Integration of proteomic and metabolomic analysis reveal distinct metabolic alterations of prostate cancer-associated fibroblasts compared to normal fibroblasts from patient's stroma samples.

The prostate gland is a complex and heterogeneous organ composed of epithelium and stroma. Whilst many studies into prostate cancer focus on epithelium, the stroma is known to play a key role in disease with the emergence of a cancer-associated fibroblasts (CAF) phenotype associated upon disease progression. In this work, we studied the metabolic rewiring of stromal fibroblasts following differentiation to a cancer-associated, myofibroblast-like, phenotype. We determined that CAFs were metabolically more active compared to normal fibroblasts. This corresponded with a heightened lipogenic metabolism, as both reservoir species and building block compounds. Interestingly, lipid metabolism affects mitochondria functioning yet the mechanisms of lipid-mediated functions are unclear. Data showing oxidised fatty acids and glutathione system are elevated in CAFs, compared to normal fibroblasts, strengthens the hypothesis that increased metabolic activity is related to mitochondrial activity. This manuscript describes mechanisms responsible for the altered metabolic flux and shows that prostate cancer-derived extracellular vesicles can increase basal respiration in normal fibroblasts, mirroring that of the disease-like phenotype. This indicates that extracellular vesicles derived from prostate cancer cells may drive an altered oxygen-dependent metabolism associated to mitochondria in CAFs.

Tuberous Sclerosis Complex cell-derived EVs have an altered protein cargo capable of regulating their microenvironment and have potential as disease biomarkers.

Hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) is a feature of many solid tumours and is a key pathogenic driver in the inherited condition Tuberous Sclerosis Complex (TSC). Modulation of the tumour microenvironment by extracellular vesicles (EVs) is known to facilitate the development of various cancers. The role of EVs in modulating the tumour microenvironment and their impact on the development of TSC tumours, however, remains unclear. This study, therefore, focuses on the poorly defined contribution of EVs to tumour growth in TSC. We characterised EVs secreted from TSC2-deficient and TSC2-expressing cells and identified a distinct protein cargo in TSC2-deficient EVs, containing an enrichment of proteins thought to be involved in tumour-supporting signalling pathways. We show EVs from TSC2-deficient cells promote cell viability, proliferation and growth factor secretion from recipient fibroblasts within the tumour microenvironment. Rapalogs (mTORC1 inhibitors) are the current therapy for TSC tumours. Here, we demonstrate a previously unknown intercellular therapeutic effect of rapamycin in altering EV cargo and reducing capacity to promote cell proliferation in the tumour microenvironment. Furthermore, EV cargo proteins have the potential for clinical applications as TSC biomarkers, and we reveal three EV-associated proteins that are elevated in plasma from TSC patients compared to healthy donor plasma.

Integrins are enriched on aberrantly fucosylated tumour-derived urinary extracellular vesicles.

Urinary extracellular vesicles (uEVs) are enriched with glycosylated proteins which have been extensively studied as putative biomarkers of urological cancers. Here, we characterized the glycosylation and integrin profile of EVs derived from urological cancer cell lines. We used fluorescent europium-doped nanoparticles coated with lectins and antibodies to identify a biomarker combination consisting of integrin subunit alpha 3 (ITGA3) and fucose. In addition, we used the same cancer cell line-derived EVs as analytical standards to assess the sensitivity of the ITGA3-UEA assay. The clinical performance of the ITGA3-UEA assay was analysed using urine samples of various urological pathologies including diagnostically challenging benign prostatic hyperplasia (BPH), prostate cancer (PCa) and bladder cancer (BlCa). The assay can significantly discriminate BlCa from all other patient groups: PCa (9.2-fold; p = 0.00038), BPH (5.5-fold; p = 0.004) and healthy individuals (and 23-fold; p = 0.0001). Our results demonstrate that aberrantly fucosylated uEVs and integrin ITGA3 can be detected with fucose-specific lectin UEA in a simple bioaffinity assay for the detection of BlCa directly from unprocessed urine.

Stroma-derived extracellular vesicle mRNA signatures inform histological nature of prostate cancer.

