Therapeutic effects of adipose-tissue-derived mesenchymal stromal cells and their extracellular vesicles in experimental silicosis.

BACKGROUND: Silicosis is an occupational disease that affects workers who inhale silica particles, leading to extensive lung fibrosis and ultimately causing respiratory failure. Mesenchymal stromal cells (MSCs) have been shown to exert therapeutic effects in lung diseases and represent an alternative treatment for silicosis. Recently, it has been suggested that similar effects can be achieved by the therapeutic use of extracellular vesicles (EVs) obtained from MSCs. The aim of this study was to investigate the effects of adipose-tissue-derived MSCs (AD-MSCs) or their EVs in a model of silicosis. METHODS: Silicosis was induced by intratracheal instillation of silica in C57BL/6 mice. After the onset of disease, animals received saline, AD-MSCs, or EVs, intratracheally. RESULTS: At day 30, AD-MSCs and EVs led to a reduction in collagen fiber content, size of granuloma, and in the number of macrophages inside granuloma and in the alveolar septa. In addition, the expression levels of interleukin 1ß and transforming growth factor beta in the lungs were decreased. Higher dose of EVs also reduced lung static elastance when compared with the untreated silicosis group. CONCLUSIONS: Both AD-MSCs and EVs, locally delivered, ameliorated fibrosis and inflammation, but dose-enhanced EVs yielded better therapeutic outcomes in this model of silicosis.

Dynamic Scaling of Exosome Sizes.

A model is proposed for characterizing exosome size distributions based on dynamic scaling of domain growth on the limiting membrane of multivesicular bodies in the established exosome biogenesis pathway. The scaling exponent in this model captures the asymmetry of exosome size distributions, which are notably right-skewed to larger vesicles, independent of the minimum detectable vesicle size. Analyses of exosome size distributions obtained by cryogenic transmission electron microscopy imaging and nanoparticle tracking show, respectively, that the scaling exponent is sensitive to the state of the cell source for exosomes in cell culture supernatants and can distinguish exosome size distributions in serum samples taken from cancer patients relative to those from healthy donors. Finally, we comment on mechanistic differences between our dynamic scaling model and random fragmentation models used to describe size distributions of synthetic vesicles.

Analyzing the miRNA content of extracellular vesicles by fluorescence nanoparticle tracking.

We present a method that takes advantage of the fluorophore loading dependence of fluorescence nanoparticle tracking (fNTA) to determine the content of specific miRNA targets in extracellular vesicles (EVs) and their stoichiometry across the entire EV population. The method is based on an assay for detecting EV miRNA by hybridization to fluorescently labeled, miRNA-specific molecular beacons encapsulated in cationic lipoplex nanoparticles that fuse non-specifically with negatively charged EVs. To demonstrate the method, we carry out a stoichiometric analysis of miR-21 in EVs released from A549 lung cancer cells. We find approximately 2.3% of the A549 EVs have an average copy number of ~44 miR-21/A549 EV and contain at least a threshold number of 33 miR-21 copies/A549 EV required for fluorescence tracking. Potential applications of sizing, enumerating, and phenotyping EVs using this method include specifying dosages for therapeutic applications and identifying specific EV subpopulations in patient samples for diagnostic applications.

Epigenetic regulation of connective tissue growth factor by MicroRNA-214 delivery in exosomes from mouse or human hepatic stellate cells.

