Extracellular vesicles: An overview of biogenesis,function,and role in breast cancer

Introduction

Extracellular vesicles (EVs) are released by a variety of cell types, including cancer cells. ¹ Over the years, researchers have successfully isolated these EVs from conditioned media and body fluids including plasma, ² urine, ³ malignant pleural effusions, ⁴ breast milk, ⁵ and saliva ⁶ and indicated their role as potential circulating biomarkers.

EVs are considered as important mediators of intercellular communication, via the horizontal transfer of biologically active cargo, like proteins, ⁷ messenger RNA (mRNA), and non-coding RNAs. ⁸ Furthermore, tumor cell–derived EVs contain both normal and abnormal molecular components, for example, oncoproteins or oncomir.

In addition, there has been increasing interest in the relevance of these EVs with breast cancer (BC) biology. In this review, we briefly summarize the recent findings on BC cell–derived EVs, and we also describe how these vesicles may be utilized in the context of gene therapy. Finally, we touch on the challenges and potential future directions for studying EV biology and therapy.

EVs: exosomes and microvesicles

EVs are a class of membrane-bound organelles secreted by various cell types. EVs include (1) exosomes: 40–100 nm diameter membranous vesicles of endocytic origin, (2) microvesicles (MVs; also referred to as ectosomes): large membranous vesicles (50–1000 nm diameter) that are shed directly from the plasma membrane, and (3) apoptotic blebs (50–5000 nm diameter): released by dying cells. Here, we focus on two major subtypes of EVs: exosomes and MVs, which are distinguished by their process of biogenesis and biophysical properties, including size and surface protein markers (Figure 1(a)).

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Figure 1.

(a) Exosome and microvesicle biogenesis. (b) An enlarged exosome showing that there are a variety of common exosomal surface markers such as tetraspanins and lipid raft-associated proteins as well as internal markers such as Alix and Tsg101. Each exosome also contains small RNAs and proteins which can be transferred to recipient cells.

The term “exosome” is the most commonly used word, and this has become a “buzz term” for EV-related science. The actual meaning of this word, however, is not universally accepted. Exosomes are homogeneous small membrane vesicles of endocytic origin secreted by most cell types in vitro ¹ and are derived from the endocytic recycling pathway (Figure 1(b)). In endocytosis, endocytic vesicles fuse with the plasma membrane to form early endosomes. Later, the early endosomes mature into late endosomes. Exosomes are formed intracellularly by invaginations of the multivesicular body’s limiting membrane. Instead of fusing with the lysosome, these multivesicular bodies directly fuse with the plasma membrane and release contents of exosomes into the extracellular space. ⁹ The mechanisms for exosome biogenesis, protein cargo sorting, and release involve the endosomal sorting complex required for transport ¹⁰ and other associated proteins such as Alix^11,12 and Tsg101. ¹³

In contrast to exosomes, MVs are a larger and more heterogeneous population of EVs. The mechanism of MV biogenesis involves directly budding off the plasma membrane into the extracellular space, and hence, their surface markers are largely dependent on the composition of the membrane of origin.

Both types of vesicles have been shown to deliver functional mRNA, miRNA, and proteins to recipient cells. To date, 13,333 proteins, 2375 mRNAs, and 764 miRNAs obtained from 146 studies have been identified in association with exosomes in the ExoCarta database (http://www.exocarta.org).

As a lack of thorough vesicle characterization has led to an overlap, the terms “exosome” and “microvesicle” have been used interchangeably in many published studies. Nonetheless, both exosomes and MVs have been shown to be promising targets in cancer treatment.

Functions of EVs

The EV field is rapidly expanding and becoming increasingly complex, especially as it overlaps with the even newer field of exRNA-mediated communication. A generic biological standard of EVs would be very useful as a baseline to compare EV preparations obtained by individual laboratories. When in vitro functional studies are performed with isolated EVs, a quantitative analysis of the dose–function relationship should be presented. And, it is also important to make use of systematic negative controls which should exhibit minimal functional effects.

Publications in high-impact journals have proposed exciting functional roles of EVs. The primary role of EVs in cell-to-cell communication is to shuttle various bioactive signaling molecules between the donor and the recipient cells. ¹⁴ EVs can transmit biological information by directly activating cell surface receptors on the recipient cells, fusing with the recipient cells plasma membrane and delivering their cargo including oncoproteins, oncogenes, and non-coding RNAs^8,15,16 into the cytoplasm of the recipient cells, which has fundamentally changed the thinking about gene regulation, as the EVs can regulate the recipient cells at a post-transcriptional level.

