Publication List

2021

• M. Gupta, E. Burns, N. Z. Georgantas, J. Thierauf, N. Nayyar, A. Gordon, S. Jones, M. Pisapia, Y. Sun, R. P. Burns, J. K. Velarde, J. T. Jordan, M. J. Frigault, B. V. Nahed, P. S. Jones, F. G. Barker, W. Curry, R. Gupta, T. T. Batchelor, J. Romero, P. Brastianos, H. Marble, M. Martinez-Lage, K. Tateishi, J. K. Lennerz, J. Dietrich, D. Cahill, B. Carter, and G. M. Shankar. A Rapid Genotyping Panel for Detection of Primary Central Nervous System Lymphoma. Blood, Mar 2021.
  [abstract▼] [details] [BibTeX▼]

  Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N=1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0-617 days. Permanent histopathology confirmed PCNSL in 142/152 biopsies (93.4%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6% sensitivity and 100% specificity (95% CI: 44.1-70.4% and 87.2-100%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8% sensitivity and 100% specificity (95% CI: 56.2-74.5% and 83.9-100%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL.

  @article{pmid33735913,
      Author = "Gupta, M. and Burns, E. and Georgantas, N. Z. and Thierauf, J. and Nayyar, N. and Gordon, A. and Jones, S. and Pisapia, M. and Sun, Y. and Burns, R. P. and Velarde, J. K. and Jordan, J. T. and Frigault, M. J. and Nahed, B. V. and Jones, P. S. and Barker, F. G. and Curry, W. and Gupta, R. and Batchelor, T. T. and Romero, J. and Brastianos, P. and Marble, H. and Martinez-Lage, M. and Tateishi, K. and Lennerz, J. K. and Dietrich, J. and Cahill, D. and Carter, B. and Shankar, G. M.",
      Title = "{{A} {R}apid {G}enotyping {P}anel for {D}etection of {P}rimary {C}entral {N}ervous {S}ystem {L}ymphoma}",
      Journal = "Blood",
      Year = "2021",
      Month = "Mar",
      Abstract = "Diagnosing primary central nervous system lymphoma (PCNSL) frequently requires neurosurgical biopsy due to nonspecific radiologic features and the low yield of cerebrospinal fluid (CSF) studies. We characterized the clinical evaluation of suspected PCNSL (N=1007 patients) and designed a rapid multiplexed genotyping assay for MYD88, TERT promoter, IDH1/2, H3F3A, and BRAF mutations to facilitate the diagnosis of PCNSL from CSF and detect other neoplasms in the differential diagnosis. Among 159 patients with confirmed PCNSL, the median time to secure a diagnosis of PCNSL was 10 days, with a range of 0-617 days. Permanent histopathology confirmed PCNSL in 142/152 biopsies (93.4\%), whereas CSF analyses were diagnostic in only 15/113 samplings (13.3\%). Among 86 archived clinical specimens, our targeted genotyping assay accurately detected hematologic malignancies with 57.6\% sensitivity and 100\% specificity (95\% CI: 44.1-70.4\% and 87.2-100\%, respectively). MYD88 and TERT promoter mutations were prospectively identified in DNA extracts of CSF obtained from patients with PCNSL and glioblastoma, respectively, within 80 minutes. Across 132 specimens, hallmark mutations indicating the presence of malignancy were detected with 65.8\% sensitivity and 100\% specificity (95\% CI: 56.2-74.5\% and 83.9-100\%, respectively). This targeted genotyping approach offers a rapid, scalable adjunct to reduce diagnostic and treatment delays in PCNSL."
  }
  

• K. Muralidharan, A. Yekula, J. L. Small, Z. S. Rosh, K. M. Kang, L. Wang, S. Lau, H. Zhang, H. Lee, C. Bettegowda, M. R. Chicoine, S. N. Kalkanis, G. M. Shankar, B. V. Nahed, W. T. Curry, P. S. Jones, D. P. Cahill, L. Balaj, and B. S. Carter. TERT Promoter Mutation Analysis for Blood-Based Diagnosis and Monitoring of Gliomas. Clin Cancer Res, 27(1):169–178, Jan 2021. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785705PMC7785705] [DOI:\href https://dx.doi.org/10.1158/1078-0432.CCR-20-308310.1158/1078-0432.CCR-20-3083] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2694140726941407].
  [abstract▼] [details] [BibTeX▼]

  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).\\ 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.\\ 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.\\ Our results demonstrate the feasibility of detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity.

  @article{pmid33051308,
      Author = "Muralidharan, K. and Yekula, A. and Small, J. L. and Rosh, Z. S. and Kang, K. M. and Wang, L. and Lau, S. and Zhang, H. and Lee, H. and Bettegowda, C. and Chicoine, M. R. and Kalkanis, S. N. and Shankar, G. M. and Nahed, B. V. and Curry, W. T. and Jones, P. S. and Cahill, D. P. and Balaj, L. and Carter, B. S.",
      Title = "{{T}{E}{R}{T} {P}romoter {M}utation {A}nalysis for {B}lood-{B}ased {D}iagnosis and {M}onitoring of {G}liomas}",
      Journal = "Clin Cancer Res",
      Year = "2021",
      Volume = "27",
      Number = "1",
      Pages = "169--178",
      Month = "Jan",
      Abstract = "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).\\ 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.\\ 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.\\ Our results demonstrate the feasibility of detecting circulating cfDNA TERT promoter mutations in patients with glioma with clinically relevant sensitivity and specificity.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785705}{PMC7785705}] [DOI:\href{https://dx.doi.org/10.1158/1078-0432.CCR-20-3083}{10.1158/1078-0432.CCR-20-3083}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/26941407}{26941407}]"
  }
  

2020

• S. L. N. Maas, T. S. van Solinge, R. Schnoor, A. Yekula, J. T. Senders, J. de Vrij, P. Robe, B. S. Carter, L. Balaj, G. J. A. Arkesteijn, E. N. M. Nolte-'t Hoen, and M. L. D. Broekman. Orally Administered 5-aminolevulinic Acid for Isolation and Characterization of Circulating Tumor-Derived Extracellular Vesicles in Glioblastoma Patients. Cancers (Basel), Nov 2020. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695169PMC7695169] [DOI:\href https://dx.doi.org/10.3390/cancers1211329710.3390/cancers12113297] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2554771925547719].
  [abstract▼] [details] [BibTeX▼]

  Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.

  @article{pmid33171819,
      Author = "Maas, S. L. N. and van Solinge, T. S. and Schnoor, R. and Yekula, A. and Senders, J. T. and de Vrij, J. and Robe, P. and Carter, B. S. and Balaj, L. and Arkesteijn, G. J. A. and Nolte-'t Hoen, E. N. M. and Broekman, M. L. D.",
      Title = "{{O}rally {A}dministered 5-aminolevulinic {A}cid for {I}solation and {C}haracterization of {C}irculating {T}umor-{D}erived {E}xtracellular {V}esicles in {G}lioblastoma {P}atients}",
      Journal = "Cancers (Basel)",
      Year = "2020",
      Volume = "12",
      Number = "11",
      Month = "Nov",
      Abstract = "Background: In glioblastoma (GB), tissue is required for accurate diagnosis and subtyping. Tissue can be obtained through resection or (stereotactic) biopsy, but these invasive procedures provide risks for patients. Extracellular vesicles (EVs) are small, cell-derived vesicles that contain miRNAs, proteins, and lipids, and possible candidates for liquid biopsies. GB-derived EVs can be found in the blood of patients, but it is difficult to distinguish them from circulating non-tumor EVs. 5-aminolevulinic acid (5-ALA) is orally administered to GB patients to facilitate tumor visualization and maximal resection, as it is metabolized to fluorescent protoporphyrin IX (PpIX) that accumulates in glioma cells. In this study, we assessed whether PpIX accumulates in GB-derived EVs and whether these EVs could be isolated and characterized to enable a liquid biopsy in GB. Methods: EVs were isolated from the conditioned media of U87 cells treated with 5-ALA by differential ultracentrifugation. Blood samples were collected and processed from healthy controls and patients undergoing 5-ALA guided surgery for GB. High-resolution flow cytometry (hFC) enabled detection and sorting of PpIX-positive EVs, which were subsequently analyzed by digital droplet PCR (ddPCR). Results: PpIX-positive EVs could be detected in conditioned cell culture media as well as in patient samples after administration of 5-ALA. By using hFC, we could sort the PpIX-positive EVs for further analysis with ddPCR, which indicated the presence of EVs and GB-associated miRNAs. Conclusion: GB-derived EVs can be isolated from the plasma of GB patients by using 5-ALA induced fluorescence. Although many challenges remain, our findings show new possibilities for the development of blood-based liquid biopsies in GB patients.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7695169}{PMC7695169}] [DOI:\href{https://dx.doi.org/10.3390/cancers12113297}{10.3390/cancers12113297}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/25547719}{25547719}]"
  }
  

• A. Yekula, K. Muralidharan, Z. S. Rosh, A. E. Youngkin, K. M. Kang, L. Balaj, and B. S. Carter. Liquid Biopsy Strategies to Distinguish Progression from Pseudoprogression and Radiation Necrosis in Glioblastomas. Adv Biosyst, 4(12):e2000029, 12 2020. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708392PMC7708392] [DOI:\href https://dx.doi.org/10.1002/adbi.20200002910.1002/adbi.202000029] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2069457320694573].
  [abstract▼] [details] [BibTeX▼]

  Liquid biopsy for the detection and monitoring of central nervous system tumors is of significant clinical interest. At initial diagnosis, the majority of patients with central nervous system tumors undergo magnetic resonance imaging (MRI), followed by invasive brain biopsy to determine the molecular diagnosis of the WHO 2016 classification paradigm. Despite the importance of MRI for long-term treatment monitoring, in the majority of patients who receive chemoradiation therapy for glioblastoma, it can be challenging to distinguish between radiation treatment effects including pseudoprogression, radiation necrosis, and recurrent/progressive disease based on imaging alone. Tissue biopsy-based monitoring is high risk and not always feasible. However, distinguishing these entities is of critical importance for the management of patients and can significantly affect survival. Liquid biopsy strategies including circulating tumor cells, circulating free DNA, and extracellular vesicles have the potential to afford significant useful molecular information at both the stage of diagnosis and monitoring for these tumors. Here, current liquid biopsy-based approaches in the context of tumor monitoring to differentiate progressive disease from pseudoprogression and radiation necrosis are reviewed.

