Identification and characterization of hADSC-derived exosome proteins from different isolation methods

Exosomes are secreted into the extracellular space by most cell types and contain various molecular constituents, which play roles in many biological processes. Adipose-derived mesenchymal stem cells (ADSCs) can differentiate into a variety of cell types and secrete a series of paracrine factors through exosomes. ADSC-derived exosomes have shown diagnostic and therapeutic potential in many clinical diseases. The molecular components are critical for their mechanisms. Several methods have been developed for exosome purification, including ultracentrifugation, ultrafiltration, density gradient purification, size-based isolation, polymer precipitation and immuno-affinity purification. Thus, we employed four methods to isolate exosomes from the hADSC culture medium, including ultracentrifugation, size exclusion chromatography, ExoQuick-TC precipitation and ExoQuick-TC ULTRA isolation. Following exosome isolation, we performed quantitative proteomic analysis of the exosome proteins using isobaric tags for relative and absolute quantification (iTRAQ) labelling, combined with 2D-LC-MS/MS. There were 599 universal and 138 stably expressed proteins in hADSC-derived exosomes. We proved that these proteins were potential hADSC-derived exosomes markers, including CD109, CD166, HSPA4, TRAP1, RAB2A, RAB11B and RAB14. From the quantitative proteomic analysis, we demonstrated that hADSC-derived exosome protein expression varied, with lipopolysaccharide (LPS) treatment, in the different isolation methods. Pathway analysis and proliferation, migration and endothelial tube formation assays showed varying effects in cells stimulated with hADSC-derived exosomes from different isolation methods. Our study revealed that different isolation methods might introduce variations in the protein composition in exosomes, which reflects their effects on biological function. The pros and cons of these methods are important points to consider for downstream research applications.     

Proteome characterization of melanoma exosomes reveals a specific signature for metastatic cell lines

Exosomes are important mediators in cell‐to‐cell communication and, recently, their role in melanoma progression has been brought to light. Here, we characterized exosomes secreted by seven melanoma cell lines with varying degrees of aggressivity. Extensive proteomic analysis of their exosomes confirmed the presence of characteristic exosomal markers as well as melanoma‐specific antigens and oncogenic proteins. Importantly, the protein composition differed among exosomes from different lines. Exosomes from aggressive cells contained specific proteins involved in cell motility, angiogenesis, and immune response, while these proteins were less abundant or absent in exosomes from less aggressive cells. Interestingly, when exposed to exosomes from metastatic lines, less aggressive cells increased their migratory capacities, likely due to transfer of pro‐migratory exosomal proteins to recipient cells. Hence, this study shows that the specific protein composition of melanoma exosomes depends on the cells’ aggressivity and suggests that exosomes influence the behavior of other tumor cells and their microenvironment.     

Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3–4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.     

Protein profile of exosomes from trabecular meshwork cells

To better understand the role of exosomes in the trabecular meshwork (TM), the site of intraocular pressure control, the exosome proteome from primary cultures of human TM cell monolayers was analyzed. Exosomes were purified from urine and conditioned media from primary cultures of human TM cell monolayers and subjected to a two dimensional HPLC separation and MS/MS analyses using the MudPIT strategy. Spectra were searched against a human protein database using Sequest. Protein profiles were compared to each other and the Exocarta database and the presence of specific protein markers confirmed by Western blot analyses of exosomes from aqueous humor and human TM cell strains (n=5) that were untreated, or exposed to dexamethasone and/or ionomycin. TM cell exosomes contained 108 of the 143 most represented exosome proteins in ExoCarta, including previously characterized markers such as membrane organizing and tetraspanin proteins. Several cell-specific proteins in TM exosomes were identified including myocilin, emilin-1 and neuropilin-1. All TM exosome proteins had flotation densities on sucrose gradients and release responses to ionomycin typical for exosomes. Taken together, TM exosomes have a characteristic exosome protein profile plus contain unique proteins, including the glaucoma-causing protein, myocilin; suggesting a role for exosomes in the control of intraocular pressure.     

Exosomes Derived from Hypoxic Leukemia Cells Enhance Tube Formation in Endothelial Cells

Hypoxia plays an important role during the evolution of cancer cells and their microenvironment. Emerging evidence suggests communication between cancer cells and their microenvironment occurs via exosomes. This study aimed to clarify whether hypoxia affects angiogenic function through exosomes secreted from leukemia cells. We used the human leukemia cell line K562 for exosome-generating cells and human umbilical vein endothelial cells (HUVECs) for exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) conditions for 24 h were isolated and quantitated by nanoparticle tracking analysis. These exosomes were then cocultured with HUVECs to evaluate angiogenic activity. The exosomes secreted from K562 cells in hypoxic conditions significantly enhanced tube formation by HUVECs compared with exosomes produced in normoxic conditions. Using a TaqMan low-density miRNA array, we found a subset of miRNAs, including miR-210, were significantly increased in exosomes secreted from hypoxic K562 cells. We demonstrated that cancer cells and their exosomes have altered miRNA profiles under hypoxic conditions. Although exosomes contain various molecular constituents such as proteins and mRNAs, altered exosomal compartments under hypoxic conditions, including miR-210, affected the behavior of endothelial cells. Our results suggest that exosomal miRNA derived from cancer cells under hypoxic conditions may partly affect angiogenic activity in endothelial cells.     

