Immunomodulatory potential of human clonal mesenchymal stem cells and their extracellular vesicle subpopulations in an inflammatory-mediated diabetic Rhesus monkey model.

AIMS: This study aimed to investigate the therapeutic potential of a homogenous clonal population of mesenchymal stem cells (cMSC) and their extracellular vesicles (cMSC-EV) subpopulations on isolated rat islets in vitro and in inflammatory-mediated type 1 diabetes (T1D) non-human primate models. MAIN METHODS: EV subpopulations were isolated from human bone marrow-derived cMSC supernatant by low- and high-speed ultracentrifuge (EV-20K and EV-U110K) and sucrose density gradient (EV-S110K). The EVs were characterized generally and for the level of albumin, acetylcholinesterase (AChE) activity, co-isolate apoptotic markers, and expression of CD63+/annexin V+. Rat islet-derived single cells (iSCs) proliferation was measured using a Ki-67 proliferation assay. Diabetes was induced by multiple low-dose administrations of streptozotocin in rhesus monkeys. The diabetic monkeys were divided into three groups: the cMSC group, received two injections of 1.5 × 106 cMSC/kg body weight; the EV group received two injections of EVs isolated from 1.5 × 106 cMSC/kg, and the vehicle group received phosphate-buffered saline. KEY FINDINGS: EV-S110K showed higher AChE activity, lower expression of CD63+/annexin V+, and lower apoptotic co-isolates. EV-S110K induced ß-cell proliferation in vitro in a dose-dependent manner. The administration of EV-S110K and/or cMSC in diabetic monkeys demonstrated no significant changes in general diabetic indices and ß-cell mass in the pancreas of the monkeys. Both treatments demonstrated a lowering trend in blood glucose levels and reduced pro-inflammatory cytokines. In contrast, regulatory T cells and anti-inflammatory cytokines were increased. SIGNIFICANCE: cMSC and cMSC-EV provided initial evidence to attenuate clinical symptoms in inflammatory-mediated T1D non-human primates through immunomodulation.

Small and Large Extracellular Vesicles Derived from Pleural Mesothelioma Cell Lines Offer Biomarker Potential.

Pleural mesothelioma, previously known as malignant pleural mesothelioma, is an aggressive and fatal cancer of the pleura, with one of the poorest survival rates. Pleural mesothelioma is in urgent clinical need for biomarkers to aid early diagnosis, improve prognostication, and stratify patients for treatment. Extracellular vesicles (EVs) have great potential as biomarkers; however, there are limited studies to date on their role in pleural mesothelioma. We conducted a comprehensive proteomic analysis on different EV populations derived from five pleural mesothelioma cell lines and an immortalized control cell line. We characterized three subtypes of EVs (10 K, 18 K, and 100 K), and identified a total of 4054 unique proteins. Major differences were found in the cargo between the three EV subtypes. We show that 10 K EVs were enriched in mitochondrial components and metabolic processes, while 18 K and 100 K EVs were enriched in endoplasmic reticulum stress. We found 46 new cancer-associated proteins for pleural mesothelioma, and the presence of mesothelin and PD-L1/PD-L2 enriched in 100 K and 10 K EV, respectively. We demonstrate that different EV populations derived from pleural mesothelioma cells have unique cancer-specific proteomes and carry oncogenic cargo, which could offer a novel means to extract biomarkers of interest for pleural mesothelioma from liquid biopsies.

Extracellular vesicles from biological fluids as potential markers in castration resistant prostate cancer.

PURPOSE: Extracellular vesicles (EV) secreted from cancer cells are present in various biological fluids, carrying distinctly different cellular components compared to normal cells, and have great potential to be used as markers for disease initiation, progression, and response to treatment. This under-utilised tool provides insights into a better understanding of prostate cancer. METHODS: EV from serum and urine of healthy men and castration-resistant prostate cancer (CRPC) patients were isolated and characterised by transmission electron microscopy, particle size analysis, and western blot. Proteomic and cholesterol liquid chromatography-mass spectrometry (LC-MS) analyses were conducted. RESULTS: There was a successful enrichment of small EV/exosomes isolated from serum and urine. EV derived from biological fluids of CRPC patients had significant differences in composition when compared with those from healthy controls. Analysis of matched serum and urine samples from six prostate cancer patients revealed specific EV proteins common in both types of biological fluid for each patient. CONCLUSION: Some of the EV proteins identified from our analyses have potential to be used as CRPC markers. These markers may depict a pattern in cancer progression through non-invasive sample collection.

