Molecular Determinants Involved in the Docking and Uptake of Tumor-Derived Extracellular Vesicles: Implications in Cancer.

Extracellular vesicles produced by tumor cells (TEVs) influence all stages of cancer development and spread, including tumorigenesis, cancer progression, and metastasis. TEVs can trigger profound phenotypic and functional changes in target cells through three main general mechanisms: (i) docking of TEVs on target cells and triggering of intra-cellular signaling; (ii) fusion of TEVs and target cell membranes with release of TEVs molecular cargo in the cytoplasm of recipient cell; and (iii) uptake of TEVs by recipient cells. Though the overall tumor-promoting effects of TEVs as well as the general mechanisms involved in TEVs interactions with, and uptake by, recipient cells are relatively well established, current knowledge about the molecular determinants that mediate the docking and uptake of tumor-derived EVs by specific target cells is still rather deficient. These molecular determinants dictate the cell and organ tropism of TEVs and ultimately control the specificity of TEVs-promoted metastases. Here, we will review current knowledge on selected specific molecules that mediate the tropism of TEVs towards specific target cells and organs, including the integrins, ICAM-1 Inter-Cellular Adhesion Molecule), ALCAM (Activated Leukocyte Cell Adhesion Molecule), CD44, the metalloproteinases ADAM17 (A Disintegrin And Metalloproteinase member 17) and ADAM10 (A Disintegrin And Metalloproteinase member 10), and the tetraspanin CD9.

Proof of concept of using a membrane-sensing peptide for sEVs affinity-based isolation.

Introduction: One main limitation in biomarker studies using EVs is the lack of a suitable isolation method rendering high yield and purity samples in a quick and easily standardized procedure. Here we report an affinity isolation method with a membrane-sensing peptide (MSP) derived from bradykinin. Methods: We designed a protocol based on agarose beads carrying cation chelates to specifically bind to the 6His-tagged membrane-sensing peptide. This approach presents several advantages: 1) cation-carrying agaroses are widely used and standardized for His-tagged protein isolation, 2) the affinity protocol can be performed in small volumes, feasible and manageable for clinical routine and 3) elution with imidazole or EDTA allows a gentle and easy recovery without EV damage, facilitating subsequent characterization and functional analyses. Results: The optimized final procedure incubates 0.5 mg of peptide for 10 min with 10 µL of Long-arm Cobalt agarose before an overnight incubation with concentrated cell conditioned medium. EV downstream analyses can be directly performed on the agarose beads adding lysis or nucleic-acid extraction buffers, or gently eluted with imidazole or EDTA, rendering a fully competent EV preparation. Discussion: This new isolation methodology is based on the recognition of general membrane characteristics independent of surface markers. It is thus unbiased and can be used in any species EV sample, even in samples from animal or plant species against which no suitable antibodies exist. Being an affinity method, the sample handling protocol is very simple, less time-consuming, does not require specialized equipment and can be easily introduced in a clinical automated routine. We demonstrated the high purity and yield of the method in comparison with other commercially available kits. This method can also be scale up or down, with the possibility of analyzing very low amounts of sample, and it is compatible with any downstream analyses thanks to the gentle elution procedure.

Two Complementary Strategies to Quantitate Extracellular Vesicle Uptake Using Bioluminescence and Non-Lipidic Dyes.

The study of the molecular mechanisms controlling extracellular vesicle uptake by a target cell is an aspect of great interest within the EV community due to EV relevance in intercellular communication for tissue homeostasis or different disease progressions such as cancer or Alzheimer's. Since the EV field is relatively young, standardization of techniques for even basic aspects such as their isolation and characterization is still under development and debate. So it is for the study of EV uptake, where the currently most used strategies have critical limitations. Newly designed techniques should try to discern the uptake events from the surface EV binding or to improve the sensitivity and fidelity of the assays. Here, we describe two different complementary methods to measure and quantify EV uptake that we believe, help to overcome certain limitations of the currently used techniques. One is based on a mEGFP-Tspn-Rluc construct, to sort these two reporters into EVs. The use of bioluminescence signal to measure EV uptake allows for a better sensitivity, discerns EV binding from uptake, and allows kinetics measurement in alive cells, being compatible with a high-throughput screen format. The second one is a flow cytometry assay based in EV staining with a maleimide conjugated with a fluorophore, a chemical compound that covalently binds to proteins within sulfhydryl residues, being a good alternative to lipidic dyes and compatible with flow cytometry sorting of cell populations that have captured the labeled EVs.