UNLABELLED: Connective tissue growth factor (CCN2) drives fibrogenesis in hepatic stellate cells (HSC). Here we show that CCN2 up-regulation in fibrotic or steatotic livers, or in culture-activated or ethanol-treated primary mouse HSC, is associated with a reciprocal down-regulation of microRNA-214 (miR-214). By using protector or reporter assays to investigate the 3'-untranslated region (UTR) of CCN2 mRNA, we found that induction of CCN2 expression in HSC by fibrosis-inducing stimuli was due to reduced expression of miR-214, which otherwise inhibited CCN2 expression by directly binding to the CCN2 3'-UTR. Additionally, miR-214 was present in HSC exosomes, which were bi-membrane vesicles, 50-150 nm in diameter, negatively charged (-26 mV), and positive for CD9. MiR-214 levels in exosomes but not in cell lysates were reduced by pretreatment of the cells with the exosome inhibitor, GW4869. Coculture of either quiescent HSC or miR-214-transfected activated HSC with CCN2 3'-UTR luciferase reporter-transfected recipient HSC resulted in miR-214- and exosome-dependent regulation of a wild-type CCN2 3'-UTR reporter but not of a mutant CCN2 3'-UTR reporter lacking the miR-214 binding site. Exosomes from HSC were a conduit for uptake of miR-214 by primary mouse hepatocytes. Down-regulation of CCN2 expression by miR-214 also occurred in human LX-2 HSC, consistent with a conserved miR-214 binding site in the human CCN2 3'-UTR. MiR-214 in LX-2 cells was shuttled by way of exosomes to recipient LX-2 cells or human HepG2 hepatocytes, resulting in suppression of CCN2 3'-UTR activity or expression of CCN2 downstream targets, including alpha smooth muscle actin or collagen. Experimental fibrosis in mice was associated with reduced circulating miR-214 levels. CONCLUSION: Exosomal transfer of miR-214 is a paradigm for the regulation of CCN2-dependent fibrogenesis and identifies fibrotic pathways as targets of intercellular regulation by exosomal miRs.

Macrophage microvesicles induce macrophage differentiation and miR-223 transfer.

Microvesicles are small membrane-bound particles comprised of exosomes and various-sized extracellular vesicles. These are released by several cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets, while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and explored their role in the differentiation of naive monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including mono cytes, endothelial cells, epithelial cells, and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation.

Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane-associated antigen critical to niche homing.

Membrane antigens are critical to the pathogenesis of chronic lymphocytic leukemia (CLL) as they facilitate microenvironment homing, proliferation, and survival. Targeting the CLL membrane and associated signaling patterns is a current focus of therapeutic development. Many tumor membrane targets are simultaneously targeted by humoral immunity, thus forming recognizable immunoglobulin responses. We sought to use this immune response to identify novel membrane-associated targets for CLL. Using a novel strategy, we interrogated CLL membrane-specific autologous immunoglobulin G reactivity. Our analysis unveiled lymphocyte cytosolic protein 1 (LCP1), a lymphocyte-specific target that is highly expressed in CLL. LCP1 plays a critical role in B-cell biology by crosslinking F-actin filaments, thereby solidifying cytoskeletal structures and providing a scaffold for critical signaling pathways. Small interfering RNA knockdown of LCP1 blocked migration toward CXCL12 in transwell assays and to bone marrow in an in vivo xenotransplant model, confirming a role for LCP1 in leukemia migration. Furthermore, we demonstrate that the Bruton's tyrosine kinase inhibitor ibrutinib or the PI3K inhibitor idelalisib block B-cell receptor induced activation of LCP1. Our data demonstrate a novel strategy to identify cancer membrane target antigens using humoral anti-tumor immunity. In addition, we identify LCP1 as a membrane-associated target in CLL with confirmed pathogenic significance. This clinical trial was registered at clinicaltrials.gov; study ID number: OSU-0025 OSU-0156.

Characterization of multiple myeloma vesicles by label-free relative quantitation.

Multiple myeloma (MM) is a hematological malignancy caused by a microenviromentally aided persistence of plasma cells in the bone marrow. The role that extracellular vesicles (EVs), microvesicles and exosomes, released by MM cells have in cell-to-cell communication and signaling in the bone marrow is currently unknown. This paper describes the proteomic content of EVs derived from MM.1S and U266 MM cell lines. First, we compared the protein identifications between the vesicles and cellular lysates of each cell line finding a large overlap in protein identifications. Next, we applied label-free spectral count quantitation to determine proteins with differential abundance between the groups. Finally, we used bioinformatics to categorize proteins with significantly different abundances into functional groups. The results illustrate the first use of label-free spectral counting applied to determine relative protein abundances in EVs.

