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Stable expression of human B7-H4 in a mouse mammary tumor model as a target for cancer immunotherapy
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Stable expression of human B7-H4 in a mouse mammary tumor model as a target for cancer immunotherapy
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Content
Stable
expression
of
Human
B7-‐H4
in
a
Mouse
Mammary
Tumor
Model
as
a
Target
for
Cancer
Immunotherapy
A
Thesis
Presented
to
the
FACULTY
OF
THE
USC
GRADUATE
SCHOOL
UNIVERSITY
OF
SOUTHERN
CALIFORNIA
In
Partial
Fulfillment
of
the
Requirements
for
the
Degree
MASTER
OF
SCIENCE
(MOLECULAR
MICROBIOLOGY
AND
IMMUNOLOGY)
May
2014
Copyright
2014
Ishwarya
Sankaranarayanan
2
ACKNOWLEDGEMENTS
Foremost,
I
would
like
to
express
my
gratitude
to
Prof.
Alan
Epstein,
for
allowing
me
to
grow
as
a
researcher
in
his
laboratory.
His
support
and
guidance
has
whet
my
knowledge
to
pursue
my
research.
I
would
like
to
specially
thank
Katrin
Tiemann
PhD,
for
having
given
me
priceless
moments
of
her
mentorship
and
motivation
not
only
for
my
research
but
also
for
my
career.
In
addition,
I
would
like
to
thank
my
fellow
lab
members
for
their
endless
support
over
the
last
two
years,
without
whom
my
research
would
not
have
been
interesting.
Last
but
not
the
least,
I
am
extremely
grateful
to
my
family
and
friends
for
all
their
sacrifices
and
prayers
and
continuous
encouragement
to
help
me
achieve
this
goal.
3
TABLE
OF
CONTENTS
Table
of
Contents
ACKNOWLEDGEMENTS
.........................................................................................................
2
TABLE
OF
CONTENTS
.............................................................................................................
3
LIST
OF
TABLES
.......................................................................................................................
6
LIST
OF
FIGURES
......................................................................................................................
7
ABBREVIATIONS
......................................................................................................................
8
ABSTRACT
..................................................................................................................................
9
CHAPTER
1
..............................................................................................................................
10
INTRODUCTION
.....................................................................................................................
10
1.1
Breast
Cancer:
............................................................................................................................................
10
1.2
Therapies
for
Breast
Cancer:
...............................................................................................................
10
1.3
Immunotherapy
Approaches:
..................................................................................................................
13
1.4
Tumor
Invasion:
........................................................................................................................................
13
1.5
B7
Family:
.........................................................................................................................................................
14
1.6
B7-‐H4:
................................................................................................................................................................
16
CHAPTER
2
..............................................................................................................................
18
2
Aim
of
this
project
........................................................................................................................................
18
4
CHAPTER
3
..............................................................................................................................
19
MATERIALS
AND
METHODS
...............................................................................................
19
3.1
Materials:
..........................................................................................................................................................
19
3.1.1
4T1
Mouse
Mammary
Carcinoma:
.....................................................................................................
19
3.1.2
Plasmid
eGFP-‐IRES
...................................................................................................................................
19
3.1.3
Buffers
and
Solutions:
.............................................................................................................................
20
3.2
Methods:
...........................................................................................................................................................
23
3.2.1
Cell
Lines
and
Cell
Culture:
....................................................................................................................
23
3.2.2
Amplification
of
h-‐B7-‐H4
from
SKBR3:
...........................................................................................
24
3.2.3
PCR
amplification
of
B7-‐H4:
.................................................................................................................
25
3.2.4
Preparation
of
Competent
Cells:
.........................................................................................................
26
3.2.5
Construction
of
Plasmid:
........................................................................................................................
26
3.2.6
Transformation:
.........................................................................................................................................
28
3.2.7
Preparation
of
Agar
plates:
...................................................................................................................
28
3.2.8
Colony
PCR:
..................................................................................................................................................
29
3.2.9
Transfection:
...............................................................................................................................................
29
3.2.10
Screening
of
transfected
cells
for
B7-‐H4
expression
and
stability:
..................................
31
3.2.11
Tumor
Implantation
studies:
.............................................................................................................
33
3.2.12
Immunohistochemistry:
......................................................................................................................
33
CHAPTER
4
..............................................................................................................................
35
RESULTS
...................................................................................................................................
35
4.1
Expression
of
B7-‐H4
on
human
tumor
cell
lines
.............................................................................
35
4.2
Amplification
of
B7-‐H4
from
SKBR3
.....................................................................................................
36
5
4.3
Plasmid
Construction:
.................................................................................................................................
36
4.4
Transformation,
Colony
PCR,
Sequencing:
.........................................................................................
38
4.5
Stable
cell
line
Transfection
using
Lipofectamine2000:
..............................................................
40
4.6
Screening
of
cells
by
Western
Blot
and
Flow
Cytometry:
............................................................
41
4.7
Tumor
Implantation
studies,
Immunohistochemistry:
................................................................
47
CHAPTER
5
..............................................................................................................................
50
DISCUSSION
.............................................................................................................................
50
FUTURE
DIRECTIONS…………………………………………………………………………………….53
CHAPTER
6
..............................................................................................................................
54
REFERENCES
...........................................................................................................................
54
6
LIST
OF
TABLES
Table1:
Classification
and
function
of
B7
family
proteins
Table2:
Cell
lines.
Summary
of
cell
lines
used
in
this
project
and
their
growth
conditions
Table3:
RNA
to
cDNA
components
Table4:
Primer
sequences
Table5:
Screening
of
in-‐house
antibodies
7
LIST
OF
FIGURES
Figure
1.1
Treatments
of
breast
cancer
Figure
1.2
Immune-‐escape
theory
Figure
1.3
Binding
of
B7-‐H4
on
T-‐cells
Figure
1.4
Nucleotide
alignment
of
mouse
and
human
B7-‐H4
Figure
3.1
Plasmid
map
of
pIRES-‐EGFP
Figure
4.1
Protein
levels
of
B7-‐H4
in
various
carcinoma
cell
line
Figure
4.2
Isolation
and
amplification
of
human
B7-‐H4
from
SKBR3
Figure
4.3
Plasmid
digestion
Figure
4.4.1
Transformation
of
ligated
pIRES-‐EGFP-‐hB7-‐H4
Figure
4.4.2
Colony
PCR
of
transformed
pIRES-‐EGFP-‐hB7-‐H4
Figure
4.4.3
nBlast
between
sequence
results
and
h-‐B7-‐H4
Figure
4.5
Transfection
of
4T1
cells
with
different
transfection
agents
Figure
4.6.1
Screening
of
hB7-‐H4
by
Western
Blot
Figure
4.6.2
Screening
of
4T1
transfected
cells
by
Flow
Cytometry
Figure
4.6.3
Binding
of
in-‐house
antibodies
to
B7-‐H4-‐Fc.