  @article{pmid32484293,
      Author = "Yekula, A. and Muralidharan, K. and Rosh, Z. S. and Youngkin, A. E. and Kang, K. M. and Balaj, L. and Carter, B. S.",
      Title = "{{L}iquid {B}iopsy {S}trategies to {D}istinguish {P}rogression from {P}seudoprogression and {R}adiation {N}ecrosis in {G}lioblastomas}",
      Journal = "Adv Biosyst",
      Year = "2020",
      Volume = "4",
      Number = "12",
      Pages = "e2000029",
      Month = "12",
      Abstract = "Liquid biopsy for the detection and monitoring of central nervous system tumors is of significant clinical interest. At initial diagnosis, the majority of patients with central nervous system tumors undergo magnetic resonance imaging (MRI), followed by invasive brain biopsy to determine the molecular diagnosis of the WHO 2016 classification paradigm. Despite the importance of MRI for long-term treatment monitoring, in the majority of patients who receive chemoradiation therapy for glioblastoma, it can be challenging to distinguish between radiation treatment effects including pseudoprogression, radiation necrosis, and recurrent/progressive disease based on imaging alone. Tissue biopsy-based monitoring is high risk and not always feasible. However, distinguishing these entities is of critical importance for the management of patients and can significantly affect survival. Liquid biopsy strategies including circulating tumor cells, circulating free DNA, and extracellular vesicles have the potential to afford significant useful molecular information at both the stage of diagnosis and monitoring for these tumors. Here, current liquid biopsy-based approaches in the context of tumor monitoring to differentiate progressive disease from pseudoprogression and radiation necrosis are reviewed.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7708392}{PMC7708392}] [DOI:\href{https://dx.doi.org/10.1002/adbi.202000029}{10.1002/adbi.202000029}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/20694573}{20694573}]"
  }
  

• L. Wang, A. Yekula, K. Muralidharan, J. L. Small, Z. S. Rosh, K. M. Kang, B. S. Carter, and L. Balaj. Novel Gene Fusions in Glioblastoma Tumor Tissue and Matched Patient Plasma. Cancers (Basel), May 2020. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281415PMC7281415] [DOI:\href https://dx.doi.org/10.3390/cancers1205121910.3390/cancers12051219] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2845378428453784].
  [abstract▼] [details] [BibTeX▼]

  Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a minimally invasive alternative to diagnose and monitor tumor-specific molecular aberrations in patient biofluids. Here, we used targeted RNA sequencing to screen GBM tissue and the matched plasma of patients (n = 9) for RNA fusion transcripts. We identified two novel fusion transcripts in GBM tissue and five novel fusions in the matched plasma of GBM patients. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both tissue and matched plasma. A longitudinal follow-up of a GBM patient with a FGFR3-TACC3 positive glioma revealed the potential of monitoring RNA fusions in plasma. In summary, we report a sensitive RNA-seq-based liquid biopsy strategy to detect RNA level fusion status in the plasma of GBM patients.

  @article{pmid32414213,
      Author = "Wang, L. and Yekula, A. and Muralidharan, K. and Small, J. L. and Rosh, Z. S. and Kang, K. M. and Carter, B. S. and Balaj, L.",
      Title = "{{N}ovel {G}ene {F}usions in {G}lioblastoma {T}umor {T}issue and {M}atched {P}atient {P}lasma}",
      Journal = "Cancers (Basel)",
      Year = "2020",
      Volume = "12",
      Number = "5",
      Month = "May",
      Abstract = "Sequencing studies have provided novel insights into the heterogeneous molecular landscape of glioblastoma (GBM), unveiling a subset of patients with gene fusions. Tissue biopsy is highly invasive, limited by sampling frequency and incompletely representative of intra-tumor heterogeneity. Extracellular vesicle-based liquid biopsy provides a minimally invasive alternative to diagnose and monitor tumor-specific molecular aberrations in patient biofluids. Here, we used targeted RNA sequencing to screen GBM tissue and the matched plasma of patients (n = 9) for RNA fusion transcripts. We identified two novel fusion transcripts in GBM tissue and five novel fusions in the matched plasma of GBM patients. The fusion transcripts FGFR3-TACC3 and VTI1A-TCF7L2 were detected in both tissue and matched plasma. A longitudinal follow-up of a GBM patient with a FGFR3-TACC3 positive glioma revealed the potential of monitoring RNA fusions in plasma. In summary, we report a sensitive RNA-seq-based liquid biopsy strategy to detect RNA level fusion status in the plasma of GBM patients.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281415}{PMC7281415}] [DOI:\href{https://dx.doi.org/10.3390/cancers12051219}{10.3390/cancers12051219}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/28453784}{28453784}]"
  }
  

• A. Yekula, K. Muralidharan, K. M. Kang, L. Wang, L. Balaj, and B. S. Carter. From laboratory to clinic: Translation of extracellular vesicle based cancer biomarkers. Methods, 177:58–66, 05 2020. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198349PMC7198349] [DOI:\href https://dx.doi.org/10.1016/j.ymeth.2020.02.00310.1016/j.ymeth.2020.02.003] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2845378428453784].
  [abstract▼] [details] [BibTeX▼]

  The past decade has witnessed a rapid growth in the field of extracellular vesicle (EV) based biomarkers for the diagnosis and monitoring of cancer. Several studies have reported novel EV based biomarkers, but the technical and clinical validation phase has been hampered by general challenges common to biomedical research field as well as specific challenges inherent to the nanoparticle field. This has led to more common failures than success stories in the biomarker discovery pipeline. As a result, more attention must be focused on the process of biomarker discovery, verification, and validation to allow for translation and application of novel EV based research to patient care. Herein, we briefly discuss the hurdles and potential solutions in EV biomarker discovery and verification and validation, and clinical translation.

  @article{pmid32061674,
      Author = "Yekula, A. and Muralidharan, K. and Kang, K. M. and Wang, L. and Balaj, L. and Carter, B. S.",
      Title = "{{F}rom laboratory to clinic: {T}ranslation of extracellular vesicle based cancer biomarkers}",
      Journal = "Methods",
      Year = "2020",
      Volume = "177",
      Pages = "58--66",
      Month = "05",
      Abstract = "The past decade has witnessed a rapid growth in the field of extracellular vesicle (EV) based biomarkers for the diagnosis and monitoring of cancer. Several studies have reported novel EV based biomarkers, but the technical and clinical validation phase has been hampered by general challenges common to biomedical research field as well as specific challenges inherent to the nanoparticle field. This has led to more common failures than success stories in the biomarker discovery pipeline. As a result, more attention must be focused on the process of biomarker discovery, verification, and validation to allow for translation and application of novel EV based research to patient care. Herein, we briefly discuss the hurdles and potential solutions in EV biomarker discovery and verification and validation, and clinical translation.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7198349}{PMC7198349}] [DOI:\href{https://dx.doi.org/10.1016/j.ymeth.2020.02.003}{10.1016/j.ymeth.2020.02.003}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/28453784}{28453784}]"
  }
  

• A. Yekula, V. R. Minciacchi, M. Morello, H. Shao, Y. Park, X. Zhang, K. Muralidharan, M. R. Freeman, R. Weissleder, H. Lee, B. Carter, X. O. Breakefield, D. Di Vizio, and L. Balaj. Large and small extracellular vesicles released by glioma cells in vitro and in vivo. J Extracell Vesicles, 9(1):1689784, 2020. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896449PMC6896449] [DOI:\href https://dx.doi.org/10.1080/20013078.2019.168978410.1080/20013078.2019.1689784] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2845378428453784].
  [abstract▼] [details] [BibTeX▼]

  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 (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII 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 U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII 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 U87EGFRvIII 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 U87EGFRvIII cells and from serum of U87EGFRvIII 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.

  @article{pmid31839905,
      Author = "Yekula, A. and Minciacchi, V. R. and Morello, M. and Shao, H. and Park, Y. and Zhang, X. and Muralidharan, K. and Freeman, M. R. and Weissleder, R. and Lee, H. and Carter, B. and Breakefield, X. O. and Di Vizio, D. and Balaj, L.",
      Title = "{{L}arge and small extracellular vesicles released by glioma cells in vitro and in vivo}",
      Journal = "J Extracell Vesicles",
      Year = "2020",
      Volume = "9",
      Number = "1",
      Pages = "1689784",
      Abstract = "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 (U87EGFRvIII) in vitro and in vivo with respect to size, morphology and the presence of tumour cargo. The two EV subpopulations purified from GBM U87EGFRvIII cancer cells, non-cancer human umbilical vein endothelial cells (HUVEC; control) and serum of U87EGFRvIII 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 U87EGFRvIII and HUVEC release a similar number of small EVs, but U87EGFRvIII glioma cells alone release a higher number of large EVs compared to non-cancer HUVEC. The EGFRvIII mRNA from the two EV subpopulations from U87EGFRvIII 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 U87EGFRvIII 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 U87EGFRvIII cells and from serum of U87EGFRvIII 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.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6896449}{PMC6896449}] [DOI:\href{https://dx.doi.org/10.1080/20013078.2019.1689784}{10.1080/20013078.2019.1689784}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/28453784}{28453784}]"
  }
  

2019

• A. Yekula, A. Yekula, K. Muralidharan, K. Kang, B. S. Carter, and L. Balaj. Extracellular Vesicles in Glioblastoma Tumor Microenvironment. Front Immunol, 10:3137, 2019. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990128PMC6990128] [DOI:\href https://dx.doi.org/10.3389/fimmu.2019.0313710.3389/fimmu.2019.03137] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2334996223349962].
  [abstract▼] [details] [BibTeX▼]

  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.

  @article{pmid32038644,
      Author = "Yekula, A. and Yekula, A. and Muralidharan, K. and Kang, K. and Carter, B. S. and Balaj, L.",
      Title = "{{E}xtracellular {V}esicles in {G}lioblastoma {T}umor {M}icroenvironment}",
      Journal = "Front Immunol",
      Year = "2019",
      Volume = "10",
      Pages = "3137",
      Abstract = "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.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6990128}{PMC6990128}] [DOI:\href{https://dx.doi.org/10.3389/fimmu.2019.03137}{10.3389/fimmu.2019.03137}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/23349962}{23349962}]"
  }
  

• P. S. Jones, A. Yekula, E. Lansbury, J. L. Small, C. Ayinon, S. Mordecai, F. H. Hochberg, J. Tigges, B. Delcuze, A. Charest, I. Ghiran, L. Balaj, and B. S. Carter. Characterization of plasma-derived protoporphyrin-IX-positive extracellular vesicles following 5-ALA use in patients with malignant glioma. EBioMedicine, 48:23–35, Oct 2019. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838454PMC6838454] [DOI:\href https://dx.doi.org/10.1016/j.ebiom.2019.09.02510.1016/j.ebiom.2019.09.025] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1249361012493610].
  [abstract▼] [details] [BibTeX▼]

  Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed.\\ We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS.\\ We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2 = 0.888).\\ Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.

