1. Thomas Brinkman
Modified Dialysis Filtration of Pancreatic Ductal Adenocarcinoma Exosomes to Prevent
Formation of Pre-Metastatic Niche
I. SPECIFIC AIMS
The objective of this study is to devise a method to remove tumor secreted exosomes
from the blood. We have chosen to extract these exosomes from the blood by binding to the
proteins found on Kupffer cells in vitro along with the use of primary antibodies specific to
PDAC exosome biomarker epidermal growth factor receptor (EGFR). Tumorigenic exosomes are
microvesicles secreted by the primary tumor and play roles in tumor development and metastasis.
They do this by promoting carcinogenesis and modifying the immune system to allow the tumor
to circulate in the blood unscathed (An et al., 2015). These microvesicles arrive at metastatic sites
by organ-specific integrins prior to tumor cell arrival. This forms a premetastatic niche for the
tumor in order to decrease exposure time in blood and increase the chances of a successful
metastasis (Offord, 2016). Although exosomes are just beginning to be fully explored, it is known
that in pancreatic ductal adenocarcinoma (PDAC) exosomes bind to proteins on Kupffer cells in
the liver. This binding triggers a cascade of events that promote favorable conditions for
metastasis (Costa-Silva, et al., 2015). EGFR is a biomarker that is commonly overexpressed in
pancreatic cancers. EGFR has been associated with many factors involved in carcinogenesis
(Adamczyk et al., 2011). The aspects of the exosome mediated metastatic pathway that can be a
target in this treatment involves the protein on Kupffer cells that bind with integrin αvβ5 along
with the common biomarker of exosomes, EGFR.
We hypothesize that PDAC exosomes can be successfully removed from the blood using
a modified dialysis treatment. We have chosen to study this pathway in metastatic PDAC cells as
a model system. Previous research suggests that PDAC derived exosomes bind to a protein on
Kupffer cells and trigger a favorable environment for metastasis. In addition, previous literature
suggests that this metastatic cascade can be reduced by the inhibition of exosomal macrophage
migratory inhibitory factor (MIF). This is a protein found in high numbers in the PDAC
exosomes (Offord, 2016), so removal of exosomes would have a similar effect. Data suggests that
PDAC derived exosomes indirectly allow metastasis by interacting with Kupffer cells through
proteins carried within the exosomes. Therefore,the specific aims for this proposal are:
1) Identify and isolate the specific protein on Kupffer cells responsible for PDAC
exosome integrin binding.
2) Create primary antibodiesthat adhere to PDAC exosome biomarker EGFR.
3) Use a modified dialysis machine to pass the patient’s blood through a microfluidic
chip to isolate circulating PDAC-exosomes fromthe blood.
Pancreatic cancer has the lowest five-year survival rate amongst solid tumors, and our
proposal works to prevent the progression of this disease. Data obtained from previous studies
support our hypothesis that exosomes will bind to the isolated receptor proteins from Kupffer
cells and primary antibodies on a microchip. Removing these exosomes from the blood is critical
for an increased survival rate for pancreatic cancer patients. Developing this treatment can allow
for a decrease of metastatic rates and lead to future exosome isolation treatments for other tumor
types using a modified dialysis treatment.

2. II. Introduction
Cancer is a highly studied yet poorly understood disease that requires continual advances
in technology to keep up with its plasticity and survivability. It is projected that in 2016 there will
be 1,685,210 new cases of cancer diagnosed and 595,690 deaths from cancer in the United States
alone (Siegel, et al., 2016). One of the key aspects to tumor survival is its ability to become
dormant and re-metastasize after some time. Dormant tumors are asymptomatic and currently
undetectable through traditional treatments. Dormancy can occur at any period of tumor
development, and may contribute to metastasis and relapse either at the site of the primary tumor
or at distant organs reportedly decades after treatment (Wang & Lin, 2013). The tumors present
upon relapse are thought to be even more malicious than the primary tumor because these cells
survived treatments and have acquired traits favorable to their survival. Pancreatic cancer, as the
main focus of this proposal, is one of the most destructive tumor types with an average survival
rate of about 6 months and a 6% five-year survival rate. (Costa-Silva, et al., 2015). Pancreatic
ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer accounting for
nearly 90% of deaths in pancreatic cancer patients (Costa-Silva, et al., 2015). It is estimated that
53,070 people will be diagnosed and 41,780 will die of pancreatic cancer in the United States in
2016 (American Cancer Society, 2016). With the survival rate almost equal to the death rate,
further study is required to reduce the invasiveness of pancreatic cancer. Fortunately, researchers
have been able to identify a mechanism for metastasis in PDAC cells that may respond to
therapeutic treatments.
