Tumor‐derived exosomes: Key players in non‐small cell lung cancer metastasis and their implication for targeted therapy

Abbreviations TDEs tumor-derived exosomes TME the tumor microenvironment NSCLC non-small cell lung cancer SCLC small cell lung carcinoma EMT epithelial-to-mesenchymal transition EVs extracellular vesicles MVB multivesicular body ILVs intraluminal vesicles miRNA microRNA ESCRT Cryo-TEM, cryo-transmission electron microscopy; endosomal sorting complex required for transport ECM extracellular matrix CAFs cancer-associated fibroblasts HBECs human bronchial epithelial cells MMP matrix metalloproteinase EGFR-TKI epidermal growth factor receptor- tyrosine kinase inhibitor 1 INTRODUCTION

Exosomes are defined as extracellular vesicles (EVs) which play a vital role in cellular communication and disease. The lipid bilayered vesicles are secreted by virtually all types of mammalian cells and carry biomolecules.1 They were first reported in 1983 by Harding et al.2 who depicted the recycling of the transferrin receptor in reticulocytes via endocytosis. In the same year, another group delineated the kinetics and internalization of transferrin receptor in a human hepatoma cell line.3 The term ‘exosomes' was first coined by Johnstone et al.4 in 1987 when this group also described the isolation of vesicles from sheep reticulocytes by ultracentrifugation.

Studies in the past decade have shown a more multifaceted role of exosomes. The functions of exosome are dependent on the cell types that secrete them, but almost all exosomes have some common physical characteristics.5 Electron microscopy analysis reveals that they are cup-shaped spheres surrounded by a lipid bilayer and range between 30 and 100 nm in diameter.6, 7 Exosomes are found in numerous body fluids including plasma, semen, urine, saliva, breast milk, amniotic fluid, ascites fluid, cerebrospinal fluid, and bile.8-16 As intercell communicators, exosome vesicles are a source of genetic material along with proteins and lipids. Exosomes carry DNA, messenger RNA (mRNA), microRNA (miRNA), and long noncoding RNAs that can be taken up by recipient cells.17 The content of exosomes is specific to the cell of origin, which enables signals transmitted from parent cells to the neighboring ones without direct cell–cell contact.18

Exosomes are released by a variety of eukaryotic cell types including cancer cells.19 Tumor-derived exosomes (TDEs) influence tumor invasion and metastasis.20-22 Consequently, exosomes have a pivotal role in cancer progression, and they can be used not only as biomarkers for cancer diagnosis and prognosis but also as drug delivery vehicles and reconfigurable therapeutic systems.

Lung cancer is one of the most frequently diagnosed cancers worldwide. Despite progress in our understanding of risk factors, pathogenesis, diagnostic markers, and therapeutic strategies for lung cancer, it remains the leading cause of cancer-related death in both males and females.23, 24 The 5-year survival of lung cancer is among the worst of all tumor types, varying from 4% to 17%, depending on different tumor stages and regions.25 At the time of diagnosis, lung carcinomas are most often in an advanced/metastatic stage.26 A better understanding of the molecular mechanisms for lung cancer progression and identification of potential therapeutic targets for curtailing metastasis will help to achieve a survival benefit for patients with this fatal disease.

Histologically lung cancer is classified into two main groups: small cell lung carcinoma (SCLC), accounting for 15% of all lung cancers, and nonsmall cell lung cancer (NSCLC), containing 85% of all lung cancers. NSCLC are subclassified into adenocarcinoma (ADC), squamous cell carcinoma, and large cell lung carcinoma.27

Tumor metastasis is a hallmark of cancer. Metastases are responsible for more than 90% of cancer-related deaths.28 The most frequently metastatic sites of lung cancer are bone 34.3%, lung 32.1%, brain 28.4%, adrenals 16.7%, and liver 13.4%.25 The metastatic cascade depicts the process in which malignant tumor cells reach distant organs from their primary site via hematogenous or/and lymphatic circulation. In these complicated processes, migration, invasion, angiogenesis, hypoxia, and epithelial-to-mesenchymal transition (EMT) are involved, which are tightly controlled by gene expression signatures, along with the interaction between tumor cells and the tumor microenvironment (TME).29 In addition, TDEs and exosomes derived from the components of the TME have been considered as key players in tumor metastasis network recently.30

This review briefly summarizes the role of TDEs in lung cancer progression and metastasis, related to the activation of the TME. Also, the involvement of TDEs in NSCLC targeted therapy resistance is addressed, and the potential application of TDEs in lung cancer treatment is discussed.

