L16 March 13 EVs

Question 1

1. What are extracellular vesicles?
2. How made/released
3. Fxn
4. What areas found in
5. What are its pathological processes?
6. 2 main classes

Answer 1

1. Lipid membrane encapsulated vesicles
2. Released by cells into intercellular space in response to cell stress
3. Function in cell-cell communication. Participate in a variety of normal physiological processes: blood coagulation, immunity, differentiation, angiogenesis, tissue regeneration
4. Found in bodily fluids including blood, urine, bile, breast milk
5. promote neurodegenerative diseases, cancer
6. exosomes (NANOvesicles) and shed MICROvesicles (ectosomes)

Question 2

Slide 4

Answer 2

NA

Question 3

1. What are exosomes
2. What is the size of exosomes
3. Where do exosomes come from?
4. How do exosomes relate to cancer
5. Fxn of exosomes

Answer 3

1. Small membrane derived nanovesicles released from many cell types. Possess lipid membranes with integral proteins; interior contains proteins, nucleic acids, metabolites.
2. S= 60-80nm; L=90-120nm
3. Derived from endosomes; intraluminal vesicles of multivesicular bodies (late endosomal compartments) that fuse with PM
4. Secreted in higher conc from cancer cells; potential biological targets and biomarker for cancer diagnosis and prognosis
5. Can travel to distant tissues and fuse with PM of target cells

Question 4

Describe exosome biogenesis

Answer 4

ESCRT = Endosomal Sorting Complex Required for Transport

Question 5

Describe the composition of exosome and cargo

Answer 5

Vesicle surface components

1. MHC molecules
2. Lipids
3. Targeting and/or adhesion molecules
4. RNA binding proteins
5. Oncoproteins
6. Membrane transport and/or fusion proteins
7. mRNA, IncRNA, miRNA

Vesicle lumen components

1. Splicing molecules
2. TFs
3. RNA binding proteins
4. Oncoproteins
5. Membrane transport and/or fusion proteins
6. mRNA, IncRNA, miRNA

Question 6

Describe how exosomes may play a role in pancreatic cancer

Answer 6

Background: Exosomes are small vesicles in the tumor microenvironment containing nucleic acids and proteins w/capacity to influence cell behaviour.
Results: Exosomes contain double-stranded genomic DNA
Conclusion: Exosomes have capacity to CARRY and TRANSPORT GENOMIC DNA spanning all chromosomes w/KRAS and p53 mutations
Significance: Exosomes can aid in identifying genomic mutations in patients w/pancreatic cancer

Question 7

Describe the mechanisms of exosome uptake by recipient cells. How are they protected from scavenging phagocytes in circulation?

Answer 7

1. Evs can be internalized by the secreting cell itself (ie. autocrine) or by other cells
2. SURFACE INTEGRINS facilitate exosome ATTACHMENT and INTAKE into RECIPIENT cells
3. Uptake occurs through various pathways, including membrane fusion, receptor-mediated uptake, and endocytosis
4. CD47 “do not eat me” signals on exosome membranes protects them from scavenging phagocytes and improves stability in circulation

Question 8

Slide 14

Answer 8

NA

Question 9

What are 3 approaches used to analyze EV-mediated transfer in vivo?

Answer 9

1. Fluorescent reporter
   - fluorescent tagged mRNA
   - search for fluorescence in recipient cells
2. Luciferase reporter (bioluminescence)
   - membrane-bound luciferase
   - search for bioluinescence in recipient cells
3. Recombination dependent (CRE recombinase) reporter gene activation
   - CRE recombinase -> unrecombined reporter cell -> recombined reporter cell

Question 10

How can exosomes be used as cancer biomarkers? Where are exosomes found? What are examples of exosome biomarkers?

Answer 10

1. Exosomes can be detected in tumor tissue and bodily fluids
2. Found in higher conc in TUMOR TISSUE and SERUM and PLASMA of cancer patients
3. Exosome mRNAs, miRNAs, proteins, and lipids all examples of candidate biomarkers
4. E.g. EGF receptor transcript, many miRNAs
5. E.g. drug transporter
6. Currently experimental; not yet implemented clinically
7. Now: exosomes as therapy delivery vehicles

Question 11

4 roles of exosomes in tumorigenesis

Answer 11

1. Remodel ECM and promote vasculogenesis and tumor cell proliferation
2. Travel to distant sites to promote generation of the pre-metastatic niche
3. Immune responses become deregulated; anti-tumor immune fxns impeded
4. Bone marrow derived cells are recruited to tumor and pre tumor tissue here they promote cancer development

Question 12

What are the EV-mediated effects on tumor progression vs. EV-mediated effect at distant sites?

Answer 12

1. Cell migration and invasiveness
2. ECM remodeling
3. Drug resistance
4. Vascular permeability
5. Pre-metastic niche establishment