Detection of extracellular RNAs in cancer and viral infection via tethered cationic lipoplex nanoparticles containing molecular beacons.

Noninvasive early detection methods have the potential to reduce mortality rates of both cancer and infectious diseases. Here, we present a novel assay by which tethered cationic lipoplex nanoparticles containing molecular beacons (MBs) can capture cancer cell-derived exosomes or viruses and identify encapsulated RNAs in a single step. A series of ultracentrifugation and Exoquick isolation kit were first used to isolate exosomes from the cell culture medium and human serum, respectively. Cationic lipoplex nanoparticles linked onto the surface of a thin glass plate capture negatively charged viruses or cell-secreted exosomes by electrostatic interactions to form larger nanoscale complexes. Lipoplex/virus or lipoplex/exosome fusion leads to the mixing of viral/exosomal RNAs and MBs within the lipoplexes. After the target RNAs specially bind to the MBs, exosomes enriched in target RNAs are readily identified by the fluorescence signals of MBs. The in situ detection of target extracellular RNAs without diluting the samples leads to high detection sensitivity not achievable by existing methods, e.g., quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Here we demonstrate this concept using lentivirus and serum from lung cancer patients.

Extracellular vesicles enhance pulmonary transduction of stably associated adeno-associated virus following intratracheal administration.

Adeno-associated virus (AAV) vector has shown multiple clinical breakthroughs, but its clinical implementation in inhaled gene therapy remains elusive due to difficulty in transducing lung airway cells. We demonstrate here AAV serotype 6 (AAV6) associated with extracellular vesicles (EVs) and secreted from vector-producing HEK-293 cells during vector preparation (EVAAV6) as a safe and highly efficacious gene delivery platform for inhaled gene therapy applications. Specifically, we discovered that EVAAV6 provided markedly enhanced reporter transgene expression in mucus-covered air-liquid interface (ALI) cultures of primary human bronchial and nasal epithelial cells as well as in mouse lung airways compared to standard preparations of AAV6 alone. Of note, AAV6 has been previously shown to outperform other clinically tested AAV serotypes, including those approved by the FDA for treating non-lung diseases, in transducing ALI cultures of primary human airway cells. We provide compelling experimental evidence that the superior performance of EVAAV6 is attributed to the ability of EV to facilitate mucus penetration and cellular entry/transduction of AAV6. The tight and stable linkage between AAV6 and EVs appears essential to exploit the benefits of EVs given that a physical mixture of individually prepared EVs and AAV6 failed to mediate EV-AAV6 interactions or to enhance gene transfer efficacy.

Rare intercellular material transfer as a confound to interpreting inner retinal neuronal transplantation following internal limiting membrane disruption.

Intercellular cytoplasmic material transfer (MT) occurs between transplanted and developing photoreceptors and ambiguates cell origin identification in developmental, transdifferentiation, and transplantation experiments. Whether MT is a photoreceptor-specific phenomenon is unclear. Retinal ganglion cell (RGC) replacement, through transdifferentiation or transplantation, holds potential for restoring vision in optic neuropathies. During careful assessment for MT following human stem cell-derived RGC transplantation into mice, we identified RGC xenografts occasionally giving rise to labeling of donor-derived cytoplasmic, nuclear, and mitochondrial proteins within recipient Müller glia. Critically, nuclear organization is distinct between human and murine retinal neurons, which enables unequivocal discrimination of donor from host cells. MT was greatly facilitated by internal limiting membrane disruption, which also augments retinal engraftment following transplantation. Our findings demonstrate that retinal MT is not unique to photoreceptors and challenge the isolated use of species-specific immunofluorescent markers for xenotransplant identification. Assessment for MT is critical when analyzing neuronal replacement interventions.

Coordination state probabilities and the solvation free energy of Zn2+ in aqueous methanol solutions.

