Exosomes in cancer: small particle, big player

Exosomes have emerged as a novel mode of intercellular communication. Exosomes can shuttle bioactive molecules including proteins, DNA, mRNA, as well as non-coding RNAs from one cell to another, leading to the exchange of genetic information and reprogramming of the recipient cells. Increasing evidence suggests that tumor cells release excessive amount of exosomes, which may influence tumor initiation, growth, progression, metastasis, and drug resistance. In addition, exosomes transfer message from tumor cells to immune cells and stromal cells, contributing to the escape from immune surveillance and the formation of tumor niche. In this review, we highlight the recent advances in the biology of exosomes as cancer communicasomes. We review the multifaceted roles of exosomes, the small secreted particles, in communicating with other cells within tumor microenvironment. Given that exosomes are cell type specific, stable, and accessible from body fluids, exosomes may provide promising biomarkers for cancer diagnosis and represent new targets for cancer therapy.


Introduction
Exosomes are small, lipid bilayer membrane vesicles of endocytic origin. Exosomes can be defined by several common characteristics, including size (50-100 nm in diameter), density (1.13-1.19 g/ml), morphology ("cup" or "dish" shaped in transmission electron microscopy), and certain enriched protein markers (tetraspanins, TSG101, Hsp70). Initially discovered as the garbage bags for removal of unwanted material from cells, the role of exosomes in immune response is gradually recognized as they function in antigen presentation. More recently, the researchers reveal that exosomes contain proteins and nucleic acids that are functional when transferred into recipient cells. Exosomes have been shown to act as shuttles between cells by transmitting signals (referred to as communicasomes). In this review, we highlight the recent advances in the roles of exosomes in cancer with an emphasis on the potential of exosomes as diagnosis biomarker and therapy target.

Biogenesis, release, and uptake of exosomes
Exosome formation is a fine-tuned process which includes four stages: initiation, endocytosis, multivesicular bodies (MVBs) formation, and exosome secretion [1]. Multivesicular bodies (MVBs) are endocytic structures formed by the budding of an endosomal membrane into the lumen of the compartment. After vesicular accumulation, the MVBs are either sorted for cargo degradation in the lysosome or released into the extracellular space as exosomes by fusing with the plasma membrane ( Fig. 1). The mechanisms underlying the sorting of cargo into the intraluminal vesicles (ILVs) are not yet fully elucidated. Both endosomal sorting complex required for transport (ESCRT)-dependent and independent signals have been suggested to determine the sorting of exosomes [2]. The formation of exosomes has been shown to be controlled by the syndecan heparan sulfate proteoglycans and their cytoplasmic adaptor syntenin [3].
The Rab guanosine triphosphatases (GTPases) have been found to critically regulate exosome secretion. Ostrowski et al. have identified that Rab27a/b affects the size and localization of MVBs [4]. Hsu et al. suggest that Rab3 regulates MVBs docking to tethering at the plasma membrane [5]. The accumulation of intracellular Ca 2+ results in increased exosome secretion [6]. In addition, intracellular and intercellular pH has been shown to affect exosome release. When the microenvironmental pH is low, exosome secretion and uptake by recipient cells increases [7]. There is evidence that oncogenes and tumor suppressors regulate exosome secretion in cancer [8]. Yu et al. demonstrate that p53-regulated protein tumor suppressor-activated pathway 6 (TSAP6) induces exosome secretion under stressed conditions [9,10]. Heparanase is an enzyme with elevated level in cancer. Overexpression of heparanase promotes exosome secretion [11]. Intriguingly, exosomes from normal mammary epithelial cells inhibit exosome secretion by breast cancer cells, implicating a feedback control to maintain dynamic equilibrium [12].
Exosomes transfer information to the target cells through three main ways: (1) receptor-ligand interaction; (2) direct fusion with plasma membrane; (3) endocytosis by phagocytosis (Fig. 1). Although the specific receptors that mediate the uptake of exosomes have not been found, there are several proteins that may act as potential receptors for exosome uptake, such as Tim1/4 for B cells [13] and ICAM-1 for APCs [14]. The uptake of exosomes by direct plasma membrane fusion mode has not been well studied. Melanoma cells could take up exosomes by fusion and low pH facilitates this process [15]. Phagocytosis is an efficient way of exosome uptake. Phagocytic cells have a greater uptake of exosomes than non-phagocytic cells [16]. The uptake of exosomes by recipient cells is energy dependent [17]. Heparan sulfate proteoglycans (HSPGs) function as internalizing receptors of cancer cell-derived exosomes. Enzymatic depletion of cell-surface HSPG or pharmacological inhibition of endogenous proteoglycan biosynthesis significantly attenuates exosome uptake [18].

