The cellular secretion of microvesicles represents a remarkable system for short and long range cell-to-cell communication. Tumor cells secrete large amounts of microvesicles containing proteins, mRNA, and miRNAs that can be delivered to other tumor and nontumor cells such as stromal, endothelial, inflammatory, and immune cells. This review will summarize the recent findings on the role of microvesicles in reprogramming tumor microenvironment and in the organization of the premetastatic niche. The potential role of the molecules within microvesicles in tumor diagnosis and treatment will also be addressed.
DIVERSITY OF CELL-DERIVED MICROVESICLES
Genes encoding the secreted proteins represent about 10% of the human genome, comprising multiple components of molecular networks interconnecting cells within their extracellular environment. Deregulation of protein secretion and of cell–microenvironment interactions underlies many pathologies including cancer . Protein secretion occurs through the conventional endoplasmic reticulum (ER)–Golgi secretory pathway or through the unconventional secretion pathways mediated by vesicular and nonvesicular translocation of proteins through the cell membrane. Vesicular export leads to the release of heterogeneous populations of stable, secreted vesicles. Initially identified as a mechanism by which cells could dispose of damaged or unnecessary proteins (as for example during reticulocyte maturation) , cell-derived microvesicles are now seen as signaling particles that contain lipids, proteins, and nucleic acids which carry molecular information both locally and at distant sites, shaping cell microenvironment during development and cancer progression [3▪▪,4,5,6▪▪,7,8].
Microvesicles recovered from cells or from body fluids are very heterogeneous in size (30 to over 200 nm) and composition [3▪▪]. A specific population of microvesicles is formed from the endosomal system resulting in multivesicular bodies (MVBs). These microvesicles are known as exosomes, classically defined as homogeneous membranous vesicles lined by a lipid bilayer, sized between 30 and 100 nm, and containing specific sets of proteins, lipids, and nucleic acids (including microRNAs) . The biogenesis of exosomes and the sorting of proteins in these microvesicles is not totally understood. It involves the endosomal sorting complexes required for transport (ESCRT), which are required for the recognition and sorting of ubiquitinated proteins into MVBs [9,10]. Syndecan heparan sulfate proteoglycans control the formation of exosomes through the ESCRT machinery [11▪]. Protein sorting is also associated with lipid rafts domains of the exosomal membrane , whereas ceramide triggers the budding of exosomes into MVBs . MVBs fuse with plasma membrane and release exosomes to the extracellular space in a process dependent on the activity of Rab27A and Rab27B GTPases . Another pathway for microvesicles biogenesis involves the budding of cytoplasmic protrusions directly from plasma membrane followed by their release in the extracellular space. These vesicles, known as shedding vesicles or ectosomes, are heterogeneous in their molecular composition and size (30–200 nm diameter) . A subgroup of budding microvesicles is driven by interaction between Tsg101 and ARRDC1 (arrestin domain-containing protein 1) proteins, resulting in the relocation of Tsg101 from endosomes to plasma membrane and the release of microvesicles containing Tsg101, ARRDC1, and other cellular proteins [16▪]. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack late endosomal markers.
Given their overlapping physicochemical properties, exosomes and other types of shedding vesicles are not well distinguished by most purification methods. Thus, there is confusion in naming secreted vesicles, some authors use the term ‘exosome’ according to the stringent criteria, whereas others use the terms ‘exosome’ and ‘microvesicle’ interchangeably. Further terminology problems arise from the confusion with apoptotic blebs, which are larger types of vesicles (50–5000 nm diameter) released by dying cells. Given the rapid development of the field, these definitions are not firmly established and may further evolve into subcategories in the coming years.
In this review, we use the term ‘microvesicle’ as generic denomination for secreted vesicles and, whenever possible, we identify ‘exosomes’ as a specific entity based on the stringent criteria summarized above. However, as many reports do not provide information on the nature of the vesicles, our naming policy is to retain the term employed in the original report. We focus on tumor-cell-derived microvesicles (TCMVs) and we briefly discuss their biogenesis, composition, signaling roles, and significance as biomarkers.
