Accepted Manuscript Title: Extracellular vesicles in lung cancer—From bench to bedside Authors: Tsukasa Kadota, Yusuke Yoshioka, Yu Fujita, Kazuyoshi Kuwano, Takahiro Ochiya PII: DOI: Reference:
S1084-9521(17)30140-4 http://dx.doi.org/doi:10.1016/j.semcdb.2017.03.001 YSCDB 2196
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Seminars in Cell & Developmental Biology
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3-10-2016 21-2-2017 2-3-2017
Please cite this article as: Kadota Tsukasa, Yoshioka Yusuke, Fujita Yu, Kuwano Kazuyoshi, Ochiya Takahiro.Extracellular vesicles in lung cancer—From bench to bedside.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.03.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Extracellular vesicles in lung cancer—from bench to bedside Tsukasa Kadota 1,2, Yusuke Yoshioka 1, Yu Fujita 1,2, Kazuyoshi Kuwano 2, Takahiro Ochiya 1* 1 Division of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan. 2 Division of Respiratory Diseases, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan * Correspondence:
[email protected]; Tel.: +81‐3‐3542‐2511 Abstract: Lung cancer is the leading cause of cancer‐related deaths worldwide. Despite significant advances in lung cancer research and novel therapies, a better understanding of the disease is crucially needed to facilitate early detection and appropriate diagnoses and to improve treatment outcomes. Extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, are released from all tested cell types and modulate cell‐cell communication. EVs transfer a wide variety of molecules, such as proteins, messenger RNAs and microRNAs. Emerging data suggest that EVs play an important role in lung cancer pathogenesis and may have potential as biomarkers and therapeutics. Here, we review current research on EVs in lung cancer. Keywords: Lung cancer; ; ; ; ; , Extracellular vesicle, Exosome, Microenvironment, Biomarker, Therapy 1. Introduction Lung cancer is the leading cause of cancer‐related deaths worldwide [1], accounting for approximately 1.6 million deaths annually. More than 80% of lung cancers are classified as non‐small cell lung cancers (NSCLCs), whereas the remaining cancers are classified as small cell lung cancers (SCLCs). Smoking has been demonstrated to be a major risk factor for the development of lung cancer. A diagnosis of lung cancer is made based on the pathologic evaluation of cytologic or histopathologic specimens. Some serum tumor markers such as carcinoembyonic antigen, cytokeratin 19 fragment, neuron specific enolase and pro‐gastrin‐ releasing peptide are used as diagnostic adjunct and monitoring the course of lung cancer in the clinical setting although the diagnostic value of these tumor markers are limited by its sensitivity and specificity. In addition, despite significant advances in lung cancer research and novel therapies such as epidermal growth factor receptor tyrosine kinase inhibitors (EGFR‐TKI), anaplastic lymphoma kinase (ALK) inhibitors and
immune checkpoint inhibitors in NSCLC, only a proportion of lung cancer patients experienced a therapeutic benefit from these drugs [2‐4]. Therefore, improvements in the understanding of molecular mechanisms, early detection, and the discovery of targeted therapies for lung cancer are crucial. Extracellular vesicles (EVs) are small membranous vesicles that are secreted by all tested cell types. EVs are categorized as exosomes, microvesicles (also known as microparticles), or apoptotic bodies according to their size, biogenesis, and secretion mechanisms [5,6]. Exosomes are generated through the endocytic pathway and are released into the extracellular space by fusion of the peripheral membrane of multivesicular bodies with the limiting plasma membrane [7,8]. This process involves several proteins such as Rab proteins (for instance, Rab27A/B) and tetraspanins (for instance, CD9, CD63, CD81, and CD82), which are recognized as exosomal markers [9,10]. Microvesicles, which are larger than exosomes, are generated from the plasma membrane via shedding or budding under physiological conditions or in response to stimuli. Microvesicles are also rich in several lipids and phosphatidylserine and contain membrane components similar to those of the parental cell membrane [11]. Apoptotic bodies are several micrometers in diameter and are released from the plasma membrane during cell apoptosis via indiscriminate blebbing. Apoptotic bodies also contain DNA fragments and RNA [12]. Of these vesicles, exosomes and microvesicles have received the most attention and have been well characterized. Although the origins of these vesicles have been defined, current technologies cannot clearly distinguish among the different types of EVs. Thus, in this review, we use the term EVs as a general term for all types of vesicles in the extracellular space, according to the recommendations of the International Society for Extracellular Vesicles (ISEV) [13]. In contrast, we refer to the vesicle types being discussed when they were specifically identified in a study Accumulating evidence highlights the role of EVs as key mediators of cell‐cell communication through the transfer of their functional contents such as proteins, messenger RNA (mRNA), microRNA (miRNA), DNA, lipids and metabolites [14,15]. These EV‐mediated interactions within the tumor microenvironment contribute to tumor initiation and progression. Given that EVs can be isolated from different bodily fluids such as plasma, pleural effusion, and urine, EVs are considered to be promising biomarkers and therapeutics in lung cancer [16]. Here, we review the pathological roles of EVs in lung cancer and discussed the potential clinical utility of EVs as biomarkers and therapeutic agents in the treatment of lung cancer.
2. The roles of EVs in the lung cancer microenvironment 2.1 Lung cancer‐derived EVs 2.1.1 Immune response regulated by lung cancer‐derived EVs Evasion of antitumor immune responses is now recognized to be a hallmark of cancer [17]. In the tumor microenvironment, various immune cell types such as T lymphocytes, B lymphocytes, macrophages, and natural killer (NK) cells regulate tumorigenesis and progression. Currently, it is believed that the immune system plays dual roles in cancer. The immune system can suppress tumor growth by destroying cancer cells or inhibiting their outgrowth. In contrast, the immune system can also promote tumor progression by supporting chronic inflammation, facilitating tumor immunogenicity, and suppressing antitumor immunity [18,19]. The roles of these immune cells depend in part on the tumor microenvironment. Recently, numerous studies have reported that EVs modulate the immune response. In the lung cancer microenvironment, Fabbri et al. have shown that lung cancer‐derived EVs contain miR‐21 and ‐29a, which bind and activate toll like receptor (TLR) 8 in immune cells, thereby triggering TLR‐mediated NF‐κB activation and prometastatic inflammatory responses [20]. This result indicates that lung cancer cell‐derived EVs can lead to tumor growth and metastasis. On the other hand, Berchem et al. found that hypoxic lung cancer cell line‐derived EVs negatively regulate NK cell cytotoxicity against different cancer cells via the transfer of transforming growth factor (TGF)‐β and miR‐23a [21]. EV‐derived miR‐23a acts as an immunosuppressive factor because it directly targets CD107a, a molecule that protects NK cells from degradation [22]. Moreover, Huang et al. have shown that EVs purified from lung biopsies contain EGFR, which suppresses CD8+ T cells by inducing tumor antigen‐specific regulatory T cells (Tregs) [23]. EGFR is a transmembrane protein with cytoplasmic kinase activity that is associated with NSCLC growth, invasion, and metastasis. EGFR is upregulated in lung cancers, and its inhibition by EGFR‐TKIs improves patient outcomes [2]. These results suggest that lung cancer cell‐derived EVs induce tumor‐promoting inflammation and suppress cancer cell cytotoxicity. 2.1.2 Epithelial–mesenchymal transition (EMT) regulated by lung cancer‐derived EVs The epithelial–mesenchymal transition (EMT), defined as the acquisition of a mesenchymal phenotype by epithelial cells, is critical in cancer progression. During this process, transformed epithelial cells acquire
invasive potential, resist apoptosis, and disseminate [17], Although the involvement of EMT in metastasis has long been a subject of debate [24‐26], these steps are considered to be required for metastasis. EMT may be triggered by a variety of mediators, including EVs. In lung cancer pathogenesis, Rahman et al. have reported that exosomes derived from highly metastatic lung cancer cells, as well as those derived from the sera of humans with late stage lung cancer, express higher vimentin levels, which can promote EMT in human bronchial epithelial cells via EV transfer [27]. Vimentin is involved in several aspects of cancer initiation and progression, including tumorigenesis, EMT, and metastasis [28], thus indicating that lung cancer‐derived exosomes can be mediators of EMT in recipient cells during lung cancer pathogenesis. 2.1.3 Cancer‐associated fibroblasts (CAFs) regulated by lung cancer‐derived EVs
