Close
About
FAQ
Home
Collections
Login
USC Login
Register
0
Selected
Invert selection
Deselect all
Deselect all
Click here to refresh results
Click here to refresh results
USC
/
Digital Library
/
University of Southern California Dissertations and Theses
/
Establishing a human EGFR expressing murine mammary carcinoma cell line-D2F2, as a syngeneic immunocompetent model
(USC Thesis Other)
Establishing a human EGFR expressing murine mammary carcinoma cell line-D2F2, as a syngeneic immunocompetent model
PDF
Download
Share
Open document
Flip pages
Contact Us
Contact Us
Copy asset link
Request this asset
Transcript (if available)
Content
Establishing a human EGFR expressing
murine mammary carcinoma cell line-
D2F2, as a syngeneic immunocompetent
model
A thesis submitted by
Vyshnavi Pachipulusu
In partial fulfillment of the requirements for the degree of
Master of Science
in
Molecular Microbiology and Immunology
Keck School of Medicine- University of Southern California
August 2019
Adviser: Dr. Alan Epstein
2
Acknowledgement
I would like to thank Dr. Alan Epstein for giving me the opportunity to work in his lab. I would also
like to thank the co-principle investigators of the lab, Dr Peisheng Hu and Dr. Leslie Khawli for
guiding me through the project and helping me complete my thesis. They have taught me how to
think scientifically, plan and execute experiments, analyze, interpret and present data.
I would like to thank the thesis committee members Dr. Keigo Machida and Dr. Lucio Comai for
their support and constructive criticism which have let me grow as a scientist and learn to look at
research from different perception.
I would like to express my gratitude to all the members of the Epstein lab, Alison Smith, Long
Zheng, Luqing Ren, Tiffany Jehng, Aida Kouhi, Maggie and Seth Epstein for helping me through
any hiccups and teaching and sharing their knowledge with me. I would like to thank the medical
students, Kevin Yu, Joseph Yoo, Gregory Stone, Daniel Cohrs who helped me over the summer. I am
very thankful to Alison Smith for guiding, planning, teaching and being an amazingly patient and
supportive mentor.
I would also like to acknowledge all the members in the Molecular Microbiology and Immunology
and the Pathology department in the Keck School of Medicine, for teaching, helping and opening my
eyes to the many opportunities provided by the school and the scientific community. I would like to
thank all my batchmates for providing a mutualistic environment despite our intensive two-year
course.
I would finally like to thank my parents and sibling for supporting, encouraging and believing in me.
I would not have been able to pursue being in the biological sciences if not for their guidance and
inspiration from my grandparents and would like to thank them.
3
Abstract
Epidermal growth factor receptor was one of the first biomarkers identified as it was seen to be
upregulated in metastatic breast cancer. The upregulation is caused due to a mutation in the tyrosine
kinase domain of the EGFR protein. The mutation alters the EGFR protein, but not sufficiently to
evoke an immune response. There have been several drugs developed against the human EGFR like
the tyrosine kinase inhibitors for example- gefitinib, erlotinib and afatinib or monoclonal antibodies
for example-cetuximab, CTL-1/ ch225, panitumumab, necitumumab and zalutumumab. These drugs
are screened in immunocompromised mice systems and hence have been seen to exhibit partial
response in the case of mAbs and toxicity in terms of the TKI. D2F2 is a murine mammary gland
carcinoma obtained from a BALB/c mouse which is a cold tumor exhibiting the characteristics of a
triple negative breast cancer along with that of metastatic advanced breast cancer. Genetically
modifying the D2F2 cell line to express human EGFR by lentiviral transduction produces the
D2F2/E1 clones. Upon screening, different clones which express different surface human EGFR
were isolated. These clones were checked for in vivo subcutaneous engraftments in BALB/c mice by
using the D2F2/E1 clones alone or the D2F2/E1 clones with Matrigel which both showed tumor
auto-regression or no engraftment respectively. The use of a coculture of the D2F2/E1 clones and
NIH-3T3 murine fibroblasts helps overcome the mouse immunogenicity and one of the cell lines
which showed low to medium surface human EGFR (D2F2/E1-A1) showed steady growth
comparable to the D2F2 unmodified cell line, while the higher hEGFR expressing clones showed
regression. The tumor from this D2F2/E1-A1 cell line was harvested, and the cells were isolated
from the successfully engrafted tumors to validate that the tumor was formed due to the transduced
cell lines as well as the fibroblasts. These cells with further evaluation can be established as a murine
mammary carcinoma D2F2 cell line which was modified to express human EGFR on the surface to
successfully be a syngeneic immunocompetent model.
4
Table of Contents
INTRODUCTION.................................................................................................................... 6
EGFR ...................................................................................................................................... 7
Function of EGFR .............................................................................................................. 7
Structure of EGFR .............................................................................................................. 9
EGFR Cellular Localization ............................................................................................. 11
EGFR- a biomarker in breast cancer ............................................................................... 12
EGFR Based Therapies .................................................................................................... 13
D2F2 CELL LINE ................................................................................................................... 16
SIGNIFICANCE OF THE PROJECT ............................................................................................. 17
OBJECTIVES ........................................................................................................................ 18
AIM 1: PREVIOUS STUDIES ..................................................................................................... 18
AIM 2: STRATEGIES TO EVADE THE IMMUNOGENICITY ......................................................... 19
MATERIALS AND METHODS .......................................................................................... 20
CELL CULTURE ..................................................................................................................... 20
VIRAL PARTICLES AND TRANSDUCTION ................................................................................ 20
SCREENING TO GET D2F2/E1 TRANSDUCED COLONIES .......................................................... 21
IMMUNOFLUORESCENCE ........................................................................................................ 22
FLOW CYTOMETRY ................................................................................................................ 23
ANIMAL STUDIES ................................................................................................................... 25
HARVESTING TUMORS AND IMMUNOHISTOCHEMISTRY ......................................................... 27
5
RESULTS ............................................................................................................................... 28
PREVIOUS STUDIES ................................................................................................................ 28
Generating a lentiviral plasmid ........................................................................................ 28
Transfection of HEK cells with the viral plasmids and producing virus.......................... 29
Transduction of the murine D2F2 cell lines with the virus .............................................. 30
Screening and selecting the transduced D2F2 cells with high surface human EGFR
expression ......................................................................................................................... 31
In vivo testing.................................................................................................................... 32
STRATEGIES TO EVADE THE IMMUNE SYSTEM ...................................................................... 33
Immunofluorescence of transfected clones ....................................................................... 33
Screening for different hEGFR expression with flow cytometry ...................................... 35
In vivo Experiments: Different Administration Strategies ............................................... 38
Validating the cell line in the tumor tissue ....................................................................... 41
Flow cytometry ............................................................................................................. 42
Immunohistochemistry ................................................................................................. 44
DISCUSSION AND CONCLUSION ................................................................................... 45
FUTURE DIRECTIONS ....................................................................................................... 48
REFERENCES ....................................................................................................................... 49
6
Introduction
In the field of cancer through the Human Genome Project, many promising targets have been
identified. Many drugs of different classes are being made by the industry and the academic fields
against the identified biomarkers. These drugs are specifically designed for the human biomarkers.
The pre-clinical and lab testing are carried out on immune-compromised lab mice and this is often
not completely translational in humans. The use of humanized mice has hence been adapted which is
effective but also has the drawbacks of being expensive, not being a complete replica of the human
immune system and involving time consuming mice experiments. Use of genetically modified
murine cells lines which produce human receptors can be the solution as it will reduce the time and
effort as well as the cost while enabling the use of an immunocompetent syngeneic model.
A study in 2017 shows that there are around 150,000 women in the United States who have
metastatic breast cancer (Printz C. 2017). Eighteen to thirty six percent of the women survive 3 to 5
years after diagnosis. EGFR is one of the common biomarkers in metastatic cancers. Due to its
stability in breast cancer, it is to be explored as a potential target for therapies.
The D2F2 is a murine mammary tumor cell line. This cell line was chosen before to transduce the
human ErbB2 receptor tyrosine kinase protein to form a tumor model which can be used in
immunocompromised mice (Whittington PJ et al. 2006). This cell line could potentially also be used
to transduce human EGFR protein. To study the immune system in the presence of a drug would be
greatly beneficial, and hence this cell line has been chosen to make a syngeneic immunocompetent
cell line which exhibits the human EGFR tyrosine kinase receptor.