Structure and contents of exosomes
Exosomes consist of a lipid bilayer membrane surrounding a small cytosol (Fig. 1). The structured lipids not only mold the exosomes but are also involved in exosome function. In addition to lipids, nucleic acids and proteins have also been detected in exosomes. Thakur et al. demonstrate that double-stranded DNA is present in exosomes from cancer cells and reflects the mutational status of the originated cells [19]. Valadi et al. demonstrate that exosomes contain mRNA and miRNA [20]. Exosome-carried RNA can shuttle between cells and thus is called "exosomal shuttle RNA" (esRNA). The protein composition of tumor cell-derived exosomes has been well characterized for a number of cancers by using different proteomic methods. The most common proteins, mRNA, and miRNAs found in exosomes have been deposited in ExoCarta (www.exocarta.org). To date, 4563 proteins, 1639 mRNAs, and 764 miRNAs have been identified in exosomes from different species and tissues by independent examinations. The exosomal contents vary between different physiological and pathological conditions and original cell types. Moreover, the composition of Fig. 1 Biogenesis, release, structure, and uptake of exosomes. Exosomes are produced from the multivesicular bodies (MVBs) (also known as late endosomes). The membrane of the MVBs bulges inward to form exosomes. During this process, proteins (e.g., receptor, cytoplasmic proteins, tetraspanin), nucleic acids (e.g., DNA, mRNA, miRNA), and lipids (e.g., cholesterol, ceramide) are packed into exosomes in a cell type-dependent manner. MVBs fuse with the cellular membrane to release exosomes into the extracellular space. Several mechanisms have been suggested to mediate the uptake of exosomes, including a exosome fusion with the cellular membrane of the recipient cell, leading to the release of the exosomal cargo into the cytoplasm, b juxtracrine signaling through receptor-ligand interactions, c and endocytosis by phagocytosis exosomes can be distinct from the originated cells due to the selective sorting of the cargo into exosomes.

Isolation, detection, and analysis of exosomes
Exosomes have been isolated and characterized from distinct cells under normal and stressed conditions. At present, the most commonly used methods for exosome isolation include ultracentrifugation, combined with sucrose gradient, and the immune-bead isolation (e.g., magnetic activated cell sorting; MACS). There are many commercial kits available for the extraction of exosomes. Transmission electron microscopy (TEM), Western blot, and FACS are frequently used to characterize the isolated exosomes based on their biochemical properties (e.g., morphology, size, exosomal markers). There is a lack of the accurate method to determine the concentration of exosomes. The researchers have to rely on inaccurate measurements of protein concentration or nanoparticle tracking analysis. Quantitative RT-PCR, nucleic acid sequencing, Western blot, or ELISA are used for exosome RNA and protein identification. The International Society for Extracellular Vesicles (ISEV) has recently released minimal experimental requirements for definition of extracellular vesicles and their functions [21].

Roles of exosomes in cancer
Accumulating evidence indicates that exosomes play important roles in cancer. Exosomes transfer oncogenic proteins and nucleic acids to modulate the activity of recipient cells and play decisive roles in tumorigenesis, growth, progression, metastasis, and drug resistance (Fig. 2). Exosomes can act on various recipient cells. The uptake of exosomes may induce a persistent and efficient modulation of recipient cells. In this section, we will discuss about the roles of exosomes in cancer and the molecular mechanisms (Table 1).

