Introduction to Exosomes

Exosomes are microscopic vesicles, 30—150 nm in diameter, which are secreted by almost all cell types and are also present in almost every biological fluid1. Lipids, proteins, and nucleic acids comprise the complex exosome structure and function as signalosomes that mediate information transfer between cells2. Exosomes also have a number of transmembrane proteins protruding from the surface envelope including tetraspanins (e.g. CD9 and CD63), lipid rafts (e.g. flotillin-1) and proteins involved in antigen presentation (e.g. MHCI and MHCII).  While exosomes were originally considered a mechanism to discarding unwanted cellular debris, today these nano-sized membrane vesicles are valued as highly relevant therapeutic targets for many diseases due their roles in disease transmission, intercellular cross-talk, angiogenesis, and inflammation3-10.  Specifically, exosomes are implicated in the progression of many diseases. For instance, exosomes are established to contribute the plaque formation associated with Alzheimer's disease via the accumulation of β-amyloid (Aβ) peptides that are released from these microvesicles11. Additionally, exosome-secreted biomolecules are confirmed to facilitate the development of many cancers through cellular communication in the tumor microenvironment12.

Isolation of exosomes

ORB can isolate exosomes using a choice of commercial kits (such as101Bio, Wako Chemicals, and Izon) as well as through classical ultracentrifugation-based isolation method which is particularly well-suited for large volume samples. Sample types suitable for exosome isolation include serum, plasma, saliva, urine, milk, and bronchial lavage, as well as cerebrospinal, lymphatic, and ascites fluids

Quantification and sizing of exosomes

Exosome sizing and quantification using NS300
Figure 1. Microvesicle characterization using Nanosight NS300. Size distribution and concentration of three exosome samples following analysis on NS300. The size of the purified exosomes are shown on the x-axis while the concentration of particles are displayed on the y-axis.

ORB can identify the size distribution and concentration of exosomes, using the powerful NanoSight 300 (NS300) instrument. . Data generated by the NS300 (Fig. 1) is compiled into a report that includes images, videos, bar-charts, and histograms showing the size distribution and concentration of nanoparticles in the samples.  Sizing and quantification allow determination of the different populations of exosomes present in the biosamples prior to subsequent analysis. This quantitative and visual analysis provide a first characterization of the exosomes potentially involved in pathological processes of diseases including tumor and neurodegenerative diseases.

RNA Profiling Exosomes

Investigations of exosome-bound genetic information, which can include long and small RNAs, can be used to identify cell– or tissue-specific markers and detect diagnostic and prognostic biomarkers useful for patient stratification or the determination of non-responders13-15. Given that exosomes are established transmitters of microRNAs that are associated with the development and metastasis of many cancers, exosomal RNAs are widely accepted as oncology biomarkers to identify the presence or severity of solid tumor and hematologic malignancies12. Reports also specifically implicate exosomal mRNA markers in drug-resistance16. 

Substantial data exists from the examinations of exosomal microRNA using microarray and real-time quantitative PCR assays, and more recently broader characterizations of exosomal RNA using sequencing technologies have become standard practice, including the interrogation of exosomal long RNA16.

Exosome RNA Profiling at ORB

ORB provides a complete service solution for exosomal RNA profiling. ORB can isolate exosomes from biofluids, perform size-distribution analysis and extract RNA from exosomes using a choice of commercial kits available from such manufacturers as Norgen and Qiagen. Alternatively, company scientists can extract RNA from exosomes prepared by clients, which typically include exosomes isolation by ultracentrifugation from large volume of conditioned media or urine. Whether extracting RNA from internally isolated exosomes or those provided by clients, ORB has the experience to design and interpret custom quality control assays to determine whether the extracted RNA is suitable for further processing. Biofluids suitable for exosomal RNA analysis include serum, plasma, saliva, urine, milk, and bronchial lavage, as well as cerebrospinal, lymphatic, and ascites fluids.

ORB’s offerings to characterize and quantitate exosomal RNA can be divided into small and long RNA profiling services. Small RNA biotypes can be investigated using microRNA microarray or sequencing technologies. To support wet-lab assays, ORB also provides thoroughly validated bioinformatic analysis and predictive modeling services to advance basic research, commercial drug  and biomarker discovery efforts or companion diagnostics projects.  Follow the links below for more information about the company's exosomal profiling capabilities and related services.


ORB's Track Record in Exosomal RNA Profiling

ORB has a large amount of experience in exosomal RNA profiling and has contributed to many important and pioneering exosome studies with microRNA microarray and small RNA sequencing services.

In an investigation which sought to determine reliable blood-based markers that can be used as surrogates for tumor biopsies in the diagnosis of ovarian cancer, ORB analyzed mature microRNA signatures of tumor-derived exosomes isolated from serum of papillary adenocarcinoma patients using ORB’s custom-developed microRNA microarray13. In another study, which utilized ORB’s proprietary microRNA microarray to interrogate tumor-derived microRNA patterns in exosomes, circulating exosome-based diagnostic biomarkers that are associated with lung adenocarcinoma were detected17. ORB also provided microarray profiling of low molecular weight RNA extracted exosomes derived from PC12 cells to determine how exosomal microRNAs contribute to the induction of neuronal differentiation in stem cells18

ORB’s small RNA sequencing service was used to examine how seminal fluid exosomal non-coding RNAs contribute to the immunoregulation of the recipient's mucosa. In addition to known microRNAs, this study determined that tRNAs, Y RNAs and protein-coding mRNA fragments may also be involved in the immunosuppressive effects associated with semen19.

