Extracellular vesicle-orchestrated crosstalk between cancer-associated fibroblasts and tumors

Communication networks in the tumor microenvironment (TME) play a crucial role in tumor progression. Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells in the TME. Bidirectional signal transduction between cancer cells and CAFs within the TME is important for cancer development and treatment responsiveness. Extracellular vesicles (EVs) carrying proteins, miRNAs, and other biomolecules are secreted into the extracellular matrix (ECM), which has been demonstrated to be an important communication medium between tumors and CAFs. Tumors regulate the activation of CAFs by secreting EVs. Conversely, CAFs can also affect tumor proliferation, metastasis, and therapeutic resistance through EVs. Here, we will classify EV cargoes and discuss the role of EV-mediated interactions between CAFs and tumors, reviewing current knowledge in combination with our confirmed results.     

Extracellular vesicle-orchestrated crosstalk between cancer-associated fibroblasts and tumors

Communication networks in the tumor microenvironment (TME) play a crucial role in tumor progression. Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells in the TME. Bidirectional signal transduction between cancer cells and CAFs within the TME is important for cancer development and treatment responsiveness. Extracellular vesicles (EVs) carrying proteins, miRNAs, and other biomolecules are secreted into the extracellular matrix (ECM), which has been demonstrated to be an important communication medium between tumors and CAFs. Tumors regulate the activation of CAFs by secreting EVs. Conversely, CAFs can also affect tumor proliferation, metastasis, and therapeutic resistance through EVs. Here, we will classify EV cargoes and discuss the role of EV-mediated interactions between CAFs and tumors, reviewing current knowledge in combination with our confirmed results.     

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

Extracellular vesicles (EVs) including exosomes and microvesicles are lipid bilayer‐encapsulated nanoparticles released by cells, ranging from 40 nm to several microns in diameter. Biological cargoes including proteins, RNAs, and DNAs can be ferried by EVs to neighboring and distant cells via biofluids, serving as a means of cell‐to‐cell communication under normal and pathological conditions, especially cancers. On the other hand, EVs have been investigated as a novel “information capsule” for early disease detection and monitoring via liquid biopsy. This review summarizes current advancements in EV subtype characterization, cancer EV capture, proteomic analysis technologies, as well as possible EV‐based multiomics for cancer diagnostics.     

Extracellular vesicles in pancreatic cancer progression and therapies

Pancreatic cancer (PC) is one of the leading causes of cancer-related death worldwide due to delayed diagnosis and limited treatments. More than 90% of all pancreatic cancers are pancreatic ductal adenocarcinoma (PDAC). Extensive communication between tumour cells and other cell types in the tumour microenvironment have been identified which regulate cancer hallmarks during pancreatic tumorigenesis via secretory factors and extracellular vesicles (EVs). The EV-capsuled factors not only facilitate tumour growth locally, but also enter circulation and reach distant organs to construct a pre-metastatic niche. In this review, we delineate the key factors in pancreatic ductal adenocarcinoma derived EVs that mediate different tumour processes. Also, we highlight the factors that are related to the crosstalk with cancer stem cells/cancer-initiating cells (CSC/CIC), the subpopulation of cancer cells that can efficiently metastasize and resist currently used chemotherapies. Lastly, we discuss the potential of EV-capsuled factors in early diagnosis and antitumour therapeutic strategies.Subject terms: Cancer microenvironment, Cancer stem cells     

Extracellular vesicles and immunogenic stress in cancer

Tumor progression requires bidirectional cell-to-cell communication within a complex tumor microenvironment (TME). Extracellular vesicles (EVs) as carriers have the capacity to shuttle regulatory molecules, including nucleic acids, proteins, and lipids, between cancer cells and multiple stromal cells, inducing remarkable phenotypic alterations in the TME. Recently proposed the concept “immunogenic stress”, which means in some stressed microenvironment, cancer cells can release EVs containing specific immunoregulatory mediators, depending on the initiating stress-associated pathway, thereby provoking the changes of immune status in the TME. Considerable evidence has revealed that the intracellular mechanisms underlying the response to diverse stresses are mainly autophagy, endoplasmic reticulum (ER) stress reactions and the DNA damage response (DDR). In addition, the activation of immunogenic stress responses endows hosts with immune surveillance capacity; in contrast, several cargoes in EVs under immunogenic stress trigger a passive immune response by mediating the function of immune cells. This review discusses the current understanding of the immunogenic stress pathways in cancer and describes the interrelation between EVs and immunogenic stress to propose potential treatment strategies and biomarkers.Subject terms: Immunosurveillance, Autophagy, Cancer     

