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Motivation: Extracellular vesicles (EVs) are produced and released by most cells and are now recognized to play a role in intercellular communication through the delivery of molecular cargo, including proteins, lipids, and RNA. Small RNA sequencing (small RNA-seq) has been widely used to characterize the small RNA content in EVs. However, there is a lack of a systematic assessment of the quality, technical biases, RNA composition, and RNA biotypes enrichment for small RNA profiling of EVs across cell types, biofluids, and conditions. Methods: We collected and reanalyzed small RNA-seq datasets for 2756 samples from 83 studies involving 55 with EVs only and 28 with both EVs and matched donor cells. We assessed their quality by the total number of reads after adapter trimming, the overall alignment rate to the host and non-host genomes, and the proportional abundance of total small RNA and specific biotypes, such as miRNA, tRNA, rRNA, and Y RNA. Results: We found that EV extraction methods varied in their reproducibility in isolating small RNAs, with effects on small RNA composition. Comparing proportional abundances of RNA biotypes between EVs and matched donor cells, we discovered that rRNA and tRNA fragments were relatively enriched, but miRNAs and snoRNA were depleted in EVs. Except for the export of eight miRNAs being context-independent, the selective release of most miRNAs into EVs was study-specific. Conclusion: This work guides quality control and the selection of EV isolation methods and enhances the interpretation of small RNA contents and preferential loading in EVs.

Supplementary Figures 1-3, Table 1, Methods from Microenvironmental Independence Associated with Tumor Progression

Supplementary Figure S3. Cleavage of EphA2 in vitro.

Supplementary Figure 1 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Supplementary Figure 2 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Free Article from Microenvironmental Independence Associated with Tumor Progression

Supplementary Figure 5 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Supplementary Figure 3 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Invadopodia are branched actin-rich structures associated with extracellular matrix (ECM) degradation that collectively form the invasive machinery of aggressive cancer cells. Cortactin is a prominent component and a specific marker of invadopodia. Amplification of cortactin is associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), possibly because of its activity in invadopodia. Although the role of cortactin in invadopodia has been attributed to signaling and actin assembly, it is incompletely understood. We made HNSCC cells deficient in cortactin by RNA interference knockdown methods. In these cortactin knockdown cells, invadopodia were reduced in number and lost their ability to degrade ECM. In the reverse experiment, overexpression of cortactin dramatically increased ECM degradation, far above and beyond the effect on formation of actin/Arp3–positive invadopodia puncta. Secretion of matrix metalloproteinases (MMP) MMP-2 and MMP-9, as well as plasma membrane delivery of MT1-MMP correlated closely with cortactin expression levels. MMP inhibitor treatment of control cells mimicked the cortactin knockdown phenotype, with abolished ECM degradation and fewer invadopodia, suggesting a positive feedback loop in which degradation products from MMP activity promote new invadopodia formation. Collectively, these data suggest that a major role of cortactin in invadopodia is to regulate the secretion of MMPs and point to a novel mechanism coupling dynamic actin assembly to the secretory machinery, producing enhanced ECM degradation and invasiveness. Furthermore, these data provide a possible explanation for the observed association between cortactin overexpression and enhanced invasiveness and poor prognosis in HNSCC patients. [Cancer Res 2007;67(9):4227–35]

Supplementary Table 1, Figures 1-3, Methods from Microenvironmental Independence Associated with Tumor Progression

Tumor-microenvironment interactions are increasingly recognized to influence tumor progression. To understand the competitive dynamics of tumor cells in diverse microenvironments, we experimentally parameterized a hybrid discrete-continuum mathematical model with phenotypic trait data from a set of related mammary cell lines with normal, transformed, or tumorigenic properties. Surprisingly, in a resource-rich microenvironment, with few limitations on proliferation or migration, transformed (but not tumorigenic) cells were most successful and outcompeted other cell types in heterogeneous tumor simulations. Conversely, constrained microenvironments with limitations on space and/or growth factors gave a selective advantage to phenotypes derived from tumorigenic cell lines. Analysis of the relative performance of each phenotype in constrained versus unconstrained microenvironments revealed that, although all cell types grew more slowly in resource-constrained microenvironments, the most aggressive cells were least affected by microenvironmental constraints. A game theory model testing the relationship between microenvironment resource availability and competitive cellular dynamics supports the concept that microenvironmental independence is an advantageous cellular trait in resource-limited microenvironments. [Cancer Res 2009;69(22):8797–806]