Cancer-associated fibroblasts (CAFs) are a heterogeneous subpopulation of cells in the tumor microenvironment. The synthesis of extracellular matrix (ECM) and basement membrane components by these cells, as well as their ability to reprogram the metabolic and immune components of the tumor microenvironment, lead to adaptive resistance to chemotherapy [29]. In response to inflammatory stimuli released by damaged organs, fibroblasts become activated and secrete growth factors and ECM. The mechanisms underlying the conversion of resident fibroblasts to CAFs are complex, but some of the phenotypic features of CAFs can be induced by TGF-β [30]. Lung cancer-derived EVs can also induce similar phenotypic changes in resident fibroblasts. Gutkin et al. have reported that lung cancer cell-derived exosomes contain human telomerase reverse transcriptase (hTERT) mRNA, the transcript of the enzyme telomerase, and can transform nonmalignant fibroblasts into telomerase-positive cells [31]. Telomerase is activated in approximately 85%–90% of all tumor biopsies and is essential for indefinite proliferation, which is often considered to be a hallmark of cancer [17,32]. This transfer creates non-malignant cells with telomerase activity, thereby facilitating increased proliferation, as well as protection from replicative senescence and DNA damage. These changes may contribute to the formation of CAFs [31]. Wysoczynski et al. have shown that human and murine lung cancer cell-derived microvesicles activate both fibroblasts and endothelial cells and serve as chemoattractants for these cell types. Fibroblasts stimulated by lung cancer cell-derived EVs express several pro-angiogenic factors such as IL-8 and vascular endothelial growth factor (VEGF), which subsequently stimulate both lung
cancer and endothelial cells. The authors have also observed that fibroblasts stimulated by EVs enhance the metastatic potential of lung cancer cells in vivo [33]. 2.1.4 Angiogenesis regulated by lung cancer‐derived EVs Tumor angiogenesis is crucial for invasive tumor growth and metastasis. Tumor angiogenesis results in the formation of abnormal blood vessels, which are characterized by aberrant structure, altered interactions between endothelial and surrounding cells, aberrant blood flow, increased permeability and delayed maturation [34,35]. These processes and features are controlled by a wide variety of signaling molecules and receptors such as VEGF [36,37]. Cancer‐derived EVs also play roles in angiogenesis by affecting endothelial cells in the tumor microenvironment. Cui et al. have demonstrated that the activation of PI3K/AKT/HIF‐ 1/miR‐210 signaling by a tissue inhibitor of metalloproteinases‐1 (TIMP‐1) supports tumor growth and cancer progression [38]. In addition, miR‐210‐enriched lung cancer cell‐derived exosomes downregulate Ephrin A3 in endothelial cells and thus promote angiogenesis. Previous reports have also shown that TIMP‐1 protects tumor cells from cell death and that miR‐210 regulates angiogenesis [39,40]. In a study by Valencia et al., miR‐192‐enriched EVs derived from miR‐192‐overexpressing cells have been found to transfer to endothelial cells and suppress angiogenesis and metastasis by repressing the pro‐angiogenic factors IL‐8, ICAM, and CXCL1 [41]. In addition, in a rat critical limb ischemia study, Sheu et al. have found that circulating microvesicles from lung cancer patients, compared with those from healthy subjects, enhance angiogenesis, vascularization, and restoration of blood flow to a greater extent [42]. Furthermore, Al‐Nedawi et al. have reported that lung cancer cell‐derived EVs contain activated EGFR, which can be absorbed by endothelial cells and elicit EGFR‐dependent autocrine VEGF expression [27,43]. These results suggest that lung cancer‐ derived EVs regulate angiogenesis, thereby probably contributing to cancer pathogenesis and metastasis. 2.1.5 Other microenviromental regulation by lung cancer‐derived EVs Lung cancer cell‐derived EVs can transfer to surrounding cells. Mesenchymal stem cells (MSCs) are multipotent non‐hematopoietic cells with the potential to differentiate into several cell types [44]. MSCs have been found to promote tumor growth and metastasis [44]. MSCs have been found to promote tumor growth and metastasis [45]. In a study by Li et al, lung cancer cell‐derived exosomes have been found to support tumor growth by educating naive MSCs and converting them into a novel type of pro‐inflammatory
MSC by activation of TLR2/NF‐κB signaling; this effect is dependent on direct interactions with the exosomal surface heat shock protein (Hsp) 70 [46]. In addition, Li et al. have reported that miR‐302b, a tumor suppressor miRNA found in exosomes from lung cancer cells with low metastatic potential, suppress the proliferation and migration of highly metastatic lung cancer cells via the TGF‐β receptor II (TGFβRII)/ERK pathway. TGFβRII is a target of miR‐302b in lung cancer cells and is activated by TGF‐β1. TGF‐β1 can activate key signaling molecules involved in Smad and non‐Smad signaling pathways, which can regulate cancer cell proliferation [47]. Moreover, Tatto et al. have reported that microvesicles from human lung cancer cell explants alter the phenotypes and genotypes of normal human bone marrow cells [48]. Although further studies are needed, these results suggest that cancer‐derived EVs in the tumor microenvironment contribute to lung cancer initiation and progression (Figure 1).
2.2 The roles of non-Lung Cancer-derived EVs Non‐cancer cells also secrete EVs and facilitate EV‐mediated intracellular communication. In the tumor microenvironment, complex interactions between activated cytotoxic T lymphocytes (CTLs) and various cell types contribute to immune suppression. CTLs are key defender against tumor cells. Activated CTLs induce and express high levels of Fas ligand (FasL). It has been reported that Fas activation initiated by FasL in CTLs promotes the tumor progression in most tumors [49,50], while, in some tumor, increased FasL expression mediates CTL tumor cell killing and auto‐apoptosis [51]. In lung cancer microenviroment, Cai et al. have reported that exosomes from activated T cells promote lung cancer cell metastasis via the Fas/FasL pathway [52]. FasL‐carrying exosomes derived from CTL cells promoted the accumulation of cellular Fas‐associated death domain–like interleukin 1β–converting enzyme (FLICE) inhibitory proteins and subsequently activated the ERK and NF‐κB pathways, which subsequently increased matrix metalloproteinase (MMP) 9 expression. The upregulation of MMP9 leads to exacerbated tumor invasion, and these tumor‐promoting exosomes have been identified in tumor‐bearing mice. These results indicate that EVs facilitate tumor immune escape. In addition, Xiao et al. have demonstrated that a human mast cell line expresses mast/stem cell growth factor receptor kit (SCFR) protein, a member of the tyrosine kinase family of growth factor receptors, as well as c‐KIT mRNA. The transfer of these factors to lung adenocarcinoma cells leads to enhanced proliferation of
recipient tumor cells via the activation of the PI3K signaling pathway[53]. Furthermore, Liang et al. have also demonstrated that the levels of miR‐223 in platelets and platelet‐derived EVs are significantly increased during the progression of disease in lung cancer patients. They have also shown that platelets from lung cancers patients are able to remotely modulate the invasion of lung cancer cells through the release of miR‐ 223‐containing platelet‐derived EVs [54]. Moreover, Janowska‐Wieczorek et al. have shown that platelet‐ derived EVs could interact with lung cancer cells and enhance their metastatic and angiogenic potential [55]. Lung epithelial cells in the airways and alveoli are the first sites of exposure to environmental stimuli such as cigarette smoke, and the subsequent release of various inflammatory mediators by these cells influences the cancer microenvironment [56]. Lung epithelial cells are also considered to be a major producer of EVs in lung and contribute to lung homeostasis and lung cancer pathogenesis [57‐59]. Several studies have analyzed how epithelial cell‐derived EVs alter the lung cancer microenvironment. For example, Liu et al. have revealed that exosomal miR‐21‐mediated angiogenesis plays a role in cigarette smoke exposure (CSE)‐ induced tumorigenesis [60]. In the study, CSE has been found to activate the IL‐6/STAT3 signaling pathway, which increases miR‐21 levels in both human bronchial epithelial cells and their exosomes. In response to CSE, exosomal miR‐21 also increases VEGF levels in endothelial cells, thereby inducing angiogenesis and consequently tumorigenesis. In addition, our group has shown that six members of the let‐7 family (let‐7a, let‐7b, let‐7c, let‐7f, let‐7g, and let‐7i) are significantly downregulated in human bronchial epithelial cell‐ derived EVs after CSE [61,62]. In lung tumors, the expression levels of let‐7 family members are generally lower than those in normal lung tissues [63]. These alterations in the levels of tumor suppressive miRNAs have been recognized to contribute to lung tumor development. Moreover, Kim et al. have reported that TGF‐β1 induced EMT changes the levels of exosomal proteins and miRNAs (for instance, miR‐23a) in A549 human adenocarcinoma alveolar basal epithelial cells, thereby inducing EMT‐like phenotypic changes in A549 cells [64]. Furthermore, Xu et al. have reported that arsenite‐transformed human bronchial epithelial cells release miR‐21‐enriched exosomes, which lead to the proliferation of neighboring normal human bronchial epithelial cells [65]. Arsenite, an environmental carcinogen, invokes cellular miR‐21 expression, which is involved in the malignant transformation of human bronchial epithelial cells [66]. Taken together,