Figure
4.7
Tumor
implantation
studies
Figure
4.8
Expression
of
hB7-‐H4
by
immunohistochemistry
8
ABBREVIATIONS
IL-‐6
Interleukin-‐6
IL-‐10
Interleukin-‐10
APC
Antigen
Presenting
Cells
TGF-‐ß
Transforming
growth
factor
ß
Tregs
T
regulatory
cells
CTLA-‐4
Cytotoxic
T-‐lymphocytes
Antigen
4
VTCN1
V-‐set
domain
containing
T
cell
activation
inhibitor
1
nBlast
Nucleotide
Blast
NCBI
National
Center
for
Biotechnology
Information
IRES
Internal
ribosomal
entry
site
EGFP
Enhanced
green
fluorescent
protein
IVS
synthetic
intron
RPMI-‐1640
Roswell
Park
Memorial
Institute-‐1640
medium
FBS
Fetal
Bovine
Serum
ATCC
American
Type
Culture
Collection
PBS
Phosphate
Buffered
Saline
NEB
New
England
Bioscience
SCBT
Santa
Cruz
Biotechnology
PLGF
Placental
like
Growth
Factor
HER2
Human
Epidermal
Growth
Factor
Receptor-‐2
SDS-‐PAGE
Sodium
dodecyl
sulphate-‐Polyacryamide
gel
electrophoresis
9
ABSTRACT
B7-‐H4
is
a
member
of
the
B7
family,
which
is
a
costimulatory
molecule
and
negatively
regulates
the
immune
response
of
T
cells.
Its
mechanism
of
tumoral
immune
escape
and
therapeutic
targeting
is
still
unknown.
B7-‐H4
is
overly
expressed
in
breast
cancer,
ovarian
cancer,
and
many
solid
tumors
and
is
being
investigated
as
a
good
immunotherapy
target
for
antibodies
or
antibody
drug
conjugates.
To
elucidate
its
immune
function
as
well
as
investigate
newly
developed
anti-‐B7-‐H4
antibodies,
the
murine
mammary
tumor
cell
line,
4T1,
was
transfected
with
the
human
B7-‐H4
gene
to
generate
a
syngeneic
tumor
model
in
immuno-‐
competent
BALB/c
mice.
To
facilitate
the
generation
of
a
stabile
cell
line
for
in
vivo
studies,
4T1
a
mouse
breast
cancer
cell
line
with
metastatic
potential
similar
to
that
seen
in
human
breast
cancer,
was
transfected
with
the
pIRES-‐eGFP
plasmid
containing
an
internal
ribosomal
entry
site
of
encephalomyocarditis
virus
(CMV),
the
gene
for
green
fluorescent
protein
(GFP),
and
the
Genetecin
resistant
gene
for
selection.
The
human
B7-‐H4
gene
was
first
amplified
from
the
SKBR3
human
breast
cancer
cell
line
known
to
be
a
high
expressor
of
B7-‐H4
and
cloned
into
pIRES-‐eGFP
via
the
EcoRI
and
BamHI
restriction
sites.
Tumors
grown
on
BALB/c
mice
with
transfected
4T1
cells
were
not
rejected
demonstrating
the
utility
of
this
approach.
10
CHAPTER
1
INTRODUCTION
1.1 Breast
Cancer:
Breast
cancer
is
one
of
the
most
common
cancers
found
in
females
1
and
represents
23%
of
all
the
tumors
worldwide.
The
breast
consists
of
glands
producing
milk,
ducts,
and
connective
tissues
that
hold
the
two
tissues
together.
2
Breast
cancer
is
subclassified
into
two
types:
ductal
carcinoma
and
lobular
carcinoma.
Ductal
carcinoma
is
the
most
common
type,
and
originates
from
the
lining
of
the
milk
duct.
They
are
subclassified
into
invasive
or
in
situ
carcinoma,
the
latter
being
metastatic
By
contrast,
lobular
carcinoma
originates
at
the
glands
that
produce
the
milk,
and
it
may
be
present
inside
the
glands
or
may
metastasizes
to
the
surrounding
tissues.
In
2014
alone,
there
have
been
over
200,000
new
cases
and
40,000
deaths
in
women
according
to
National
Cancer
Institute
and
National
Institutes
of
Health
(http://www.cancer.gov/cancertopics/types/breast)
Breast
cancer
can
also
occur
in
males
although
it
is
rare.
1.2 Therapies
for
Breast
Cancer:
Invasive
lobular
and
ductal
carcinoma
may
be
fatal,
but
some
current
treatments
of
breast
cancer
are
beneficial.
Treatments
are
characterized
as
either
local
or
systemic
in
design
as
shown
in
Figure
1.1.
11
1.2.1
Surgery:
Surgery
is
performed
to
remove
partial
breast
tissue
bearing
tumor
(partial
mastectomy)
or
the
entire
breast
(mastectomy).
Partial
mastectomy
depends
on
the
size,
location,
and
usually
is
augmented
by
radiation
therapy.
Mastectomies
involve
in
removal
of
the
entire
breast
along
with
surrounding
tissues.
Side
effects
of
this
treatment
are
pain,
swelling,
and
infection.
3
1.2.2
Radiation:
This
treatment
is
with
high-‐energy
radiation
to
prevent
spread
of
cancerous
cells
to
neighboring
areas.
It
is
usually
given
after
surgery.
GAMMA
knife
radiotherapy
is
used
to
irradiate
only
the
tumor
maximally
while
sparing
the
surrounding
healthy
tissues.
By
contrast,
external
beam
radiation
treats
both
healthy
tissues
and
tumor.
4
Treatment
option
for
Breast
Cancer
Local
Therapies
Surgery
Radiation
Systemic
Therapies
Chemotherapy
Hormone
Therapy
Targeted
Therapy
Figure
1.1
Treatments
of
breast
cancer
12
1.2.3
Chemotherapy:
Chemotherapy
is
administered
systemically
to
deliver
cytotoxic
drugs.
Adjuvant
chemotherapy
is
used
after
surgery
to
destroy
residual
tumor
and
neoadjuvant
therapy
is
used
prior
to
surgery
to
shrink
the
size
of
the
tumor.
These
drugs
generally
are
cytotoxic
to
rapidly
growing
cells
in
the
tumor
and
around
tissues
such
as
gut
and
hair
follicles.
5
1.2.4
Hormone
Therapy:
This
treatment
usually
is
used
along
with
adjuvant
therapy.
It
is
based
upon.
It
is
based
upon
the
dependence
of
breast
cancer
cells
for
estrogen
to
grow.