Bacterial extracellular vesicles and their novel therapeutic applications in health and cancer.

Bacterial cells communicate with host cells and other bacteria through the release of membrane vesicles known as bacterial extracellular vesicles (BEV). BEV are established mediators of intracellular signaling, stress tolerance, horizontal gene transfer, immune stimulation and pathogenicity. Both Gram-positive and Gram-negative bacteria produce extracellular vesicles through different mechanisms based on cell structure. BEV contain and transfer different types of cargo such as nucleic acids, proteins and lipids, which are used to interact with and affect host cells such as cytotoxicity and immunomodulation. The role of these membranous microvesicles in host communication, intra- and inter-species cell interaction and signaling, and contribution to various diseases have been well demonstrated. Due to their structure, these vesicles can be easily engineered to be utilized for clinical application, as shown with its role in vaccine therapy, and could be used as a diagnostic and cancer drug delivery tool in the future. However, like other novel therapeutic approaches, further investigation and standardization is imperative for BEV to become a routine vector or a conventional treatment method.

Chloroquine treatment induces secretion of autophagy-related proteins and inclusion of Atg8-family proteins in distinct extracellular vesicle populations.

Chloroquine (CQ), a lysosomotropic agent, is commonly used to inhibit lysosomal degradation and macroautophagy/autophagy. Here we investigated the cell-extrinsic effects of CQ on secretion. We showed that lysosomal and autophagy inhibition by CQ altered the secretome, and induced the release of Atg8 orthologs and autophagy receptors. Atg8-family proteins, in particular, were secreted inside small extracellular vesicles (sEVs) in a lipidation-dependent manner. CQ treatment enhanced the release of Atg8-family proteins inside sEVs. Using full-length ATG16L1 and an ATG16L1 mutant that enables Atg8-family protein lipidation on double but not on single membranes, we demonstrated that LC3B is released in two distinct sEV populations: one enriched with SDCBP/Syntenin-1, CD63, and endosomal lipidated LC3B, and another that contains LC3B but is not enriched with SDCBP/Syntenin-1 or CD63, and which our data supports as originating from a double-membrane source. Our findings underscore the context-dependency of sEV heterogeneity and composition, and illustrate the integration of autophagy and sEV composition in response to lysosomal inhibition.Abbreviations: ACTB: actin beta; ANOVA: analysis of variance; ATG4B: autophagy related 4B cysteine peptidase; Atg8: autophagy related 8; ATG16L1: autophagy related 16 like 1; ATP5F1A/ATP5a: ATP synthase F1 subunit alpha; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CASP7: caspase 7; CQ: chloroquine; CD9: CD9 molecule; CD63: CD63 molecule; DAPI: 4',6-diamidino-2-phenylindole; DQ-BSA: dye quenched-bovine serum albumin; ER: endoplasmic reticulum; ERN1/IRE1a: endoplasmic reticulum to nucleus signaling 1; EV: extracellular vesicles; FBS: fetal bovine serum; FDR: false discovery rate; GABARAP: GABA type A receptor-associated protein; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; GO: gene ontology; HCQ: hydroxychloroquine; HSP90AA1: heat shock protein 90 alpha family class A member 1; IP: immunoprecipitation; KO: knockout; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; LMNA: lamin A/C; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MS: mass spectrometry; NBR1: NBR1 autophagy cargo receptor; NCOA4: nuclear receptor coactivator 4; NTA: nanoparticle tracking analysis; PE: phosphatidylethanolamine; PECA: probe-level expression change averaging; SDCBP/syntenin-1: syndecan binding protein; SD: standard deviation; SE: secreted; sEV: small extracellular vesicles; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TEM: transmission electron microscopy; TMT: tandem-mass tag; TSG101: tumor susceptibility 101; ULK1: unc-51 like autophagy activating kinase 1; WC: whole cell.

Exploring the role of extracellular vesicles and their protein cargo in lung cancer metastasis: A review.