MOLECULAR COMPOSITION OF MICROVESICLES AND EXOSOMES
Because of their selective mechanisms of production, microvesicles are composed of selected types of protein, lipids, and nucleic acids. The Exocarta website maintains a database of identified components of exosomes (http://www.exocarta.org/). As per end of August 2012, this database has compiled data from 146 studies containing annotations on 4563 proteins, 1639 mRNA, 764 miRNA, and 194 entries on lipids [17▪]. Exosomes are enriched in proteins involved in cellular transport and membrane fusion (flotilins and annexins), in MVB biogenesis (Tsg101 and Allix), in integrins and tetraspanins (CD9, CD63, CD81, and CD82), in heat shock proteins (Hsp70 and Hsp90), in posttranslational modifier proteins (kinases, phosphatases, and glycosyltransferases), and in lipid rafts, cholesterol, sphingolipids, and ceramides [3▪▪,17▪]. The lipid composition of exosomes is primordial for fusogenic properties and cellular signaling in the modified pH environment of cancer cells [18,19].
Microvesicles contain a wide contingent of functional RNA including mRNA and noncoding small RNA (miRNA) . Functional mRNAs can transfer to target cells where they are translated into proteins that modulate cellular signaling, such as transcriptional regulators, protein kinases, or metabolic enzymes [21–23]. A large number of mRNAs encoding cell-cycle-related proteins have been identified in blood-circulating microvesicles from patients with colorectal cancer, which can be transferred to endothelial cells and promote their proliferation in vitro . Similarly, many of the identified miRNAs either enhance or inhibit signaling pathways relevant for cancer development and progression, targeting a wide range of factors that includes Ras, Wnt, Myc, Bcl2, mTor, Sirt1, VEGF2, FGF1, β1 integrin, TGFβ, Notch, EGF, p53, and many components of their signaling pathways (for comprehensive list of esmiRNA, see http://www.exocarta.org).
TUMOR-CELL-DERIVED MICROVESICLES AS PLATFORMS FOR INTERCELLULAR SIGNALING
Microvesicles contain a diversity of proteins on their outer surface that are organized within specific lipid compartments (lipid rafts) and their specific interaction with cellular membrane proteins can modulate cellular signaling. In addition, intravesicular molecules, in particular miRNA, can transfer to target cells and modulate their protein content, which also interferes with cellular signaling. These structural and molecular characteristics of microvesicles confer them the capacity to function as platforms for highly efficient intercellular signaling (Fig. 1). For example, exosomes are responsible for the long-range signaling through Wnt proteins, a class of hydrophobic growth factors regulating beta-catenin-dependent gene expression. During development, Wnt proteins exert their function over a distance to induce patterning and cell differentiation decisions. A recent study has shown that Wnts are secreted on exosomes both during Drosophila development and in human cells, and induce Wnt signaling activity in target cells [25▪▪].
TCMVs (sometimes called ‘oncosomes’) can be isolated from bodily fluids such as plasma, saliva, or urine of cancer patients. TCMVs can induce tumor-specific immunity reverting tumor development, thus representing the potential source for therapeutic tumor vaccination . On the other hand, TCMVs can modulate tumor escape by generating an immunosuppressive microenvironment permissive for tumor growth, invasion, and metastasis [27,28]. These effects imply a signaling effect of TCMVs toward cells involved in processes such as cell adhesion, proliferation, survival, invasion, inflammation, innate or adaptative immune response, blood coagulation, education to a prometastatic behavior, vascular leakiness at premetastatic sites, thrombosis, and angiogenesis (Table 1 [29–44,45▪▪,46]).
TCMVs modulate the interactions between cell surface and extracellular matrix (ECM) components. TCMVs from ovarian or breast increase the secretion of matrix remodeling metalloproteases (MPP2 and MPP9) and urokinase activator plasminogen (uPA) [29,30]. Detachment of breast tumor cells from ECM increases the secretion of exosomes which are subsequently used to enhance the reattachment and spread of these cells. This mechanism depends upon cellular annexin A1 for exosome immobilization and upon annexin A6 in lipid rafts for uptake, internalization, and recycling of microvesicles . TCMVs carry high levels of tissue factor; the main trigger of coagulation cascade and presence of tissue factor in circulating TCMVs from patients with breast and pancreas cancer is associated to venous thromboembolism .
TCMVs operate as signaling factors that enhance proliferation in an autocrine or paracrine fashion. Glioblastoma and breast cancer cells secrete exosomes containing tissue transglutaminase that crosslinks fibronectin, which are both transferred to recipient cells to activate mitogenic signaling and to stimulate cellular transformation . Exosomes from stomach cancer cells exert an autocrine effect by activating PI3K/AKT and ERK1/2 cascades . Exosomes from nasopharyngeal carcinoma infected by Epstein-Barr virus contain latent membrane protein 1 (LMP1) that increases PI3K/AKT paracrine signaling in endothelial (HUVEC) cells . However, the precise molecular mechanisms by which TCMV targets these cascades are not understood.