2 EXOSOMES: FORMATION, RELEASE, AND UPTAKE

EVs represent vesicles that are released into the extracellular space that form small bodies surrounded by a lipid membrane containing cytosol, proteins, and nucleic acids. These vesicles are released by almost all eukaryotic cells and their size ranges between 30 nm to a few micrometers. EVs are very heterogeneous, and their classification is complex. EVs are engaged in various pathophysiological processes in humans including cancer and inflammatory as well as cardiovascular diseases. These vesicles mainly comprise exosomes, apoptotic bodies released during programmed cell death, and microvesicles released from the plasma membrane of cells.31-33

Exosomes are small EVs of endocytic origin and have a specific size range of 30–100 nm. A representative image of exosomes isolated from normal lung tissues and analyzed under cryo-transmission electron microscopy is shown in Figure 1A. Exosomes are usually formed by exocytosis of a particular type of late endosome called multivesicular body (MVB). MVBs consist of intraluminal vesicles (ILVs) which are formed by the outward budding of the membrane of late endosomes.31, 34 MVBs have two fates: either they fuse with lysosomes and undergo degradation or they fuse with the plasma membrane and are released into the extracellular space. The formation, secretion and sorting of MVBs are handled by the endosomal sorting complex required for transport (ESCRT).5 Five different ESCRT complexes exist with distinct functions, namely ESCRT-0, -I, -II, - III, and the Vps4 complex. The ESCRT-0 initiates the MVB pathway.35 The signal for entering the MVB pathway is ubiquitination and only ESCRT-III does not recognize ubiquitin. ESCRT-I and -II are involved in recognizing ubiquitinated cargo, formation of ILVs, and provide stability. In contrast, ESCRT-III recruits de-ubiquitinating enzymes and removes ubiquitin from the cargo before assembling it into the ILVs. This complex also enables the disassembly of the ESCRT machinery from the endosomal membrane.36 ESCRT complexes are not exclusively responsible for the cargo sorting and ILV formation. The sphingolipid ceramide is also required along with proteins ALIX, clathrin, and TSG101.37, 38 In addition, an ESCRT-independent pathway exists which mostly relies on tetraspanins. In this respect, absence of CD9 can cause defective exosome secretion from bone marrow dendritic cells. In another study, CD63 was shown to be directly involved in the formation of lysosome related organelles.39, 40 CD9 and CD63 are considered as exosome markers. Expression of CD9 and CD63 detected by Western blot analysis is shown in Figure 1B.

image Characterization of exosomes secreted by normal lung tissues. (A) Cryo-transmission electron microscopic (Cryo-TEM) analysis detecting membranous vesicles in the diameter range of 30–100 nm. For Cryo-TEM, a small droplet (5 μl) of the isolated exosomes was placed on a TEM grid Quantifoil UltrAuFoil holey gold film (R 1.2/1.3, 400 mesh; Quantifoil Micro Tools GmbH). Subsequently, the sample was rapidly plunge-frozen in liquid ethane (cooled to about −180°C by liquid nitrogen) in a Zeiss Cryobox (Carl Zeiss GmbH) and transferred into a pre-cooled CM120 cryo-TEM (Philips) using a Gatan 626-DH cryo-holder (Gatan). The cryo-TEM was operated at 120 kV and the digital image was taken using a 2 K CMOS camera (F216 and EMMENU V4.0 software, TVIPS GmbH). (B) Detection of the exosome markers CD63 and CD9 in the exosomes isolated from the normal lung tissues by Western blot analysis. Detailed information about exosome isolation and Western blot analysis was descried previously.41 Exo, exosomes; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; TEM, transmission electron microscopy