Tumorigenesis
Normal cells are transformed into cancer cells in the process of tumorigenesis. Exosomes from malignant cells have shown the potential to induce normal cell transformation. For instance, prostate cancer cell-derived exosomes could induce neoplastic transformation of adipose-derived stem cells (ASCs) [22], which is associated with trafficking of oncogenic proteins (Ras superfamily of GTPases), mRNA (K-ras and H-ras), as well as miRNAs (miR-125b, miR-130b, and miR-155) by exosomes. In addition, Melo et al. suggest that breast cancer cell-derived exosomes contain precursor microRNAs  (pre-miRNAs) associated with RNA-induced silencing complex (RISC)-loading complex proteins, which could induce a rapid and efficient silencing of mRNAs in nontumorigenic epithelial cells, resulting in transcriptome reprogramming and oncogenic transformation [23]. They further demonstrate that the exosomes from serum specimen from breast cancer patients but not those from healthy donors induce tumor formation in mice when coinjected with the nontumorigenic epithelial cells, suggesting a potential mechanism for exosome in tumorigenesis. Cancer is composed of heterogeneous cell populations. Side population (SP) cells are a sub-population of cells that exhibit stem cell-like characteristics and can be isolated in cancer by adapting the Hoechst33342 staining method. Koch et al. demonstrate that in diffuse large B-cell lymphoma, side population cells could export Wnt3a via exosomes to neighboring cells, thus modulating SP-non-SP transitions and maintaining population equilibrium [24]. Altogether, these findings indicate that exosomes may contribute to tumor development and uncontrolled tumor progression by acting as a mediator in the transformation of normal cells to malignant cells and a modulator for the balance between cancer stem cells (CSCs) and non-CSCs.

Tumor growth
The promoting effects of exosomes from distinct sources on tumor cell proliferation have been widely reported. Cancer cells uptake exosomes that contain survivin, an anti-apoptotic protein, to protect them from genotoxic stress-induced cell death [25]. Exosomes from serum of glioblastoma patients contain EGFRvIII mRNA, which stimulate the proliferation of human glioma cells through a self-promoting way [26]. Colon cancer cellderived exosomes are enriched in ΔNp73 mRNA. The proliferation potential of target cells is greatly enhanced Linc-ROR HCC cells HCC cells Reduces chemotherapy sensitivity [82] by incubation with ΔNp73-containing exosomes [27]. The interaction between tumor stromal cells and tumor cells also efficiently promote tumor growth. Exosomes from chronic myelogenous leukemia (CML) cells stimulate bone marrow stromal cells to produce IL-8, which in turn promote the growth of leukemia cells [28]. Bone marrow mesenchymal stromal cells (BM-MSCs) from multiple myeloma (MM) patients release exosomes that express increased levels of oncogenic proteins, cytokines, and adhesion molecules to facilitate the growth of MM cells [29]. Thus, exosomes from tumor cells and microenvironment could act coordinately to promote tumor growth.

Tumor angiogenesis
The formation of new blood vessels is required for tumor growth and progression. Proteomic analysis has revealed that abundant angiogenic factors are present in malignant mesothelioma-derived exosomes [30]. Exosome uptake induces upregulation of angiogenesisrelated genes and results in enhanced endothelial cell proliferation, migration, and sprouting [31]. Exosomes derived from hypoxic glioblastoma cells are more potent to induce angiogenesis [32]. Exosomes from metastatic breast cancer cells contain miR-105. Exosome-mediated transfer of miR-105 degrades ZO-1 protein, disturbs tight junctions, and induces vascular permeability in distant organs [33]. Exosomal miR-92a from K562 leukemia cells targets integrin α5 to enhance endothelial cell migration and tube formation [34]. MiR-210 is significantly enriched in exosomes from hypoxic K562 cells, which promotes the angiogenic activity of endothelial cells [35]. Multiple myeloma cells grown under hypoxic condition produce more exosomes containing miR-135b, which directly suppresses FIH-1, an inhibitor of HIF-1, to enhance endothelial tube formation in endothelial cells [36]. Exosomes are critically involved in tumor angiogenesis by directly delivering angiogenic proteins into endothelial cells or modulating the angiogenic function of endothelial cells by exosomal miRNAs.