Kupffer cells (KC) bind to integrins found on PDAC exosomes, and stimulate the
formation of the premetastatic niche. KC are found in the lumen of liver sinusoids and are known
to activate macrophages (Bilzer, Roggel, & Gerbes, 2006). They are a critical aspect of the host’s
innate immunity. Unfortunately they also induce favorable metastatic environments through the
production of hepatocyte growth factor known to aid in tumor proliferation, and increase
angiogenesis by secreting proteases that change the extracellular matrix (Bilzer, Roggel, &
Gerbes, 2006). It has been found that these events occur upon the binding with integrin αvβ5 on
PDAC exosomes (Offord, 2016). Filtration of integrin αvβ5 may decrease the pro-metastatic
factors secreted by Kupffer cells and lead to a decrease in metastasis for PDAC patients.
Metastasis is defined as migration of tumor cells from the primary tumor to another
location in the body. It is thought that tumors do this in an organ-specific manner. This organ
specificity is known as metastatic organotropism and was first predicted by Stephen Paget in
1889 (Hoshino, et al., 2015). Tumors secrete molecules to aid in their metastasis, and exosomes
may be the pivotal molecule in organ specificity. Tumor secreted exosomes are thought to play a
key role in intercellular communication of cancer cells with future metastatic sites through
signaling molecules, DNA transfer, and microRNA secretion (Costa-Silva, et al., 2015).
Migratory inhibitory factor (MIF) is one of these proteins found in high numbers in PDAC
isolated exosomes. It has been noted that the knockdown of MIF expression decreases the
tumor’s ability to metastasize, suggesting MIF plays a role in exosome-mediated metastasis
(Costa-Silva, et al., 2015). It was found that exosomal integrins direct this metastatic
organotropism by binding to target cells in a tissue-specific manner (Hosino, et al., 2015). It is
known that integrin αvβ5 has a specific binding site in the liver on Kupffer cells (Offord, 2016).
This integrin is known as an upstream regulator of transforming growth factor β (TGFβ). TGFβ is
commonly overexpressed in PDAC patients and known to promote epithelial-to-mesenchymal
transition, angiogenesis, and suppress the immune system’s ability to detect tumors (Hezel, el al.,
2015). TGFβ also promotes the formation of fibronectin by hepatic stellate cells. Fibronectin is a
cell adhesion molecule that creates favorable environment for tumor arrival (Costa-Silva, et al.,
2015). If PDAC exosomes can be removed altogether it would be harder for the tumor to create a
premetastatic niche by removing these metastatic factors. Figure 1 depicts the cascade of events
caused by PDAC exosomes.

3. A PDAC exosomal biomarker called epidermal growth factor receptor (EGFR) is found
overexpressed in pancreatic cancer. It has known effects for carcinogenesis, increased tumor
aggressiveness, and potentially a trigger for pathways involving cell proliferation, differentiation,
and migration (Adamczyk, et al., 2011). This is highly characterized and primary antibodies can
be created for this protein. Primary antibodies can be synthesized by injecting an antigen of
interest into a host animal and its immune system makes an antibody against that specific protein.
A portion of the host animal’s spleen is removed and fused with a myeloma cell. This creates a
hybridoma that will produce continually antibodies for future experimental purposes. A
monoclonal antibody only binds to one epitope on an antigen target, which creates high
specificity for only one type of protein.