Demonstrations of ORB’s Exosomal RNA Profiling Capabilities

Demonstration studies are described and data presented on both the long RNA (mRNA) and small RNA exosome RNA sequencing pages. 

 

Contact us to discuss your specific exosome-related research project today.

References
  1. Zhang, J., Li, S., Li, L., Li, M., Guo, C., Yao, J., & Mi, S. (2015). Exosome and exosomal microRNA: trafficking, sorting, and function. Genomics, proteomics & bioinformatics13(1), 17-24.
  2. Raposo, G., & Stoorvogel, W. (2013). Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol200(4), 373-383.
  3. Alvarez-Erviti, L., Seow, Y., Schapira, A. H., Gardiner, C., Sargent, I. L., Wood, M. J., & Cooper, J. M. (2011). Lysosomal dysfunction increases exosome-mediated alpha-synuclein release and transmission. Neurobiology of disease42(3), 360-367.
  4. Bukong, T. N., Momen-Heravi, F., Kodys, K., Bala, S., & Szabo, G. (2014). Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90. PLoS Pathog10(10), e1004424.
  5. Munich, S., Sobo-Vujanovic, A., Buchser, W. J., Beer-Stolz, D., & Vujanovic, N. L. (2012). Dendritic cell exosomes directly kill tumor cells and activate natural killer cells via TNF superfamily ligands. Oncoimmunology1(7), 1074-1083.
  6. Mulcahy, Laura Ann, Ryan Charles Pink, and David Raul Francisco Carter. "Routes and mechanisms of extracellular vesicle uptake." Journal of extracellular vesicles 3 (2014).
  7. van Balkom, B. W., De Jong, O. G., Smits, M., Brummelman, J., den Ouden, K., de Bree, P. M., & Verhaar, M. C. (2013). Endothelial cells require miR-214 to secrete exosomes that suppress senescence and induce angiogenesis in human and mouse endothelial cells. Blood121(19), 3997-4006.
  8. Lee, J. K., Park, S. R., Jung, B. K., Jeon, Y. K., Lee, Y. S., Kim, M. K., & Kim, C. W. (2013). Exosomes derived from mesenchymal stem cells suppress angiogenesis by down-regulating VEGF expression in breast cancer cells. PloS one8(12), e84256.
  9. Bala, S., Petrasek, J., Mundkur, S., Catalano, D., Levin, I., Ward, J., & Szabo, G. (2012). Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug‐induced, and inflammatory liver diseases. Hepatology56(5), 1946-1957.
  10. Kim, S. H., Lechman, E. R., Bianco, N., Menon, R., Keravala, A., Nash, J., & Robbins, P. D. (2005). Exosomes derived from IL-10-treated dendritic cells can suppress inflammation and collagen-induced arthritis. The Journal of Immunology174(10), 6440-6448.
  11. Rajendran, L., Honsho, M., Zahn, T. R., Keller, P., Geiger, K. D., Verkade, P., & Simons, K. (2006). Alzheimer's disease β-amyloid peptides are released in association with exosomes. Proceedings of the National Academy of Sciences103(30), 11172-11177.
  12. Azmi, A. S., Bao, B., & Sarkar, F. H. (2013). Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review. Cancer and Metastasis Reviews32(3-4), 623-642.
  13. Taylor, D. D., & Gercel-Taylor, C. (2008). MicroRNA signatures of tumor-derived exosomes as diagnostic biomarkers of ovarian cancer. Gynecologic oncology110(1), 13-21.
  14. Nilsson, J., Skog, J., Nordstrand, A., Baranov, V., Mincheva-Nilsson, L., Breakefield, X. O., & Widmark, A. (2009). Prostate cancer-derived urine exosomes: a novel approach to biomarkers for prostate cancer. British journal of cancer100(10), 1603-1607.
  15. Brock, G., Castellanos-Rizaldos, E., Hu, L., Coticchia, C., & Skog, J. (2015). Liquid biopsy for cancer screening, patient stratification and monitoring. Translational Cancer Research4(3), 280-290.
  16. Shao, H., Chung, J., Lee, K., Balaj, L., Min, C., Carter, B. S., ... & Weissleder, R. (2015). Chip-based analysis of exosomal mRNA mediating drug resistance in glioblastoma. Nature communications6.
  17. Rabinowits, G., Gerçel-Taylor, C., Day, J. M., Taylor, D. D., & Kloecker, G. H. (2009). Exosomal microRNA: a diagnostic marker for lung cancer. Clinical lung cancer10(1), 42-46.
  18. Takeda, Y. S., & Xu, Q. (2015). Neuronal differentiation of human mesenchymal stem cells using exosomes derived from differentiating neuronal cells. PloS one10(8), e0135111.
  19. Vojtech, L., Woo, S., Hughes, S., Levy, C., Ballweber, L., Sauteraud, R. P., ... & Hladik, F. (2014). Exosomes in human semen carry a distinctive repertoire of small non-coding RNAs with potential regulatory functions. Nucleic acids research, gku347.