Tumor metastasis
Exosomes contribute to tumor metastasis by enhancing tumor cell migration and invasion, establishing premetastatic niche, and remodeling the extracellular matrix. EBV-positive nasopharyngeal carcinoma (NPC) cell-derived exosomes contain HIF-1α, which increases migration and invasiveness of EBV-negative NPC cells [37]. Metastatic cancer cells secrete increased level of miRNA with tumor-suppressor function, which may suggest another mechanism for the role of exosomes in metastasis [38]. The formation of pre-metastatic niche is a prerequisite for tumor metastasis. Exosomes from highly metastatic melanoma enhance the metastatic ability of primary tumors by converting bone marrow progenitor cells to a pro-vasculogenic and pre-metastatic phenotype via the MET receptor [39]. Gastrointestinal stromal tumor cells release exosomes containing protein tyrosine kinase to convert progenitor smooth muscle cells to a premetastatic phenotype [40]. Suetsugu et al. show that highly metastatic breast cancer cells can transfer their own exosomes to other cancer cells and normal lung tissue cells in vitro and in vivo by using fluorescent protein imaging method [41], which provides direct evidence for the involvement of exosomes from highly metastatic cancer cells in educating stromal cells. Luga and colleagues have shown that exosomes produced by stromal cells are taken up by breast cancer cells and are then loaded with Wnt11, which is associated with stimulation of the invasiveness and metastasis of the breast cancer cells [42]. Exosomes from activated CD8+ T cells promote cancer cell invasion and lung metastasis via the Fas/FasL pathway [43], which adds another layer of mechanism for the role of tumor-infiltrating lymphocytes in cancer metastasis. Exosome-mediated transfer of oncogenic microRNAs into cancer cells is associated with enhanced metastatic potential. IL-4-activated macrophage-derived exosomes transfer miR-223 to co-cultivated breast cancer cells, leading to increase of cell invasion [44]. Exosomemediated delivery of miR-221/222 from MSCs to gastric cancer cells greatly enhances gastric cancer cell migration [45]. Fabbri et al. suggest that miRNAs in tumor-secreted exosomes can directly bind toll-like receptor (TLR) in immune cells to promote tumor metastasis [46]. Recently, Costa-Silva and colleagues demonstrate that MIF-containing exosomes from pancreatic ductal adenocarcinoma (PDAC) cells induce TGF-β production in liver Kupffer cells, which in turn upregulates fibronectin (FN) expression by hepatic stellate cells and enhances recruitment of bone marrowderived cells, finally leading to the formation of liver pre-metastatic niche [47], suggesting a complicated network that involves cancer cells, stromal cells, and immune cells in exosome-initiated pre-metastatic niche formation. Intriguingly, Zomer et al. use the Cre-LoxP system to visualize extracellular vesicle (EV) exchange between tumor cells in living mice [48]. They show that the less malignant tumor cells that take up EVs released by malignant tumor cells display enhanced migratory behavior and metastatic capacity, indicating that the metastatic behavior can be phenocopied through extracellular vesicle exchange. Taken together, these findings reveal that the intercellular communication mediated by exosomes may be an important mechanism for tumor metastasis.

Tumor drug resistance
Exosomes contribute to the development of therapy resistance in tumor cells through a variety of mechanisms.
Tumor-derived exosomes can transfer multi-drug resistance (MDR)-associated proteins and miRNAs to target cells [49,50]. In addition, exosomes participate in the process of tumor resistance by mediating drug efflux. The drugs and their metabolites can be encapsulated and exported by exosomes [51,52]. Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas [53]. Moreover, exosomes may counteract the effect of antibody drugs by modulating their binding to tumor cells. Lymphoma exosomes carry CD20, which bind therapeutic anti-CD20 antibodies and protect target cells from antibody attack [54]. Exosomes from HER2-overexpressing breast cancer cells express active HER2 and can bind to the HER2 antibody trastuzumab to inhibit its activity [55]. Exosomes secreted by stromal cells also contribute to tumor drug resistance. BM-MSC-derived exosomes induce multiple myeloma cells resistant to bortezomib through the activation of several survival relevant pathways [56]. Therefore, exosomes released by cancer cells and stromal cells may have a potential to modulate sensitivity of cancer cells to distinct therapies.

