Cerebrospinal Fluid Extracellular Vesicles Research Articles

Recommendations for reproducibility of cerebrospinal fluid extracellular vesicle studies.

Cerebrospinal fluid (CSF) is a clear, transparent fluid derived from blood plasma that protects the brain and spinal cord against mechanical shock, provides buoyancy, clears metabolic waste and transports extracellular components to remote sites in the brain. Given its contact with the brain and the spinal cord, CSF is the most informative biofluid for studies of the central nervous system (CNS). In addition to other components, CSF contains extracellular vesicles (EVs) that carry bioactive cargoes (e.g., lipids, nucleic acids, proteins), and that can have biological functions within and beyond the CNS. Thus, CSF EVs likely serve as both mediators of and contributors to communication in the CNS. Accordingly, their potential as biomarkers for CNS diseases has stimulated much excitement for and attention to CSF EV research. However, studies on CSF EVs present unique challenges relative to EV studies in other biofluids, including the invasive nature of CSF collection, limited CSF volumes and the low numbers of EVs in CSF as compared to plasma. Here, the objectives of the International Society for Extracellular Vesicles CSF Task Force are to promote the reproducibility of CSF EV studies by providing current reporting and best practices, and recommendations and reporting guidelines, for CSF EV studies. To accomplish this, we created and distributed a world-wide survey to ISEV members to assess methods considered 'best practices' for CSF EVs, then performed a detailed literature review for CSF EV publications that was used to curate methods and resources. Based on responses to the survey and curated information from publications, the CSF Task Force herein provides recommendations and reporting guidelines to promote the reproducibility of CSF EV studies in seven domains: (i) CSF Collection, Processing, and Storage; (ii) CSF EV Separation/Concentration; (iii) CSF EV Size and Number Measurements; (iv) CSF EV Protein Studies; (v) CSF EV RNA Studies; (vi) CSF EV Omics Studies and (vii) CSF EV Functional Studies.

Viral Immune signatures from cerebrospinal fluid extracellular vesicles and particles in HAM and other chronic neurological diseases.

Extracellular vesicles and particles (EVPs) are released from virtually all cell types, and may package many inflammatory factors and, in the case of infection, viral components. As such, EVPs can play not only a direct role in the development and progression of disease but can also be used as biomarkers. Here, we characterized immune signatures of EVPs from the cerebrospinal fluid (CSF) of individuals with HTLV-1-associated myelopathy (HAM), other chronic neurologic diseases, and healthy volunteers (HVs) to determine potential indicators of viral involvement and mechanisms of disease. We analyzed the EVPs from the CSF of HVs, individuals with HAM, HTLV-1-infected asymptomatic carriers (ACs), and from patients with a variety of chronic neurologic diseases of both known viral and non-viral etiologies to investigate the surface repertoires of CSF EVPs during disease. Significant increases in CD8+ and CD2+ EVPs were found in HAM patient CSF samples compared to other clinical groups (p = 0.0002 and p = 0.0003 compared to HVs, respectively, and p = 0.001 and p = 0.0228 compared to MS, respectively), consistent with the immunopathologically-mediated disease associated with CD8+ T-cells in the central nervous system (CNS) of HAM patients. Furthermore, CD8+ (p < 0.0001), CD2+ (p < 0.0001), CD44+ (p = 0.0176), and CD40+ (p = 0.0413) EVP signals were significantly increased in the CSF from individuals with viral infections compared to those without. These data suggest that CD8+ and CD2+ CSF EVPs may be important as: 1) potential biomarkers and indicators of disease pathways for viral-mediated neurological diseases, particularly HAM, and 2) as possible meditators of the disease process in infected individuals.