  @article{pmid31628025,
      Author = "Jones, P. S. and Yekula, A. and Lansbury, E. and Small, J. L. and Ayinon, C. and Mordecai, S. and Hochberg, F. H. and Tigges, J. and Delcuze, B. and Charest, A. and Ghiran, I. and Balaj, L. and Carter, B. S.",
      Title = "{{C}haracterization of plasma-derived protoporphyrin-{I}{X}-positive extracellular vesicles following 5-{A}{L}{A} use in patients with malignant glioma}",
      Journal = "EBioMedicine",
      Year = "2019",
      Volume = "48",
      Pages = "23--35",
      Month = "Oct",
      Abstract = "Malignant gliomas are rapidly progressive brain tumors with high mortality. Fluorescence guided surgery (FGS) with 5-aminolevulinic acid (5-ALA) provides fluorescent delineation of malignant tissue, which helps achieve maximum safe resection. 5-ALA-based fluorescence is due to preferential accumulation of the fluorophore protoporphyrin-IX (PpIX) in malignant glioma tissue. Additionally, gliomas cells release extracellular vesicles (EVs) which carry biomarkers of disease. Herein, we performed animal and human studies to investigate whether 5-ALA dosed glioma cells, in vitro and in vivo, release PpIX positive EVs in circulation which can be captured and analyzed.\\ We used imaging flow cytometry (IFC) to characterize PpIX-positive EVs released from 5-ALA-dosed glioma cells, glioma-bearing xenograft models, as well as patients with malignant glioma undergoing FGS.\\ We first show that glioma cells dosed with 5-ALA release 247-fold higher PpIX positive EVs compared to mock dosed glioma cells. Second, we demonstrate that the plasma of glioma-bearing mice (n = 2) dosed with 5-ALA contain significantly higher levels of circulating PpIX-positive EVs than their pre-dosing background (p = 0.004). Lastly, we also show that the plasma of patients with avidly fluorescent tumors (n = 4) undergoing FGS contain circulating PpIX-positive EVs at levels significantly higher than their pre-dosing background (p = 0.00009) and this rise in signal correlates with enhancing tumor volumes (r 2 = 0.888).\\ Our findings highlight the potential of plasma-derived PpIX-positive EV-based diagnostics for malignant gliomas, offering a novel liquid biopsy platform for confirming and monitoring tumor status.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6838454}{PMC6838454}] [DOI:\href{https://dx.doi.org/10.1016/j.ebiom.2019.09.025}{10.1016/j.ebiom.2019.09.025}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/12493610}{12493610}]"
  }
  

• S. Das, K. M. Ansel, M. Bitzer, X. O. Breakefield, A. Charest, D. J. Galas, M. B. Gerstein, M. Gupta, A. Milosavljevic, M. T. McManus, T. Patel, R. L. Raffai, J. Rozowsky, M. E. Roth, J. A. Saugstad, K. Van Keuren-Jensen, A. M. Weaver, L. C. Laurent, A. B. Abdel-Mageed, C. Adamidi, P. D. Adelson, K. M. Akat, E. Alsop, K. M. Ansel, J. Arango, N. Aronin, S. K. Avsaroglu, A. Azizian, L. Balaj, I. Z. Ben-Dov, K. Bertram, M. Bitzer, R. Blelloch, K. A. Bogardus, X. O. Breakefield, G. A. Calin, B. S. Carter, A. Charest, C. C. Chen, T. Chitnis, R. J. Coffey, A. Courtright-Lim, S. Das, A. Datta, P. DeHoff, T. G. Diacovo, D. J. Erle, A. Etheridge, M. Ferrer, J. L. Franklin, J. E. Freedman, D. J. Galas, T. Galeev, R. Gandhi, A. Garcia, M. B. Gerstein, V. Ghai, I. C. Ghiran, M. D. Giraldez, A. Goga, T. Gogakos, B. Goilav, S. J. Gould, P. Guo, M. Gupta, F. Hochberg, B. Huang, M. Huentelman, C. Hunter, E. Hutchins, A. R. Jackson, M. Y. S. Kalani, P. Kanlikilicer, R. A. Karaszti, K. Van Keuren-Jensen, A. Khvorova, Y. Kim, H. Kim, T. K. Kim, R. Kitchen, R. P. Kraig, A. M. Krichevsky, R. Y. Kwong, L. C. Laurent, M. Lee, N. L'Etoile, S. E. Levy, F. Li, J. Li, X. Li, G. Lopez-Berestein, R. Lucero, B. Mateescu, A. C. Matin, K. E. A. Max, M. T. McManus, T. R. Mempel, C. Meyer, A. Milosavljevic, D. Mondal, K. J. Mukamal, O. D. Murillo, T. Muthukumar, D. A. Nickerson, C. J. O'Donnell, D. J. Patel, T. Patel, J. G. Patton, A. Paul, E. R. Peskind, M. A. Phelps, C. Putterman, P. J. Quesenberry, J. F. Quinn, R. L. Raffai, S. Ranabothu, S. J. Rao, C. Rodriguez-Aguayo, A. Rosenzweig, M. E. Roth, J. Rozowsky, M. S. Sabatine, N. A. Sakhanenko, J. A. Saugstad, T. D. Schmittgen, N. Shah, R. Shah, K. Shedden, J. Shi, A. K. Sood, A. Sopeyin, R. M. Spengler, R. Spetzler, S. Srinivasan, S. L. Subramanian, M. Suthanthiran, K. Tanriverdi, Y. Teng, M. Tewari, W. Thistlethwaite, T. Tuschl, K. K. Urbanowicz, K. C. Vickers, O. Voinnet, K. Wang, A. M. Weaver, Z. Wei, H. L. Weiner, Z. R. Weiss, Z. Williams, D. T. W. Wong, P. G. Woodruff, X. Xiao, I. K. Yan, A. Yeri, B. Zhang, and H. G. Zhang. The Extracellular RNA Communication Consortium: Establishing Foundational Knowledge and Technologies for Extracellular RNA Research. Cell, 177(2):231–242, 04 2019. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601620PMC6601620] [DOI:\href https://dx.doi.org/10.1016/j.cell.2019.03.02310.1016/j.cell.2019.03.023] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2160996421609964].
  [abstract▼] [details] [BibTeX▼]

  The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

  @article{pmid30951667,
      Author = "Das, S. and Ansel, K. M. and Bitzer, M. and Breakefield, X. O. and Charest, A. and Galas, D. J. and Gerstein, M. B. and Gupta, M. and Milosavljevic, A. and McManus, M. T. and Patel, T. and Raffai, R. L. and Rozowsky, J. and Roth, M. E. and Saugstad, J. A. and Van Keuren-Jensen, K. and Weaver, A. M. and Laurent, L. C. and Abdel-Mageed, A. B. and Adamidi, C. and Adelson, P. D. and Akat, K. M. and Alsop, E. and Ansel, K. M. and Arango, J. and Aronin, N. and Avsaroglu, S. K. and Azizian, A. and Balaj, L. and Ben-Dov, I. Z. and Bertram, K. and Bitzer, M. and Blelloch, R. and Bogardus, K. A. and Breakefield, X. O. and Calin, G. A. and Carter, B. S. and Charest, A. and Chen, C. C. and Chitnis, T. and Coffey, R. J. and Courtright-Lim, A. and Das, S. and Datta, A. and DeHoff, P. and Diacovo, T. G. and Erle, D. J. and Etheridge, A. and Ferrer, M. and Franklin, J. L. and Freedman, J. E. and Galas, D. J. and Galeev, T. and Gandhi, R. and Garcia, A. and Gerstein, M. B. and Ghai, V. and Ghiran, I. C. and Giraldez, M. D. and Goga, A. and Gogakos, T. and Goilav, B. and Gould, S. J. and Guo, P. and Gupta, M. and Hochberg, F. and Huang, B. and Huentelman, M. and Hunter, C. and Hutchins, E. and Jackson, A. R. and Kalani, M. Y. S. and Kanlikilicer, P. and Karaszti, R. A. and Van Keuren-Jensen, K. and Khvorova, A. and Kim, Y. and Kim, H. and Kim, T. K. and Kitchen, R. and Kraig, R. P. and Krichevsky, A. M. and Kwong, R. Y. and Laurent, L. C. and Lee, M. and L'Etoile, N. and Levy, S. E. and Li, F. and Li, J. and Li, X. and Lopez-Berestein, G. and Lucero, R. and Mateescu, B. and Matin, A. C. and Max, K. E. A. and McManus, M. T. and Mempel, T. R. and Meyer, C. and Milosavljevic, A. and Mondal, D. and Mukamal, K. J. and Murillo, O. D. and Muthukumar, T. and Nickerson, D. A. and O'Donnell, C. J. and Patel, D. J. and Patel, T. and Patton, J. G. and Paul, A. and Peskind, E. R. and Phelps, M. A. and Putterman, C. and Quesenberry, P. J. and Quinn, J. F. and Raffai, R. L. and Ranabothu, S. and Rao, S. J. and Rodriguez-Aguayo, C. and Rosenzweig, A. and Roth, M. E. and Rozowsky, J. and Sabatine, M. S. and Sakhanenko, N. A. and Saugstad, J. A. and Schmittgen, T. D. and Shah, N. and Shah, R. and Shedden, K. and Shi, J. and Sood, A. K. and Sopeyin, A. and Spengler, R. M. and Spetzler, R. and Srinivasan, S. and Subramanian, S. L. and Suthanthiran, M. and Tanriverdi, K. and Teng, Y. and Tewari, M. and Thistlethwaite, W. and Tuschl, T. and Urbanowicz, K. K. and Vickers, K. C. and Voinnet, O. and Wang, K. and Weaver, A. M. and Wei, Z. and Weiner, H. L. and Weiss, Z. R. and Williams, Z. and Wong, D. T. W. and Woodruff, P. G. and Xiao, X. and Yan, I. K. and Yeri, A. and Zhang, B. and Zhang, H. G.",
      Title = "{{T}he {E}xtracellular {R}{N}{A} {C}ommunication {C}onsortium: {E}stablishing {F}oundational {K}nowledge and {T}echnologies for {E}xtracellular {R}{N}{A} {R}esearch}",
      Journal = "Cell",
      Year = "2019",
      Volume = "177",
      Number = "2",
      Pages = "231--242",
      Month = "04",
      Abstract = "The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601620}{PMC6601620}] [DOI:\href{https://dx.doi.org/10.1016/j.cell.2019.03.023}{10.1016/j.cell.2019.03.023}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/21609964}{21609964}]"
  }
  

• S. Roy, H. Y. Lin, C. Y. Chou, C. H. Huang, J. Small, N. Sadik, C. M. Ayinon, E. Lansbury, L. Cruz, A. Yekula, P. S. Jones, L. Balaj, and B. S. Carter. Navigating the Landscape of Tumor Extracellular Vesicle Heterogeneity. Int J Mol Sci, Mar 2019. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471355PMC6471355] [DOI:\href https://dx.doi.org/10.3390/ijms2006134910.3390/ijms20061349] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/98657029865702].
  [abstract▼] [details] [BibTeX▼]

  The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.