Coordination state probabilities for the [Zn(H(2)O)(n)(CH(3)OH)(m)](2+) complex in aqueous methanol solutions are calculated as a function of the bulk solution concentration, and the number of methanol ligands, m = 0, 1, ..., 6 with n+m = 6. Zinc ion solvation free energies, which serve to normalize these probabilities, also reproduce the methanol concentration dependence of the experimentally derived free energy of zinc ion transfer from water to aqueous methanol solutions. Coordination state probabilities, p(n, m), are derived by extending quasi-chemical theory of ion hydration to solvent mixtures and mixed ligands. Free energy contributions to p(n, m) include the free energy of forming the mixed-ligand complex in the ideal gas, obtained by quantum chemical calculations, and the solvation free energy of the complex, approximated by a dielectric continuum model. We find that replacing water ligands with methanol ligands preferentially stabilizes methanol-rich complexes in the ideal gas. Conversely, water-rich complexes are stabilized by the solvation free energy contribution, such that the [Zn(H(2)O)(6)](2+) complex is the dominant species in solution for all methanol concentrations considered. Stabilization of the methanol-rich complexes is a consequence of the local coordination chemistry, dominated by the delocalization of charge on the zinc ion, while the stabilization of water-rich complexes is a consequence of favorable ion-solvent electrostatic interactions and smaller dielectric cavities for the water-rich complexes at fixed total charge in the dielectric continuum model. Our analysis also highlights an entropic contribution associated with the reversible work required to remove n water and m methanol molecules from bulk solution to form the [Zn(H(2)O)(n)(CH(3)OH)(m)](2+) complex, which captures the methanol concentration dependence of the solvation free energy of the zinc ion.

Analysis of Cooperativity and Group Additivity in the Hydration of 1,2-Dimethoxyethane.

We calculate hydration free energies of 1,2-dimethoxyethane (DME) conformations in water at 298 K and 1 bar. We find that the preference for the two most abundant tgt and tgg conformations derives from favorable nonspecific (i.e., long-range) solute-water interactions that are partially offset by unfavorable free energies of forming cavities in water to accommodate these conformations. The much lower population of the third most abundant tg+g- conformation, the most abundant conformation in the ideal gas at 298 K, is attributed to less favorable long-range solute-water interactions. We also find that long-range methyl/methylene group-water and ether oxygen-water interactions make significant nonadditive contributions to the free energy of DME hydration and propose a method based on quasichemical theory for reducing these nonadditive contributions by identifying constituent groups of DME that minimize the covariance in the long-range methyl/methylene group-water and ether oxygen-water interactions. We apply this method to show that the decomposition of DME into its constituent dimethyl ether groups is a better approximation of group additivity than decompositions based on distinguishing hydrophobic/hydrophilic constituent groups.

Thermodynamics of Hydration from the Perspective of the Molecular Quasichemical Theory of Solutions.

The quasichemical organization of the potential distribution theorem, molecular quasichemical theory (QCT), enables practical calculations and also provides a conceptual framework for molecular hydration phenomena. QCT can be viewed from multiple perspectives: (a) as a way to regularize an ill-conditioned statistical thermodynamic problem; (b) as an introduction of and emphasis on the neighborship characteristics of a solute of interest; or (c) as a way to include accurate electronic structure descriptions of near-neighbor interactions in defensible statistical thermodynamics by clearly defining neighborship clusters. The theory has been applied to solutes of a wide range of chemical complexity, ranging from ions that interact with water with both long-ranged and chemically intricate short-ranged interactions, to solutes that interact with water solely through traditional van der Waals interations, and including water itself. The solutes range in variety from monatomic ions to chemically heterogeneous macromolecules. A notable feature of QCT is that, in applying the theory to this range of solutes, the theory itself provides guidance on the necessary approximations and simplifications that can facilitate the calculations. In this Perspective, we develop these ideas and document them with examples that reveal the insights that can be extracted using the QCT formulation.

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

Communication: Direct observation of a hydrophobic bond in loop closure of a capped (-OCH2CH2-)n oligomer in water.

