Biological Cargo: Exosomes and their Role in Cancer Progression and Metastasis.

Cancer cells are among the many types of cells that release exosomes, which are nanovesicles. Because of their many potential applications, exosomes have recently garnered much attention from cancer researchers. The bioactive substances that exosomes release as cargo have been the subject of several investigations. The substances in question may operate as biomarkers for diagnosis or affect apoptosis, the immune system, the development and spread of cancer, and other processes. Others have begun to look at exosomes in experimental therapeutic trials because they believe they may be useful in the treatment of cancer. This review started with a short description of exosome biogenesis and key features. Next, the potential of tumor-derived exosomes and oncosomes to influence the immune system throughout the development of cancer, as well as alter tumor microenvironments (TMEs) and pre-metastatic niche creation, was investigated. Finally, there was talk of exosomes' possible use in cancer treatment. Furthermore, there is emerging consensus about the potential application of exosomes to be biological reprogrammers of cancer cells, either as carriers of naturally occurring chemicals, including anticancer medications, or as carriers of anticancer vaccines for immunotherapy as well as boron neutron capture therapy (BNCT). We briefly review the key ideas and logic behind this intriguing therapy recommendation.

Cancer stem cells release interleukin-33 within large oncosomes to promote immunosuppressive differentiation of macrophage precursors.

In squamous cell carcinoma (SCC), macrophages responding to interleukin (IL)-33 create a TGF-ß-rich stromal niche that maintains cancer stem cells (CSCs), which evade chemotherapy-induced apoptosis in part via activation of the NRF2 antioxidant program. Here, we examined how IL-33 derived from CSCs facilitates the development of an immunosuppressive microenvironment. CSCs with high NRF2 activity redistributed nuclear IL-33 to the cytoplasm and released IL-33 as cargo of large oncosomes (LOs). Mechanistically, NRF2 increased the expression of the lipid scramblase ATG9B, which exposed an "eat me" signal on the LO surface, leading to annexin A1 (ANXA1) loading. These LOs promoted the differentiation of AXNA1 receptor+ myeloid precursors into immunosuppressive macrophages. Blocking ATG9B's scramblase activity or depleting ANXA1 decreased niche macrophages and hindered tumor progression. Thus, IL-33 is released from live CSCs via LOs to promote the differentiation of alternatively activated macrophage, with potential relevance to other settings of inflammation and tissue repair.

Composition of Conditioned Media from Radioresistant and Chemoresistant Cancer Cells Reveals miRNA and Other Secretory Factors Implicated in the Development of Resistance.

Resistance to chemo- or radiotherapy is the main obstacle to consistent treatment outcomes in oncology patients. A deeper understanding of the mechanisms driving the development of resistance is required. This review focuses on secretory factors derived from chemo- and radioresistant cancer cells, cancer-associated fibroblasts (CAFs), mesenchymal stem cells (MSCs), and cancer stem cells (CSCs) that mediate the development of resistance in unexposed cells. The first line of evidence considers the experiments with conditioned media (CM) from chemo- and radioresistant cells, CAFs, MSCs, and CSCs that elevate resistance upon the ionizing radiation or anti-cancer drug exposure of previously untreated cells. The composition of CM revealed factors such as circular RNAs; interleukins; plasminogen activator inhibitor; and oncosome-shuttled lncRNAs, mRNAs, and miRNAs that aid in cellular communication and transmit signals inducing the chemo- and radioresistance of sensitive cancer cells. Data, demonstrating that radioresistant cancer cells become resistant to anti-neoplastic drug exposure and vice versa, are also discussed. The mechanisms driving the development of cross-resistance between chemotherapy and radiotherapy are highlighted. The secretion of resistance-mediating factors to intercellular fluid and blood brings attention to its diagnostic potential. Highly stable serum miRNA candidates were proposed by several studies as prognostic markers of radioresistance; however, clinical studies are needed to validate their utility. The ability to predict a treatment response with the help of the miRNA resistance status database will help with the selection of an effective therapeutic strategy. The possibility of miRNA-based therapy is currently being investigated with ongoing clinical studies, and such approaches can be used to alleviate resistance in oncology patients.

Small and Large Extracellular Vesicles Derived from Pleural Mesothelioma Cell Lines Offer Biomarker Potential.