  @article{pmid30889795,
      Author = "Roy, S. and Lin, H. Y. and Chou, C. Y. and Huang, C. H. and Small, J. and Sadik, N. and Ayinon, C. M. and Lansbury, E. and Cruz, L. and Yekula, A. and Jones, P. S. and Balaj, L. and Carter, B. S.",
      Title = "{{N}avigating the {L}andscape of {T}umor {E}xtracellular {V}esicle {H}eterogeneity}",
      Journal = "Int J Mol Sci",
      Year = "2019",
      Volume = "20",
      Number = "6",
      Month = "Mar",
      Abstract = "The last decade has seen a rapid expansion of interest in extracellular vesicles (EVs) released by cells and proposed to mediate intercellular communication in physiological and pathological conditions. Considering that the genetic content of EVs reflects that of their respective parent cell, many researchers have proposed EVs as a source of biomarkers in various diseases. So far, the question of heterogeneity in given EV samples is rarely addressed at the experimental level. Because of their relatively small size, EVs are difficult to reliably isolate and detect within a given sample. Consequently, standardized protocols that have been optimized for accurate characterization of EVs are lacking despite recent advancements in the field. Continuous improvements in pre-analytical parameters permit more efficient assessment of EVs, however, methods to more objectively distinguish EVs from background, and to interpret multiple single-EV parameters are lacking. Here, we review EV heterogeneity according to their origin, mode of release, membrane composition, organelle and biochemical content, and other factors. In doing so, we also provide an overview of currently available and potentially applicable methods for single EV analysis. Finally, we examine the latest findings from experiments that have analyzed the issue at the single EV level and discuss potential implications.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6471355}{PMC6471355}] [DOI:\href{https://dx.doi.org/10.3390/ijms20061349}{10.3390/ijms20061349}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/9865702}{9865702}]"
  }
  

2018

• C. Théry, K. W. Witwer, E. Aikawa, M. J. Alcaraz, J. D. Anderson, R. Andriantsitohaina, A. Antoniou, T. Arab, F. Archer, G. K. Atkin-Smith, D. C. Ayre, J. M. Bach, D. Bachurski, H. Baharvand, L. Balaj, S. Baldacchino, N. N. Bauer, A. A. Baxter, M. Bebawy, C. Beckham, A. Bedina Zavec, A. Benmoussa, A. C. Berardi, P. Bergese, E. Bielska, C. Blenkiron, S. Bobis-Wozowicz, E. Boilard, W. Boireau, A. Bongiovanni, F. E. Borràs, S. Bosch, C. M. Boulanger, X. Breakefield, A. M. Breglio, M. Á. Brennan, D. R. Brigstock, A. Brisson, M. L. Broekman, J. F. Bromberg, P. Bryl-Górecka, S. Buch, A. H. Buck, D. Burger, S. Busatto, D. Buschmann, B. Bussolati, E. I. Buzás, J. B. Byrd, G. Camussi, D. R. Carter, S. Caruso, L. W. Chamley, Y. T. Chang, C. Chen, S. Chen, L. Cheng, A. R. Chin, A. Clayton, S. P. Clerici, A. Cocks, E. Cocucci, R. J. Coffey, A. Cordeiro-da-Silva, Y. Couch, F. A. Coumans, B. Coyle, R. Crescitelli, M. F. Criado, C. D'Souza-Schorey, S. Das, A. Datta Chaudhuri, P. de Candia, E. F. De Santana, O. De Wever, H. A. Del Portillo, T. Demaret, S. Deville, A. Devitt, B. Dhondt, D. Di Vizio, L. C. Dieterich, V. Dolo, A. P. Dominguez Rubio, M. Dominici, M. R. Dourado, T. A. Driedonks, F. V. Duarte, H. M. Duncan, R. M. Eichenberger, K. Ekström, S. El Andaloussi, C. Elie-Caille, U. Erdbrügger, J. M. Falcón-Pérez, F. Fatima, J. E. Fish, M. Flores-Bellver, A. Försönits, A. Frelet-Barrand, F. Fricke, G. Fuhrmann, S. Gabrielsson, A. Gámez-Valero, C. Gardiner, K. Gärtner, R. Gaudin, Y. S. Gho, B. Giebel, C. Gilbert, M. Gimona, I. Giusti, D. C. Goberdhan, A. Görgens, S. M. Gorski, D. W. Greening, J. C. Gross, A. Gualerzi, G. N. Gupta, D. Gustafson, A. Handberg, R. A. Haraszti, P. Harrison, H. Hegyesi, A. Hendrix, A. F. Hill, F. H. Hochberg, K. F. Hoffmann, B. Holder, H. Holthofer, B. Hosseinkhani, G. Hu, Y. Huang, V. Huber, S. Hunt, A. G. Ibrahim, T. Ikezu, J. M. Inal, M. Isin, A. Ivanova, H. K. Jackson, S. Jacobsen, S. M. Jay, M. Jayachandran, G. Jenster, L. Jiang, S. M. Johnson, J. C. Jones, A. Jong, T. Jovanovic-Talisman, S. Jung, R. Kalluri, S. I. Kano, S. Kaur, Y. Kawamura, E. T. Keller, D. Khamari, E. Khomyakova, A. Khvorova, P. Kierulf, K. P. Kim, T. Kislinger, M. Klingeborn, D. J. Klinke, M. Kornek, M. M. Kosanović, Á. F. Kovács, E. M. Krämer-Albers, S. Krasemann, M. Krause, I. V. Kurochkin, G. D. Kusuma, S. Kuypers, S. Laitinen, S. M. Langevin, L. R. Languino, J. Lannigan, C. Lässer, L. C. Laurent, G. Lavieu, E. Lázaro-Ibáñez, S. Le Lay, M. S. Lee, Y. X. F. Lee, D. S. Lemos, M. Lenassi, A. Leszczynska, I. T. Li, K. Liao, S. F. Libregts, E. Ligeti, R. Lim, S. K. Lim, A. Linē, K. Linnemannstöns, A. Llorente, C. A. Lombard, M. J. Lorenowicz, Á. M. Lörincz, J. Lötvall, J. Lovett, M. C. Lowry, X. Loyer, Q. Lu, B. Lukomska, T. R. Lunavat, S. L. Maas, H. Malhi, A. Marcilla, J. Mariani, J. Mariscal, E. S. Martens-Uzunova, L. Martin-Jaular, M. C. Martinez, V. R. Martins, M. Mathieu, S. Mathivanan, M. Maugeri, L. K. McGinnis, M. J. McVey, D. G. Meckes, K. L. Meehan, I. Mertens, V. R. Minciacchi, A. Möller, M. Møller Jørgensen, A. Morales-Kastresana, J. Morhayim, F. Mullier, M. Muraca, L. Musante, V. Mussack, D. C. Muth, K. H. Myburgh, T. Najrana, M. Nawaz, I. Nazarenko, P. Nejsum, C. Neri, T. Neri, R. Nieuwland, L. Nimrichter, J. P. Nolan, E. N. Nolte-'t Hoen, N. Noren Hooten, L. O'Driscoll, T. O'Grady, A. O'Loghlen, T. Ochiya, M. Olivier, A. Ortiz, L. A. Ortiz, X. Osteikoetxea, O. Østergaard, M. Ostrowski, J. Park, D. M. Pegtel, H. Peinado, F. Perut, M. W. Pfaffl, D. G. Phinney, B. C. Pieters, R. C. Pink, D. S. Pisetsky, E. Pogge von Strandmann, I. Polakovicova, I. K. Poon, B. H. Powell, I. Prada, L. Pulliam, P. Quesenberry, A. Radeghieri, R. L. Raffai, S. Raimondo, J. Rak, M. I. Ramirez, G. Raposo, M. S. Rayyan, N. Regev-Rudzki, F. L. Ricklefs, P. D. Robbins, D. D. Roberts, S. C. Rodrigues, E. Rohde, S. Rome, K. M. Rouschop, A. Rughetti, A. E. Russell, P. Saá, S. Sahoo, E. Salas-Huenuleo, C. Sánchez, J. A. Saugstad, M. J. Saul, R. M. Schiffelers, R. Schneider, T. H. Schøyen, A. Scott, E. Shahaj, S. Sharma, O. Shatnyeva, F. Shekari, G. V. Shelke, A. K. Shetty, K. Shiba, P. R. Siljander, A. M. Silva, A. Skowronek, O. L. Snyder, R. P. Soares, B. W. Sódar, C. Soekmadji, J. Sotillo, P. D. Stahl, W. Stoorvogel, S. L. Stott, E. F. Strasser, S. Swift, H. Tahara, M. Tewari, K. Timms, S. Tiwari, R. Tixeira, M. Tkach, W. S. Toh, R. Tomasini, A. C. Torrecilhas, J. P. Tosar, V. Toxavidis, L. Urbanelli, P. Vader, B. W. van Balkom, S. G. van der Grein, J. Van Deun, M. J. van Herwijnen, K. Van Keuren-Jensen, G. van Niel, M. E. van Royen, A. J. van Wijnen, M. H. Vasconcelos, I. J. Vechetti, T. D. Veit, L. J. Vella, É. Velot, F. J. Verweij, B. Vestad, J. L. Viñas, T. Visnovitz, K. V. Vukman, J. Wahlgren, D. C. Watson, M. H. Wauben, A. Weaver, J. P. Webber, V. Weber, A. M. Wehman, D. J. Weiss, J. A. Welsh, S. Wendt, A. M. Wheelock, Z. Wiener, L. Witte, J. Wolfram, A. Xagorari, P. Xander, J. Xu, X. Yan, M. Yáñez-Mó, H. Yin, Y. Yuana, V. Zappulli, J. Zarubova, V. Žėkas, J. Y. Zhang, Z. Zhao, L. Zheng, A. R. Zheutlin, A. M. Zickler, P. Zimmermann, A. M. Zivkovic, D. Zocco, and E. K. Zuba-Surma. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles, 7(1):1535750, 2018. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC6322352] [DOI:\href https://dx.doi.org/10.1080/20013078.2018.153575010.1080/20013078.2018.1535750] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2990457229904572].
  [abstract▼] [details] [BibTeX▼]