these results suggest that non‐cancer cell‐derived EVs facilitate communication between lung cancer cells and other non‐cancer cells via EV transfer, thereby contributing to lung cancer pathogenesis (Figure 2). 2.3 The roles of EVs in drug resistance Drug resistance is associated with multifactorial problems, and exosomes are involved in the underlying mechanism. Several studies have investigated the involvement of EVs in the resistance of lung cancer to drug (cisplatin and EGFR‐TKIs) and radiation therapy. For example, Xiao et al. have shown that exposure of A549 cells to cisplatin enhances exosome release and influences cisplatin resistance in a paracrine manner [67]. This result suggests that changes in the contents of EVs may mediate the resistance of A549 cells to cisplatin. In addition, Li et al. have demonstrated the roles of lung cancer‐derived EVs in the resistance to concomitant administration of cisplatin and the EGFR‐TKI gefitinib [68]. The authors have shown that exosomes derived from gefitinib‐treated lung cancer cells (GEVs) significantly reduce cisplatin‐induced apoptosis, growth arrest and autophagic activity. In contrast, exosomes derived from cisplatin‐treated lung cancer cells have no significant effects on gefitinib‐induced apoptosis, growth arrest or autophagy. Inhibition of exosome secretion also exhibit synergistic effects with concomitant gefitinib and cisplatin. These results suggest that GEVs may play an important role in the antagonistic effects of gefitinib and cisplatin. In addition, work by Kwang Pyo Kim’s group has identified the contents of gefitinib‐resistant lung cancer cell‐derived EVs. The researchers first characterized the proteome of EVs associated with gefitinib resistance and identified AKT/mTOR signaling mediators as key EV‐derived factors that enhanced drug resistance [69]. They then characterized the lipidomic profile of EVs associated with gifitinib resistance and found 35 differentially regulated lipids. These findings indicate that EVs may serve as biomarkers for predicting clinical response to chemotherapy. Furthermore, Mijn et al. have measured the levels of AKT and ERK1/2 kinase phosphorylation in circulating EVs to evaluate their biomarker potential [70]. Although the authors found increased amounts of proteins in EVs derived from cancer patients compared with those from healthy controls, no significant differences were observed in the levels of AKT and ERK1/2 phosphorylation. In addition, in a subgroup of 12 patients with Kirsten rat sarcoma viral oncogene homolog (KRAS) mutant NSCLC treated with sorafenib/metformin, high AKT levels were associated with treatment failure and poor
survival. Together, these results suggest that although EVs have potential as biomarkers of drug resistance, further investigation is needed to assess their effectiveness. 3. Cancer detection by using exosomal proteins Lung cancer has high prevalence and mortality rates [1,53]. According to the American Cancer Society, the five‐year survival rates of NSCLC are related to tumor stage at diagnosis, and range from over 45% for stage IA and IB disease, to less than 5% for stage IV disease [71]. Most lung cancer patients with symptoms of local or metastatic disease are difficult to cure, thus suggesting a potential benefit of early lung cancer detection. To date, lung cancer screening with low‐dose computed tomography (LDCT) significantly reduces lung cancer burden [72,73]. However, LDCT also has potentially harmful drawbacks including overdiagnosis, radiation exposure, and patient distress. In addition, in recent years, the identification of oncogenic activation of tyrosine kinases such as EGFR and ALK in some advanced NSCLC tumors has led to a paradigm shift and the development of specific molecular treatments for patients [2,3]. At present, the identification of these patient subsets requires invasive sampling of tissue or pleural fluids. For these reasons, intensive searches have been conducted for biomarkers of lung cancer. Therefore, non‐ or minimally invasive diagnostic biomarkers with high sensitivities and specificities for lung cancer are a crucial clinical need. Recently, EVs have been identified as novel disease biomarkers for several reasons. First, EVs reflect the physiological state and microenvironment of their parental cells, and most cells secrete EVs that contain specific proteins, nucleic acids and lipids [74,75]. Second, EVs are found in blood and other bodily fluids. Third, EVs are very stable in the extracellular environment because of their phospholipid bilayers. To date, numerous circulating EV‐derived miRNAs and proteins have been investigated as potentially useful diagnostic and/or prognostic biomarkers for various diseases, especially for cancer [15,76,77]. For example, a pioneering study by Taylor et al. have demonstrated that the levels of eight miRNAs within circulating tumor derived EVs have diagnostic value in ovarian cancer [78]. Furthermore, Ogata‐Kawata et al. have showed that serum levels of seven EV miRNAs are significantly higher in primary colorectal cancer patients compared with healthy controls and are downregulated after surgical resection of tumors [79]. Huang et al. have shown that levels of miR‐1290 and miR‐375 significantly associate with survival in prostate cancer [80]. EVs also have potential as biomarkers for the detection of oncogenic mutations. Whole genome sequencing
have revealed that circulating EVs are enriched with high molecular weight double‐stranded DNA. Importantly, driver mutations have been identified in the circulating EVs [81,82]. In addition to nucleic acids, EV‐derived proteins have been the subject of intense research. Yoshioka et al. have shown that CD147 and CD9 double‐positive EVs are significantly higher in serum from colorectal cancer patients compared with healthy controls [9]. Similar to other cancer types, a number of EV lung cancer biomarkers have been identified in recent years. In this section, we focus on EV‐derived proteins (Table 1), as details regarding EV‐ derived miRNA biomarkers of lung cancer have been described in our past review articles [83]. Previous studies have revealed that cancer‐derived EVs contain various proteins that play a critical role in the pathogenesis of lung cancer. To identify EV‐derived protein biomarkers of lung cancer, proteomic analyses can be conducted on cancer cell lines and bodily fluids, such as plasma, pleural effusion, urine, and saliva, of lung cancer patients. Using Nano‐ELS‐MS/MS, Clark et al. have examined the differential protein profiles of exosomes from a normal bronchial epithelial cell line and two NSCLC cell lines harboring mutations in KRAS or EGFR [84]. The authors have shown that cell adhesion proteins, extracellular matrix proteins, proteases, and cell signaling molecules are enriched in NSCLC exosomes. In addition, several proteins such as EGFR and KRAS known to be involved in lung tumorigenesis have been identified in lung cancer‐derived exosomes. To confirm whether exosomal EGFR in conditioned medium and plasma can be detected and used to facilitate lung cancer diagnoses, Yamashita et al. have evaluated exosomal EGFR expression with an ELISA with an anti‐CD81 capture antibody. Whereas levels of soluble EGFR in plasma were not significantly different between lung cancer patients and normal controls, remarkably high exosomal EGFR expression levels were observed in five of nine cancer patients [85]. Moreover, Huang et al. have investigated the expression of EGFR in exosomes purified from lung biopsies. A western blot analysis showed that greater than 80% of exosomes from patients with lung cancer were EGFR‐positive, whereas less than 2% of exosomes from patients with chronic inflammation were EGFR positive [23]. These results indicate that EV‐derived proteins such as EGFR may be potential biomarkers that may facilitate cancer detection and may serve as companion diagnostics for response to EGFR‐TKIs. On the basis of these results, several studies have explored the potential of serum exosomes as diagnostic biomarkers of lung cancer. In 2014, Ueda et al. examined EV‐derived protein biomarkers in sera from lung
cancer patients [86]. First, they analyzed EVs isolated from 46 serum samples by anti‐CD9‐mass spectrometric immunoassay tips and LC‐MS/MS. This analysis identified 1,369 proteins, of which 18 were exosome‐derived lung cancer biomarker candidates. After further validation of 212 samples with a CD9‐ CD91 sandwich ELISA, the authors next showed that CD91 is significantly elevated in exosomes. With a cutoff value of 2.04 U/exosome, the area under the curve (AUC), sensitivity, and specificity of exosomal CD91 were 0.724, 0.60, and 0.89, respectively. In 2015, Jakobsen et al. have used an EV array to examine plasma exosomes from 109 NSCLC patients and 110 non‐cancer patients [87]. The results showed that a 30‐marker model had the largest AUC of 0.83, a sensitivity of 0.75 and a specificity of 0.76. In 2016, Sandfeld‐Paulsen et al. also used an EV array to analyze plasma from 581 lung cancer patients (431 with lung cancer and 150 controls) [88]. CD151, CD171, and tetraspanin 8 (TSPAN8) were the markers that most strongly differentiated cancer patients with any histologic subtype from individuals without cancer (CD151: AUC = 0.68, CD171: AUC = 0.60, and TSPAN8: AUC = 0.60). The AUC of 10 markers was 0.74 [70,88]. Several EV‐derived proteins in other bodily fluids have been shown to be putative biomarkers of lung cancer. Park et al. have analyzed EVs from the pleural effusion of NSCLC patients by using one‐dimensional SDS‐PAGE followed by nano‐LC‐MS/MS [89]. Bioinformatic analyses of the MS data identified pathologically relevant proteins and potential diagnostic markers for NSCLC, including proteins related to EGFR and KRAS signaling. In addition, Li et al. have investigated the proteome of lung cancer‐derived EVs in urine samples of normal controls and NSCLC patients by using one‐dimensional LC‐MS/MS [90]. They identified 18 proteins and focused on leucine‐rich alpha‐2‐glycoprotein (LRG1). The authors then validated LRG1 in the lung tissue of NSCLC patients. LRG1 is an essential physiological and pathological protein involved in protein‐protein interactions, signal transduction, cell adhesion, development, and angiogenesis [91,92]. LRG1 has also been detected in the sera of patients with lung cancer [93]. Moreover, Sun et al. have analyzed the proteome of salivary EVs in three lung cancer patients and normal controls by shotgun proteomic analysis [94]. In that study, 113 and 95 proteins were identified in the cancer group and the control group, respectively. Among these proteins, 63 proteins were discovered only in the cancer patients. Twelve of these proteins were lung cancer‐related markers, including 6 annexin protein family members. The annexin proteins are thought to be involved in cell migration [95].