Supplementary Methods, Figure Legends and References

Supplementary Table 1 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Supplementary Figure Legends 1-5 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Supplementary Figure 4 from Cortactin Is an Essential Regulator of Matrix Metalloproteinase Secretion and Extracellular Matrix Degradation in Invadopodia

Small Cell Lung Cancer (SCLC) tumors are made up of distinct cell subpopulations, including neuroendocrine (NE) and non-NE cells. While secreted factors from non-NE SCLC cells have been shown to support the growth of the NE cells, the underlying molecular factors are not well understood. Here, we show that exosome-type small extracellular vesicles (SEVs) secreted from non-NE SCLC cells promote adhesion and survival of NE SCLC cells. Proteomic analysis of purified small EVs revealed that extracellular matrix (ECM) proteins and integrins are highly enriched in small EVs of non-NE cells whereas nucleic acid-binding proteins are enriched in small EVs purified from NE cells. Addition of select purified ECM proteins identified in purified EVs, specifically fibronectin, laminin 411, and laminin 511, were able to substitute for the role of non-NE-derived SEVs in promoting adhesion, survival, and tumorigenicity of NE SCLC cells. Those same proteins were differentially expressed by human SCLC subtypes. These data suggest that ECM-carrying SEVs secreted by non-NE cells play a key role in supporting SCLC tumor growth and survival.

Cell migration is essential for the development and maintenance of multicellular organisms, contributing to embryogenesis, wound healing, immune response, and other critical processes. It is also involved in the pathogenesis of many diseases, including immune deficiency disorders and cancer metastasis. Recently, extracellular vesicles (EVs) have been shown to play important roles in cell migration. Here, we review recent studies describing the functions of EVs in multiple aspects of cell motility, including directional sensing, cell adhesion, extracellular matrix (ECM) degradation, and leader-follower behavior. We also discuss the role of EVs in migration during development and disease and the utility of imaging tools for studying the role of EVs in cell migration.

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes., A prior live-cell exosome reporter showed dim fluorescence and could not be expressed stably, limiting its usefulness. Here the authors stabilise the reporter to allow long-term tracking of exosomes, and incorporate a second fluorophore to visualise the entire exosome lifecycle.

During the last decade, there has been great interest in elucidating the biological role of extracellular vesicles (EVs), particularly, their hormone-like role in cell-to-cell communication. The field of endocrinology is uniquely placed to provide insight into the functions of EVs, which are secreted from all cells into biological fluids and carry endocrine signals to engage in paracellular and distal interactions. EVs are a heterogeneous population of membrane-bound vesicles of varying size, content, and bioactivity. EVs are specifically packaged with signaling molecules, including lipids, proteins, and nucleic acids, and are released via exocytosis into biofluid compartments. EVs regulate the activity of both proximal and distal target cells, including translational activity, metabolism, growth, and development. As such, EVs signaling represents an integral pathway mediating intercellular communication. Moreover, as the content of EVs is cell-type specific, it is a “fingerprint” of the releasing cell and its metabolic status. Recently, changes in the profile of EV and bioactivity have been described in several endocrine-related conditions including diabetes, obesity, cardiovascular diseases, and cancer. The goal of this statement is to highlight relevant aspects of EV research and their potential role in the field of endocrinology.