  The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

  @article{pmid30637094,
      Author = "Théry, C. and Witwer, K. W. and Aikawa, E. and Alcaraz, M. J. and Anderson, J. D. and Andriantsitohaina, R. and Antoniou, A. and Arab, T. and Archer, F. and Atkin-Smith, G. K. and Ayre, D. C. and Bach, J. M. and Bachurski, D. and Baharvand, H. and Balaj, L. and Baldacchino, S. and Bauer, N. N. and Baxter, A. A. and Bebawy, M. and Beckham, C. and Bedina Zavec, A. and Benmoussa, A. and Berardi, A. C. and Bergese, P. and Bielska, E. and Blenkiron, C. and Bobis-Wozowicz, S. and Boilard, E. and Boireau, W. and Bongiovanni, A. and Borràs, F. E. and Bosch, S. and Boulanger, C. M. and Breakefield, X. and Breglio, A. M. and Brennan, M. Á. and Brigstock, D. R. and Brisson, A. and Broekman, M. L. and Bromberg, J. F. and Bryl-Górecka, P. and Buch, S. and Buck, A. H. and Burger, D. and Busatto, S. and Buschmann, D. and Bussolati, B. and Buzás, E. I. and Byrd, J. B. and Camussi, G. and Carter, D. R. and Caruso, S. and Chamley, L. W. and Chang, Y. T. and Chen, C. and Chen, S. and Cheng, L. and Chin, A. R. and Clayton, A. and Clerici, S. P. and Cocks, A. and Cocucci, E. and Coffey, R. J. and Cordeiro-da-Silva, A. and Couch, Y. and Coumans, F. A. and Coyle, B. and Crescitelli, R. and Criado, M. F. and D'Souza-Schorey, C. and Das, S. and Datta Chaudhuri, A. and de Candia, P. and De Santana, E. F. and De Wever, O. and Del Portillo, H. A. and Demaret, T. and Deville, S. and Devitt, A. and Dhondt, B. and Di Vizio, D. and Dieterich, L. C. and Dolo, V. and Dominguez Rubio, A. P. and Dominici, M. and Dourado, M. R. and Driedonks, T. A. and Duarte, F. V. and Duncan, H. M. and Eichenberger, R. M. and Ekström, K. and El Andaloussi, S. and Elie-Caille, C. and Erdbrügger, U. and Falcón-Pérez, J. M. and Fatima, F. and Fish, J. E. and Flores-Bellver, M. and Försönits, A. and Frelet-Barrand, A. and Fricke, F. and Fuhrmann, G. and Gabrielsson, S. and Gámez-Valero, A. and Gardiner, C. and Gärtner, K. and Gaudin, R. and Gho, Y. S. and Giebel, B. and Gilbert, C. and Gimona, M. and Giusti, I. and Goberdhan, D. C. and Görgens, A. and Gorski, S. M. and Greening, D. W. and Gross, J. C. and Gualerzi, A. and Gupta, G. N. and Gustafson, D. and Handberg, A. and Haraszti, R. A. and Harrison, P. and Hegyesi, H. and Hendrix, A. and Hill, A. F. and Hochberg, F. H. and Hoffmann, K. F. and Holder, B. and Holthofer, H. and Hosseinkhani, B. and Hu, G. and Huang, Y. and Huber, V. and Hunt, S. and Ibrahim, A. G. and Ikezu, T. and Inal, J. M. and Isin, M. and Ivanova, A. and Jackson, H. K. and Jacobsen, S. and Jay, S. M. and Jayachandran, M. and Jenster, G. and Jiang, L. and Johnson, S. M. and Jones, J. C. and Jong, A. and Jovanovic-Talisman, T. and Jung, S. and Kalluri, R. and Kano, S. I. and Kaur, S. and Kawamura, Y. and Keller, E. T. and Khamari, D. and Khomyakova, E. and Khvorova, A. and Kierulf, P. and Kim, K. P. and Kislinger, T. and Klingeborn, M. and Klinke, D. J. and Kornek, M. and Kosanović, M. M. and Kovács, Á. F. and Krämer-Albers, E. M. and Krasemann, S. and Krause, M. and Kurochkin, I. V. and Kusuma, G. D. and Kuypers, S. and Laitinen, S. and Langevin, S. M. and Languino, L. R. and Lannigan, J. and Lässer, C. and Laurent, L. C. and Lavieu, G. and Lázaro-Ibáñez, E. and Le Lay, S. and Lee, M. S. and Lee, Y. X. F. and Lemos, D. S. and Lenassi, M. and Leszczynska, A. and Li, I. T. and Liao, K. and Libregts, S. F. and Ligeti, E. and Lim, R. and Lim, S. K. and Linē, A. and Linnemannstöns, K. and Llorente, A. and Lombard, C. A. and Lorenowicz, M. J. and Lörincz, Á. M. and Lötvall, J. and Lovett, J. and Lowry, M. C. and Loyer, X. and Lu, Q. and Lukomska, B. and Lunavat, T. R. and Maas, S. L. and Malhi, H. and Marcilla, A. and Mariani, J. and Mariscal, J. and Martens-Uzunova, E. S. and Martin-Jaular, L. and Martinez, M. C. and Martins, V. R. and Mathieu, M. and Mathivanan, S. and Maugeri, M. and McGinnis, L. K. and McVey, M. J. and Meckes, D. G. and Meehan, K. L. and Mertens, I. and Minciacchi, V. R. and Möller, A. and Møller Jørgensen, M. and Morales-Kastresana, A. and Morhayim, J. and Mullier, F. and Muraca, M. and Musante, L. and Mussack, V. and Muth, D. C. and Myburgh, K. H. and Najrana, T. and Nawaz, M. and Nazarenko, I. and Nejsum, P. and Neri, C. and Neri, T. and Nieuwland, R. and Nimrichter, L. and Nolan, J. P. and Nolte-'t Hoen, E. N. and Noren Hooten, N. and O'Driscoll, L. and O'Grady, T. and O'Loghlen, A. and Ochiya, T. and Olivier, M. and Ortiz, A. and Ortiz, L. A. and Osteikoetxea, X. and Østergaard, O. and Ostrowski, M. and Park, J. and Pegtel, D. M. and Peinado, H. and Perut, F. and Pfaffl, M. W. and Phinney, D. G. and Pieters, B. C. and Pink, R. C. and Pisetsky, D. S. and Pogge von Strandmann, E. and Polakovicova, I. and Poon, I. K. and Powell, B. H. and Prada, I. and Pulliam, L. and Quesenberry, P. and Radeghieri, A. and Raffai, R. L. and Raimondo, S. and Rak, J. and Ramirez, M. I. and Raposo, G. and Rayyan, M. S. and Regev-Rudzki, N. and Ricklefs, F. L. and Robbins, P. D. and Roberts, D. D. and Rodrigues, S. C. and Rohde, E. and Rome, S. and Rouschop, K. M. and Rughetti, A. and Russell, A. E. and Saá, P. and Sahoo, S. and Salas-Huenuleo, E. and Sánchez, C. and Saugstad, J. A. and Saul, M. J. and Schiffelers, R. M. and Schneider, R. and Schøyen, T. H. and Scott, A. and Shahaj, E. and Sharma, S. and Shatnyeva, O. and Shekari, F. and Shelke, G. V. and Shetty, A. K. and Shiba, K. and Siljander, P. R. and Silva, A. M. and Skowronek, A. and Snyder, O. L. and Soares, R. P. and Sódar, B. W. and Soekmadji, C. and Sotillo, J. and Stahl, P. D. and Stoorvogel, W. and Stott, S. L. and Strasser, E. F. and Swift, S. and Tahara, H. and Tewari, M. and Timms, K. and Tiwari, S. and Tixeira, R. and Tkach, M. and Toh, W. S. and Tomasini, R. and Torrecilhas, A. C. and Tosar, J. P. and Toxavidis, V. and Urbanelli, L. and Vader, P. and van Balkom, B. W. and van der Grein, S. G. and Van Deun, J. and van Herwijnen, M. J. and Van Keuren-Jensen, K. and van Niel, G. and van Royen, M. E. and van Wijnen, A. J. and Vasconcelos, M. H. and Vechetti, I. J. and Veit, T. D. and Vella, L. J. and Velot, É. and Verweij, F. J. and Vestad, B. and Viñas, J. L. and Visnovitz, T. and Vukman, K. V. and Wahlgren, J. and Watson, D. C. and Wauben, M. H. and Weaver, A. and Webber, J. P. and Weber, V. and Wehman, A. M. and Weiss, D. J. and Welsh, J. A. and Wendt, S. and Wheelock, A. M. and Wiener, Z. and Witte, L. and Wolfram, J. and Xagorari, A. and Xander, P. and Xu, J. and Yan, X. and Yáñez-Mó, M. and Yin, H. and Yuana, Y. and Zappulli, V. and Zarubova, J. and Žėkas, V. and Zhang, J. Y. and Zhao, Z. and Zheng, L. and Zheutlin, A. R. and Zickler, A. M. and Zimmermann, P. and Zivkovic, A. M. and Zocco, D. and Zuba-Surma, E. K.",
      Title = "{{M}inimal information for studies of extracellular vesicles 2018 ({M}{I}{S}{E}{V}2018): a position statement of the {I}nternational {S}ociety for {E}xtracellular {V}esicles and update of the {M}{I}{S}{E}{V}2014 guidelines}",
      Journal = "J Extracell Vesicles",
      Year = "2018",
      Volume = "7",
      Number = "1",
      Pages = "1535750",
      Abstract = {The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.},
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352}{PMC6322352}] [DOI:\href{https://dx.doi.org/10.1080/20013078.2018.1535750}{10.1080/20013078.2018.1535750}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/29904572}{29904572}]"
  }
  

• F. L. Ricklefs, Q. Alayo, H. Krenzlin, A. B. Mahmoud, M. C. Speranza, H. Nakashima, J. L. Hayes, K. Lee, L. Balaj, C. Passaro, A. K. Rooj, S. Krasemann, B. S. Carter, C. C. Chen, T. Steed, J. Treiber, S. Rodig, K. Yang, I. Nakano, H. Lee, R. Weissleder, X. O. Breakefield, J. Godlewski, M. Westphal, K. Lamszus, G. J. Freeman, A. Bronisz, S. E. Lawler, and E. A. Chiocca. Immune evasion mediated by PD-L1 on glioblastoma-derived extracellular vesicles. Sci Adv, 4(3):eaar2766, 03 2018. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842038PMC5842038] [DOI:\href https://dx.doi.org/10.1126/sciadv.aar276610.1126/sciadv.aar2766] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2361331723613317].
  [abstract▼] [details] [BibTeX▼]

  Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.

