Proteomics Analysis of A33 Immunoaffinity-purified Exosomes Released from the Human Colon Tumor Cell Line LIM1215 Reveals a Tissue-specific Protein Signature

Exosomes are 40–100-nm-diameter nanovesicles of endocytic origin that are released from diverse cell types. To better understand the biological role of exosomes and to avoid confounding data arising from proteinaceous contaminants, it is important to work with highly purified material. Here, we describe an immunoaffinity capture method using the colon epithelial cell-specific A33 antibody to purify colorectal cancer cell (LIM1215)-derived exosomes. LC-MS/MS revealed 394 unique exosomal proteins of which 112 proteins (28%) contained signal peptides and a significant enrichment of proteins containing coiled coil, RAS, and MIRO domains. A comparative protein profiling analysis of LIM1215-, murine mast cell-, and human urine-derived exosomes revealed a subset of proteins common to all exosomes such as endosomal sorting complex required for transport (ESCRT) proteins, tetraspanins, signaling, trafficking, and cytoskeletal proteins. A conspicuous finding of this comparative analysis was the presence of host cell-specific (LIM1215 exosome) proteins such as A33, cadherin-17, carcinoembryonic antigen, epithelial cell surface antigen (EpCAM), proliferating cell nuclear antigen, epidermal growth factor receptor, mucin 13, misshapen-like kinase 1, keratin 18, mitogen-activated protein kinase 4, claudins (1, 3, and 7), centrosomal protein 55 kDa, and ephrin-B1 and -B2. Furthermore, we report the presence of the enzyme phospholipid scramblase implicated in transbilayer lipid distribution membrane remodeling. The LIM1215-specific exosomal proteins identified in this study may provide insights into colon cancer biology and potential diagnostic biomarkers.Exosomes represent a distinct class of membrane nanovesicles (40–100-nm diameter) of endocytic origin that are released from diverse cell types under both normal and pathological conditions (1). Although initial studies focused on exosomes released from various cell types in vitro, exosomes have also been reported in diverse body fluids such as urine (2), amniotic fluid (3, 4), malignant ascites (5–7), bronchoalveolar lavage fluid (8), synovial fluid (9), platelets (10), breast milk (11), and blood (12). Exosomes are formed through the inward budding of late endosomal membranes that give rise to intraluminal vesicles (ILVs)1 within intracellular multivesicular bodies (MVBs). MVBs have a well known intermediary function in the degradation of either proteins internalized from the cell surface (e.g. cell surface receptors) or intracellular proteins sorted from the trans-Golgi network. Proteins destined for degradation are sorted, typically in a ubiquitin-dependent manner, into the ILVs of the nascent MVBs, which then fuse with pre-existing lysosomes (13). An alternate fate for MVBs involves their fusion with the plasma membrane and ensuing release of ILVs into the extracellular environment as exosomes. The biogenesis of exosomes has been linked to the protein complex ESCRT machinery, which is required for both formation of MVBs and the recruitment of their endosome-derived cargo proteins (14).Exosomes exhibit pleiotropic biological functions including immunomodulatory activity, mediation of cell-cell communication, and, possibly, the transport and propagation of infectious cargo such as prions and retroviruses (1, 15, 16). Despite these advances in our understanding of exosome function, the physiological significance of exosomes is still not fully understood. The observation that exosomes contains inactive RNA and microRNAs that can be transferred to another cell and be translated in the recipient suggest that exosomes may provide a novel vehicle for genetic exchange between cells (17). More recently, the finding of glioblastoma tumor cell-derived exosomes that contain mRNA mutant/variants and microRNAs characteristic of the glioma coupled with the finding of these microvesicles in serum of glioblastoma patients suggests that blood-based exosomes may provide important diagnostic information and aid in therapeutic decisions for cancer patients (18).The molecular composition of exosomes purified from the cell culture medium from various cell types and diverse body fluids has been analyzed by proteomics as well as fluorescence-activated cell sorting, Western blot analysis, and immunohistochemistry (1, 19). In addition to displaying a protein composition that reflects their endosomal origin, these proteome profiling studies also indicate a unique protein fingerprint that reflects their cellular origin as well as possible physiological role and targeting properties. However, interpretation of exosomal proteome profiles in a biological context also highlights a cautionary note, especially if exosomes are not highly purified. For example, retroviruses such as HIV particles that bud from the cell surface using the same endocytic pathway machinery as exosomes to egress from hematopoietic cells can be a confounding