EXTRACELLULAR VESICLES IN CANCER

Guide:Dr. K. N. Gujar

Co-Guide: Dr. M. S. Gambhire

Presented By:Aniket A. Vaidhya M. Pharm, Sem.- II

(Pharmaceutics) Roll.no:522

Sinhgad College of Pharmacy Vadgaon (Bk), Pune (41)

Seminar on

A Novel Platform For Cancer Therapy Using Extracellular Vesicles

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(2016)12/ 04/2016

2 Extracellular Vesicles

Cell-Derived Vesicles Both Eukaryotic and Prokaryotic cells release vesicles. Evs are spherical particles enclosed by a phospholipid

bilayer. The diameter of vesicles typically ranges from 30 nm to 1

µm. Most vesicles have specialized functions and play a key

role in, intercellular signaling, waste management, and coagulation.

Nowadays Vesicles can potentially be used for therapy, prognosis, and biomarkers for health and disease.

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Ref.: Andaloussi Samir EL .Extracellular vesicles: biology and emerging therapeutic opportunities, Nature Reviews Drug Discovery .2015: Vol 12.347-358.

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Contain several types of function molecules, such as proteins, mRNAs, and miRNAs.

Emerged as potential tools for a Drug Delivery system that can target organs or cells.

Have a function of Organ Tropism . They are Laturally occurring from cells. Have a Low side effect. Delivered drugs to specific organ.

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Ref.: Tominaga Naoomi .A novel platform for cancer therapy using extracellular vesicles ,Advanced Drug Delivery Reviews.2015:Vol. 10.1-6.

4 History

The discovery of cell-derived vesicles 1940, when preliminary studies were performed, addressing the

“biological significance of the thrombo plastin protein of blood” (Chargaff and West, 1946).

that cell-free plasma contains a subcellular factor that promotes clotting of blood (Chargaff and West, 1946).

More than 20 years later, in 1967, this subcellular fraction was identified by electron microscopy and was shown to consist of small vesicles, originating from platelets and termed platelet dust” (Wolf, 1967).

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Ref.: Pol Edwin vander .“Classification, Functions, and Clinical Relevance of Extracellular Vesicles” The American Society for Pharmacology and Experimental Therapeuics . 2012: Vol. 64, No. 3 pg no. 676–705.

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These vesicles diameter between 20 and 50 nm and density of 1.020 to 1.025 g/ml (Wolf, 1967). One decade later, fetal calf serum was also shown to

contain “numerous microvesicles” ranging in diameter from 30 to 60 nm (Dalton, 1975).

the term “Exosomes” was introduced when vesicles were isolated from conditioned culture medium of sheep reticulocytes. (John stone , 1987).

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Cont..

Ref.: Pol Edwin vander .“Classification, Functions, and Clinical Relevance of Extracellular Vesicles” The American Society for Pharmacology and Experimental Therapeuics . 2012: Vol. 64, No. 3 pg no. 676–705.

6Different types of Evs A

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Microvesicles

Apoptotic bodies

Exosome

Ref.: Tominaga Naoomi .A novel platform for cancer therapy using extracellular vesicles ,Advanced Drug Delivery Reviews.2015:Vol. 10.1-6.

7 Microvesicles (MVs)

MVs are structures surrounded by a phospholipid bilayer. ( 100–1,000 nm in diameter )

Their size range overlaps that of bacteria. They are formed by regulated release by budding/ blebbing of the

plasma membrane. They have been characterized as products of Platelets, Red blood cells

and Endothelial cells. Isolation and analytical methods include Differential Centrifugation ,

Flow Cytometry (FC) and Capture-based assays.

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Ref.: Zaborowski Mikołaj P. “Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study,”BioScience Advance Access, 2015. Vol.20.1-15.

8 Apoptotic bodies

The term ‘‘apoptotic body’’ was coined by Kerr in 1972 . Apoptotic bodies are 1–5 µm in diameter (approximately

the size range of platelets) Apoptotic bodies are released as blebs of cells undergoing

apoptosis. key functions of apoptotic bodies are horizontal transfer of

oncogenes , horizontal transfer of DNA. Due to Uptake of apoptotic bodies it lead to immuno

suppression .

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9 Exosomes

First described by Trams in 1983 They are vesicles surrounded by a phospholipid bilayer

(approximately 50–100 nm in diameter), their size range roughly overlaps that of the viruses.

They are released by exocytosis of multivesicular bodies. Exosomes have been predominantly characterized in the

case of immune cells (dendritic cells, T cell, B cells, macrophages) and tumors.

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Key mechanisms by which exosomes may exert their biological functions on cells include

(1) Direct contact between surface molecules of vesicles and cells,

(2) Endocytosis of vesicles,

(3) Vesicle-cell membrane fusion . Exosomes may horizontally transfer mRNA and miRNA .

