1

The role of exosomes in cancer metastasis and early detection through plasmon

resonance in aggressive strains of cancers

BIOM 8004 Portfolio

Melody Williams

11/30/2016

2

Introduction:

Exosomes are small extra cellular organelles that are vesicles utilized in intra cellular

communication. These vesicles contain biological macromolecules of the original cell. These

macromolecules consist of lipids, proteins, RNA, and DNA. [1] Exosomes are a recent topic of interest in

cancer research in that they function differently from other cellular micro vesicles. These other micro

vesicles (apoptotic blebs, vesicles for protein transport, etc.) have a different makeup from exosomes.

While many micro vesicles have distinct cargo related to their purpose, cancerous exosomes carry

proteins, RNA, lipids, DNA, as well as components within the cytosol as they are being formed. [2] The

importance of exosome research lies in that these macromolecules give insight to characteristics of the

origin cancer cell.

Exosomes are generated within the cell through the formation of early endosomes. Early

endosomes are larger cellular vesicles that harbor many exosomes. As part of the lipid bilayer of the

membrane invaginates to form an endosome, proteins on the membrane are incorporated into the internal

space. Molecules within the cell cytosol are also present within this endosome. The exosomes form within

the endosome incorporating membrane proteins into their membranes as well as cytosol contents (RNA,

lipids, proteins) into their interiors. [1,2] These endosomes then have two potential destinations. They can

either go to lysosomes for digestion and recycling of macromolecules or exit the cell and released in the

extra cellular matrix. The exosomes destined to leave the cell exit via an exocytic vesicle (endosome now

transitions to an exiting vesicle). The exocytic vesicle merges with the plasma membrane of the cell and

opens towards the extracellular matrix to release the exosomes. [1] (Figure 1)

Exosomes once released into extra cellular space, can undergo cellular uptake in nearby cells.

Exosomes tend to have a high expression of antigens on their membrane. This allows for more effective

binding on cell plasma membranes. Once they bind, they open while in the cytosol releasing their

contents. [1,2] Since the exosomes often contain genetic material, cells that receive exosomes gain protein

coding instructions to allow protein expression within the cell. It has been shown that cancerous

3

exosomes can

convert benign cells

into cancerous ones

through miRNA

(micro RNAs). These

RNAs have various

functions (depending

on which type is in

the exosome)

including gene

silencing. [3] Cancer

cells often become

cancerous from having their tumor suppressor genes silenced or altered causing uncontrolled/unregulated

growth. [4] If exosomes were to carry such silencing RNA’s capable of turning off tumor suppressor

genes, it would be valuable to understand which strains of cancerous exosomes carry such genetic coding.

Therefore, the ability to analyze exosomes further to gain understanding of the contents of the origin cell

could give insight to the metastasis of aggressive strains of epithelial cancers. This could be applied to

early detection of cancer metastasis finding cancerous exosomes before tumors form in a patient.

There are various obstacles involved in exosome research. The size of exosomes range from 40-

100 nm. This makes them difficult to detect through standard, recognized laboratory methods. Most

standard laboratory instrumentation can only efficiently and accurately detect intact vesicles at > 250 nm

(Flow cytometry). Higher concentrations of exosomes tend to be required in order to detect them through

standard methods. [1] Larger samples are needed for detection in bio-organic samples. This leads to

increased patient discomfort for effective cancerous exosome detection. Samples including exosomes

tend to have other types of extra cellular vesicles. These vesicles can be similar sizes to exosomes. Light

4

scattering techniques are customarily used to determine concentration and size of smaller biological

molecules including extra cellular vesicles. While dynamic light scattering (DLS) produces some results

in exosomes’ average size, exosomes customarily do not scatter light well. [2] More sophisticated

instrumentation is needed to definitively show size and concentration of such small transport vesicles.

Other methods of detection have involved antigen expression profiling through Western Blot and

enzyme-linked immunosorbent assay (ELISA). [3] These methods quantitatively determine the presence

of specific antigens expressed on the selected lines of exosomes. Size and concentration characterization

of exosomes have also been determined using instrumentation involving transmission electron

microscopy (TEM) and Nanosight dynamic light scattering (NDLS) [5,7]. These detection methods could

be improved through targeted analysis and isolation of exosomes from other extra cellular vesicles.