factor in biochemical and physiological analyses of exosomes. Furthermore, exosomes and HIV-1 particles have similar biophysical properties such as size (40–100 and 100 nm, respectively) and buoyant density (1.13–1.21 g/liter (20) and 1.13–1.21 g/liter (21), respectively) as well as molecular composition and their ability to activate immune cells. Although earlier studies describe exosomes carrying virion cargo (22–24), recent exosome purification strategies deploying immunoaffinity capture (25) or a combination of immunoaffinity capture and density gradient centrifugation (26) demonstrate that exosomes from hematopoietic cells can be purified free of virions like HIV-1.In-depth proteomics studies with large data sets that might contribute to the understanding of the biological function of exosomes are, to date, limited (2, 17). Moreover, strategies used to purify exosomes differ between laboratories (1) with little consensus concerning criteria of purity. Isolation strategies typically involve a combination of differential centrifugation, filtration, concentration, and flotation density gradient followed by characterization using electron microscopy, flow cytometry, and Western blotting (for a review, see Simpson et al. (1)). As a first step toward understanding the physiological role of exosomes in colon cancer biology, we describe here a robust strategy to isolate and characterize exosomes released from LIM1215 colorectal carcinoma cells (27) for the purpose of proteome analysis. This isolation strategy utilized the colon epithelial cell-specific A33 antibody (28–31) to immunoaffinity capture A33-containing exosomes using microbeads. Here, we report for the first time an in-depth proteomics analysis of A33-containing exosomes released from the LIM1215 colon carcinoma cell line. Using these data, we performed a comparative bioinformatics analysis with human urinary and mast cell-derived exosomes.     

Proteomics Analysis of Bladder Cancer Exosomes

Exosomes are nanometer-sized vesicles, secreted by various cell types, present in biological fluids that are particularly rich in membrane proteins. Ex vivo analysis of exosomes may provide biomarker discovery platforms and form non-invasive tools for disease diagnosis and monitoring. These vesicles have never before been studied in the context of bladder cancer, a major malignancy of the urological tract. We present the first proteomics analysis of bladder cancer cell exosomes. Using ultracentrifugation on a sucrose cushion, exosomes were highly purified from cultured HT1376 bladder cancer cells and verified as low in contaminants by Western blotting and flow cytometry of exosome-coated beads. Solubilization in a buffer containing SDS and DTT was essential for achieving proteomics analysis using an LC-MALDI-TOF/TOF MS approach. We report 353 high quality identifications with 72 proteins not previously identified by other human exosome proteomics studies. Overrepresentation analysis to compare this data set with previous exosome proteomics studies (using the ExoCarta database) revealed that the proteome was consistent with that of various exosomes with particular overlap with exosomes of carcinoma origin. Interrogating the Gene Ontology database highlighted a strong association of this proteome with carcinoma of bladder and other sites. The data also highlighted how homology among human leukocyte antigen haplotypes may confound MASCOT designation of major histocompatability complex Class I nomenclature, requiring data from PCR-based human leukocyte antigen haplotyping to clarify anomalous identifications. Validation of 18 MS protein identifications (including basigin, galectin-3, trophoblast glycoprotein (5T4), and others) was performed by a combination of Western blotting, flotation on linear sucrose gradients, and flow cytometry, confirming their exosomal expression. Some were confirmed positive on urinary exosomes from a bladder cancer patient. In summary, the exosome proteomics data set presented is of unrivaled quality. The data will aid in the development of urine exosome-based clinical tools for monitoring disease and will inform follow-up studies into varied aspects of exosome manufacture and function.Bladder cancer is one of the eight most frequent cancers in the Western world, and the frequency of transitional cell carcinoma (TCC),¹ which accounts for 90% of bladder cancers, is second only to prostate cancer as a malignancy of the genitourinary tract. Urine cytology and cystoscopy remain the predominant clinical tools for diagnosing and monitoring the disease, but cytology is poorly sensitive, particularly for low grade tumors, and does not serve as a prognostic tool. Cystoscopy is an invasive procedure, and there is pressing need to identify informative molecular markers that can be used to replace it.Recently, small cell-derived vesicles termed exosomes that are present in body fluids (1–5) have been proposed as a potential source of diagnostic markers (2, 6–8). These nanometer-sized vesicles, which are secreted by most cell types, originate