Histological assessment of prostate cancer is the key diagnostic test and can predict disease outcome. This is however an invasive procedure that carries associated risks, hence non-invasive assays to support the diagnostic pathway are much needed. A key feature of disease progression, and subsequent poor prognosis, is the presence of an altered stroma. Here we explored the utility of prostate stromal cell-derived vesicles as indicators of an altered tumour environment. We compared vesicles from six donor-matched pairs of adjacent-normal versus disease-associated primary stromal cultures. We identified 19 differentially expressed transcripts that discriminate disease from normal stromal extracellular vesicles (EVs). EVs isolated from patient serum were investigated for these putative disease-discriminating mRNA. A set of transcripts including Caveolin-1 (CAV1), TMP2, THBS1, and CTGF were found to be successful in discriminating clinically insignificant (Gleason = 6) disease from clinically significant (Gleason > 8) prostate cancer. Furthermore, correlation between transcript expression and progression-free survival suggests that levels of these mRNA may predict disease outcome. Informed by a machine learning approach, combining measures of the five most informative EV-associated mRNAs with PSA was shown to significantly improve assay sensitivity and specificity. An in-silico model was produced, showcasing the superiority of this multi-modal liquid biopsy compared to needle biopsy for predicting disease progression. This proof of concept highlights the utility of serum EV analytics as a companion diagnostic test with prognostic utility, which may obviate the need for biopsy.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Prostate Cancer Cell Extracellular Vesicles Increase Mineralisation of Bone Osteoblast Precursor Cells in an In Vitro Model.

Skeletal metastases are the most common form of secondary tumour associated with prostate cancer (PCa). The aberrant function of bone cells neighbouring these tumours leads to the devel-opment of osteoblastic lesions. Communication between PCa cells and bone cells in bone envi-ronments governs both the formation/development of the associated lesion, and growth of the secondary tumour. Using osteoblasts as a model system, we observed that PCa cells and their conditioned medium could stimulate and increase mineralisation and osteoblasts' differentiation. Secreted factors within PCa-conditioned medium responsible for osteoblastic changes included small extracellular vesicles (sEVs), which were sufficient to drive osteoblastogenesis. Using MiR-seq, we profiled the miRNA content of PCa sEVs, showing that miR-16-5p was highly ex-pressed. MiR-16 was subsequently higher in EV-treated 7F2 cells and a miR-16 mimic could also stimulate mineralisation. Next, using RNA-seq of extracellular vesicle (EV)-treated 7F2 cells, we observed a large degree of gene downregulation and an increased mineralisation. Ingenuity® Pathway Analysis (IPA®) revealed that miR-16-5p (and other miRs) was a likely upstream effec-tor. MiR-16-5p targets in 7F2 cells, possibly involved in osteoblastogenesis, were included for val-idation, namely AXIN2, PLSCR4, ADRB2 and DLL1. We then confirmed the targeting and dow-regulation of these genes by sEV miR-16-5p using luciferase UTR (untranslated region) reporters. Conversely, the overexpression of PLSCR4, ADRB2 and DLL1 lead to decreased osteoblastogene-sis. These results indicate that miR-16 is an inducer of osteoblastogenesis and is transmitted through prostate cancer-derived sEVs. The mechanism is a likely contributor towards the for-mation of osteoblastic lesions in metastatic PCa.

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6-9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane.

Targeting Antigen to the Surface of EVs Improves the In Vivo Immunogenicity of Human and Non-human Adenoviral Vaccines in Mice.

Adenoviral (Ad) vectors represent promising vaccine platforms for infectious disease. To overcome pre-existing immunity to commonly used human adenovirus serotype 5 (Ad5), vectors based on rare species or non-human Ads are being developed. However, these vectors often exhibit reduced potency compared with Ad5, necessitating the use of innovative approaches to augment the immunogenicity of the encoded antigen (Ag). To achieve this, we engineered model Ag, enhanced green fluorescent protein (EGFP), for targeting to the surface of host-derived extracellular vesicles (EVs), namely exosomes. Exosomes are nano-sized EVs that play important roles in cell-to-cell communication and in regulating immune responses. Directed targeting of Ag to the surface of EVs/exosomes is achieved by "exosome display," through fusion of Ag to the C1C2 domain of lactadherin, a protein highly enriched in exosomes. Herein, we engineered chimpanzee adenovirus ChAdOx1 and Ad5-based vaccines encoding EGFP, or EGFP targeted to EVs (EGFP_C1C2), and compared vaccine immunogenicity in mice. We determined that exosome display substantially increases Ag-specific humoral immunity following intramuscular and intranasal vaccination, improving the immunological potency of both ChAdOx1 and Ad5. We propose that this Ag-engineering approach could increase the immunogenicity of diverse Ad vectors that exhibit desirable manufacturing characteristics, but currently lack the potency of Ad5.

Summary of the ISEV workshop on extracellular vesicles as disease biomarkers, held in Birmingham, UK, during December 2017.