Towards optimised extracellular vesicle proteomics from cerebrospinal fluid

The proteomic profile of extracellular vesicles (EVs) from cerebrospinal fluid (CSF) can reveal novel biomarkers for diseases of the brain. Here, we validate an ultrafiltration combined with size-exclusion chromatography (UF-SEC) method for isolation of EVs from canine CSF and probe the effect of starting volume on the EV proteomics profile. First, we performed a literature review of CSF EV articles to define the current state of art, discovering a need for basic characterisation of CSF EVs. Secondly, we isolated EVs from CSF by UF-SEC and characterised the SEC fractions by protein amount, particle count, transmission electron microscopy, and immunoblotting. Data are presented as mean ± standard deviation. Using proteomics, SEC fractions 3–5 were compared and enrichment of EV markers in fraction 3 was detected, whereas fractions 4–5 contained more apolipoproteins. Lastly, we compared starting volumes of pooled CSF (6 ml, 3 ml, 1 ml, and 0.5 ml) to evaluate the effect on the proteomic profile. Even with a 0.5 ml starting volume, 743 ± 77 or 345 ± 88 proteins were identified depending on whether ‘matches between runs’ was active in MaxQuant. The results confirm that UF-SEC effectively isolates CSF EVs and that EV proteomic analysis can be performed from 0.5 ml of canine CSF.

C1q is increased in cerebrospinal fluid-derived extracellular vesicles in Alzheimer's disease: A multi-cohort proteomics and immuno-assay validation study.

Extracellular vesicles (EVs) may propagate and modulate Alzheimer's disease (AD) pathology. We aimed to comprehensively characterize the proteome of cerebrospinal fluid (CSF) EVs to identify proteins and pathways altered in AD. CSF EVs were isolated by ultracentrifugation (Cohort 1) or Vn96 peptide (Cohort 2) fromnon-neurodegenerative controls (n=15, 16) and AD patients (n=22, 20, respectively). EVs were subjected to untargeted quantitative mass spectrometry-based proteomics. Results were validated by enzyme-linked immunosorbent assay (ELISA) in Cohorts 3 and 4, consisting of controls (n=16, n=43, (Cohort3, Cohort4)), and patients with AD (n=24, n=100). We found>30 differentially expressed proteins in AD CSF EVs involved in immune-regulation. Increase of C1q levels in AD compared to non-demented controls was validated by ELISA(∼ 1.5 fold, p (Cohort 3)=0.03, p (Cohort 4)=0.005). EVs may be utilized as a potential biomarker and may play a so far unprecedented role in immune-regulation in AD.

Phosphoproteome analysis of cerebrospinal fluid extracellular vesicles in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare but highly aggressive extra-nodal non-Hodgkin's lymphoma, mostly of the diffuse large B-cell lymphoma (DLBCL) type. The present invasive diagnosis and poor prognosis of PCNSL propose an urgent need to develop molecular markers for early detection, real-time monitoring and treatment evaluation. Cerebrospinal fluid (CSF)-derived extracellular vesicles (EVs) are promising biomarker carriers for liquid biopsy of CNS diseases and brain tumors; however, research remains challenging due to the low concentration of EVs in the limited available volume of CSF from each individual patient and the low efficiency of existing methods for EV enrichment. Here, we introduce functionalized magnetic beads called EVTRAP (extracellular vesicles total recovery and purification) for rapid and efficient EV isolation from CSF. By coupling with high-performance mass spectrometry, over 19 000 peptides representing 1841 proteins were identified from just 30 μL of CSF. Furthermore, up to 3000 phosphopeptides representing over 1000 phosphoproteins were identified from about 2 mL of CSF. Finally, we analyzed the EV phosphoproteomics of CSF samples from PCNSL patients and non-PCNSL controls. Among them, multiple phosphoproteins related to PCNSL, including SPP1, MARCKS, NPM1 and VIM, were shown to be up-regulated in the PCNSL group. These results demonstrated the feasibility of the EVTRAP-based analytical strategy in CSF EV phosphoproteomic analysis of PCNSL molecular markers.

Differential Effects of APOE Genotype on MicroRNA Cargo of Cerebrospinal Fluid Extracellular Vesicles in Females With Alzheimer's Disease Compared to Males.