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin‐1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non–small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin‐1 and flotillin‐2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin‐1 as well as its EV‐to‐cellular ratio vary drastically depending on cell type.     

Proteomic analysis of melanoma-derived exosomes by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry

Exosomes are 40-100 nm vesicles released by numerous cell types and are thought to have a variety of roles depending on their origin. Exosomes derived from antigen presenting cells have been shown to be capable of initiating immune responses in vivo and eradicating established tumours in murine models. Tumour-derived exosomes can be utilised as a source of tumour antigen for cross-priming to T-cells and are thus of interest for use in anti-tumour immunotherapy. Further exploration into the protein composition of exosomes may increase our understanding of their potential roles in vivo and this study has examined the proteome of exosomes purified from cell supernatants of the melanoma cell lines MeWo and SK-MEL-28. The vesicular nature and size (30-100 nm) of the purified exosomes was confirmed by electron microscopy and sucrose density gradient centrifugation. Western blotting demonstrated the absence of calnexin and cytochrome c, verifying the purity of the exosome preparations, as well as enrichment of MHC class I and the tumour-associated antigens Mart-1 and Mel-CAM. The two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) protein profiles of exosomes from the two cell lines were highly comparable and strikingly different from the profiles of the total cell lysates. Mass spectrometric sequencing identified proteins present in 49 protein spots in the exosome lysates. Several of these have been identified previously in exosomes but some are novel, including p120 catenin, radixin, and immunoglobulin superfamily member 8 (PGRL). Proteins present in whole-cell lysates that were significantly reduced or excluded from exosomes were also identified and included several mitochondrial and lysosomal proteins, again confirming the proposed endosomal origin of exosomes. This study presents a starting point for future more in-depth protein studies of tumour-derived exosomes which will aid the understanding of their biogenesis and targeting for use in anti-tumour immunotherapy protocols.     

Isolation and proteomic analysis of exosomes secreted by human cancer cells in vitro

Exosomes are 20-100 nm membrane vesicles of endocytic origin secreted by most cell types in vitro and in vivo. Since exosomes contain both RNA (mRNA and microRNA) and proteins, which can be transferred to another cell, and be functional in that new environment, these vesicles may be involved in the communication between cells. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo suggest their participation in pathological situations. Our purpose here is to describe methods for the production, purification, and proteomic characterization of exosomes derived from human cancer cells in vitro. Based on exosomes' unique lipidic composition, we have developed the new approach to increase production of exosomes by cells in vitro. Secondly, we have developed quality control by laser correlation spectroscopy for exosomal assays based on the amount of MHC class I and CD63 molecules on their surface. At last, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry was used after 2D electrophoresis for the proteomic analysis of exosomes derived from cancer cell lines. This study describes the protein composition of brain tumor cell-derived exosomes in more detail.     

Characterization of Human Thymic Exosomes

Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.     

Up-regulation of interferon-stimulated gene 15 and its conjugation machinery,UbE1L and UbcH8 expression by tumor necrosis factor-α through p38 MAPK and JNK signaling pathways in human lung carcinoma

Exosomes are nanometer-sized vesicles involved in intercellular communication, and they are released by various cell types. To learn about exosomes produced by Schwann cells (SCs) and to explore their potential function in repairing the central nervous system (CNS), we isolated exosomes from supernatants of SCs by ultracentrifugation, characterized them by electron microscopy and immunoblotting and determined their protein profile using proteomic analysis. The results demonstrated that Schwann cell-derived exosomes (SCDEs) were, on average, 106.5 nm in diameter, round, and had cup-like concavity and expressed exosome markers CD9 and Alix but not tumor susceptibility gene (TSG) 101. We identified a total of 433 proteins, among which 398 proteins overlapped with the ExoCarta database. According to their specific functions, we identified 12 proteins that are closely related to CNS repair and classified them by different potential mechanisms, such as axon regeneration and inflammation inhibition. Gene Oncology analysis indicated that SCDEs are mainly involved in signal transduction and cell communication. Biological pathway analysis showed that pathways are mostly involved in exosome biogenesis, formation, uptake and axon regeneration. Among the pathways, the neurotrophin, PI3K-Akt and cAMP signaling pathways played important roles in CNS repair. Our study isolated SCDEs, unveiled their contents, presented potential neurorestorative proteins and pathways and provided a rich proteomics data resource that will be valuable for future studies of the functions of individual proteins in neurodegenerative diseases.