7
EGFR
EGFR was discovered in 1975 and was identified as a 170kDa protein which induces
phosphorylation upon stimulation (Carpenter G, 1978). EGFR/HER1/ErbB1 is part of the tyrosine
kinase family HER family receptors which has three other members HER2/ErbB2, HER3/ErbB3, and
HER4/ErbB4.
As defined by the World Health Organization “A biomarker is any substance, structure or process
that can be measured in the body or its products and influence or predict the incidence of outcome or
disease” (Goossens N, 2015). One of the first identified biomarkers was the epidermal growth factor
receptor (EGFR) which was seen to be upregulated in many cancers like non-small-cell lung cancer,
metastatic colorectal cancer, glioblastoma, head and neck cancer, pancreatic cancer, and breast
cancer (Wee P et al. 2004).
Function of EGFR
EGFR is a receptor tyrosine kinase. Upon binding with ligands like extracellular domain-binding
EGF (epidermal growth factor) ligand or a short transmembrane domain TGF-α (transforming
growth factor-α), the receptor engages in the phosphorylation of its tyrosine kinase domain
(Gschwind A et al. 2004).
The role of EGFR in normal cells is to regulate the cell proliferation, cell survival and angiogenesis.
The ligands EGF or TGF- α bind to the extracellular domain of the EGF-receptor. This induces the
ErbB-1 to homo or heterodimerize with the other members of the ErbB family (ErbB 1-4). This
results in the phosphorylation of the intracellular domain of the receptor, which in turn leads to the
activation of the intracellular signaling pathways responsible for cell survival like the
8
phosphotidylinosol-3 kinase (PI3K) pathway for cell survival and inhibition of apoptosis and the
mitogen activated protein kinase (MAPK) pathway which ensures proliferation and angiogenesis
(Berg J. M et al. Biochemistry 6th ed 2007) (Fig.1).
Fig.1: The EGFR signaling pathway resulting in cell replication thereby prolonging the life of the
cell (Ladanyi M et al. 2008)
9
Structure of EGFR
The human epidermal growth factor receptor (EGFR) belongs to the receptor tyrosine kinase family
to the ErbB lineage of proteins (ErbB1-4). EGFR is also called ErbB1 or Her1 protein. These
proteins are transmembrane proteins that include an extracellular, a transmembrane and an
intracellular domain (Roskoski R Jr, 2013).
The extracellular part of the receptor consists of 2 homologous ligand binding domains (Domains I,
III). Domains II and IV are cysteine-rich and have many di-sulphide bonds (Cys1-Cys3, Cys2-Cys4)
(Fig.2). There are many ligands specific to EGFR (ErbB1) and these ligands bind to domains I and
III and this is mediated by domain II (Table.1). These interactions induced the beta hairpin in the
second domain called the dimerization arm to extend and enable interaction with domain I of another
ErbB family (ErbB I, II, III, IV). The fourth domain in EGFR is in close proximity of the
transmembrane domain. Not much is known about the transmembrane domain and the 30-residue
Fig.2: The Domains of EGFR (Ferguson KM, 2008)
10
anchoring juxtamembrane which is part of the intracellular region of the EGF receptor. The
intracellular part of the protein also consists of the tyrosine kinase domain and the C-terminal. The
EGFR is special and unique from the other receptor tyrosine kinases as it is the TK domain is
activated by receptor dimerization through an allosteric mechanism. The C-terminus consists of 190
amino acids and has multiple phosphorylation sites (Ferguson KM, 2008) (Fig.3).
Fig.3: EGFR-ligand binding mechanism (Ferguson KM, 2008)
11
EGFR Cellular Localization
EGFR is present on many of the cell organelles as a membrane protein. It is widely seen on the the
cell membrane, nucleus and endosomes. It acts as a receptor tyrosine kinase when present
intracellularly following the same mechanism of action. The EGF or the corresponding ligand is
translocated from the cell membrane to the nucleus via the golgi bodies or the endoplasmic reticulum
(Genecards) (Fig.4).
Table.1: Listed here are the
seven mammalian ligands of the
ErbB receptor family and their
binding specificity.
(http://chemistry.berea.edu/~bio
chemistry/2009/Alisha_Kayla/)
Fig.4: Cellular localization of EGFR (Genecards)
12
EGFR- a biomarker in breast cancer
Her2/ErbB2 has been a prominent biomarker in breast cancer and is the most commonly targeted
receptor tyrosine kinase. But it has been observed that there in a role of EGFR/Her1/ErbB1 in the
development of hormone-resistant breast cancer (Chan SK et al. 2006).
Chromosome 7p11.2 contains the EGFR gene which has 28 exons which code for the extracellular
ligand binding sites (exons 5-7 and 13-16), transmembrane domain (exon 17), intracellular tyrosine
kinase domain (exon 18-24) and the intracellular C-terminus which is involved in
autophosphorylation (exon-25-28). In lung cancer the main mutations are seen in the tyrosine kinase
domain which are the in-frame deletion in exon 19 and the point mutation in exon 21. These
mutations are correlated with the amplification of EGFR (Reddi.V. 2013) (Fig.5). These mutations in
the cancer cells make EGFR a neoantigen (Yi M et al. 2018). Due to the nature of these neoantigens,
their identification by the immune system is obscure, which make them targets of the cancer cells
(Schumacher TN et al. 2015).
Fig.5: Spectrum of EGFR mutations (Ladanyi M et al. 2008)
13
Many reports suggest the presence of EGFR positive breast cancer cases. A clinical study carried out
in breast cancer shows that around 40-50% of the tumors in a sample of 5232 patients with breast
cancer are EGFR positive (Klijn JG et al. 1992). There were indications that showed the presence of
EGFR mutations in exons 18-21 which are usually seen in cases BRCA positive with sporadic breast
carcinoma, indicating them to be elevated in hereditary breast cancer (D Generali et al. 2007). In a
Norwegian study, they found cases of EGFR positive early breast cancer with T790 mutation
(Bemanian V et al. 2015). These clinical studies along with others indicated the EGFR positive breast
cancers to be present based on the degree of differentiation and distant metastasis (Meche A. 2009).
In addition, this subset of cases showed poor prognosis and resistance to tyrosine kinase inhibitor
therapy indicating the need to focus on therapy against EGFR/ErbB1 receptor. Most of these studies
indicate the higher expression of EGFR positive breast cancer in estrogen negative, progesterone
negative and Her2 protein negative i.e., triple negative breast cancer (TNBC) which is known to be
more aggressive and resistant to current therapies. EGFR is frequently overexpressed in this subset of
TNBC. Immunohistochemical studies using the PharmDx to check the mRNA expression have
indicated a significant increase in the expression of EGFR in TNBC (Nakai K et al. 2016).
EGFR Based Therapies
EGFR has become an important biomarker in triple negative breast cancer. There are unfortunately
no drugs approved against EGFR in breast cancer. However, there are small molecule drugs which
are made against ErbB1 receptor called tyrosine kinase inhibitors (TKI) like gefitinib, erlotinib and
afatinib which have been approved for colorectal, squamous head and neck, squamous skin and non-
small cell lung cancers.
The tyrosine kinase inhibitors can act in the following 4 ways:
• ATP active site competitive inhibition
14
• ATP non-active site binding, to render activation energetically unfavorable
• Allosteric inhibition, to change the conformation and prevent ATP and kinase domain
interaction
• Covalent inhibition of the ATP-binding site, making the kinase domain unavailable
There are 11 TKI’s approved by FDA, but there are mutations in the tyrosine kinase domain of
EGFR which makes the specificity of the drug change. For example, the T790 mutation was induced
by treatment with getifinib (Yi-fan Chen et al. 2011).
In recent years, anti-EGFR monoclonal antibodies like cetuximab and panitumumab have been
developed against colorectal cancer and squamous cell head and neck cancer. The antagonistic
mechanism of action of mAbs are by competitive inhibition of the ligands that bind to domain III of
the EGFR extracellular region. The antibody binds to only domain III and does not try to bind to
domain I, which does not cause conformational changes in domain II, preventing the dimerization of
the EGFR with other ErbB family domain I regions. This competitive inhibition does not lead to the
activation of the tyrosine kinase domain to account for its antagonistic effects (Li S et al. 2005)
(Fig.6).
Cetuximab was been found to be only partially effective in patients with colorectal cancer, head and
neck cancer and non-small cell lung cancer (Zhang W et al 2007). It was later shown that
combination therapy with irinotecan or afatinib was more effective in these cancers (Cunningham D
et al. 2004) (Ribeiro Gomes J et al. 2015).