Tamoxifen
a
commonly
used
drug
blocks
the
binding
of
estrogen
to
its
receptor
to
inhibit
tumor
cell
growth.
This
drug
causes
blood
clots,
and
also
Osteoporosis.
6
1.2.5
Targeted
Therapy:
Targeted
therapy
is
specific
to
genes
and/or
proteins
that
cause
tumor
cells
to
grow
rapidly
and
abnormally.
For
cells
to
be
cancerous,
normal
cells
undergo
carcinogenesis
by
mutations
in
a
wide
variety
of
genes
such
as
BRACA2
and
MGMT
7
.
One
such
targeted
therapy
is
for
the
HER2
protein,
which
is
also
known
as
growth-‐
promoting
protein
and
is
overly
expressed
in
breast
cancer.
Targeting
HER2
with
Herceptin
helps
to
slow
the
growth
of
cancer
cells
and
stimulate
the
immune
system
to
attack
them.
8
13
1.3
Immunotherapy
Approaches:
Cancerous
cells
disguise
themselves
to
evade
the
immune
system
9
,
often
by
losing
their
HLA
expression.
In
addition,
tumor
may
express
inhibitory
proteins
such
as
B7-‐H4,
PD-‐L1,
and
others
to
block
immune
activation
to
the
tumor
10
.
1.4 Tumor
Invasion:
T-‐lymphocytes
play
an
important
role
in
protecting
the
normal
tissues
by
its
ability
to
distinguish
foreign
from
native
antigens.
For
tumors
to
grow
and
survive,
they
must
disguise
themselves
in
order
to
escape
from
the
host’s
immune
system.
The
tumor
microenvironment
plays
an
important
role
in
this
mechanism
immune
suppression
by
secreting
immunosuppressive
cytokines,
or
dysfunctioning
the
immune
cells
in
order
to
evade
the
cancerous
cells
11
.
This
process
of
evasion
increases
the
expression
of
inhibitory
B7
molecules
and
subsequently
secretes
immunosuppressive
cytokines
like
TGF-‐β
and
down
regulates
T-‐cell
proliferation
by
interfering
with
the
cell
cycle
12
.
T-‐
regulatory
cells
are
cells
that
suppress
the
immune
system
by
inducing
IL-‐6
and
IL-‐10
production
by
macrophages,
by
secreting
immunosuppressive
adenosine,
and
by
causing
antigen-‐presenting
cells
(APC)
to
express
B7-‐H4.
APC
cells
also
secrete
TGF-‐ß,
which
in
turn
increases
the
up-‐regulation
of
B7-‐H4
and
T-‐cell
inhibition
via
interference
with
the
cell
cycle.
The
role
of
B7
family
and
TGF-‐ß
secretion
regulates
anti-‐tumor
immunity
12
.
14
Fig
1.2:
“Immune-‐
escape”
theory,
B7H4-‐
associated
with
Antigen
Presenting
Cells
(APC)
after
transformation
by
T
regulatory
cells
(Tregs)
produce
high
levels
of
IL-‐6,
IL-‐10,
and
immunosuppressive
cytokines
such
as
TGF-‐
β.
The
secretion
of
TGF-‐ß
and
B7-‐H4
leads
to
T-‐
cell
inhibition
via
cell
cycle
arrest
12
.
1.5
B7
Family:
The
B7
family
consists
of
membrane
proteins
that
are
present
on
the
antigen
presenting
cells
(APC)
and
negatively
regulate
the
T-‐cell
response.
The
B7
family
consists
of
B7-‐1,
B7-‐2,
B7-‐DC,
B7-‐H1,
B7-‐H2,
and
the
newly
discovered
B7-‐H3
and
B7-‐H4.
The
first
group
of
the
B7
family
is
the
B7-‐1
and
B7-‐2
that
bind
to
CD28
(cluster
of
differentiation
28)
and
Cytotoxic
T-‐lymphocytes
Antigen
4
(CTLA-‐4)
on
APC.
The
second
group
comprising
of
B7-‐H1
and
B7-‐DC
contains
ligands
involved
in
the
T-‐cell
dysfunctioning.
The
last
group
of
the
B7
family
is
the
B7-‐H3
and
B7-‐H4
which
involves
costimulation
to
inhibit
T-‐cell
proliferation.
For
B7-‐H4,
the
receptor
Gynecologic Oncology, Volume 127, Issue 2, 2012, 420 - 425
15
is
still
unknown
(see
Table
1).
Costimulation
begins
with
the
interaction
of
T-‐cells
with
the
major
histocompatibility
complex
in
order
to
activate
the
T-‐cells
followed
by
binding
of
tumor
receptor
B7-‐H4
to
its
unknown
receptor
on
the
T-‐cell
as
a
costimulatory
signal
causing
it
to
produce
a
down
regulation
of
the
T-‐cell
response
12,
13
.
Figure
1.3:
Binding
of
B7-‐H4
to
T-‐cells.
B7-‐H4
on
tumor
(Beige)
binds
to
its
counter
receptor
on
T-‐cells
(red)
and
inhibits
proliferation
and
cytokine
production
by
CD4+
and
CD8+
T-‐cells.
Table
1
Classification
and
function
of
B7
family:
B7
Molecule
Alternative
Name
Receptor
Function
B7-‐1
CD-‐80
CD-‐28,
CTLA-‐4
Stimulatory
B7-‐2
CD-‐86
CD-‐28,
CTLA-‐4
Stimulatory
B7-‐H1
PD-‐L1,
CD274
CD-‐80,
CD-‐279
Inhibitory
B7-‐H2
PD-‐L2,
CD273
Programmed
cell
death-‐1
Inhibitory
B7-‐H3
CD276
NK
subsets
Inhibitory/Stimulatory
B7-‐H4
VTCN1
Unknown
Inhibitory/Stimulatory
Table
1
Classification
and
function
of
B7
family
protein
13
.
16
1.6
B7-‐H4:
B7-‐H4
is
part
of
the
B7
family
and
also
known
as
V-‐set
domain-‐containing
T-‐cell
activation
inhibitor
1
or
VTCN1.
It
is
found
overly
expressed
in
breast,
ovarian
and
lung
cancers
14
.
It
is
a
membrane
protein
that
plays
an
important
role
in
T-‐cell
inhibition
13
.
The
increased
expression
of
B7-‐H4
in
breast
cancer
has
shown
to
decrease
the
number
of
tumor
immune
cell
infiltrates
thus,
preventing
tumor
cells
from
undergoing
apoptosis
15
.
SKBR3,
a
human
mammary
carcinoma
cell
line,
overexpresses
B7-‐H4
on
its
cell
surface.