Pleural mesothelioma, previously known as malignant pleural mesothelioma, is an aggressive and fatal cancer of the pleura, with one of the poorest survival rates. Pleural mesothelioma is in urgent clinical need for biomarkers to aid early diagnosis, improve prognostication, and stratify patients for treatment. Extracellular vesicles (EVs) have great potential as biomarkers; however, there are limited studies to date on their role in pleural mesothelioma. We conducted a comprehensive proteomic analysis on different EV populations derived from five pleural mesothelioma cell lines and an immortalized control cell line. We characterized three subtypes of EVs (10 K, 18 K, and 100 K), and identified a total of 4054 unique proteins. Major differences were found in the cargo between the three EV subtypes. We show that 10 K EVs were enriched in mitochondrial components and metabolic processes, while 18 K and 100 K EVs were enriched in endoplasmic reticulum stress. We found 46 new cancer-associated proteins for pleural mesothelioma, and the presence of mesothelin and PD-L1/PD-L2 enriched in 100 K and 10 K EV, respectively. We demonstrate that different EV populations derived from pleural mesothelioma cells have unique cancer-specific proteomes and carry oncogenic cargo, which could offer a novel means to extract biomarkers of interest for pleural mesothelioma from liquid biopsies.

Melanoma Cells Produce Large Vesicular-Bodies That Cause Rapid Disruption of Brain Endothelial Barrier-Integrity and Disassembly of Junctional Proteins.

It is known that many cells produce extracellular vesicles, and this includes a range of different cancer cell types. Here we demonstrate the profound effects of large vesicular-like bodies produced by melanoma cells on the barrier integrity of human brain endothelial cells. These vesicular-bodies have not been fully characterised but range in size from ~500 nm to >10 µm, are surrounded by membrane and are enzymatically active based on cell-tracker incorporation. Their size is consistent with previously reported large oncosomes and apoptotic bodies. We demonstrate that these melanoma-derived vesicular-bodies rapidly affect brain endothelial barrier integrity, measured using ECIS biosensor technology, where the disruption is evident within ~60 min. This disruption involves acquisition of the vesicles through transcellular uptake into the endothelial cells. We also observed extensive actin-rearrangement, actin removal from the paracellular boundary of the endothelial cells and envelopment of the vesicular-bodies by actin. This was concordant with widespread changes in CD144 localisation, which was consistent with the loss of junctional strength. High-resolution confocal imaging revealed proximity of the melanoma vesicular-bodies juxtaposed to the endothelial nucleus, often containing fragmented DNA themselves, raising speculation over this association and potential delivery of nuclear material into the brain endothelial cells. The disruption of the endothelial cells occurs in a manner that is faster and completely distinct to that of invasion by intact melanoma cells. Given the clinical observation of large vesicles in the circulation of melanoma patients by others, we hypothesize their involvement in weakening or priming the brain vasculature for melanoma invasion.

Blebs and former blebs: From surface protrusions to extracellular vesicles in cancer signalling, anoikis resistance and beyond.

Associations between plasma membrane blebbing and metastatic progression have been widely reported. There are also reports of increased extracellular vesicle release from cancer cells. Yet the ties between these closely related phenomena are incompletely understood. In this commentary, we remark on a recent finding on cellular membrane blebs in melanoma signaling. We discuss possible implications for cancer biology and draw parallels to knowns and unknowns in the relationships of extracellular vesicles and cancer progression.

Defining A Liquid Biopsy Profile of Circulating Tumor Cells and Oncosomes in Metastatic Colorectal Cancer for Clinical Utility.

Metastatic colorectal cancer (mCRC) is characterized by its extensive disease heterogeneity, suggesting that individualized analysis could be vital to improving patient outcomes. As a minimally invasive approach, the liquid biopsy has the potential to longitudinally monitor heterogeneous analytes. Current platforms primarily utilize enrichment-based approaches for epithelial-derived circulating tumor cells (CTC), but this subtype is infrequent in the peripheral blood (PB) of mCRC patients, leading to the liquid biopsy's relative disuse in this cancer type. In this study, we evaluated 18 PB samples from 10 mCRC patients using the unbiased high-definition single-cell assay (HDSCA). We first employed a rare-event (Landscape) immunofluorescence (IF) protocol, which captured a heterogenous CTC and oncosome population, the likes of which was not observed across 50 normal donor (ND) samples. Subsequent analysis was conducted using a colorectal-targeted IF protocol to assess the frequency of CDX2-expressing CTCs and oncosomes. A multi-assay clustering analysis isolated morphologically distinct subtypes across the two IF stains, demonstrating the value of applying an unbiased single-cell approach to multiple assays in tandem. Rare-event enumerations at a single timepoint and the variation of these events over time correlated with progression-free survival. This study supports the clinical utility of an unbiased approach to interrogating the liquid biopsy in mCRC, representing the heterogeneity within the CTC classification and warranting the further molecular characterization of the rare-event analytes with clinical promise.