Interestingly, EVs have been analyzed in a phase 1/2 lung cancer clinical trial. Yu et al. have assessed the effects of combination therapy with a JAK and an EGFR inhibitor for patients with EGFR‐mutant NSCLC with acquired resistance to EGFR‐TKIs [96]. JAK signaling has been implicated in modulating a response and resistance to EGFR‐TKIs [97]. In this study, combination therapy was well tolerated but ineffective. Study of plasma exosomes from the plasma of NSCLC patients indicated that EGFR levels decreased after the initiation of therapy in 4 out of 17 samples. Remarkably, the three patients with the longest progression‐free survival (PFS) experienced a reduction in exosomal EGFR levels two weeks into the study. Although further investigation is needed, and several technical and scientific obstacles must be overcome before clinical applications for EVs can developed, these data demonstrate that EV‐derived proteins in various bodily fluids have potential as lung cancer biomarkers. 4. EVs are Therapeutic Targets for Lung Cancer Numerous studies have demonstrated that EVs have crucial roles in cancer biology and may have therapeutic applications. There are two potential therapeutic strategies using EVs: (1) eliminating the EVs that contain nucleic acids or proteins that mediate intracellular communication and disease pathogenesis and (2) using EVs as a source of lung regenerating or immunomodulatory agents or as a drug delivery vehicles. Eliminating EVs may be achieved by the following therapeutic approaches: capturing circulating EVs, disrupting EV uptake by recipient cells, and inhibiting EV production or secretion [77]. For instance, Kosaka et al. have revealed that inhibiting exosomes secretion via nSMase2 knockdown in cancer cells suppresses tumor metastasis in a xenograft breast cancer model [14]. Recently Nishida‐Aoki et al have also shown that administration of human‐specific anti‐CD9 and anti‐CD63 antibodies, which are consider to be enriched at the surface of exosomes, decreases metastasis in a xenograft breast cancer model [98]. However, there are as yet no available data on EV elimination in lung cancer. Under the latter strategies, the use of dendritic cell‐derived exosomes (Dex) as anticancer vaccinations is one possibility. Dex can present tumor‐associated antigens and trigger T cell activation. Dex membranes harbor a wide range of immune response proteins such as antigen presentation (e.g., MHC I, II and CD1) molecules and co‐stimulatory (e.g., CD86 and CD40) molecules [99‐101]. Over the past decade, studies have sought to use Dex in clinical cell‐free cancer vaccines [102]. In lung cancer, phase I and II clinical trials using
Dex have been performed. In 2005, Morse et al. performed a phase I study to test the safety, feasibility and efficacy of IFN‐γ in free DEX loaded with MAGE tumor antigens in patients with NSCLC. Thirteen patients were enrolled, and 9 completed therapy. The Dex therapy was feasible and well‐tolerated. Some patients experienced long term disease stability. Besse et al. have conducted a phase II clinical trial to test the clinical benefits of INF‐γ‐Dex loaded with MHC class I‐ and II‐restricted cancer antigens in 22 NSCLC patients. Grade three hepatotoxicity occurred in one patient. The median overall survival (OS) for all patients was 15 months, with 6‐month, 1‐year, and 2‐year survival rates of 86%, 55% and 25%, respectively. The median PFS for all 22 patients was 2.2 months, indicating no objective tumor response. Although EVs can stimulate immune responses and promote anti‐tumor responses, these results indicate limited clinical outcomes. The use of MSC‐derived EVs is another example of EV‐mediated cancer therapy. Recently, MSCs have been found to orchestrate tissue regeneration, anti‐inflammation and/or immunosuppression. Therefore, much attention has been paid to identifying the roles and the potential clinical uses of MSCs in cancer progression and treatment. To date, numerous studies have suggested that MSC‐derived EVs (MSC‐EVs) may be an alternative to MSCs, and extensive research has investigated the use of MSC‐EVs for cancer therapy. Although no studies have administered MSC‐EVs to lung cancer patients, Kalimuthu et al. have recently developed bioluminescent EVs using Renilla luciferase (Rluc)‐expressing MSCs [103]. When the EVs from MSC/Rluc cells (EV‐MSC/Rluc) were visualized in a murine lung cancer model, EV‐MSC/Rluc exerted a therapeutic effect on LLC cells both in vitro and in vivo. EVs have also attracted considerable attention as drug delivery vehicles, because EVs have unique characteristics such as a lipid bilayer architecture, decreased immunogenicity and toxicity, nano‐scale size and a specific composition that minimizes recognition by the mononuclear phagocyte system and stability in the blood circulation and tissue [102]. To date, EV‐mediated delivery of chemotherapeutic agents such as doxorubicin and paclitaxel has been attempted [104,105]. Recently, Aqil et al. have reported the chemotherapeutic potential of EVs loaded with Celastrol (CEL) in lung cancer [106]. CEL isolated from the herb Tripterygium wilfordii is a terpenoid known to inhibit Hsp90 and NF‐κB activation, thereby exerting anti‐proliferative and anti‐tumor effects [107]. The authors have demonstrated that bovine raw milk‐derived
EVs loaded with CEL exhibits in vitro and in vivo antitumor activities via inhibition of cell‐cycle progression and NF‐κB activation, as well as via the induction of chronic ER stress‐mediated apoptosis. 5. Conclusions EVs are important regulators of cell‐cell communication in both physiologic and pathologic processes. The findings described herein confirm that EVs play a pivotal role in the pathogenesis of lung cancer. Although the use of EVs as lung cancer biomarkers and therapeutics is promising, further research and development are needed. We hope that the clinical use of EVs will help patients with lung cancer management and treatment in the future. Acknowledgements: This work was supported in part by a Grant‐in‐Aid for the Japan Science and Technology Agency (JST), administered through the Center of Open Innovation Network for Smart Health (COINS) and initiated by the Council for Science. This work was also funded by a Grant‐in‐Aid from the Basic Science and Platform Technology Program for Innovative Biological Medicine and the Project for Cancer Research and Therapeutic Evolution (P‐CREATE). Conflicts of Interest: The authors declare no conflicts of interest.
Reference [1]
Global Burden of Disease Cancer Collaboration, C. Fitzmaurice, D. Dicker, A. Pain, H. Hamavid, M. Moradi‐Lakeh, et al., The Global Burden of Cancer 2013, JAMA Oncol. 1 (2015) 505–527. doi:10.1001/jamaoncol.2015.0735.
[2]
M. Maemondo, A. Inoue, K. Kobayashi, Gefitinib or chemotherapy for non–small‐cell lung cancer with mutated EGFR, N. Engl. J. Med. 362 (2010) 2380–2388. doi: 10.1056/NEJMoa0909530.