Monoclonal antibody resistance has been seen in colorectal cancer. The mutation of RAS, and
downstream mutations by BRAF activation and PTEN deletion have shown to be the cause for
resistance against the monoclonal antibodies against EGFR. In breast cancer, the activating mutations
are rare and hence these anti-EGFR therapies can be used.
15
Dr. Epstein’s lab has made a chimeric antibody called CTL-1 or ch225 which has similar activity as
that of cetuximab (Hu P et al. 2017). To use this antibody as a monotherapy or combination therapy,
the establishment of a model would help screen the drug.
Fig.6: Cetuximab- Mechanism of Action - Cetuximab binds to domain III of a
tethered EGFR molecule (Li S et al. 2005)
16
D2F2 Cell Line
D2F2 is a murine mammary gland carcinoma which is obtained from a prolactin-induced BALB/c
hyperplastic alveolar nodule line (Whittington, P. J. et al. 2008). It is an adherent cell line which is
known to produce a very low level of the mouse erbb-2 receptor (Marie P. Piechocki et al. 2001).
D2F2 is a cold tumor which means that these tumors are very hard to infiltrate with the immune cells,
making them a hard target for monoclonal antibodies. The reason for the T-cell deficit in the cold
tumors could be due to loss of MHC expression, low neoantigen burden and defects in antigen
presenting cells or the trafficking of T cells to the tumor site (Bonaventura P et al. 2019) (Fig.7).
The therapeutic approaches to overcome these cold tumors include upregulation of MHC,
combination therapy and CAR-T cell therapy. Among others, D2F2 cell line was chosen as it has
been previously used to establish a tumor model which expresses the human ErbB-2 receptor called
D2F2/E2 which has also served as a cell-based vaccine (Whittington, P. J. et al. 2008).
Fig.7 Reversing a cold into a hot tumor (Bonaventura P et al. 2019)
17
Significance of the project
There have been many breast cells lines made like the D2F2/E2 and a 4T1/Her2 cell line which have
been successfully transduced with the human ErbB-2 receptor tyrosine kinase. These have been used
successfully in immune-compromised mice for studies. By contrast, a model for human EGFR is
needed which can be used in immune-competent syngeneic hosts. 4T1 cell line is a triple negative
breast cancer cell line but is known to be a hot tumor. Thus, a cold tumor cell line like D2F2 which
expresses low amounts of murine Her2, is an ideal cell line to produce human EGFR. D2F2 also
causes the formation of metastatic advanced mammary tumors and studying the immune
environment in an immune-competent animal system can help our understanding of immunogenicity
and susceptibility of the tumor.
The purpose of making a D2F2/E1 model is as follow:
• It provides a model to screen the CTL-1 antibody (a biosimilar of the anti-EGFR monoclonal
antibody Cetuximab).
• The model will help us understand the response of the immune system to the neoantigen that
have been introduced.
- It will help us understand the functions of the neoantigen (hEGFR) without any
compounded effects.
- It helps us monitor the adaptive as well as effector responses of the mouse system
towards the neoantigen.
18
Objectives
Aim 1: Previous Studies
1) Generation of a stable murine mammary cell line (D2F2) which exhibits surface expression
of the human protein- EGFR.
• Generating a lentiviral plasmid
• Transfection of HEK cells with the viral plasmids and producing virus.
• Transduction of the murine D2F2 cell lines with the virus.
• Screening and selecting the transduced D2F2 cells with high surface human
EGFR expression.
2) Subcutaneous tumor forming ability of these cell lines in vivo in immunocompetent BALB/c
mice and demonstration of the stable expression of human EGFR in vivo.
Previous studies showed that the higher human EGFR expressing clones were recognized by the
mouse immune system and hence were unable to grow progressively. To prevent this from occurring,
immunogenicity the D2F2/E1 cell lines with a range of hEGFR surface expression-low medium and
high expressing clones were studied. In addition, two modifications, namely the use of Matrigel
which is a gelatinous protein mixture secreted by the Engelbreth-Holm-Swarm (EHS) mouse
sarcoma cells produced by BD sciences along with the D2F2/E1 and the coadministration of feeder
NIH-3T3 fibroblasts, were tested to support the continuous growth of implanted cell lines.
19
Developing tumors were then harvested and stained using immunohistochemistry for the expression
of human EGFR. In addition, isolated tumor cells were subjected to flow cytometry to determine the
extent of intracellular as well as membrane bound hEGFR expressed in the transduced tumors.
Aim 2: Strategies to Evade the Immunogenicity
1) Transducing the virus and selecting a range of hEGFR expressing clones (low, medium and
high) of murine mammary gland cancer D2F2 (D2F2/E1).
2) Compare different administration strategies to try and prevent the immunogenic response in
the mouse model and check for successful engraftment of the D2F2/E1 clones.
3) Harvesting the tumors to check positivity for human EGFR on membrane or intracellularly
by IHC and flow cytometry.
20
Materials and Methods
Cell Culture
D2F2 cells are an adherent malignant cell line of the mouse mammary gland of BALB/c mice [D2F2
(RRID: CVCL_0I91)]. D2F2 cell line is cultured in complete media (RPMI-1640 supplemented with
10% fetal bovine serum, nonessential amino acids, penicillin G, and streptomycin). The cells are
incubated at 37ºC in a humidified, 5% CO 2 incubator. The transduced D2F2 cell lines which are
D2F2/E1-#10, D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49, D2F2/E1-A1, D2F2/E1-A2, D2F2/E1-
A3, D2F2/E1-B1, D2F2/E1-B2 and D2F2/E1-B3 are also cultured in complete media. NIH-3T3 cells
[NIH 3T3 (RRID: CVCL_0594)] are adherent fibroblasts which were isolated from embryo tissue of
BALB/c mice. These cells are cultured in complete media as above. The human colorectal carcinoma
cell line is DiFi [DiFi (RRID: CVCL_6895)] which is positive for human EGFR and used as a
positive control in the in vitro experiments. Since all these lines are adherent, the cells were passaged
with the help of trypsin to detach the cells from the flask. The cells were then centrifugated and the
supernatant is discarded so the cells were devoid of trypsin when reintroduced into a new flask with
complete media.
Viral Particles and Transduction
The viral particles made for the previous study were stored in the freezer. This was used for the
transduction of the D2F2 cell line. The D2F2 cells were thawed and cultured in complete media and
21
the cells were split thrice to ensure the cells ability to replicate. The cells were counted and assessed
for viability before diluting to obtained 100,000 cells per well of a 24 well plate. They were allowed
to grow and adhere onto the tissue cultured well plate overnight. The next day half the media was
removed and replaced with fresh media. Ten microliters of HEPES buffer was added to stabilize the
pH of the media. Different volumes (15μl, 10μl, 4μl, 1μl) of virus were added to wells along with 6μl
of lenti A and 2μl of lenti B which help with transduction. The EGFR virus contained 8μl/ml of
polybrene which helps with transduction by its cationic charge to reduce the electrostatic repulsion
between the lentivirus and the cell (Denning W et al. 2013). Medium was replaced the following day
and the cells were checked under the fluorescence microscope 3 days after addition of the virus
mixture.
Screening to get D2F2/E1 Transduced Colonies
To select transduced clones
with different human EGFR
expression levels, samples
were serially diluted to get
one cell per 100μl of media.
The diluted culture was
pipetted into a 96 well plate
and incubated at 37ºC in a
humidified, 5% CO 2
incubator overnight. The wells were checked for colonies after 2 or 3 days. The wells with single
22
colonies were scaled up in a 24 well plate. These were checked under the microscope for ZsGreen
expression indicating successful transduction under the fluorescent microscope. The single
transduced colonies were scaled up in a 6 well plate and the ZsGreen and surface hEGFR expression
levels were determined using flow cytometry. The clones of different expression levels were selected
and cultured in flasks.
Immunofluorescence
Immunofluorescence microscopy was used to detect for the presence of the transduced hEGFR
protein which was in the same promoter as a fluorescent green protein ZsGreen1. This protein is
produced with the human EGFR protein due to the presence of the IRES site between the genes
which recruits a different ribosome to produce and fold the ZsGreen1. EGFR staining was performed
by using anti-hEGFR antibodies produced in the laboratory.
