The potential diagnostic and prognostic role of extracellular vesicles in glioma: current status and future perspectives

Figures & data

Table 1. Selected EV-derived molecules and their roles in glioma.

Molecules	Biological source	Biological significance	Method of detection	References
miR-301a	Serum	Upregulated in GB patients, promote proliferation and invasion of glioma-derived H4 cells, levels downregulated after tumor removal and independently associated with overall survival	qRT-PCR	[104]
miR-124a	Mesenchymal stem cells	Viability and clonogenicity in glioma stem cells reduced in vitro, in vivo transfer of miR-124a showed that 50% of animals lived long	qRT-PCR	[148]
miR-373	U87 cell culture medium	Elicit proangiogenic response, cell proliferation	Affymetrix microarrays	[83]
miR-1587	Mesenchymal stem cells	Increase proliferation and clonogenicity	Illumina next-generation sequencing	[107]
VEGF-A	Patient-derived glioma stem cells	Promote angiogenesis, increase endothelial permeability	qRT-PCR, ELISA	[61]
EGFRvIII	1. Serum2. Cerebrospinal fluid	1. Egfrviii expressed both in serum exosome and tumor tissue samples (the sensitivity and specificity in serum were 79.31% and 51.58% respectively), expression level inversely correlated with survival.2. Egfrviii mutation present in CSF Extracellular vesicles but not in normal tissue (the sensitivity and specificity are 61% and 98% respectively), amplication of wild-type EGFR present in both CSF derived EVS and normal tissue.	qRT-PCR	[63,119]
miR-221	Cell culture	Positively correlated with proliferation, migration and temozolomide resistance	qRT-PCR	[111]
Annexin A1	Cell culture	GB tumors have higher level of annexin A1 compared with normal brain in silico	quantitative high-resolution mass spectrometry	[88]
RBM11	Cell culture	Apoptotic EVS transfer of RBM11 to recipient cells transform Cyclin D1 and MDM4 to pro-oncogenic Cyclin D1a and MDM4s isoforms, which indicate worse survival of GB patients.	qRT-PCR	[149]
CRYAB	Cell culture	Upon stimulation by cytokine, U373 cells produce and secrete more CRYAB and increase the cancer progression related proteins	ELISA	[150]
miR-151a	CSF	miR-151a overexpression correlates with improved prognosis, whereas reduced expression indicates poor prognosis	FISH	[115]
RNU6-1	serum	Small noncoding RNA (RNU6-1) at higher levels in GB patients	qRT-PCR	[151]
miR-148a	serum	miR-148a levels elevated in GB patients, affects cell proliferation and metastasis	RT-PCR	[152]
PTRF	serum	PTRF/CD63 ratio significantly higher in Grade IV glioma	Western blot	[153]
miR-21	CSF	EV miR-21 levels higher than control group	qRT-PCR	[154]
miR-10b	CSF	miR-10b levels significantly higher in GB patients compared with non-neoplastic control group	qRT-PCR	[141]
IDH1	CSF	Mutant IDH1 mrna detected in GB CSF samples	ddPCR, BEAMing	[105]
miR-24, miR-103, miR-125	CSF	Levels of miR-24, miR-103, miR-125 significantly higher in experimental group	qRT-PCR	[120]
cric-TTBK2	Tissue, cell culture	Expression level of cric-TTBK2 higher in GB tissue and cell culture; promotes cell proliferation, migration, and invasion, while inhibits apoptosis	qRT-PCR, in situ hybridization	[155]

Table 2. Overview of extracellular vesicles isolation.

Technique name	Isolation procedure	Benefits	Demerits
Ultracentrifugation	EVs separated based on their size, shape and density	Cost effective in terms of reagents, high-throughput	Expensive in terms of instrumentation, extensive manual labor, long procedure, may interfere with downstream analysis
Ultrafiltration	EV isolation is exclusively based on their size or molecular weight	Rapid, simple instrumentation, easy to operate, direct RNA extraction possible	Purity not so high, clogging and vesicle trapping may reduce the yield
Precipitation	Precipitation done through altering their solubility by tying up water molecules	Easy to handle, requires simple instrumentation, sizable sample capacity	Frequent contamination, time-consuming, requires pre and post cleanup
Immuno-affinity capture-based techniques	Interaction between surface antigens of EVs and immobilized antibodies	Able to isolate specific EVs, purity of EVs are extremely high, able to sub-categorize.	Costly, need to design immobilized antibody every time, low quantity and low output, binding capacity hampered by tumor microenvironment
Microfluidics-based approaches	Micro/Nano scale isolation based on physical and biochemical properties of EVs	Quick, inexpensive, handy, easy to operate, high portability	Need standardization on large cohort of clinical samples, low input and low yield may affect downstream analysis
Size exclusion chromatography	EVs are separated on the basis of size on a single column	High recovery rate, fast, no EVs-aggregation, high purity and integrity	Concentration of isolated EVs are too low, similar sized particles may co-isolate
Density gradient centrifugation	EVs are isolated in a sucrose and iodixanol density gradient according to their size and mass density	Separation efficiency and purity are higher than ultracentrifugation, samples remains in intact form	Laborious, time-consuming, low throughput, isolated samples may be contaminated by same size particles, complex instrumentation process.

Table 3. Techniques currently used for extracellular vesicles research with their test indexes.

Experiment name	Test Indexes	Advantages	Disadvantages
Flow cytometry	Marker proteins, concentration	Able to determine the cellular origin, high-throughput, it’s possible to analyze thousands exosomes in one sample.	Low sensitivity, limited resolution, a significant number of particles remain undetected, sometimes EVs are counted as a single event signal.
Nanoparticle tracking analysis	Concentration	Can precisely measure small particles, samples remain intact during experimental period, sample preparation is very fast and easy, fast measurement, recovery of samples possible after measurements.	It’s very tough to get proper dilution, larger particles may mask the smaller ones.
Transmission electron microscopy	Concentration	High resolution, it’s possible to detect heterogeneous population	Extensive and multistep sample preparation required, the electron beam may also cause damages to biological samples.
Western blot	Marker proteins	Able to detect as little as 0.1 ng, selectively detect a target protein.	Time consuming, high cost and high technical demand, need to optimize the process, prone to false or subjective result.
ELISA	Concentration, marker protein	Highly sensitive and specific, quick and convenient, it is possible to measure a particular surface marker protein both qualitatively and quantitatively.	Requires large amount of EVs, low detection limit.
Quantitative real time PCR	Mutation, RNA	Can be performed easily in most labs, Inexpensive, well-established method, no post PCR processing required.	Less sensitive in compare to ddPCR, complexity arises due to simultaneous thermal cycling and fluorescence detection, it’s quite challenging to quantify EV miRNAs.
Digital droplet PCR	Mutation, DNA methylation, copy number variation	Superior in sensitivity and feasibility in compare qRT-PCR, highly tolerant to inhibitor, no need to rely on references and standards, able to detect copy number variation.	It requires highly allele-specific probes, overestimation may be caused due to DNA denaturation during partitioning, molecular dropout and sample inhomogeneity causes underestimation, the initial cost of equipment are quite high, expensive in terms of consumables.
Next generation sequencing	RNA, Mutation	It can distinguish miRNAs at resolution of single base, able to analyze hundreds of miRNAs individually or in panel in a single experiment, it’s possible to detect abnormalities across the entire genome.	Library preparation for RNA-Seq could be a challenging job, sophisticated bioinformatics systems required, highly expensive and time consuming, the sensitivity of identification of rare mutations are quite low.

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