5-Fluorouracil treatment represses pseudouridine-containing miRNA export into extracellular vesicles.

5-Fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. We examined the impact of 5-FU on post-transcriptional small RNA modifications (PTxMs) and the expression and export of RNA into small extracellular vesicles (sEVs). EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. We found that treatment of colorectal cancer (CRC) cells with 5-FU represses sEV export of miRNA and snRNA-derived RNAs, but promotes export of snoRNA-derived RNAs. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and sEV small RNA profiles. In contrast, 5-FU exposure led to increased levels of cellular small RNAs containing a variety of methyl-modified bases. These unexpected findings show that 5-FU exposure leads to altered RNA expression, base modification, and aberrant trafficking and localization of small RNAs.

Extracellular RNA in oncogenesis, metastasis and drug resistance.

Extracellular vesicles and nanoparticles (EVPs) are now recognized as a novel form of cell-cell communication. All cells release a wide array of heterogeneous EVPs with distinct protein, lipid, and RNA content, dependent on the pathophysiological state of the donor cell. The overall cargo content in EVPs is not equivalent to cellular levels, implying a regulated pathway for selection and export. In cancer, release and uptake of EVPs within the tumour microenvironment can influence growth, proliferation, invasiveness, and immune evasion. Secreted EVPs can also have distant, systemic effects that can promote metastasis. Here, we review current knowledge of EVP biogenesis and cargo selection with a focus on the role that extracellular RNA plays in oncogenesis and metastasis. Almost all subtypes of RNA have been identified in EVPs, with miRNAs being the best characterized. We review the roles of specific miRNAs that have been detected in EVPs and that play a role in oncogenesis and metastasis.

miR-100 and miR-125b Contribute to Enhanced 3D Growth and Invasiveness and can be Functionally Transferred to Silence Target Genes in Recipient Cells.

Extracellular communication via the transfer of vesicles and nanoparticles is now recognized to play an important role in tumor microenvironment interactions. Cancer cells upregulate and secrete abundant levels of miR-100 and miR-125b that can alter gene expression by both cell- and non-cell-autonomous mechanisms. We previously showed that these miRNAs activate Wnt signaling in colorectal cancer (CRC) through noncanonical pairing with 5 negative regulators of Wnt signaling. To identify additional targets of miR-100 and miR-125b , we used bioinformatic approaches comparing multiple CRC cell lines, including knockout lines lacking one or both of these miRNAs. From an initial list of 96 potential mRNA targets, we tested 15 targets with 8 showing significant downregulation in the presence of miR-100 and miR-125b . Among these, Cingulin (CGN) and Protein tyrosine phosphatase receptor type-R (PTPRR) are downregulated in multiple cancers, consistent with regulation by increased levels of miR-100 and miR-125b. We also show that increased cellular levels of miR-100 and miR-125b enhance 3D growth and invasiveness in CRC and glioblastoma cell lines. Lastly, we demonstrate that extracellular transfer of miR-100 and miR-125b can silence both reporter and endogenous mRNA targets in recipient cells and also increase the invasiveness of recipient spheroid colonies when grown under 3D conditions in type I collagen.

Multiparametric Single-Vesicle Flow Cytometry Resolves Extracellular Vesicle Heterogeneity and Reveals Selective Regulation of Biogenesis and Cargo Distribution.

Mammalian cells release a heterogeneous array of extracellular vesicles (EVs) that contribute to intercellular communication by means of the cargo that they carry. To resolve EV heterogeneity and determine if cargo is partitioned into select EV populations, we developed a method named "EV Fingerprinting" that discerns distinct vesicle populations using dimensional reduction of multiparametric data collected by quantitative single-EV flow cytometry. EV populations were found to be discernible by a combination of membrane order and EV size, both of which were obtained through multiparametric analysis of fluorescent features from the lipophilic dye Di-8-ANEPPS incorporated into the lipid bilayer. Molecular perturbation of EV secretion and biogenesis through respective ablation of the small GTPase Rab27a and overexpression of the EV-associated tetraspanin CD63 revealed distinct and selective alterations in EV populations, as well as cargo distribution. While Rab27a disproportionately affects all small EV populations with high membrane order, the overexpression of CD63 selectively increased the production of one small EV population of intermediate membrane order. Multiplexing experiments subsequently revealed that EV cargos have a distinct, nonrandom distribution with CD63 and CD81 selectively partitioning into smaller vs larger EVs, respectively. These studies not only present a method to probe EV biogenesis but also reveal how the selective partitioning of cargo contributes to EV heterogeneity.