16
Understanding
the
mechanism
and
tumor
immune
response
of
B7-‐H4
is
important
in
defining
it
as
a
new
target
for
cancer
immunotherapy
12,
1718
.
The
human
B7-‐H4
gene
is
found
on
chromosome
1p11.1,
consisting
of
five
introns
and
six
exons.
About
87%
homology
has
been
observed
between
mouse
and
human
B7-‐H4
and
a
25%
homology
with
other
B7
family
members
19
.
It
is
around
800bps
and
consists
of
a
type
I
transmembrane
domain,
a
large
hydrophobic
transmembrane,
and
a
very
short
intracellular
domain.
17
M
B7-‐H4
H
B7-‐H4
Fig
1.4
Nucleotide
alignment
of
mouse
and
human
B7-‐H4
via
nBlast
(NCBI)
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasth
ome)
M
B7-‐H4
H
B7-‐H4
M
B7-‐H4
H
B7-‐H4
M
B7-‐H4
H
B7-‐H4
M
B7-‐H4
H
B7-‐H4
M
B7-‐H4
H
B7-‐H4
18
CHAPTER
2
2 Aim
of
this
project
The
overall
goal
for
this
project
is
to
create
a
stabile
cell
line
expressing
the
human
B7-‐H4
membrane
protein
in
a
4T1
mouse
mammary
carcinoma
in
order
to
target
human
B7-‐H4
in
immunocompetent
Balb/C
mice.
This
tumor
model
will
then
be
used
further
to
test
in-‐house
anti-‐human-‐B7H4
antibodies
as
potential
therapeutics
or
delivery
vehicle
by
biodistribution
and
imaging
technologies.
Specifically,
human
carcinoma
cell
line
with
high
expression
of
B7-‐H4
will
be
used
to
amplify
the
gene.
Furthermore,
our
goal
is
to
detect
the
best
in-‐house
antibody
to
B7-‐H4
by
screening
via
Western
Blotting,
Flow
Cytometry,
and
Immunohistochemistry.
In
addition,
we
plan
on
studying
tumor
growth/rejection
in
BALB/c
mice
by
newly
developed
reagents
in
immunocompetent
mice
bearing
hu-‐
B7-‐H4
positive
syngeneic
tumor,
such
as
the
4T1
murine
breast
tumor
model.
19
CHAPTER
3
MATERIALS
AND
METHODS
3.1
Materials:
3.1.1
4T1
Mouse
Mammary
Carcinoma:
Tumor
growth
and
metastasis
are
important
characteristic
of
tumors
that
need
to
be
addressed
cancer
immunotherapy
because
of
HLA
expressory
human
cells
cannot
be
grown
in
mice.
By
contrast,
tumors
syngeneic
to
the
host
are
able
to
grow
and
in
some
models,
metastasize
to
lung,
liver,
and
brain
are
seen
in
cancer
patients.
The
4T1
murine
mammary
carcinoma
is
highly
metastatic
and
closely
mimics
that
of
human
breast
cancer
20
.
They
are
adherent,
epithelial
cells
that
were
first
isolated
from
a
BALB/cfC3H
mouse
and
do
not
express
B7-‐H4.
3.1.2
Plasmid
eGFP-‐IRES
The
plasmid
pIRES-‐eGFP
(see
Fig.3.2)
contains
Internal
Ribosomal
Entry
Site
(IRES)
of
the
Encephalomycarditis
virus
(ECMV)
and
an
enhanced
green
fluorescent
protein
(eGFP)
21
.
The
IRES
is
positioned
between
the
multiple
cloning
site
and
the
eGFP,
which
enables
the
expression
of
both
the
gene
of
interest
and
the
eGFP
gene
simultaneously
from
one
promoter.
The
presence
of
eGFP
protein
provides
an
efficient
selection
process
using
fluorescence
microscopy
or
flow
cytometry.
The
IRES-‐eGFP
plasmid
contains
a
synthetic
intron
(IVS),
which
enhances
the
stability
of
20
mRNA.
This
plasmid
also
contains
an
ampicillin
resistant
gene
and
a
neo
gene
resistant
to
geneticin
enabling
selection
in
cell
culture
22,23,24
.
IRES
EcoRI
BamHI
MSC
CMV
pIRES-‐EGFP
5.2kb
NEO
Fig
3.1
Plasmid
map
of
pIRES-‐eGFP.
It
contains
a
CMV
promoter
(blue),
a
multiple
cloning
sites
with
restriction
enzymes
BamHI
and
EcoRI,
an
internal
ribosomal
entry
site
(red),
green
fluorescent
protein
for
detection
using
green
signal
(green),
and
ampicillin
and
neomycin
resistant
genes
(orange)
needed
for
selection.
GFP
21
3.1.3
Buffers
and
Solutions:
2x
Western
Blot
Protein
Loading
Solution
20%
Glycerol
0.02%
Bromophenol
Blue
125mM
TrisCl
(pH
6.8)
5%
SDS
10x
TBS
(pH
7.4)
1M
Tris
Base
5M
NaCl
5x
Western
Blot
Running
Buffer
250mM
Tris
Base
(pH
8.3)
1.92M
Glycine
0.5%
SDS
10mM
EDTA
1x
Western
Transfer
Buffer
20mM
Tris
Base
194mM
Glycine
0.005%
SDS
20%
Methanol
1x
Western
Blot
Wash
Solution
(TBS-‐T
or
PBS-‐T)
1x
TBS,
0.1%
Tween-‐20
1x
PBS,
0.1%
Tween-‐20
1x
Western
Blot
Blocking
Solution
(TBS-‐T-‐BSA
or
TBS-‐T-‐milk)
22
TBS-‐T
with
5%
BSA
or
5%
instant
dry
milk
RFI
Buffer:
100mM
Rubidium
Chloride
50mM
Manganese
Chloride
30mM
Potassium
acetate
10mM
Calcium
Chloride
RFII
Buffer
(pH
6.8)
10mM
Rubidium
Chloride
30mM
Calcium
Chloride
15%
Glycerol
10mM
3-‐(N-‐morpholino)
propanesulfonic
acid
500ml
LB
Broth
10g
LB
broth
powder
500ml
of
MilliQ
23
3.2
Methods:
3.2.1
Cell
Lines
and
Cell
Culture:
Table
2:
Cell
lines.
Summary
of
cell
lines
used
in
this
project
and
their
growth
conditions.
Cell
line
Type
of
Carcinoma
Growth
Properties
Culture
Medium
Split
Ratio
Source
4T1
Mouse
mammary
Adherent
RPMI-‐1640
with
10%
FBS
and
1%
L-‐
glutamine
1:5
ATCC
SKBR3
Human
mammary
Adherent
Same
1:5
ATCC
JAR
Human
trophoblastic
Adherent
Same
1:5
ATCC
T47D
Human
mammary
Adherent
Same
1:5
ATCC
MCF7
Human
mammary
Adherent
Same
1:5
ATCC
SKOV3
Human
ovarian
Adherent
Same
1:5
ATCC
MDA
MB
468
Human
mammary
Adherent
Same
1:3
ATCC
HT29
Human
colorectal
Adherent
Same
1:5
ATCC
Table
2.