Extracellular vesicles in ovarian cancer: applications to tumor biology,immunotherapy and biomarker discovery

In recent years there has been tremendous interest in both the basic biology and applications of extracellular vesicles (EVs) in translational cancer research. This includes a better understanding of their biogenesis and mechanisms of selective cargo packaging, their precise roles in horizontal communication, and their application as non-invasive biomarkers. The rapid advances in next-generation omics technologies are the driving forces for these discoveries. In this review, the authors focus on recent results of EV research in ovarian cancer. A deeper understanding of ovarian cancer-derived EVs, the types of cargo molecules and their biological roles in cancer growth, metastases and drug resistance, could have significant impact on the discovery of novel biomarkers and innovative therapeutics. Insights into the role of EVs in immune regulation could lead to novel approaches built on EV-based immunotherapy.     

Tumor‐Derived Extracellular Vesicles as a Novel Source of Protein Biomarkers for Cancer Diagnosis and Monitoring

“Liquid biopsies” have received attention as a complementary tool for traditional tissue biopsies that may enhance the spectrum of analysis for tumor‐derived factors. One such factor gaining prominence in the liquid biopsy field is extracellular vesicles (EVs), membrane‐bound nanovesicles which are secreted by cells into biofluids such as blood, urine, and saliva. EVs are released in both physiological and pathological conditions and can transport a variety of molecules, including proteins, metabolites, RNA, microRNAs, and DNA, to distant sites throughout the body. As such, they are emerging as a promising source of tumor biomarkers for the noninvasive diagnosis, prognosis, and monitoring of cancer patients. In particular, the wealth of tumor‐related information that can be gleaned from the EV proteomic cargo has become apparent through mass spectrometric analysis, which has provided new benchmarks for clinically focused biomarker research. In this review, the current achievements in the use of MS for identifying potential EV‐derived protein biomarkers of cancer are explored, and the techniques and challenges involved in this pursuit are summarized.     

Opposing functions for retromer and Rab11 in extracellular vesicle traffic at presynaptic terminals

Neuronal extracellular vesicles (EVs) play important roles in intercellular communication and pathogenic protein propagation in neurological disease. However, it remains unclear how cargoes are selectively packaged into neuronal EVs. Here, we show that loss of the endosomal retromer complex leads to accumulation of EV cargoes including amyloid precursor protein (APP), synaptotagmin-4 (Syt4), and neuroglian (Nrg) at Drosophila motor neuron presynaptic terminals, resulting in increased release of these cargoes in EVs. By systematically exploring known retromer-dependent trafficking mechanisms, we show that EV regulation is separable from several previously identified roles of neuronal retromer. Conversely, mutations in rab11 and rab4, regulators of endosome-plasma membrane recycling, cause reduced EV cargo levels, and rab11 suppresses cargo accumulation in retromer mutants. Thus, EV traffic reflects a balance between Rab4/Rab11 recycling and retromer-dependent removal from EV precursor compartments. Our data shed light on previous studies implicating Rab11 and retromer in competing pathways in Alzheimer’s disease, and suggest that misregulated EV traffic may be an underlying defect.     

Recent advances in extracellular vesicle research for urological cancers: From technology to application

Urological malignancies, including prostate cancer, bladder cancer and kidney cancer, are major causes of morbidity and mortality worldwide. Because of the high incidence, diversity in biology, and especially direct interaction with urine, urological cancers are an important resource for both scientists and clinicians for novel diagnostic and therapeutic discovery. Extracellular vesicles (EVs) are lipid bilayer encapsulated particles released by cells into the extracellular space. Since EVs work as a safe way to transport important biological information through the whole body, they are now recognized as an important mechanism of cell–cell communication and have opened a new window for us to gain a better understanding of cancer biology, novel diagnostics, and therapeutic options. In recent years, numerous evolutions in EV technologies and novel biological and clinical findings continue to be reported in the research field of urological cancers. This comprehensive review aims to give an update of recent advances in EV technologies and summarize the state-of-the-art knowledge of EVs related to prostate cancer, bladder cancer and kidney cancer, particularly focusing on the potential of EV as biomarkers and their biological roles in promoting cancer and metastasis.     