from multivesicular bodies of the endocytic tract and reflect a subproteome of the cell. Exosomes are enriched in membrane and cytosolic proteins, and this molecular repertoire appears to be of particular functional importance to the immune system (9). Exosomes also comprise an array of lipids, mRNA, and microRNA, which are likely involved in conveying intercellular communication processes (10). Importantly, many exosomal components are simply not present as free soluble molecules in body fluids, such as certain microRNA species, which are encapsulated within the exosome lumen (6, 10). Therefore, the ability to isolate exosomes from urine (2), plasma (1), saliva (11), or other physiological sources (3) holds significant potential for obtaining novel and complex sets of biomarkers in a non-invasive manner. Exosome analysis may therefore be of value in disease diagnosis and monitoring in a variety of settings (6, 7, 12–14).Exosomes as indicators of pathology were first documented in the context of renal injury where a differential proteomics approach revealed changes in urinary exosome phenotype following renal injury (7). The researchers identified exosomally expressed Fetuin-A as a marker that became elevated 50-fold within hours following nephrotoxin exposure in rodents. Exosomal Fetuin-A elevation was also apparent in patients with acute renal injury before changes in urinary creatinine were observed (7). Clinical exosome analysis may also prove useful for solid cancers, such as ovarian or lung cancer, where the quantity of epithelial cell adhesion molecule-positive serum exosomes may correlate with tumor stage/grade. Such disease-associated exosomes express microRNA species not detected in healthy subjects (6, 12), although in this respect, there is little correlation between microRNA and disease bulk (6, 12). Other recent examples include studies of urinary exosomes in prostate cancer with exosomes expressing protein markers 5T4 (15), prostate cancer gene 3 (PCA-3) (8), or mRNA (TMPRSS2-ERG) (8, 16) associated with prostate cancer. To our knowledge, exosomes have not yet been studied in the context of other urological malignancies such as renal cancer, and to date, only one report describes the urine-derived microparticles from bladder cancer patients (17). In that report, they examined the proteome of a highly complex mixture of microvesicles, exosomes, and other urinary constituents that can be pelleted by high speed ultracentrifugation, identifying eight proteins that may be elevated in cancer. However, given the nature of the sample analyzed, it is unknown whether these proteins are exosomally expressed.Identification of the principal and most relevant molecular markers in these and other clinical scenarios remains a major challenge. In part, this is because exosomes present within complex body fluids originate from heterogeneous cell types. For example, plasma exosomes may be derived from platelets, lymphocytes, or endothelial cells (1), and a proportion may arise from well perfused organs such as the liver (18) and likely other organs as well (16). Similarly, exosomes present in urine arise from urothelial cells of the kidney and downstream of the renal tract (2, 8, 15).Importantly, all proteomics studies of exosomes isolated from body fluids are unavoidably complicated by the presence of high abundance non-exosomal proteins contaminating the preparations. Examples include albumin, immunoglobulin, and complement components present in exosomes prepared from malignant effusions (5) and Tamm-Horsfall protein present in exosomes purified from urine (2). As such, great care must be taken in the interpretation of the large data sets produced by proteomics studies, requiring careful validation of the proteins of interest. The protein composition of exosomes using a single homogenous cell type is one approach that may be used to uncover the protein components of exosomes produced by various cell types.There remain two major issues in the realm of exosome proteomics that complicate our interpretation of lists of identified proteins. Foremost are the diverse methods chosen for exosome purification that in some studies have involved attempts to remove contaminants through a key biophysical property of the vesicles, i.e. their capacity to float on sucrose (19, 20) or other dense media (21). Not all published studies, however, have taken such steps, preferring a far simpler pellet (or pellet and wash) approach. These latter preparations may be significantly contaminated by components of the cellular secretome, cell fragments, and other components. All of these factors could lead to false positive identifications of exosome proteins. The second key issue centers on the MS approaches utilized in various exosome proteomics studies. Many early examples relied only on a peptide mass fingerprinting approach, lacking robust peptide sequence data (22, 23), and more recently, search criteria that are generally recommended for MS-derived sequence data have not been specified in all studies. In this