Extracellular vesicles-mediated signaling in the osteosarcoma microenvironment: Roles and potential therapeutic targets.

Osteosarcoma (OS) is the most common non-hematologic malignant tumor of bone in children. It is usually characterized by a high risk of developing lung metastasis and poor prognosis. Extracellular vesicles (EVs) are cell-derived nanoparticles with a small size of 50-200 nm in diameter. As a communicator, the contents of the EVs secreted via either fusing with lysosomes for degradation and recycling or fusing with the cell plasma membrane into the extracellular environment, which play an important role in regulating the tumor microenvironment of OS and mediating the Wnt/β-catenin and TGF-β signalings. Increasing evidences suggest that EVs have significant role in OS growth, progression, metastasis and drug resistance. In this study, the roles of EVs in the physiology and pathogenesis of OS and the potential attractive therapeutic target for the treatment of OS were reviewed.

Introduction

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. Current treatment for newly diagnosed OS includes three aspects: preoperative chemotherapy, surgical resection and postoperative chemotherapy. These management strategies have improved the outcomes of patients with localized OS. However, patients with advanced, metastatic and recurrent OS continue to experience a quite poor prognosis. Although current multidisciplinary treatments have been used for OS, there is still no drastic change in the overall prognosis during the past two decades. The 5-year survival rate of OS patients with metastases is 20% compared with 65% of patients with localized disease [1].

Extracellular vesicles are lipid bilayer membrane vesicles with a small size of 50–200 nm in diameter. As a communicator in the cancer microenvironment, previous evidences revealed that extracellular vesicles can directly stimulate target cells with their membrane molecules or deliver their contents into multiple types of cells for direct influence [2]. Extracellular vesicles are released by all types of cells, including OS cells. Indeed, recent studies revealed that extracellular vesicles secreted by tumor cells played a critical role in cancer cell development, survival, metastasis and drug resistance [3-5]. However, the role of extracellular vesicles in the biological and pathophysiological processes of OS was still not clear. In this review, we provide an overview regard to the currently available data to illustrate the role of extracellular vesicles in OS.

Biogenesis and functions of extracellular vesicles

Extracellular vesicles are produced by all normal and pathological cells and secreted from the internal vesicles. The diameters of them are 50–200 nm. Extracellular vesicles are derived from cells via a multivesicular body endocytic process [6], and are found in nearly all extracellular space and body fluids, including blood plasma, cerebrospinal fluid, saliva, breast milk, urine and semen. Also, extracellular vesicles are observed abundantly in tumor microenvironment [7].

After extracellular vesicles are formed, a variety of molecules, such as multiple proteins, nucleic acids, enzymes and other soluble factors are contained in them. Extracellular vesicles may differ according to the tissue birthplace and specific cell type from which they originate, and may be subjected to the stimulation and physiological variation that the cells experience. The components of extracellular vesicles could partly reflect the contents of the original cells [8]. Study indicated that double‑stranded genomic DNA contained in extracellular vesicles derived from cancer cells could partly reflect the mutational status of the originate cells [9]. Also, Ismail et al. [10] reported that RNAs contained in extracellular vesicles can exchange genetic information with target cells, and the expression of genes and intercellular communication in the target cells was influenced by extracellular vesicles. Notably, a significantly higher expression of extracellular vesicles was found in tumor cells than normal cells, which meant extracellular vesicles may play a special role in cancer development and drug resistance [11].

The contents of the extracellular vesicles secreted via either fusing with lysosomes for degradation and recycling or fusing with the cell plasma membrane into the extracellular environment. Notably, extracellular vesicles production and release are signal and stimuli dependent, and various proteins are associated with the process of extracellular vesicles secretion. Members of the Rab family are demonstrated to accurately regulate the secretion of extracellular vesicles, especially Rab27a and Rab27b affecting the size and localization of extracellular vesicles [12]. Also the factor p53 is shown to be involved in the extracellular vesicles release [13]. Previous studies revealed that elevated intracellular calcium concentration, acidosis, cAMP levels and P2 × 7 receptor activation modulated the pool of extracellular vesicles output [3]. After extracellular vesicles secreted into the extracellular space, they may be taken up by the target cells via direct fusion with the plasma membrane; receptor-ligand interaction; endocytosis by phagocytosis and degradation in the lysosome [14].

Extracellular vesicles in the microenvironment of OS

As a communicator, the main function of extracellular vesicles in intercellular communication is to exchange information with target cells. Increasing studies revealed that extracellular vesicles had significant roles in tumor development, progression, metastasis and chemo-resistance [3]. Detection of extracellular vesicles in osteoblastic and osteoclastic lesions provided a strong rationale to study the function of extracellular vesicles in messaging OS bone microenvironment [15]. Studies have reported the characterization of extracellular vesicles derived from OS cells and its potential implications on the bone marrow stroma. It clearly reported that abundant of the extracellular vesicles have diameters within 50 to 200 nm [16].