Extracellular vesicles (EV) are membrane-enclosed structures of varying size released from all cells and contain a variety of cargo including proteins, lipids, and nucleic acids. They are postulated to play a pivotal role in cancer metastasis through delivery of oncogenic material to neighbouring and distant cells to promote development of a metastatic niche and tumour seeding. Here we reviewed protein data in relevant literature to determine whether specific proteins known to be involved in metastasis can be reliably identified in lung cancer EV, whether these proteins are important in all or specific lung cancers, and whether results from in-vitro cell studies are supported by research examining EV in human biofluids. Our analysis suggests that specific proteins may be more important for individual lung cancers, but interpretation of the literature is currently limited by a relative lack of research investigating EV proteins in some cancers and in clinical studies using biofluids.

Application of extracellular vesicles in the diagnosis and treatment of prostate cancer: Implications for clinical practice.

Extracellular vesicles (EV) are cell-derived lipid bilayer-delimited structures providing an important means of intercellular communication. Recent studies have shown that EV, particularly exosomes and large-oncosomes contain miRNA and proteins crucial in prostate cancer (PCa) progression, metastasis and treatment resistance. This includes not just EV released from PCa cells, but also from other cells in the tumor microenvironment. PCa patient derived EV have a unique composition compared to healthy and benign prostatic diseases. As such, EV show promise as diagnostic liquid biopsy biomarkers, both as an adjunct and alternative to the invasive current gold-standard. EV could also be utilized to stratify patients' risk and predict response to hormonal, chemo, immune- and targeted therapy, which will direct future treatment decisions in PCa. We present a summary of the current evidence on the role of EV in PCa and the application of EV in PCa diagnosis and treatment to optimize patient outcomes.

Extracellular Vesicles from Mesenchymal Stromal Cells for the Treatment of Inflammation-Related Conditions.

Over the past two decades, mesenchymal stromal cells (MSCs) have demonstrated great potential in the treatment of inflammation-related conditions. Numerous early stage clinical trials have suggested that this treatment strategy has potential to lead to significant improvements in clinical outcomes. While promising, there remain substantial regulatory hurdles, safety concerns, and logistical issues that need to be addressed before cell-based treatments can have widespread clinical impact. These drawbacks, along with research aimed at elucidating the mechanisms by which MSCs exert their therapeutic effects, have inspired the development of extracellular vesicles (EVs) as anti-inflammatory therapeutic agents. The use of MSC-derived EVs for treating inflammation-related conditions has shown therapeutic potential in both in vitro and small animal studies. This review will explore the current research landscape pertaining to the use of MSC-derived EVs as anti-inflammatory and pro-regenerative agents in a range of inflammation-related conditions: osteoarthritis, rheumatoid arthritis, Alzheimer's disease, cardiovascular disease, and preeclampsia. Along with this, the mechanisms by which MSC-derived EVs exert their beneficial effects on the damaged or degenerative tissues will be reviewed, giving insight into their therapeutic potential. Challenges and future perspectives on the use of MSC-derived EVs for the treatment of inflammation-related conditions will be discussed.

Extracellular vesicles as biomarkers in malignant pleural mesothelioma: A review.

Extracellular vesicles (EV) are secreted by all cells, including cancer cells, as a mode of intercellular transport and communication. The main types of EV known to date include exosomes, microvesicles and apoptotic bodies, as well as oncosomes and large oncosomes, which are specific to cancer cells. These different EV populations carry specific cargo from one cell to another to stimulate a specific response. They can be found in all body fluids and can be detected in liquid biopsies. EV released from mesothelioma cells can reveal important information about the molecules and signalling pathways involved in the development and progression of the tumour. The presence of tumour-derived EV in circulating body fluids makes them potential novel biomarkers for early diagnosis, prognostication and surveillance of cancer. In this review, we explore the characteristics and functional roles of EV reported in the literature, with a focus on their role in malignant pleural mesothelioma.

Retrospective Evaluation of the Use of Pembrolizumab in Malignant Mesothelioma in a Real-World Australian Population.