Intracellular vesicle trafficking is primarily regulated by Rab GTPases which are associated with intracellular membranes.42 Using an RNA interference screen, Ostrowski et al.43 identified several Rab GTPases which promote exosome secretion including Rab2b, Rab5a, Rab9a, Rab27a, and Rab27b. Most of these Rab proteins have been previously associated with endocytic functions, consistent with the postulated endosomal origin of exosomes.42, 44, 45

After release, exosomes then bind to recipient cells and get internalized. In binding to surface receptors, they can trigger intercellular signaling.46, 47 The internalization of exosomes can be conducted by clathrin-mediated endocytosis and macropinocytosis48 or clathrin-independent pinocytosis.49 Alternatively, exosomes can also be taken up by caveolae formation and lipid rafts.50 Visualizing of the cellular uptake of exosomes by live-cell microscopy revealed that exosome vesicles can be directly endocytosed by cells and their contents internalized.51

3 TUMOR-DERIVED EXOSOMES

The functional role of exosomes released by most mammalian cells is to allow crosstalk between the cells and their microenvironment. Exosomes carry many biological active molecules and can transfer them from cell to cell to establish intercellular communication. Cancer cells, like other cells, also secrete exosomes, called tumor-derived exosomes (TDEs). TDEs are widely studied to understand the immune activities surrounding a tumor and the interaction between tumor cells and their microenvironment. Recently TDEs have gained much attention. Research on TDEs can help to gain insights into tumor metastasis, progression, and antitumor immune responses. Lung cancer is the most frequently diagnosed form of cancer worldwide causing the greatest number of deaths up to date. It turned out that TDEs are potential diagnostic and prognostic biomarkers of lung cancer, and they can be applied to comprehend the mechanisms responsible for antitumor therapy resistance.52

3.1 Lung TDEs influence the TME

The tumor and its microenvironment consist of several components which directly or indirectly affect tumor progression and metastasis. The major components are cancer cells, immune cells, extracellular matrix (ECM), and the stroma. The nature of the TME influences tumor development and progression by creating an imbalance between tumor-suppressive and oncogenic responses. Manipulation of the microenvironment to revitalize antitumor immune responses has already been proven to be an essential tool in cancer treatment and patient-specific therapeutic strategy.52-54 TDEs are the central means of information transfer between cancer cells and the TME. Several studies have disclosed the role of exosomes in regulating immune responses, EMT, activation of cancer-associated fibroblasts (CAFs), and angiogenesis.51

TDEs can induce immune suppression and change the TME to favor tumor progression. Studies have shown that lung TDEs can suppress maturation of immune cells, impair NK cell activation, and induce myeloid-derived suppressor cells.55 Lung TDEs can be taken up by macrophages and facilitate tumor progression and immune suppression.56 TDEs can release transforming growth factor beta (TGF-β) to enhance regulatory T cell proliferation and induce effector T cell apoptosis by interacting with Fas/FasL.57 Lung TDEs may also regulate tumor cell migration via TGF-β and interleukin (IL)10.58 Drug-induced COX-2 overexpression could be transferred from lung cancer cells to neighbor cells via exosomes, which resulted in TDE-induced upregulation of PGE2 and VEGF in TDE-binding cells, and induction of inflammatory reactions.59

CAFs also secrete exosomes that can alter cellular metabolism. These exosomes can inhibit mitochondrial oxidative phosphorylation and shifting the cancer cell metabolism to glycolysis and glutamine-dependent reductive carboxylation. Moreover, exosomes can carry and supply amino acids, lipids, and tricarboxylic acid cycle intermediates to cancer cells for metabolism.60 Exosomes derived from NSCLC cells inhibit apoptosis and enhance cell proliferation by delivering alpha smooth muscle actin in normal lung fibroblasts and NSCLC cells.61

