Glioblastomas (GBM) are highly aggressive primary brain tumors. Complex and dynamic tumor microenvironment (TME) plays a crucial role in the sustained growth, proliferation, and invasion of GBM. Several means of intercellular communication have been documented between glioma cells and the TME, including growth factors, cytokines, chemokines as well as extracellular vesicles (EVs). EVs carry functional genomic and proteomic cargo from their parental cells and deliver that information to surrounding and distant recipient cells to modulate their behavior. EVs are emerging as crucial mediators of establishment and maintenance of the tumor by modulating the TME into a tumor promoting system. Herein we review recent literature in the context of GBM TME and the means by which EVs modulate tumor proliferation, reprogram metabolic activity, induce angiogenesis, escape immune surveillance, acquire drug resistance and undergo invasion. Understanding the multifaceted roles of EVs in the niche of GBM TME will provide invaluable insights into understanding the biology of GBM and provide functional insights into the dynamic EV-mediated intercellular communication during gliomagenesis, creating new opportunities for GBM diagnostics and therapeutics.

Purpose: Liquid biopsy offers a minimally invasive tool to diagnose and monitor the heterogeneous molecular landscape of tumors over time and therapy. Detection of TERT promoter mutations (C228T, C250T) in cfDNA has been successful for some systemic cancers but has yet to be demonstrated in gliomas, despite the high prevalence of these mutations in glioma tissue (>60% of all tumors). Experimental Design: Here, we developed a novel digital droplet PCR (ddPCR) assay that incorporates features to improve sensitivity and allows for the simultaneous detection and longitudinal monitoring of two TERT promoter mutations (C228T and C250T) in cfDNA from the plasma of patients with glioma. Results: In baseline performance in tumor tissue, the assay had perfect concordance with an independently performed clinical pathology laboratory assessment of TERT promoter mutations in the same tumor samples [95% confidence interval (CI), 94%–100%]. Extending to matched plasma samples, we detected TERT mutations in both discovery and blinded multi-institution validation cohorts with an overall sensitivity of 62.5% (95% CI, 52%–73%) and a specificity of 90% (95% CI, 80%–96%) compared with the gold-standard tumor tissue–based detection of TERT mutations. Upon longitudinal monitoring in 5 patients, we report that peripheral TERT-mutant allele frequency reflects the clinical course of the disease, with levels decreasing after surgical intervention and therapy and increasing with tumor progression. Conclusions: Our results demonstrate the feasibility of detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity.

Tumour cells release diverse populations of extracellular vesicles (EVs) ranging in size, molecular cargo, and function. We sought to characterize mRNA and protein content of EV subpopulations released by human glioblastoma (GBM) cells expressing a mutant form of epidermal growth factor receptor (U87 ^EGFRvIII ) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87 ^EGFRvIII cancer cells, non‐cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87 ^EGFRvIII glioma‐bearing mice using differential centrifugation (EVs that sediment at 10,000 × g or 100,000 × g are termed large EVs and small EVs, respectively) were characterized using transmission electron microscopy (TEM), confocal microscopy, nanoparticle tracking analysis (NTA), flow cytometry, immunofluorescence (IF), quantitative‐polymerase chain reaction (qPCR), droplet digital polymerase chain reaction (ddPCR) and micro‐nuclear magnetic resonance (μNMR). We report that both U87 ^EGFRvIII and HUVEC release a similar number of small EVs, but U87 ^EGFRvIII glioma cells alone release a higher number of large EVs compared to non‐cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87 ^EGFRvIII glioma cells was comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Similarly, EGFRvIII mRNA in large and small EVs isolated from the serum of U87 ^EGFRvIII glioma‐bearing mice is comparable, while the EGFR protein (wild type + vIII) levels are significantly higher in large EVs. Here we report for the first time a direct comparison of large and small EVs released by glioma U87 ^EGFRvIII cells and from serum of U87 ^EGFRvIII glioma‐bearing mice. Both large and small EVs contain tumour‐specific EGFRvIII mRNA and proteins and combining these platforms may be beneficial in detecting rare mutant events in circulating biofluids.

Purpose: Liquid biopsy offers an attractive platform for noninvasive tumor diagnosis, prognostication, and prediction of glioblastoma clinical outcomes. Prior studies report that 30% to 50% of GBM lesions characterized by EGFR amplification also harbor the EGFRvIII mutation. Experimental Design: A novel digital droplet PCR (ddPCR) assay for high GC content amplicons was developed and optimized for sensitive detection of EGFRvIII in tumor tissue and circulating extracellular vesicle RNA (EV RNA) isolated from the plasma of patients with glioma. Results: Our optimized qPCR assay detected EGFRvIII mRNA in 81% [95% confidence interval (CI), 68%–94%] of EGFR-amplified glioma tumor tissue, indicating a higher than previously reported prevalence of EGFRvIII in glioma. Using the optimized ddPCR assay in discovery and blinded validation cohorts, we detected EGFRvIII mutation in 73% (95% CI, 64%–82%) of patients with a specificity of 98% (95% CI, 87%–100%), compared with qPCR tumor tissue analysis. In addition, upon longitudinal monitoring in 4 patients, we report detection of EGFRvIII in the plasma of patients with different clinical outcomes, rising with tumor progression, and decreasing in response to treatment. Conclusions: This study demonstrates the feasibility of detecting EGFRvIII mutation in plasma using a highly sensitive and specific ddPCR assay. We also show a higher than previously reported EGFRvIII prevalence in glioma tumor tissue. Several features of the assay are favorable for clinical implementation for detection and monitoring of EGFRvIII-positive tumors.