INTRODUCTION: We investigated the efficacy and toxicity of pembrolizumab in patients with mesothelioma from a real-world Australian population. We aimed to determine clinical factors and predictive biomarkers that could help select patients who are likely to benefit from pembrolizumab. METHOD: Patients with mesothelioma who were treated with pembrolizumab as part of the Insurance and Care New South Wales compensation scheme were included. Clinical information was collected retrospectively. Tumor biomarkers such as programmed death-ligand 1 (PD-L1), BAP1, and CD3-positive (CD3+) tumor-infiltrating lymphocytes (TILs) were examined using archival formalin-fixed paraffin-embedded tumor samples. RESULTS: A total of 98 patients were included with a median age of 70 years (range, 46-91 y); 92% were men; 76% had epithelioid subtype; 21% had an Eastern Cooperative Oncology Group (ECOG) performance status of 0. Pembrolizumab was used as second-line or subsequent-line treatment in 94 patients and as first-line treatment in four patients. The overall response rate was 18%, and the disease control rate was 56%. The median progression-free survival (PFS) was 4.8 months (95% confidence interval: 3.6-6.2), and the median overall survival (OS) was 9.5 months (95% confidence interval: 6.6-13.7). Immune-related adverse events occurred in 27% of patients, of which nine (9%) were of grade 3 or higher. In the multivariable analysis, factors independently associated with longer PFS included baseline ECOG status of 0 (median PFS: 12 mo versus 4 mo, p < 0.01) and PD-L1 tumor proportion score of greater than or equal to 1% (median PFS: 6 mo versus 4 mo, p < 0.01). Baseline platelet count of less than or equal to 400 × 109/liter was independently associated with longer PFS and OS (median PFS: 6 mo versus 2 mo, p = 0.05; median OS: 10 mo versus 4 mo, p = 0.01), whereas lack of pretreatment dexamethasone was independently associated with OS but not PFS (median OS: 10 mo versus 3 mo, p = 0.01). The odds of response were higher for patients with baseline ECOG status of 0 (p = 0.02) and with greater than or equal to 5% CD3+ TILs in the tumor (p < 0.01). PD-L1 expression, BAP1 loss, and CD3+ TILs in the stroma were not significantly associated with the overall response rate. CONCLUSIONS: Immunotherapy is a reasonable treatment option for patients with mesothelioma. Our results are comparable to other clinical trials investigating pembrolizumab in mesothelioma in terms of response. Good performance status assessment remains the most robust predictor for patient outcomes. CD3+ TILs in the tumor may help select patients that are likely to respond to pembrolizumab, whereas factors such as PD-L1 expression, baseline platelet count, and lack of pretreatment dexamethasone may help predict survival outcomes from pembrolizumab treatment.

Stem Cell-Derived Extracellular Vesicles for Treating Joint Injury and Osteoarthritis.

Extracellular vesicles (EVs) are nanoscale particles secreted by almost all cell types to facilitate intercellular communication. Stem cell-derived EVs theoretically have the same biological functions as stem cells, but offer the advantages of small size, low immunogenicity, and removal of issues such as low cell survival and unpredictable long-term behaviour associated with direct cell transplantation. They have been an area of intense interest in regenerative medicine, due to the potential to harness their anti-inflammatory and pro-regenerative effects to induce healing in a wide variety of tissues. However, the potential of using stem cell-derived EVs for treating joint injury and osteoarthritis has not yet been extensively explored. The pathogenesis of osteoarthritis, with or without prior joint injury, is not well understood, and there is a longstanding unmet clinical need to develop new treatments that provide a therapeutic effect in preventing or stopping joint degeneration, rather than merely relieving the symptoms of the disease. This review summarises the current evidence relating to stem cell-derived EVs in joint injury and osteoarthritis, providing a concise discussion of their characteristics, advantages, therapeutic effects, limitations and outlook in this exciting new area.

Extracellular Fatty Acids Are the Major Contributor to Lipid Synthesis in Prostate Cancer.