This report summarises the presentations and activities of the ISEV Workshop on extracellular vesicle biomarkers held in Birmingham, UK during December 2017. Among the key messages was broad agreement about the importance of biospecimen science. Much greater attention needs to be paid towards the provenance of collected samples. The workshop also highlighted clear gaps in our knowledge about pre-analytical factors that alter extracellular vesicles (EVs). The future utility of certified standards for credentialing of instruments and software, to analyse EV and for tracking the influence of isolation steps on the structure and content of EVs were also discussed. Several example studies were presented, demonstrating the potential utility for EVs in disease diagnosis, prognosis, longitudinal serial testing and stratification of patients. The conclusion of the workshop was that more effort focused on pre-analytical issues and benchmarking of isolation methods is needed to strengthen collaborations and advance more effective biomarkers.

Dominant immunosuppression of dendritic cell function by prostate-cancer-derived exosomes.

Exosomes are a distinct population of extracellular vesicles of endocytic origin with a protein repertoire similar to the parent cell. Although tumour-derived exosomes harbour immunosuppressive characteristics, they also carry tumour antigens and thus potentially contribute to immune activation. The aim of this study was to examine the impact of prostate cancer exosomes on tumour antigen cross-presentation. DU145 cells, transduced with shRNA to knockdown Rab27a (DU145KD) that inhibits exosome secretion, triggered significantly stronger tumour-antigen-specific T cell responses when loaded onto dendritic cells (DC) than control DU145 cells. Enhanced T cell response was prevented by adding purified exogenous DU145 exosomes to DU145KD cells, demonstrating that the dominant effect of tumour exosomes is immunosuppression and not antigen delivery. CD8+ T cell responses were impaired via exosomal regulation of DC function; exosomes triggered the expression of CD73, an ecto-5-nucleotidase responsible for AMP to adenosine hydrolysis, on DC. CD73 induction on DC that constitutively express CD39 resulted in an ATP-dependent inhibition of TNFα- and IL-12-production. We identified exosomal prostaglandin E2 (PGE2) as a potential driver of CD73 induction, as inhibition of PGE2 receptors significantly reduced exosome-dependent CD73 induction. The results reveal a hitherto unknown suppression of DC function via exosomal PGE2, adding a new element to tumour exosome-immune cell cross-talk. Abbreviations: AMP: adenosine monophosphate; ATP: adenosine triphosphate; BLCL: B lymphoblastoid cell line; CME: exosomes enriched from cell line conditioned media; DC: dendritic cell; DMSO: dimethyl-sulfoxide; DU145C: DU145 cells with irrelevant knockdown control; DU145KD: DU145 cells with Rab27a knockdown; ELISA: enzyme-linked immunosorbent assay; FBS: fetal bovine serum; GM-CSF: granulocyte-monocyte colony stimulating factor; HLA: human lymphocyte antigen; IL: interleukin; LPS: lipopolysaccharide; mfi: mean fluorescence intensity; PBMC: peripheral blood mononuclear cells; PBS: phosphate buffer solution; PGE2: prostaglandin E2; TRF: time-resolved fluorescence.

Proteomics analysis of vesicles isolated from plasma and urine of prostate cancer patients using a multiplex, aptamer-based protein array.

Proteomics analysis of biofluid-derived vesicles holds enormous potential for discovering non-invasive disease markers. Obtaining vesicles of sufficient quality and quantity for profiling studies has, however, been a major problem, as samples are often replete with co-isolated material that can interfere with the identification of genuine low abundance, vesicle components. Here, we used a combination of ultracentrifugation and size-exclusion chromatography to isolate and analyse vesicles of plasma or urine origin. We describe a sample-handling workflow that gives reproducible, quality vesicle isolations sufficient for subsequent protein profiling. Using a semi-quantitative aptamer-based protein array, we identified around 1,000 proteins, of which almost 400 were present at comparable quantities in plasma versus urine vesicles. Significant differences were, however, apparent with elements like HSP90, integrin αVß5 and Contactin-1 more prevalent in urinary vesicles, while hepatocyte growth factor activator, prostate-specific antigen-antichymotrypsin complex and many others were more abundant in plasma vesicles. This was also applied to a small set of specimens collected from men with metastatic prostate cancer, highlighting several proteins with the potential to indicate treatment refractory disease. The study provides a practical platform for furthering protein profiling of vesicles in prostate cancer, and, hopefully, many other disease scenarios.

Prostate stromal cell proteomics analysis discriminates normal from tumour reactive stromal phenotypes.