Exosome removal techniques are an active area of research and various methods have
been developed. A table of these methods can be seen in Table 1 below. A microfluidic apparatus
has been developed that extracts microvesicles from blood. This was determined to be faster and
cheaper than traditional centrifugation and filtration techniques. It only requires small quantities
of microvesicles in the fluid for successful isolation. The microfluidic technique extracts
exosomes in an antigen-specific manner using a microchip containing antibodies. It was
determined that the efficiency of isolating microvesicles was 42-94% in this microfluidic
approach (Chen, et al., 2010). This was done on a small scale, but this idea can be expanded into
a body-wide treatment through the use of a modified dialysis machine. Dialysis involves
circulating a patient’s blood into a machine. The blood is then passed through a filter and waste
products are cleared to return back to the patient. This can be modified in many ways by using
different combinations of biomarkers commonly found in PDAC exosomes for successful
filtration. This treatment is most beneficial for patients diagnosed with PDAC early on to prevent
PDAC metastasis, and for patients whose tumors have went into remission to prevent future
metastasis. Upon success of this treatment, it can also be expanded to other tumor types by using
their common biomarkers. Success of this apparatus may save millions from potential metastasis
or relapse of cancer.
Source: Offord, 2016

4. Figure 1: Mechanism of PDAC exosomes on Kupffer cells in the liver. The Kupffer cells are
attached to the liver and bind to PDAC exosomes via integrin specific binding. Kupffer cells are
macrophages and upon interaction with PDAC exosomes emits Transforming Growth Factor β
(TGFβ). At this point, the liver’s stellate cells begin secreting fibronectin that recruits bone
marrow derived macrophages. Additional secretions include epithelial cells and fibroblasts. All of
these secretions are important to a premetastatic niche formation favorable for the tumor’s arrival
during metastasis (Offord, 2016).
Table 1: Analysis of exosome removal techniques. Techniques have been performed in various
experiments. Affinity isolation technique appears to be the best. This method has very high
specificity and purity, is relatively cheap, and contamination is not a concern. These reasons
together form the basis of our experimental procedure. (An et al., 2015).
Figure 2:Microchip used for filtration of exosomes. This image depicts the experimental setup
of one microchip used to filter exosomes from the blood. In our experiment there will be 10
microchips hooked up side-by-side creating a parallel circuit for the blood to travel though. See
methods part 3 for further description.
III. Experimental Design
1) Identify and isolate the specific protein on Kupffer cells responsible for PDAC
exosome integrin binding.
i. Experimental procedure: A tissue culture containing a primary cell line of Kupffer
cells will be cultured and maintained on DMEM. A binding assay will be conducted to
determine which receptor binds the αVβ5 integrin found on PDAC exosomes.
Radioactive PDAC exosomes will be placed into tissue cultures and binding will be
Source: An et al, 2015
Source: Chen et al., 2010

5. detected through autoradiography. Nonspecific binding is calculated by doing the
binding assay in the presence of an excess of unlabeled PDAC exosomes. Protocol
modified from Lodish et al.
Once the receptor is determined the Thermo Scientific Cell Surface Protein
Isolation Kit will be used to remove these receptors. Once these receptors are removed
a western blot will be performed to determine if the correct protein has been isolated.
The control group will apply same antibodies used to bind Kupffer cell integrin
receptor onto a different type of tissue such as epithelial cells. This will assure that
there will be no nonspecific binding by these antibodies. A control protein for the
Western Blot will be GAPDH due to its constitutive expression. It will act as a loading
control and verification of the successfulprotein isolation.
ii. Data Analysis: The first part of the data analysis is determining which receptor the
PDAC exosomes are binding to and ultimately the expected number of exosomes each
Kupffer cell can bind to. Calculating the specific and nonspecific binding present will
quantify this. Total binding will be determined by the amount of radiation given off
upon the addition of a known amount of radiolabeled exosome. Nonspecific binding is
calculated by the amount of labeled PDAC exosomes bound in the presence of pre-
added unlabeled PDAC exosomes. Since there are only a certain number of receptors
per cell, they will get saturated by the unlabeled PDAC exosomes. Then with addition
of labeled exosome, the nonspecific binding sights will be labeled since they will not
be saturated. Specific binding will be determined by finding the difference between
total binding and nonspecific binding. With this data a curve can be generated.