Multiple biological factors, including age, sex, and genetics, influence Alzheimer’s disease (AD) risk. Of the 6.2 million Americans living with Alzheimer’s dementia in 2021, 3.8 million are women and 2.4 million are men. The strongest genetic risk factor for sporadic AD is apolipoprotein E-e4 (APOE-e4). Female APOE-e4 carriers develop AD more frequently than age-matched males and have more brain atrophy and memory loss. Consequently, biomarkers that are sensitive to biological risk factors may improve AD diagnostics and may provide insight into underlying mechanistic changes that could drive disease progression. Here, we have assessed the effects of sex and APOE-e4 on the miRNA cargo of cerebrospinal fluid (CSF) extracellular vesicles (EVs) in AD. We used ultrafiltration (UF) combined with size exclusion chromatography (SEC) to enrich CSF EVs (e.g., Flotillin+). CSF EVs were isolated from female and male AD or controls (CTLs) that were either APOE-e3,4 or -e3,3 positive (n = 7/group, 56 total). MiRNA expression levels were quantified using a custom TaqMan™ array that assayed 190 miRNAs previously found in CSF, including 25 miRNAs that we previously validated as candidate AD biomarkers. We identified changes in the EV miRNA cargo that were affected by both AD and sex. In total, four miRNAs (miR-16-5p, -331-3p, -409-3p, and -454-3p) were significantly increased in AD vs. CTL, independent of sex and APOE-e4 status. Pathway analysis of the predicted gene targets of these four miRNAs with identified pathways was highly relevant to neurodegeneration (e.g., senescence and autophagy). There were also three miRNAs (miR-146b-5p, -150-5p, and -342-3p) that were significantly increased in females vs. males, independent of disease state and APOE-e4 status. We then performed a statistical analysis to assess the effect of APOE genotype in AD within each sex and found that APOE-e4 status affects different subsets of CSF EV miRNAs in females vs. males. Together, this study demonstrates the complexity of the biological factors associated with AD risk and the impact on EV miRNAs, which may contribute to AD pathophysiology.

Cerebrospinal Fluid EV Concentration and Size Are Altered in Alzheimer's Disease and Dementia with Lewy Bodies.

Alzheimer’s disease (AD), dementia with Lewy bodies (DLB) and frontotemporal dementia (FTD) represent the three major neurodegenerative dementias characterized by abnormal brain protein accumulation. In this study, we investigated extracellular vesicles (EVs) and neurotrophic factors in the cerebrospinal fluid (CSF) of 120 subjects: 36 with AD, 30 with DLB, 34 with FTD and 20 controls. Specifically, CSF EVs were analyzed by Nanoparticle Tracking Analysis and neurotrophic factors were measured with ELISA. We found higher EV concentration and lower EV size in AD and DLB groups compared to the controls. Classification tree analysis demonstrated EV size as the best parameter able to discriminate the patients from the controls (96.7% vs. 3.3%, respectively). The diagnostic performance of the EV concentration/size ratio resulted in a fair discrimination level with an area under the curve of 0.74. Moreover, the EV concentration/size ratio was associated with the p-Tau181/Aβ42 ratio in AD patients. In addition, we described altered levels of cystatin C and progranulin in the DLB and AD groups. We did not find any correlation between neurotrophic factors and EV parameters. In conclusion, the results of this study suggest a common involvement of the endosomal pathway in neurodegenerative dementias, giving important insight into the molecular mechanisms underlying these pathologies.

Molecular Signature of Extracellular Vesicular Small Non-Coding RNAs Derived from Cerebrospinal Fluid of Leptomeningeal Metastasis Patients: Functional Implication of miR-21 and Other Small RNAs in Cancer Malignancy.