Changes within interstitial stromal compartments often accompany carcinogenesis, and this is true of prostate cancer. Typically, the tissue becomes populated by myofibroblasts that can promote progression. Not all myofibroblasts exhibit the same negative influence, however, and identifying the aggressive form of myofibroblast may provide useful information at diagnosis. A means of molecularly defining such myofibroblasts is unknown. We compared protein profiles of normal and diseased stroma isolated from prostate cancer patients to identify discriminating hallmarks of disease-associated stroma. We included the stimulation of normal stromal cells with known myofibroblast inducers namely soluble TGFß and exosome-associated-TGFß and compared the function and protein profiles arising. In all 6-patients examined, diseased stroma exhibited a pro-angiogenic influence on endothelial cells, generating large multicellular vessel-like structures. Identical structures were apparent following stimulation of normal stroma with exosomes (5/6 patients), but TGFß-stimulation generated a non-angiogenic stroma. Proteomics highlighted disease-related cytoskeleton alterations such as elevated Transgelin (TAGLN). Many of these were also changed following TGFß or exosome stimulation and did not well discriminate the nature of the stimulus. Soluble TGFß, however triggered differential expression of proteins related to mitochondrial function including voltage dependent ion channels VDAC1 and 2, and this was not found in the other stromal types studied. Surprisingly, Aldehyde Dehydrogenase (ALDH1A1), a stem-cell associated protein was detected in normal stromal cells and found to decrease in disease. In summary, we have discovered a set of proteins that contribute to defining disease-associated myofibroblasts, and emphasise the similarity between exosome-generated myofibroblasts and those naturally arising in situ.

Ready-made chromatography columns for extracellular vesicle isolation from plasma.

Proteomic studies of circulating vesicles are hampered by difficulties in purifying vesicles from plasma and serum. Isolations are contaminated with high-abundance blood proteins that may mask genuine vesicular-associated proteins and/or simply provide misleading data. In this brief report, we explored the potential utility of a commercially available size exclusion chromatography column for rapid vesicle purification. We evaluated the performance of the column, with cancer cell line conditioned medium or healthy donor plasma, in terms of removing non-vesicular protein and enriching for vesicles exhibiting exosome characteristics. Serial fractions revealed a peak for typical exosomal proteins (CD9, CD81 etc.) that preceded the peak for highly abundant proteins, including albumin, for either sample type, and harvesting only this peak would represent elimination of >95% of protein from the sample. The columns showed good reproducibility, and streamlining the workflow would allow the exosome-relevant material to be collected in less than 10 minutes. Surprisingly, however, subsequent post-column vesicle concentration steps whilst resulting in some protein loss also lead to low vesicle recoveries, with a net effect of reducing sample purity (assessed by the particle-to-protein ratio). The columns provide a convenient, reproducible and highly effective means of eliminating >95% of non-vesicular protein from biological fluid samples such as plasma.

Extracellular vesicles as modulators of the cancer microenvironment.

The tumour microenvironment is a highly complex and dynamic tissue. It comprises not only neoplastic cells, but also other resident cells within the milieu such as stroma and vascular cells in addition to a variable cellular infiltrate from the periphery. A host of soluble factors such as growth factors, chemokines, eicosanoids soluble metabolites and extracellular matrix components have been extensively documented as factors which modulate this environment. However, in recent years there has also been growing interests in the potential roles of extracellular vesicles (EV) in many of the processes governing the nature of cancerous tissue. In this brief review, we have assembled evidence describing several distinct functions for extracellular vesicles in modulating the microenvironment with examples that include immune evasion, angiogenesis and stromal activation. Whilst there remains a great deal to be learnt about the interplay between vesicles and the cancerous environment, it is becoming clear that vesicle-mediated communication has a major influence on key aspects of cancer growth and progression. We conclude that the design of future therapeutics should acknowledge the existence and roles of extracellular vesicles, and seriously consider strategies for circumventing their effects in vivo.

Cancer exosomes trigger mesenchymal stem cell differentiation into pro-angiogenic and pro-invasive myofibroblasts.

Stromal fibroblasts become altered in response to solid cancers, to exhibit myofibroblastic characteristics, with disease promoting influence. Infiltrating mesenchymal stem cells (MSC) may contribute towards these changes, but the factors secreted by cancer cells that impact MSC differentiation are poorly understood. We investigated the role of nano-metre sized vesicles (exosomes), secreted by prostate cancer cells, on the differentiation of bone-marrow MSC (BM-MSC), and the subsequent functional consequences of such changes. Purified exosomes impaired classical adipogenic differentiation, skewing differentiation towards alpha-smooth muscle actin (αSMA) positive myofibroblastic cells. A single exosomes treatment generated myofibroblasts secreting high levels of VEGF-A, HGF and matrix regulating factors (MMP-1, -3 and -13). Differentiated MSC had pro-angiogenic functions and enhanced tumour proliferation and invasivity assessed in a 3D co-culture model. Differentiation was dependent on exosomal-TGFß, but soluble TGFß at matched dose could not generate the same phenotype. Exosomes present in the cancer cell secretome were the principal factors driving this phenotype. Prostate cancer exosomes dominantly dictate a programme of MSC differentiation generating myofibroblasts with functional properties consistent with disease promotion.