Biomechanical stress in the bone marrow stroma can elevated intracellular calcium concentration, which in turn accelerates the production of extracellular vesicles, and up-regulate the expression of extracellular remodeling enzymes, such as matrix metalloproteinases (MMPs). The significantly higher expression of MMPs and down-regulation of miRNA143 are correlated with the poor prognostic outcomes in patients with OS. Therefore, detection of MMPs in extracellular vesicles is a valuable finding for predicting OS prognosis [16], [17]. Casimiro et al. [18] identified RANKL as the important regulatory factor for osteoclast differentiation due to it playing a special role in the activation of MMPs and stimulation of osteoclastogenesis. Lim et al. observed that the transfer of nucleic acid from bone microenvironment to breast cancer cells through extracellular vesicles may have a significant role in the quiescence of bone metastases [19].

CD-9 belongs to tetraspanin protein family and is found enriched in extracellular vesicles. It can regulate osteoclast differentiation and suppress the formation of mature polykaryons. In osteotropic cancers, CD-9 not only induces the homing of cancer cells in the bone microenvironment, but also enhances osteoclastic bone resorption via overexpression [20]. Herr et al. [21] reported that blocking of CD-9 by KMC8 would suppress multinucleated osteoclast formation and mediate osteoclast differentiation. Yi et al. [22] also indicated the regulatory function of CD-9 in the mediation of MMP-9 induced migration and invasion in cancer.

The expression of TGF-β is highly in the serum of patients with OS compared with those without OS. TGF-β can regulate the bone marrow stroma and stimulate migration of OS cells directly [23]. TGF-β contained in extracellular vesicles can increases the accumulation of immature myeloid cells, and the function of immature myeloid cells from the osteolytic bone marrow stroma in accelerating osteoclastic bone resorption was demonstrated [24].Thus, targeting the OS bone microenvironment and inhibiting extracellular vesicles secretion may prevent tumorigenesis.

Wnt signaling and extracellular vesicles in OS

The Wingless (Wnt) signaling pathway plays an important role in cell proliferation and tumorigenesis. Previous studies revealed that maintenance of cancer cells are regulated by the Wnt/β‑catenin signaling pathway in several cancers. And aberrantly activated the Wnt/β-catenin pathway is correlated with the progression of OS [25]. Chen et al. established that activation of autocrine Wnt/β-catenin signaling in the tumor cell-derived extracellular vesicles would enhance the development and survival of OS cells [26].

The function of Wnt signaling in OS remains controversial. Some studies suggested an oncogenic role for this pathway, but other studies supported an anti-tumorigenic role for it. Goldstein et al. found that treatment with BHQ880 (an antibody against the Wnt signaling inhibitor) would induce increased nuclear localization of β-catenin, which resulted in elevated expression of a number of Wnt target genes released from extracellular vesicles and inhibited OS metastasis. These studies indicated that Wnt signaling pathway promoted bone differentiation in OS, and prevented tumor progression and metastasis [27].

Several valuable molecular strategies for therapeutic intervention by targeting Wnt signaling in OS have been reported. Two groups of secreted Wnt antagonists are characterized by their inhibition mechanisms. The first group of antagonists directly bind to Wnt ligands and promot inhibitory reaction, such as sFRP family and Wnt inhibitory factor-1. CD82 and CD9 were abundantly found in extracellular vesicles, which would suppress β-catenin-mediated Wnt signaling activity. It revealed that the function of CD82 and CD9 in mediating the down-regulation of Wnt signaling induced discharge of β-catenin [28].The second group of antagonists suppress the Wnt signaling pathway by binding to transmembrane receptors, such as the Dickkopf (Dkk) family and sclerostin. The Dkk family comprises four secretory proteins, which could mediate Wnt signaling pathway via binding to the transmembrane receptors LRP5/6 [29]. Dkk-3 can suppress the motility of β-catenin nuclear in OS cells, and the decreased expression of Dkk-3 was shown to prevent progression and migration of OS cells [30].

Recently, a number of microRNAs were found to be played important roles in the pathogenesis of OS. These microRNAs were detected in tumor-derived extracellular vesicles and acted as oncogenic [31], [32] or suppressive RNAs [33]. For example, miR-370 could suppress the invasion of OS cell by inhibiting the Wnt/β-catenin signaling pathway [34].

Based on the findings above, the Wnt signaling pathway plays a significant role in the progression and metastasis of OS. Thus, preventing autocrine activation of Wnt/β-catenin signaling by regulating tumor cells extracellular vesicles may be an effective therapeutic method for inhibiting OS development and metastasis.

TGF-β signaling and extracellular vesicles in OS

Transforming growth factor β (TGF-β) super family plays significance roles in the development of various diseases. It is one of the most abundant molecules in the tumor microenvironment. Study revealed that tumor cells derived from extracellular vesicles are able to enhance the proliferation and migration of tumor cells through activating an anti-apoptotic pathway regulated by exosome-associated TGF- β [35]. In particular, TGF-β is strongly associated with the development and progression of OS. Increased expression of TGF-β was found in the serum of patients with OS compared with those without OS [36]. And a significantly higher expression of TGF-β was found in OS patients with metastasis than those without metastasis [37]. Also the elevated serum TGF-β level was correlated with high-grade OS [38].