What is the blood concentration of extracellular vesicles? Implications for the use of extracellular vesicles as blood-borne biomarkers of cancer

Circulating biomarkers have a great potential in diagnosing cancer diseases at early stages, where curative treatment is a realistic possibility. In the recent years, using extracellular vesicles (EVs) derived from blood as biomarkers has gained widespread popularity, mainly because they are thought to be easy to isolate and carry a vast variety of biological cargos that can be analyzed for biomarker purposes. However, our current knowledge on the plasma EV concentration in normophysiological states is sparse. Here, we provide the very first mean estimate of the plasma EV concentration based on values obtained from a thorough literature review. The different estimates obtained from the literature are correlated to the isolation techniques used to obtain them, illustrating how some methodologies may over- or underestimate the plasma EV concentration. We also show that the estimated plasma EV concentration (approximately 10¹⁰ EVs per mL) defines EVs as a minority population compared to other colloidal particles of the systemic circulation, namely the lipoproteins, which are known contaminants in EV isolates and carry biomarker molecules themselves. Lastly, we introduce the possibility of regarding EVs and lipoproteins as a continuum of lipid-containing particles to which biomarker molecules can be associated. Using such a holistic approach, increased strength of plasma-derived cancer biomarkers may soon be revealed.     

Cerebrospinal Fluid Extracellular Vesicles Undergo Age Dependent Declines and Contain Known and Novel Non-coding RNAs

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.     

The biology of extracellular microvesicles

The study of extracellular vesicles (EVs) is a rapidly evolving field, owing in large part to recent advances in the realization of their significant contributions to normal physiology and disease. Once discredited as cell debris, these membrane vesicles have now emerged as mediators of intercellular communication by interaction with target cells, drug and gene delivery, and as potentially versatile platforms of clinical biomarkers as a result of their distinctive protein, nucleic acid and lipid cargoes. While there are multiple classes of EVs released from almost all cell types, here we focus primarily on the biogenesis, fate and functional cargoes of microvesicles (MVs). MVs regulate many important cellular processes including facilitating cell invasion, cell growth, evasion of immune response, stimulating angiogenesis, drug resistance and many others.      

miR-365 secreted from M2 Macrophage-derived extracellular vesicles promotes pancreatic ductal adenocarcinoma progression through the BTG2/FAK/AKT axis

Clinical and experimental evidence indicates that tumour-associated macrophages support cancer progression. Moreover, macrophage-derived extracellular vesicles (EVs) are involved in pathogenesis of multiple cancers, yet the functions of molecular determinants in which have not been fully understood. Herein, we aim to understand whether macrophage modulates pancreatic ductal adenocarcinoma (PDAC) progression in an EV-dependent manner and the underlying mechanisms. microRNA (miR)-365 was experimentally determined to be enriched in the EVs from M2 macrophages (M2-EVs), which could be transferred into PDAC cells. Using a co-culture system, M2-EVs could enhance the proliferating, migrating and invading potentials of PDAC cells, while inhibition of miR-365 in M2-EVs could repress these malignant functions. B-cell translocation gene 2 (BTG2) was identified to be a direct target of miR-365, while the focal adhesion kinase (F/ATP)-dependent tyrosine kinase (AKT) pathway was activated by miR-365. We further demonstrated that overexpression of BTG2 could delay the progression of PDAC in vitro, whereas by impairing BTG2-mediated anti-tumour effect, M2-EV-miR-365 promoted PDAC progression. For validation, a nude mouse model of tumorigenesis was established, in which we found that targeting M2-EV-miR-365 contributed to suppression of tumour growth. Collectively, M2-EVs carry miR-365 to suppress BTG2 expression, which activated FAK/AKT pathway, thus promoting PDAC development.     

The protein interaction network of extracellular vesicles derived from human colorectal cancer cells

Various mammalian cells including tumor cells secrete extracellular vesicles (EVs), otherwise known as exosomes and microvesicles. EVs are nanosized bilayered proteolipids and play multiple roles in intercellular communication. Although many vesicular proteins have been identified, their functional interrelationships and the mechanisms of EV biogenesis remain unknown. By interrogating proteomic data using systems approaches, we have created a protein interaction network of human colorectal cancer cell-derived EVs which comprises 1491 interactions between 957 vesicular proteins. We discovered that EVs have well-connected clusters with several hub proteins similar to other subcellular networks. We also experimentally validated that direct protein interactions between cellular proteins may be involved in protein sorting during EV formation. Moreover, physically and functionally interconnected protein complexes form functional modules involved in EV biogenesis and functions. Specifically, we discovered that SRC signaling plays a major role in EV biogenesis, and confirmed that inhibition of SRC kinase decreased the intracellular biogenesis and cell surface release of EVs. Our study provides global insights into the cargo-sorting, biogenesis, and pathophysiological roles of these complex extracellular organelles.     