EVPsort: An Atlas of Small ncRNA Profiling and Sorting in Extracellular Vesicles and Particles.

Extracellular vesicles and particles (EVPs) play a crucial role in mediating cell-to-cell communication by transporting various molecular cargos, with small non-coding RNAs (ncRNAs) holding particular significance. A thorough investigation into the abundance and sorting mechanisms of ncRNA within EVPs is imperative for advancing their clinical applications. We have developed EVPsort, which not only provides an extensive overview of ncRNA profiling in 3,162 samples across various biofluids, cell lines, and disease contexts but also seamlessly integrates 19 external databases and tools. This integration encompasses information on associations between ncRNAs and RNA-binding proteins (RBPs), motifs, targets, pathways, diseases, and drugs. With its rich resources and powerful analysis tools, EVPsort extends its profiling capabilities to investigate ncRNA sorting, identify relevant RBPs and motifs, and assess functional implications. EVPsort stands as a pioneering database dedicated to comprehensively addressing both the abundance and sorting of ncRNA within EVPs. It is freely accessible at https://bioinfo.vanderbilt.edu/evpsort/.

5-Fluorouracil Treatment Represses Pseudouridine-Containing Small RNA Export into Extracellular Vesicles.

5-fluorouracil (5-FU) has been used for chemotherapy for colorectal and other cancers for over 50 years. The prevailing view of its mechanism of action is inhibition of thymidine synthase leading to defects in DNA replication and repair. However, 5-FU is also incorporated into RNA causing toxicity due to defects in RNA metabolism, inhibition of pseudouridine modification, and altered ribosome function. Here, we examine the impact of 5-FU on the expression and export of small RNAs (sRNAs) into small extracellular vesicles (sEVs). Moreover, we assess the role of 5-FU in regulation of post-transcriptional sRNA modifications (PTxM) using mass spectrometry approaches. EVs are secreted by all cells and contain a variety of proteins and RNAs that can function in cell-cell communication. PTxMs on cellular and extracellular sRNAs provide yet another layer of gene regulation. We found that treatment of the colorectal cancer (CRC) cell line DLD-1 with 5-FU led to surprising differential export of miRNA snRNA, and snoRNA transcripts. Strikingly, 5-FU treatment significantly decreased the levels of pseudouridine on both cellular and secreted EV sRNAs. In contrast, 5-FU exposure led to increased levels of cellular sRNAs containing a variety of methyl-modified bases. Our results suggest that 5-FU exposure leads to altered expression, base modifications, and mislocalization of EV base-modified sRNAs.

Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.

Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid, and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein vesicle associated protein associated protein A (VAP-A) drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and upregulated proteins in small EVs (SEVs) purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. Search Tool for the Retrieval of Reciprocity Genes (STRING) protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD SEVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.

Extracellular vesicles from non-neuroendocrine SCLC cells promote adhesion and survival of neuroendocrine SCLC cells.

Small cell lung cancer (SCLC) tumors are made up of distinct cell subpopulations, including neuroendocrine (NE) and non-neuroendocrine (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 SEVs revealed that extracellular matrix (ECM) proteins and integrins are highly enriched in SEVs of non-NE cells whereas nucleic acid-binding proteins are enriched in SEVs purified from NE cells. Addition of select purified ECM proteins identified in purified extracellular vesicles (EVs), specifically fibronectin, laminin 411, and laminin 511, were able to substitute for the role of non-NE-derived SEVs in promoting adhesion and survival 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 the growth and survival of NE SCLC cells.