ALCAM/CD166 Is Involved in the Binding and Uptake of Cancer-Derived Extracellular Vesicles.

Colorectal cancer (CRC) and ovarian cancer (OvC) patients frequently develop peritoneal metastasis, a condition associated with a very poor prognosis. In these cancers, tumor-derived extracellular vesicles (EVs) cause immunosuppression, facilitate the direct attachment and invasion of cancer cells through the mesothelium, induce the conversion of peritoneal mesothelial cells (PMCs) into cancer-associated fibroblasts (CAFs) and transfer a more aggressive phenotype amongst cancer cells. Although the promoting role of EVs in CRC and OvC peritoneal metastasis is well established, the specific molecules that mediate the interactions between tumor-derived EVs and immune and non-immune target cells remain elusive. Here, we employed the SKOV-3 (ovarian adenocarcinoma) and Colo-320 (colorectal adenocarcinoma) human cell lines as model systems to study the interactions and uptake of EVs produced by ovarian carcinoma and colorectal carcinoma cells, respectively. We established that the adhesion molecule ALCAM/CD166 is involved in the interaction of cancer-derived EVs with recipient cancer cells (a process termed "EV binding" or "EV docking") and in their subsequent uptake by these cells. The identification of ALCAM/CD166 as a molecule mediating the docking and uptake of CRC and OvC-derived EVs may be potentially exploited to block the peritoneal metastasis cascade promoted by EVs in CRC and OvC patients.

Tetraspanins interweave EV secretion, endosomal network dynamics and cellular metabolism.

Tetraspanin proteins organize membrane nanodomains related to cell adhesion and migration. An essential feature conserved along the superfamily is their cone-shaped tertiary structure, which allows tetraspanins to be enriched in highly curved membrane structures. Their conical shape, together with their ability to associate to transmembrane receptors and to bind to cystoskeletal and signaling scaffolds, are key in their ability to regulate endosomal network dynamics and Extracellular Vesicle biogenesis and cargo selection. Recent evidence suggests that tetraspanins have a relevant impact in mitochondria turnover and regulation of cellular metabolism. In this review we highlight those reports that point to tetraspanins as key regulators in the communication between the endosomal network, EVs and the cellular metabolism.

A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma.

BACKGROUND: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. RESULTS: Here we describe a method that, using just a few microliters of patient's plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. CONCLUSIONS: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

Cellular Integrin α5ß1 and Exosomal ADAM17 Mediate the Binding and Uptake of Exosomes Produced by Colorectal Carcinoma Cells.

Approximately 25% of colorectal cancer (CRC) patients develop peritoneal metastasis, a condition associated with a bleak prognosis. The CRC peritoneal dissemination cascade involves the shedding of cancer cells from the primary tumor, their transport through the peritoneal cavity, their adhesion to the peritoneal mesothelial cells (PMCs) that line all peritoneal organs, and invasion of cancer cells through this mesothelial cell barrier and underlying stroma to establish new metastatic foci. Exosomes produced by cancer cells have been shown to influence many processes related to cancer progression and metastasis. In epithelial ovarian cancer these extracellular vesicles (EVs) have been shown to favor different steps of the peritoneal dissemination cascade by changing the functional phenotype of cancer cells and PMCs. Little is currently known, however, about the roles played by exosomes in the pathogenesis and peritoneal metastasis cascade of CRC and especially about the molecules that mediate their interaction and uptake by target PMCs and tumor cells. We isolated exosomes by size-exclusion chromatography from CRC cells and performed cell-adhesion assays to immobilized exosomes in the presence of blocking antibodies against surface proteins and measured the uptake of fluorescently-labelled exosomes. We report here that the interaction between integrin α5ß1 on CRC cells (and PMCs) and its ligand ADAM17 on exosomes mediated the binding and uptake of CRC-derived exosomes. Furthermore, this process was negatively regulated by the expression of tetraspanin CD9 on exosomes.

