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.

IntroductionDendritic cells (DCs) are highly specialized to integrate diverse stimuli for the generation of antigen-specific immunity and lifelong immunologic memory. As immature cells, they are ubiquitously distributed in peripheral tissues where they serve as immune sensors, sampling foreign and self antigens. 1,2 Microbial antigens recognized by pattern recognition receptors, 3 and self molecules released by dead and dying cells, 4 trigger the migration of DCs. While en route to secondary lymphoid tissues, DCs up-regulate their expression of cell surface major histocompatibility complex (MHC) and costimulatory molecules and the secretion of cytokines and chemokines necessary for the initiation of adaptive immune responses. 1,2 The intimate and dynamic interactions that occur between DCs and cognate lymphocytes in secondary lymphoid tissues are equally specialized. It is now clear that T-cell activation requires a zone of adhesive, direct contact with antigen-presenting cells (APCs), popularly known as the immune synapse. 5 From the DC vantage, the formation of immune synapses involves cytoskeletal reorganization and the focal redistribution of antigen-MHCs and accessory molecules. [6][7][8][9] The immune synapse appears to maximize the physical interactions between DCs and lymphocytes and forms a stable platform for the recruitment and concentration of signaling molecules. 10,11 The immune synapse exhibits many similarities with classical neuronal synapses, including a requirement for cell-to-cell adhesion and close membrane apposition. In addition, triggering of cell surface receptors leads to intracellular signal transduction in both structures. 10,11 The tight synaptic cleft between neurons (20-40 nm) and the neuromuscular junction (ϳ100 nm) facilitates the rapid diffusion of neurotransmitters. 11 Although a relatively transient structure, the immune synapse also features a synaptic cleft (15-40 nm). 9,10,12 We therefore considered the possibility that the immune synapse may also support the regulated secretion of labile molecules that could contribute to signaling between DCs and lymphocytes.Langerhans cells, the archetypal DCs, are known to accumulate neurotransmitters. 13 ) is a classical neurotransmitter and vasoactive amine involved in signaling a variety of behavioral and physiologic states including appetite, sleep, mood, and pain. 14 Moreover, 5-HT has significant effects on inflammation and immunity. Mast cells and platelets both express the 5-HT-specific transporter (SERT), which enables them to sequester 5-HT from the microenvironment. In turn, this 5-HT is released in response to injury or inflammatory signals such as IgE complexes, complement components, and platelet-activating factor. 15,16 Exocytosed 5-HT regulates platelet aggregation and modulates leukocyte motility and airway hyperresponsiveness. 16,17 5-HT is also reported to modulate T-cell activation and differentiation. 16,18,19 The mechanisms by which T cells may encounter For personal use only. on May 12, 2018. by guest www.bloodjou...

Transient receptor potential melastatin 8 (TRPM8) and transient receptor potential vanilloid 1 (TRPV1) are ion channels that detect cold and hot sensations, respectively. Their activation depolarizes the peripheral nerve terminals resulting in action potentials that propagate to brain via the spinal cord. These receptors also play a significant role in synaptic transmission between dorsal root ganglion (DRG) and dorsal horn (DH) neurons. Here, we show that TRPM8 is functionally downregulated by activation of protein kinase C (PKC) resulting in inhibition of membrane currents and increases in intracellular Ca 2ϩ compared with upregulation of TRPV1 in cloned and native receptors. Bradykinin significantly downregulates TRPM8 via activation of PKC in DRG neurons. Activation of TRPM8 or TRPV1 at first sensory synapse between DRG and DH neurons leads to a robust increase in frequency of spontaneous/miniature EPSCs. PKC activation blunts TRPM8-and facilitates TRPV1-mediated synaptic transmission. Significantly, downregulation is attributable to PKC-mediated dephosphorylation of TRPM8 that could be reversed by phosphatase inhibitors. These findings suggest that inflammatory thermal hyperalgesia mediated by TRPV1 may be further aggravated by downregulation of TRPM8, because the latter could mediate the much needed cool/soothing sensation.

