Apr 29, 2015 - Introduction to extracellular vesicles (Exosomes and microvesicles) and clinical .... Proteomics, lipidomics, transcriptomics, genomics.
Introduction to extracellular vesicles (Exosomes and microvesicles) and clinical implications
Dr. Muhammad Nawaz Department of Pathology and Forensic Medicine Ribeirao Preto Medical School – USP
Background and history • Discovery of vesicles, and the journey • Need for Extracellular vesicles Society • Discussing issues, nomenclature, standardizing the protocols, training workshops and so forth
• Establishing Extracellular vesicles journal (JEV) • Extracellular vesicle meetings (Society meeting each year) • Databases and portals
• Chapters in different regions • And the success story (exciting and revolutionized field of research with new/alternative solutions)
Nobel Laureate in the field: James Rothman: Yale, 2013
Society
Official journal of the Society
Database, Community Portal
Database
Database
(ASEMV)
UK forum for extracellular Vesicles
Nobel Laureate in the field: James Rothman: Yale, 2013
Useful links International Society of Extracellular Vesicles (ISEV) http://isev.org/ Journal of Extracellular Vesicles (JEV) http://isev.org/jev/ (Official journal of the Society)
EVpedia - Extracellular vesicle database for high-throughput analysis http://student4.postech.ac.kr/evpedia2_xe/xe/ Vesiclepedia http://microvesicles.org/ American Society of Exosomes and Microvesicles http://www.asemv.org/ UK Extracellular Vesicles Forum http://www.ukev.org.uk/
Exocarta http://www.exocarta.org/
Extracellular vesicles (EVs):
Nano-sized vesicles which, based on their size, origin, morphology, and mode of release, are categorized into exosomes (40-200 nm), microvesicles (50-1000 nm), apoptotic bodies (50-5000 nm). • are the major classes, most often studied and well characterized • However, there are several other classes/populations of vesicles
Several other classes of EVs classified and named based on their origin, size, morphology and functions
Biogenesis and secretion/release of EVS Well described are Exosomes and microvesicles
Nawaz et al. 2014. PMID: 25403245
Principal classes
Exosomes (40-200nm)
Microvesicles/ Ectosomes (50-1000nm)
Apoptotic Bodies (50-5000nm)
Produced through Apoptosis Produced from outer membrane shedding Produced through Endoctytic/ exocytic pathway
Nawaz et al., 2014: Nature Reviews Urology, 11(12): 688-701. PMID: 25403245
Exosomes
Nawaz et al. 2014. PMID: 25403245
Masyuk. et al., 2010
MVBs (multivesicular bodies) and intraluminal vesicles (ILVs) Pegtel et al ., 2010
Multivesiclular body (MVB) containing ILVs
Raposo and Stoorvogel 2013. JCB, 200 (4): 373-383
PubMed hits, searched on – 12-06-2015 Extracellular Vesicles
Exosomes
Ectosomes
Microvesicles
Exosomes in Brazil
What are the components of Extracellular vesicles? Typical vesicle (EV), carry a plethora of biological molecules: mRNAs, miRNA, LncRNAs, gDNA, mtDNA, proteins, lipids, glycans Complete data sets are available at EV databases (EVpedia, vesiclepedia, ExoCarta etc.)
What are the most often recognized functions of EVs • • • • • • • • •
• • • •
Intercellular communication (cross-talk), signalling Exchange of genetic material (Horizontally), proteins, lipids etc. Induction of novel phenotypes into recipient cells Modulation of microenvironment Cellular migration, invasion, Tumour progression Tumor angiogenesis Immune responses, immune surveillance Inflammation (acute and chronic Inflammatory disorders, cardiovascular diseases and vascular disorders), Stem cell biology, development, tissue homeostatic, tissue remodeling, cellular differentiation, self renewal, tissue regeneration, repair Nervous system Infectious diseases (viral, parasitic, bacterial and fungal infections) Fertility, reproduction & pregnancy In fact, diverse functions
Communication: Surface interactions (cell-to-cell and/or exosome-to-cell) Vesicle based Cell-to-cell communication and horizontal transfer of biological material (RNAs, protein, lipids) to recipient cells
Exosome uptake by recipient cells
Raposo and Stoorvogel 2013. JCB, 200 (4): 373-383
Cell-to-cell communication and transfer of signals, molecules to recipient cell via Extracellular vesicles (Exosomes and microvesicles)
Transfer of genetic material: First report on mRNA transfer Ratajczak et al., 2006
First systematic and comprehensive report on microRNA, mRNAs, along with several other classes of coding and non coding RNAs presence in exosomes and their shuttling (esRNA) to recipient cells. (Valadi et al., 2007) from the lab of Jan O. Lotvall – Gothenburg, Sweden This study has proven a milestone to develop strategies for the delivery of therapeutic miRNAs and siRNAs using exosomes as delivery vectors. (authors have some patents)
Indeed RNAs shuttled to recipient cells are functional and could be translated into proteins within recipient cells.
Selectively enriched (customized) exosomal miRNAs and their transfer to other cells
Source of extracellular vesicles? Mostly from the body fluids: • Blood (plasma, serum) • Saliva • Spinal Fluid • Breast Milk • Lymph • Sweat • Urine • Prostatic secretions • Cysts • Cell cultures • Can also be obtained directly from Tissues
Analysis: • Preparative strategies • Isolation, purification • Characterization • Downstream analysis • Proteomics, lipidomics, transcriptomics, genomics •
Nawaz et al. 2014. PMID: 25403245
From Urine
From cultured media
From Blood
Characterization and downstream analysis of exosomes
Nawaz et al. 2014. PMID: 25403245
Could this knowledge be used for prospective therapeutics and clinical settings?