Prostate cancer cells exhibit altered cellular metabolism but, notably, not the hallmarks of Warburg metabolism. Prostate cancer cells exhibit increased de novo synthesis of fatty acids (FA); however, little is known about how extracellular FAs, such as those in the circulation, may support prostate cancer progression. Here, we show that increasing FA availability increased intracellular triacylglycerol content in cultured patient-derived tumor explants, LNCaP and C4-2B spheroids, a range of prostate cancer cells (LNCaP, C4-2B, 22Rv1, PC-3), and prostate epithelial cells (PNT1). Extracellular FAs are the major source (∼83%) of carbons to the total lipid pool in all cell lines, compared with glucose (∼13%) and glutamine (∼4%), and FA oxidation rates are greater in prostate cancer cells compared with PNT1 cells, which preferentially partitioned extracellular FAs into triacylglycerols. Because of the higher rates of FA oxidation in C4-2B cells, cells remained viable when challenged by the addition of palmitate to culture media and inhibition of mitochondrial FA oxidation sensitized C4-2B cells to palmitate-induced apoptosis. Whereas in PC-3 cells, palmitate induced apoptosis, which was prevented by pretreatment of PC-3 cells with FAs, and this protective effect required DGAT-1-mediated triacylglycerol synthesis. These outcomes highlight for the first-time heterogeneity of lipid metabolism in prostate cancer cells and the potential influence that obesity-associated dyslipidemia or host circulating has on prostate cancer progression. IMPLICATIONS: Extracellular-derived FAs are primary building blocks for complex lipids and heterogeneity in FA metabolism exists in prostate cancer that can influence tumor cell behavior.

Extracellular vesicles as mediators of immunopathology in infectious diseases.

In the last decades, extracellular vesicles have emerged as important elements in cell-cell communication and as key players in disease pathogenesis via transmission of their cargo between different cells. Various works have described different subpopulations of these membrane structures, based on their cell of origin, biogenesis, size, biophysical properties and cargo. In addition to their pathophysiological role in the development and progression of different diseases including infectious diseases, neurodegenerative disorders and cancer, extracellular vesicles are now recognized for their potential as novel therapeutic targets and intelligent drug delivery system. Here, we have reviewed the most recent data on different subtypes of extracellular vesicles, focusing on microvesicles and exosomes and their subpopulations, their involvement in immune-mediated pathogenesis of various infectious diseases and their role as potential therapeutic targets.

Correction: Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes.

[This corrects the article DOI: 10.1371/journal.pone.0154967.].

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.

Exosomes confer pro-survival signals to alter the phenotype of prostate cells in their surrounding environment.

Prostate cancer (PCa) is the most frequently diagnosed cancer in men. Current research on tumour-related extracellular vesicles (EVs) suggests that exosomes play a significant role in paracrine signaling pathways, thus potentially influencing cancer progression via multiple mechanisms. In fact, during the last decade numerous studies have revealed the role of EVs in the progression of various pathological conditions including cancer. Moreover, differences in the proteomic, lipidomic, and cholesterol content of exosomes derived from PCa cell lines versus benign prostate cell lines confirm that exosomes could be excellent biomarker candidates. As such, as part of an extensive proteomic analysis using LCMS we previously described a potential role of exosomes as biomarkers for PCa. Current evidence suggests that uptake of EV's into the local tumour microenvironment encouraging us to further examine the role of these vesicles in distinct mechanisms involved in the progression of PCa and castration resistant PCa. For the purpose of this study, we hypothesized that exosomes play a pivotal role in cell-cell communication in the local tumour microenvironment, conferring activation of numerous survival mechanisms during PCa progression and development of therapeutic resistance. Our in vitro results demonstrate that PCa derived exosomes significantly reduce apoptosis, increase cancer cell proliferation and induce cell migration in LNCaP and RWPE-1 cells. In conjunction with our in vitro findings, we have also demonstrated that exosomes increased tumor volume and serum PSA levels in vivo when xenograft bearing mice were administered DU145 cell derived exosomes intravenously. This research suggests that, regardless of androgen receptor phenotype, exosomes derived from PCa cells significantly enhance multiple mechanisms that contribute to PCa progression.

Large oncosomes contain distinct protein cargo and represent a separate functional class of tumor-derived extracellular vesicles.

Large oncosomes (LO) are atypically large (1-10 µm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep(TM)) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

Regulation of local steroidogenesis in the brain and in prostate cancer: lessons learned from interdisciplinary collaboration.