Fibroblasts in pancreatic cancer: molecular and clinical perspectives

Pancreatic stellate cells (PSCs) and cancer-associated fibroblasts (CAFs) are highly abundant cells in the pancreatic tumor microenvironment (TME) that modulate desmoplasia. The formation of a dense stroma leads to immunosuppression and therapy resistance that are major causes of treatment failure in pancreatic ductal adenocarcinoma (PDAC). Recent evidence suggests that several subpopulations of CAFs in the TME can interconvert, explaining the dual roles (antitumorigenic and protumorigenic) of CAFs in PDAC and the contradictory results of CAF-targeted therapies in clinical trials. This highlights the need to clarify CAF heterogeneity and their interactions with PDAC cells. This review focuses on the communication between activated PSCs/CAFs and PDAC cells, as well as on the mechanisms underlying this crosstalk. CAF-focused therapies and emerging biomarkers are also outlined.     

Unique Protein Profiles of Extracellular Vesicles as Diagnostic Biomarkers for Early and Advanced Non‐Small Cell Lung Cancer

Extracellular vesicles (EVs) mediate intercellular communication via transferring proteins and other biological molecules and have been recently investigated as biomarkers of disease. Sensitive and specific biomarkers are required for lung cancer diagnosis and prognosis. The present study screens for abnormal EV proteins in non‐small cell lung cancer (NSCLC) using a quantitative proteomics strategy involving LC‐MS/MS to identify ideal biomarkers for NSCLC diagnosis. EVs are enriched from the sera of early and advanced NSCLC patients and healthy controls and from cell culture supernatants of lung adenocarcinoma and bronchial epithelial cell lines. In the sera and supernatants, 279 and 632 differentially expressed proteins, respectively, are associated with signaling pathways including extracellular membrane–receptor interaction, focal adhesion, and regulation of the actin cytoskeleton. Thirty‐two EV proteins are identified at the intersection of differentially expressed proteins between the NSCLC groups and cell lines. Based on bioinformatics analysis, in silico immunohistochemical, and PRM verification, fibronectin is selected for following in vitro studies and validation with an independent cohort. Fibronectin on EVs is estimated to perform well in the diagnosis of NSCLC patients based on AUC, showing great potential for clinical use and demonstrating the efficacy of this method for EV‐associated biomarker screening.     

Effect of mesenchymal stem cells-derived exosomes on tumor microenvironment: Tumor progression versus tumor suppression

Mesenchymal stem cells (MSCs) are multipotent cells with the potential to differentiate into different cell types. Owing to their immunosuppressive and anti-inflammatory properties, they are widely used in regenerative medicine, but they have a dual effect on cancer progression and exert both growth-stimulatory or -inhibitory effects on different cancer types. It has been proposed that these controversial effects of MSC in tumor microenvironment (TME) are mediated by their polarization to proinflammatory or anti-inflammatory phenotype. In addition, they can polarize the immune system cells that in turn influence tumor progression. One of the mechanisms involved in the TME communications is extracellular vesicles (EVs). MSCs, as one of cell populations in TME, produce a large amount of EVs that can influence tumor development. Similar to MSC, MSC-EVs can exert both anti- or protumorigenic effects. In the current study, we will investigate the current knowledge related to MSC role in cancer progression with a focus on the MSC-EV content in limiting tumor growth, angiogenesis, and metastasis. We suppose MSC-EVs can be used as safe vehicles for delivering antitumor agents to TME.     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.     

Optimizing Size Exclusion Chromatography for Extracellular Vesicle Enrichment and Proteomic Analysis from Clinically Relevant Samples

The field of extracellular vesicle (EV) research has rapidly expanded in recent years, with particular interest in their potential as circulating biomarkers. Proteomic analysis of EVs from clinical samples is complicated by the low abundance of EV proteins relative to highly abundant circulating proteins such as albumin and apolipoproteins. To overcome this, size exclusion chromatography (SEC) has been proposed as a method to enrich EVs whilst depleting protein contaminants; however, the optimal SEC parameters for EV proteomics have not been thoroughly investigated. Here, quantitative evaluation and optimization of SEC are reported for separating EVs from contaminating proteins. Using a synthetic model system followed by cell line‐derived EVs, it is found that a 10 mL Sepharose 4B column in PBS produces optimal resolution of EVs from background protein. By spiking‐in cancer cell‐derived EVs to healthy plasma, it is shown that some cancer EV‐associated proteins are detectable by nano‐LC‐MS/MS when as little as 1% of the total plasma EV number are derived from a cancer cell line. These results suggest that an optimized SEC and nanoLC‐MS/MS workflow may be sufficiently sensitive for disease EV protein biomarker discovery from patient‐derived clinical samples.