Saturation values will be determine will allow the determination of the amount of
PDAC exosome present that these receptors can successfully bind to.
The Western Blot analysis will be analyzed after isolation of the receptor cells.
Band presence in experimental group will be compared to control for presence of
isolated receptor. GAPDH will be used as a loading control. The proteins are known
since the antibody used for the blot is specific to a certain protein.
iii. Expected Results: We expect to see a high presence of specific binding when using
the radioactive exosomes. A positive result would also include a very low amount of
nonspecific binding. We expect a curve that levels off showing saturable binding of
the Kupffer cells with the radiolabeled exosomes. The Western Blot is expected to
detect the presence of a band in the experimental, but the absence of this band in the
control section. When the antibodies are used on epithelial cells it is expected that
there will be no binding because there will be no Kupffer cells present. Constitutive
expression is expected in the GAPDH section to assure the loading was equal and the
cell is still alive and metabolizing.
iv. Anticipated Complications: This experiment may show problems in the specificity
of the radioactive exosomes used to identify the receptors. If there is low specificity
for the PDAC exosomes, a blocking solution will be applied prior to the addition of
the radiolabeled exosomes. This will allow most proteins in the cell to be saturated
and decrease nonspecific binding. Another possible complication is the structural
integrity of receptors for the PDAC integrin on the surface of Kupffer cells. If they do
not maintain their structure in vitro, then this experiment would have to be done in
vivo.

6. 2) Create primary antibodies that adhere to PDACexosome biomarker EGFR.
i. Experimental Procedure: Epidermal Growth Factor Receptor (EGFR) will be
purchased from Abcam (ab89746). Rabbits will first be immunized with the EGFR
protein and left for three weeks to allow antibodies to form. A booster shot will be
administered 3 weeks after the initial immunization and then 3 additional days will
pass before fusion. After this, the spleen will be removed from the animals. A
screening assay will be performed to assure the correct antibodies are produced from
the B cells. The screening assay and the assay for making a hybridoma were
modified from Lerner, 1981. Cells from the spleen will be fused with a human
myeloma cell because most spleen cells will be making the antibody of interest.
Spleen and myeloma cells will be fused using the fusing agent polyethylene glycol.
This creates the hybridoma that will secrete the antibody of interest. Once the
hybridoma colonies are formed and isolated, the cells are then screened again to
ensure the production of the antibodies. Antibodies will be tested for quality by
staining tissue with EGFR. Control will consist of mixing antibodies with EGFR
antigen in a tube then staining tissue with EGFR through immunohistochemistry to
test specificity of antibodies. Another control will mix the antibody made with
antigens that are similar to EGFR and staining tissue-containing EGFR again.
ii. Data Analysis: During the screening assay to assure the correct antibodies are
produced, a gamma counter will be used. Background counts will be compared to
the positive antibody containing wells to verify the presence of antibodies. The
presence of staining will be searched for when observing the tests of the quality of
the antibody and specificity.
iii. Expected Results: The gamma counter should read around 100 cpm for the
background control. Then with the positive wells the gamma read should be
anywhere from 200 to 2,000 cpm. This should be the result for both screens
performed to ensure a successful hybridoma has been created. This would indicate
the presence of our antibody against the EGFR, and allow for further analysis. There
should be staining present in the experimental antibody staining. Then for the
control when the antibody is mixed with the EGFR antigen, it should not stain any
tissue to indicate these antibodies created are specific to EGFR. The other control
with similar antignes should have staining found on the tissue with EGFR to
indicate the antibody for EGFR does not bind to any similar antigens.
iv. Anticipated Complications: The creation of the hybridoma is based off of the
spleen cells creating the antibody in question. If the host’s immune response is weak
it will result in little to no antibody production. In this case, the experiment would
need to be restarted using a new host. This is why screening is crucial for the
experiment to proceed. In creating the hybridoma, the perfect myeloma cell line
must be chosen. The human myeloma should work, but it is not certain that this will
fuse and create the hybridoma. If the spleen cells and the myeloma do not fuse then
a more careful selection must be made for which myeloma will be used for
production of the hybridoma. If specificity is a problem in these experiments, a new
antibody will have be created for a different biomarker on PDAC exosomes.