An innovative method is being explored in exosome detection implementing plasmon resonance.

[6] Plasmon resonance is the oscillation of electrons on a metallic surface that syncs with a specified

wavelength of light. The signal is read according to the specific wavelength for the metallic surface and

its intensity. Utilizing surface plasmon resonance, protein binding can be determined when there is an

increased intensity from the initial, un-bound wavelength intensity. [6] Surface plasmon resonance is

regularly used to confirm binding affinities for antigens and antibodies. Using spectroscopy, the incident

light is delivered through a light source (usually a laser) causing the electron oscillation and then detected

through instrumentation. If binding has occurred, the intensity of the wavelength reflected to the detector

increases. [5,6] The use of this type of technology has been repurposed to detect exosomes from solution

and bound to a gold coated surface. It can also be further applied for targeted exosome analysis as well as

binding affinity.

Cancer and Exosomes:

Cancer is unregulated as well as uncontrolled cell growth and division of nonfunctioning cells.

[4] Chances of survival of a patient decrease with the presence of invasive tumors. Cancer cell

communication and metastasis research is a growing field in that early detection prior to the formation of

5

a tumor increases the chances of patient survival allowing earlier medical intervention. Exosomes have

been a recent topic of interest in that they have been shown to play a role in cancer metastasis. [1,2]

Research of the metastasis involving aggressive and invasive strains of cancers can be furthered through

exosome studies and improved detection. Exosomes contain various components of the origin cancer cell.

Studies have shown biological macromolecules including mRNA, miRNA, oncoproteins, transcriptional

regulators, and DNA to be incorporated into the cell genome are found within these extracellular vesicles

[3,8] It is important to take note that these molecules influence protein expression in the recipient cell as

well as incorporation of oncoproteins and cancerous genetic code which can permanently alter the cell if

the DNA is incorporated into the cell’s genome. The presence of cancerous RNA within a normal cell

would still be transcribed by ribosomes giving the expression of cancerous proteins. Incorporating

cancerous DNA into a healthy cell’s genome is problematic in that the future messenger RNA’s (mRNA)

released from the nucleus would likely involve this new placed cancerous genetic code. [4] Therefore,

cancerous proteins would likely be expressed and gene regulation could be altered. Researching

cancerous exosomes and their contents provide a unique insight into the heterogeneity of the origin cancer

cell as well as the methods of metastasis.

Exosomes are small (40-100 nm), customarily round, and have a lipid bilayer similar to the origin

cell. These extra cellular vesicles carry high protein antigen expressions on their membranes. [1-3] The

antigens presented on exosomes are related to the type of cancer from which they originated. For

example, MCF7 cells (a breast cancer line) carry a high expression of the antigen CD63 on their lipid

membranes. It has been shown that exosomes from MCF7 carry a high expression of CD63 on their

membranes as well. [3,5] Since they have such a high expression of antigens, this could be useful in

developing a highly specific detection system allowing the diagnosis of cancer.

The macromolecule contents of exosomes can be linked to cancer progression due to their various

functions. The types of DNA found in exosomes include mitochondrial, single stranded DNA, as well as

double stranded DNA. Out of the three, double stranded DNA has shown to be the prevalent form in

6

pancreatic exosomes. [3] The exosomes in a study presented similar genetic mutations in the DNA to the

original cancer cells and are indicative of pancreatic cancer. The mRNA’s (messenger RNA’s) received

by normal cells can influence their translational profile and tumor progression. Angiogenic proteins have

also been found in exosomes which contribute to cell proliferation and tubule formation. The tubule

formation is essential in cell division moving cellular components to each pole and prepare for cell

division. [3] Short non-coding RNA’s called miRNA (micro RNA’s) suppress gene expression by

destabilizing, inhibiting, and degrading mRNA. Highly metastatic gastric cancer cells have an increased

amount of miRNA’s as opposed to less invasive forms of gastric cancer. Genetic coding and the proteins

related to cellular proliferation as well as gene suppression appear to have a strong correlation to cancer

metastasis and tumor development. [3]

Exocarta is a database utilized for exosome study to offer a comparative perspective of various

types of cancerous and normal exosomes undergoing research. The database offers insight to various

organisms involving their exosome makeup, membrane composition, antigen expression, as well as

contents (including genetic coding). [1] It is a compilation of various studies involving exosomes linking

to papers based on options searched. Exosome study is an emerging field. This database is a good tool in

finding highly specific studies on exosomes of interest. It is another means to further research the

functions and structures involved with exosomes. Understanding further the roles of these exosomes and

detecting them as well as their contents will aid in early intervention of medical treatment possibly

increasing the chance of survival.