Canine B Cell Lymphoma- and Leukemia-Derived Extracellular Vesicles Moderate Differentiation and Cytokine Production of T and B Cells In Vitro.

Extracellular vesicles (EVs) are formed in physiological and pathological conditions by almost all mammalian cells. They are known as submicron "molecules" that transport and horizontally transfer their cargo from maternal cells to donor cells. Moreover, cancer cells produce tumor-derived EVs (TEVs), which are present in blood of patients with solid tumors and those with hematological malignancies. Their role in evading immune system surveillance and induction of immunosuppression in hematological cancer is limited. According to the authors' best knowledge, there is no information about the impact of TEVs from canine lymphoma (CLBL-1) and leukemia (CLB70) on lymphocytes isolated from peripheral blood mononuclear cells (PBMCs). In conclusion, we demonstrate in in vitro experiments that CLBL-1 EVs and CLB70 EVs are effectively taken up by T and B lymphocytes. TEVs decrease the percentage of B lymphocytes and increase that of T lymphocytes, and change T cells' phenotype into the effector memory (EM) or terminally differentiated effector memory (TEMRA) subtype after in vitro co-culturing. Moreover, CLBL70 EVs have pro-tumorogenic properties by inhibiting the production of CD8+IL-17+ cells.

Exploring the role of extracellular vesicles and their protein cargo in lung cancer metastasis: A review.

Extracellular vesicles (EV) are membrane-enclosed structures of varying size released from all cells and contain a variety of cargo including proteins, lipids, and nucleic acids. They are postulated to play a pivotal role in cancer metastasis through delivery of oncogenic material to neighbouring and distant cells to promote development of a metastatic niche and tumour seeding. Here we reviewed protein data in relevant literature to determine whether specific proteins known to be involved in metastasis can be reliably identified in lung cancer EV, whether these proteins are important in all or specific lung cancers, and whether results from in-vitro cell studies are supported by research examining EV in human biofluids. Our analysis suggests that specific proteins may be more important for individual lung cancers, but interpretation of the literature is currently limited by a relative lack of research investigating EV proteins in some cancers and in clinical studies using biofluids.

miR-1227 Targets SEC23A to Regulate the Shedding of Large Extracellular Vesicles.

Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Application of extracellular vesicles in the diagnosis and treatment of prostate cancer: Implications for clinical practice.

Extracellular vesicles (EV) are cell-derived lipid bilayer-delimited structures providing an important means of intercellular communication. Recent studies have shown that EV, particularly exosomes and large-oncosomes contain miRNA and proteins crucial in prostate cancer (PCa) progression, metastasis and treatment resistance. This includes not just EV released from PCa cells, but also from other cells in the tumor microenvironment. PCa patient derived EV have a unique composition compared to healthy and benign prostatic diseases. As such, EV show promise as diagnostic liquid biopsy biomarkers, both as an adjunct and alternative to the invasive current gold-standard. EV could also be utilized to stratify patients' risk and predict response to hormonal, chemo, immune- and targeted therapy, which will direct future treatment decisions in PCa. We present a summary of the current evidence on the role of EV in PCa and the application of EV in PCa diagnosis and treatment to optimize patient outcomes.

Current Search through Liquid Biopsy of Effective Biomarkers for Early Cancer Diagnosis into the Rich Cargoes of Extracellular Vesicles.

There exist many different human cancers, but regardless of the cancer type, an early diagnosis is a necessary condition for further optimal outcomes from the disease. Therefore, efficient specific and sensitive cancer biomarkers are urgently needed. This is especially true for the cancers depicting a silent progression, and those only diagnosed in an already metastatic state with a poor survival prognostic. After a rapid overview of the previous methods for cancer diagnosis, the outstanding characteristics of extracellular vesicles (EVs) will be presented, as new interesting candidates for early cancer diagnosis in human biofluid non-invasive liquid biopsy. The present review aims to give the state-of-the-art of the numerous searches of efficient EV-mediated cancer diagnosis. The corresponding literature quest was performed by means of an original approach, using a powerful Expernova Questel big data platform, which was specifically adapted for a literature search on EVs. The chosen collected scientific papers are presented in two parts, the first one drawing up a picture of the current general status of EV-mediated cancer diagnosis and the second one showing recent applications of such EV-mediated diagnosis for six important human-specific cancers, i.e., lung, breast, prostate, colorectal, ovary and pancreatic cancers. However, the promising perspective of finally succeeding in the worldwide quest for the much-needed early cancer diagnosis has to be moderated by the many remaining challenges left to solve before achieving the efficient clinical translation of the constantly increasing scientific knowledge.

Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer.

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

Biogenesis of Extracellular Vesicles.

Extracellular vesicles (EVs) refer to vesicles that are released by cells into the extracellular space. EVs mediate cell-to-cell communication via delivery of functional biomolecules between host and recipient cells. EVs can be categorised based on their mode of biogenesis and secretion and include apoptotic bodies, ectosomes or shedding microvesicles and exosomes among others. EVs have gained immense interest in recent years owing to their implications in pathophysiological conditions. Indeed, EVs have been proven useful in clinical applications as potential drug delivery vehicles and as source of diagnostic biomarkers. Despite the growing body of evidence supporting the clinical benefits, the processes involved in the biogenesis of EVs are poorly understood. Hence, it is critical to gain a deeper understanding of the underlying molecular machineries that ultimately govern the biogenesis and secretion of EVs. This chapter discusses the current knowledge on molecular mechanisms involved in the biogenesis of various subtypes of EVs.

Exosomes and Cell Communication: From Tumour-Derived Exosomes and Their Role in Tumour Progression to the Use of Exosomal Cargo for Cancer Treatment.

Exosomes are nano-vesicle-shaped particles secreted by various cells, including cancer cells. Recently, the interest in exosomes among cancer researchers has grown enormously for their many potential roles, and many studies have focused on the bioactive molecules that they export as exosomal cargo. These molecules can function as biomarkers in diagnosis or play a relevant role in modulating the immune system and in promoting apoptosis, cancer development and progression. Others, considering exosomes potentially helpful for cancer treatment, have started to investigate them in experimental therapeutic trials. In this review, first, the biogenesis of exosomes and their main characteristics was briefly described. Then, the capability of tumour-derived exosomes and oncosomes in tumour microenvironments (TMEs) remodelling and pre-metastatic niche formation, as well as their interference with the immune system during cancer development, was examined. Finally, the potential role of exosomes for cancer therapy was discussed. Particularly, in addition, their use as carriers of natural substances and drugs with anticancer properties or carriers of boron neutron capture therapy (BNCT) and anticancer vaccines for immunotherapy, exosomes as biological reprogrammers of cancer cells have gained increased consensus. The principal aspects and the rationale of this intriguing therapeutic proposal are briefly considered.

Extracellular Vesicles and Bone-Associated Cancer.

PURPOSE OF REVIEW: In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers. RECENT FINDINGS: Crosstalk between tumor and bone has been demonstrated to promote tumor and metastatic progression. In addition to direct cell-to-cell contact and soluble factors, such as cytokines, EVs mediate crosstalk between tumor and bone. EVs are composed of a heterogenous group of membrane-delineated vesicles of varying size range, mechanisms of formation, and content. These include apoptotic bodies, microvesicles, large oncosomes, and exosomes. EVs derived from primary tumors have been shown to alter bone remodeling and create formation of a pre-metastatic niche that favors development of bone metastasis. Similarly, EVs from marrow stromal cells have been shown to promote tumor progression. Additionally, EVs can act as therapeutic delivery vehicles due to their low immunogenicity and targeting specificity. EVs play critical roles in intercellular communication. Multiple classes of EVs exist based on size on mechanism of formation. In addition to a role in pathophysiology, EVs can be exploited as therapeutic delivery vehicles.

Proteomic Characterization of Two Extracellular Vesicle Subtypes Isolated from Human Glioblastoma Stem Cell Secretome by Sequential Centrifugal Ultrafiltration.

Extracellular vesicles (EVs) released from tumor cells are actively investigated, since molecules therein contained and likely transferred to neighboring cells, supplying them with oncogenic information/functions, may represent cancer biomarkers and/or druggable targets. Here, we characterized by a proteomic point of view two EV subtypes isolated by sequential centrifugal ultrafiltration technique from culture medium of glioblastoma (GBM)-derived stem-like cells (GSCs) obtained from surgical specimens of human GBM, the most aggressive and lethal primary brain tumor. Electron microscopy and western blot analysis distinguished them into microvesicles (MVs) and exosomes (Exos). Two-dimensional electrophoresis followed by MALDI TOF analysis allowed us to identify, besides a common pool, sets of proteins specific for each EV subtypes with peculiar differences in their molecular/biological functions. Such a diversity was confirmed by identification of some top proteins selected in MVs and Exos. They were mainly chaperone or metabolic enzymes in MVs, whereas, in Exos, molecules are involved in cell-matrix adhesion, cell migration/aggressiveness, and chemotherapy resistance. These proteins, identified by EVs from primary GSCs and not GBM cell lines, could be regarded as new possible prognostic markers/druggable targets of the human tumor, although data need to be confirmed in EVs isolated from a greater GSC number.