The different transduced cell lines along with the unmodified D2F2 (negative control) were counted
and checked for viability. The cells were serially diluted to 5000 cells/ml of media and 1ml of the
culture was pipetted into the 8-transwell slides which are tissue culture treated. The slides were
incubated at 37ºC in a humidified, 5% CO 2 incubator till the cells adhere to the slide and were up to
70-80% confluent. The trans-well slides were then fixed in 4% paraformaldehyde and incubated at
room temperature for an hour. The slides were dismounted from the wells and washed in the wash
buffer (Phosphate buffer saline+0.1% Bovine serum albumin) for 3-5 minutes. They were
permeabilized with the permeabilization buffer (PBS+0.5% Triton-X) for 5 minutes. The slides were
washed with dilution buffer (PBS+2%BSA+2% fetal bovine serum+ Sodium azide +0.3% Triton-X)
for 5 minutes. The slides were then incubated in blocking buffer (PBS+1%BSA+2% fetal bovine
23
serum+0.3% Triton-X). The primary and secondary antibodies were added before adding the nuclear
counterstain. A drop of diluted 0.1μg/ml DAPI was added on top of each of the slide wells and
incubated at room temperature for 2-5 minutes before washing with PBS. The slides were then
mounted with a glass cover slip using Prolong Diamond Mountant (Thermofisher).
The slides were observed under the fluorescent microscope in the molecular imaging core in
Hoffman Medical Research Building and the data acquired with the help of the software MetaMorph.
The images were then modified with the help of ImageJ.
Flow Cytometry
The cells from the different clones were characterized based on their size and level of expression of
ZsGreen as well as surface and internal hEGFR expression by flow cytometry. Flow cytometry is a
quantitative immunophenotyping technique which can analyze the living cells with the help of
multicolor fluorescent antibody labelling. The experiment was done in parallel with the D2F2 cells as
a negative control for both ZsGreen and hEGFR and DiFi cells as the positive control for hEGFR
expression. D2F2/E1-#10 was a high expressing cell line which was used as a positive control for
both ZsGreen and hEGFR.
24
The cells were treated
with detachin rather than
trypsin to preserve
antigen structure of
membrane proteins. The
cell lines were counted
and checked for
viability. If the cell
viability was less than
95%, they were subjected to ficoll-hypaque density centrifugation to remove dead cells. The ficoll
was carefully added to the bottom of the tube of cell suspension with the help of a syringe. Upon
centrifugation the live cells were trapped between the layer of ficoll and media whereas the dead
cells form a pellet at the bottom of the tube. The supernatant is collected and again centrifugated for
the cells to remove the ficoll. These cells were then added to flow cytometry tubes and washed with
PBS+10% FBS. The Fc block for mouse/human for the respective cell lines were added before
incubating with the primary antibody. The primary antibody against CTL-1 was made in Dr.
Epstein’s lab (Hu. P et al. 2017) and 5μg/test was used for flow cytometry experiment and incubated
for 40 minutes with shaking in the dark at 4°C. The cells were then washed with PBS and stained
with the secondary antibody, a goat fragment anti-mouse biotinylated antibody for 20 minutes as
above. After washing the unbound secondary, the tertiary APC streptavidin was added and incubated
in the dark 4°C with shaking for 20 minutes. The samples were washed with PBS before analyzed by
flow cytometry for surface staining. For intracellular staining, the cells were first fixed in 2% PFA
for an hour at room temperature. They were then washed with PBS and permeabilized with
PBS+10% FBS+0.5% Tween and blocked with PBS+10%FBS. The Attune flow cytometer in Dr.
Epstein’s lab was used to obtain the data.
25
Animal Studies
Animal studies were carried out in 6-week-old BALB/c mice which were ordered from Jackson
Laboratories. The mice were shaved on the right or both flanks in accordance to the experiment and
all tumors were injected subcutaneously using a 25-gauge needle and a 0.2ml inoculum.
The cells were cultured in complete media and were treated with trypsin to detach from the surface.
The live cells were isolated by density gradient ficoll dead cell exclusion method as any dead cells
results in necrosis and ulceration when injected subcutaneously into the mice.
The first round of in vivo experiments had 4
groups with n=5. The tumor cell lines used in
this round were of D2F2/E1-#10, D2F2/E1-
A1, D2F2/E1-A2 and D2F2/E1-B1. Matrigel
is a mixture of extracellular membrane
proteins like collagen, laminin, heparin and
growth factors. The tumor cells (2×10
6
cells)
and Matrigel were mixed in the ratio of 5:1 on
ice. The syringes were prepared before
injecting the mice to prevent settling of the
cells or hardening of the Matrigel.
26
The second round of in vivo experiments had 9
groups with n=3. The tumor cell lines used in
this round were of D2F2, D2F2/E1-#10,
D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49,
D2F2/E1-A1, D2F2/E1-A2, D2F2/E1-B1 and
D2F2/E1-B2. NIH-3T3 fibroblasts have been
shown to enhance the engraftment of tumors as
they provide the required extracellular matrix
environment with the necessary growth factors
and proteins to successfully engraft and form a
subcutaneous tumor. NIH-3T3 fibroblasts are
BALB/c derived and were used in co-culture
with the tumor cell lines in a 1:4 (5×10
5
fibroblasts: 2×10
6
tumor cells) and 1:8 (5×10
5
fibroblasts:
4×10
6
tumor cells) ratios for the left and right flank hind tumors respectively.
27
Harvesting Tumors and Immunohistochemistry
Tumors grown in the coculture of the D2F2/E1 clones and fibroblasts were banked at the end-point
or along the way before regression of the tumor. Sterilely excised tumors were cut into small
fragments that would fit into the tissue processing embedding cassettes. After 24 hours of fixation in
10% buffered formalin at room temperature, the cassettes were then sent to the histology lab in the
Histology and Pathology Core at USC for paraffin embedding, hematoxylin and eosin staining, and
immunohistochemical staining.
In addition, flow cytometry was performed on single cell preparations of the tumor slices to
quantitate intracellular and membrane hEGFR expression.
28
Results
Previous Studies
Generating a lentiviral plasmid
Recombinant DNA were made by inserting the human EGFR gene construct into the lentiviral pLVX
plasmid. The gene is a variant protooncogene called epidermal growth factor receptor isoform g
precursor and is devoid of an exon in the 5’coding area, which results in a shorter isoform of the
human EFGR protein (Uniprot ID: Q504U8). This gene was inserted into the plasmids multiple
cloning site (MCS) with the help of restriction enzymes sites Spe1 and Not1.
Spe1 Not1
The plasmid has a mammalian EF-1 alpha (elongation factor-1 alpha) promoter which has been
shown to be stable and efficient for generating cancer cell lines for homogenous expression of the
gene of interest (Teschendorf C et al. 2002). The multiple cloning site holds the hEGFR gene
construct. It has an internal ribosome entry site (IRES) which helps co-express genes under the same
promoter as different functional proteins, which in this case in the ZsGreen1, a green fluorescent
protein derived from Zoanthus sp. which is an indicator for the transduction of the plasmid into the
D2F2 cell line. The plasmid also has the woodchuck hepatitis post-transcriptional regulatory element
(WPRE), which can enhance the protein expression as well as viral titres (Klein R1 et al. 2006). The
plasmids have the ampicillin resistance gene and have the pUC origin which helps produce a high
copy number of the plasmid (Fig.8).
29
Transfection of HEK cells with the viral plasmids and producing
virus
To produce the virus, the pLVX_EGFR plasmid along with the packaging and transfer plasmids were
introduced into the HEK 293 cells. Human embryonic kidney 293 cells are generally used for virus
transfection and viral production. The lentiviral packaging vectors are psPAX2 and envelope vector
pMD2.G are responsible to form the functional lentiviral particles. The transfer plasmids are required
for the engraftment of the gene into the host genome. The HEK 293 cells were incubated with the
plasmid mixture for 18 hours before changing the media. The media supernatant of the HEK293 cells
contains the viral particles after 48,72 and 96 hours. The virus was collected from the supernatant
and filtered through a PES filter.
Fig. 8:
Vector map of the
pLVX_EGFR plasmid
construct.
30
Transduction of the murine D2F2 cell lines with the virus
Transduction is a process where a gene of interest can be transferred to a cell through a virus. This is
a process seen in bacteria for horizontal gene transfer and has been used to genetically modify cells
in laboratories (Griffiths AJF et al. 2000). The D2F2 murine cells were counted and diluted to get a
100,000 cells per well of a 24 well plate. Since the cell line is adherent, it was left overnight to fix
onto the plate. The media in the plate was replaced by HEPES buffer, EGFR virus and lentivirus A
and B. The HEPES buffer helps stabilize the pH. The lentivirus A and B help with the transduction
process. Polybrene is a cationic polymer which is used to reduce the repulsive forces between the cell
and the lentivirus. It is known to increase the transduction efficiency by 2 to10 fold. The virus titre
contained 8µl of polybrene per ml (Denning W et al. 2013). Media was regularly changed, and the
cells were checked under the fluorescent microscope for the ZsGreen to indicate successful
transduction and expression of the virally transferred genes.