The small r variation of the probability density P(r) for end-to-end separations of a -CH(2)CH(3) capped (-OCH(2)CH(2)-)(n) oligomer in water is computed to be closely similar to the CH(4)···CH(4) potential of mean force under the same circumstances. Since the aqueous solution CH(4)···CH(4) potential of mean force is the natural physical definition of a primitive hydrophobic bond, the present result identifies an experimentally accessible circumstance for direct observation of a hydrophobic bond which has not been observed previously because of the low solubility of CH(4) in water. The physical picture is that the soluble chain molecules carry the capping groups into aqueous solution, and permits them to find one another with reasonable frequency. Comparison with the corresponding results without the solvent shows that hydration of the solute oxygen atoms swells the chain molecule globule. This supports the view that the chain molecule globule might have a secondary effect on the hydrophobic interaction that is of first interest here. The volume of the chain molecule globule is important for comparing the probabilities with and without solvent because it characterizes the local concentration of capping groups. Study of other capping groups to enable x-ray and neutron diffraction measurements of P(r) is discussed.

Hydrophilic Interactions Dominate the Inverse Temperature Dependence of Polypeptide Hydration Free Energies Attributed to Hydrophobicity.

We address the association of the hydrophobic driving forces in protein folding with the inverse temperature dependence of protein hydration, wherein stabilizing hydration effects strengthen with increasing temperature in a physiological range. All-atom calculations of the free energy of hydration of aqueous deca-alanine conformers, holistically including backbone and side-chain interactions together, show that attractive peptide-solvent interactions and the thermal expansion of the solvent dominate the inverse temperature signatures that have been interpreted traditionally as the hydrophobic stabilization of proteins in aqueous solution. Equivalent calculations on a methane solute are also presented as a benchmark for comparison. The present study calls for a reassessment of the forces that stabilize folded protein conformations in aqueous solutions and of the additivity of hydrophobic/hydrophilic contributions.

Quasichemical theory with a soft cutoff.

In view of the wide success of molecular quasichemical theory of liquids, this paper develops the soft-cutoff version of that theory. This development allows molecular dynamics simulations to be used for the calculation of solvation free energy, whereas the hard-cutoff version of the theory needs Monte Carlo simulations. This development also shows how fluids composed of molecules with smooth repulsive interactions can be treated analogously to the molecular-field theory of the hard-sphere fluid. In the treatment of liquid water, quasichemical theory with soft-cutoff conditioning does not change the fundamental convergence characteristics of the theory using hard-cutoff conditioning. In fact, hard cutoffs are found here to work better than softer ones in that case.

Insights into protein structure and function from disorder-complexity space.

Intrinsically disordered proteins have a wide variety of important functional roles. However, the relationship between sequence and function in these proteins is significantly different than that for well-folded proteins. In a previous work, we showed that the propensity to be disordered can be recognized based on sequence composition alone. Here that analysis is furthered by examining the relationship of disorder propensity to sequence complexity, where the metrics for these two properties depend only on composition. The distributions of 40 amino acid peptides from both ordered and disordered proteins are graphed in this disorder-complexity space. An analysis of Swiss-Prot shows that most peptides have high complexity and relatively low disorder. However, there are also an appreciable number of low complexity-high disorder peptides in the database. In contrast, there are no low complexity-low disorder peptides. A similar analysis for peptides in the PDB reveals a much narrower distribution, with few peptides of low complexity and high disorder. In this case, the bounds of the disorder-complexity distribution are well defined and might be used to evaluate the likelihood that a peptide can be crystallized with current methods. The disorder-complexity distributions of individual proteins and sets of proteins grouped by function are also examined. Among individual proteins, there is an enormous variety of distributions that in some cases can be rationalized with regard to function. Groups of functionally related proteins are found to have distributions that are similar within each group but show notable differences between groups. Finally, a pattern matching algorithm is used to search for proteins with particular disorder-complexity distributions. The results suggest that this approach might be used to identify relationships between otherwise dissimilar proteins.