List
of
cell
lines
used
in
this
project
and
their
media
demands.
The
cells
were
cultured
at
37ºC
in
a
humidified,
5%
CO2
atmosphere.
To
passage
these
cell
lines,
cells
were
washed
with
phosphate-‐buffered
saline,
and
trypsinized
with
1X
to
detach
the
cells
from
the
surface
of
the
flask,
centrifuged
in
a
falcon
tube
at
370xg
for
10
minutes,
and
were
split
a
1:5
for
subpasaging.
24
3.2.2
Amplification
of
h-‐B7-‐H4
from
SKBR3:
One
hundred
thousand
cells
were
used
to
extract
the
RNA
using
the
Qiagen
RNeasy
mini
kit
(Qiagen)
according
to
the
manufacture’s
protocol.
In
short,
a
QIAshredder
homogenizer
column
(Qiagen)
was
first
used
to
lyse
and
disrupt
the
cells
followed
by
the
addition
of
1
volume
of
70%
ethanol.
The
sample
along
with
their
precipitates
were
added
to
RNeasy
spin
column,
followed
by
multiple
wash
steps
before
eluting
the
RNA.
The
RNA
concentration
was
measured
using
a
Nanodrop
2000
(Thermo
Scientific).
RNA
was
then
reverse
transcribed
to
cDNA
using
High-‐capacity
RNA-‐to-‐cDNA
Kit
(Life
technology)
according
to
manufacturer’s
protocol
and
a
C1000
Touch
Thermal
Cycler
(Biorad)
at
37ºC
for
60
minutes,
and
95ºC
for
5
minutes
for
34
cycles.
Table
3:
RNA
to
cDNA
components
Two
sets
of
primers
were
designed,
one
with
both
restriction
sites
attached
to
the
primer
and
the
other
with
just
EcoRI.
The
primers
were
designed
using
the
APE
software
(http://biologylabs.utah.edu/jorgensen/wayned/ape/)
online
with
the
plasmid
map
sequence
and
the
sequence
of
B7-‐H4
from
NCBI.
Unique
restriction
Component
Volume/Reaction
(µl)
2X
RT
Buffer
10.0
20X
RT
Enzyme
Mix
1.0
Nuclease-‐free
H20
6.5
Sample
(1000ng)
3.5
Total
per
Reaction
20
25
sites
in
the
plasmid
were
chosen
such
that
they
were
not
present
in
the
B7-‐H4
sequence.
Approximately
24
nucleotide
sequences
with
GC
content
up
to
60%
were
selected
and
the
melting
temperature
was
calculated
(See
Table
5).
The
primer
sequences
were
then
sent
to
Integrated
DNA
technologies
at
the
University
of
Southern
California
for
synthesis.
Table
4:
Primer
sequences
3.2.3
PCR
amplification
of
B7-‐H4:
Gene
amplification
was
performed
using
Phusion®
High-‐Fidelity
PCR
master
Mix
with
HF
buffer
(NEB)
with
appropriate
forward
and
reverse
primers
at
0.5µM
concentration
each.
Twenty
microliter
per
reaction
were
prepared
in
PCR
tubes
for
the
C1000
Touch
Thermal
Cycler
(Biorad).
To
each
tube
the
following
was
added:
6.8µl
of
nuclease
free
water,
2µl
primer
mix
(0.5µM
of
forward
and
reverse
primer)
(see
Table
4)
and
1µl
of
cDNA
(5ng/µl).
The
PCR
was
run
with
the
following
program:
The
initialization
step
98ºC
for
3
minutes,
followed
by
35
times
repeated
step
of
denaturation
98ºC
for
10
seconds,
annealing
at
65ºC
for
15
seconds,
extension
at
72ºC
for
20
seconds.
Afterwards
the
final
extension
was
carried
out
for
5
minutes
at
72ºC.
Finally
the
samples
were
cooled
to
12ºC
before
further
usage.
Primer
Melting
Temp(ºC)
Strand
Sequence
B7-‐H4-‐EcoRI
65
Sense
CATCCGGAATTCATGGCTTCCCTGGGG
B7-‐H4-‐BamHI
65
Anti-‐sense
TACCGCGGATCCATGGCTTCCCTGGG
B7-‐H4-‐EcoRI
61
Sense
GGCGCGCCTCGAATTATGGCTTCCCTG
B7-‐H4-‐EcoRI
59
Anti-‐sense
CAAAAGGGCGGAATTTTATTTTAGCAT
26
After
the
completion
of
the
run,
1%
agarose
gel
was
prepared
with
1xTBE
and
5µl
of
ethidium
bromide.
Five
microliters
of
sample
was
mixed
with
5x
DNA
loading
dye
to
a
final
concentration
of
1x
and
loaded
into
the
well.
Ten
microliters
of
pre-‐stained
1kb
DNA
ladder
(New
England
Biolabs)
was
also
loaded
on
the
gel.
The
gel
was
run
at
a
voltage
of
100V
for
approximately
35
min
and
bands
were
observed
under
the
UV
light.
3.2.4
Preparation
of
Competent
Cells:
Novablue
cells
obtained
from
Millipore
were
grown
overnight
in
5ml
of
LB
broth
at
37ºC
in
a
shaker
incubator.
This
overnight
culture
was
then
inoculated
into
200
ml
of
LB
and
incubated
at
37ºC
in
the
shaker
incubator
until
the
OD600
reached
0.5
measured
using
a
spectrophotometer
(Biorad).
The
culture
was
incubated
on
ice
for
15mins
and
centrifuged
at
2800xg
for
15
minutes.
After
which
the
supernatant
was
discarded
and
the
pellet
was
resuspended
in
70ml
of
RFI
solution
and
incubated
on
ice
for
1
hour.
Centrifugation
step
was
followed
and
the
pellet
was
suspended
in
17ml
of
RFII.
Aliquots
of
competent
cells
(100ul
each)
were
flash
frozen
in
liquid
nitrogen
and
stored
at
-‐80ºC.
3.2.5
Construction
of
Plasmid:
3.2.5.1
Plasmid
preparation
Five
nanograms
per
microliter
of
pIRES-‐eGFP
plasmid
(Invitrogen)
were
retransformed
using
50
µl
of
freshly
prepared
competent
cells
and
were
plated
on
a
35µg/ml
kanamycin
containing
agar
plate.