Activation of the microenvironment is closely associated with EMT. Exosomes derived from highly metastatic lung cancer can induce vimentin expression and EMT in human bronchial epithelial cells (HBECs) and in addition, these TDEs enhance cell migration, proliferation, invasion, and metastasis.62 ZEB1, a master EMT transcription factor, can induce a mesenchymal phenotype in normal cells. It was observed that exosomes derived from transformed mesenchymal HBECs increased ZEB1 expression in parental HBECs, thereby stimulating a mesenchymal phenotype and rendering the HBECs chemoresistant.63 A study by Wu et al.64 showed that TGF-β-mediated exosomal lnc-MMP2-2 promotes lung cancer cell migration and invasion through upregulation of matrix metalloproteinase (MMP2) which is involved in degradation of the extracellular matrix. Exosomes from irradiated lung cancer cells can increase the expression of glycolytic enzymes and glycolytic activity in recipient cells, which in turn controls the motility of these cells via signaling proteins ALDOA and ALDH3A1.65

Accumulating evidence indicates that lung cancers produce more exosomes under hypoxic conditions compared to normoxic ones.66, 67 Hypoxic lung cancer-secreted exosomal miRNAs influence the TME and promote tumor metastasis. Hypoxic lung-cancer-derived exosomal miR-103a increases the oncogenic effects of macrophages by enhancing M2 polarization and targeting the tumor suppressor gene PTEN.68 Hypoxic lung cancer-derived exosomal miR-23a inhibits the tight junction protein ZO-1 and induces accumulation of HIF1α in endothelial cells, thus increasing vascular permeability and facilitating tumor cell spread.69 Hypoxic lung tumor-derived exosomal miR-150 decreases anticancer activity of NK cell by targeting CD226,70 a member of the immunoglobulin superfamily. Collectively, these data suggest that lung TDEs, interacting with the TME, have a tremendous impact on tumor progression and development (Figure 2).

image Interaction of lung tumor-derived exosomes (TDEs) with the tumor microenvironment (TME) has a tremendous impact on tumor progression and development. Under normoxia, lung TDEs enhance cell proliferation, EMT and angiogenesis while inhibit apoptosis to promote lung cancer metastasis (left); under hypoxic conditions, exosomal miRNAs affect TME components for example macrophages, endothelial cells, and immune cells to facilitate lung cancer metastasis (right). EMT, epithelial-to-mesenchymal transition; miRNAs, microRNA [Color figure can be viewed at wileyonlinelibrary.com] 3.2 Tumor-derived exosomal miRNAs in lung cancer metastasis

miRNAs are small noncoding RNAs with length between 21 and 24 nucleotides that have a key role as regulators of gene expression at posttranscriptional level.71 Exosome-derived miRNAs (exosomal miRNAs) are associated with many pathophysiological processes in lung cancer like EMT, proliferation, migration, invasion, and angiogenesis, which ultimately lead to tumor progression and metastasis.72