  @article{pmid29532035,
      Author = "Ricklefs, F. L. and Alayo, Q. and Krenzlin, H. and Mahmoud, A. B. and Speranza, M. C. and Nakashima, H. and Hayes, J. L. and Lee, K. and Balaj, L. and Passaro, C. and Rooj, A. K. and Krasemann, S. and Carter, B. S. and Chen, C. C. and Steed, T. and Treiber, J. and Rodig, S. and Yang, K. and Nakano, I. and Lee, H. and Weissleder, R. and Breakefield, X. O. and Godlewski, J. and Westphal, M. and Lamszus, K. and Freeman, G. J. and Bronisz, A. and Lawler, S. E. and Chiocca, E. A.",
      Title = "{{I}mmune evasion mediated by {P}{D}-{L}1 on glioblastoma-derived extracellular vesicles}",
      Journal = "Sci Adv",
      Year = "2018",
      Volume = "4",
      Number = "3",
      Pages = "eaar2766",
      Month = "03",
      Abstract = "Binding of programmed death ligand-1 (PD-L1) to programmed cell death protein-1 (PD1) leads to cancer immune evasion via inhibition of T cell function. One of the defining characteristics of glioblastoma, a universally fatal brain cancer, is its profound local and systemic immunosuppression. Glioblastoma has also been shown to generate extracellular vesicles (EVs), which may play an important role in tumor progression. We thus hypothesized that glioblastoma EVs may be important mediators of immunosuppression and that PD-L1 could play a role. We show that glioblastoma EVs block T cell activation and proliferation in response to T cell receptor stimulation. PD-L1 was expressed on the surface of some, but not of all, glioblastoma-derived EVs, with the potential to directly bind to PD1. An anti-PD1 receptor blocking antibody significantly reversed the EV-mediated blockade of T cell activation but only when PD-L1 was present on EVs. When glioblastoma PD-L1 was up-regulated by IFN-γ, EVs also showed some PD-L1-dependent inhibition of T cell activation. PD-L1 expression correlated with the mesenchymal transcriptome profile and was anatomically localized in the perinecrotic and pseudopalisading niche of human glioblastoma specimens. PD-L1 DNA was present in circulating EVs from glioblastoma patients where it correlated with tumor volumes of up to 60 cm3. These results suggest that PD-L1 on EVs may be another mechanism for glioblastoma to suppress antitumor immunity and support the potential of EVs as biomarkers in tumor patients.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5842038}{PMC5842038}] [DOI:\href{https://dx.doi.org/10.1126/sciadv.aar2766}{10.1126/sciadv.aar2766}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/23613317}{23613317}]"
  }
  

2017

• Z. Wei, A. O. Batagov, S. Schinelli, J. Wang, Y. Wang, R. El Fatimy, R. Rabinovsky, L. Balaj, C. C. Chen, F. Hochberg, B. Carter, X. O. Breakefield, and A. M. Krichevsky. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nat Commun, 8(1):1145, 10 2017. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658400PMC5658400] [DOI:\href https://dx.doi.org/10.1038/s41467-017-01196-x10.1038/s41467-017-01196-x] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2518495125184951].
  [abstract▼] [details] [BibTeX▼]

  Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.

  @article{pmid29074968,
      Author = "Wei, Z. and Batagov, A. O. and Schinelli, S. and Wang, J. and Wang, Y. and El Fatimy, R. and Rabinovsky, R. and Balaj, L. and Chen, C. C. and Hochberg, F. and Carter, B. and Breakefield, X. O. and Krichevsky, A. M.",
      Title = "{{C}oding and noncoding landscape of extracellular {R}{N}{A} released by human glioma stem cells}",
      Journal = "Nat Commun",
      Year = "2017",
      Volume = "8",
      Number = "1",
      Pages = "1145",
      Month = "10",
      Abstract = "Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reflecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal fluid.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5658400}{PMC5658400}] [DOI:\href{https://dx.doi.org/10.1038/s41467-017-01196-x}{10.1038/s41467-017-01196-x}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/25184951}{25184951}]"
  }
  

• G. M. Shankar, L. Balaj, S. L. Stott, B. Nahed, and B. S. Carter. Liquid biopsy for brain tumors. Expert Rev Mol Diagn, 17(10):943–947, 10 2017. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856481PMC5856481] [DOI:\href https://dx.doi.org/10.1080/14737159.2017.137485410.1080/14737159.2017.1374854] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2689775226897752].
  [abstract▼] [details] [BibTeX▼]

  Minimally invasive methods will augment the clinical approach for establishing the diagnosis or monitoring treatment response of central nervous system tumors. Liquid biopsy by blood or cerebrospinal fluid sampling holds promise in this regard. Areas covered: In this literature review, the authors highlight recent studies describing the analysis of circulating tumor cells, cell free nucleic acids, and extracellular vesicles as strategies to accomplish liquid biopsy in glioblastoma and metastatic tumors. The authors then discuss the continued efforts to improve signal detection, standardize the liquid biopsy handling and preparation, develop platforms for clinical application, and establish a role for liquid biopsies in personalized medicine. Expert commentary: As the technologies used to analyze these biomarkers continue to evolve, we propose that there is a future potential to precisely diagnose and monitor treatment response with liquid biopsies.

  @article{pmid28875730,
      Author = "Shankar, G. M. and Balaj, L. and Stott, S. L. and Nahed, B. and Carter, B. S.",
      Title = "{{L}iquid biopsy for brain tumors}",
      Journal = "Expert Rev Mol Diagn",
      Year = "2017",
      Volume = "17",
      Number = "10",
      Pages = "943--947",
      Month = "10",
      Abstract = "Minimally invasive methods will augment the clinical approach for establishing the diagnosis or monitoring treatment response of central nervous system tumors. Liquid biopsy by blood or cerebrospinal fluid sampling holds promise in this regard. Areas covered: In this literature review, the authors highlight recent studies describing the analysis of circulating tumor cells, cell free nucleic acids, and extracellular vesicles as strategies to accomplish liquid biopsy in glioblastoma and metastatic tumors. The authors then discuss the continued efforts to improve signal detection, standardize the liquid biopsy handling and preparation, develop platforms for clinical application, and establish a role for liquid biopsies in personalized medicine. Expert commentary: As the technologies used to analyze these biomarkers continue to evolve, we propose that there is a future potential to precisely diagnose and monitor treatment response with liquid biopsies.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856481}{PMC5856481}] [DOI:\href{https://dx.doi.org/10.1080/14737159.2017.1374854}{10.1080/14737159.2017.1374854}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/26897752}{26897752}]"
  }
  

• J. M. Figueroa, J. Skog, J. Akers, H. Li, R. Komotar, R. Jensen, F. Ringel, I. Yang, S. Kalkanis, R. Thompson, L. LoGuidice, E. Berghoff, A. Parsa, L. Liau, W. Curry, D. Cahill, C. Bettegowda, F. F. Lang, E. A. Chiocca, J. Henson, R. Kim, X. Breakefield, C. Chen, K. Messer, F. Hochberg, and B. S. Carter. Detection of wild-type EGFR amplification and EGFRvIII mutation in CSF-derived extracellular vesicles of glioblastoma patients. Neuro Oncol, 19(11):1494–1502, Oct 2017. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737576PMC5737576] [DOI:\href https://dx.doi.org/10.1093/neuonc/nox08510.1093/neuonc/nox085] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2012925120129251].
  [abstract▼] [details] [pdf] [BibTeX▼]

  RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR).\\ CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs.\\ EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61% and a specificity of 98% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM.\\ Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.

  @article{pmid28453784,
      Author = "Figueroa, J. M. and Skog, J. and Akers, J. and Li, H. and Komotar, R. and Jensen, R. and Ringel, F. and Yang, I. and Kalkanis, S. and Thompson, R. and LoGuidice, L. and Berghoff, E. and Parsa, A. and Liau, L. and Curry, W. and Cahill, D. and Bettegowda, C. and Lang, F. F. and Chiocca, E. A. and Henson, J. and Kim, R. and Breakefield, X. and Chen, C. and Messer, K. and Hochberg, F. and Carter, B. S.",
      Title = "{{D}etection of wild-type {E}{G}{F}{R} amplification and {E}{G}{F}{R}v{I}{I}{I} mutation in {C}{S}{F}-derived extracellular vesicles of glioblastoma patients}",
      Journal = "Neuro Oncol",
      Year = "2017",
      Volume = "19",
      Number = "11",
      Pages = "1494--1502",
      Month = "Oct",
      Abstract = {RNAs within extracellular vesicles (EVs) have potential as diagnostic biomarkers for patients with cancer and are identified in a variety of biofluids. Glioblastomas (GBMs) release EVs containing RNA into cerebrospinal fluid (CSF). Here we describe a multi-institutional study of RNA extracted from CSF-derived EVs of GBM patients to detect the presence of tumor-associated amplifications and mutations in epidermal growth factor receptor (EGFR).\\ CSF and matching tumor tissue were obtained from patients undergoing resection of GBMs. We determined wild-type (wt)EGFR DNA copy number amplification, as well as wtEGFR and EGFR variant (v)III RNA expression in tumor samples. We also characterized wtEGFR and EGFRvIII RNA expression in CSF-derived EVs.\\ EGFRvIII-positive tumors had significantly greater wtEGFR DNA amplification (P = 0.02) and RNA expression (P = 0.03), and EGFRvIII-positive CSF-derived EVs had significantly more wtEGFR RNA expression (P = 0.004). EGFRvIII was detected in CSF-derived EVs for 14 of the 23 EGFRvIII tissue-positive GBM patients. Conversely, only one of the 48 EGFRvIII tissue-negative patients had the EGFRvIII mutation detected in their CSF-derived EVs. These results yield a sensitivity of 61\% and a specificity of 98\% for the utility of CSF-derived EVs to detect an EGFRvIII-positive GBM.\\ Our results demonstrate CSF-derived EVs contain RNA signatures reflective of the underlying molecular genetic status of GBMs in terms of wtEGFR expression and EGFRvIII status. The high specificity of the CSF-derived EV diagnostic test gives us an accurate determination of positive EGFRvIII tumor status and is essentially a less invasive "liquid biopsy" that might direct mutation-specific therapies for GBMs.},
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737576}{PMC5737576}] [DOI:\href{https://dx.doi.org/10.1093/neuonc/nox085}{10.1093/neuonc/nox085}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/20129251}{20129251}]",
      customlinkpdf = "https://s3.amazonaws.com/cerespapers/egfrviiicsf.pdf"
  }
  

