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.

The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research.

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.

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.

Inhibition of αvß3 integrin impairs adhesion and uptake of tumor-derived small extracellular vesicles.

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 αvß3 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 αvß3 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 αvß3 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 αvß3 integrin in cell-cell communication through SEVs. Video Abstract.

Quantitative Proteomic Analysis of Small and Large Extracellular Vesicles (EVs) Reveals Enrichment of Adhesion Proteins in Small EVs.

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 might have unique functional properties. In order to identify protein cargos that may contribute to different functions of SEVs and LEVs, we used isobaric tags for relative and absolute quantitation (iTRAQ)-liquid chromatography (LC) tandem mass spectrometry (MS) on EVs isolated from a colon cancer cell line. Bioinformatics 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, processing, and metabolism. Western blot analysis of EVs purified from two 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.

Regulation of invadopodia by mechanical signaling.

Mechanical rigidity in the tumor microenvironment is associated with a high risk of tumor formation and aggressiveness. Adhesion-based signaling driven by a rigid microenvironment is thought to facilitate invasion and migration of cancer cells away from primary tumors. Proteolytic degradation of extracellular matrix (ECM) is a key component of this process and is mediated by subcellular actin-rich structures known as invadopodia. Both ECM rigidity and cellular traction stresses promote invadopodia formation and activity, suggesting a role for these structures in mechanosensing. The presence and activity of mechanosensitive adhesive and signaling components at invadopodia further indicates the potential for these structures to utilize myosin-dependent forces to probe and remodel their ECM environments. Here, we provide a brief review of the role of adhesion-based mechanical signaling in controlling invadopodia and invasive cancer behavior.

Activating PIK3CA Mutations Induce an Epidermal Growth Factor Receptor (EGFR)/Extracellular Signal-regulated Kinase (ERK) Paracrine Signaling Axis in Basal-like Breast Cancer.

Mutations in PIK3CA, the gene encoding the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K) have been shown to transform human mammary epithelial cells (MECs). These mutations are present in all breast cancer subtypes, including basal-like breast cancer (BLBC). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identified 72 protein expression changes in human basal-like MECs with knock-in E545K or H1047R PIK3CA mutations versus isogenic MECs with wild-type PIK3CA. Several of these were secreted proteins, cell surface receptors or ECM interacting molecules and were required for growth of PIK3CA mutant cells as well as adjacent cells with wild-type PIK3CA. The proteins identified by MS were enriched among human BLBC cell lines and pointed to a PI3K-dependent amphiregulin/EGFR/ERK signaling axis that is activated in BLBC. Proteins induced by PIK3CA mutations correlated with EGFR signaling and reduced relapse-free survival in BLBC. Treatment with EGFR inhibitors reduced growth of PIK3CA mutant BLBC cell lines and murine mammary tumors driven by a PIK3CA mutant transgene, all together suggesting that PIK3CA mutations promote tumor growth in part by inducing protein changes that activate EGFR.

Signaling inputs to invadopodia and podosomes.

Remodeling of extracellular matrix (ECM) is a fundamental cell property that allows cells to alter their microenvironment and move through tissues. Invadopodia and podosomes are subcellular actin-rich structures that are specialized for matrix degradation and are formed by cancer and normal cells, respectively. Although initial studies focused on defining the core machinery of these two structures, recent studies have identified inputs from both growth factor and adhesion signaling as crucial for invasive activity. This Commentary will outline the current knowledge on the upstream signaling inputs to invadopodia and podosomes and their role in governing distinct stages of these invasive structures. We discuss invadopodia and podosomes as adhesion structures and highlight new data showing that invadopodia-associated adhesion rings promote the maturation of already-formed invadopodia. We present a model in which growth factor stimulation leads to phosphoinositide 3-kinase (PI3K) activity and formation of invadopodia, whereas adhesion signaling promotes exocytosis of proteinases at invadopodia.

3D collagen alignment limits protrusions to enhance breast cancer cell persistence.

Patients with mammographically dense breast tissue have a greatly increased risk of developing breast cancer. Dense breast tissue contains more stromal collagen, which contributes to increased matrix stiffness and alters normal cellular responses. Stromal collagen within and surrounding mammary tumors is frequently aligned and reoriented perpendicular to the tumor boundary. We have shown that aligned collagen predicts poor outcome in breast cancer patients, and postulate this is because it facilitates invasion by providing tracks on which cells migrate out of the tumor. However, the mechanisms by which alignment may promote migration are not understood. Here, we investigated the contribution of matrix stiffness and alignment to cell migration speed and persistence. Mechanical measurements of the stiffness of collagen matrices with varying density and alignment were compared with the results of a 3D microchannel alignment assay to quantify cell migration. We further interpreted the experimental results using a computational model of cell migration. We find that collagen alignment confers an increase in stiffness, but does not increase the speed of migrating cells. Instead, alignment enhances the efficiency of migration by increasing directional persistence and restricting protrusions along aligned fibers, resulting in a greater distance traveled. These results suggest that matrix topography, rather than stiffness, is the dominant feature by which an aligned matrix can enhance invasion through 3D collagen matrices.

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.

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.

Establishment and validation of computational model for MT1-MMP dependent ECM degradation and intervention strategies.

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.

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.

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.

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.

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.

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.