Embryonic Trophectoderm Secretomics Reveals Chemotactic Migration and Intercellular Communication of Endometrial and Circulating MSCs in Embryonic Implantation.

Embryonic implantation is a key step in the establishment of pregnancy. In the present work, we have carried out an in-depth proteomic analysis of the secretome (extracellular vesicles and soluble proteins) of two bovine blastocysts embryonic trophectoderm primary cultures (BBT), confirming different epithelial-mesenchymal transition stages in these cells. BBT-secretomes contain early pregnancy-related proteins and angiogenic proteins both as cargo in EVs and the soluble fraction. We have demonstrated the functional transfer of protein-containing secretome between embryonic trophectoderm and maternal MSC in vitro using two BBT primary cultures eight endometrial MSC (eMSC) and five peripheral blood MSC (pbMSC) lines. We observed that eMSC and pbMSC chemotax to both the soluble fraction and EVs of the BBT secretome. In addition, in a complementary direction, we found that the pattern of expression of implantation proteins in BBT-EVs changes depending on: (i) their epithelial-mesenchymal phenotype; (ii) as a result of the uptake of eMSC- or pbMSC-EV previously stimulated or not with embryonic signals (IFN-); (iii) because of the stimulation with the endometrial cytokines present in the uterine fluid in the peri-implantation period.

CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells.

Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.

An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles.

Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.

Bovine peripheral blood MSCs chemotax towards inflammation and embryo implantation stimuli.

Mesenchymal stem cells (MSCs) have a great potential in regenerative medicine because of their multipotential and immunoregulatory capacities, while in early pregnancy they could participate in the immunotolerance of the mother towards the embryo. Peripheral blood constitutes an accessible source of MSCs. We successfully isolated peripheral blood MSC (pbMSCs) lines, with or without previous bone marrow mobilization. All pbMSCs lines obtained in both conditions presented classical MSC markers and properties, alkaline phosphatase activity and multipotent capacity to differentiate among adipogenic, osteogenic or chondrogenic lineages, and suppressed the proliferation of T cells. pbMSCs showed migratory capacity without cytokine stimulation while increasing their migration rate in the presence of inflammatory or embryo implantation stimuli. Interestingly, in contrast to MSCs derived from endometrial tissue, three pbMSCs lines also showed increased migration towards the IFN-τ implantation cytokine. Moreover, the secretome produced by an early implantation stage embryonic trophectoderm cell line showed a chemoattractant effect in pbMSCs. Our results suggest that circulating MSCs are present in the peripheral blood under healthy conditions. The fact that both the inflammation and implantation signals induced pbMSCs chemotaxis highlights MSC heterogeneity and suggests that their migratory capacity may differ according to their tissue of origin and would suggest the possible active recruitment of MSCs from bone marrow during pregnancy to repress the immune response to prevent the embryo rejection by the maternal organism.

One-year dietary supplementation with walnuts modifies exosomal miRNA in elderly subjects.

PURPOSE: Epidemiological studies and clinical trials support the association of nut consumption with a lower risk of prevalent non-communicable diseases, particularly cardiovascular disease. However, the molecular mechanisms underlying nut benefits remain to be fully described. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and play a pivotal role in health and disease. Exosomes are extracellular vesicles released from cells and mediate intercellular communication. Whether nut consumption modulates circulating miRNAs (c-miRNAs) transported in exosomes is poorly described. METHODS: Cognitively healthy elderly subjects were randomized to either control (n = 110, abstaining from walnuts) or daily supplementation with walnuts (15% of their total energy, ≈30-60 g/day, n = 101) for 1-year. C-miRNAs were screened in exosomes isolated from 10 samples, before and after supplementation, and identified c-miRNA candidates were validated in the whole cohort. In addition, nanoparticle tracking analysis and lipidomics were assessed in pooled exosomes from the whole cohort. RESULTS: Exosomal hsa-miR-32-5p and hsa-miR-29b-3p were consistently induced by walnut consumption. No major changes in exosomal lipids, nanoparticle concentration or size were found. CONCLUSION: Our results provide novel evidence that certain c-miRNAs transported in exosomes are modulated by walnut consumption. The extent to which this finding contributes to the benefits of walnuts deserves further research.

