Johnny C. Akers

Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies

Recent studies suggest both normal and cancerous cells secrete vesicles into the extracellular space. These extracellular vesicles (EVs) contain materials that mirror the genetic and proteomic content of the secreting cell. The identification of cancer-specific material in EVs isolated from the biofluids (e.g., serum, cerebrospinal fluid, urine) of cancer patients suggests EVs as an attractive platform for biomarker development. It is important to recognize that the EVs derived from clinical samples are likely highly heterogeneous in make-up and arose from diverse sets of biologic processes. This article aims to review the biologic processes that give rise to various types of EVs, including exosomes, microvesicles, retrovirus like particles, and apoptotic bodies. Clinical pertinence of these EVs to neuro-oncology will also be discussed.

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.

RNA-seq of 272 gliomas revealed a novel, recurrent PTPRZ1-MET fusion transcript in secondary glioblastomas

Studies of gene rearrangements and the consequent oncogenic fusion proteins have laid the foundation for targeted cancer therapy. To identify oncogenic fusions associated with glioma progression, we catalogued fusion transcripts by RNA-seq of 272 gliomas. Fusion transcripts were more frequently found in high-grade gliomas, in the classical subtype of gliomas, and in gliomas treated with radiation/temozolomide. Sixty-seven in-frame fusion transcripts were identified, including three recurrent fusion transcripts: FGFR3-TACC3, RNF213-SLC26A11, and PTPRZ1-MET (ZM). Interestingly, the ZM fusion was found only in grade III astrocytomas (1/13; 7.7%) or secondary GBMs (sGBMs, 3/20; 15.0%). In an independent cohort of sGBMs, the ZM fusion was found in three of 20 (15%) specimens. Genomic analysis revealed that the fusion arose from translocation events involving introns 3 or 8 of PTPRZ and intron 1 of MET. ZM fusion transcripts were found in GBMs irrespective of isocitrate dehydrogenase 1 (IDH1) mutation status. sGBMs harboring ZM fusion showed higher expression of genes required for PIK3CA signaling and lowered expression of genes that suppressed RB1 or TP53 function. Expression of the ZM fusion was mutually exclusive with EGFR overexpression in sGBMs. Exogenous expression of the ZM fusion in the U87MG glioblastoma line enhanced cell migration and invasion. Clinically, patients afflicted with ZM fusion harboring glioblastomas survived poorly relative to those afflicted with non-ZM-harboring sGBMs (P < 0.001). Our study profiles the shifting RNA landscape of gliomas during progression and reveled ZM as a novel, recurrent fusion transcript in sGBMs.

Differential Expression of miR-145 in Children with Kawasaki Disease

Background Kawasaki disease is an acute, self-limited vasculitis of childhood that can result in structural damage to the coronary arteries. Previous studies have implicated the TGF-β pathway in disease pathogenesis and generation of myofibroblasts in the arterial wall. microRNAs are small non-coding RNAs that modulate gene expression at the post-transcriptional level and can be transported between cells in extracellular vesicles. To understand the role that microRNAs play in modifying gene expression in Kawasaki disease, we studied microRNAs from whole blood during the acute and convalescent stages of the illness. Methodology/Principal Findings RNA isolated from the matched whole blood of 12 patients with acute and convalescent Kawasaki disease were analyzed by sequencing of small RNA. This analysis revealed six microRNAs (miRs-143, -199b-5p, -618, -223, -145 and -145* (complementary strand)) whose levels were significantly elevated during the acute phase of Kawasaki disease. The result was validated using targeted qRT-PCR using an independent cohort (n = 16). miR-145, which plays a critical role in the differentiation of neutrophils and vascular smooth muscle cells, was expressed at high levels in blood samples from acute Kawasaki disease but not adenovirus-infected control patients (p = 0.005). miR-145 was also detected in small extracellular vesicles isolated from acute Kawasaki disease plasma samples. Pathway analysis of the predicted targets of the 6 differentially expressed microRNAs identified the TGF-β pathway as the top pathway regulated by microRNAs in Kawasaki disease. Conclusion Sequencing of small RNA species allowed discovery of microRNAs that may participate in Kawasaki disease pathogenesis. miR-145 may participate, along with other differentially expressed microRNAs, in regulating expression of genes in the TGF-β pathway during the acute illness. If the predicted target genes are confirmed, our findings suggest a model of Kawasaki disease pathogenesis whereby miR-145 modulates TGF-β signaling in the arterial wall.