study, we have listed only those proteins identified by good quality MS/MS data for two or more peptides. Variability in the robustness and bias in bioinformatics analysis of data sets and in the steps taken to validate identified proteins is an additional factor that impacts the confidence in the identification lists produced.In this study, we aimed to perform the first proteomics analysis of human bladder cancer exosomes. We took extensive steps to produce high purity and quality-assured exosome preparations prior to beginning proteomics workflows. Solubilizing the sample with SDS and a reducing agent (DTT) was a critical step that allowed for global protein identification using nanoscale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. In this study, we present the identification of a significant number of exosomally expressed proteins (353 in total) of unrivaled quality. Critical manual examination of these identifications revealed issues with multiple (physiologically impossible) MHC Class I identifications that were attributed to a misdesignation of nomenclature by MASCOT due to peptide (and target protein) homology. The data were subjected to unbiased overrepresentation analysis (examining ExoCarta and Gene Ontology databases) to reveal a proteome consistent with exosomes, particularly of carcinoma origin. Validation of several identified proteins, by combining ultracentrifugation on a linear sucrose gradient with Western blotting and/or analysis of exosome-coated latex beads, demonstrated correct surface orientation of several MS-identified membrane proteins at densities consistent with exosomes.The robust approaches taken emphasize our confidence in the validity of the identifications generated and highlight that 72 (of 353) proteins have not been previously shown to be exosomally expressed by other human proteomics studies. The data will be useful for future studies in this underinvestigated disease and will form a platform not only for future clinical validation of some of these putative markers but also to aid further investigations into novel aspects of exosome function and manufacture.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Sip1, a Novel RS Domain-Containing Protein Essential for Pre-mRNA Splicing

Previous studies have shown that protein-protein interactions among splicing factors may play an important role in pre-mRNA splicing. We report here identification and functional characterization of a new splicing factor, Sip1 (SC35-interacting protein 1). Sip1 was initially identified by virtue of its interaction with SC35, a splicing factor of the SR family. Sip1 interacts with not only several SR proteins but also with U1-70K and U2AF65, proteins associated with 5′ and 3′ splice sites, respectively. The predicted Sip1 sequence contains an arginine-serine-rich (RS) domain but does not have any known RNA-binding motifs, indicating that it is not a member of the SR family. Sip1 also contains a region with weak sequence similarity to the Drosophila splicing regulator suppressor of white apricot (SWAP). An essential role for Sip1 in pre-mRNA splicing was suggested by the observation that anti-Sip1 antibodies depleted splicing activity from HeLa nuclear extract. Purified recombinant Sip1 protein, but not other RS domain-containing proteins such as SC35, ASF/SF2, and U2AF65, restored the splicing activity of the Sip1-immunodepleted extract. Addition of U2AF65 protein further enhanced the splicing reconstitution by the Sip1 protein. Deficiency in the formation of both A and B splicing complexes in the Sip1-depleted nuclear extract indicates an important role of Sip1 in spliceosome assembly. Together, these results demonstrate that Sip1 is a novel RS domain-containing protein required for pre-mRNA splicing and that the functional role of Sip1 in splicing is distinct from those of known RS domain-containing splicing factors.Pre-mRNA splicing takes place in spliceosomes, the large RNA-protein complexes containing pre-mRNA, U1, U2, U4/6, and U5 small nuclear ribonucleoprotein particles (snRNPs), and a large number of accessory protein factors (for reviews, see references 21, 22, 37, 44, and 48). It is increasingly clear that the protein factors are important for pre-mRNA splicing and that studies of these factors are essential for further understanding of molecular mechanisms of pre-mRNA splicing.Most mammalian splicing factors have been identified by biochemical fractionation and purification (3, 15, 19, 31–36, 45, 69–71, 73), by using antibodies recognizing splicing factors (8, 9, 16, 17, 61, 66, 67, 74), and by sequence homology (25, 52, 74).Splicing factors containing arginine-serine-rich (RS) domains have emerged as important players in pre-mRNA splicing. These include members of the SR family, both subunits of U2 auxiliary factor (U2AF), and the U1 snRNP protein U1-70K (for reviews, see references 18, 41, and 59). Drosophila alternative splicing regulators transformer (Tra), transformer 2 (Tra2), and suppressor of white apricot (SWAP) also contain RS domains (20, 40, 42). RS