Simple SummaryLeptomeningeal metastasis (LM) is a lethal complication in which cancer metastasizes to the meninges. Currently, there are neither definitive treatments nor diagnosis methods for LM patients. In this study, we suggest the examination of small non-coding RNA (smRNA) populations of extracellular vesicles (EVs) derived from the cerebrospinal fluid (CSF) as a potential vehicle for diagnosis and treatment strategies. Systemic and quantitative analysis of smRNA subpopulations from LM CSF EVs showed unique expression patterns between LM patients and healthy donors. In addition, LM CSF EVs smRNAs appeared to be associated with LM pathogenesis suggesting they may be viable targets for novel diagnostic and treatment strategies.Leptomeningeal metastasis (LM) is a fatal and rare complication of cancer in which the cancer spreads via the cerebrospinal fluid (CSF). At present, there is no definitive treatment or diagnosis for this deleterious disease. In this study, we systemically and quantitatively investigated biased expression of key small non-coding RNA (smRNA) subpopulations from LM CSF extracellular vesicles (EVs) via a unique smRNA sequencing method. The analyzed subpopulations included microRNA (miRNA), Piwi-interacting RNA (piRNA), Y RNA, small nuclear RNA (snRNA), small nucleolar RNAs (snoRNA), vault RNA (vtRNA), novel miRNA, etc. Here, among identified miRNAs, miR-21, which was already known to play an essential oncogenic role in tumorigenesis, was thoroughly investigated via systemic biochemical, miR-21 sensor, and physiological cell-based approaches, with the goal of confirming its functionality and potential as a biomarker for the pathogenesis and diagnosis of LM. We herein uncovered LM CSF extravesicular smRNAs that may be associated with LM-related complications and elucidated plausible pathways that may mechanistically contribute to LM progression. In sum, the analyzed smRNA subpopulations will be useful as targets for the development of therapeutic and diagnostic strategies for LM and LM-related complications.

BIOM-09. MULTIPLEX ANALYSIS OF CSF EXTRACELLULAR VESICLES OF INTRASPINAL TUMORS

Abstract BACKGROUND: Extracellular vesicles (EVs) play an important role in cell-cell communication in different types of tumors, carrying multiple layers of biological functional molecules, including proteins, RNA, DNA and lipids. We previously demonstrated that extracellular vesicles (EV) from central nervous system tumors reflect the molecular subtype of the original tumor and mediate an exchange of pro-oncogenic signals. Their implication as biomarkers in tumor disease is under current investigation. It is unclear, however, to what extent cerebrospinal fluid (CSF) EVs from intraspinal tumors are utilizable for diagnostical purposes and how their marker profiles overlap with EVs derived from non tumorous EVs. We analyzed CSF EVs of intraspinal tumors to define CSF EV profiles that allow tumor subtype classification. METHODS: EVs were isolated from CSF of patients suffering from intraspinal meningioma (n=5), ependymoma (n=7) and neurinoma (n=5). Patients suffering from normal pressure hydrocephalus were used as controls (n=5). EVs were analyzed by multiplex bead based assay, immunoblotting, electron microscopy and NTA. RESULTS: CSF EVs were 97.21 ± 3.37nm (intraspinal tumor patients) and 101.6 ± 3.68nm (controls) in sizes and showed vesicular structures by electron microscopy. Particle number were not significantly different between both groups (p = 0.103). Using our 37 protein mutliplex EV profiling kit we found 29 proteins to be expressed in a sufficient manner on CSF EVs. CSF EVs of intraspinal meningioma showed elevated CD62P, HLA-DR, CD40, CD42a and CD45 expression levels, while ependymoma showed decreased levels of CD9, CD63, CD81, whereas neurinomas had elevated levels of SSEA-3 and CD25. CONCLUSION: This is the first comprehensive analysis of CSF EV of intraspinal tumor patients. CSF EV display distinct subpopulations that may allow tumor classification and long-term surveillance. However as tumor-specific EVs may be rare, there is still the need to identify markers that can enrich tumor-specific EVs for molecular profiling.

Proteomic Profiling of Extracellular Vesicles Derived from Cerebrospinal Fluid of Alzheimer's Disease Patients: A Pilot Study.

