as fibronectin, fibrinogen, vitronectin, and laminin as well as collagens . The attachment to the ECM is adjusted by integrin adhesion receptors ; integrins operate as bidirectional transducer molecules by matching signals from both, the outside to the inside of the cell, or from the inside to the outside of the cell. Both signaling directions are tightly regulated in focal adhesion to support cell adhesion, spreading, and motility of endothelial cells. Both endothelial cell integrins and ECM proteins have been implicated in cancer associated angiogenesis and may represent important therapeutic targets .
Integrins have to interact with several kinases as well as related adaptor proteins since they have no intrinsic kinase activity. Integrins can activate Focal Adhesion Kinase (FAK) signaling thereby communicating signals that can induce cell migration or cell proliferation . FAK activation then leads to activation of src family kinases which control not only the activity of Rho GTPases but also downstream kinases such as AKT, thereby affecting endothelial cell proliferation, migration, and survival . All these downstream kinases may represent important therapeutic targets for the development of new antiangiogenic drugs. Finally, tumor microenvironment, which consists of several cytotypes including fibroblasts, pericytes, mesenchymal-stem cells, and inflammatory-immune cells, also contributes to angiogenesis via secretion of angiogenic molecules and cell-to-cell interactions [15, 23].
Given the molecular and biological complexity of angiogenesis in cancer, the understanding of how cancer and non cancer derived exosomes participate in this process represents an important challenge that can open new paths for the development of novel and effective anticancer drugs able to inhibit the neoangiogenic process during cancer development.
5 Exosomes in Cancer-Associated Angiogenesis
Numerous studies have identified that exosomes can be released from various cell types: dendritic cells, B lymphocytes, stem cells, mesenchymal stem cells, tumor cell lines, platelets, cardiomyocytes, endothelial cells have all been implicated in the release of exosomes which in turn then mediate important biological functions mediating distant cell-to-cell interactions .
Endothelial cells can release different types of membrane vesicles, including microvesicles, exosomes, and apoptotic bodies, in response to cellular activation or apoptosis . Defining the specific biological functions mediated by the specific types of vesicles remains an important methodological challenge, as previously discussed. Data reported in literature needs to be carefully interpreted after taking into consideration methodological aspects.
It has been shown that endothelial exosomes might be involved in vascular development through incorporation and transfer of Delta-like ligand 4 (Dll4; Delta 4) protein to neighboring endothelial cells. This leads to an inhibition of Notch signaling and an increased capillary-like structure formation in vitro and in vivo . This suggests that the Delta like ligand/Notch pathway may not require the direct cell–cell contact and that exosome release by endothelial cells may work in the place of cell-to-cell contact thus expanding the range of cell signaling potential for angiogenesis regulation. Other studies have also shown that endothelial-derived exosomes contain proteins, microRNA, and mRNA with pro-angiogenic potential. For example, it has been reported that matrix metalloproteinases harbored by exosomes from endothelial cells are functionally active and can mediate endothelial cell invasion and capillary-like formation . All together it seems that endothelial cell-derived exosomes containing proteins and mRNAs/microRNAs which may function as paracrine or autocrine factors thus having the ability to facilitate cancer associated angiogenesis and metastasis [24, 27]. For example, it has been shown that exosomes from LAMA84 chronic myeloid leukemia (CML) cells affect vascular remodeling in vitro through an IL-8 mediated activation of VCAM-1 . Umezu et al.  have found that miRNA-enclosed exosomes have a critical role in mediating leukemia cell-to-endothelial cell communication. Exosomes, collected from miR-92a-overexpressing leukemia cells (K562 cells), did enter into endothelial cells, resulting in increased endothelial cell migration and tube formation.
Glioblastoma cell-derived exosomes have been shown to interact with endothelial cells and thereby stimulate endothelial cell proliferation [30, 31]. In this same disease it has been observed that exosomes derived from tumor cells grown in hypoxia as compared to those derived by cells grown in normoxic conditions significantly stimulated angiogenesis .
King et al.  reported that hypoxia promoted the release of exosomes from breast cancer cells, and the hypoxically regulated miR-210 was presented at elevated levels in hypoxic exosomes.
Cancer cells can indirectly stimulate angiogenesis via platlets-activation and subsequent release of platelet derived endothelial-stimulating exosomes. For example, Janowska-Wieczorek et al.  have shown that exosomes released from human platelet α-granules could contribute to tumormetastasis and angiogenesis. Finally, cancer cells can also stimulate the release of pro-angiogenic exosomes from cells of the tumor microenvironment, i.e., fibroblasts, macrophages, other immune cells, and mesenchymal stem cells (ref. 24 for review). In summary this evidence supports the hypothesis that exosomes contribute to cancer angiogenesis via several mechanisms and may represent a target for future anti-angiogenic drugs. However studies in this field are still limited so far.
Exosomes are small nanovesicles secreted by all cell types in the body, especially tumor cells, and are involved in cell-to-cell communications. They represent attractive biomarkers in cancer as they are accessible in the peripheral blood and represent a mirror of the tumor cell’s content. Functionally, recent evidence has shown the primordial role of exosomes in tumor metastasis. In fact, exosomes are involved in priming the distant niche before the arrival of the first metastatic cell, thus initiating what is referred to as the pre-metastatic niche. Tumor-derived exosomes release a specific content of miRNA, mRNA, and proteins in recipient cells, therefore modifying their properties. This phenomenon leads to angiogenesis that promotes tumor progression and metastasis. The knowledge of these mechanisms could lead to therapeutic advances, especially in regards to the use of exosomes to deliver drugs or small interfering RNA to specific targeted cells.
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