Tetraspanin CD81 regulates HSV-1 infection.

Different members of the tetraspanin superfamily have been described to regulate different virus infectious cycles at several stages: viral entry, viral replication or virion exit or infectivity. In addition, tetraspanin CD81 regulates HIV reverse transcription through its association with the dNTP hydrolase SAMHD1. Here we aimed at analysing the role of CD81 in Herpes simplex virus 1 infectivity using a neuroblastoma cell model. For this purpose, we generated a CD81 KO cell line using the CRISPR/Cas9 technology. Despite being CD81 a plasma membrane protein, CD81 KO cells showed no defects in viral entry nor in the expression of early protein markers. In contrast, glycoprotein B and C, which require viral DNA replication for their expression, were significantly reduced in CD81 KO infected cells. Indeed, HSV-1 DNA replication and the formation of new infectious particles were severely compromised in CD81 KO cells. We could not detect significant changes in SAMHD1 total expression levels, but a relocalization into endosomal structures was observed in CD81 KO cells. In summary, CD81 KO cells showed impaired viral DNA replication and produced greatly diminished viral titers.

Selected Tetraspanins Functionalized Niosomes as Potential Standards for Exosome Immunoassays.

Quantitative detection of exosomes in bio-fluids is a challenging task in a dynamic research field. The absence of a well-established reference material (RM) for method development and inter-comparison studies could be potentially overcome with artificial exosomes: lab-produced biomimetic particles with morphological and functional properties close to natural exosomes. This work presents the design, development and functional characteristics of fully artificial exosomes based on tetraspanin extracellular loops-coated niosomes, produced by bio-nanotechnology methods based on supra-molecular chemistry and recombinant protein technology. Mono- and double-functionalized particles with CD9/CD63 tetraspanins have been developed and characterized from a morphological and functional point of view. Produced bio-particles showed close similarities with natural entities in terms of physical properties. Their utility for bioanalysis is demonstrated by their detection and molecular-type discrimination by enzyme-linked immunosorbent assays (ELISAs), one of the most frequent bio-analytical method found in routine and research labs. The basic material based on streptavidin-coated niosomes allows the surface functionalization with any biotinylated protein or peptide, introducing versatility. Although promising results have been reported, further optimizations and deeper characterization will help this innovative biomaterial become a robust RM for validation and development of diagnostic tools for exosomes determination.

RIAM-VASP Module Relays Integrin Complement Receptors in Outside-In Signaling Driving Particle Engulfment.

The phagocytic integrins and complement receptors αMß2/CR3 and αXß2/CR4 are classically associated with the phagocytosis of iC3b-opsonized particles. The activation of this receptor is dependent on signals derived from other receptors (inside-out signaling) with the crucial involvement of the Rap1-RIAM-Talin-1 pathway. Here, we analyze the implication of RIAM and its binding partner VASP in the signaling events occurring downstream of ß2 integrins (outside-in) during complement-mediated phagocytosis. To this end, we used HL-60 promyelocytic cell lines deficient in RIAM or VASP or overexpressing EGFP-tagged VASP to determine VASP dynamics at phagocytic cups. Our results indicate that RIAM-deficient HL-60 cells presented impaired particle internalization and altered integrin downstream signaling during complement-dependent phagocytosis. Similarly, VASP deficiency completely blocked phagocytosis, while VASP overexpression increased the random movement of phagocytic particles at the cell surface, with reduced internalization. Moreover, the recruitment of VASP to particle contact sites, amount of pSer157-VASP and formation of actin-rich phagocytic cups were dependent on RIAM expression. Our results suggested that RIAM worked as a relay for integrin complement receptors in outside-in signaling, coordinating integrin activation and cytoskeletal rearrangements via its interaction with VASP.

Novel nonclassic progesterone receptor PGRMC1 pulldown-precipitated proteins reveal a key role during human decidualization.