Pathological hallmarks of Alzheimer’s disease (AD) are deposits of amyloid beta (Aβ) and hyper-phosphorylated tau aggregates in brain plaques. Recent studies have highlighted the importance of Aβ and tau-containing extracellular vesicles (EVs) in AD. We therefore examined EVs separated from cerebrospinal fluid (CSF) of AD, mild cognitive impairment (MCI), and control (CTRL) patient samples to profile the protein composition of CSF EV. EV fractions were separated from AD (n = 13), MCI (n = 10), and CTRL (n = 10) CSF samples using MagCapture Exosome Isolation kit. The CSF-derived EV proteins were identified and quantified by label-free and tandem mass tag (TMT)-labeled mass spectrometry. Label-free proteomics analysis identified 2546 proteins that were significantly enriched for extracellular exosome ontology by Gene Ontology analysis. Canonical Pathway Analysis revealed glia-related signaling. Quantitative proteomics analysis, moreover, showed that EVs expressed 1284 unique proteins in AD, MCI and CTRL groups. Statistical analysis identified three proteins—HSPA1A, NPEPPS, and PTGFRN—involved in AD progression. In addition, the PTGFRN showed a moderate correlation with amyloid plaque (rho = 0.404, p = 0.027) and tangle scores (rho = 0.500, p = 0.005) in AD, MCI and CTRL. Based on the CSF EV proteomics, these data indicate that three proteins, HSPA1A, NPEPPS and PTGFRN, may be used to monitor the progression of MCI to AD.

Proteomic analysis of cerebrospinal fluid extracellular vesicles reveals synaptic injury, inflammation, and stress response markers in HIV patients with cognitive impairment

BackgroundExtracellular vesicles (EVs) are nano-sized particles present in most body fluids including cerebrospinal fluid (CSF). Little is known about CSF EV proteins in HIV+ individuals. Here, we characterize the CSF EV proteome in HIV+ subjects and its relationship to neuroinflammation, stress responses, and HIV-associated neurocognitive disorders (HAND).MethodsCSF EVs isolated from 20 HIV+ subjects with (n = 10) or without (n = 10) cognitive impairment were characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting, and untargeted LC/MS/MS mass spectrometry. Functional annotation was performed by gene ontology (GO) mapping and expression annotation using Biobase Transfac and PANTHER software. Cultured astrocytic U87 cells were treated with hydrogen peroxide for 4 h to induce oxidative stress and EVs isolated by ultracentrifugation. Selected markers of astrocytes (GFAP, GLUL), inflammation (CRP), and stress responses (PRDX2, PARK7, HSP70) were evaluated in EVs released by U87 cells following induction of oxidative stress and in CSF EVs from HIV+ patients by immunoblotting.ResultsMass spectrometry identified 2727 and 1626 proteins in EV fractions and EV-depleted CSF samples, respectively. CSF EV fractions were enriched with exosomal markers including Alix, syntenin, tetraspanins, and heat-shock proteins and a subset of neuronal, astrocyte, oligodendrocyte, and choroid plexus markers, in comparison to EV-depleted CSF. Proteins related to synapses, immune/inflammatory responses, stress responses, metabolic processes, mitochondrial functions, and blood-brain barrier were also identified in CSF EV fractions by GO mapping. HAND subjects had higher abundance of CSF EVs and proteins mapping to GO terms for synapses, glial cells, inflammation, and stress responses compared to those without HAND. GFAP, GLUL, CRP, PRDX2, PARK7, and HSP70 were confirmed by immunoblotting of CSF EVs from subjects with HAND and were also detected in EVs released by U87 cells under oxidative stress.ConclusionsThese findings suggest that CSF EVs derived from neurons, glial cells, and choroid plexus carry synaptic, immune/inflammation-related, and stress response proteins in HIV+ individuals with cognitive impairment, representing a valuable source for biomarker discovery.

Cerebrospinal fluid extracellular vesicles and neurofilament light protein as biomarkers of central nervous system injury in HIV-infected patients on antiretroviral therapy.

