Extra cellular vesicles

• Extracellular Vesicles


  Structures derived from the cell and expelled into peri-cellular space
• Microvesicles (ectosomes)


  • Derived from the outward budding of the plasma membrane
  • Membranous

  
• Exosomes


  • Synthesised from endocytic compartments of the cell
  • Released via exocytosis

  
• Migrasomes


  • Structurally similar to MVE
  • Contains multiple small vesicles
  • Deposited on cell projections (membranous stalk - kind of like footprints to follow)
  • Can be taken by other cells
• Oncosomes


  • Oncogenic vesicles found in cancer
• Formation of intraluminal vesicles

  1. ESCRT recognise ubiquitied proteins and transport them into the budding/folding membrane in the endosome

  2. Ubiquitination can mark proteins for transfer into endosome and hence an enrichment is possible

  
• Sphingomyelinase


  1. Sphingomyelin found on the plasma membrane of the endosome

  2. It is broken down into ceramide > spontaneous folding of membrane and vesicle formation

  
• Expulsion of MVE content

  1. Dependent on cell and function, cells can pack target molecules for release/expulsion into pre-exosomes (molecular enrichment)

  2. Fusion of MVB with the plasma membrane

  3. Release of vesicles into the EC space

  
• Diversity and heterogeneity of extracellular vesicles

  • Different shapes and sizes
  • Different internal content
  • Differences in molecular phenotype

  
• Plasma membrane derived vesicles

  • Membrane bleb - spherical membrane protrusion that is Ca regulated with involvement of a range of enzymes
  • Phosphatidylserine PS molecule found predominantly on the inner leaflet of the plasma membrane
  • As bleb begins, PS can be seen distributed evenly on both sides of the membrane (important feature - usually for macrophages to identify apoptotic cells)

  
• EV budding in microvilli

  • Vesicle structures: Tubule-like structures that could have cargo
  • Vesicle within vesicle (multi laminar structures)
  • Degradative compound carrying high quantities of cargo
  • Single, uni laminar vesicles are predominant, taking up 80% of the population

  
• Exosome biogenesis and secretion: regulation by endogenous factors

  1. Biogenesis: ESCRT dependent, ESCRT independent, Tetraspanins

  2. Transportation and docking to the plasma membrane: Rab proteins, Myosin, Microtubules

  3. Fusion and liberation of intra luminal vesicle cargo: SNAREs

  
• Theory of different vesicle formation
• Exogenous factors impact secretion and cargo
• Process of isolation


  1. Ultracentrifugation coupled with sucrose gradient

  2. Affinity methods: beads, plates or columns

  3. Chromatography

  4. Precipitation methods

  5. Filtration methods
• Functions in activating T cells

  • Exosomes can carry MHC II which is a molecule involved in the activation of CD4 T cells into T helper cells
  • B cell derived exosomes are shown to be able to stimulate proliferation in CD4 T cells
• DC is stimulated with antigenic peptides from cancer cells


  Immunisation with live cell or derived exosome > exosome immunisation was able to induce protective, long term immunity against that cancer cell, a much stronger response than the parent cell
• Possible reason for stronger immune response with exosome immunisation

  • DC respond to the immunosuppressive environment induced by the tumour and elicit a weak immune response
  • Exosome, a cell product, however lacks that ability to adapt and change. This is perhaps it is still able to induce a large immune response irrespective of the immunosuppressive environment
• DC exosome based vaccination therapy in cancer
• Functions in inhibiting immune cells

  • Cancer exosomes were able to prevent proliferation in T cells in the presence of an artificial/normal physiological stimuli. The interaction appears to be independent of the stimulus
  • Exosomes elicit a range of mechanisms to inhibit the proliferation of T cells: Delivery of TGFB stimulation of Treg cells > inhibits other T cell responses, Fas-L on surface > contact with Fas on T cell T cell apoptosis, NKG2DL decrease cytokine secretion from T cells and decrease NK cell responses, CD39 and 73 enzymes that convert ATP to adenosine > regulation of TCR-dependent activation
• Functions in remodeling TME (stroma)