Colonies
were
picked
and
grown
in
round
bottom
falcon
tube
(VWR)
with
5
ml
agar
broth
containing
kanamycin
at
37ºC
27
in
a
shaker
incubator
overnight.
The
sample
was
centrifuged
at
1500xg
for
25
minutes
to
pellet
the
bacterial
cells.
The
plasmid
was
isolated
using
the
QIAprep
Spin
Miniprep
kit
(Qiagen)
according
to
manufacturer’s
protocol.
The
pellet
was
resuspended
and
washed
with
buffers
provided
by
the
manufacturer.
Plasmid
DNA
was
eluted
using
30ul
of
Elution
Buffer
(EB)
(Qiagen)
and
the
concentration
was
measured
using
the
Nanodrop
Spectrophotometer.
3.2.5.2
Plasmid
Digestion:
The
plasmid
was
cut
using
restriction
sites
EcoR1
and
BamH1
in
1x
Cutsmart
Buffer
(NEB)
at
37ºC
overnight.
The
plasmid
was
digested
using
either
one
enzyme
(restriction
digest
controls)
or
both.
The
components
for
single
digest
are
as
follows:
0.8µl
of
EcoRI
or
BamHI
(10
units/ml),
1µg
of
plasmid,
2µl
of
10X
cutsmart
buffer
in
a
total
reaction
volume
of
20µl.
The
double
digest
components
were
0.8µl
of
each
restriction
enzyme
EcoRI
and
BamHI
(10
units/ml),
2µl
of
10X
cutsmart
buffer,
1µg
of
the
purified
plasmid
to
a
reaction
volume
of
50µl
with
water.
The
inserts
(PCR
products)
were
also
digested
with
the
same
restriction
enzymes
to
obtain
compatible
ends.
An
agaorose
gel
electrophoresis
was
followed
on
to
investigate
whether
the
digest
was
successful.
As
a
negative
control,
undigested
plasmid
was
included
on
the
agarose
gel.
28
3.2.5.3
Plasmid
Ligation:
Quick
ligation
kit
(NEB)
was
used
for
this
step.
A
1:3
ratio
of
the
double
digested
plasmid
to
the
digested
PCR
amplified
B7-‐H4
products
was
used
along
with
2µl
of
10X
ligation
buffer
and
1µl
of
the
enzyme,
and
the
final
volume
was
made
up
to
20µl
with
autoclaved
water.
Samples
were
incubated
for
5
min
at
room
temperature.
This
step
proceeded
transformation
methods
described
below.
3.2.6
Transformation:
Competent
cells
were
thawed
10
minutes
on
ice
and
2-‐4µl
of
the
ligated
mixture
or
the
plasmid
were
added
to
the
them.
The
mixture
was
incubated
on
ice
for
30
minutes
followed
by
a
heat
shock
at
42ºC
for
30
seconds.
LB
media
(750ul)
was
added
to
the
cells
and
they
were
incubated
at
37ºC
in
a
shaker
incubator
for
1
hour
and
100µl
of
this
culture
were
spread
on
to
the
plates
using
glass
beads
(Novagen).
The
plates
were
incubated
overnight
at
37ºC.
3.2.7
Preparation
of
Agar
plates:
LB
Agar
(Sigma)
was
prepared
and
autoclaved
according
to
the
manufacture’s
protocol.
Kanamycin
was
added
to
the
autoclaved
agar
to
a
final
concentration
of
35
µg/ml
and
agar
was
poured
into
petri
plates
(roughly
20ml
per
plate)
allowing
it
to
cool
to
solidify.
29
3.2.8
Colony
PCR:
Colonies
grown
on
the
agar
plates
were
picked
and
added
to
50µl
of
water
of
which,
25µl
were
added
to
an
eppendroff
tube
containing
500µl
of
LB
media.
This
was
incubated
at
37ºC
for
approximately
2
hours.
The
remaining
25µl
were
boiled
in
a
mini
digital
dry
bath
(Southwest
science)
at
100ºC
for
5-‐10
minutes
and
snapped
cooled
on
ice
for
2
minutes
post-‐incubation.
Seven
microliters
of
this
mixture
was
used
as
template
for
the
colony
PCR
using
the
same
set
of
primers
and
conditions
as
mentioned
previously
(see
Table
4).
On
confirmation
of
insert
by
agarose
gel
electrophoresis,
the
positive
samples
(containing
a
band)
were
grown
in
5ml
of
LB
media
containing
35µl/ml
of
kanamycin
at
37ºC
in
a
shaker
incubator
over
night.
Plasmids
were
extracted
with
a
Qiagen
Miniprep
kit
(see
also
3.2.5.1)
and
were
sent
for
sequencing
to
Genewiz.Inc.
The
sequenced
results
were
blasted
using
a
freely
available
web
program
nBLAST
(NCBI)
to
check
for
the
similarity
between
the
sequenced
results
and
known
B7-‐H4
sequence.
3.2.9
Transfection:
3.2.9.1
Estimation
of
Antibiotic
concentration:
4T1
cells
were
treated
with
Geneticin
(G418
(Santa
Cruz
Biotechnology))
at
different
concentrations
(0.5,
1.5,
10,
100,
500
and
1000µg/ml)
and
incubated
for
3
days
to
determine
the
minimum
antibiotic
concentration
required
to
kill
the
cells.
The
cells
that
remained
stabile
survived
the
minimum
concentration
of
antibiotics
and
were
used
for
screening.
30
3.2.9.2
Transfection
of
4T1
cells
with
B7-‐H4:
4T1
mouse
mammary
carcinoma
cells
were
seeded
into
a
24
well
plate
at
a
density
of
3x10
5
cells/well
and
incubated
at
37ºC
in
a
humidified
5%
CO2
atmosphere
overnight.
The
cells
were
treated
with
Opti-‐MEM
(serum
free
media)
4
hours
prior
to
the
transfections
and
the
manufacture’s
protocol
was
followed.
Lipofectamine2000
(3µl)
(Invitrogen)
was
mixed
with
247µl
of
Opti-‐MEM
and
incubated
for
5
minutes
at
room
temperature.
This
mixture
was
added
to
an
eppendroff
containing
500ng
of
sequenced
plasmid
in
250ul
Opti-‐MEM
and
incubated
for
15
minutes
at
room
temperature.
The
mixture
was
added
drop
wise
to
the
4T1
cells
and
incubated
for
48
hours
at
37ºC
at
5%
CO2.
The
medium
was
replaced
with
fresh
RPMI1640
containing
10%
FBS.
Post
48h,Geneticin
was
added
at
a
final
concentration
of
1µg/ml,
to
select
colonies
that
were
resistant
to
the
antibiotics
because
of
the
presence
of
the
inserted
target
gene.
