Comparative Analysis of Technologies for Quantifying Extracellular Vesicles (EVs) in Clinical Cerebrospinal Fluids (CSF)

Extracellular vesicles (EVs) have emerged as a promising biomarker platform for glioblastoma patients. However, the optimal method for quantitative assessment of EVs in clinical bio-fluid remains a point of contention. Multiple high-resolution platforms for quantitative EV analysis have emerged, including methods grounded in diffraction measurement of Brownian motion (NTA), tunable resistive pulse sensing (TRPS), vesicle flow cytometry (VFC), and transmission electron microscopy (TEM). Here we compared quantitative EV assessment using cerebrospinal fluids derived from glioblastoma patients using these methods. For EVs 150 nm in diameter), NTA consistently detected lower number of EVs relative to TRPS. These results unveil the strength and pitfalls of each quantitative method alone for assessing EVs derived from clinical cerebrospinal fluids and suggest that thoughtful synthesis of multi-platform quantitation will be required to guide meaningful clinical investigations

Genome-wide shRNA screen revealed integrated mitogenic signaling between dopamine receptor D2 (DRD2) and epidermal growth factor receptor (EGFR) in glioblastoma

Glioblastoma remains one of the deadliest of human cancers, with most patients succumbing to the disease within two years of diagnosis. The available data suggest that simultaneous inactivation of critical nodes within the glioblastoma molecular circuitry will be required for meaningful clinical efficacy. We conducted parallel genome-wide shRNA screens to identify such nodes and uncovered a number of G-Protein Coupled Receptor (GPCR) neurotransmitter pathways, including the Dopamine Receptor D2 (DRD2) signaling pathway. Supporting the importance of DRD2 in glioblastoma, DRD2 mRNA and protein expression were elevated in clinical glioblastoma specimens relative to matched non-neoplastic cerebrum. Treatment with independent si-/shRNAs against DRD2 or with DRD2 antagonists suppressed the growth of patient-derived glioblastoma lines both in vitro and in vivo. Importantly, glioblastoma lines derived from independent genetically engineered mouse models (GEMMs) were more sensitive to haloperidol, an FDA approved DRD2 antagonist, than the premalignant astrocyte lines by approximately an order of magnitude. The pro-proliferative effect of DRD2 was, in part, mediated through a GNAI2/Rap1/Ras/ERK signaling axis. Combined inhibition of DRD2 and Epidermal Growth Factor Receptor (EGFR) led to synergistic tumoricidal activity as well as ERK suppression in independent in vivo and in vitro glioblastoma models. Our results suggest combined EGFR and DRD2 inhibition as a promising strategy for glioblastoma treatment

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

Molecular Mechanism of Tryptophan-Dependent Transcriptional Regulation in Chlamydia trachomatis

Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis

Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid

BackgroundTumor specific genetic material can be detected in extracellular vesicles (EVs) isolated from blood, cerebrospinal fluid (CSF), and other biofluids of glioblastoma patients. As such, EVs have emerged as a promising platform for biomarker discovery. However, the optimal procedure to transport clinical EV samples remains poorly characterized.MethodsWe examined the stability of EVs isolated from CSF of glioblastoma patients that were stored under different conditions. EV recovery was determined by Nanoparticle tracking analysis, and qRT-PCR was performed to determine the levels of miRNAs.ResultsCSF EVs that were lyophilized and stored at room temperature (RT) for seven days exhibited a 37-43% reduction in EV number. This reduction was further associated with decreased abundance of representative miRNAs. In contrast, the EV number and morphology remained largely unchanged if CSF were stored at RT. Total RNA and representative miRNA levels were well-preserved under this condition for up to seven days. A single cycle of freezing and thawing did not significantly alter EV number, morphology, RNA content, or miRNA levels. However, incremental decreases in these parameters were observed after two cycles of freezing and thawing.ConclusionsThese results suggest that EVs in CSF are stable at RT for at least seven days. Repeated cycles of freezing/thawing should be avoided to minimize experimental artifacts

Prediction of conversion from mild cognitive impairment to dementia with neuronally derived blood exosome protein profile.

IntroductionLevels of Alzheimer's disease (AD)-related proteins in plasma neuronal derived exosomes (NDEs) were quantified to identify biomarkers for prediction and staging of mild cognitive impairment (MCI) and AD.MethodsPlasma exosomes were extracted, precipitated, and enriched for neuronal source by anti-L1CAM antibody absorption. NDEs were characterized by size (Nanosight) and shape (TEM) and extracted NDE protein biomarkers were quantified by ELISAs. Plasma NDE cargo was injected into normal mice, and results were characterized by immunohistochemistry to determine pathogenic potential.ResultsPlasma NDE levels of P-T181-tau, P-S396-tau, and Aβ1-42 were significantly higher, whereas those of neurogranin (NRGN) and the repressor element 1-silencing transcription factor (REST) were significantly lower in AD and MCI converting to AD (ADC) patients compared to cognitively normal controls (CNC) subjects and stable MCI patients. Mice injected with plasma NDEs from ADC patients displayed increased P-tau (PHF-1 antibody)-positive cells in the CA1 region of the hippocampus compared to plasma NDEs from CNC and stable MCI patients.ConclusionsAbnormal plasma NDE levels of P-tau, Aβ1-42, NRGN, and REST accurately predict conversion of MCI to AD dementia. Plasma NDEs from demented patients seeded tau aggregation and induced AD-like neuropathology in normal mouse CNS