Tumor immune escape
Initially reported as tumor-associated antigens and tumor immune response stimulators, the recent studies have shown that tumor-derived exosomes might rather perform immunosuppressive functions. Tumor exosomes block the differentiation of murine myeloid precursor cells into dendritic cells (DC) [57]. Tumor exosome-carried TGF-β1 skews IL-2 responsiveness in favor of regulatory T cells and away from cytotoxic cells [58]. Human nasopharyngeal carcinoma-derived exosomes recruit, expand, and regulate the function of regulatory T cells through CCL20 [59]. NPC cell-derived exosomes impair T cell function, which is associated with upregulated miRNAs in the exosomes [60]. Tumor cell-derived exosomes switch the differentiation of myeloid cells to myeloid-derived suppressor cells (MDSCs) and induce accelerated lung metastasis in a MyD88-dependent manner [61,62]. Hsp72 on tumor-derived exosomes promotes the immunosuppressive activity of MDSCs via autocrine activation of IL-6/STAT3 pathway [63]. Breast cancer cell-derived exosomes simulate the activation of NF-κB and enhance the secretion of pro-inflammatory cytokines in macrophages [64]. Exosomes from human prostate cancer cells express ligands for NKG2D on their surface and downregulate NKG2D expression on natural killer (NK) and CD8+ T cells, leading to the impairment of their cytotoxic function [65]. Collectively, these data suggest that tumor-derived exosomes interfere on multiple levels with the immune system to drive tumor immune evasion.

Tumor-stroma interaction
Tumor stroma is believed to be critically involved in tumor development and progression. Webber et al. suggest that prostate cancer cells could trigger differentiation of fibroblasts into myofibroblasts through exosomal TGF-β [66]. In addition, prostate cancer exosomes triggered TGFβ1dependent fibroblast differentiation resemble stromal cells isolated from cancerous prostate tissue [67], which accelerates tumor growth by supporting angiogenesis. MSCs function as precursors for tumor myofibroblast. The research from our lab suggests that tumor cellderived exosomes could induce differentiation of human MSCs to carcinoma-associated fibroblasts (CAFs) [68]. Adipose tissue-derived MSCs treated with breast cancerderived exosomes also display the characteristics of myofibroblasts [69].  [42]. Therefore, exosomes may mediate a reciprocal interplay between tumor cells and stromal cells to synergistically promote tumor progression.

Tumor thrombosis
Tissue factor (TF) overexpression is closely associated with tumor progression. TF can get incorporated into tumor-derived exosomes. The hypercoagulable state in cancer patients may be partially influenced by the release of TF-bearing exosomes from tumor cells. Garnier et al. demonstrate that exosomes link the procoagulant status with metastatic phenotype in cancer. Induction of EMT changes in epithelial cancer cells results in the release of exosomes containing elevated level of tissue factor. Importantly, TF-rich exosomes can be transferred to endothelial cells and cause their exaggerated procoagulant conversion [71], suggesting that EMT influences tumor-vascular interaction through altered TF-containing exosomes. However, the exact roles of exosomes in tumor thrombosis and consequent impact on tumor growth, progression, and metastasis remain to be further explored.

Exosomes as cancer biomarkers and targets
The findings that exosomes play critical roles in almost all aspects of cancer provide opportunities for the development of exosomes as ideal diagnostic biomarkers and therapeutic targets. Exosome-shuttled proteins and nucleic acids have been suggested as novel diagnostic and prognostic indicators for a variety of cancers. Moreover, utilizing tumor-derived exosomes as vaccines and exosomes Urine [133] from distinct sources as carriers for drugs and small molecules have been proved to be effective in pre-clinical studies and clinical trials.

Exosomes as cancer diagnostic biomarkers
Exosomes are readily accessible in nearly all body fluids including blood, urine, saliva, and ascites. Exosomes contain bioactive molecules that reflect the pathological state of the originated cells, thus providing an enriched source of biomarkers ( Table 2). The level of exosomes is elevated in the plasma of some cancer patients as compared to healthy controls. There is a positive correlation between the abundance of tumor exosomes and tumor stage in ovarian cancer patients [72]. Tumor is characterized by a specific miRNA profile. The majority of circulating microRNAs is concentrated in exosomes [73]. Exosomal miRNAs have been suggested as diagnostic and prognostic indicators for lung cancer, esophageal squamous cell carcinoma, prostate cancer, breast cancer, glioblastoma, ovarian cancer, and other cancer types [74][75][76][77][78][79][80]. Exosomal miRNAs are positively correlated with the stage and degree of cancer progression. In addition to miRNAs, long non-coding RNAs (LncRNAs) are also detected in exosomes [81,82]. LncRNA from serum of gastric cancer patients is defined as a novel exosomal biomarker [83,84].