3) Use a modified dialysis machine to pass the patient’s blood through a
microfluidic chip to isolate circulating PDAC-exosomes from the blood.

7. i. Experimental Procedure: Microfluidic chips that were created by Chen et al. found
in references below will be used for filtration of PDAC exosomes. A sample of how
one microchip assay looks can be found in Figure 3 above. The difference in our
chip is the presence of the receptors on Kupffer cells that bind integrin αvβ5 of
PDAC exosomes along with the antibody created for EGFR. The control group will
have chips containing anti-EGFR and anti- Kupffer integrin receptor antibodies as
negative controls. An additional control will be non-cancerous mouse blood running
through experimental chips. These chips have an average flow rate of 16 uL/min, so
using ten chips in a parallel circuit would cause a flow rate of 160 uL/min. The initial
tests would be on mice with known PDAC cancer for experimental purposes. A
mouse has an average blood volume of 1.5 mL, so it would take about 10 minutes for
one round of blood to circulate through the machine. The protocol was modified from
Chen et al., 2010. This will go on for 2 hours to ensure blood has circulated through
microchips multiple times. Standard dialysis cannulation protocol would be followed
to get blood circulating through the machine and back into the patient. The dialysis
machine is used to extract the blood and create the external circuit for blood to go
through the chips. When the treatment has concluded, electron micrograph (EM)
images will be taken to visualize exosomes. Then DNA and RNA will be isolated
from the microvessicles in the chips. Subsequent PCR and RT-qPCR will be
performed using primers of known genes found in PDAC exosomes. DNA marker
tested will be KRAS, and the miRNA genes will be miR-17-5p and miR-21 (An, et
al. 2015). These are biomarkers found in pancreatic cancer microvessicles. Products
of PCR will be run on gel electrophoresis for analysis.
ii. Data Analysis: EM images will allow for the visualization of the presence of
exosomes and determination of the size specs of the exosomes isolated. Microchips
will be analyzed for the treatment and control groups. Gel electrophoresis will
separate DNA based on molecular size to indicate the presence of our genes of
interest.
iii. Expected Results: EM analysis should show the appearance of tiny beads
(exosomes) throughout the experimental chips. The control chips containing anti-
EGFR and anti-Kupffer Cells should not contain exosomes. Size of these exosomes
will be determined from these images. For the PCR gel electrophoresis results there
should be bands found where the DNA and miRNA is specific to PDAC exosomes.
Bands should not be found in the noncancerous blood dialysis treatment to confirm
specificity of the isolation. Since the current of blood was divided equally, the
number of exosomes and quantity of isolated DNA/miRNA per chip should be equal
per chip.
iv. Anticipated Complications: If the EM images show little to no PDAC exosomes
isolated, the time period the treatment is run will be adjusted. The primary concern
with prolonged dialysis running is that blood current may sweep some exosomes off
of the chip and bring it back into the blood. If this is the case and shorter running
times do resolve this, then more specific antibodies and binding proteins will need to
be designed for higher specificity. If the normal blood treatment control shows
isolated exosomes, then nonspecific binding is occurring. Proteins used in the assay
to bind the exosomes would have to be changed. If bands from the PCR of the
miRNA biomarkers do not appear, then the DNA will be the only isolation performed
because miRNA is unstable and easily degraded with RNases found ubiquitously. If

8. the amount of DNA/miRNA isolated from the chips is not near equal, a modification
in the number or quality of chips will be changed to enhance results.
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