31
Screening and selecting the transduced D2F2 cells with high
surface human EGFR expression
The D2F2 transduced cells
(D2F2/E1 cells) need to be
selected. The transduction
does not ensure a 100%
transduction rate or a
similar rate of expression of
the transduced gene in all
cells. Hence the transduced
cell lines need to be
screened. This is done by diluting the transduced cells to obtain one cell per well of the 96 well plate.
The plates are incubated at 37ºC overnight in a humidified, 5% CO 2 incubator. The plates are
monitored for colony formation. The wells with a single colony of cells are selected and grown on a
24 well plate. These cells are checked under the fluorescent microscope and the ones that are positive
for ZsGreen indicating successful transduction are then selected and scaled up in a 6 well plate. The
cells from the 6 well plates were checked for ZsGreen expression by flow cytometry and the ones
showing the highest levels were chosen on the basis that more the ZsGreen produced more human
EGFR was present in the cells. These cell lines were named as D2F2/E1-#10, D2F2/E1-#23,
D2F2/E1-#33 and D2F2/E1-#49.
32
In vivo testing
The selected cell lines were then tested in
immunocompetent BALB/c mice. The mice were
subcutaneously injected with 1-2× 10
6
cells for each
of the 4 clones selected in 3 mice on the right and
left hind flanks. The concentrations of 2.5× 10
6
cells
and 5× 10
6
cells were subcutaneously injected in
another set of 3 mice in the left and right flanks
respectively. Tumor volumes were measured, and it
was found that the tumors grew but they were found
to regress by themselves shortly after
transplantation. This was expected due to the
expression of a human EGFR protein on the surface of the mouse D2F2 cell.
33
Strategies to Evade the Immune System
Immunofluorescence of transfected clones
The D2F2/E1 transduced clones were selected based on immunofluorescence of the ZsGreen1
protein. The ZsGreen1 is a tetramer which is excited at 493nm and shows emission at 505nm. It has a
relative brightness of 117% as compared to the EGFP fluorescent protein (Day RN, 2011). The cells
were counter-stained with DAPI, a fluorescent dye which binds to the adenine-thymine regions of the
DNA or chromosomes (Tarnowski BI et al. 1991). The fluorescence microscope produces a
differential interference contrast (DIC) image which gives the outline of the cell in a three-
dimensional appearance.
A B
C D
Fig.9:
Immunofluorescence
staining of negative
control unmodified D2F2
cell line.
A. DAPI- nucleus
B. ZsGreen- no
signal
C. DIC image-
cytoskeleton
D. Overlay of the
stains
34
The unmodified D2F2 cell line was used as a negative control for ZsGreen (Fig.9). The cell lines
which showed positive for transduction and expression of the hEGFR protein were- D2F2/E1-#10
(Fig.10), D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49, D2F2/E1-A1, D2F2/E1-A2, D2F2/E1-B1 and
D2F2/E1-B2. The cells showed distribution of the protein throughout the cell and was bright in the
nucleus and endosomes.
A B
C D
Fig.10:
Immunofluorescence
staining of representative
positively transduced
D2F2/E1-#10 cell line.
A. DAPI- nucleus
B. ZsGreen- positive
(Green bright
specs are
endosomes)
C. DIC image-
cytoskeleton
D. Overlay of the
stains
35
Screening for different hEGFR expression with flow cytometry
The cells were run on the flow cytometer to check for surface human EGFR expression. The cells
were run after staining with the lab anti-hEGFR antibody. The transduced cell lines were all run on
the flow cytometer- D2F2/E1-#10, D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49, D2F2/E1-A1,
D2F2/E1-A2, D2F2/E1-B1 and D2F2/E1-B2. D2F2 was used as a negative control for both ZsGreen
and h-EGFR, DiFi was used as a positive for h-EGFR and a previously transduced, high hEGFR
expressing clone, D2F2/E1-#10 was used as a positive for ZsGreen. The cells were isolated for
singlets by plotting a dot plot for forward scatter based on height and area (Fig.11-A). The live cells
were gated for in the side scatter area vs forward scatter area dot plot (Fig.11-B). The cells were
screened by checking for their positivity in BL1-A channel which detects the ZsGreen (488nm) and
the RL1-A channel which detects the APC (638nm) which is the tertiary antibody for the anti-
hEGFR antibody (Attune® Acoustic Focusing Cytometer-User Guide). Most of the transduced cells
showed positive for both surface h-EGFR and ZsGreen (Fig.11-C-F). To confirm and see the variable
surface h-EGFR expression histograms were plotted. The histograms showed the variable expression
of surface hEGFR and ZsGreen (Fig.12). There was no correlation seen between the ZsGreen
expression and hEGFR expression as the hEGFR is also distributed intracellularly. As hoped for a
range of D2F2/E1 clones were developed which had different levels of surface hEGFR expression
(Herzenberg LA et al. 2006).
36
Fig.11: Flow cytometry- Dot plots
A. Singlets in FSC-H vs FSC-A dot plot.
B. Live cells in SSC-A vs FSC-A dot plot.
C. D2F2- negative for ZsGreen as well as ch225
D. Difi- positive for surface ch225
E. D2F2/E1-#10 -positive for ZsGreen and surface ch225
F. D2F2/E1-A1- positive for ZsGreen and surface ch225
37
Fig.12:
A. Histogram of range of ZsGreen expression by the D2F2/E1 clones
B. Histogram of range of human EGFR expression by the D2F2/E1 clones
A
B
38
In vivo Experiments: Different Administration Strategies
The previous studies showed that the tumors formed by the
D2F2/E1-#10, D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49 cell
lines trigger immunogenicity in the BALB/c immunocompetent
mice and showed natural tumor regression. To avoid this, Matrigel
was used along with the cell lines since prior literature has shown
better engraftment of the breast cancer cells in mice using this
method (Bao L et al. 1994). The cell lines used were D2F2/E1-A1,
D2F2/E1-A2, D2F2/E1-B1 and D2F2/E1-B2 along with D2F2 as a
control. The subcutaneous injection of Matrigel along with the
2×10
6
cells of the transduced cell lines showed no tumor growth
after 10 days. The subcutaneous injection of Matrigel and the
unmodified D2F2 cell line showed tumor growth as expected.
Between days 11 and 14 the mice had a layer of fat at the site of
injection indicating that the cells were present at the site of
injection and then showed distinctive hair regrowth only at the injection site which indicates that
there is clearance of the tumor cells and restoration of the epidermal hair follicles (Harel S et al.
2015) (Cui Z et al. 2003) (Fig.13). The mice were kept in observation and were re-challenged with
the D2F2 tumors which also showed clearance which indicates that they were immunized against the
cell line.
There are studies which show that use of fibroblasts in co-culture with breast cancer cells show better
progression in vivo (Sung KE et al. 2013). Human/rat/mouse fibroblasts co-injection with cancer
cells of human or animal origins in immunocompromised mice showed better engraftment of the
Fig.13:
BALB/c mouse which cleared
the transduced cell line
showing a distinct regrowth
of hair as a path above the
injection site.
39
tumor cells (Picard O et al. 1986) (Kojima M et al. 2014). Therefore, the transduced cell lines-
D2F2/E1-#10, D2F2/E1-#23, D2F2/E1-#33, D2F2/E1-#49, D2F2/E1-A1, D2F2/E1-A2, D2F2/E1-B1
and D2F2/E1-B2 and D2F2 were co-injected with mouse NIH-3T3 fibroblasts. The tumors in these
mice grew rapidly after injection. Each mouse was injected with 2.5×10
6
cells (cancer cells:
fibroblasts=4:1) and 4.5×10
6
cells (cancer cells: fibroblasts=8:1) on the left and right flank
respectively. The 4.5×10
6
cells tumor showed higher tumor volumes in shorter time frame compared
to the 2.5×10
6
cells injection. The cell lines which showed growth of tumors which were steady and
comparable to that of the D2F2 cell line were the D2F2/E1-A1, D2F2/E1-B1 and D2F2/E1-B2.
These were the cell lines which showed low or medium ZsGreen and surface hEGFR expression
(Fig.14,15).