Objective:The relationship of cerebrospinal fluid (CSF) extracellular vesicles to neurocognitive impairment (NCI) in HIV-infected individuals is unclear. Here, we characterize CSF extracellular vesicles and their association with central nervous system (CNS) injury related biomarkers [neurofilament light (NFL), S100B, neopterin] and NCI in HIV-positive individuals on combination antiretroviral therapy (cART).Design:A cross-sectional and longitudinal study of CSF samples from HIV-positive individuals on cART.Methods:NFL, S100B and neopterin were measured by ELISA in 190 CSF samples from 112 individuals (67 HIV-positive and 45 HIV-negative). CSF extracellular vesicles were isolated and characterized by electron microscopy, nanoparticle tracking analysis, immunoblotting for exosome markers (CD9, CD63, CD81, FLOT-1) and ELISA for HLA-DR.Results:HIV-positive individuals had median age 52 years, 67% with suppressed plasma viral load (< 50 copies/ml), median CD4+ nadir 66 cells/μl and CD4+ cell count 313 cells/μl. CSF NFL, S100B and neopterin levels were higher in HIV-positive vs. HIV-negative individuals, and nonsuppressed vs. suppressed HIV-positive individuals. Although CSF NFL and S100B levels were higher in NCI vs. unimpaired HIV-positive individuals (P < 0.05), only NFL was associated with NCI in adjusted models (P < 0.05). CSF extracellular vesicles were increased in HIV-positive vs. HIV-negative individuals, and NCI vs. unimpaired HIV-positive individuals (P < 0.05), and correlated positively with NFL (P < 0.001). HLA-DR was enriched in CSF extracellular vesicles from HIV-positive individuals with NCI (P < 0.05), suggesting that myeloid cells are a potential source of CSF extracellular vesicles during HIV infection.Conclusion:Increased CSF extracellular vesicles correlate with neuronal injury biomarker NFL in cART-treated HIV-positive individuals with neurocognitive impairment, suggesting potential applications as novel biomarkers of CNS injury.

Sexual dimorphism in inflammasome-containing extracellular vesicles and the regulation of innate immunity in the brain of reproductive senescent females

A woman's risk for stroke increases exponentially following the onset of menopause; however, the underlying mechanisms responsible for the increased risk remain unknown. The depletion of endogenous estrogen at menopause is known to activate the inflammatory response. Therefore, in this study we have used reproductively senescent (RS) rats to test the hypotheses that (1) inflammasome activation is significantly higher in the brain of RS females (RSF) as compared to their younger counterparts and age-matched senescent male rats, and that (2) RS triggers an innate immune response mediated in part by inflammasome-containing extracellular vesicles (EV) that originate in the female reproductive organs and then spreads to the brain. We tested these hypotheses using male and female Sprague–Dawley rats (Young: 6–7 months and RS: 9–13 months). Hippocampus, gonads and serum were collected. Additionally, cerebrospinal fluid (CSF) of pre- and post-menopausal women (ages 23 to 37 and 52 to 68) was purchased and extracellular vesicles (EV) were isolated from serum and CSF. The Inflammasome proteins caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and IL-1β were then resolved by immunoblotting. We found that inflammasome protein expression increased significantly in the analyzed tissues in RSF as compared to young females (YF), such difference was not present in age-matched male rat brains. Interestingly, we found that Nik-related kinase (NRK), which is present in female reproductive organs was present in the CSF and serum-derived EV, suggesting that the source of the EV seen in the brain during RS/menopause originate, in part, in the female reproductive organs. Thus, this study shows for the first time an involvement of the inflammasome originating in the female reproductive system as a contributor to inflammation in the brain that makes the peri-menopausal women's brain more susceptible to neurodegenerative diseases such as stroke.

A cerebrospinal fluid microRNA signature as biomarker for glioblastoma.

PurposeTo develop a cerebrospinal fluid (CSF) miRNA diagnostic biomarker for glioblastoma.Experimental DesignGlioblastoma tissue and matched CSF from the same patient (obtained prior to tumor manipulation) were profiled by TaqMan OpenArray® Human MicroRNA Panel. CSF miRNA profiles from glioblastoma patients and controls were created from three discovery cohorts and confirmed in two validation cohorts.ResultsmiRNA profiles from clinical CSF correlated with those found in glioblastoma tissues. Comparison of CSF miRNA profiles between glioblastoma patients and non-brain tumor patients yielded a tumor “signature” consisting of nine miRNAs. The “signature” correlated with glioblastoma tumor volume (p=0.008). When prospectively applied to cisternal CSF, the sensitivity and specificity of the ‘signature’ for glioblastoma detection were 67% and 80%, respectively. For lumbar CSF, the sensitivity and specificity of the signature were 28% and 95%, respectively. Comparable results were obtained from analyses of CSF extracellular vesicles (EVs) and crude CSF.ConclusionWe report a CSF miRNA signature as a “liquid biopsy” diagnostic platform for glioblastoma.