TCMVs exert a wide range of direct or indirect effects on cell survival. For example, TCMVs from chronic lymphocytic leukemia (CLL) modulate bone marrow stromal cells via AKT/mTOR/p70S6K/HIF-1 and β-catenin pathways, inducing the production of VEGF, which is in turn a survival factor for CLL B-cells . In contrast, TCMVs from pancreas cancer cells interact with lipid rafts containing Notch-1 partners to downregulate cyclin D and poly (ADP-ribose) polymerase (PARP), enhancing apoptosis through the mitochondrial pathway [41,42].
One of the most remarkable properties of TCMV is the capacity to operate as protein cargo between cells to enhance oncogenic signaling [44,45▪▪,49–51]. TCMVs from breast and colorectal cancer cell lines contain growth factor ligands such as EGF, amphiregulin, or TGFalpha. The combination of these molecules within TCMVs contributes to invasion [49,50]. TCMVs also contain growth factor receptors such as EGFR, which has been identified in exosomes from pancreas cancer cell lines and from primary brain tumors [44,51]. TCMVs from melanomas carry IL-1a, FGF, GCS-F, TNFα, Leptin, TGFα, VEGF, TNF receptors 1 and 2, and Met, which increase angiogenesis and T-cell death [45▪▪]. Furthermore, TCMVs may carry activated oncogenes. For example, TCMVs from glioma and squamous cell carcinoma cells can transfer truncated epidermal growth factor receptor EGFRvIII to other tumor cells or to endothelial cells, which upon incorporating the mutant protein induce the expression of EGFRvIII-regulated genes [35,37]. Similarly, the Notch ligand Delta-like 4 (Dll4) can be transferred by glioblastoma cell exosomes to endothelial cells, enhancing neoangiogenesis . Remarkably, several breast cancer cell lines release exosomes that contain HER2 which binds the trastuzumab antibody in vivo, reducing its therapeutic availability and increasing tumor aggressiveness . Recent results demonstrate that exosomes derived from breast cancer cells can also differentiate adipose tissue-derived mesenchymal stem cells into myofibroblast-like cells via TGFβ receptor-mediated SMAD pathway .
MICROVESICLES, SIGNALING OF METASTASIS, AND PRIMING OF THE METASTATIC NICHE
Microvesicles play critical roles in either enhancing or decreasing metastasis by operating as signaling platforms that shuttle molecular information between tumor cells and their environment. Tetraspanins, a family of transmembrane proteins commonly detected in TCMVs, can recruit a variety of proteins and modulate their cognate signaling pathways . For example, the tetraspanins CD82 and CD9 sequester E-cadherin/beta-catenin complexes within exosomes, decreasing their cellular availability and thus downregulate epithelial-to-mesenchymal transition (EMT) leading to metastasis . In contrast, TCMVs may also operate as presenting particles for metastasis-promoting factors such as amphiregulin, which is at least five times more potent in activating EGFR when associated to microvesicles than in its soluble form .
The concept of premetastatic niche is based on the notion that metastatic cells require a receptive microenvironment supporting a temporal sequence of events that favors their arrival, engraftment, and survival in the metastatic site . In recent years, evidence has accumulated that TCMVs have a key role in the initiation of the premetastatic niche through their capacity to promote communication between tumor and nontumor cells (Fig. 2). TCMVs contain molecules recognized by T-cells, which may exert immunosuppressive effects facilitating immune escape of metastatic cells . TCMVs inhibit the differentiation of CD11b+ myeloid precursors and monocytes into dentritic cells, thus suppressing antigen-specific T-cell response [58,59]. TCMVs from melanoma or renal carcinoma stimulate the growth of normal endothelial cells and promote neoangiogenesis in vivo, enhancing metastasis in distant organs such as lungs [45▪▪,60▪]. Similarly, ovarian cancer cells secrete TCMVs and promote an angiogenic phenotype in endothelial cells in vitro through a mechanism mediated by CD147 (matrix metalloproteinase MMP inducer or basigin) . TCMVs from prostate and lung carcinoma cells activate stromal fibroblasts and endothelial cells to upregulate MMP-9 increasing the motility, resistance to apoptosis, and production of angiogenic factors [62,63]. Leukemic B-cells deliver TCMVs that interact with stromal cells promoting cyclin D1, c-myc, and VEGF expression . EMMPRIN, a transmembrane glycoprotein component of microvesicles, can stimulate matrix metalloproteinase expression in fibroblasts and consequently facilitate tumor invasion and metastasis .