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Tumor Volume (mm
3
)
Days After Injection
D2F2/E1- 4.5 ×10
6
cells per injection
D2
F2
#10
#23
#33
#49
A1
A2
B2
B1
Fig.14:
Average tumor volume of the D2F2/E1 clones (4 ×10
6
cells per injection) plotted till day 21
after injection
40
When tabulated and compared, it was observed that the D2F2/E1-A1 cell line was the only one
which had considerable surface hEGFR expression whereas the other two cell lines showed almost
negligible surface hEGFR expression (Table.2). This indicates that the cell line D2F2/E1-A1 could
possibly be a syngeneic tumor model to evade the immunocompetent mouse system while producing
a human receptor on its surface
0
20
40
60
80
100
120
140
160
180
200
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Tumor Volume (mm
3
)
Days After Injection
D2F2/E1- 2.5×10
6
cells per injection
D2F2
#10
#23
#33
#49
A1
A2
B1
B2
End
Point
Fig.15:
Average tumor volume of the D2F2/E1 clones (2.5 ×10
6
cells per injection) plotted till day 21
after injection
41
Table.2:
Comparative Analysis of the D2F2/E1 cell lines in terms of expression and tumor growth
patterns on Day-21
(D2F2 tumor volume= 410.4 mm
3
)
Zs Green (MFI)
EGFR Expression
(MFI)
Tumor Volume
(mm
3
) Tumor growth trend
B2 0.9 B2 0.5 B2 505.3 B2 Growing
B1 274.9 B1 0.7 B1 390.1 B1 Growing
A1 345.8 #23 76.3 A1 358.5 A1 Growing
#23 567.7 A1 114.1 #33 44.9 #33 Stable
#33 672.2 #33 116.9 A2 21.0 A2 Regressing
#10 696.9 #49 117.5 #23 18.3 #23 Stable
#49 858.3 #10 306.9 #49 16.2 #49 No engraftment
A2 895.1 A2 349.7 #10 4.1 #10 No engraftment
Validating the cell line in the tumor tissue
Due to the presence of the fibroblasts, validation is required to know if the tumor was formed by the
fibroblasts or the transduced D2F2 cells. To check this the tumor tissue was banked and tumor cells
were isolated and cultured from the desired cell line D2F2/E1-A1.
42
Flow cytometry
The cells from the D2F2/E1-A1-tumor were quantitated for hEGFR expression by flow cytometer to
check for surface and intracellular expression. DiFi cell line and D2F2/E1-#10 cell line were run in
parallel as positive control for hEGFR and ZsGreen expression respectively. Unmodified D2F2 cell
line was used as the negative control. The surface hEGFR expression of D2F2/E1-A1-tumor cells
was low but we can see that the internal hEGFR expression was high (Fig.16,17,18).
Fig.16:
Histogram of the ZsGreen expression in D2F2/E1-A1-tumor cells along with controls
43
Fig.17:
Histogram of the surface hEGFR expression in D2F2/E1-A1-tumor cells along with
controls
Fig.18:
Histogram of the intracellular hEGFR expression in D2F2/E1-A1-tumor cells along with
controls
44
Immunohistochemistry
To check the composition of the tumor and arrangement of cells the tumors harvested were used for
immunohistochemical staining with an anti-hEGFR antibody from Abcam. The D2F2 tumor was
used as a negative control for these studies. D2F2/E1-A1-tumor and D2F2/E1-#10-tumor were
stained to check for hEGFR expression. As we can see the D2F2/E1-#10-tumor tissue is highly
stained for the hEGFR. We see that the D2F2/E1-A1-tumor tissue also showed positive for hEGFR
but not as intense or with as much coverage as the D2F2/E1-#10-tumor tissue. There was unexpected
background staining of the D2F2-tumor tissue. These experiments are therefore being repeated with a
different antibody at different concentrations (Fig.19).
A B
Fig.19:
Immunohistochemical staining of slides
An anti-hEGFR antibody from Abcam was used for the staining.
A) D2F2/E1-#10-tumor tissue
B) D2F2/E1-A1-tumor tissue
A negative antibody is currently being purchased since the antibody chosen
for this study from Abcam was unsuitable.
45
Discussion and Conclusion
EGFR is one of the most common biomarkers targeted in advanced cancers. Although, there are
several cell lines made which express human carcinoma or mouse carcinoma expressing human
protein receptors, no hEGFR expressing model currently in common use.
The successful transduction of the viral pLVX_EGFR plasmid into the D2F2 cell line to form the
D2F2/E1 clones was validated by immunofluorescence by the detection of ZsGreen. However, the
EGFR staining was not accomplished by immunofluorescence. This may be due to the distortion of
the hEGFR molecule by the reagent Triton-X which has been shown to decrease the functional
stability of EGFR (Mi LZ et al. 2008). The use of a different detergent like Tween20 (used for
intracellular flow cytometry) or a lower concentration (less than 0.1% Triton-X) could help resolve
this problem regarding indirect immunofluorescence microscopy.
Because of the above results, the clones of D2F2/E1 were successfully chosen with the help of flow
cytometry which showed different surface hEGFR expression compared to the ZsGreen detected.
The clones with low to medium surface hEGFR were selected and cultured to minimize expected
immunogenicity in mice.
The mice were injected with the selected D2F2/E1 tumor cell lines along with Matrigel to promote
engraftment. This combination, however, showed no engraftment and rapid clearance of the tumor
cells in vivo. Matrigel is used for in vitro models successfully but as stated in vivo it does show
immunogenicity (Serban MA et al. 2008). By contrast, co-culture injections of fibroblasts and the
D2F2/E1 cell lines were successful. Cells lines that showed a higher surface expression of hEGFR
showed immune regression whereas the low surface hEGFR expressing clone D2F2/E1-A1 clone
46
was successfully engrafted subcutaneously in the BALB/c mice. The NIH-3T3 fibroblasts produce a
nutrient rich extracellular scaffold for the tumor to form. These cells exhibit mouse EGFR proteins
on their surface (Zhou Y et al. 2012) which might have been masking the hEGFR expressed on the
surface of D2F2/E1-A1. The tumors which grew showed angiogenesis and expansion along with
ulceration due to necrosis. D2F2/E1-A1 tumor growth was validated by demonstrating the cells
positivity for ZsGreen and hEGFR. Flow cytometry of the D2F2/E1-A1-tumor cells showed lower
surface hEGFR expression compared to the transduced D2F2/E1-A1 cells. The internal hEGFR
expression in the D2F2/E1-A1-tumor cells was moderately high and this might indicate either a rapid
turnover of transmembrane expression or suppressed expression. The tumors grew large enough to
reach the end-point of the experiment. The remainder of the tumors which had higher surface hEGFR
expression showed high growth but regressed over time. These tumors also showed necrotic
ulceration before complete regression occurred in mice.
Immunohistochemical staining of the harvested tumor tissue was positive for hEGFR staining with
the help of the anti-human EGFR antibody from Abcam. The product showed some background
staining for the concentration recommended. Increasing the dilution of the antibody and using the
CTL-1 or ch225 antibody made in Dr. Epstein’s lab can help increase the staining efficiency. The use
of mouse Fc-block can also help reduce the background staining. The staining showed small pockets
of NIH-3T3 fibroblasts and larger sheets of invasive D2F2/E1-A1 cells within the tumor.
47
In conclusion, screening by flow cytometry and in vivo tumor engraftment studies, provided
convincing evidence of tumor growth of the human EGFR producing murine mammary carcinoma
D2F2 cell line. This cell line can help with large scale in vivo drug screening as well as immune-
oncology therapeutic studies including CAR-T cell experiments. The side effects of experimental
drugs can also be better studied to aid investigators in pre-clinical studies to generate predictive data
to support future Investigational New Drug (IND) applications.
48
Future directions
The use of the cell line D2F2/E1-A1 will be very useful as a model to understand the immune system
and its response in an immunocompetent system without any compounding effects. However, the cell
line needs to be validated before it can be used for other studies. Repetition of mice experiment with
the ideal co-injection of fibroblasts and D2F2/E1-A1 cells will validate the reproducibility of the cell
line. The harvested D2F2/E1-A1-tumor cells can be screened to isolate a single cell colony derived
population of cells. These can be injected into BALB/c mice with and without the aid of fibroblasts
to check if the cells can engraft subcutaneously. The cells distribution of protein must be determined
by standardizing the immunofluorescence protocol to be devoid of Triton-X which will allow the
CTL-1 to stain the cells. The immunohistochemistry stains also need to be improved to confirm if the
cells stained are the transduced D2F2/E1-A1 cell line by either modifying the concentration, use of
mouse Fc-blocks or by using another specific antibody. Along with these experiments western blots
can be run to check the integrity of the hEGFR protein produced in the transduced D2F2/E1-A1 cells.