Standard Methods of Exosome Detection:

Exosomes can be detected and characterized through various methods including nanosight

dynamic light scattering (NSDLS), transmission electron microscopy (TEM), Western blot (WB),

enzyme-linked immunosorbent assay (ELISA), as well as genetic profiling. [5, 8-11] The properties

primarily used for detection involve size, concentration, shape, RNA/DNA presence, and antigen

expression. Each are useful in characterization of exosomes. Most studies utilize multiple methods to

7

fully characterize the chosen line of exosomes for research. The more methods used, the stronger the data

for thorough characterization and quantification.

NSDLS and TEM are primarily used for determining the size of exosomes. NSDLS characterizes

exosomes using dynamic light scattering and Brownian motion capturing real time data through video and

photography. Most nano sized particles scatter light, even if not very well (ie exosomes). Since all atoms

are moving, ‘Brownian motion’ is the term for the observed atomic movement of particles. NSDLS

operates using an incident light that is passed through a solution of exosomes. The light scatters and size

is calculated using the Stokes- Einstein equation. NSDLS not only shows the size of exosomes, but can

also give the concentration with camera and software analysis technology. [7] Other forms of DLS only

measure size and do not give a concentration due to lack of camera and software. TEM is a method to

show mono/polydispersity of exosomes involving their shape and size. [5] TEM operates through using

an electron beam to pass through a sample of exosomes. The preparation of the sample takes three to four

days making it time intensive. TEM is useful in its high resolution which surpasses light microscopes. [4]

While NSDLS does offer real time video, exosomes only show as mere pixels on a screen. Software is

required to analyze the data. Given the very small size of exosomes, TEM is an effective method in

definitively determining the size and shape of exosomes.

WB and ELISA are methods to determine antigen expression on the exosome membrane. Both

WB and ELISA utilize antibodies to bind to antigens to confirm antigen expression. [3,8] The difference

lies in the mechanism. WB uses gel electrophoresis to separate the proteins requiring lysis of the

exosomes. The antigens then can be tested using antibodies and “blotted” onto a separate membrane

where the antibodies have a specific enzyme labeled probe to show the presence of these antigens. [9]

ELISA can be performed with intact exosomes in solution added to a 96 well plate. The presence of

antigens is shown through introduction of antibodies in exosome solution or having antigens already

bound to the surface of each testing well. [10] Markers are then added that are antibody specific to show

effective binding of exosome to antibody.

8

miRNA/DNA isolation kits are widely used to extract genetic coding from exosomes. The

exosomes are lysed and the genetic material is removed. These processes are usually coupled with

polymerase chain reaction (PCR) to amplify the desired genes found within exosomes. [8,11] Since

exosomes are small and can be low in concentration, PCR is effective at increasing the amount of genetic

material involving the genes being researched. PCR uses a bacterial enzyme to effectively produce

millions of copies of genetic coding within a few cycles. This gives a benefit of having a larger sample

size of DNA/miRNA for sequencing and analysis. [4] After the coding amplification occurs,

identification of the gene and code sequencing can be determined through a service.

Surface Plasmon Resonance and Exosome Detection:

Surface Plasmon resonance has been utilized in the past to show antigen binding on gold (Au)

coated surface materials. Grasso et. al have made an extension of this process to involve cancer derived

exosomes through antibody/antigen binding on Au surfaces and detection with surface plasmon resonance

(SPR). Using ELISA, dynamic light scattering (DLS), Cryo-EM (TEM at cryogenic temperatures) they

were also able to characterize both cell cultured exosomes as well as donor derived exosomes. [5]

A. Materials and Methods

1. Breast cancer cell lines: MDA-MB-231, MCF-7, and BT-474 exosomes were studied from

cell culture. Exosomes were also studied from healthy donors. They were isolated via

ultracentrifugation, filtration through a .22 um filter as well as size exclusion

chromatography. The donor derived exosomes were first centrifuged through standard means

to remove cells and cell debris.