Colonies
(about
100
cells)
were
picked
using
3mm
Scienceware
®
cloning
discs
(Sigma)
that
were
immersed
in
trypsin.
The
discs
with
cells
were
placed
in
24-‐well
plate
containing
RPMI-‐1640
media
with
10%
FBS
and
1µg/ml
of
Geneticin.
The
colonies
were
grown
until
confluency
in
the
24-‐well
plate
was
achieved
before
screening
them
via
Western
blot
and
flow
cytometry.
31
3.2.10
Screening
of
transfected
cells
for
B7-‐H4
expression
and
stability:
3.2.10.1
Flow
Cytometry:
The
cells
were
detached
from
a
6
well
plate
using
700µl
of
0.5%
trypsin
and
3.5x10
5
cells/test
were
washed
and
resuspended
in
PBS
to
remove
residual
RPMI
media.
The
positive
control
(SKBR3),
a
tube
of
untransfected
4T1
cells
(negative
control),
as
well
as
the
test
clones
were
incubated
with
2.5µl
of
B7-‐H4-‐APC
antibody
(BD
Pharmingen)
and
as
an
additional
negative
control
4T1
cells
were
mixed
with
20µl
of
APC
Mouse
IgG1
κ
isotype
control
(BD
Pharmingen).
To
all
samples,
50µl
of
PBS
containing
2%
FBS
were
added
and
they
were
incubated
at
4ºC
on
a
shaker
for
45
minutes
in
the
dark.
Post-‐incubation,
cells
were
washed
twice
with
PBS
and
centrifuged
at
370xg
for
10
minutes.
The
cell
pellets
were
resuspended
in
500ul
PBS
and
analyzed
with
an
Attune
FACS
machine
(Life
Technologies)
at
638nm.
The
cell
population
was
screened
using
RL1
channel
red
laser
(APC).
Medium
fluorescence
of
samples
was
calculated
relative
to
the
isotype
control.
One
million
cells
were
treated
using
the
same
parameter
as
above
for
the
sorting
of
cells
at
the
FACS
CORE
(University
of
Southern
California)
to
retrieve
the
highest
expressing
cells
by
gating
for
the
highest
5%
in
the
positive
population.
32
3.2.10.2
Western
Blot:
Approximately
1.2x10
6
cells
from
a
6-‐well
plate
were
lysed
using
300µl
of
cytobuster
(Novagen)
according
to
standard
protocols
and
centrifuged
at
13000xg
for
10
minutes.
To
this,
4X-‐
non-‐reducing
loading
dye
was
added
and
boiled
at
100ºC
in
a
dry
water
bath
for
5-‐10
minutes
in
order
to
denature
the
proteins.
The
samples
were
then
loaded
on
a
10%
SDS-‐PAGE
gel
(Biorad).
The
Precision
Plus
Protein
Kaleidoscope
Ladder
(Biorad)
was
employed
for
size
identification.
The
SDS-‐PAGE
was
run
at
150V
for
45
minutes.
As
negative
control,
4T1
proteins
were
used
and
SKBR3
samples
from
the
cell
line
were
used
as
positive
control.
The
samples
on
the
gel
were
transferred
to
a
nitrocellulose
membrane
(Biorad)
using
transfer
buffer
with
the
gel
facing
the
cathode
with
a
constant
voltage
of
100
for
45
minutes
at
4ºC.
The
membrane
was
blocked
with
5%
milk
powder
(Nestle)
in
TBS-‐T
and
incubated
with
primary
antibody
B7-‐H4
(G-‐18)
(Santa
Cruz
Biotechnology)
at
1:1000
overnight
at
4ºC.
The
membrane
was
washed
thrice
for
10
min
with
TBS-‐T
before
incubating
with
secondary
antibody
goat
anti-‐rabbit
IgG-‐HRP
(Santa
Cruz
Biotechnology)
at
1:5000
for
45
minutes
at
RT.
Subsequently,
it
was
washed
thrice
to
remove
excess
antibody.
Target
proteins
were
detected
using
Immobilon
Western
chemiluminescent
HRP
substrate
(Millipore).
ß-‐actin
(Santa
Cruz
Biotechnology)
was
used
as
an
internal
control
by
stripping
the
blot
using
stripping
buffer
(Millipore),
followed
by
an
incubation
step
for
45
minutes
at
RT
and
then
detection.
33
To
test
the
binding
of
various
in-‐house
antibodies,
B7-‐H4-‐Fc
(positive
control),
a
human
fusion
protein
consisting
of
an
extracellular
domain
of
hB7-‐H4
fused
to
that
of
the
Fc
region
of
human
IgG1,
and
placental-‐like
growth
factor-‐
Fc
(PLGF-‐Fc)
(negative
control)
prepared
in
a
similar
way
in
the
laboratory
were
used.
Five
microgram
per
milliliter
of
the
samples
were
loaded
on
a
10%
SDS-‐PAGE
gel
with
4X
non-‐reducing
loading
dye.
The
samples
were
transferred
onto
a
nitrocellulose
membrane
and
various
in-‐house
antibodies
were
tested
at
1:1000
dilutions
with
goat
anti-‐rabbit
IgG-‐HRP
(Santa
Cruz
Biotechnology)
as
the
secondary
signal
diluted
at
1:5000.
3.2.11
Tumor
Implantation
studies:
BALB/c
mice
were
injected
subcutaneously
with
1.5x10
6
cells/200µl
of
4T1
cells
alone
and
4T1-‐B7-‐H4-‐IRES-‐eGFP
on
either
rear
flank
of
BALB/c
mice.
The
tumor
growth
was
measured
for
2
weeks
using
a
vernier
caliper
to
check
for
any
tumor
rejections
before
euthanizing.
Mice
were
sacrificed
by
overdose
inhalation
of
Isoflurane
followed
by
cervical
dislocation.
The
tumors
were
fixed
in
10%
formaldehyde
overnight
before
sending
them
for
tissue
sectioning
at
the
University
of
Southern
California
(Pathology
core
facility).
3.2.12
Immunohistochemistry:
Tissue
sections
were
baked
for
30mins
at
60ºC
and
deparafinized
in
xylene
twice
for
5
minutes.
Slides
were
then
hydrated
twice
in
100%
ethanol
for
3
minutes
followed
by
95%
and
80%
ethanol
for
1
minute
each.
The
sections
were
rinsed
with
distilled
water
for
2
minutes
before
proceeding
to
antigen
retrieval
step.
The
steamer
was
34
pre-‐heated
with
10mM
sodium
citrate
and
the
tissue
sections
were
placed
on
a
blank
slide
in
order
to
provide
a
capillary
upwards
action
for
25
minutes
and
cooled
down
for
another
20
minutes.