OBJECTIVE: To investigate PGRMC1-precipitating proteins in human endometrial stromal cells (ESC) to understand its role during in vitro decidualization. DESIGN: Prospective observational study. SETTING: Academic fertility center. PATIENT(S): Fifteen fertile oocyte donors. INTERVENTION(S): Isolated ESCs decidualized in vitro and used in pulldown assays. MAIN OUTCOME MEASURE(S): GST-PGRMC1-precipitated proteins identified in nondecidualized ESC (ndESC) and ESC decidualized via a long (8 days) or short (4 days) decidualization protocol (dESC). RESULT(S): Using pulldown assays and mass spectrometry, decidualization was evaluated by prolactin secretion (ELISA) and cytoskeleton morphology (F-actin staining). The protein interactions were validated by colocalization and coimmunoprecipitation. The pulldown and mass spectrometry analysis identified 21, 24, and 24 new significant GST-PGRMC1-precipitated proteins in ndESC, long dESC, and short dESC, respectively, compared with controls. The functional annotation analysis categorized these proteins mainly into endomembrane system and mitochondria cellular components, both related to adenosine triphosphate (ATP) generation and transport activity, protein biosynthesis and posttranslational processing, vesicle trafficking, and protection against oxidative stress activities. Monoamine oxidase B (MAOB) and B-cell receptor-associated protein 31 (BAP31) were identified in dESC from both decidualization protocols. PGRMC1-MAOB/BAP31 interactions were confirmed by immunofluorescence and coimmunoprecipitation in dESC. CONCLUSION(S): Novel GST-PGRMC1-precipitated proteins discovered in ESC suggest that this protein is implicated in deep remodeling of ESC during decidualization and aggregates mainly with proteins involved in biosynthesis, intracellular transport, and mitochondrial activity.

Regulation of MT1-MMP Activity through Its Association with ERMs.

Membrane-bound proteases play a key role in biology by degrading matrix proteins or shedding adhesion receptors. MT1-MMP metalloproteinase is critical during cancer invasion, angiogenesis, and development. MT1-MMP activity is strictly regulated by internalization, recycling, autoprocessing but also through its incorporation into tetraspanin-enriched microdomains (TEMs), into invadopodia, or by its secretion on extracellular vesicles (EVs). We identified a juxtamembrane positively charged cluster responsible for the interaction of MT1-MMP with ERM (ezrin/radixin/moesin) cytoskeletal connectors in breast carcinoma cells. Linkage to ERMs regulates MT1-MMP subcellular distribution and internalization, but not its incorporation into extracellular vesicles. MT1-MMP association to ERMs and insertion into TEMs are independent phenomena, so that mutation of the ERM-binding motif in the cytoplasmic region of MT1-MMP does not preclude its association with the tetraspanin CD151, but impairs the accumulation and coalescence of CD151/MT1-MMP complexes at actin-rich structures. Conversely, gene deletion of CD151 does not impact on MT1-MMP colocalization with ERM molecules. At the plasma membrane MT1-MMP autoprocessing is severely dependent on ERM association and seems to be the dominant regulator of the enzyme collagenolytic activity. This newly characterized MT1-MMP/ERM association can thus be of relevance for tumor cell invasion.

Extracellular Vesicles From Liver Progenitor Cells Downregulates Fibroblast Metabolic Activity and Increase the Expression of Immune-Response Related Molecules.

Extracellular vesicles (EVs) mediate cell-to-cell crosstalk whose content can induce changes in acceptor cells and their microenvironment. MLP29 cells are mouse liver progenitor cells that release EVs loaded with signaling cues that could affect cell fate. In the current work, we incubated 3T3-L1 mouse fibroblasts with MLP29-derived EVs, and then analyzed changes by proteomics and transcriptomics. Results showed a general downregulation of protein and transcript expression related to proliferative and metabolic routes dependent on TGF-beta. We also observed an increase in the ERBB2 interacting protein (ERBIN) and Cxcl2, together with an induction of ribosome biogenesis and interferon-related response molecules, suggesting the activation of immune system signaling.