2. Size distribution of exosomes was performed using DLS over 12 x 10 second readings 3

times using a Zetasizer Nano ZS.

3. Exosomes were processed for Cryo-EM for definitive size and shape characterization.

4. Exosomes were molecular screened using SPR (Biocore 3000) through modifying the Au

sensor with carboxylated polyethylene glycol polymer (PEG) and bound with Neutravidin

9

through an amine bond. Unbound proteins were removed via sonication and biotinylated

antibodies were injected to bind to the Neutravidin. Finally, exosome solutions were injected

to the Biocore instrument flowing over the antibody functionalized surface to determine

Au/antibody/exosome binding.

5. ELISA was used to further determine molecular screening of antigen expression on the lines

of cancerous exosomes. Exosomes incubated overnight (4 degrees C) in a 96 well plate,

washed with D-PBS (phosphate buffer solution), blocking solution (reduces non-specific

binding) was added and incubated at room temperature for 1.5 and 1 hours each time

followed by 3x PBS washes. Lastly, HRP- conjugated anti mouse IgG antibodies were added

to each well and incubated at room temperature for an hour. The antibodies were detected

using 3,3’,5,5’ tetramethylbenzidine. Optical densities were then recorded at 450 nm using

Spectra Max 360 multi-well reader.

B. Results and Discussion

1. Figure 2 and 3 below shows the Cryo-EM micrograph and the DLS size distribution. Both the

DLS and Cryo-EM show an average exosome size at approximately 80 nm

10

2. Schematic of the SPR analysis of exosomes (Figure 4) and the results for MCF7 with CD44

specific binding on the SPR schematic. The results showed a positive signal for the presence

of exosomes vs. the IgG control.

11

3. Binding using antibodies for antigen expression of CD 44, CD 63, CD 24, CD 9, EPCAM,

and HER-2 with MCF-7 is shown in Figure 5a. Figure 5b shows the binding with CD63

antibodies for the 3 breast cancer lines studied. Figure 5c offers the strength of expression to

be found through molecular profiling. Both CD 44 and CD 63 appear in the MCF7 line of

exosomes. The highest affinity for CD44 is present with MDA-MB-231.

4. Clinical application results involving healthy donors is listed in Figure 6. The exosomes were

collected per the protocol previously discussed. SPR and Cryo-EM were performed. The

normal exosomes had an expression of CD 44 and CD 24. The SPR response was not as high

as the cancerous exosomes suggesting a lesser expression for the antigens.

C. Conclusion

The authors showed the effectiveness of using surface plasmon resonance in the detection of

exosomes involving 3 different cancer lines. They efficiently immobilized the exosomes through

antigen/antibody binding and confirmed antigen presence on various lines of cancerous exosomes

through the SPR binding studies and ELISA. The healthy donor exosome studies show a potential

12

for clinical use giving results of binding within an hour. However, it is uncertain as to the origin

of the exosomes in the healthy donors. Further characterization would need to be explored to

determine standard levels of exosomes in concentration, genetic material presence, and antigen

expression. Cancer patient application of this method would need to be further explored to

determine effectiveness given exosome concentration can vary with individuals.

Expanding on Surface Plasmon Resonance and Exosome Detection

Targeted analysis through immobilization and antigen specific binding will further the specificity

of SPR. A proposed method that would increase specificity and potentially generate a high throughput

test to give results in an hour utilizes surface modified Au slides and a Raman spectrometer.

Immobilization of the exosomes would be performed through a similar polymer based method as Grasso

et al effectively have shown. Individual exosomes could be characterized and detected allowing for a

smaller patient sample. Clinical application would fall into generating a template to provide a tight seal to

allow multiple individual wells for samples from many patients to be analyzed. Once exosomes are

immobilized, antibody specific conjugated Au nano particles would be introduced to each well. Each type

of particle would contain the antibody specific to the antigen expression for the cancer being detected.