Culture conditions greatly impact the levels of vesicular and extravesicular Ago2 and RNA in extracellular vesicle preparations.

Extracellular vesicle (EV)-carried miRNAs can influence gene expression and functional phenotypes in recipient cells. Argonaute 2 (Ago2) is a key miRNA-binding protein that has been identified in EVs and could influence RNA silencing. However, Ago2 is in a non-vesicular form in serum and can be an EV contaminant. In addition, RNA-binding proteins (RBPs), including Ago2, and RNAs are often minor EV components whose sorting into EVs may be regulated by cell signaling state. To determine the conditions that influence detection of RBPs and RNAs in EVs, we evaluated the effect of growth factors, oncogene signaling, serum, and cell density on the vesicular and nonvesicular content of Ago2, other RBPs, and RNA in small EV (SEV) preparations. Media components affected both the intravesicular and extravesicular levels of RBPs and miRNAs in EVs, with serum contributing strongly to extravesicular miRNA contamination. Furthermore, isolation of EVs from hollow fiber bioreactors revealed complex preparations, with multiple EV-containing peaks and a large amount of extravesicular Ago2/RBPs. Finally, KRAS mutation impacts the detection of intra- and extra-vesicular Ago2. These data indicate that multiple cell culture conditions and cell states impact the presence of RBPs in EV preparations, some of which can be attributed to serum contamination.

Systematic Assessment of Small RNA Profiling in Human Extracellular Vesicles.

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.

Analysis of small EV proteomes reveals unique functional protein networks regulated by VAP-A.

Extracellular vesicles (EVs) influence cell phenotypes and functions via protein, nucleic acid and lipid cargoes. EVs are heterogeneous, due to diverse biogenesis mechanisms that remain poorly understood. Our previous study revealed that the endoplasmic reticulum (ER) membrane contact site (MCS) linker protein VAP-A drives biogenesis of a subset of RNA-enriched EVs. Here, we examine the protein content of VAP-A-regulated EVs. Using label-free proteomics, we identified down- and up-regulated proteins in sEVs purified from VAP-A knockdown (KD) colon cancer cells. Gene set enrichment analysis (GSEA) of the data revealed protein classes that are differentially sorted to SEVs dependent on VAP-A. STRING protein-protein interaction network analysis of the RNA-binding protein (RBP) gene set identified several RNA functional machineries that are downregulated in VAP-A KD EVs, including ribosome, spliceosome, mRNA surveillance, and RNA transport proteins. We also observed downregulation of other functionally interacting protein networks, including cadherin-binding, unfolded protein binding, and ATP-dependent proteins.

Context-specific regulation of extracellular vesicle biogenesis and cargo selection.

To coordinate, adapt and respond to biological signals, cells convey specific messages to other cells. An important aspect of cell-cell communication involves secretion of molecules into the extracellular space. How these molecules are selected for secretion has been a fundamental question in the membrane trafficking field for decades. Recently, extracellular vesicles (EVs) have been recognized as key players in intercellular communication, carrying not only membrane proteins and lipids but also RNAs, cytosolic proteins and other signalling molecules to recipient cells. To communicate the right message, it is essential to sort cargoes into EVs in a regulated and context-specific manner. In recent years, a wealth of lipidomic, proteomic and RNA sequencing studies have revealed that EV cargo composition differs depending upon the donor cell type, metabolic cues and disease states. Analyses of distinct cargo 'fingerprints' have uncovered mechanistic linkages between the activation of specific molecular pathways and cargo sorting. In addition, cell biology studies are beginning to reveal novel biogenesis mechanisms regulated by cellular context. Here, we review context-specific mechanisms of EV biogenesis and cargo sorting, focusing on how cell signalling and cell state influence which cellular components are ultimately targeted to EVs.