TRPV1, the archetypal member of the vanilloid TRP family, was initially identified as the receptor for capsaicin, the pungent ingredient in hot chili peppers. The receptor has a diverse tissue distribution, with high expression in sensory neurons. TRPV1 is a nonselective cation channel with significant permeability to calcium, protons, and large polyvalent cations. It is the most polymodal TRP channel, being activated by numerous stimuli, including heat, voltage, vanilloids, lipids, and protons/cations. TRPV1 acts as a molecular integrator of physical and chemical stimuli in peripheral nociceptor terminals and plays a critical role in thermal inflammatory hyperalgesia. In addition, TRPV1 may regulate a variety of physiological functions in different organ systems. Various second messenger systems regulate TRPV1 activity, predominantly by serine-threonine phosphorylation. In this review, we provide a concise summary of the information currently available about this channel.

Background:The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk.Methods:We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations.Results:In vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells.Conclusions:Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

BackgroundPrimary and secondary brain cancers are highly treatment resistant, and their marked angiogenesis attracts interest as a potential therapeutic target. Recent observations reveal that the microvascular endothelium of primary high-grade gliomas expresses prostate specific membrane antigen (PSMA). Breast cancers express PSMA and they frequently form secondary brain tumors. Hence we report here our pilot study addressing the feasibility of PSMA targeting in brain and metastatic breast tumors, by examining PSMA levels in all glioma grades (19 patients) and in breast cancer brain metastases (5 patients).MethodsTumor specimens were acquired from archival material and normal brain tissues from autopsies. Tissue were stained and probed for PSMA, and the expression levels imaged and quantified using automated hardware and software. PSMA staining intensities of glioma subtypes, breast tumors, and breast tumor brain metastases were compared statistically versus normals.ResultsNormal brain microvessels (4 autopsies) did not stain for PSMA, while a small proportion (<5%) of healthy neurons stained, and were surrounded by an intact blood brain barrier. Tumor microvessels of the highly angiogenic grade IV gliomas showed intense PSMA staining which varied between patients and was significantly higher (p < 0.05) than normal brain. Grade I gliomas showed moderate vessel staining, while grade II and III gliomas had no vessel staining, but a few (<2%) of the tumor cells stained. Both primary breast cancer tissues and the associated brain metastases exhibited vascular PSMA staining, although the intensity of staining was generally less for the metastatic lesions.ConclusionsOur results align with and extend previous data showing PSMA expression in blood vessels of gliomas and breast cancer brain metastases. These results provide a rationale for more comprehensive studies to explore PSMA targeted agents for treating secondary brain tumors with PSMA expressing vasculature. Moreover, given that PSMA participates in angiogenesis, cell signaling, tumor survival, and invasion, characterizing its expression may help guide later investigations of the poorly understood process of low grade glioma progression to glioblastoma.

Aim: Genomically matched trials in primary brain tumors (PBTs) require recent tumor sequencing. We evaluated whether circulating tumor DNA (ctDNA) could facilitate genomic interrogation in these patients. Methods: Data from 419 PBT patients tested clinically with a ctDNA NGS panel at a CLIA-certified laboratory were analyzed. Results: A total of 211 patients (50%) had ≥1 somatic alteration detected. Detection was highest in meningioma (59%) and gliobastoma (55%). Single nucleotide variants were detected in 61 genes, with amplifications detected in ERBB2, MET, EGFR and others. Conclusion: Contrary to previous studies with very low yields, we found half of PBT patients had detectable ctDNA with genomically targetable off-label or clinical trial options for almost 50%. For those PBT patients with detectable ctDNA, plasma cfDNA genomic analysis is a clinically viable option for identifying genomically driven therapy options.

Purpose Non-invasive drug biomarkers for the early assessment of tumor response can enable adaptive therapeutic decision-making and proof-of-concept studies for investigational drugs. Circulating tumor DNA (ctDNA) is released into the circulation by tumor cell turnover and has been shown to be detectable in urine. Experimental Design We tested the hypothesis that dynamic changes in epidermal growth factor receptor (EGFR) activating (exon 19del and L858R) and resistance (T790M) mutation levels detected in urine could inform tumor response within days of therapy for advanced non-small cell lung cancer (NSCLC) patients receiving osimertinib, a second line third generation anti-EGFR tyrosine kinase inhibitor. Results Eight of nine evaluable NSCLC patients had detectable T790M-mutant DNA fragments in pre-treatment baseline samples. Daily monitoring of mutations in urine indicated a pattern of intermittent spikes throughout week 1 suggesting apoptosis with an overall decrease in fragment numbers between baselines to day 7 preceding radiographic response assessed at 6-12 weeks. Conclusions These findings suggest drug-induced tumor apoptosis within days of initial dosing. Daily sampling of ctDNA may enable early assessment of patient response and proof-of-concept studies for drug development.