The role of TCMVs in promoting metastasis has recently received spectacular in-vivo support when it was demonstrated that exosomes derived from melanoma cells can home to sentinel lymph nodes, modifying the microenvironment and promoting the expression of sets of genes that facilitate the recruitment, trapping, and growth of tumor cells [65▪▪]. TCMVs from melanoma carry the tyrosine kinase receptor Met which ‘educates’ bone marrow progenitors, mobilizing and recruiting them to the premetastatic niche where they are reprogrammed toward a vasculogenic phenotype [65▪▪]. These exosomes also increase vascular leakiness in premetastatic niches influencing the intravasation of tumor cells and their metastatic growth. These observations underscore an exosome-specific melanoma signature including Met, which is highly expressed in circulating bone marrow progenitors from patients with metastatic melanoma. The concept emerging from these studies is that, in combination with a number of cytokines and soluble factors, TCMVs act as systemic messengers influencing the route of dissemination of metastatic cells and contributing to tumor-specific patterns of secondary cancer lesions .
The microvesicle-mediated signaling processes that promote metastasis are not only supported by TCMVs, but also involve signaling through microvesicles secreted by stromal, immune response, inflammatory, and vascular cells in the microenvironment (Fig. 2). For example, in a mouse model carrying xenografts of cancer cell lines, exosomes produced by the activated T-cells promote the invasive ability of B16 murine melanoma cells and their migration to the lung. This process is mediated through interaction between FasL at the exosome surface and Fas on tumor cells, increasing the amount of intracellular FLICE apoptosis inhibitory protein and activating intracellular pathways that lead to MMP-9 release . Thus, microvesicle-mediated signaling that promotes metastasis involves multidirectional trafficking of microvesicles between normal, activated, and tumor cells, defining a complex interactome of microvesicles that primes the metastatic niche, enhances migration, and facilitates immune escape.
TUMOR-CELL-DERIVED MICROVESICLES STRESS RESPONSES, AND TUMOR CELL METABOLISM
In recent years, cancer has emerged as a disease of metabolism and cell bioenergetics, highlighted by the capacity to ferment glucose into lactate regardless of the presence of oxygen, an effect known as ‘aerobic glycolysis’ (Warburg effect ). This tumor metabolic phenotype has an impact on protein import and export through secretion of microvesicles. So far, however, there is still only fragmented information on how tumor-associated metabolic changes may influence the production and biological activity of TCMVs.
Oxygen deprivation is a limiting factor for solid tumor growth, and mild-to-severe hypoxia is responsible for aberrant neoangiogenesis. The secretion of TCMVs is largely increased during hypoxia and contributes to tumor progression. Under oxygen deprivation, ovarian and endometrial carcinomas secrete large amounts of TCMVs containing the adhesion molecule L1, a potent inducer of cell migration . Increased levels of TCMVs also occur in hypoxic lung carcinoma cells . These vesicles activate and chemoattract stromal fibroblasts, and endothelial cells induce the expression of angiopoietic factors in stromal cells and stimulate the latter cells to secrete factors that enhance the metastatic potential of tumor cells in vivo.
Thermal and oxidative stresses increase the exosomal secretion of ligands of NKG2D (NK cell receptor natural killer group 2, member D) by T-cell and B-cell leukemia/lymphoma which downregulates NKG2D receptor-mediated response, impairs NK-cell cytotoxicity, and contribute to immune escape . Conversely, TCMVs from heat-stressed tumor cells contain chemokines such as CCL2, CCL3, CCL4, CCL5, and CCL20 that chemoattract CD11c (+) DC and CD4(+)/CD8(+) T-cells which activate a specific antitumor immune response in tumor-bearing mice . Other stress conditions such as treatment of breast cancer and erythroleukemic cells with INFγ induce the secretion of exosomes containing the heat shock protein HSP72, which upregulates CD83 expression and stimulate IL-12 release by naive dendritic cells, enhancing tumor recognition .