domains in these proteins play important roles in pre-mRNA splicing (7, 71, 75), in nuclear localization of these splicing proteins (23, 40), and in protein-RNA interactions (56, 60, 64). Previous studies by us and others have demonstrated that one mechanism whereby SR proteins function in splicing is to mediate specific protein-protein interactions among spliceosomal components and between general splicing factors and alternative splicing regulators (1, 1a, 6, 10, 27, 63, 74, 77). Such protein-protein interactions may play critical roles in splice site recognition and association (for reviews, see references 4, 18, 37, 41, 47 and 59). Specific interactions among the splicing factors also suggest that it is possible to identify new splicing factors by their interactions with known splicing factors.Here we report identification of a new splicing factor, Sip1, by its interaction with the essential splicing factor SC35. The predicted Sip1 protein sequence contains an RS domain and a region with sequence similarity to the Drosophila splicing regulator, SWAP. We have expressed and purified recombinant Sip1 protein and raised polyclonal antibodies against the recombinant Sip1 protein. The anti-Sip1 antibodies specifically recognize a protein migrating at a molecular mass of approximately 210 kDa in HeLa nuclear extract. The anti-Sip1 antibodies sufficiently deplete Sip1 protein from the nuclear extract, and the Sip1-depleted extract is inactive in pre-mRNA splicing. Addition of recombinant Sip1 protein can partially restore splicing activity to the Sip1-depleted nuclear extract, indicating an essential role of Sip1 in pre-mRNA splicing. Other RS domain-containing proteins, including SC35, ASF/SF2, and U2AF65, cannot substitute for Sip1 in reconstituting splicing activity of the Sip1-depleted nuclear extract. However, addition of U2AF65 further increases splicing activity of Sip1-reconstituted nuclear extract, suggesting that there may be a functional interaction between Sip1 and U2AF65 in nuclear extract.     

Algorithms for Finding Small Attractors in Boolean Networks

A Boolean network is a model used to study the interactions between different genes in genetic regulatory networks. In this paper, we present several algorithms using gene ordering and feedback vertex sets to identify singleton attractors and small attractors in Boolean networks. We analyze the average case time complexities of some of the proposed algorithms. For instance, it is shown that the outdegree-based ordering algorithm for finding singleton attractors works in time for , which is much faster than the naive time algorithm, where is the number of genes and is the maximum indegree. We performed extensive computational experiments on these algorithms, which resulted in good agreement with theoretical results. In contrast, we give a simple and complete proof for showing that finding an attractor with the shortest period is NP-hard.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer.Prostate cancer (PCa)¹ is the leading type of cancer diagnosed in men. The American Cancer Society reported 217,730 new cases of PCa in the United States last year. Death from PCa follows its incidence profile closely as the third leading cause of cancer-related death in men (1). In the early stages, the disease is locally confined to the prostate and is hormone or androgen-dependent. It can be managed at this stage by surgical intervention or radiation treatment. However, over time (varying from months to years), many prostate cancers metastasize and, even with aggressive hormone deprivation therapy, progress to castration resistant prostate cancer (CRPC), which ultimately results in death. During early metastasis, a response to androgen deprivation therapy (ADT) is usually observed. Nonetheless, despite the reduction in androgen levels after ADT, androgen receptor (AR) remains active and contributes to CRPC progression (2–4).The routine screening test for PCa diagnosis in North America includes measurement of prostate specific antigen (PSA) in the blood, digital rectal examination and a prostate biopsy (5). PSA screening for PCa detection is controversial because certain activities can induce the production of PSA, unrelated to the presence of cancer (6). Consequently prostate biopsy, albeit an invasive procedure, remains the only definitive diagnostic test for PCa. There is an urgent current need, therefore, for the discovery of relevant biomarkers to replace the existing diagnostic tests for better and earlier detection of PCa (7).One possible source of biomarkers which could be used as part of a diagnostic test are exosomes. All cells produce and release exosomes, which are often found in different body fluids such as plasma (8), serum (9, 10) malignant ascites (11, 12) urine (13), amniotic fluid (14), bronchoalveolar lavage fluid (15, 16), and breast milk (17, 18). Recent studies suggest however that cancer cells produce exosomes, which may be differentiated from those derived from normal cells primarily based upon their cargo. Exosomes are