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Cont..


11 Size Ranges of Membrane Vesicles

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Ref.: Gyorgy Bence .“Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles”, Cellular and Molecular Life Sciences.Springer.2011: 68:2667–2688.

12Schematic Representation of EVS

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Ref.: Gyorgy Bence .“Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles”, Cellular and Molecular Life Sciences.Springer.2011: 68:2667–2688.

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14 Characteristics of Different Types of EVs

Ref.: Vader Pieter .“Extracellular vesicles: emerging targets for cancer therapy”, Trends in Molecular Medicine.2014: Vol. 20. 385-393.

15 Exosomes

Natural Nanoparticles for use as Drug Delivery vesicles. Exosomes are smallest extracellular vesicles. Size=30–100 nm originates from late endosomes. Formed in multivesicular bodies inside the cells. Released in the extracellular medium by a broad array of

cells.

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First identified in sheep red blood cell ( 1983) -Believed to be a mechanism for the exchange of proteins between cells.

16 Composition

Exosomes are composed of a wide range of contents including:

Proteins ( integrins, selectins, Rab proteins, tetraspanins like CD9, CD81, CD63 etc)

Lipids (e.g, steroids, sphingolipids, glycerophospholipids) Nucleic acids (mRNA, miRNA, sRNA, DNA) Growth receptors Soluble factors

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Ref.: Gupta Archana .“Exosome as a Mediators of Neuroinflamation,” Journal Of Neuroinflamation,2014.Vol.15.11-68.

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18 Functions

Different biological functions both in normal and pathophysiological conditions.

Elimination of un-necessary proteins & molecules from cell and blood coagulation.

Exchange of materials between cells, intercellular communication.

Propagation of pathogens, immune responses (inhibitory and regulatory).

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19 Biogenesis and Release of Exosome A

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Ref.: Raposo Graça. “Extracellular vesicles: Exosomes, microvesicles, and friends”The Journal Of Cell Biology, 2013. Vol. 200.373-383.

20 Biogenesis and Release of Exosome

Released by B‐cells, dendritic cells (DCs), T‐cells, epithelial cells, platelets

present in physiological fluids serum, urine, breast milk, cerebrospinal fluid, saliva

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21 Synthesis and Secretion:

The process of exosome biogenesis is divided into four stages Initiation Endocytosis Multivesicular bodies (MVBs) formation and Exosome secretion

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22 Roles of exosomes in cancer

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Ref.: Zhang XU.“Exosomes in cancer: small particle”, big player Journal of Hematology & Oncology . 2015:8:83.

23 CASE STUDY 1

(i) Prepare and characterize a new formulation of exosomes loaded with PTX (exoPTX), (ii) Assess the feasibility using exoPTX for MDR-related anticancer therapy.

Objective

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Graphical Abstract

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Materials

Paclitaxel (PTX) and Doxorubicin (DOX) , Lipophilic fluorescent dyes, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine

perchlorate (DIL), Rhodamine 123 (R123), Cell culture medium and fetal bovine serum (FBS), ExoQuick-TC™ Exosome Precipitation Solution.

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Exosome harvested from the conditioned media of RAW 264.7 cells cultured in exosome depleted media using the ExoQuick-TC kit

Exosome Characterization Nanoparticle tracking analysis Dynamic light scattering Atomic force microscopy Western blot analysis

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Harvesting of Exosome

27 Drug Loading into Exosomes

METHOD

Incubation at room temperature (RT)

Electroporation

Sonication

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28Incubation method

Purified exosomes were first mixed with PTX in 1 mL PBS.

Admixture was incubated at 37°C for 1 hour with shaking.

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29 Eelectroporation method

Exosomes were mixed with PTX

Added to a chilled 4 mm electroporation cuvette.

The mixture was then electroporated using an Eppendorf Eporator at 1000 kV for 5 ms,

Then incubated at 37°C for 30 min to allow for recovery of the exosomal membrane.

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30 Sonication Method

PTX-exosome mixture was sonicated using a Model 505 Sonic Dismembrator .(settings: 20% amplitude, 6 cycles of 30 s on/off for three minutes with a two minute cooling period between each cycle.)

After sonication, exoPTX solution were incubated at 37°C for 60 min to allow for recovery of the exosomal membrane.

Excess free drug was separated from exoPTX by size exclusion chromatography.

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31 Characterization

•Accumulation of exosomes and exosome-incorporated PTX in cancer cells

•In vitro Cytotoxicity Assay

•Biodistribution of Exosomes in Mice with Pulmonary Metastases

•Therapeutic Efficacy of exoPTX against Pulmonary Metastases

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32 Characterization of PTX exosomal formulations

RESULTSA

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A) The size of exoPTX was measured by NTA and DLS

B) The exosome protein content was confirmed by western blot

D) The morphology of drug-loaded exosomes was examined by AFM

33 A profound accumulation of exosomes in 3LL-M27 cells in vitro

3LL-M27 cells were incubated with fluorescently-labeled (red) exosomes, or liposomes, NPs for various times and the amount of accumulated nanocarriers was examined by confocal microscopy (A), and spectrophotometry (B). Bar: 10 μm.