As one of the most widely studied pathways in OS, variations in the TGF-β signaling pathway involved in cell differentiation, growth and apoptosis. And it was closely related with the development and progression of OS in an autocrine manner [39]. Previous studies indicated that the osteoblast differentiation was mediated by the TGF-β signaling pathway, and evidences suggested the role of TGF-β1 in the pro-migratory effect of OS [40]. Loss of TGF-β signaling is important for tumorigenesis of many cancers. Aberrant signaling in a variety of pathways has shown to be related with OS progression [41]. Elevated expression of TGF-β1 and TGF-β3 secreted from extracellular vesicles was demonstrated in OS patients, and it was closely correlated with tumor progression [42].

Angiogenesis plays a significant role in tumor development and progression. It's established as an indicator for high risk of metastasis and poor prognosis. Tumor cells under hypoxia can elevate secretion of extracellular vesicles, which leads to the activation of vascular cells during tumor angiogenesis. Extracellular vesicles-associated TGF-β secreted from injured cells promotes original tissue recovery and regenerative [43]. Bhattacharya et al. found that TGF-β1-containing extracellular vesicles promoted tumor development and metastasis via angiogenesis in OS [44]. The therapeutic effects of an anti-TGF-β antibody depend on the recovery of the immune response in OS [45]. Mohammad et al. showed that SD-208 forcefully affected the ability of the primary OS developing lung metastases by blocking the capacity of TGF-β1 [46]. Therefore, preventing TGF-β signaling via modulation of extracellular vesicles could be a novel therapeutic target for OS.

Extracellular vesicles and immunotherapy for OS

Currently, personalized medicine strategies are widely used for the treatment of patients with cancer. The primary function of extracellular vesicles in intercellular communication is to exchange information with target cells. Thus, specific bioactive molecules contained in extracellular vesicles would design and select to deliver anti-tumor drugs for the treatment of cancer. Researchers have designed drug-loaded magnetic exosome-based vehicle, which can be delivered directly to the target tumors cells under an external magnetic field and suppress tumor progression [47]. The abundant of drug-loaded extracellular vesicles around tumors can significantly improve the anti-tumor efficiency and limit their side effects. Therefore, extracellular vesicles worked as delivery vehicles could be an attractive therapeutic for cancer. And this novel therapeutic has gained increasing interest due to the effective biocompatibility and biodistribution of extracellular vesicles [4].

Down-regulation immunoreaction or escaping from immune surveillance is important for cancer development and metastasis. Zitvogel and colleagues have indicated the immune therapeutic benefits of extracellular vesicles in tumor. They revealed vaccination of mice with extracellular vesicles secreted from dendritic cells (DCs) had functional MHC, T-cell and costimulatory molecules [48]. The DCs were shown to stimulate an antitumor immune response and suppressed tumor growth in a T cell-dependent manner. Therefore, DC vaccination secreted antibodies mediate certain immunoregulatory molecules can improve the therapeutic efficiency in OS. Furthermore, recent studies indicated that using DC immunotherapy may induce cytotoxic T cell response in OS [49]. Bacterial products injected into unresectable tumors was able to stimulate human’ immune response and suppressed tumor progression [50]. Therefore, strategy for specifically targeting extracellular vesicles is a promising treatment for cancer.

As a delivery system, extracellular vesicles are extensively working as vehicles for a variety of cancers therapeutic cargos. The insulin-like growth factor (IGF) well established as an important factor on oncogenesis, tumor progression, tumor metastasis and chemoresistance. And the increased expression of IGF-1, IGF-2 and IGF-1 receptor (IGF-1R) is found in the serum of patients with OS [51]. IGF pathway has become the target of novel therapeutics. Ganitumab worked as a monoclonal antibody for IGF-1R, is able to suppress the proliferation of OS and mediates OS tumor progression [52]. High level of IGFBP3 is related with a poor prognosis and high grade tumors, which is expressed in extracellular vesicles from human osteoblasts [53]. Therefore, this molecule may be new pathway and potential therapeutic target for the treatment of OS.

Conclusion

OS is a malignant tumor of the bone with poor outcome and high risk of lung metastasis. Communication between tumor cells and other organs is crucial for cancer progression. Extracellular vesicles emerge as major players in this communication and play an important role in regulating the tumor microenvironment of OS. They also regulated the Wnt/β-catenin and TGF-β signaling pathways. Tumor-derived extracellular vesicles contribute to the failure of cancer treatment, and eliminating these extracellular vesicles seems helpful for tumor therapy. However, the detailed molecular mechanisms of extracellular vesicles assisting tumor progression and metastasis also was not fully elucidated. Additional studies are necessary to determine the exact roles of extracellular vesicles in the pathogenesis of OS.

Conflict of interest

The authors declare that they have no conflict of interest.