EVs participate in the maintenance of normal physiology—for example, stem cell maintenance, ¹⁷ tissue repair, ¹⁸ and immune surveillance. ¹⁹ The best understood role of EVs in disease is their role in tumor biology: EVs have been implicated in carcinogenesis, tumor progression, molding the tumor microenvironment, and immune modulation.^20,21

The mechanisms of EVs secretion

Tumor-derived EVs promote tumor progression through intercommunication between the tumor cells and their surrounding cells. ²² The factors associated with EVs release are not well understood, though roles have been reported for p53,^23,24 ceramide synthesis, ²⁵ calcium, and acidosis.^26,27 Nevertheless, fewer papers on the regulating mechanisms of EVs release by BC cells have been reported. EVs releasing was accelerated in the hypoxia environment, and this hypoxic reaction may be mediated by hypoxia-inducible factor 1-alpha (HIF-1α). ²⁸ For instance, when BC cells were cultured under hypoxic conditions (1% to 0.1% O₂), their EVs release was significantly induced, while inhibition of HIF-1α before hypoxic exposure suppressed EVs release. This has great significance for understanding the hypoxic tumor features, but with hypoxic cancer cells may release more EVs into their surrounding environment to promote their own growth and invasion. Tumors are complex tissues that include various kinds of cells such as vascular endothelial cells, mesenchymal cells, and immune cells. Consequently, the communication between cancer cells and their surrounding cells has been widely studied.

Mechanical changes can also affect exosome secretion. For example, it was recently shown that detachment of adherent BC cells from various surfaces could induce rapid exosome secretion. ²⁹ Furthermore, a feedback regulatory mechanism is that exosome release is regulated by the presence of exosome concentration in the extracellular environment. ³⁰

EVs in cell-to-cell communication

Dynamic analysis reveals the dynamic mechanism for the phenomenon of cell-to-cell communication. EVs released by different cell types that may serve as a mediator of cell-to-cell communication has been proved. ⁴ Exosomes contain genetic materials—mRNAs and miRNAs, therefore allowing transmission of biological information among cells. In recent years, scientists have shown that many kinds of cell types can reprogram their neighboring cells by releasing EVs. ³⁰

Human mammary gland is composed of epithelial cells and surrounding mesenchymal cells, containing fibroblasts, endothelial cells, and leukocytes. Both epithelial and stromal cells are exosome-releasing and/or exosome-targeting cells. ³¹ Epithelium- and mesenchyme-derived EVs are involved in epithelium to mesenchyme, mesenchyme to mesenchyme, and epithelium to epithelium cell communication, which are crucial for breast normal development and physiologic functions. The non-lactating human mammary gland releases EVs containing a number of anti-microbial peptides such as β-defensins, the cathelicidin LL37, lactoferrin, and adrenomedullin, which inhibit bacterial growth in the well-developed duct system. Breast milk exosomes contain HLA-DR, MUC1, CD63, and immune-related miRNA and hence play a vital role in the development of the child’s immune system.^32,33

The roles of EVs in BC

Exosomes are nano-vesicles involved in the physiologic regulation of mammary gland development (e.g. lactation), but are also emerging as important mediators of breast tumorigenesis. ³⁴ The BC cells produce increased numbers of exosomes relative to the same number of normal mammary epithelial cells. Ras-related RAB proteins (RAB27A) have been shown to be required for EV secretion in BC cells, and knocking down RAB27A reduces growth due to impaired recruitment of bone-marrow-derived pro-tumoral immune cells. ³⁵ Lower incidence of metastasis was also observed. ³⁵ Evidence from patients with BC indicate that there are increased numbers of MVs in the blood of these patients. ³⁶ In addition, researchers suggest that exosomes transported miRNA and proteins could promote neoplastic transformation and widely participate in different stages of BC development.^37,38