As already discussed, crosstalk between tumor cells and the TME plays a critical role in tumor progression. A study by Fang et al.73 showed that liver cancer derived exosomal miR-1247-3p activated CAFs to facilitate formation of a premetastatic niche in the lung.72 It was found that exosomal miR-1247-3p directly targeted B4GALT3, resulting in activation of the β1-integrin–nuclear factor kappa B pathway in CAFs.73 Lung ADC-derived exosomal miR-21 was involved in bone metastasis through facilitating osteoclastogenesis via targeting Pdcd4, a known regulator of osteoclastogenesis.74 miR-192 was identified as a repressor of tumor metastasis by comparative transcriptomic profiling using an in vivo murine model of bone metastasis.75 Treatment of NSCLC cell line A549 with miR-192-enriched exosome-like vesicles abrogates angiogenesis by inhibition of IL-8, ICAM, and CXCL1 in vitro and reduces the metastatic burden and tumor colonization in the bone in vivo.75 NSCLC-derived exosomal miR-619-5p promotes metastasis through modulation of angiogenesis and inhibition of the regulator of calcineurin 1 gene isoform 4 (RCAN1.4),76 a tumor suppressor in various cancer cells. Exosomal miR-494 and miR-542-3p derived from metastatic rat ADC BSp73ASML modulate draining lymph nodes and lung tissue to support tumor spread via targeting cadherin-17 and upregulation of MMP2, MMP3, and MMP14.77 Recently accumulating research data have implied that exosomal miRNAs influence NSCLC progression and metastasis through interacting with the Wnt/β-catenin signaling pathway. Liu et al. observed that the plasma exosomal miR-433 level was lower in NSCLC patients with chemoresistance compared to patients with chemosensitive NSCLC, which was negatively associated with distant metastasis. Furthermore, it is shown that miR-433 inhibited NSCLC progression via incremental infiltration of CD4 and CD8 cells and inactivation of the Wnt/β-catenin signaling pathway.78 Exosomal miR-1260b derived from NSCLC promotes tumor metastasis via targeting homeodomain-interacting protein kinase-2,79 and previously, this miR-1260b was found to be able to promote tumor cell invasion in lung ADC through activation of Wnt/β-catenin signaling pathway.80 Analysis of the association between exosomal microRNA clusters and bone metastasis from NSCLC revealed that miR-574-5p, a suppressor of Wnt/β-catenin pathway, was downregulated, while miR-328-3p and miR-423-3p, two activators of this pathway, were upregulated in NSCLC patients with bone metastasis.81 Evidence supporting the notion that TDEs facilitate metastasis in the context of lung cancer is still coming. For example, miR-499a-5p promotes lung ADC cell proliferation and EMT and, therefore, facilitates tumor cell metastasis via mTOR signaling.82 Exosomal miR-106b acts as a novel biomarker for lung cancer and promotes cancer metastasis through inhibition of the tumor suppressor gene PTEN.83 Chen et al.84 found that exosomal miR-3180-3p inhibits proliferation and metastasis of non-small cell lung cancer by downregulating FOXP4.84 Breast cancer-derived exosome transfected with miR-126 could inhibit A549 NSCLC cell proliferation and migration through interrupting the PTEN/PI3K/AKT pathway and suppress lung tumor metastasis in vivo.85 Exosomal miRNAs from hypoxic bone marrow-derived mesenchymal stem/stromal cells enhance lung cancer metastasis via STAT3-induced EMT (Table 1).86

3.3 The role of tumor-derived exosomal proteins in lung cancer metastasis

It is widely believed that TDE proteins carrying oncogenic proteins are involved in lung cancer progression and metastasis. Taverna et al.87 observed that NSCLC-derived exosomes containing amphiregulin activated the EGFR pathway in preosteoclasts that in turn fostered bone metastasis by upregulation of RANKL. Another study showed that exosomes derived from the highly metastatic lung cancer cell line 95D promoted metastasis in the lung cancer cell line A549, the lung fibroblast cell line MRC-5, and the poorly metastatic cell line 95C through activation of the HGF/c-Met pathway. Additionally, quantitative proteomics analysis revealed that 268 exosomal proteins differentially expressed in 95D cells might contribute to the enhanced metastatic behavior.88 Wnt proteins have been identified as exosomal cargoes, contributing to tumorigenesis and metastasis. Golgi phosphoprotein 3 was found to be interacted with cytoskeleton-associated protein 4, which enhanced the secretion of exosomal WNT3A and promoted NSCLC cell metastasis.89 Tumor metastasis is closely associated with EMT. The main features of EMT related to tumor invasiveness and metastasis are the loss of epithelial cell properties and gain of mesenchymal phenotype.90 A study by Kim et al.91 reported that the exosomal β-catenin protein was upregulated in A549, stimulated by the EMT inducer TGF-β, and autologous treatment of exosomes led to a significantly increased TCF/LET transcriptional activity in A549 cells, indicating that exosomes might induce phenotypic switches via autocrine signaling. In addition, exosomes derived from highly metastatic lung cancer cells and advanced stage patient serum induced a mesenchymal phenotype alteration and increased expression of the mesenchymal marker protein vimentin in normal HBECs.62