Cerebrospinal fluid extracellular vesicles undergo age dependent declines and contain known and novel non-coding RNAs.

Brain development requires precise orchestration of cellular events through the coordinate exchange of information between distally located cells. One mechanism by which intercellular communication is achieved is through the transfer of extracellular vesicles (EVs). Exosomes are EVs that carry lipids, nucleic acids, and proteins and are detectable in most biological fluids including cerebrospinal fluid (CSF). Here we report that CSF EV concentrations undergo age dependent fluctuations. We characterized EV RNA content by next generation small RNA sequencing and miRNA microarray analysis and identified a temporal shift in CSF EV content. CSF EVs encapsulated miRNAs that contain a conserved hnRNPA2/B1 recognition sequence. We found that hnRNPA2/B1-containing EVs were produced by choroid plexus epithelial cells and that hnRNPA2/B1 containing EVs decreased with age. These results provide insight into EV exchange of miRNAs within the central nervous system and a framework to understand how changes in EVs may have an important impact on brain development.

Abstract 5316: Quantitative cancer analysis using digital PCR: Absolute counting of DNA (solid tumors and liquid biopsies in glioma, breast, and colon cancer) and RNA (mRNA and miRNA using One-Step RT-dPCR)

Abstract Background: There is a need for more accurate and sensitive methods to quantify nucleic acids present in precious samples and non-invasively collected “liquid biopsy” material. Digital PCR (dPCR) is one technology with recent advances in automation, enabling researchers to sensitively quantify a sample's nucleic acids by adding up digital fluorescent counts of single target molecules. Particularly exciting are the emerging uses of cell-free nucleic acids found in peripheral body fluids for dynamic measurements of total body mutational burden of heterogeneous cancers, potentially reducing the need for multiple invasive tumor biopsies. In addition, combined measurement of mRNA and miRNA RNA biomarkers from a single sample, for example from cell-free exosomes, is of clinical interest. Here we present results from several studies using dPCR for DNA and cDNA molecule counting with various cancers, as well as in a first time demonstration quantifying RNA directly with multiple miRNA species, and mRNA together with miRNA. Method &amp; Results: Picoliter droplet digital PCR was used to perform single molecule counting. Standard qPCR assay reagents for each target were combined in duplex or multiplex formats together with samples, and partitioned into millions of 5 picoliter-sized droplets on a RainDance RainDropTM dPCR system. Each sample was then thermal cycled prior to digital fluorescent quantification. In separate studies we show quantification of mutant IDH1 mRNA (cDNA converted) in glioma patient cerebrospinal fluid extracellular vesicles, PIK3CA mutations and methylation of CyclinB2 and Retinoic Acid Receptor promoters in breast tumor DNA, KRAS and BRAF mutations in colorectal cancer plasma cell-free DNA, and multiplexed miRNA biomarkers from plasma (cDNA converted). In addition, we show direct quantification of mRNA molecules in highly precise multiplex One-Step RT-dPCR measurements across a wide dynamic range. Finally, we show simultaneous One-Step RT dPCR direct digital counting of miRNA and mRNA molecules. Conclusion: Droplet digital PCR is a highly sensitive and precise approach to quantify nucleic acids without the need for standard curves. With the RainDance RainDrop dPCR platform there are sufficient partitions and compatibility with various robust chemistries to apply dPCR to DNA and RNA analyses from a variety of biofluids and neoplasms, enabling accurate absolute counting of multiple target molecules across a wide dynamic range. Citation Format: Michael L. Samuels, Valerie Taly, Leonora Balaj, Saumya Das, Ben Ho Park. Quantitative cancer analysis using digital PCR: Absolute counting of DNA (solid tumors and liquid biopsies in glioma, breast, and colon cancer) and RNA (mRNA and miRNA using One-Step RT-dPCR). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5316. doi:10.1158/1538-7445.AM2014-5316

Proteomic analysis of cerebrospinal fluid extracellular vesicles: a comprehensive dataset.