Growing evidence suggests the potential involvement of EVs in human BC initiation and progression. A recent study utilized green fluorescent protein (GFP)-tagged CD63 expressing BC cells to show that BC cells could transfer their exosomes to other cancer cells and normal lung tissue in vitro and into the tumor microenvironment and the circulation of mice with BC metastases in vivo, suggesting a role for tumor-derived exosomes in the BC progression. ³⁹ Table 1 lists some characterizations of EVs in BC. It is noteworthy that exosomes are derived from the claudin-low triple-negative breast cancer (TNBC) cell line Hs578T and its more invasive Hs578Ts(i)8 variant, and subsequent analysis of recipient cells in response to exosomes showed that Hs578Ts(i)8 exosomes versus Hs578T exosomes significantly increased the proliferation, migration, and invasion capacity of three recipient cell lines (i.e. SKBR3, MDA-MB-231, and HCC1954). ⁴⁰ Furthermore, their clinical study also showed that exosomes from TNBC patients’ sera significantly increased recipient cells’ invasion when compared to those healthy control sera. Additionally, miRNA-10b was found to be expressed at significantly higher in the TNBC MDA-MB-231 cells and, in turn, the noninvasive mammary epithelial cell line HMLE cell invasion was increased, when MDA-MB-231-derived exosomal miRNA-10b was applied to HMLE cells. ⁴¹ Similarly, MDA-MB-231-derived exosomal miRNA-105 was transferred to an endothelial cell line, human dermal microvascular endothelial cell (HMVEC), in vitro, causing impaired endothelial monolayer tight junctions and promoted migration. ⁴² Advancing to in vivo studies, mice were pretreated with these MDA-MB-231-derived, miRNA-105-loaded exosomes, following subsequent intracardiac injection of MDA-MB-231 cells, lung and brain metastases were observed in these mice. ⁴² Specifically, MDA-MB-231-derived exosomes have been shown to transfer miRNA-10b and miRNA-21 to MCF10A cells, resulting in cell proliferation and colony-forming capacity to be increased in the MCF10A cells. ⁴³ The successful delivery of MDA-MB-231-derived exosomes resulted in promotion of tumor development in vivo. In addition, Breast tumor–derived exosomes can induce increased expression of tumor-promoting factors, like stromal cell-derived factor 1 (SDF-1), vascular endothelial growth factor (VEGF), chemokine ligand 5 (CCL5), and transforming growth factor beta (TGF-β), which contribute to progression and malignancy of breast tumor cells by converting mesenchymal stem cells (MSCs) within tumor stroma into tumor-associated myofibroblasts in the tumor microenvironment via the SMAD-mediated signaling pathway. ⁴⁴ Conversely, BC cells are also targets for EVs derived from the cells of other tissues. For example, MSC-derived exosomes (MSCs) carry miRNA-16, a miRNA known to target VEGF, and significantly suppress tumor angiogenesis in vitro and in vivo by communicating directly with the BC cells via down-regulation of VEGF in tumor cells. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules. ⁴⁵ In a recent study, exosomes that secreted from interleukin (IL)-4-activated macrophages shuttle miRNA-223 into BC cells, and miRNA-223 promotes BC cell invasion, highlighting a novel communication mechanism between tumor-associated macrophages and cancer cells. ⁴⁶ Last but not least, fibroblast-secreted exosomes carry Cd81 and promote BC metastasis in a mouse model via Wnt-planar cell polarity (PCP) signaling. ⁴⁷

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Table 1.

Characterization of EVs in breast cancer.

Cell line	EVs content	Recipient cell	Function	Study	Ref.
Hs578T Hs578Ts(i)8	No special instructions	SKBR3, MDA-MB-231 and HCC1954	Cell invasion was promoted	In vitro and in vivo	Suetsugu et al., 39 O’Brien et al. 40
MDA-MB-231	miR-10b	HMLE	Cell invasion was promoted	In vitro	Singh et al. 41
MDA-MB-231	miR-105	HMLE	Metastasis was promoted	In vitro and in vivo	Zhou et al. 42
MDA-MB-231	miR-10b and miR-21	MCF10A	Cell proliferation was promoted	In vitro and in vivo	Melo et al. 43
Mesenchymal stem cells	miR-16	MCF-7	Tumor angiogenesis was suppressed	In vitro and in vivo	Cho et al., 44 Lee et al. 45
Macrophages	miR-223	MCF-7	Cell invasion was promoted	In vitro and in vivo	Yang et al. 46
Fibroblast	Cd81	MCF-7	Metastasis was promoted	In vivo	Luga et al. 47
MCF-7	Notch3, CD44, miR-27b and miR-130b	MCF-7 stem cells	Breast cancer stem cell was promoted	In vitro and in vivo	Papi et al. 48

EVs: extracellular vesicles.

The roles of stem cell–derived exosomes in BC progression and as potential therapeutics are becoming evident. The human hypoxic MCF-7 BC cell–derived exosomes induce a specific subset of the stem cell regulatory gene such as Notch3, CD44, and two oncomiRs (miRNA27b and miRNA130b) in modulating BC stem cells formation, suggesting potential role in tumor progression. ⁴⁸ Further research into exosomal cell communication and gene transport is necessary to greater understanding of their roles in invasion and metastasis and to their possible exploitation for BC therapies.