Visualization of Exosome Release and Uptake During Cell Migration Using the Live Imaging Reporter pHluorin_M153R-CD63.

Exosome secretion and uptake regulate cell migration through autocrine and paracrine mechanisms. Monitoring exosome secretion and uptake during cell migration is critical for investigation of these mechanisms. Exosomes can be visualized by direct labeling with fluorescent dyes or by tagging intrinsic markers with fluorescent proteins for live imaging. Due to several limitations of fluorescent dye-labeled exosomes, we created two bright genetically encoded reporters of exosome secretion, pHluorin_M153R-CD63 and pHluorin_M153R-CD63-mScarlet. Here, we describe how to visualize secretion and uptake of exosomes labeled with these pH-sensitive and pH-insensitive fluorescent protein-tagged exosomal markers during cell migration using time-lapse fluorescent microscopy.

Archetype tasks link intratumoral heterogeneity to plasticity and cancer hallmarks in small cell lung cancer.

Small cell lung cancer (SCLC) tumors comprise heterogeneous mixtures of cell states, categorized into neuroendocrine (NE) and non-neuroendocrine (non-NE) transcriptional subtypes. NE to non-NE state transitions, fueled by plasticity, likely underlie adaptability to treatment and dismal survival rates. Here, we apply an archetypal analysis to model plasticity by recasting SCLC phenotypic heterogeneity through multi-task evolutionary theory. Cell line and tumor transcriptomics data fit well in a five-dimensional convex polytope whose vertices optimize tasks reminiscent of pulmonary NE cells, the SCLC normal counterparts. These tasks, supported by knowledge and experimental data, include proliferation, slithering, metabolism, secretion, and injury repair, reflecting cancer hallmarks. SCLC subtypes, either at the population or single-cell level, can be positioned in archetypal space by bulk or single-cell transcriptomics, respectively, and characterized as task specialists or multi-task generalists by the distance from archetype vertex signatures. In the archetype space, modeling single-cell plasticity as a Markovian process along an underlying state manifold indicates that task trade-offs, in response to microenvironmental perturbations or treatment, may drive cell plasticity. Stifling phenotypic transitions and plasticity may provide new targets for much-needed translational advances in SCLC. A record of this paper's Transparent Peer Review process is included in the supplemental information.

Extracellular Vesicles and Their Emerging Roles as Cellular Messengers in Endocrinology: An Endocrine Society Scientific Statement.

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.

VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites.

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.

ER membrane contact sites: key platforms for biogenesis of RNA-containing extracellular vesicles.

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.

Sunitinib and Axitinib increase secretion and glycolytic activity of small extracellular vesicles in renal cell carcinoma.

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.

Depletion of METTL3 alters cellular and extracellular levels of miRNAs containing m6A consensus sequences.

Extracellular vesicles (EVs) are capable of transferring cargo from donor to recipient cells, but precisely how cargo content is regulated for export is mostly unknown. For miRNA cargo, we previously showed that when compared to isogenic colorectal cancer (CRC) cells expressing wild-type KRAS, a distinct subset of miRNAs are differentially enriched in EVs from KRAS mutant active CRC cells, with miR-100 being one of the most enriched. The mechanisms that could explain how miR-100 and other miRNAs are differentially exported into EVs have not been fully elucidated. Here, we tested the effect of N6-methyladenosine (m6A) modification on miRNA export into EVs by depletion of METTL3 and ALKBH5, a writer and eraser of m6A modification, respectively. While the effects of ALKBH5 knockdown were quite modest, decreased levels of METTL3 led to reduced cellular and extracellular levels of a subset of miRNAs that contain consensus sequences for m6A modification. Functional testing of EVs prepared from cells expressing shRNAs against METTL3 showed that they were less capable of conferring colony growth in 3D to wild-type KRAS cells and were also largely incapable of conferring the spread of cetuximab resistance. Our data support a role for METTL3 modification on cellular miRNA levels and export of specific miRNAs.

Extracellular vesicles: Critical players during cell migration.

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.