Sex steroids play critical roles in the regulation of the brain and many other organs. Traditionally, researchers have focused on sex steroid signaling that involves travel from the gonads via the circulation to intracellular receptors in target tissues. This classic concept has been challenged, however, by the growing number of cases in which steroids are synthesized locally and act locally within diverse tissues. For example, the brain and prostate carcinoma were previously considered targets of gonadal sex steroids, but under certain circumstances, these tissues can upregulate their steroidogenic potential, particularly when circulating sex steroid concentrations are low. We review some of the similarities and differences between local sex steroid synthesis in the brain and prostate cancer. We also share five lessons that we have learned during the course of our interdisciplinary collaboration, which brought together neuroendocrinologists and cancer biologists. These lessons have important implications for future research in both fields.

Next-generation steroidogenesis inhibitors, dutasteride and abiraterone, attenuate but still do not eliminate androgen biosynthesis in 22RV1 cells in vitro.

Castration resistant prostate cancer (CRPC) is often lethal and inevitably develops after androgen ablation therapy. However, in the majority of cases it remains androgen dependent. CRPC tumors have the ability to synthesize their own androgens from cholesterol by engaging in de novo steroidogenesis. We investigated the potential of 22RV1 prostate cancer cells to convert the supplemented steroid precursors within this pathway under the effects of current clinical steroidogenesis inhibitors such as abiraterone and dutasteride, either alone or in combination. Under steroid starved conditions, enzymes responsible for de novo steroidogenesis were upregulated. Testosterone and dihydrotestosterone (DHT) were formed by using both dehydroepiandrosterone (DHEA) and progesterone as substrates. Formation of testosterone and DHT was higher following incubation with DHEA compared to progesterone. Progesterone decreased the mRNA expression of enzymes responsible for steroidogenesis. Abiraterone treatment decreased testosterone production but increased several precursor steroids in both classical and backdoor pathways in the presence of progesterone. In contrast, the DHT levels were elevated following treatment with abiraterone when progesterone was absent. Dutasteride decreased the formation of testosterone, DHT and precursor steroids in the backdoor pathway but increased steroid precursors in the classical steroidogenesis pathway. The combination of abiraterone and dutasteride decreased testosterone and DHT in the presence of progesterone but increased DHT in the absence of progesterone. Abiraterone inhibited androgen receptor (AR) activation but not to the same extent as MDV3100. However, abiraterone and dutasteride treatment, either alone or in combination, were more effective in decreasing prostate specific antigen secretion into the media than MDV3100. Thus, while interventions with these drugs alone or in combination fail to completely inhibit steroidogenesis in the 22RV1 cells, the combined inhibition of androgen production and blockade of AR can exceed the effect of MDV3100. Further characterization of bypass mechanisms that may develop as a response to these inhibitors is necessary to achieve optimal suppression of testosterone and DHT synthesis as a part of therapeutic regimens for the treatment of CRPC.

Reproducibility and efficiency of serum-derived exosome extraction methods.

OBJECTIVES: Exosomes are emerging as a source of biomarkers with putative prognostic and diagnostic value. However, little is known about the efficiency, reproducibility and reliability of the protocols routinely used to quantify exosomes in the human serum. DESIGN AND METHODS: We used increasing amounts of the same serum sample to isolate exosomes using two different methods: ultracentrifugation onto a sucrose cushion and ExoQuick™. Quantitative analysis of serum-derived exosomes was performed by determining protein concentration (BCA assay) and the number of nanoparticles (Nanosight™ technology). Exosome quality was assessed by Coomassie staining and Western blotting for CD9, LAMP2 exosomal markers and a negative marker Grp94. RESULTS: Correlation between serum volume and the number of isolated exosomes is significant for both methods when exosomes are quantified using protein concentration. However, when the number of nanoparticles is used to quantify exosomes, ExoQuick™ is the only reproducible and efficient method. CD9, LAMP2 and Grp94 exosomal markers are equivalently expressed in both methods. However, exosomes isolated using ultracentrifuge method are strongly contaminated with albumin and IgG. CONCLUSION: ExoQuick™ is an efficient and reproducible method to isolate exosomes for quantitative studies, whereas ultracentrifugation is not. Moreover, high albumin contamination of ultracentrifuged-derived exosomes impairs the use of protein concentration as a mean to quantify serum-derived exosomes.