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 detected more EVs than TRPS in three of the four samples tested. VFC particle counts are consistently 2–3 fold lower than NTA and TRPS, suggesting contribution of protein aggregates or other non-lipid particles to particle count by these platforms. While TEM yield meaningful data in terms of the morphology, its particle count are consistently two orders of magnitude lower relative to counts generated by NTA and TRPS. For larger particles (>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.

Neuro-oncologic applications of exosomes, microvesicles, and other nano-sized extracellular particles

The discovery that tumor-derived proteins and nucleic acids can be detected in nano-sized vesicles in the plasma and cerebrospinal fluid of patients afflicted with brain tumors has expanded opportunities for biomarker and therapeutic discovery. Through delivery of their contents to surrounding cells, exosomes, microvesicles, and other nano-sized extracellular vesicles secreted by tumors modulate their environment to promote tumor growth and survival. In this review, we discuss the biological processes mediated by these extracellular vesicles and their applications in terms of brain tumor diagnosis, monitoring, and therapy. We review the normal physiology of these extracellular vesicles, their pertinence to tumor biology, and directions for research in this field.

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

Levels of Alzheimers 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.

Optimizing preservation of extracellular vesicular miRNAs derived from clinical cerebrospinal fluid

BACKGROUND Tumor 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. METHODS We 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. RESULTS CSF 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. CONCLUSIONS These 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.

Synthesis and delivery of short, noncoding RNA by B lymphocytes

Significance Short, noncoding RNAs are a powerful way to regulate gene expression. As such, they are potential candidates for new forms of therapy. However, clinical translation is still hampered by lack of suitable and cost-effective delivery methods. Here we demonstrate that primary B lymphocytes can be programmed with plasmid DNA, effectively and easily, for the synthesis and delivery of antisense sequences (anti-microRNA) in vitro and in vivo. We also demonstrate that anti-microRNAs secreted by primary B lymphocytes in vivo are taken up by T lymphocytes during antigen activation. These initial observations predict that microRNA-based intervention to modulate immune responses or to target cancer cell growth may be possible through the type of cell-based therapy disclosed here. Evolutionarily conserved short (20–30 nucleotides) noncoding RNAs (microRNAs) are powerful regulators of gene expression in a variety of physiological and pathological processes. As such, means to efficiently modulate microRNA function constitute an important therapeutic opportunity. Here we demonstrate that primary B lymphocytes can be genetically programmed with nonviral plasmid DNA for the biogenesis and delivery of antisense sequences (anti-microRNA) against microRNA-150 (miR-150). Within 18 h of transfection with an anti-miR-150 construct, primary B lymphocytes secrete ∼3,000 copies of anti-miR-150 molecules per cell. Anti-miR-150 molecules released by B lymphocytes were internalized by CD8 T lymphocytes during cross-priming in vitro and in vivo, resulting in marked down-regulation of endogenous miR-150. However, such internalization was not observed in the absence of cross-priming. These results suggest that shuttling anti-miR-150 molecules from B lymphocytes to T cells requires the activation of receiver T cells via the antigen receptor. Finally, anti-miR-150 synthesized in B cells were secreted both as free and extracellular vesicle-associated fractions, but only extracellular vesicle-associated anti-miR-150 were apparently taken up by CD8 T cells. Collectively, these data indicate that primary B lymphocytes represent an efficient platform for the synthesis and delivery of short, noncoding RNA, paving the way for an approach to immunogenomic therapies.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 μL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.