The mechanisms by which cytoplasmic cargoes such as RNAs are incorporated into extracellular vesicles (EVs) are poorly understood. In a recent article published in Developmental Cell, we describe a novel function of endoplasmic reticulum membrane contact sites (ER MCS) in regulating biogenesis of RNA-containing EVs (Barman et al., 2022). We identified the ER MCS tether protein VAP-A and the ceramide transporter CERT as key drivers of this process. VAP-A depletion and overexpression produced corresponding changes in the overall number and RNA content of secreted EVs. Further sub-fractionation of small EVs from VAP-A depleted cells revealed a distinct loss in a specific subset of dense, RNA-loaded small EVs that are critical for the transfer of miR-100 to recipient cells. Cell imaging data confirmed the loss of RNA and RNA binding proteins (RBPs) in VAP-A-knockdown multivesicular bodies. Lipid analysis of VAP-A-knockdown EVs revealed decreases in ceramides, which are known to affect EV biogenesis. Depletion of the ceramide transfer protein CERT, which interacts with its binding partner VAP-A at ER MCS, leads to similar defects in EV number and RNA content as VAP-A-knockdown. These data suggest a model for ER MCS as platforms for biogenesis of a key EV population via ceramide transfer and RNA loading.

Extracellular vesicles (EVs) encompass a wide range of vesicles that are released by all cell types. They package protein, nucleic acids, metabolites, and other cargo that can be delivered to recipient cells and affect their phenotypes. However, little is known about how pharmaceutical agents can alter EV secretion, protein and metabolic cargo, and the active biological processes taking place in these vesicles. In this study, we isolated EVs from human renal cell carcinoma (RCC) cells treated with tyrosine kinase inhibitors (TKIs) Sunitinib and Axitinib. We found these TKIs increase the number of large (lEVs) and small extracellular vesicles (sEVs) secreted from RCC cells in a dose-dependent manner. In addition, quantitative proteomics revealed that metabolic proteins are enriched in sEVs secreted from Sunitinib-treated cells. In particular, the glucose transporter GLUT1 was enriched in sEVs purified from TKI-treated cells. These sEVs displayed increased glucose uptake and glycolytic metabolism compared to sEVs released from vehicle-treated cells. Overexpression of GLUT1 in RCC cells augmented GLUT1 levels in sEVs, which subsequently displayed higher glucose uptake and glycolytic activity. Together, these findings suggest that these TKIs alter metabolic cargo and activity in RCC sEVs. Proposed model of the effect of Sunitinib and Axitinib on extracellular vesicle secretion and metabolism. Upon treatment with Sunitinib or Axitinib, RCC cells secrete more extracellular vesicles that contain GLUT1, leading to increased glycolytic metabolism in their vesicles. Figure created with BioRender.com

RNA transfer via extracellular vesicles (EVs) influences cell phenotypes; however, lack of information regarding biogenesis of RNA-containing EVs has limited progress in the field. Here, we identify endoplasmic reticulum membrane contact sites (ER MCSs) as platforms for the generation of RNA-containing EVs. We identify a subpopulation of small EVs that is highly enriched in RNA and regulated by the ER MCS linker protein VAP-A. Functionally, VAP-A-regulated EVs are critical for miR-100 transfer between cells and in vivo tumor formation. Lipid analysis of VAP-A-knockdown EVs revealed reductions in the EV biogenesis lipid ceramide. Knockdown of the VAP-A-binding ceramide transfer protein CERT led to similar defects in EV RNA content. Imaging experiments revealed that VAP-A promotes luminal filling of multivesicular bodies (MVBs), CERT localizes to MVBs, and the ceramide-generating enzyme neutral sphingomyelinase 2 colocalizes with VAP-A-positive ER. We propose that ceramide transfer via VAP-A-CERT linkages drives the biogenesis of a select RNA-containing EV population.