Antibody/antigen binding would be detected through SPR on the Raman spectrometer to show an

increase of intensity on the initial peak. After detection through SPR, NSDLS could show the binding

through light scattering. Additionally, if a test were to show positive through SPR, exosomes could be

further studied for their biological macromolecule contents with WB, miRNA kits, and PCR. This would

allow a clinical application using isolated patient derived exosomes showing a highly specific

antigen/antibody binding. The results could be cross referenced with current data (Exocarta) for

comparative studies to definitively confirm the presence of cancerous exosomes. This method is only

limited due to the fact exosome research is still very new and further research is needed to understand

distinct trends in antigen expression as well as macromolecule contents exclusive to specific cancerous

exosomes.

13

Conclusion:

Cancerous exosomes have been shown to play a role in metastasis. Detecting these exosomes

before the presence of tumors can increase the chance of survival for a patient. Exosome research is a

relatively new field of study. Generating more effective methods of single exosome analysis through

targeted methods allow for clinical applications, but also a greater understanding of the heterogeneity of

the cancer involved. SPR has been shown to be effective in immobilizing singular exosomes which gives

way to expanding to more specific detection methods including antibody/nanoparticle research. Further

research is required to fully develop a database of antigen expression trends and biological

macromolecule components. SPR coupled with genetic studies and ELISA will aid in further contributing

to such databases like Exocarta to have these specific trends identified.

References:

1. Mathivanan, Suresh, Hong Ji, and Richard J. Simpson. "Exosomes: Extracellular Organelles Important in Intercellular Communication." Journal of Proteomics 73.10 (2010): 1907-920. Web.

2. Raposo, Graça, and Willem Stoorvogel. "Extracellular Vesicles: Exosomes, Microvesicles, and Friends." J Cell Biol The Journal of Cell Biology 200.4 (2013): 373-83. Web.

3. Greening, David W., Shashi K. Gopal, Rommel A. Mathias, Lin Liu, Jingyi Sheng, Hong-Jian Zhu, and Richard J. Simpson. "Emerging Roles of Exosomes during Epithelial - mesenchymal Transition and Cancer Progression." Seminars in Cell & Developmental Biology 40 (2015): 60-71. Web.

4. Alberts, Bruce. Essential Cell Biology. New York, NY: Garland Science, 2014. Print.

5. Grasso, Luigino, Romain Wyss, Lorenz Weidenauer, Ashwin Thampi, Davide Demurtas, Michel Prudent, Niels Lion, and Horst Vogel. "Molecular Screening of Cancer-derived Exosomes by Surface Plasmon Resonance Spectroscopy." Anal Bioanal Chem Analytical and Bioanalytical Chemistry 407.18 (2015): 5425-432. Web.

14

6. Patching, Simon G. "Surface Plasmon Resonance Spectroscopy for Characterisation of Membrane Protein-ligand Interactions and Its Potential for Drug Discovery." Biochimica Et Biophysica Acta (BBA) - Biomembranes 1838.1 (2014): 43-55. Web.

7. Dragovic, Rebecca A., Christopher Gardiner, Alexandra S. Brooks, Dionne S. Tannetta, David J.p. Ferguson, Patrick Hole, Bob Carr, Christopher W.g. Redman, Adrian L. Harris, Peter J. Dobson, Paul Harrison, and Ian L. Sargent. "Sizing and Phenotyping of Cellular Vesicles Using Nanoparticle Tracking Analysis." Nanomedicine: Nanotechnology, Biology and Medicine 7.6 (2011): 780-88. Web.

8. Hannafon, Bethany N., Yvonne D. Trigoso, Cameron L. Calloway, Y. Daniel Zhao, David H. Lum, Alana L. Welm, Zhizhuang J. Zhao, Kenneth E. Blick, William C. Dooley, and W. Q. Ding. "Plasma Exosome MicroRNAs Are Indicative of Breast Cancer." Breast Cancer Research Breast Cancer Res 18.1 (2016): n. pag. Web.

9. "Probe the Western Blot for Your Target Proteins Using Primary and Secondary Antibodies." Thermo Fisher Scientific. Thermo Fisher Scientific, n.d. Web. 29 Nov. 2016.

10. "10 Day Lentivirus Packaging Service." Exosome ELISA Kits. System Bio Sciences Inc., 2016. Web. 29 Nov. 2016.

11. "MiRNeasy Mini Kit." MiRNA Isolation Kit: MiRNeasy Mini Kit - QIAGEN Online Shop. Qiagen, 2016. Web. 29 Nov. 2016.