2015

• L. C. Laurent, A. B. Abdel-Mageed, P. D. Adelson, J. Arango, L. Balaj, X. Breakefield, E. Carlson, B. S. Carter, B. Majem, C. C. Chen, E. Cocucci, K. Danielson, A. Courtright, S. Das, Z. Y. Abd Elmageed, D. Enderle, A. Ezrin, M. Ferrer, J. Freedman, D. Galas, R. Gandhi, M. J. Huentelman, K. Van Keuren-Jensen, Y. Kalani, Y. Kim, A. M. Krichevsky, C. Lai, M. Lal-Nag, C. D. Laurent, T. Leonardo, F. Li, I. Malenica, D. Mondal, P. Nejad, T. Patel, R. L. Raffai, R. Rubio, J. Skog, R. Spetzler, J. Sun, K. Tanriverdi, K. Vickers, L. Wang, Y. Wang, Z. Wei, H. L. Weiner, D. Wong, I. K. Yan, A. Yeri, and S. Gould. Meeting report: discussions and preliminary findings on extracellular RNA measurement methods from laboratories in the NIH Extracellular RNA Communication Consortium. J Extracell Vesicles, 4:26533, 2015. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553263PMC4553263] [DOI:\href https://dx.doi.org/10.3402/jev.v4.2653310.3402/jev.v4.26533] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2553693425536934].
  [abstract▼] [details] [BibTeX▼]

  Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.

  @article{pmid26320937,
      Author = "Laurent, L. C. and Abdel-Mageed, A. B. and Adelson, P. D. and Arango, J. and Balaj, L. and Breakefield, X. and Carlson, E. and Carter, B. S. and Majem, B. and Chen, C. C. and Cocucci, E. and Danielson, K. and Courtright, A. and Das, S. and Abd Elmageed, Z. Y. and Enderle, D. and Ezrin, A. and Ferrer, M. and Freedman, J. and Galas, D. and Gandhi, R. and Huentelman, M. J. and Van Keuren-Jensen, K. and Kalani, Y. and Kim, Y. and Krichevsky, A. M. and Lai, C. and Lal-Nag, M. and Laurent, C. D. and Leonardo, T. and Li, F. and Malenica, I. and Mondal, D. and Nejad, P. and Patel, T. and Raffai, R. L. and Rubio, R. and Skog, J. and Spetzler, R. and Sun, J. and Tanriverdi, K. and Vickers, K. and Wang, L. and Wang, Y. and Wei, Z. and Weiner, H. L. and Wong, D. and Yan, I. K. and Yeri, A. and Gould, S.",
      Title = "{{M}eeting report: discussions and preliminary findings on extracellular {R}{N}{A} measurement methods from laboratories in the {N}{I}{H} {E}xtracellular {R}{N}{A} {C}ommunication {C}onsortium}",
      Journal = "J Extracell Vesicles",
      Year = "2015",
      Volume = "4",
      Pages = "26533",
      Abstract = "Extracellular RNAs (exRNAs) have been identified in all tested biofluids and have been associated with a variety of extracellular vesicles, ribonucleoprotein complexes and lipoprotein complexes. Much of the interest in exRNAs lies in the fact that they may serve as signalling molecules between cells, their potential to serve as biomarkers for prediction and diagnosis of disease and the possibility that exRNAs or the extracellular particles that carry them might be used for therapeutic purposes. Among the most significant bottlenecks to progress in this field is the lack of robust and standardized methods for collection and processing of biofluids, separation of different types of exRNA-containing particles and isolation and analysis of exRNAs. The Sample and Assay Standards Working Group of the Extracellular RNA Communication Consortium is a group of laboratories funded by the U.S. National Institutes of Health to develop such methods. In our first joint endeavour, we held a series of conference calls and in-person meetings to survey the methods used among our members, placed them in the context of the current literature and used our findings to identify areas in which the identification of robust methodologies would promote rapid advancements in the exRNA field.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4553263}{PMC4553263}] [DOI:\href{https://dx.doi.org/10.3402/jev.v4.26533}{10.3402/jev.v4.26533}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/25536934}{25536934}]"
  }
  

• H. Shao, J. Chung, K. Lee, L. Balaj, C. Min, B. S. Carter, F. H. Hochberg, X. O. Breakefield, H. Lee, and R. Weissleder. Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nat Commun, 6:6999, May 2015. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430127PMC4430127] [DOI:\href https://dx.doi.org/10.1038/ncomms799910.1038/ncomms7999] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2553693425536934].
  [abstract▼] [details] [BibTeX▼]

  Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.

  @article{pmid25959588,
      Author = "Shao, H. and Chung, J. and Lee, K. and Balaj, L. and Min, C. and Carter, B. S. and Hochberg, F. H. and Breakefield, X. O. and Lee, H. and Weissleder, R.",
      Title = "{{C}hip-based analysis of exosomal m{R}{N}{A} mediating drug resistance in glioblastoma}",
      Journal = "Nat Commun",
      Year = "2015",
      Volume = "6",
      Pages = "6999",
      Month = "May",
      Abstract = "Real-time monitoring of drug efficacy in glioblastoma multiforme (GBM) is a major clinical problem as serial re-biopsy of primary tumours is often not a clinical option. MGMT (O(6)-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are key enzymes capable of repairing temozolomide-induced DNA damages and their levels in tissue are inversely related to treatment efficacy. Yet, serial clinical analysis remains difficult, and, when done, primarily relies on promoter methylation studies of tumour biopsy material at the time of initial surgery. Here we present a microfluidic chip to analyse mRNA levels of MGMT and APNG in enriched tumour exosomes obtained from blood. We show that exosomal mRNA levels of these enzymes correlate well with levels found in parental cells and that levels change considerably during treatment of seven patients. We propose that if validated on a larger cohort of patients, the method may be used to predict drug response in GBM patients.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4430127}{PMC4430127}] [DOI:\href{https://dx.doi.org/10.1038/ncomms7999}{10.1038/ncomms7999}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/25536934}{25536934}]"
  }
  

2014

• F. H. Hochberg, N. A. Atai, D. Gonda, M. S. Hughes, B. Mawejje, L. Balaj, and R. S. Carter. Glioma diagnostics and biomarkers: an ongoing challenge in the field of medicine and science. Expert Rev Mol Diagn, 14(4):439–452, May 2014. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451266PMC5451266] [DOI:\href https://dx.doi.org/10.1586/14737159.2014.90520210.1586/14737159.2014.905202] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1679121416791214].
  [abstract▼] [details] [BibTeX▼]

  Glioma is the most common brain tumor. For the more aggressive form, glioblastoma, standard treatment includes surgical resection, irradiation with adjuvant temozolomide and, on recurrence, experimental chemotherapy. However, the survival of patients remains poor. There is a critical need for minimally invasive biomarkers for diagnosis and as measures of response to therapeutic interventions. Glioma shed extracellular vesicles (EVs), which invade the surrounding tissue and circulate within both the cerebrospinal fluid and the systemic circulation. These tumor-derived EVs and their content serve as an attractive source of biomarkers. In this review, we discuss the current state of the art of biomarkers for glioma with emphasis on their EV derivation.

  @article{pmid24746164,
      Author = "Hochberg, F. H. and Atai, N. A. and Gonda, D. and Hughes, M. S. and Mawejje, B. and Balaj, L. and Carter, R. S.",
      Title = "{{G}lioma diagnostics and biomarkers: an ongoing challenge in the field of medicine and science}",
      Journal = "Expert Rev Mol Diagn",
      Year = "2014",
      Volume = "14",
      Number = "4",
      Pages = "439--452",
      Month = "May",
      Abstract = "Glioma is the most common brain tumor. For the more aggressive form, glioblastoma, standard treatment includes surgical resection, irradiation with adjuvant temozolomide and, on recurrence, experimental chemotherapy. However, the survival of patients remains poor. There is a critical need for minimally invasive biomarkers for diagnosis and as measures of response to therapeutic interventions. Glioma shed extracellular vesicles (EVs), which invade the surrounding tissue and circulate within both the cerebrospinal fluid and the systemic circulation. These tumor-derived EVs and their content serve as an attractive source of biomarkers. In this review, we discuss the current state of the art of biomarkers for glioma with emphasis on their EV derivation.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5451266}{PMC5451266}] [DOI:\href{https://dx.doi.org/10.1586/14737159.2014.905202}{10.1586/14737159.2014.905202}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/16791214}{16791214}]"
  }
  

2013

• W. W. Chen, L. Balaj, L. M. Liau, M. L. Samuels, S. K. Kotsopoulos, C. A. Maguire, L. Loguidice, H. Soto, M. Garrett, L. D. Zhu, S. Sivaraman, C. Chen, E. T. Wong, B. S. Carter, F. H. Hochberg, X. O. Breakefield, and J. Skog. BEAMing and Droplet Digital PCR Analysis of Mutant IDH1 mRNA in Glioma Patient Serum and Cerebrospinal Fluid Extracellular Vesicles. Mol Ther Nucleic Acids, 2:e109, Jul 2013. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732870PMC3732870] [DOI:\href https://dx.doi.org/10.1038/mtna.2013.2810.1038/mtna.2013.28] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1679121416791214].
  [abstract▼] [details] [BibTeX▼]

  Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013.