[3]
A.T. Shaw, D.‐W. Kim, K. Nakagawa, T. Seto, L. Crinó, M.‐J. Ahn, et al., Crizotinib versus Chemotherapy in Advanced ALK‐Positive Lung Cancer, N. Engl. J. Med. 368 (2013) 2385–2394. doi:10.1056/NEJMoa1214886.
[4]
H. Borghaei, L. Paz‐Ares, L. Horn, D.R. Spigel, M. Steins, N.E. Ready, et al., Nivolumab versus Docetaxel in Advanced Nonsquamous Non–Small‐Cell Lung Cancer, N. Engl. J. Med. 373 (2015) 1627–1639. doi:10.1056/NEJMoa1507643.
[5]
M. Yáñez‐Mó, P.R.M. Siljander, Z. Andreu, A.B. Zavec, F.E. Borràs, E.I. Buzas, et al., Biological properties of extracellular vesicles and their physiological functions, Journal of Extracellular Vesicles. 4 (2015) 27066. doi:10.3402/jev.v4.27066.
[6]
G. Raposo, W. Stoorvogel, Extracellular vesicles: exosomes, microvesicles, and friends, J Cell Biol. 200 (2013) 373–383. doi:10.1083/jcb.201211138.
[7]
P.D. Robbins, A.E. Morelli, Regulation of immune responses by extracellular vesicles, Nat Rev Immunol. 14 (2014) 195–208. doi:10.1038/nri3622.
[8]
M. Colombo, G. Raposo, C. Théry, Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles, Annu. Rev. Cell Dev. Biol. 30 (2014) 255–289. doi:10.1146/annurev‐cellbio‐101512‐122326.
[9]
Y. Yoshioka, Y. Konishi, N. Kosaka, T. Katsuda, T. Kato, T. Ochiya, Comparative marker analysis of extracellular vesicles in different human cancer types, Journal of Extracellular Vesicles. 2 (2013). doi:10.3402/jev.v2i0.20424.
[10]
R.J. Simpson, J.W. Lim, R.L. Moritz, S. Mathivanan, Exosomes: proteomic insights and diagnostic potential, Expert Rev Proteomics. 6 (2009) 267–283. doi:10.1586/epr.09.17.
[11]
V. Muralidharan‐Chari, J.W. Clancy, A. Sedgwick, C. D'Souza‐Schorey, Microvesicles: mediators of extracellular communication during cancer progression, Journal of Cell Science. 123 (2010) 1603– 1611. doi:10.1242/jcs.064386.
[12]
B. György, T.G. Szabó, M. Pásztói, Z. Pál, P. Misják, B. Aradi, et al., Membrane vesicles, current state‐of‐the‐art: emerging role of extracellular vesicles, Cell. Mol. Life Sci. 68 (2011) 2667–2688. doi:10.1007/s00018‐011‐0689‐3.
[13]
S.J. Gould, G. Raposo, As we wait: coping with an imperfect nomenclature for extracellular vesicles, Journal of Extracellular Vesicles. 2 (2013). doi:10.3402/jev.v2i0.20389.
[14]
N. Kosaka, H. Iguchi, Y. Yoshioka, F. Takeshita, Y. Matsuki, T. Ochiya, Secretory mechanisms and intercellular transfer of microRNAs in living cells, J. Biol. Chem. 285 (2010) 17442–17452. doi:10.1074/jbc.M110.107821.
[15]
Y. Fujita, Y. Yoshioka, T. Ochiya, Extracellular vesicle transfer of cancer pathogenic components, Cancer Science. 107 (2016) 385–390. doi:10.1111/cas.12896.
[16]
M.P. Caby, Exosomal‐like vesicles are present in human blood plasma, International Immunology. 17 (2005) 879–887. doi:10.1093/intimm/dxh267.
[17]
D. Hanahan, R.A. Weinberg, Hallmarks of Cancer: The Next Generation, Cell. 144 (2011) 646–674. doi:10.1016/j.cell.2011.02.013.
[18]
R.D. Schreiber, L.J. Old, M.J. Smyth, Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion, Science. 331 (2011) 1565–1570. doi:10.1126/science.1203486.
[19]
M.W.L. Teng, J. Galon, W.‐H. Fridman, M.J. Smyth, From mice to humans: developments in cancer immunoediting, J. Clin. Invest. 125 (2015) 3338–3346. doi:10.1172/JCI80004.
[20]
M. Fabbri, A. Paone, F. Calore, R. Galli, E. Gaudio, R. Santhanam, et al., MicroRNAs bind to Toll‐like receptors to induce prometastatic inflammatory response, Proc. Natl. Acad. Sci. U.S.a. 109 (2012) E2110–6. doi:10.1073/pnas.1209414109.
[21]
G. Berchem, M.Z. Noman, M. Bosseler, J. Paggetti, S. Baconnais, E. Le Cam, et al., Hypoxic tumor‐ derived microvesicles negatively regulate NK cell function by a mechanism involving TGF‐b and miR23a transfer, OncoImmunology. 5 (2016) 1–13. doi:10.1080/2162402X.2015.1062968.
[22]
A. Cohnen, S.C. Chiang, A. Stojanovic, H. Schmidt, M. Claus, P. Saftig, et al., Surface CD107a/LAMP‐1 protects natural killer cells from degranulation‐associated damage, Blood. 122 (2013) 1411–1418. doi:10.1182/blood‐2012‐07‐441832.
[23]
S.‐H. Huang, Y. Li, J. Zhang, J. Rong, S. Ye, Epidermal Growth Factor Receptor‐Containing Exosomes Induce Tumor‐Specific Regulatory T Cells, Cancer Investigation. 31 (2013) 330–335. doi:10.3109/07357907.2013.789905.
[24]
M. Yu, A. Bardia, B.S. Wittner, S.L. Stott, M.E. Smas, D.T. Ting, et al., Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition, Science. 339 (2013) 580– 584. doi:10.1126/science.1228522.
[25]
K.R. Fischer, A. Durrans, S. Lee, J. Sheng, F. Li, S.T.C. Wong, et al., Epithelial‐to‐mesenchymal transitionis not required for lung metastasis butcontributes to chemoresistance, Nature. 527 (2015) 472–476. doi:10.1038/nature15748.
[26]
X. Zheng, J.L. Carstens, J. Kim, M. Scheible, J. Kaye, H. Sugimoto, et al., Epithelial‐to‐mesenchymal transitionis dispensable for metastasis but induceschemoresistance in pancreatic cancer, Nature. 527 (2015) 525–530. doi:10.1038/nature16064.
[27]
M.A. Rahman, J.F. Barger, F. Lovat, M. Gao, G.A. Otterson, P. Nana‐Sinkam, Lung cancer exosomes as drivers of epithelial mesenchymal transition, Oncotarget. (2016). doi:10.18632/oncotarget.10243.
[28]
M.E. Kidd, D.K. Shumaker, K.M. Ridge, The Role of Vimentin Intermediate Filaments in the Progression of Lung Cancer, Am J Respir Cell Mol Biol. (2013) 130827094923003. doi:10.1165/rcmb.2013‐0314TR.
[29]
R. Kalluri, The biology and function offibroblasts in cancer, Nature Publishing Group. 16 (2016) 582–598. doi:10.1038/nrc.2016.73.
[30]
L. Rønnov‐Jessen, O.W. Petersen, Induction of alpha‐smooth muscle actin by transforming growth factor‐beta 1 in quiescent human breast gland fibroblasts. Implications for myofibroblast generation in breast neoplasia, Lab. Invest. 68 (1993) 696–707.
[31]
A. Gutkin, O. Uziel, E. Beery, J. Nordenberg, M. Pinchasi, H. Goldvaser, et al., Tumor cells derived exosomes contain hTERT mRNA and transform nonmalignant fibroblasts into telomerase positive cells, Oncotarget. (2016). doi:10.18632/oncotarget.10384.
[32]
J.W. Shay, W.E. Wright, Role of telomeres and telomerase in cancer, Semin. Cancer Biol. 21 (2011) 349–353. doi:10.1016/j.semcancer.2011.10.001.
[33]
M. Wysoczynski, M.Z. Ratajczak, Lung cancer secreted microvesicles: Underappreciated modulators of microenvironment in expanding tumors, Int. J. Cancer. 125 (2009) 1595–1603. doi:10.1002/ijc.24479.
[34]
P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other diseases, Nature. 407 (2000) 249–257. doi:10.1038/35025220.
[35]
G. Bergers, L.E. Benjamin, Angiogenesis: Tumorigenesis and the angiogenic switch, Nat. Rev. Cancer. 3 (2003) 401–410. doi:10.1038/nrc1093.