Table 1. Main findngs of tumor-derived exosomal miRNAs (Exo-miRNAs) in lung cancer metastasis Exo-miRNAs Origin of exosomes Recipent cells Targets Functions Ref. miR-21 NSCLC cells A549 BMMs from mice PDCD4 facilitating bone metastasis 74 miR-192 A549 A549 IL-8, ICAM, CXCL1 reducing osseous colonization 75 miR-619-5p NSCLC cells A549, H460 HUVECs RCAN1.4 promoting angiogenesis 76 and metastasis miR-494 metastatic rat ADC EC and LuFb cadherin-17 modulating premetastatic 77 miR-542-3p organ cells miR-433 A549 and H1299 transfected with A549, H1299 Wnt/β-catenin signaling inhibiting tumor progression 78 miR-433 CD4, CD8 miR-1260b A549 transfected with miR-1260b PC-9 HIPK2 promoting metastasis 79 H1299 transfected with miR-1260b A549 Wnt/β-catenin signaling promoting tumor invasion 80 miR-574-5p NSCLC patients Wnt/β-catenin signaling dsyregulation in NSCLC 81 miR-328-3p, miR-423-3p with bone metastasis miR-499-5p NSCLC cells SPC-A-1 A549, PC-A-1-BM mTOR promoting metastasis via EMT 82 miR-106b NSCLC cells SPC-A-1 SPC-A-1 PTEN upregulation of MMP2 83 transfected with miR-210-3p and MMP-3 and MMP-9 miR-3180-3p NSCLC stransfected with miR-3180-3p A549, H460 FOXP4 inhibiting metastasis 84 miR-126 breast cancer cells MDA-MB-231 A549 PTEN/PI3K/AKT inhibiting the formulation 85 of metastasis miR-193a-3p BMSC NSCLC cells H358, STAT3 promoting metastasis via EMT 86 miR-210-3p A549, H460 miR-5100 Abbreviations: ADC, adenocarcinoma; BMMs, bone marrow-derived macrophages; BMSC, bone marrow-derived mesenchymal stem/stromal cells; EC, rat aortic endothelial cell line; HUVECs, human umbilical veinendothelial cells; LuFb, rat fibroblast line; miRNA, microRNA; NSCLC, nonsmall cell lung cancer; Ref, reference.

Exosomes from nonmalignant cells also affect lung metastasis. For example, exosomes derived from adipocytes increase MMP activity by transferring MMP3 to lung cancer cells, thereby promoting lung cancer metastasis.92 Recently, it was found that exosomal PD-L1 could promote tumor growth through immune escape in NSCLC.93 The main findings of tumor-derived exosomal proteins in lung cancer metastasis are summarized in Table 2.

Table 2. Main findings of tumor-derived exosomal proteins (Exo-proteins) in lung cancer progression and metastasis Exo-protein Origin of exosomes Recepient cell Target/mechanism Function Ref amphiregulin plasma of NSCLC patients primary osteoclasts EGFR triggering osteolytic bone metastasis 87 268 differentially expressed NSCLC cells 95D A549, MRC-5 HGF/c-Met promoting metastasis 88 exosomal proteins exosomal-WNT3A GOLPH3 overexpressing A549 and H460 Wnt/β-catenin promoting metastasis 89 A549 and H460 cells proteins involved in EMT TGF-β1 treated A549 cells A549, MRC-5 β-catenin promoting metastasis via EMT 91 proteins involved in EMT serum from NSCLC patients HBEC EMT enhancing migration and invasion 61 MMP3 3T3-L1 adipocyte 3LL NSCLC cells MMP9 promoting lung cancer metastasis 92 PD-L1 NSCLC cells H460, H1975 T cell immune escape promoting tumor growth 93 Abbreviations: EMT, epithelial-to-mesenchymal transition; HBEC, human bronchial epithelial cells; MRC-5, lung fibroblast cells; NSCLC, nonsmall cell lung cancer; TGF,  transforming growth factor.

The potential role of tumor-derived exosomal proteins in diagnosis of metastatic lung cancer was revealed by several studies.94, 95 The concentration of exosomes isolated from metastatic NSCLC was found to be significantly higher in comparison with those from healthy individuals, and additionally, the exosomal levels of alpha-2-HS-glycoprotein and ECM1 increased significantly in the metastatic NSCLC patients.95 In a study by Wang et al.,

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