Once the cell line is established to use, antibody screening can be done for standardized drugs like
cetuximab to compare with lab developed immunotherapies like the CTL-1. Studying the immune
system of the mice while running these immunotherapies could set guidelines for future therapies
against human EGFR.
49
References
1. Printz, C. (2017). Scientists estimate number of U.S. women living with metastatic breast cancer.
Cancer, 123(16), 2995-2996. doi:10.1002/cncr.30886
2. Whittington, P. J., Radkevich-Brown, O., Jacob, J. B., Jones, R. F., Weise, A. M., & Wei, W.
(2008). Her-2 DNA versus cell vaccine: Immunogenicity and anti-tumor activity. Cancer
Immunology, Immunotherapy, 58(5), 759-767. doi:10.1007/s00262-008-0599-x
3. Goossens, N., Nakagawa, S., Sun, X., & Hoshida, Y. (n.d.). Cancer biomarker discovery and
validation. Transl Cancer Res, 4(3), 256–269. https://doi.org/10.3978/j.issn.2218-
676X.2015.06.04
4. Paul, M. K., & Mukhopadhyay, A. K. (2004). Tyrosine kinase - Role and significance in
Cancer. International journal of medical sciences, 1(2), 101–115
5. Wee, P., & Wang, Z. (2017). Epidermal Growth Factor Receptor Cell Proliferation Signaling
Pathways. Cancers, 9(5), 52. doi:10.3390/cancers9050052
6. Gschwind, A., Fischer, O., & Ullrich, A. (2004). The discovery of receptor tyrosine kinases:
targets for cancer therapy. Nature Reviews., 4(5), 361–370. https://doi.org/10.1038/nrc1360
7. Carpenter, G., King, L., & Cohen, S. (1978). Epidermal growth factor stimulates phosphorylation
in membrane preparations in vitro. Nature., 276(5686), 409–410.
50
8. Klijn, J., Berns, P., Schmitz, P., & Foekens, J. (n.d.). The clinical significance of epidermal
growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocrine
Reviews, 13(1), 3–17. https://doi.org/10.1210/edrv-13-1-3
9. Chan, S., Hill, M., & Gullick, W. (n.d.). The role of the epidermal growth factor receptor in breast
cancer. Journal of Mammary Gland Biology and Neoplasia., 11(1), 3–11.
https://doi.org/10.1007/s10911-006-9008-2Berg, J. M.; Tymoczko, J. L.; Stryer, L. Biochemistry,
6th ed.; W.H. Freeman and Company: New York, 2007.
10. Ladanyi, M., & Pao, W. (n.d.). Lung adenocarcinoma: guiding EGFR-targeted therapy and
beyond. Modern Pathology., 21 Suppl 2, S16–S22. https://doi.org/10.1038/modpathol.3801018
11. Roskoski, R. (n.d.). The ErbB/HER family of protein-tyrosine kinases and cancer.
Pharmacological Research, 79, 34–74. https://doi.org/10.1016/j.phrs.2013.11.002
12. Ferguson K. M. (2008). Structure-based view of epidermal growth factor receptor regulation.
Annual review of biophysics, 37, 353–373. doi:10.1146/annurev.biophys.37.032807.125829
13. (n.d.). Retrieved from http://chemistry.berea.edu/~biochemistry/2009/Alisha_Kayla/
14. (n.d.). Retrieved from https://www.genecards.org/cgi-bin/carddisp.pl?gene=EGFR
15. Mutations in the EGFR Pathway. (n.d.). Retrieved from
https://www.aacc.org/publications/cln/articles/2013/october/egfr-mutations
51
16. Bemanian, V., Sauer, T., Touma, J., Lindstedt, B. A., Chen, Y., Ødegård, H. P., Geisler, J.
(2015). The epidermal growth factor receptor (EGFR / HER-1) gatekeeper mutation T790M is
present in European patients with early breast cancer. PloS one, 10(8), e0134398.
doi:10.1371/journal.pone.0134398 Rom J Morphol Embryol. 2009;50(2):217-21.
17. Meche, A., Cîmpean, A., & Raica, M. (2009).Immunohistochemical expression and significance
of epidermal growth factor receptor (EGFR) in breast cancer. Romanian Journal of Morphology
and Embryology = Revue Roumaine de Morphologie et Embryologie /, 50(2), 217–221.
18. Klijn, J., Berns, P., Schmitz, P., & Foekens, J. (n.d.). The clinical significance of epidermal
growth factor receptor (EGF-R) in human breast cancer: a review on 5232 patients. Endocrine
Reviews, 13(1), 3–17. https://doi.org/10.1210/edrv-13-1-3
19. Yi, M., Qin, S., Zhao, W., Yu, S., Chu, Q., & Wu, K. (2018). The role of neoantigen in immune
checkpoint blockade therapy. Experimental hematology & oncology, 7, 28. doi:10.1186/s40164-
018-0120-y
20. Schumacher, T., & Schreiber, R. (n.d.). Neoantigens in cancer immunotherapy. Science.,
348(6230), 69–74. https://doi.org/10.1126/science.aaa4971
21. Nakai, K., Hung, M. C., & Yamaguchi, H. (2016). A perspective on anti-EGFR therapies
targeting triple-negative breast cancer. American journal of cancer research, 6(8), 1609–1623.
22. Bielecka, Z. F., Czarnecka, A. M., Solarek, W., Kornakiewicz, A., & Szczylik, C. (2013).
Mechanisms of Acquired Resistance to Tyrosine Kinase Inhibitors in Clear - Cell Renal Cell
52
Carcinoma (ccRCC). Current signal transduction therapy, 8(3), 218–228.
doi:10.2174/1574362409666140206223014
23. Li, S., Schmitz, K., Jeffrey, P., Wiltzius, J., Kussie, P., & Ferguson, K. (n.d.). Structural basis for
inhibition of the epidermal growth factor receptor by cetuximab. Cancer Cell, 7(4), 301–311.
https://doi.org/10.1016/j.ccr.2005.03.003
24. Ribeiro Gomes, J., & Cruz, M. R. (2015). Combination of afatinib with cetuximab in patients
with EGFR-mutant non-small-cell lung cancer resistant to EGFR inhibitors. OncoTargets and
therapy, 8, 1137–1142. doi:10.2147/OTT.S75388
25. Cunningham, D., Humblet, Y., Siena, S., Khayat, D., Bleiberg, H., Santoro, A., … Van Cutsem,
E. (n.d.). Cetuximab monotherapy and cetuximab plus irinotecan in irinotecan-refractory
metastatic colorectal cancer. The New England Journal of Medicine., 351(4), 337–345.
https://doi.org/10.1056/NEJMoa033025
26. Zhang, W., Gordon, M., Schultheis, A., Yang, D., Nagashima, F., Azuma, M., … Lenz, H. (n.d.).
FCGR2A and FCGR3A polymorphisms associated with clinical outcome of epidermal growth
factor receptor expressing metastatic colorectal cancer patients treated with single-agent
cetuximab. Journal of Clinical Oncology : Official Journal of the American Society of Clinical
Oncology., 25(24), 3712–3718. https://doi.org/10.1200/JCO.2006.08.8021
27. Piechocki, M., Pilon, S., & Wei, W. (n.d.). Complementary antitumor immunity induced by
plasmid DNA encoding secreted and cytoplasmic human ErbB-2. The Journal of Immunology :
Official Journal of the American Association of Immunologists., 167(6), 3367–3374.
53
28. Bonaventura, P., Shekarian, T., Alcazer, V., Valladeau-Guilemond, J., Valsesia-Wittmann, S.,
Amigorena, S., … Depil, S. (2019). Cold Tumors: A Therapeutic Challenge for
Immunotherapy. Frontiers in immunology, 10, 168. doi:10.3389/fimmu.2019.00168
29. JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Molecular
Cloning. JoVE, Cambridge, MA, (2019).
30. Teschendorf, C., Warrington, K., Siemann, D., & Muzyczka, N. (n.d.). Comparison of the EF-1
alpha and the CMV promoter for engineering stable tumor cell lines using recombinant adeno-
associated virus. Anticancer Research., 22(6A), 3325–3330.