Background Extracellular vesicles (EVs) are lipid-bound particles that are naturally released from cells and mediate cell-cell communication. Integrin adhesion receptors are enriched in small EVs (SEVs) and SEV-carried integrins have been shown to promote cancer cell migration and to mediate organ-specific metastasis; however, how integrins mediate these effects is not entirely clear and could represent a combination of EV binding to extracellular matrix and cells. Methods To probe integrin role in EVs binding and uptake, we employed a disintegrin inhibitor (DisBa-01) of integrin binding with specificity for αvβ3 integrin. EVs were purified from MDA-MB-231 cells conditioned media by serial centrifugation method. Isolated EVs were characterized by different techniques and further employed in adhesion, uptake and co-culture experiments. Results We find that SEVs secreted from MDA-MB-231 breast cancer cells carry αvβ3 integrin and bind directly to fibronectin-coated plates, which is inhibited by DisBa-01. SEV coating on tissue culture plates also induces adhesion of MDA-MB-231 cells, which is inhibited by DisBa-01 treatment. Analysis of EV uptake and interchange between cells reveals that the amount of CD63-positive EVs delivered from malignant MDA-MB-231 breast cells to non-malignant MCF10A breast epithelial cells is reduced by DisBa-01 treatment. Inhibition of αvβ3 integrin decreases CD63 expression in cancer cells suggesting an effect on SEV content. Conclusion In summary, our findings demonstrate for the first time a key role of αvβ3 integrin in cell-cell communication through SEVs. Graphical abstract

Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is coopted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50–150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-to-cell signaling receptor ephrin type B receptor 2 (EPHB2) as a promising candidate cargo to promote angiogenesis. Analysis of patient data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulated angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to nonadjacent endothelial cells to induce ephrin reverse signaling.

Argonaute-2 (Ago2) is a key component of the RNA-induced silencing complex that mediates downregulation of mRNA by miRNAs. Its presence in extracellular vesicles (EV) has been postulated to be important for the activity of EV-carried miRNA in modulating gene expression in recipient cells. However, whether it is in fact contained within EVs or is instead an extravesicular contaminant is controversial. In this opinion piece, we argue that the ability to detect Ago2 in EVs is a result of multiple factors, including cell source, cell signaling control of Ago2 trafficking to EVs, experimental conditions, and detection methods.

Cancer is a complex, dynamic disease that despite recent advances remains mostly incurable. Inter- and intratumoral heterogeneity are generally considered major drivers of therapy resistance, metastasis, and treatment failure. Recent advances in high-throughput experimentation have produced a wealth of data on tumor heterogeneity and researchers are increasingly turning to mathematical modeling to aid in the interpretation of these complex datasets. In this mini-review, we discuss three important classes of approaches for modeling cellular dynamics within heterogeneous tumors: agent-based models, population dynamics, and multiscale models. An important new focus, for which we provide an example, is the role of intratumoral cell-cell interactions.

© 2019 Elsevier Inc. The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data. The Extracellular RNA Communication Consortium (ERCC) presents progress toward understanding the biology of extracellular RNAs and their use as biomarkers and therapeutics.

Small extracellular vesicles (EVs) called exosomes affect a variety of autocrine and paracrine cellular phenotypes, including cellular migration, immune activation, and neuronal function. Understanding the function of exosomes in these processes requires a variety of tools, including live cell imaging. We previously constructed a live-cell reporter, pHluorin-CD63, that allowed dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, there were some caveats to its use, including relatively low fluorescent expression in cells and the inability to make cell lines that stably express the protein. By incorporating a stabilizing mutation in the pHluorin moiety, M153R, pHluorin-CD63 now exhibits higher and stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we demonstrate visualization of exosome secretion in 3D culture and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D collective migration. By incorporating a further non-pH-sensitive red fluorescent tag, this reporter allows visualization of the entire exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This new reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.

Extracellular vesicles (EVs) are important mediators of cell-cell communication due to their cargo content of proteins, lipids and RNAs. We previously reported that small EVs (SEVs) called exosomes promote directed and random cell motility, invasion, and serum-independent growth. In contrast, larger EVs (LEVs) were not active in those assays, but may have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tag for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD010840. Bioinformatic analyses revealed that SEVs are enriched in proteins associated with cell-cell junctions, cell-matrix adhesion, exosome biogenesis machinery and various signaling pathways. In contrast, LEVs are enriched in proteins associated with ribosome and RNA biogenesis and processing, and metabolism. Western blot analysis of EVs purified from 2 different cancer cell types confirmed the enrichment of cell-matrix and cell-cell adhesion proteins in SEVs. Consistent with those data, we found that cells exhibit enhanced adhesion to surfaces coated with SEVs compared to an equal protein concentration of LEVs. These data suggest that a major function of SEVs is to promote cellular adhesion.