[36]
J. Plouët, J. Schilling, D. Gospodarowicz, Isolation and characterization of a newly identified endothelial cell mitogen produced by AtT‐20 cells, Embo J. 8 (1989) 3801–3806.
[37]
N. Ferrara, W.J. Henzel, Pituitary follicular cells secrete a novel heparin‐binding growth factor specific for vascular endothelial cells, Biochem. Biophys. Res. Commun. 161 (1989) 851–858.
[38]
H. Cui, B. Seubert, E. Stahl, H. Dietz, U. Reuning, L. Moreno‐Leon, et al., Tissue inhibitor of metalloproteinases‐1 induces a pro‐tumourigenic increase of miR‐210 in lung adenocarcinoma cells and their exosomes, Oncogene. 34 (2015) 3640–3650. doi:10.1038/onc.2014.300.
[39]
X.‐W. Liu, M.E. Taube, K.‐K. Jung, Z. Dong, Y.J. Lee, S. Roshy, et al., Tissue inhibitor of metalloproteinase‐1 protects human breast epithelial cells from extrinsic cell death: a potential oncogenic activity of tissue inhibitor of metalloproteinase‐1, Cancer Research. 65 (2005) 898–906.
[40]
P. Fasanaro, Y. D'Alessandra, V. Di Stefano, R. Melchionna, S. Romani, G. Pompilio, et al., MicroRNA‐210 modulates endothelial cell response to hypoxia and inhibits the receptor tyrosine kinase ligand Ephrin‐A3, J. Biol. Chem. 283 (2008) 15878–15883. doi:10.1074/jbc.M800731200.
[41]
K. Valencia, D. Luis‐Ravelo, N. Bovy, I. Antón, S. Martínez‐Canarias, C. Zandueta, et al., ScienceDirect, Molecular Oncology. 8 (2014) 689–703. doi:10.1016/j.molonc.2014.01.012.
[42]
J.‐J. Sheu, F.‐Y. Lee, C.G. Wallace, T.‐H. Tsai, S. Leu, Y.‐L. Chen, et al., Administered circulating microparticles derived from lung cancer patients markedly improved angiogenesis, blood flow and ischemic recovery in rat critical limb ischemia, J Transl Med. 13 (2015) 59. doi:10.1186/s12967‐015‐0381‐8.
[43]
K. Al‐Nedawi, B. Meehan, R.S. Kerbel, A.C. Allison, J. Rak, Endothelial expression of autocrine VEGF upon the uptake of tumor‐derived microvesicles containing oncogenic EGFR, Proc. Natl. Acad. Sci. U.S.a. 106 (2009) 3794–3799. doi:10.1073/pnas.0804543106.
[44]
A. Uccelli, L. Moretta, V. Pistoia, Mesenchymal stem cells in health and disease, Nat Rev Immunol. 8 (2008) 726–736. doi:10.1038/nri2395.
[45]
A.E. Karnoub, A.B. Dash, A.P. Vo, A. Sullivan, M.W. Brooks, G.W. Bell, et al., Mesenchymal stem cells within tumour stroma promote breast cancer metastasis, Nature. 449 (2007) 557–563. doi:10.1038/nature06188.
[46]
X. Li, S. Wang, R. Zhu, H. Li, Q. Han, R.C. Zhao, Lung tumor exosomes induce a pro‐ inflammatory phenotype in mesenchymalstem cells via NFκB‐TLR signaling pathway, Journal of Hematology & Oncology. (2016) 1–12. doi:10.1186/s13045‐016‐0269‐y.
[47]
C. Neuzillet, A. Tijeras‐Raballand, R. Cohen, J. Cros, S. Faivre, E. Raymond, et al., Pharmacology & Therapeutics, Pharmacology and Therapeutics. 147 (2015) 22–31. doi:10.1016/j.pharmthera.2014.11.001.
[48]
M. Del Tatto, T. Ng, J.M. Aliotta, G.A. Colvin, M.S. Dooner, D. Berz, et al., Marrow cell genetic phenotype change induced by human lung cancer cells, Experimental Hematology. 39 (2011) 1072–1080. doi:10.1016/j.exphem.2011.08.008.
[49]
Y. Zhang, Q. Liu, M. Zhang, Y. Yu, X. Liu, X. Cao, Fas Signal Promotes Lung Cancer Growth by Recruiting Myeloid‐Derived Suppressor Cells via Cancer Cell‐Derived PGE2, J. Immunol. 182 (2009) 3801–3808. doi:10.4049/jimmunol.0801548.
[50]
L. Chen, S.‐M. Park, A.V. Tumanov, A. Hau, K. Sawada, C. Feig, et al., CD95 promotes tumour growth, Nature. 465 (2010) 492–496. doi:10.1038/nature09075.
[51]
S.L. Peng, M.E. Robert, A.C. Hayday, J. Craft, A tumor‐suppressor function for Fas (CD95) revealed in T cell‐deficient mice, J. Exp. Med. 184 (1996) 1149–1154.
[52]
Z. Cai, F. Yang, L. Yu, Z. Yu, L. Jiang, Q. Wang, et al., Activated T Cell Exosomes Promote Tumor Invasion via Fas Signaling Pathway, J. Immunol. 188 (2012) 5954–5961. doi:10.4049/jimmunol.1103466.
[53]
H. Xiao, C. Lässer, G.V. Shelke, J. Wang, M. Rådinger, T.R. Lunavat, et al., Mast cell exosomes promote lung adenocarcinoma cell proliferation ‐ role of KIT‐stem cell factor signaling, Cell Commun. Signal. 12 (2014) 64. doi:10.1186/s12964‐014‐0064‐8.
[54]
H. Liang, X. Yan, Y. Pan, Y. Wang, N. Wang, L. Li, et al., MicroRNA‐223 delivered by platelet‐derived microvesicles promotes lung cancer cell invasion via targeting tumor suppressor EPB41L3, Mol. Cancer. 14 (2015) 58. doi:10.1186/s12943‐015‐0327‐z.
[55]
A. Janowska‐Wieczorek, M. Wysoczynski, J. Kijowski, L. Marquez‐Curtis, B. Machalinski, J. Ratajczak, et al., Microvesicles derived from activated platelets induce metastasis and angiogenesis in lung cancer, Int. J. Cancer. 113 (2004) 752–760. doi:10.1002/ijc.20657.
[56]
A. Houghton, Mechanistic links between COPD and lung cancer, Nat. Rev. Cancer. (2013). doi:10.1038/nrc3477.
[57]
A. Kulshreshtha, T. Ahmad, A. Agrawal, B. Ghosh, Proinflammatory role of epithelial cell‐derived exosomes in allergic airway inflammation, J. Allergy Clin. Immunol. 131 (2013) 1194–203– 1203.e1–14. doi:10.1016/j.jaci.2012.12.1565.
[58]
Y. Fujita, N. Kosaka, J. Araya, K. Kuwano, T. Ochiya, Extracellular vesicles in lung microenvironment and pathogenesis, Trends in Molecular Medicine. 21 (2015) 533–542. doi:10.1016/j.molmed.2015.07.004.
[59]
T. Kadota, Y. Fujita, Y. Yoshioka, J. Araya, K. Kuwano, T. Ochiya, Extracellular Vesicles in Chronic Obstructive Pulmonary Disease, Int J Mol Sci. 17 (2016) 1801. doi:10.3390/ijms17111801.
[60]
Y. Liu, F. Luo, B. Wang, H. Li, Y. Xu, X. Liu, et al., Cancer Letters, Cancer Letters. 370 (2016) 125– 135. doi:10.1016/j.canlet.2015.10.011.
[61]
Y. Fujita, J. Araya, S. Ito, K. Kobayashi, N. Kosaka, Y. Yoshioka, et al., Suppression of autophagy by extracellular vesicles promotes myofibroblast differentiation in COPD pathogenesis, Journal of Extracellular Vesicles. 4 (2015). doi:10.1083/jcb.200412022.
[62]
Y. Fujita, J. Araya, T. Ochiya, Extracellular vesicles in smoking‐related lung diseases, Oncotarget. 6 (2015) 43144–43145. doi:10.18632/oncotarget.6556.
[63]
H. Osada, T. Takahashi, let‐7 and miR‐17‐92: Small‐sized major players in lung cancer development, Cancer Science. 102 (2010) 9–17. doi:10.1111/j.1349‐7006.2010.01707.x.
[64]
J. Kim, T.Y. Kim, M.S. Lee, J.Y. Mun, C. Ihm, S.A. Kim, Exosome cargo reflects TGF‐β1‐mediated epithelial‐to‐mesenchymal transition (EMT) status in A549 human lung adenocarcinoma cells, Biochem. Biophys. Res. Commun. 478 (2016) 643–648. doi:10.1016/j.bbrc.2016.07.124.