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles.

Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, frequently called palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyse the palmitoyl-proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L- and S-EVs harbour proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABCC4 as prostate cancer-specific palmitoyl-proteins abundant in both EV populations. Importantly, localization of the above proteins in EVs was reduced upon inhibition of palmitoylation in the producing cells. Our results suggest that this post-translational modification may play a role in the sorting of the EV-bound secretome and possibly enable selective detection of disease biomarkers.

Large Extracellular Vesicles-A New Frontier of Liquid Biopsy in Oncology.

Extracellular Vesicles (EVs) are emerging as pivotal elements in cancer. Many studies have focused on the role of Small- (S)-EVs but in recent years Large-(L)-EVs have progressively gained increasing interest due to their peculiar content and functions. Tumor-derived L-EVs carry a lot of oncogenic proteins, nucleic acids and lipids to recipient cells and are involved in the reshaping of the tumor microenvironment as well as in the metabolic rewiring and the promotion of the pro-metastatic attitude of cancer cells. Several techniques have been developed for the isolation of L-EVs and commercial kits are also available for efficient and easy recovery of these vesicles. Also, the improvement in DNA sequencing and "omics sciences" profoundly changed the way to analyze and explore the molecular content of L-EVs, thus providing novel and potentially useful cancer biomarkers. Herein, we review the most recent findings concerning the role of L-EVs in cancer and discuss their possible use in oncology as "liquid biopsy" tools as compared to the other classes of EVs.

Epstein-Barr Virus LMP1 Promotes Syntenin-1- and Hrs-Induced Extracellular Vesicle Formation for Its Own Secretion To Increase Cell Proliferation and Migration.

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication that are involved in both normal processes and pathological conditions. Latent membrane protein 1 (LMP1) is a major viral oncogene that is expressed in most Epstein-Barr virus (EBV)-associated cancers and secreted in EVs. LMP1-modified EVs have the ability to influence recipient cell growth, migration, and differentiation and regulate immune cell function. Despite the significance of LMP1-modified EVs in EBV malignancies, very little is understood about how this protein hijacks the host EV pathway for secretion. Using the biotin identification (BioID) method, we identified LMP1-proximal interacting proteins that are known to play roles in EV formation and protein trafficking. Analysis of the identified LMP1-interacting proteins revealed an enrichment in the ESCRT pathway and associated proteins, including CD63, Syntenin-1, Alix, TSG101, Hrs, and charged multivesicular body proteins (CHMPs). LMP1 transcriptionally upregulated and increased the protein expression of EV biogenesis and secretion genes. Nanoparticle tracking and immunoblot analysis revealed reduced levels of LMP1 EV packaging and of vesicle production following the knockdown of Syntenin-1, Alix, Hrs, and TSG101, with altered endolysosomal trafficking observed when Syntenin-1 and Hrs expression was reduced. Knockdown of specific ESCRT-III subunits (CHMP4B, -5, and -6) impaired LMP1 packaging and secretion into EVs. Finally, we demonstrate that the efficient secretion of LMP1-modified EVs promotes cell attachment, proliferation, and migration and tumor growth. Together, these results begin to shed light on how LMP1 exploits host ESCRT machinery to direct the incorporation of the viral oncoprotein into the EV pathway for secretion to alter the tumor microenvironment.IMPORTANCE LMP1 is a notable viral protein that contributes to the modification of EV content and tumor microenvironment remodeling. LMP1-modified EVs enhance tumor proliferation, migration, and invasion potential and promote radioresistance. Currently, the mechanisms surrounding LMP1 incorporation into the host EV pathways are not well understood. This study revealed that LMP1 utilizes Hrs, Syntenin-1, and specific components of the ESCRT-III complex for release from the cell, enhancement of EV production, and metastatic properties of cancer cells. These findings begin to unravel the mechanism of LMP1 EV trafficking and may provide new targets to control EBV-associated cancers.