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34 Effect of Pgp inhibition on Dox in MDR and sensitive cancer cells

The accumulation of free Dox or exoDox in MDCK MDR1 and MDCK WT cells was studied in celllysates. The Dox incorporation into exosomes significantly increased accumulation in sensitiveand resistant cells, while no effect of verapamil on exoDOX accumulation was found in both celllines.

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The inhibition of metastases growth in mouse lungs upon exoPTX treatment.

C57Bl/6 mice were i.v. injected with 8FlmC-FLuc-3LL-M27 (red) cells to establish pulmonarymetastases. 48 hour later mice were treated with exoPTX, or Taxol, or saline, or empty sonicatedexosomes as a control, and the treatment was repeated every other day, totally seven times.

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36Mechanistic studies of exoPTX cytotoxic effects

RRI for exoPTX in MDCK MDR1 and MDCK WT was 53.33 and 18.38, respectively. In contrast, RRI for Taxol in both resistant and sensitive cancer cells . Noteworthy, empty sonicated exosomes did not show any cytotoxicity in all studied cell lines. Thus, the increase in PTX cytotoxicity afforded by exoPTX was greater in Pgp-overexpressing cells than their sensitive counterparts.

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37 Conclusion

Exosome carriers can provide advantages of cell based drug delivery.

In this case study they utilise three methods in that mild sonication of exosomes in the presence of PTX provided the greatest loading capacity.

Also long term stability. Incorporation of PTX into exosomes may not only

increase its solubility, but also allow for overcoming of Pgp-mediated drug efflux.

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38 CASE STUDY 2

39 Method

The pEGFP-C1-RVG-Lamp2b expressing vector . Reengineered the vector by replacing the RVG fragment

with iRGD (pEGFP-C1-iRGD-Lamp2b)

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40 Conclusion

Shown that iRGD-targeted exosomes, a simple and natural nanoscale drug delivery platform

Are highly efficient in targeting chemotherapeutic drug to tumors in mice,

Thus they represent a promising approach for cancer therapy in humans.

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41 Reference1) Tominaga Naoomi .A novel platform for cancer therapy using extracellular

vesicles ,Advanced Drug Delivery Reviews.2015:Vol. 10.1-6.

2) Andaloussi Samir EL .Extracellular vesicles: biology and emerging therapeutic opportunities, Nature Reviews Drug Discovery .2015: Vol 12.347-358.

3) Johnny C. Akers .Biogenesis of extracellular vesicles (EV): exosomes, microvesicles, retrovirus-like vesicles, and apoptotic bodies, J Neurooncol , Springer .2013:Vol.113,1-11.

4) Pol Edwin vander .“Classification, Functions, and Clinical Relevance of Extracellular Vesicles” The American Society for Pharmacology and Experimental Therapeuics . 2012: Vol. 64, No. 3 pg no. 676–705.

5) Gyorgy Bence .“Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles”, Cellular and Molecular Life Sciences.Springer.2011: 68:2667–2688.

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426) Mar Gudbergsson Johann. “Systematic review of factors influencing

extracellular vesicle yield from cell cultures” Cytotechnolog, Springer.2013:Vol. 10.1007-1016.

7) Vader Pieter .“Extracellular vesicles: emerging targets for cancer therapy”, Trends in Molecular Medicine.2014: Vol. 20. 385-393.

8) Raposo Graça. “Extracellular vesicles: Exosomes, microvesicles, and friends”The Journal Of Cell Biology, 2013. Vol. 200.373-383.

9) Zhang XU.“Exosomes in cancer: small particle”, big player Journal of Hematology & Oncology . 2015:8:83.

10) Kate M. Candelario .The role of EVS in the Progression of cancer, Trends in molecular medicine 2015:Vol.20. 368-374.

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11) Myung soo kim. “Development of Exosome-encapsulated paclitaxel to Overcome MDR in cancer cells”. Journal of Nanomedicine.2015.10.012.

12) Yanhua Tian. “A doxorubicin delivery platform using engineered natural membrane vesicle exosomes for targeted tumor therapy” Journal of Biomaterials . 2014:35 .383-2390.

13) Zaborowski Mikołaj P. “Extracellular Vesicles: Composition, Biological Relevance, and Methods of Study,”BioScience Advance Access, 2015. Vol.20.1-15.

14) Gupta Archana .“Exosome as a Mediators of Neuroinflamation,” Journal Of Neuroinflamation,2014.Vol.15.11-68.

Cont..

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