MVs as diagnostic and prognostic BC biomarkers

One of the major goals in cancer research is to find the early presence of biomarkers in human fluids. Currently, MVs from human nipple fluid and blood are considered as noninvasive sources of molecular biomarkers for early detection and prognosis of various types of cancers, including BC. ⁴⁹ This approach is based on the content of MVs, and the molecular content of MVs is dependent on their cell origin and strongly associates with the original cellular conditions. For example, the concentration of MVs is significantly increased in the plasma of BC patients compared with healthy donors. On the other hand, the focal adhesion kinase (FAK) and epidermal growth factor receptor (EGFR) proteins are markedly enriched in BC-derived MVs, which are related to the presence of BC. ³⁶ The analysis of the MVs from nipple aspirate fluid (NAF) represents a very promising approach able to detect BC-related proteins mirroring the tissue-specific tumor microenvironment. A recent study ⁵⁰ reports a combinatory analysis of three well-known predictive biomarkers (Urokinase-dependent plasminogen activator system, Plasminogen activator inhibitor, and the Thomsen-Friedenreich antigen: uPA, PAI-1, and TF) in NAF collected noninvasively from healthy women and patients with breast atypia and cancer. Researchers demonstrated that the combination of TF, uPA, and PAI-1 provides predictive ability near to 100% allowing the correct prediction of both atypia and cancer disease in women requiring surgery because of suspicious breast lesions. Another research showed that metastatic BC-derived EVs secrete miR-105, which targets ZO-1—a tight junction protein, and thus destroyed the monolayer endothelial barriers and promoted tumor metastasis. In addition, the high miR-105 expression in early-stage BC-derived EVs can frequently suggest tumor metastasis and was a strong predictor for patient prognosis. ⁴² Overall, these studies provide evidence that EVs can serve as a biomarker of diagnosis and prognosis in BC. Although substantial work has to be done to further investigate this finding, these studies suggest that NAF-derived exosomes may be useful in a diagnostic and/or therapeutic setting as BC biomarkers.

Exosomes as novel therapeutics for BC

BC is the most common malignant tumor in women. Recently, progress has been made in developing novel, EV-based therapeutic means for treating different kinds of cancers, ⁵¹ including BC. For instance, EVs released by HER2-positive BC cells could bind to trastuzumab and result in a suppression of the anti-proliferative effects of trastuzumab. These findings show that HER2-positive exosomes may disturb anticancer therapy of trastuzumab and promote HER2-positive tumor progression, ⁵² which could lead to a novel treatment strategy for exosome removal to make those drugs work better. In especial, Aethlon Medical, Inc. (San Diego, USA) has introduced the HER2osome as a new therapeutic strategy to inhibit the progression of HER2-positive BC, which shows high invasive and poor prognosis. HER2-positive BC accounts for about 25% of new BC diagnosis. HER2osome, as an adjunct to therapy, enhances the effects of Herceptin and chemotherapies without adding drug adverse effect.

Overexpression of the multidrug efflux transporter ABCG2 in the plasma membrane of cancer cells confers resistance to various anticancer drugs, including mitoxantrone. EVs can mediate the ABCG2-dependent extraction of intracellular drug, thereby serving as cytotoxic drug disposal chambers. ⁵³ This implicates the role of MVs in the resistance of BC to chemotherapy. Furthermore, because exosomes are cell-derived nucleic acid carriers, they also hold the potential to function as biological therapeutic delivery systems. For example, exosomes engineered to express the transmembrane domain of the platelet-derived growth factor (PDGF) receptor fused to the GE11 peptide were shown to successfully deliver the miRNA-let-7a to EGFR-expressing BC cells in vitro and EGFR-expressing xenograft BC tissue in vivo, thus demonstrating the potential of exosomes to be used therapeutically to specifically target cancer cells with nucleic acid drug targets. ⁵⁴

In addition to deliver antitumor miRNA, MVs can also deliver chemotherapeutics such as doxorubicin (Dox) specifically to tumor tissues, ⁵⁵ leading to inhibition of tumor growth without overt toxicity, thus having great potential value for clinical applications. A different consequence of EV-associated RNA transfer in the tumor microenvironment has been recently described. ⁵⁶ EV-borne RNAs are recognized in the recipient cell cytosol, which induces development of an interferon response (including expression of genes like STAT1) similar to that induced upon viral infection. This response was shown to participate in BC cell resistance to chemotherapy. ⁵⁶ Although in vivo studies and more extensive analysis of relevant patient samples are necessary to further investigate this activity, these studies support a broad range of important roles held by exosomes in BC and highlight their potential in a therapeutic setting. ⁵⁷

Conclusion

EVs have now been implicated in numerous biological and pathological processes, and current knowledge suggests that they can play an important role in BC. EVs hold great potential as novel diagnostic and prognostic molecular biomarkers of BC and as novel therapeutic approaches. Targeting EVs directly to inhibit their deleterious effects or to deliver nucleic acids and other drug cargoes across major biological barriers are emerging as important novel therapeutic strategies. However, although significant advances have been made, many questions remain. Very little is known about the precise molecular mechanisms of EVs biogenesis, release, targeting, and interactions with target cells as well as the specific content of EVs transport. Of vital importance to these questions is how to identify and isolate these vesicles, thus appreciating the exact clinical values and the biological functions of EVs.