[65]
Y. Xu, F. Luo, Y. Liu, Le Shi, X. Lu, W. Xu, et al., Exosomal miR‐21 derived from arsenite‐ transformed human bronchial epithelial cells promotes cell proliferation associated with arsenite carcinogenesis, Archives of Toxicology. (2015) 1–12. doi:10.1007/s00204‐014‐1291‐x.
[66]
F. Luo, Y. Xu, M. Ling, Y. Zhao, W. Xu, X. Liang, et al., Toxicology and Applied Pharmacology, Toxicology and Applied Pharmacology. 273 (2013) 27–34. doi:10.1016/j.taap.2013.08.025.
[67]
X. Xiao, S. Yu, S. Li, J. Wu, R. Ma, H. Cao, et al., Exosomes: decreased sensitivity of lung cancer A549 cells to cisplatin, PLoS ONE. (2014). doi:10.1371/journal.pone.0089534.g001.
[68]
X.‐Q. Li, J.‐T. Liu, L.‐L. Fan, Y. Liu, L. Cheng, F. Wang, et al., Exosomes derived from gefitinib‐ treated EGFR‐mutant lung cancer cells alter cisplatin sensitivity via up‐regulating autophagy, Oncotarget. 7 (2016) 24585–24595. doi:10.18632/oncotarget.8358.
[69]
D.‐Y. Choi, S. You, J.H. Jung, J.C. Lee, J.K. Rho, K.Y. Lee, et al., Extracellular vesicles shed from gefitinib‐resistant nonsmall cell lung cancer regulate the tumor microenvironment, Proteomics. 14 (2014) 1845–1856. doi:10.1002/pmic.201400008.
[70]
J.C. van der Mijn, N. Sol, W. Mellema, C.R. Jimenez, S.R. Piersma, H. Dekker, et al., Analysis of AKT and ERK1/2 protein kinases in extracellular vesicles isolated from blood of patients with cancer, Journal of Extracellular Vesicles. 3 (2014) 569. doi:10.1016/j.cell.2010.03.018.
[71]
Non‐small cell lung cancer survival rates, by stage, Cancer.org. (2016) 1–3. http://www.cancer.org/cancer/lungcancer‐non‐smallcell/detailedguide/non‐small‐cell‐lung‐ cancer‐survival‐rates (accessed September 30, 2016).
[72]
National Lung Screening Trial Research Team, D.R. Aberle, A.M. Adams, C.D. Berg, W.C. Black, J.D. Clapp, et al., Reduced lung‐cancer mortality with low‐dose computed tomographic screening, N. Engl. J. Med. 365 (2011) 395–409. doi:10.1056/NEJMoa1102873.
[73]
L.L. Humphrey, M. Deffebach, M. Pappas, C. Baumann, K. Artis, J.P. Mitchell, et al., Screening for lung cancer with low‐dose computed tomography: a systematic review to update the US Preventive services task force recommendation, Ann. Intern. Med. 159 (2013) 411–420. doi:10.7326/0003‐4819‐159‐6‐201309170‐00690.
[74]
de Jong. OG, Verhaar. MC, Chen. Y, Vader. P, Gremmels. H, Posthuma. G, et al., Cellular stress conditions are reflected in the protein and RNA content of endothelial cell‐derived exosomes, (2012). doi:10.1586/epr.09.17.
[75]
L.A. Beninson, M. Fleshner, Exosomes: an emerging factor in stress‐induced immunomodulation, Semin. Immunol. 26 (2014) 394–401. doi:10.1016/j.smim.2013.12.001.
[76]
Y. Yoshioka, N. Kosaka, Y. Konishi, H. Ohta, H. Okamoto, H. Sonoda, et al., Ultra‐sensitive liquid biopsy of circulating extracellular vesicles using ExoScreen, Nat Commun. 5 (2014) 3591. doi:10.1038/ncomms4591.
[77]
N. Kosaka, Y. Yoshioka, Y. Fujita, T. Ochiya, Versatile roles of extracellular vesicles in cancer, Journal of Clinical Investigation. 126 (2016) 1163–1172. doi:10.1172/JCI81130.
[78]
D.D. Taylor, C. Gerçel‐Taylor, MicroRNA signatures of tumor‐derived exosomes as diagnostic biomarkers of ovarian cancer, Gynecol. Oncol. 110 (2008) 13–21. doi:10.1016/j.ygyno.2008.04.033.
[79]
H. Ogata‐Kawata, M. Izumiya, D. Kurioka, Y. Honma, Y. Yamada, K. Furuta, et al., Circulating Exosomal microRNAs as Biomarkers of Colon Cancer, PLoS ONE. 9 (2014) e92921. doi:10.1371/journal.pone.0092921.s012.
[80]
X. Huang, T. Yuan, M. Liang, M. Du, S. Xia, R. Dittmar, et al., Exosomal miR‐1290 and miR‐375 as prognostic markers in castration‐resistant prostate cancer, Eur. Urol. 67 (2015) 33–41. doi:10.1016/j.eururo.2014.07.035.
[81]
C. Kahlert, S.A. Melo, A. Protopopov, J. Tang, S. Seth, M. Koch, et al., Identification of Double‐ stranded Genomic DNA Spanning All Chromosomes with Mutated KRASand p53DNA in the Serum
Exosomes of Patients with Pancreatic Cancer, J. Biol. Chem. 289 (2014) 3869–3875. doi:10.1074/jbc.C113.532267. [82]
F.A. San Lucas, K. Allenson, V. Bernard, J. Castillo, D.U. Kim, K. Ellis, et al., Minimally invasive genomic and transcriptomic profiling of visceral cancers by next‐generation sequencing of circulating exosomes, Annals of Oncology. 27 (2016) 635–641. doi:10.1093/annonc/mdv604.
[83]
Y. Fujita, K. Kuwano, T. Ochiya, F. Takeshita, The Impact of Extracellular Vesicle‐Encapsulated Circulating MicroRNAs in Lung Cancer Research, BioMed Research International. 2014 (2014) 1–8. doi:10.1016/j.ccr.2014.03.007.
[84]
D.J. Clark, W.E. Fondrie, A. Yang, L. Mao, Triple SILAC quantitative proteomic analysis reveals differential abundance of cell signaling proteins between normal and lung cancer‐derived exosomes, J Proteomics. 133 (2016) 161–169. doi:10.1016/j.jprot.2015.12.023.
[85]
T. Yamashita, H. Kamada, S. Kanasaki, Y. Maeda, K. Nagano, Y. Abe, et al., Epidermal growth factor receptor localized to exosome membranes as a possible biomarker for lung cancer diagnosis, Pharmazie. 68 (2013) 969–973.
[86]
K. Ueda, N. Ishikawa, A. Tatsuguchi, N. Saichi, R. Fujii, H. Nakagawa, Antibody‐coupled monolithic silica microtips for highthroughput molecular profiling of circulating exosomes, Sci. Rep. 4 (2014) 6232. doi:10.1038/srep06232.
[87]
K.R. Jakobsen, B.S. Paulsen, R. Bæk, K. Varming, B.S. Sorensen, M.M. Jørgensen, Exosomal proteins as potential diagnostic markers in advanced non‐small cell lung carcinoma, Journal of Extracellular Vesicles. 4 (2015) 197. doi:10.1371/journal.pone.0005219.
[88]
B.S.‐P. MD, K.R.J. MSc, R.B. MSc, B.H.F.M. PhD, T.R.R.M. PhD, P.M.M. PhD, et al., Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer, Journal of Thoracic Oncology. (2016) 1–10. doi:10.1016/j.jtho.2016.05.034.
[89]
J.O. Park, D.‐Y. Choi, D.‐S. Choi, H.J. Kim, J.W. Kang, J.H. Jung, et al., Identification and characterization of proteins isolated from microvesicles derived from human lung cancer pleural effusions, Proteomics. 13 (2013) 2125–2134. doi:10.1002/pmic.201200323.
[90]
Y. Li, Y. Zhang, F. Qiu, Z. Qiu, Proteomic identification of exosomal LRG1: A potential urinary biomarker for detecting NSCLC, Electrophoresis. 32 (2011) 1976–1983. doi:10.1002/elps.201000598.
[91]
L.C. O'Donnell, L.J. Druhan, B.R. Avalos, Molecular characterization and expression analysis of leucine‐rich alpha2‐glycoprotein, a novel marker of granulocytic differentiation, J. Leukoc. Biol. 72 (2002) 478–485.