Proteomic characterization of circulating extracellular vesicles identifies novel serum myeloma associated markers.

Multiple myeloma (MM) is a hematological malignancy of clonal plasma cells in the bone marrow (BM). The microenvironment plays a key role in MM cell survival and drug resistance through release of soluble factors, expression of adhesion molecules and release of extracellular vesicles (EVs). The aim of this manuscript is to use proteomic profiling of EVs as a tool to identify circulating tumor associated markers in MM patients. First, we characterized the EV protein content obtained from different MM cell lines. Then, we established differences in protein abundance among EVs isolated from MM patient serum and BM and the serum of healthy donors. These data show that the Major Histocompatibility Complex Class I is highly enriched in EVs of MM cell lines and MM patient's serum. Next, we show that CD44 is highly expressed in the EVs isolated from the corticosteroid resistant MM cell line, MM.1R. Furthermore, CD44 was found to be differentially expressed in EVs isolated from newly diagnosed MM patients. Finally through ELISA analysis, we establish the potential of serum CD44 as a predictive biomarker of overall survival. These results support the analysis of EVs as an easily accessible source for MM biomarkers. BIOLOGICAL SIGNIFICANCE: Extracellular vesicles are becoming a research focus due to their roles in cancer cell biology such as immune evasion, therapeutic resistance, proliferation and metastases. While numerous studies of vesicle characterization and biology have been conducted in many cancer models, the role of EV in MM remains relatively unstudied. Here we found that EVs isolated from MM cells are enriched in MHC-1 antigen presenting complex and its binding protein ß2-MG, this observation is compatible with the enhanced proteasome activity of MM cells compared to other cancers and the ability of functional MHC-1 to bind and present peptides, generated from protein degradation by the proteasome. Additionally, our experiments show that CD44 is particularly enriched in the EV fraction of corticosteroid resistant MM.1R cells and is differentially expressed in the EV fraction of MM patients. This is of high significance due to the established role of CD44 in adhesion of MM cells to BMSC and induction of IL-6, the primary cytokine for MM cell survival, secretion by the BMSC. Furthermore, ELISA assays for CD44 content from the serum of 254 newly diagnosed MM patients enrolled in a Phase 3 randomized trial show highly variable CD44 levels and those patients with >280 ng/mL serum CD44 showing a reduced overall survival time. These results suggest the potential use of CD44 as a prognostic biomarker in MM.

Breast Cancer-Specific miR Signature Unique to Extracellular Vesicles Includes "microRNA-like" tRNA Fragments.

UNLABELLED: Extracellular vesicles (EV), including exosomes and shed vesicles, have been implicated in intercellular communication; however, their biomarker potential is less clear. Therefore, EVs derived from MCF7 and MCF10A cells were analyzed to identify unique miRNA (miR) profiles that distinguish their origin. One characteristic common to the miR profiles of MCF7 EVs and their parent cells is the high abundance of miR-21, let-7a, miR-100, and miR-125b, and low levels of miR-205. A second characteristic is the high abundance of "miRNA-like" tRNA fragments, which is unique to the MCF7 EVs, and is not found in comparing the cellular profiles. In addition, correlations were examined in the MCF7 cellular expression levels of these five miRs and two tRNA-derived miRNAs, miR-720 and miR-1274b, and compared with the correlations in MCF7 EV levels. Interestingly, correlations in the cellular expression of miR-125b, miR-100, and let-7a are mirrored in the EVs. In contrast, correlations in tRNA-derived miRNA levels are found only in the EVs. The findings suggest that EV miR clusters can be defined based on functional miR interactions related to correlated cellular expression levels or physical miR interactions, for example, aggregation due to comparable binding affinities to common targets. IMPLICATIONS: These results point to using high levels of tRNA-derived small RNA fragments in combination with known miR signatures of tumors to distinguish tumor-derived EVs in circulation from EVs derived from other cell sources. Such biomarkers would be unique to the EVs where high abundances of tRNA fragments are amplified with respect to their cellular levels.