Extracellular vesicles (EVs) are present in human cerebrospinal fluid (CSF), yet little is known about their protein composition. The aim of this study is to provide a comprehensive analysis of the proteome of CSF EVs by electron microscopy and high resolution tandem mass spectrometry (MS/MS) in conjunction with bioinformatics. We report an extensive catalog of 1315 proteins identified in EVs isolated from two different CSF pools by ultracentrifugation, including 230 novel EV proteins. Out of 1315 proteins, 760 were identified in both CSF pools and about 30% of those were also quantitatively enriched in the EV fraction versus the soluble CSF fraction. The proteome of CSF EVs was enriched in exosomal markers such as alix and syntenin-1, heat shock proteins and tetraspanins and contained a high proportion of brain-derived proteins (n=373). Interestingly, several known biomarkers for neurodegenerative diseases such as the amyloid precursor protein, the prion protein and DJ-1 were identified in the EV fractions. Our dataset represents the first comprehensive inventory of the EV proteome in CSF, underscoring the biomarker potential of this organelle. Further comparative studies on CSF EVs isolated from patients diagnosed with neurological disorders are warranted. Data are available via ProteomeXchange with identifier PXD000608. Biological significance In this study we analyzed the protein composition of extracellular vesicles isolated from pooled samples of human cerebrospinal fluid (CSF). CSF is a colorless fluid surrounding the brain and the spinal cord, important for the physiology of the central nervous system, ensuing mechanical protection, regulation of brain blood flow and elimination of byproducts of the brain. Since brain (patho)physiology is reflected in CSF, this biological fluid represents an ideal source of soluble and vesicle-based biomarkers for neurological diseases. Here we confirm the presence of exosome-like extracellular vesicles in CSF, underscoring a potential role in the physiology of the brain. These extracellular vesicles provide a rich source of candidate biomarkers, representing a brain "fluid biopsy". Most interestingly, the involvement of extracellular vesicles in transferring toxic proteins such as α-synuclein and β-amyloid has been postulated as one of the mechanisms involved in the spreading of neurodegeneration to different brain areas. In line with this, we show that human CSF extracellular vesicles contain prionogenic proteins such as the amyloid precursor protein and the prion protein. Delineating the protein composition of extracellular vesicles in CSF is a first and crucial step to comprehend their origin and their function in the central nervous system and to establish their biomarker potential.

MiR-21 in the extracellular vesicles (EVs) of cerebrospinal fluid (CSF): a platform for glioblastoma biomarker development.

Glioblastoma cells secrete extra-cellular vesicles (EVs) containing microRNAs (miRNAs). Analysis of these EV miRNAs in the bio-fluids of afflicted patients represents a potential platform for biomarker development. However, the analytic algorithm for quantitative assessment of EV miRNA remains under-developed. Here, we demonstrate that the reference transcripts commonly used for quantitative PCR (including GAPDH, 18S rRNA, and hsa-miR-103) were unreliable for assessing EV miRNA. In this context, we quantitated EV miRNA in absolute terms and normalized this value to the input EV number. Using this method, we examined the abundance of miR-21, a highly over-expressed miRNA in glioblastomas, in EVs. In a panel of glioblastoma cell lines, the cellular levels of miR-21 correlated with EV miR-21 levels (p<0.05), suggesting that glioblastoma cells actively secrete EVs containing miR-21. Consistent with this hypothesis, the CSF EV miR-21 levels of glioblastoma patients (n=13) were, on average, ten-fold higher than levels in EVs isolated from the CSF of non-oncologic patients (n=13, p<0.001). Notably, none of the glioblastoma CSF harbored EV miR-21 level below 0.25 copies per EV in this cohort. Using this cut-off value, we were able to prospectively distinguish CSF derived from glioblastoma and non-oncologic patients in an independent cohort of twenty-nine patients (Sensitivity=87%; Specificity=93%; AUC=0.91, p<0.01). Our results suggest that CSF EV miRNA analysis of miR-21 may serve as a platform for glioblastoma biomarker development.