Exosomes as cancer therapy targets Exosome-based immunotherapy
Dendritic cell-derived exosomes (dexosomes) have been developed as immunotherapeutic anticancer agents [85]. Tumor peptide-pulsed DC-derived exosomes suppress growth of established murine tumors in a T celldependent manner [86]. Exosomes secreted by living tumor cells contain and transfer tumor antigens to dendritic cells and induce potent CD8+ T cell-dependent antitumor effects on mouse tumors [87]. Dexosomes have entered clinical trials for colorectal cancer, metastatic melanoma, and non-small cell lung cancer and have achieved modest therapeutic effects [88].

Exosome removal for cancer therapy
The removal of exosomes from advanced cancer patients is a novel strategy to treat cancer [89]. Exosome depletion by dimethyl amiloride (DMA) in mice restores the antitumor efficacy of cyclophosphamide (CTX) through the inhibition of MDSC functions. Amiloride, a drug used to treat high blood pressure, inhibits exosome formation and blunts MDSC suppressor functions in colorectal cancer patients [63]. The biotechnology company Aethlon Medical has developed an adjunct therapeutic method HER2osome, which is able to reduce tumor-secreted HER2 positive exosomes in the circulation and thus inhibit HER2-positive breast cancer progression. However, further work is needed to evaluate the clinical safety of such a treatment strategy based on exosome removal.

Exosomes as anti-cancer drug delivery vehicles
The use of exosomes as nucleic acid or drug delivery vehicles has gained considerable interest due to their excellent biodistribution and biocompatibility [90]. Exosome-mediated delivery of therapeutic short interfering RNA (siRNA) to the target cells has been tested. The exosome-delivered siRNA is effective at causing posttranscriptional gene silencing and inducing cell death in recipient cancer cells [91][92][93]. To improve drug delivery efficacy to tumors, the researchers have modified exosomes with targeting ligands such as iRGD-Lamp2b. The modified exosomes show highly efficient targeting to αV integrin-positive breast cancer cells, and intravenous injection of these exosomes obviously inhibits tumor growth [94]. In addition, exosomes have been utilized as effective vehicle for drug delivery [95]. Exosomes from MSCs have been tested as the vehicle to package and deliver active drugs such as paclitaxel [96].

Conclusion
The rapid expansion of the number of published studies on exosomes clearly shows that research on exosomes and their functions is now a very exciting field. Exosomes are small particles with big roles and are emerging as major players in intercellular communication. Exosomes have been suggested as active transporters for proteins, DNA, mRNA, and non-coding RNAs. The roles of exosomes in cancer have been gradually realized. Although some reports have suggested anti-tumor roles of exosomes due to their potential to elicit immune response, most of the reports have revealed the various pro-tumor effects of exosomes, which is further supported by the observations that the level of circulating exosomes is increased in cancer patients and correlated with tumor progression. In this review, we discussed several aspects of exosome biology in cancer. Cancer cells communicate with the surrounding and distant cells via exosomes, which constitutes a LncRNA-p21 Prostate cancer qRT-PCR Higher level of exosomal lncRNA-p21 in patients with prostate cancer than those with benign hyperplasia Plasma [135] bi-directional interaction network to synergistically promote cancer development, progression, metastasis, and drug resistance. However, the exact mechanisms mediating the complex roles of exosomes in cancer have not yet fully elucidated. Exosomes would be ideal biomarkers for cancer diagnosis and targeted therapy because they closely represent the state of their parental cells and are relatively stable in the circulation and could be easily collected from body fluids. The potential of exosomal contents for diagnostic and prognostic biomarkers have been investigated in various cancers. It is required to develop faster and more convenient methods for validating the proposed exosomal cargos as biomarkers in specimens from human cancer patients. The use of nanotechnology to load exosomes with small molecules or drugs for cancer therapy has also been exploited. Improvements in developing new strategies to obtain a large amount of exosomes from appropriate donor cells, efficiently introducing the therapeutic agents into exosomes, and optimizing the targeted delivery of exosomes to particular tissues will facilitate the use of exosomes as natural carrier in clinical therapy. Future studies of exosomes will not only shed lights on their roles in the pathogenesis of cancer but will open new avenues for cancer diagnosis and therapeutics.

Competing interests
The authors declare that they have no competing interest Authors' contributions XZ and WRX were responsible for the conception and design of the manuscript. All authors participated in the drafting of the manuscript and approved its final version. All authors read and approved the final manuscript.