     

Multidrug resistant tumour cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells

BackgroundMultidrug resistance (MDR) is a serious impediment to cancer treatment, with overexpression of drug efflux pumps such as P-glycoprotein (P-gp) playing a significant role. In spite of being a major clinical challenge, to date there is no simple, minimally invasive and clinically validated method for diagnosis of the MDR phenotype using non-tumour biological samples. Recently, P-gp has been found in extracellular vesicles (EVs) shed by MDR cancer cells. This study aimed to compare the EVs shed by MDR cells and their drug-sensitive cellular counterparts, in order to identify biomarkers of MDR.MethodsTwo pairs of MDR and drug-sensitive counterpart tumour cell lines were studied as models. EVs were characterized in terms of size and molecular markers and their protein content was investigated by proteomic analysis and Western blot.ResultsWe found that MDR cells produced more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart. EVs from MDR cells contained P-gp and presented a different content of proteins known to be involved in the biogenesis of EVs, particularly in the biogenesis of exosomes.ConclusionsThe determination of the size and of this particular protein content of EVs shed by tumour cells may allow the development of a minimally-invasive simple method of detecting and predicting MDR.General significanceThis work describes for the first time that cancer multidrug resistant cells shed more microvesicle-like EVs and less exosomes than their drug-sensitive counterpart cells, carrying a specific content of proteins involved in EV biogenesis that could be further studied as biomarkers of MDR.     

Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes

Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa). However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR) as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight^™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.     

A web-based tool for in silico biomarker discovery based on tissue-specific protein profiles in normal and cancer tissues

Here we report the development of a publicly available Web-based analysis tool for exploring proteins expressed in a tissue- or cancer-specific manner. The search queries are based on the human tissue profiles in normal and cancer cells in the Human Protein Atlas portal and rely on the individual annotation performed by pathologists of images representing immunohistochemically stained tissue sections. Approximately 1.8 million images representing more than 3000 antibodies directed toward human proteins were used in the study. The search tool allows for the systematic exploration of the protein atlas to discover potential protein biomarkers. Such biomarkers include tissue-specific markers, cell type-specific markers, tumor type-specific markers, markers of malignancy, and prognostic or predictive markers of cancers. Here we show examples of database queries to generate sets of candidate biomarker proteins for several of these different categories. Expression profiles of candidate proteins can then subsequently be validated by examination of the underlying high resolution images. The present study shows examples of search strategies revealing several potential protein biomarkers, including proteins specifically expressed in normal cells and in cancer cells from specified tumor types. The lists of candidate proteins can be used as a starting point for further validation in larger patient cohorts using both immunological approaches and technologies utilizing more classical proteomics tools.     

Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA

Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 10⁴–10⁶ copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10³ copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS.     

Neuronal Differentiation of Human Mesenchymal Stem Cells Using Exosomes Derived from Differentiating Neuronal Cells

Exosomes deliver functional proteins and genetic materials to neighboring cells, and have potential applications for tissue regeneration. One possible mechanism of exosome-promoted tissue regeneration is through the delivery of microRNA (miRNA). In this study, we hypothesized that exosomes derived from neuronal progenitor cells contain miRNAs that promote neuronal differentiation. We treated mesenchymal stem cells (MSCs) daily with exosomes derived from PC12 cells, a neuronal cell line, for 1 week. After the treatment with PC12-derived exosomes, MSCs developed neuron-like morphology, and gene and protein expressions of neuronal markers were upregulated. Microarray analysis showed that the expression of miR-125b, which is known to play a role in neuronal differentiation of stem cells, was much higher in PC12-derived exosomes than in exosomes from B16-F10 melanoma cells. These results suggest that the delivery of miRNAs contained in PC12-derived exosomes is a possible mechanism explaining the neuronal differentiation of MSC.     

Proteomic identification of the contents of small extracellular vesicles from in vivo Plasmodium yoelii infection

Small extracellular vesicles, including exosomes, are formed by the endocytic pathway and contain genetic and protein material which reflect the contents of their cells of origin. These contents have a role in vesicle-mediated information transfer, as well as physiological and pathological functions. Thus, these vesicles are of great interest as therapeutic targets, or as vehicles for immunomodulatory control. In Plasmodium spp. infections, vesicles derived from the parasite or parasite-infected cells have been shown to induce the expression of pro-inflammatory elements, which have been correlated with manifestations of clinical disease. Herein, we characterised the protein cargo of naturally occurring sEVs in the plasma of P. yoelii-infected mice. After in vivo infections, extracellular vesicles in the size range of exosomes were collected by sequential centrifugation/ultracentrifugation followed by isopycnic gradient separation. Analysis of the vesicles was performed by transmission electron microscopy, dynamic light scattering, SDS–PAGE and flow cytometry. LC-MS analysis followed by bioinformatics analysis predicted parasite protein cargo associated with exosomes. Within these small extracellular vesicles, we identified proteins of interest as vaccine candidates, uncharacterized proteins which may be targets of T cell immunoreactivity, and proteins involved in metabolic processes, regulation, homeostasis and immunity. Importantly, the small extracellular vesicles studied in our work were obtained from in vivo infection rather than from the supernatant of in vitro cultures. These findings add to the growing interest in parasite small extracellular vesicles, further our understanding of the interactions between host and parasite, and identify novel proteins which may represent potential targets for vaccination against malaria.     

Hydrogel microchip as a tool for studying exosomes in human serum

Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.