cup-shaped (19) encapsulated by a bi-layer lipid membrane (20) with a membrane-bound compartment varying between 30–100 nm in size (19). As mentioned above, they are secreted from both normal cells and tumor cells (21) and although the underlying mechanism of exosome function is not fully understood it is known that exosomes are formed in the endosomal compartment of cells and are secreted upon fusion of multivesicular bodies (MVB) with the plasma membrane (21). The schematic cartoon in Fig. 1 depicts early endosome (EE) formation as a result of the invagination of specific regions of the plasma membrane. In addition, endocytotic cargo transported out of the cell is sorted from EE into intraluminal vesicles (ILV). Mechanisms involved in protein sorting into ILVs are still under investigation however there is evidence supporting the involvement of ubiquitin and endosomal sorting complex required for transport (ESCRT machinery) in this process. Finally, fusion of late endosome or MVB with plasma membrane releases ILVs into the extracellular matrix or the tissue microenvironment. Accumulating evidence suggests that induction of intracellular calcium (22–25), overexpression of Rab11 or citron kinase (26) as well as a reduction in membrane cholesterol, or inhibition of cholesterol biosynthesis (27), could stimulate the release of exosomes into the microenvironment.Open in a separate windowFig. 1.Mechanism involved in exosome formation and trafficking in the microenvironment.As shown in Fig. 1, once released, exosomes will interact with recipient target cells via different mechanisms such as fusion with the plasma membrane or adhesion to corresponding receptors on the plasma membrane (25).Although, the mechanisms underlying exosome formation and secretion is still under investigation, it is well-known that factors such as cell type, cell cycle, and stage of cancer, could affect the amount and composition of exosomes formed and secreted from various cells (19). It has been shown that exosomes are secreted in a multitude of cell types and though it is postulated that they are involved in membrane trafficking as communication vesicles, their relevance in cancer initiation and specifically prostate tumor growth and progression has yet to be determined (28–30). Studies on tumor-related microvesicles suggest that exosomes play a significant role in cell communication thus potentially influencing cancer progression via different mechanisms (31). Exosomes contain and protect the integrity of various proteins and an array of lipids, mRNA and miRNA which would otherwise be hydrolytically or enzymatically broken down if they existed as free soluble molecules in the extracellular microenvironment. The presence of differential exosomal protein markers involved in cancer progression combined with the presence of exosomes in accessible biological fluids highlights a potential role of exosomes as clinical biomarkers for PCa at diagnosis and progression (32, 33). Therefore isolation, purification and characterization of exosomes derived from different body fluids is an essential first step in identifying novel biomarkers from this source.In addition, exosomes may also present novel therapeutic strategies. If in fact implicated in cancer progression, exosomes present a new target set for development of novel therapeutics. Hence, a better understanding of the mechanisms involved in formation and secretion of exosomes for therapeutic targeting as well as investigating the relevance of the presence of different proteins in these membrane vesicles is required.Therefore the main purpose of the present study was to observe the release of exosomes by prostate cells, and determine characteristic differences between exosomes released by parent cells of different characteristic AR phenotypes. In order to answer this question, in addition to one nonmalignant cell line, we used five different PCa cell lines which contain/lack AR and were representative of different stages of PCa.We then confirmed the transfer of exosomes to target cells in culture using confocal microscopy of fluorescence labeled exosomes. We subsequently performed a comprehensive proteomic analysis of all six different prostate cell lines using mass spectrometry to understand differences between the protein profiles released via exosome externalization in different prostate cell lines. The final part of this study was to investigate the difference in broad classes of lipids and cholesterol as constituents of different prostate cell lines and their exosomes.Taken together the comprehensive characterization of exosomes derived from prostate cell lines which have distinct AR ±ve phenotypes, provides a basis for evaluating transfer of identified composite exosome proteins between different PCa cells as part of a recognized cell communication phenomenon. In addition this study forms a platform for future clinical validation research using exosomes as biomarkers for PCa diagnosis as well as potential therapeutic targets which could be important in the treatment of CRPC.