[92]
X. Wang, S. Abraham, J.A.G. McKenzie, N. Jeffs, M. Swire, V.B. Tripathi, et al., LRG1 promotes angiogenesis by modulating endothelial TGF‐β signalling, Nature. 499 (2013) 306–311. doi:10.1038/nature12345.
[93]
T. Okano, T. Kondo, T. Kakisaka, K. Fujii, M. Yamada, H. Kato, et al., Plasma proteomics of lung cancer by a linkage of multi‐dimensional liquid chromatography and two‐dimensional difference gel electrophoresis, Proteomics. 6 (2006) 3938–3948. doi:10.1002/pmic.200500883.
[94]
Y. Sun, Z. Xia, Z. Shang, K. Sun, X. Niu, L. Qian, et al., Facile preparation of salivary extracellular vesicles for cancer proteomics, Nature Publishing Group. 6 (2016) 24669. doi:10.1038/srep24669.
[95]
V. Gerke, S.E. Moss, Annexins: From Structure to Function, Physiological Reviews. 82 (2002) 331– 371. doi:10.1152/physrev.00030.2001.
[96]
H.A. Yu, L. Perez, Q. Chang, S.P. Gao, M.G. Kris, G.J. Riely, et al., A phase 1/2 trial of ruxolitinib and erlotinib in patients with EGFR‐mutant lung adenocarcinomas with acquired resistance to erlotinib, J Thorac Oncol. (2016). doi:10.1016/j.jtho.2016.08.140.
[97]
S.P. Gao, Q. Chang, N. Mao, L.A. Daly, R. Vogel, T. Chan, et al., JAK2 inhibition sensitizes resistant EGFR‐mutant lung adenocarcinoma to tyrosine kinase inhibitors, Sci Signal. 9 (2016) ra33. doi:10.1126/scisignal.aac8460.
[98]
N. Nishida‐Aoki, N. Tominaga, F. Takeshita, H. Sonoda, Y. Yoshioka, T. Ochiya, Disruption of Circulating Extracellular Vesicles as a Novel Therapeutic Strategy against Cancer Metastasis, Mol. Ther. 25 (2017) 181–191. doi:10.1016/j.ymthe.2016.10.009.
[99]
C. Théry, M. Ostrowski, E. Segura, Membrane vesicles as conveyors of immune responses, Nat Rev Immunol. 9 (2009) 581–593. doi:10.1038/nri2567.
[100]
D.W. Greening, S.K. Gopal, R. Xu, R.J. Simpson, W. Chen, Exosomes and their roles in immune regulation and cancer, Semin. Cell Dev. Biol. 40 (2015) 72–81. doi:10.1016/j.semcdb.2015.02.009.
[101]
G. Raposo, H.W. Nijman, W. Stoorvogel, R. Liejendekker, C.V. Harding, C.J. Melief, et al., B lymphocytes secrete antigen‐presenting vesicles, J. Exp. Med. 183 (1996) 1161–1172.
[102]
T. Lener, M. Gimona, L. Aigner, V. Börger, E. Buzas, G. Camussi, et al., Applying extracellular vesicles based therapeutics in clinical trials ‐ an ISEV position paper, Journal of Extracellular Vesicles. 4 (2015) 30087. doi:10.1084/jem.183.3.1161.
[103]
S. Kalimuthu, P. Gangadaran, X.J. Li, J.M. Oh, H.W. Lee, S.Y. Jeong, et al., therapeutic potential of mesenchymal stem cell‐derived extracellular vesicles with optical imaging reporter in tumor mice model, Sci. Rep. (2016) 1–11. doi:10.1038/srep30418.
[104]
Y. Tian, S. Li, J. Song, T. Ji, M. Zhu, G.J. Anderson, et al., Biomaterials, Biomaterials. 35 (2014) 2383–2390. doi:10.1016/j.biomaterials.2013.11.083.
[105]
M.S. Kim, M.J. Haney, Y. Zhao, V.M. PhD, I. Deygen, N.L.K. PhD, et al., Development of exosome‐ encapsulated paclitaxel to overcome MDR in cancer cells, Nanomedicine: Nanotechnology, Biology, and Medicine. 12 (2016) 655–664. doi:10.1016/j.nano.2015.10.012.
[106]
F. Aqil, H. Kausar, A.K. Agrawal, J. Jeyabalan, A.‐H. Kyakulaga, R. Munagala, et al., Experimental and Molecular Pathology, Experimental and Molecular Pathology. 101 (2016) 12–21. doi:10.1016/j.yexmp.2016.05.013.
[107]
H. Yang, Celastrol, a Triterpene Extracted from the Chinese “Thunder of God Vine,” Is a Potent Proteasome Inhibitor and Suppresses Human Prostate Cancer Growth in Nude Mice, Cancer Research. 66 (2006) 4758–4765. doi:10.1158/0008‐5472.CAN‐05‐4529.
Figure1: The roles of lung cancer‐derived extracellular vesicles The lung tumor microenvironment is dependent on complex interactions between cancer cells and surrounding non‐cancerous cells such as endothelial cells, immune cells, fibroblasts, epithelial cells, and MSCs. Lung cancer‐derived EVs have the following roles (1) miR‐21 and ‐29a containing EVs can induce tumor‐promoting inflammation via TLR‐mediated NF‐kB activation, whereas miR‐23a, TGF‐β or EGFR containing EVs can suppress cytotoxicity. (2) lung cancer‐derived EVs mediate EMT in epithelial cells via transfer of vimentin. (3) Lung cancer‐derived EVs may contain hTERT and may induce CAF differentiation. (4) Lung cancer‐derived EVs regulate the angiogenesis via miRNA (miR‐210 and ‐192) and protein transfer (EGFR) transfer. (5) Lung cancer cell‐derived exosomes can educate naive MSCs into a novel type of pro‐ inflammatory MSC via direct interactions with exosomal Hsp70, which can support tumor growth. (6) Lung cancer‐derived exosome transfer (for instance, miR‐302b) regulate the proliferation and migration of other cancer cells in the tumor microenvironment. EVs: extracellular vesicles. Figure2: The roles of non‐cancer cell‐derived extracellular vesicles Non‐cancer cells such as endothelial cells, immune cells, epithelial cells, and mast cells contribute to the lung cancer microenvironment via EVs transfer. (1) T cell‐derived EVs can promote tumor immune escape via transfer of FasL. (2) miR‐223 enriched platelet‐derived EVs can modulate the invasion of lung cancer. (3) Mast cells‐derived EVs can enhance proliferation of lung cancer through SCFR protein and c‐KIT mRNA transfer. (4) cigarette smoke extract‐stimulated epithelial cell‐derived EVs can promote angiogenesis via miR‐21 transfer. (5) TGF‐β1 induced EMT can change the exosomal protein and miRNA (for instance, miR‐ 23a) in human alveolar basal epithelial cells, thereby inducing EMT‐like phenotypic changes in other human alveolar basal epithelial cells. EVs: extracellular vesicles, SCFR: mast/stem cell growth factor receptor kit, EMT: epithelial‐mesenchymal transition Table1: Exosomal proteins as potential biomarkers for lung cancer EGFR: epidermal growth factor receptor,KRAS: kirsten rat sarcoma viral oncogene homolog,MSIA: mass spectrometric immunoassay
Figure 1
Figure 2
Tabl Exosomal proteins as potential biomarkers for lung e 1 cancer Bod Purpose Sensitivi y Potential of the ty and Detection Referen ces Flui Biomarker(s) biomark Specifici method ty ds er(s) Sn: 60.0, anti-CD9 Seru Diagnosi Sp: 89.0, CD91 (CD317) MSIA [80] m s AUC tips 0.72 30 exosomal proteins Sn: 75, (Flotilin-1, Diagnosi Sp: 76, The EV Seru ErbB4, [81] AUC: array s m EGFRvIII, N0.83 Cadherin and CD163 etc.) 10 marker models Seru ( CD151, Diagnosi AUC: The EV [82] m CD171, s 0.74 array tetraspanin 8 etc.) ELISA Seru Diagnosi with antiEGFR [79] m s CD81 antibody Treatme Seru Western EGFR nt [90] m blot renponse Drug Seru Western AKT, ERK1/2 resistanc [69] m blot e
Pleu ral EGFR signaling, effu KRAS etc sion
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Urin leucine-rich a-2e glycoprotein
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saliv Annexin family a members etc
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Diagnosi s
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Lun g biop sy
EGFR
Mass spectrome [83] try Mass spectrome [84] try Mass spectrome [88] try Western blot
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