Proteomics analysis of cancer exosomes using a novel modified aptamer-based array (SOMAscan™) platform.

We have used a novel affinity-based proteomics technology to examine the protein signature of small secreted extracellular vesicles called exosomes. The technology uses a new class of protein binding reagents called SOMAmers® (slow off-rate modified aptamers) and allows the simultaneous precise measurement of over 1000 proteins. Exosomes were highly purified from the Du145 prostate cancer cell line, by pooling selected fractions from a continuous sucrose gradient (within the density range of 1.1 to 1.2 g/ml), and examined under standard conditions or with additional detergent treatment by the SOMAscan™ array (version 3.0). Lysates of Du145 cells were also prepared, and the profiles were compared. Housekeeping proteins such as cyclophilin-A, LDH, and Hsp70 were present in exosomes, and we identified almost 100 proteins that were enriched in exosomes relative to cells. These included proteins of known association with cancer exosomes such as MFG-E8, integrins, and MET, and also those less widely reported as exosomally associated, such as ROR1 and ITIH4. Several proteins with no previously known exosomal association were confirmed as exosomally expressed in experiments using individual SOMAmer® reagents or antibodies in micro-plate assays. Western blotting confirmed the SOMAscan™-identified enrichment of exosomal NOTCH-3, L1CAM, RAC1, and ADAM9. In conclusion, we describe here over 300 proteins of hitherto unknown association with prostate cancer exosomes and suggest that the SOMAmer®-based assay technology is an effective proteomics platform for exosome-associated biomarker discovery in diverse clinical settings.

Tumor stroma-derived factors skew monocyte to dendritic cell differentiation toward a suppressive CD14+ PD-L1+ phenotype in prostate cancer.

Tumor-associated stromal myofibroblasts are essential for the progression and metastatic spread of solid tumors. Corresponding myeloid cell infiltration into primary tumors is a negative prognostic factor in some malignancies. The aim of this study was to define the exact role of stromal myofibroblasts and stromal factors in early prostate carcinoma (PCa) regulating monocyte infiltration and differentiation into dendritic cells (DCs). Epithelial and stromal primary cultures were generated from PCa biopsies and their purity confirmed. Stromal cells produced significantly more of the (C-C) motif chemokine ligand 2 (CCL2), interleukin 6 (IL-6) and transforming growth factor ß (TGFß) than epithelial cells. Monocyte chemoattraction was predominantly due to stromal-derived factors, mainly CCL2. DCs generated in the presence of stromal (but not epithelial) factors upregulated CD209, but failed to downregulate the monocyte marker CD14 in a signal transducer and activator of transcription 3 (STAT3)-dependent manner. Monocytes exposed to stromal factors did not produce detectable amounts of IL-10, however, upon lipopolysaccharide stimulation, stromal factor generated dendritic cells (sDC) produced significantly more IL-10 and less IL-12 than their conventional DC counterparts. sDC failed to cross-present tumor-antigen to CD8+ T cells and suppressed T-cell proliferation. Most importantly, sDC expressed significantly elevated levels of programmed cell death ligand-1 (PD-L1) in a primarily STAT3 and IL-6-dependent manner. In parallel with our findings in vitro, tumor-infiltrating CD14+ cells in situ were found to express both PD-L1 and CD209, and a higher percentage of tumor-associated CD3+ T cells expressed programmed cell death-1 (PD-1) molecules compared to T cells in blood. These results demonstrate a hitherto undescribed, fundamental contribution of tumor-associated stromal myofibroblasts to the development of an immunosuppressive microenvironment in early PCa.

How pure are your vesicles?

We propose a straightforward method to estimate the purity of vesicle preparations by comparing the ratio of nano-vesicle counts to protein concentration, using tools such as the increasingly available NanoSight platform and a colorimetric protein assay such as the BCA-assay. Such an approach is simple enough to apply to every vesicle preparation within a given laboratory, assisting researchers as a routine quality control step. Also, the approach may aid in comparing/standardising vesicle purity across diverse studies, and may be of particular importance in evaluating vesicular biomarkers. We herein propose some criteria to aid in the definition of pure vesicles.