An important metabolic process influencing microvesicles biogenesis is autophagy, the main physiological pathway for lysosomal degradation of malfunctioning macromolecules and obsolete or damaged organelles in eukaryotic cells . Stress-mediated autophagy triggers either protumorigenic or antitumorigenic effects, depending on cellular and environmental context. Under nutrient deprivation, autophagy recycles proteins into bioenergetic metabolism and contributes to cell survival. In other contexts, autophagy promotes stress tolerance facilitating cell survival, tumor growth, and therapeutic resistance . One of the intracellular vesicle structures of the autophagic pathway, the autophagosome, can fuse to MVB and other endocytic structures to generate the amphysome, which fuses to lysosomes. Thus, extracellular signals that increase the interactions between autophagosomes and MVBs can block exosomal secretion [76,77]. Whether regulation of exosomes secretion by autophagy prevents tumor and metastasis signaling by TCMVs remains to be demonstrated.
The p53 protein, the product of the tumor suppressor gene TP53, interconnects stress responses, metabolism, and exosomal secretion. The p53 protein is activated in response to many forms of stress that induce DNA damage and control the transcription of a series of genes involved in cell cycle, apoptosis, senescence, differentiation, and basal energy metabolism, thus contributing to multifaceted antiproliferative effects . Tumor suppressor activated pathway 6 (TSAP6) is a transcriptional target of p53, which encodes a multipass membrane protein involved in exosome secretion. Mice deficient for TSAP6 exhibit severely compromised exosome production . There is a paradox, however, in the fact that the levels of exosomes in the plasma are apparently higher in patients with colorectal cancer containing TP53 mutations than in matched patients with wild-type TP53, despite the fact that the former have lost transactivation of TSAP6 by p53.
PERSPECTIVES: EXOSOMES AS BIOMARKERS FOR CANCER DETECTION AND THERAPY
The relative stability of TCMVs in bodily fluids confers them an exceptional interest as source of biomarkers for detecting and monitoring cancer . There is clear evidence that TCMVs release in plasma, urine, and other bodily fluids is enhanced in many cancers (Table 2 and references therein [30,36,45▪▪,81–95]). On the basis of the concept that TCMVs contain ‘molecular signatures’ of their cell of origin, the systematic mapping of proteins, lipids, and nucleic acids in TCMVs may provide a shortcut to identify specific biomarkers of cancer occurrence and metastasis. To date, however, there has not yet been any significant prospective study on the value of quantitative or qualitative exosome analysis as a biomarker of early carcinogenesis.
TCMVs may also represent important therapeutic targets on their own. Assuming that they serve as signaling particles that reprogram the microenvironment in a way that facilitates tumor growth and metastasis, it would be of great interest to develop therapeutic approaches for neutralizing or trapping TCMVs, thus preventing the signaling processes they may initiate. Furthermore, there is still a great deal of research to conduct on microvesicles signaling associated with stem cells and with epithelial-to-mesenchymal transition (EMT), the two fundamental biological processes that underlie cancer initiation and development. A recent study has reported that normal human endothelial cells can be stimulated to form vessels in vivo by TCMVs derived from a subset of tumor-initiating cells expressing the mesenchymal stem-cell marker CD105 in human renal cell carcinoma [60▪]. These TCMVs enhanced lung metastasis and contained a specific set of proangiogenic mRNAs and microRNAs implicated in tumor progression and metastasis. Clearly, more studies are needed to unravel the characteristics of such tumor stem-cell-derived microvesicles, which may hold one of the keys that will unlock innovative approaches for future cancer treatment.
Tumor cell microvesicles contain molecular signatures of the tumor cell of origin and operate as platforms for intercellular signaling. Some of their cargo molecules are instrumental in enhancing tumor evasion from immune system, cancer-associated thrombosis, tumor cells migration to lymph nodes, recruitment of bone marrow precursors to the premetastatic niche, and in increasing angiogenesis at the metastatic site. Bodily fluids, in particular peripheral blood, are the most reliable source of tumor-associated vesicles for the diagnosis and identification of new therapeutic targets in cancer.
The authors are grateful to Professor Ricardo R. Brentani, who through his support and vision initiated this review, and dedicate this work to his memory. They also acknowledge São Paulo State Foundation (FAPESP) and National Institute for Oncogenomics (INCITO) MCT/CNPq/FAPESP for their financial support.
Conflicts of interest
The authors declare no conflict of interest.
M.S.D. is a fellow from FAPESP.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
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