31. Klein, R., Ruttkowski, B., Knapp, E., Salmons, B., Günzburg, W., & Hohenadl, C. (n.d.). WPRE-
mediated enhancement of gene expression is promoter and cell line specific. Gene., 372, 153–
161. https://doi.org/10.1016/j.gene.2005.12.018
32. Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New
York: W. H. Freeman; 2000. Transduction. Available from:
https://www.ncbi.nlm.nih.gov/books/NBK21760/
33. Denning, W., Das, S., Guo, S., Xu, J., Kappes, J. C., & Hel, Z. (2013). Optimization of the
transductional efficiency of lentiviral vectors: effect of sera and polycations. Molecular
biotechnology, 53(3), 308–314. doi:10.1007/s12033-012-9528-5
34. Cellosaurus: ExPASy: Cell line:
i. NIH 3T3 (RRID:CVCL 0594)
ii. D2F2 (RRID:CVCL 0I91)
54
iii. DiFi (RRID:CVCL_6895)
35. Hu, P., Graff, R., Zheng, L., Khawli, L.A., & Epstein, A.L. (2017). Preclinical Characterization
of CTL-1 , A Biosimilar Anti-EGFR Monoclonal Antibody for Cetuximab. SM J Bioequiv
Availab. 2017; 1(1): 1003.
36. Bao, L., Matsumura, Y., Baban, D., Sun, Y., & Tarin, D. (1994). Effects of inoculation site and
Matrigel on growth and metastasis of human breast cancer cells. British journal of cancer, 70(2),
228–232.
37. Day, R. N., & Davidson, M. W. (2009). The fluorescent protein palette: tools for cellular
imaging. Chemical Society reviews, 38(10), 2887–2921. doi:10.1039/b901966a
38. Tarnowski, B., Spinale, F., & Nicholson, J. (1991). DAPI as a useful stain for nuclear
quantitation. Biotechnic & Histochemistry., 66(6), 297–302.
39. Herzenberg, L., Tung, J., Moore, W., Herzenberg, L., & Parks, D. (n.d.). Interpreting flow
cytometry data: a guide for the perplexed. Nature Immunology., 7(7), 681–685.
https://doi.org/10.1038/ni0706-681
40. Harel, S., Higgins, C. A., Cerise, J. E., Dai, Z., Chen, J. C., Clynes, R., & Christiano, A. M.
(2015). Pharmacologic inhibition of JAK-STAT signaling promotes hair growth. Science
advances, 1(9), e1500973. doi:10.1126/sciadv.1500973
41. Kojima, M., Higuchi, Y., Yokota, M., Ishii, G., Saito, N., Aoyagi, K., … Ochiai, A. (2014).
Human subperitoneal fibroblast and cancer cell interaction creates microenvironment that
55
enhances tumor progression and metastasis. PloS one, 9(2), e88018.
doi:10.1371/journal.pone.0088018
42. Sung, K. E., Su, X., Berthier, E., Pehlke, C., Friedl, A., & Beebe, D. J. (2013). Understanding the
impact of 2D and 3D fibroblast cultures on in vitro breast cancer models. PloS one, 8(10),
e76373. doi:10.1371/journal.pone.0076373
43. Picard, O., Rolland, Y., & Poupon, M. (n.d.). Fibroblast-dependent tumorigenicity of cells in
nude mice: implication for implantation of metastases. Cancer Research., 46(7), 3290–3294.
44. Mi, L. Z., Grey, M. J., Nishida, N., Walz, T., Lu, C., & Springer, T. A. (2008). Functional and
structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid
nanodiscs. Biochemistry, 47(39), 10314–10323. doi:10.1021/bi801006s
45. Serban, M. A., & Prestwich, G. D. (2008). Modular extracellular matrices: solutions for the
puzzle. Methods (San Diego, Calif.), 45(1), 93–98. doi:10.1016/j.ymeth.2008.01.010
46. Zhou, Y., Lee, J. Y., Lee, C. M., Cho, W. K., Kang, M. J., Koff, J. L., … Lee, C. G. (2012).
Amphiregulin, an epidermal growth factor receptor ligand, plays an essential role in the
pathogenesis of transforming growth factor-β-induced pulmonary fibrosis. The Journal of
biological chemistry, 287(50), 41991–42000. doi:10.1074/jbc.M112.356824
Abstract (if available)
Abstract
Epidermal growth factor receptor was one of the first biomarkers identified as it was seen to be upregulated in metastatic breast cancer. The upregulation is caused due to a mutation in the tyrosine kinase domain of the EGFR protein. The mutation alters the EGFR protein, but not sufficiently to evoke an immune response. There have been several drugs developed against the human EGFR like the tyrosine kinase inhibitors for example- gefitinib, erlotinib and afatinib or monoclonal antibodies for example-cetuximab, CTL-1/ ch225, panitumumab, necitumumab and zalutumumab. These drugs are screened in immunocompromised mice systems and hence have been seen to exhibit partial response in the case of mAbs and toxicity in terms of the TKI. D2F2 is a murine mammary gland carcinoma obtained from a BALB/c mouse which is a cold tumor exhibiting the characteristics of a triple negative breast cancer along with that of metastatic advanced breast cancer. Genetically modifying the D2F2 cell line to express human EGFR by lentiviral transduction produces the D2F2/E1 clones. Upon screening, different clones which express different surface human EGFR were isolated. These clones were checked for in vivo subcutaneous engraftments in BALB/c mice by using the D2F2/E1 clones alone or the D2F2/E1 clones with Matrigel which both showed tumor auto-regression or no engraftment respectively. The use of a coculture of the D2F2/E1 clones and NIH-3T3 murine fibroblasts helps overcome the mouse immunogenicity and one of the cell lines which showed low to medium surface human EGFR (D2F2/E1-A1) showed steady growth comparable to the D2F2 unmodified cell line, while the higher hEGFR expressing clones showed regression. The tumor from this D2F2/E1-A1 cell line was harvested, and the cells were isolated from the successfully engrafted tumors to validate that the tumor was formed due to the transduced cell lines as well as the fibroblasts. These cells with further evaluation can be established as a murine mammary carcinoma D2F2 cell line which was modified to express human EGFR on the surface to successfully be a syngeneic immunocompetent model.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
PDF
Stable expression of human B7-H4 in a mouse mammary tumor model as a target for cancer immunotherapy
PDF
Tri-specific T cell engager immunotherapy targeting tumor initiating cells
PDF
Immune signature of murine solid tumor models
PDF
Polycomb repressive complex 2 subunit stabilizes NANOG to maintain self-renewal in hepatocellular carcinoma tumor-initiating stem-like cells
PDF
Roles of epithelial-mesenchymal transition and niche in tumorigenesis of tumor-initiating cells
PDF
c-JUN mediated alteration of SLC2A2 expression in hepatoma cell line HepG2
PDF
Role of a novel transmembrane protein, TMEM56, in tumorigenic growth of human PC3 prostate cancer cell line
PDF
Identification of molecular mechanism for cell-fate decision in liver; &, SARS-CoV replicon inhibitor high throughput drug screening
PDF
Novel approaches of mobilizing human iNKT cells for cancer immunotherapies
PDF
Design, synthesis and validation of Axl-targeted monoclonal antibody probe for microPET imaging of human lung cancer
PDF
Characterization of invariant natural killer T cells in a novel humanized HBV-transgenic model
PDF
Human papillomavirus type 16 entry via the annexin A2 heterotetramer leads to infection and immune evasion
PDF
The analysis and modeling of signaling pathways induced by the interactions of the SARS-CoV-2 spike protein with cellular receptors
PDF
Optimizing an immortalized human alveolar epithelial cell line model system to recapitulate lung adenocarcinoma development in vitro
Asset Metadata
Creator
Pachipulusu, Vyshnavi
(author)
Core Title
Establishing a human EGFR expressing murine mammary carcinoma cell line-D2F2, as a syngeneic immunocompetent model
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
12/27/2020
Defense Date
05/07/2019
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
D2F2 cell line,genetic modification,human EGFR,lentiviral transduction,murine mammary gland carcinoma,OAI-PMH Harvest,syngeneic immunocompetent tumor model
Format
application/pdf
(imt)
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Machida, Keigo (
committee chair
), Comai, Lucio (
committee member
), Epstein, Alan (
committee member
)
Creator Email
pachipul@usc.edu,vyshnavip19@gmail.com
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c89-179201
Unique identifier
UC11660327
Identifier
etd-Pachipulus-7514.pdf (filename),usctheses-c89-179201 (legacy record id)
Legacy Identifier
etd-Pachipulus-7514-0.pdf
Dmrecord
179201
Document Type
Thesis
Format
application/pdf (imt)
Rights
Pachipulusu, Vyshnavi
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
D2F2 cell line
genetic modification
human EGFR
lentiviral transduction
murine mammary gland carcinoma
syngeneic immunocompetent tumor model