  • Fibroblast Myofibroblast (by TGFB shown by expression of aSMA)
  • Myofibroblasts have contractile activity (able to pull things together), release a range of matrix-modulating factors and changes collagen deposition > physical change and architecture alteration in the tissue
  • Exosome treated cells are able to interact with endothelial cells and promote their survival and organisation in a hostile environment similar to disease (similar to angiogenesis)
  • TGFB-treated stromal cells inserted into mice with tumour cells are tumour protective and prevented growth. However, the EV treated stroma was able to accelerate tumour growth almost to the level of tumour treated stroma
  • Hypoxic stimulation can also cause increased packing of VEGF and IL8 (pro-angiogenic factors). In vivo model also demonstrated increased endothelial cell and blood vessel formation and organisation compared to normoxic conditions
• Activation of therapeutic resistant angiogenesis

  • Soluble VEGF delivery is able to induce angiogenesis in the tumour but inhibited by Bevacizumab (mAb targeting all isoforms of VEGF)
  • sEV-delivery is similar in induction of angiogenesis but Bevacizumab has no effect. The VEGF might be enclosed in the vesicle, protected by the membrane, therefore the Ab has no access. Another explanation could be VEGF is associated with something on the outer membrane that prevents access (steric hindrance)
• Standard model for metastasis
• Exosomal integrins may act to control organ-tropism and metasis

  • Researchers isolated cancer cells known to metastasise to the lung and liver. They extracted exosomes from these cells and swapped the exosomes with each other. Lung cell with liver exosome and liver cell with lung exosome). The lung cells now metastasise to the liver and vice versa. The integrins on exosomes allow targeting of the organ and change the environment for easier tumour invasion
• Examples in subverting treatments
• Expulsion of MVE contents (ILLVʼs)

  1. Fusion of MVB (multi vesicular bodies) with plasma membrane

  2. Release of vesicles into the extracellular space
• Molecular enrichment


  Extracellular Vesicles

  
• Diversity & Heterogeneity of vesicles

  • Structural features of small vesicles
  • Different shapes and sizes
  • Different internal "cargoˮ
  • Differences in molecular phenotype

  
• Migrosomes


  • Structurally similar to MVB
  • Contains multiple small vesicles
  • Deposited on cell projections
  • Can be taken up by other cells
• Plasma membrane derived vesicles - ectosome

  • Calcium regulated
  • Switch of phosphatidyl serine on inner leaflet of normal cells
  • Even distribution of PS (phosphotidylserine) on inner and outer membrane
  • Important feature - usually for macrophages to identify apoptotic cells
  • Hard to distinguish if its a endosome or a plasma membrane derived entity

  
• Evidence for EV budding at microvilli
• Oncosomes


  Skeptical on this
• Other vesicle (or vesicle-like) structures

  • Irregular shape
  • Vesicles within vesicles
  • With or withour cargo
• Exosome Biogenesis & Secretion: Regulation by endogenous factors


  1. Biogenesis: ESCRT dependent, ESCRT independent, Tetraspanins

  2. Transportation and docking to the plasma membrane: Rab proteins (molecular switches), Myosin, Microtubules

  3. Fusion and liberation of intra luminal vesicle cargo: SNAREs

  
• How might we isolate EV to study their content and functions

  1. Ultracentrifugation coupled with sucrose gradient

  2. Affinity methods: beads, plates or columns

  3. Chromatography

  4. Precipitation methods

  5. Filtration methods
• Emergence of MHC2 at 70000g


  Other methods for vesicle isolation
• Functions of vesicles in activating T cells

  • Some vesicles high in antigen presenting machinery
  • Direct stimulation of T cells (CD4+ T cells)
  • Exosomes can carry MHC II which is a molecule involved in the activation of CD4 T cells into T helper cells
  • B cell derived exosomes are shown to be able to stimulate proliferation in CD4 T cells
• MHC Class II stimulate helper CD4 T cell
• Direct stimulation of T cells

  (CD4+ and CD8+ T cells)
• DC is stimulated with antigenic peptides from cancer cells


  1. Exosome immunisation was able to induce protective, long term immunity against that cancer

  2. Possible reason: DC respond to the immunosuppressive environment induced by the tumour and elicit a weak immune response

  3. Exosome, a cell product, however lacks that ability to adapt and change. This is perhaps it is still able to induce a large immune response irrespective of the immunosuppressive environment