  @article{pmid23881452,
      Author = "Chen, W. W. and Balaj, L. and Liau, L. M. and Samuels, M. L. and Kotsopoulos, S. K. and Maguire, C. A. and Loguidice, L. and Soto, H. and Garrett, M. and Zhu, L. D. and Sivaraman, S. and Chen, C. and Wong, E. T. and Carter, B. S. and Hochberg, F. H. and Breakefield, X. O. and Skog, J.",
      Title = "{{B}{E}{A}{M}ing and {D}roplet {D}igital {P}{C}{R} {A}nalysis of {M}utant {I}{D}{H}1 m{R}{N}{A} in {G}lioma {P}atient {S}erum and {C}erebrospinal {F}luid {E}xtracellular {V}esicles}",
      Journal = "Mol Ther Nucleic Acids",
      Year = "2013",
      Volume = "2",
      Pages = "e109",
      Month = "Jul",
      Abstract = "Development of biofluid-based molecular diagnostic tests for cancer is an important step towards tumor characterization and real-time monitoring in a minimally invasive fashion. Extracellular vesicles (EVs) are released from tumor cells into body fluids and can provide a powerful platform for tumor biomarkers because they carry tumor proteins and nucleic acids. Detecting rare point mutations in the background of wild-type sequences in biofluids such as blood and cerebrospinal fluid (CSF) remains a major challenge. Techniques such as BEAMing (beads, emulsion, amplification, magnetics) PCR and droplet digital PCR (ddPCR) are substantially more sensitive than many other assays for mutant sequence detection. Here, we describe a novel approach that combines biofluid EV RNA and BEAMing RT-PCR (EV-BEAMing), as well droplet digital PCR to interrogate mutations from glioma tumors. EVs from CSF of patients with glioma were shown to contain mutant IDH1 transcripts, and we were able to reliably detect and quantify mutant and wild-type IDH1 RNA transcripts in CSF of patients with gliomas. EV-BEAMing and EV-ddPCR represent a valuable new strategy for cancer diagnostics, which can be applied to a variety of biofluids and neoplasms.Molecular Therapy-Nucleic Acids (2013) 2, e109; doi:10.1038/mtna.2013.28; published online 23 July 2013.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732870}{PMC3732870}] [DOI:\href{https://dx.doi.org/10.1038/mtna.2013.28}{10.1038/mtna.2013.28}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/16791214}{16791214}]"
  }
  

2012

• H. Shao, J. Chung, L. Balaj, A. Charest, D. D. Bigner, B. S. Carter, F. H. Hochberg, X. O. Breakefield, R. Weissleder, and H. Lee. Protein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy. Nat Med, 18(12):1835–1840, Dec 2012. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518564PMC3518564] [DOI:\href https://dx.doi.org/10.1038/nm.299410.1038/nm.2994] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/2152756321527563].
  [abstract▼] [details] [BibTeX▼]

  Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell-derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.

  @article{pmid23142818,
      Author = "Shao, H. and Chung, J. and Balaj, L. and Charest, A. and Bigner, D. D. and Carter, B. S. and Hochberg, F. H. and Breakefield, X. O. and Weissleder, R. and Lee, H.",
      Title = "{{P}rotein typing of circulating microvesicles allows real-time monitoring of glioblastoma therapy}",
      Journal = "Nat Med",
      Year = "2012",
      Volume = "18",
      Number = "12",
      Pages = "1835--1840",
      Month = "Dec",
      Abstract = "Glioblastomas shed large quantities of small, membrane-bound microvesicles into the circulation. Although these hold promise as potential biomarkers of therapeutic response, their identification and quantification remain challenging. Here, we describe a highly sensitive and rapid analytical technique for profiling circulating microvesicles directly from blood samples of patients with glioblastoma. Microvesicles, introduced onto a dedicated microfluidic chip, are labeled with target-specific magnetic nanoparticles and detected by a miniaturized nuclear magnetic resonance system. Compared with current methods, this integrated system has a much higher detection sensitivity and can differentiate glioblastoma multiforme (GBM) microvesicles from nontumor host cell-derived microvesicles. We also show that circulating GBM microvesicles can be used to analyze primary tumor mutations and as a predictive metric of treatment-induced changes. This platform could provide both an early indicator of drug efficacy and a potential molecular stratifier for human clinical trials.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518564}{PMC3518564}] [DOI:\href{https://dx.doi.org/10.1038/nm.2994}{10.1038/nm.2994}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/21527563}{21527563}]"
  }
  

• M. Noerholm, L. Balaj, T. Limperg, A. Salehi, L. D. Zhu, F. H. Hochberg, X. O. Breakefield, B. S. Carter, and J. Skog. RNA expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls. BMC Cancer, 12:22, Jan 2012. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329625PMC3329625] [DOI:\href https://dx.doi.org/10.1186/1471-2407-12-2210.1186/1471-2407-12-22] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1577798315777983].
  [abstract▼] [details] [BibTeX▼]

  RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients.\\ Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups).\\ Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production.\\ Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.

  @article{pmid22251860,
      Author = "Noerholm, M. and Balaj, L. and Limperg, T. and Salehi, A. and Zhu, L. D. and Hochberg, F. H. and Breakefield, X. O. and Carter, B. S. and Skog, J.",
      Title = "{{R}{N}{A} expression patterns in serum microvesicles from patients with glioblastoma multiforme and controls}",
      Journal = "BMC Cancer",
      Year = "2012",
      Volume = "12",
      Pages = "22",
      Month = "Jan",
      Abstract = "RNA from exosomes and other microvesicles contain transcripts of tumour origin. In this study we sought to identify biomarkers of glioblastoma multiforme in microvesicle RNA from serum of affected patients.\\ Microvesicle RNA from serum from patients with de-novo primary glioblastoma multiforme (N = 9) and normal controls (N = 7) were analyzed by microarray analysis. Samples were collected according to protocols approved by the Institutional Review Board. Differential expressions were validated by qRT-PCR in a separate set of samples (N = 10 in both groups).\\ Expression profiles of microvesicle RNA correctly separated individuals in two groups by unsupervised clustering. The most significant differences pertained to down-regulated genes (121 genes > 2-fold down) in the glioblastoma multiforme patient microvesicle RNA, validated by qRT-PCR on several genes. Overall, yields of microvesicle RNA from patients was higher than from normal controls, but the additional RNA was primarily of size < 500 nt. Gene ontology of the down-regulated genes indicated these are coding for ribosomal proteins and genes related to ribosome production.\\ Serum microvesicle RNA from patients with glioblastoma multiforme has significantly down-regulated levels of RNAs coding for ribosome production, compared to normal healthy controls, but a large overabundance of RNA of unknown origin with size < 500 nt.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329625}{PMC3329625}] [DOI:\href{https://dx.doi.org/10.1186/1471-2407-12-22}{10.1186/1471-2407-12-22}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/15777983}{15777983}]"
  }
  

2010

• C. Chen, J. Skog, C. H. Hsu, R. T. Lessard, L. Balaj, T. Wurdinger, B. S. Carter, X. O. Breakefield, M. Toner, and D. Irimia. Microfluidic isolation and transcriptome analysis of serum microvesicles. Lab Chip, 10(4):505–511, Feb 2010. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136803PMC3136803] [DOI:\href https://dx.doi.org/10.1039/b916199f10.1039/b916199f] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1511132715111327].
  [abstract▼] [details] [BibTeX▼]

  Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.

  @article{pmid20126692,
      Author = "Chen, C. and Skog, J. and Hsu, C. H. and Lessard, R. T. and Balaj, L. and Wurdinger, T. and Carter, B. S. and Breakefield, X. O. and Toner, M. and Irimia, D.",
      Title = "{{M}icrofluidic isolation and transcriptome analysis of serum microvesicles}",
      Journal = "Lab Chip",
      Year = "2010",
      Volume = "10",
      Number = "4",
      Pages = "505--511",
      Month = "Feb",
      Abstract = "Microvesicles (exosomes) shed from both normal and cancerous cells may serve as means of intercellular communication. These microvesicles carry proteins, lipids and nucleic acids derived from the host cell. Their isolation and analysis from blood samples have the potential to provide information about state and progression of malignancy and should prove of great clinical importance as biomarkers for a variety of disease states. However, current protocols for isolation of microvesicles from blood require high-speed centrifugation and filtration, which are cumbersome and time consuming. In order to take full advantage of the potential of microvesicles as biomarkers for clinical applications, faster and simpler methods of isolation will be needed. In this paper, we present an easy and rapid microfluidic immunoaffinity method to isolate microvesicles from small volumes of both serum from blood samples and conditioned medium from cells in culture. RNA of high quality can be extracted from these microvesicles providing a source of information about the genetic status of tumors to serve as biomarkers for diagnosis and prognosis of cancer.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3136803}{PMC3136803}] [DOI:\href{https://dx.doi.org/10.1039/b916199f}{10.1039/b916199f}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/15111327}{15111327}]"
  }
  

2008

• J. Skog, T. Würdinger, S. van Rijn, D. H. Meijer, L. Gainche, M. Sena-Esteves, W. T. Curry, B. S. Carter, A. M. Krichevsky, and X. O. Breakefield. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol, 10(12):1470–1476, Dec 2008. [PubMed Central:\href https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423894PMC3423894] [DOI:\href https://dx.doi.org/10.1038/ncb180010.1038/ncb1800] [PubMed:\href https://www.ncbi.nlm.nih.gov/pubmed/1842511418425114].
  [abstract▼] [details] [pdf] [BibTeX▼]

  Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.

  @article{pmid19011622,
      Author = "Skog, J. and Würdinger, T. and van Rijn, S. and Meijer, D. H. and Gainche, L. and Sena-Esteves, M. and Curry, W. T. and Carter, B. S. and Krichevsky, A. M. and Breakefield, X. O.",
      Title = "{{G}lioblastoma microvesicles transport {R}{N}{A} and proteins that promote tumour growth and provide diagnostic biomarkers}",
      Journal = "Nat Cell Biol",
      Year = "2008",
      Volume = "10",
      Number = "12",
      Pages = "1470--1476",
      Month = "Dec",
      Abstract = "Glioblastoma tumour cells release microvesicles (exosomes) containing mRNA, miRNA and angiogenic proteins. These microvesicles are taken up by normal host cells, such as brain microvascular endothelial cells. By incorporating an mRNA for a reporter protein into these microvesicles, we demonstrate that messages delivered by microvesicles are translated by recipient cells. These microvesicles are also enriched in angiogenic proteins and stimulate tubule formation by endothelial cells. Tumour-derived microvesicles therefore serve as a means of delivering genetic information and proteins to recipient cells in the tumour environment. Glioblastoma microvesicles also stimulated proliferation of a human glioma cell line, indicating a self-promoting aspect. Messenger RNA mutant/variants and miRNAs characteristic of gliomas could be detected in serum microvesicles of glioblastoma patients. The tumour-specific EGFRvIII was detected in serum microvesicles from 7 out of 25 glioblastoma patients. Thus, tumour-derived microvesicles may provide diagnostic information and aid in therapeutic decisions for cancer patients through a blood test.",
      Note = "[PubMed Central:\href{https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3423894}{PMC3423894}] [DOI:\href{https://dx.doi.org/10.1038/ncb1800}{10.1038/ncb1800}] [PubMed:\href{https://www.ncbi.nlm.nih.gov/pubmed/18425114}{18425114}]",
      customlinkpdf = "https://s3.amazonaws.com/cerespapers/ncb1800.pdf"
  }