Exosomes from Stem cells?????
Exosomes as nanocarriers for drug delivery/ cancer therapy
Exosomes: vehicles of gene therapy
Exosomes as nanocarriers for drug delivery
Exosomes as nanocarriers for miRNAs delivery
Exosomes as nanocarriers for miRNAs delivery
Exosomes vehicles for delivery of siRNA
Exosomes vehicles for delivery of siRNA
How it come true, transferring our miRNAs/siRNAs to target cells?
Two major strategies Electroporation
Transfection
Target gene silencing
Exosome / nanovesicle or liposome Electroporation siRNA
miRNA or siRNA
Transfection Co-culture these siRNA loaded exosomes with cancer cells
siRNA entered to cancer cells siRNA
Targets?????
PLoS One. 2015; 10(4): e0122991. Published online 2015 Apr 29.
What should be considered most critical??? • • • • • • • •
RNA loading efficacy Protection against degradation Lack of immunogenicity Targeting possibilities Cellular uptake Cytotoxicity RNA release into the cytoplasm Gene silencing efficiency
Advantages of exosomes for gene delivery??? • Since exosomes naturally transport genetic materials between cells, and derived naturally from the body (i.e. the body’s own vector), these vesicles from human might be ideal candidates as vector in gene therapy. • Without toxic effects. • No immunogenic concerns Although, exosome based siRNA delivery system shows many advantages over conventional transfection agents. However, some crucial issues need further optimization before broad clinical application can be realized???
Nanovesicles: The mini creature for better tomorrow??
Predictive/prognostic and diagnostic biomarkers Biopsies are invasive procedures and are associated with significant health problems (bleeding, infection, etc.) EVs as non-invasive liquid biopsies: Transition from tissue to liquid biopsies
• The identification of molecular signatures in biological fluids could create ‘liquid biopsies’, which would effectively overcome many of the challenges associated with traditional tissue sampling (such as invasiveness and tumour heterogeneity). • These Vesicles (EVs), could minimize potential safety risks as being non-invasive entities • EVs are thought to reflect molecular composition of the secreting cell, therefore could serve as attractive source of biomarkers. • Concentration and expression is grade specific, subtype specific (some express during early stage of the disease while some are expressed at late stage), therefore could serve as predictive/ prognostic biomarkers. • Could provide information regarding Relapse, recurrence, chemoresistance
Predictive/prognostic and diagnostic biomarkers
• The transcriptomic and genetic profiling of EVs
• EVs associated molecular signatures in combination with previously existing biomarkers. • Strategies to capture and develop biomarkers? • Proteomics of EVs, Transcriptomics, Lipidomics, genomics • High throughput proteomic approaches? • Suitable combination of high throughput technology
Nawaz et al. 2014. PMID: 25403245
Useful links International Society of Extracellular Vesicles (ISEV) http://isev.org/ Journal of Extracellular Vesicles (JEV) http://isev.org/jev/ (Official journal of the Society)
EVpedia - Extracellular vesicle database for high-throughput analysis http://student4.postech.ac.kr/evpedia2_xe/xe/ Vesiclepedia http://microvesicles.org/ American Society of Exosomes and Microvesicles http://www.asemv.org/ UK Extracellular Vesicles Forum http://www.ukev.org.uk/
Exocarta http://www.exocarta.org/
References 1. Nawaz, M., Camussi, G., Valadi, H., Nazarenko, I., Ekstrom, K., Wang, X., Principe, S., Shah, N., Ashraf, N.M., Fatima, F. et al. The emerging role of extracellular vesicles as biomarkers for urogenital cancers. Nature reviews. Urology, 2014. 11, 688-701. 2. Nawaz, M., Fatima, F., Nazarenko, I., Ekstrom, K., Murtaza, I., Anees, M., Sultan, A., Neder, L., Camussi, G., Valadi, H. et al. Extracellular vesicles in ovarian cancer: applications to tumor biology, immunotherapy and biomarker discovery. Expert review of proteomics, 2016, 13, 395-409. 3. Fatima, F. and Nawaz, M. Stem cell-derived exosomes: roles in stromal remodeling, tumor progression, and cancer immunotherapy. Chinese journal of cancer, 2015, 34, 541-553. 4. Nawaz, M., Fatima, F., Zanetti, B., Martins, I., Schiavotelo, N., Mendes, N., Silvestre, R. and Neder, L. Microvesicles in Gliomas and Medulloblastomas: An Overview. Journal of Cancer Therapy, 2014, 5, 10. 5. Fatima, F. and Nawaz, M. Vesiculated Long Non-Coding RNAs: Offshore Packages Deciphering Trans-Regulation between Cells, Cancer Progression and Resistance to Therapies. Non-Coding RNA, 2017, 3, 10. 6. Nawaz, M., Fatima, F., Vallabhaneni, K.C., Penfornis, P., Valadi, H., Ekstrom, K., Kholia, S., Whitt, J.D., Fernandes, J.D., Pochampally, R. et al. Extracellular Vesicles: Evolving Factors in Stem Cell Biology. Stem cells international, 2016, 2016:1073140. 7. Fatima, F. and Nawaz, M. Nexus between extracellular vesicles, immunomodulation and tissue remodeling: for good or for bad?. Ann Transl Med, 2017, 5(6):139. 8. The 150 most important questions in cancer research and clinical oncology series: questions 6–14: Edited by Chinese Journal of Cancer. Chinese J cancer, 2017, 36(1):31.