The
blank
slides
were
pulled
off
and
the
tissue
section
was
rinsed
with
PBS-‐T
(PBS+1%
Tween)
before
blocking
with
rabbit
serum
(Vectastain)
and
incubating
with
the
rabbit
B7-‐H4
polyclonal
antibody
(Abbiotec)
and
in-‐house
B7-‐H4
35-‐8
antibody
overnight.
Secondary
antibody
present
in
the
Vectastain
kit
was
used
for
30
minutes
at
RT.
Three
drops
of
VECTASTAIN
ABC
reagent
was
added
to
the
slides
and
incubated
for
30
minutes
and
subsequently
rinsed
with
PBS-‐T.
Further,
the
sections
were
incubated
with
DAB
peroxidase
as
a
chromogen
to
achieve
the
desired
intensity
and
rinsed
with
PBS-‐T.
For
counterstaining,
the
slides
were
immersed
in
hematoxylin
for
4
minutes,
and
then
acid
solution
followed
by
0.2%
ammonia
water
solution
for
1
minute
and
rinsed
with
water
after
every
step.
Finally
the
sections
were
dehydrated
with
95%,
100%
ethanol,
and
xylene
for
2
minutes
each.
Two
drops
of
mounting
medium
(Richard
Allan
scientific)
was
added
to
each
slide
and
the
coverslips
were
placed
on
top
before
analyzing
by
light
microscopy.
35
CHAPTER
4
RESULTS
4.1
Expression
of
B7-‐H4
on
human
tumor
cell
lines
The
results
of
screening
seven
carcinoma
cell
lines
including
SKOV3
(human
ovarian
carcinoma),
HT29
(colorectal
carcinoma),
SKBR3
(mammary
carcinoma),
T47D
(mammary
carcinoma),
MDA
MB
468
(mammary
carcinoma),
MCF7
(mammary
carcinoma),
JAR
(trophoblastic
carcinoma)
by
western
blot
for
the
protein
expression
of
B7-‐H4
are
shown
in
Fig
4.1,
using
a
commercially
available
antibody.
To
evaluate
protein
expression,
30µg
of
cell
lysate
was
loaded
in
each
lane
of
a
10%
SDS
gel.
SKBR3
mammary
carcinoma
showed
the
highest
expression
of
B7-‐H4
at
50
kDa,
followed
by
MDA-‐MB-‐468
another
human
mammary
carcinoma.
Other
cell
lines
had
no
B7-‐H4
present
or
showed
very
low
expression.
B7h4
expression
(Fig.
4.1).
SKOV3
MDA
MB
468
SKBR3
T47D
HT29
MCF7
JAR
Figure
4.1
Protein
levels
of
B7-‐H4
in
various
cell
line
carcinomas.
Western
Blot
showing
the
B7-‐H4
protein
level
in
SKOV3
(Lane
1),
MDA-‐MB-‐468
(Lane
2),
SKBR3
(Lane3),
T47D
(Lane
4),
HT29
(Lane
5),
MCF7
(Lane
6),
and
JAR
(Lane
7).
Thirty
micrograms
of
lysates
were
loaded
in
each
well.
SKBR3
demonstrated
the
highest
expression
followed
by
MDA-‐MB-‐468
at
50kDa.
SKOV3
showed
no
band,
and
others
were
faint
compared
to
the
two
very
positive
cell
lines.
36
4.2
Amplification
of
B7-‐H4
from
SKBR3
Since
SKBR3
showed
the
highest
expression
of
B7-‐H4
(see
Figure
4.1),
it
was
used
for
B7-‐H4
amplification.
The
first
step
in
this
procedure
was
to
extract
total
RNA
of
the
cell
line
(see
section
3.2.2).
The
cDNA
was
obtained
by
reverse
transcription
of
1µg
of
total
RNA
followed
by
DNA
amplification
using
PCR
(see
section
3.2.3).
Samples
were
analyzed
on
1%
agarose
gel
and
bands
were
observed
at
800bps
(see
Figure
Fig
4.2
which
demonstrates
bands
in
lanes
2,
3,
4
and
5
of
amplified
human
B7-‐H4
exacted
from
SKBR3
human
mammary
carcinoma).
Figure
4.2
PCR
amplification
of
human
B7-‐H4
from
SKBR3.
Bands
representing
amplified
B7-‐H4
using
primers
from
Table
4
in
a
1%
agarose
gel.
Lane
1
represents
the
1kb
ladder
at
800bp,
lane
2,
3
bands
of
amplified
B7-‐H4
using
primers
containing
EcoRI/
BamHI
restriction
sites,
and
lanes
4,
5
consist
of
B7-‐H4
amplified
using
primers
containing
one
restriction
enzyme.
(See
Table
4)
4.3
Plasmid
Construction:
One
microgram
pIRES-‐eGFP
plasmid
was
digested
at
EcoRI
and
BamHI
restriction
sites
using
the
smartcut
buffer,
at
37ºC
overnight.
The
insert
was
also
digested
using
the
same
restriction
enzymes
to
get
sticky
ends
in
order
to
perform
the
ligation.
Figure
4.3
shows
the
digested
samples
on
a
1%
agarose
gel,
with
undigested
plasmid
as
a
negative
control
in
lane
2,
single
digest
BamHI/EcoRI
in
lane
3
and
4
as
controls,
and
double
digestion
with
both
restriction
enzymes
in
lane
5.
Both
single
digests
as
well
as
the
double
digest
were
successful..
Single
digestion
was
done
in
1
2
3
4
5
37
order
to
check
if
the
samples
were
digested
by
both
enzymes,
since
the
two
restriction
sites
are
a
only
few
nucleotides
apart.
Fifty
nanograms
of
double
digested
plasmid
was
ligated
with
3-‐
fold
molar
excess
of
digested
amplified
human
B7-‐H4
at
restriction
sites
BamHI
and
EcoRI
using
quick
ligase
for
5
minutes
at
room
temperature
for
the
ligation
reaction.
Figure
4.3
Plasmid
digestions.
The
plasmid
was
digested
using
restriction
sites
EcoRI
and
BamHI;
as
shown
in
this
figure,
lane
2
is
undigested
plasmid,
lane
3,4
shows
single
digestion
with
either
BamHI
or
EcoRI,
and
lane
5
shows
digestions
with
both
restriction
enzymes.
Since
the
two
restriction
sites
are
about
20bp
apart
therefore,
there
is
not
much
differentiation
between
single
and
double
digested
plasmids.
Ladder
Undigested
Plasmid
Single
Digestion
EcoRI
Single
Digestion
Plasmid
BamHI
Double
Digestion
plasmid
EcoRI
and
BamHI
38
4.4
Transformation,
Colony
PCR,
Sequencing:
Five
nanograms
of
the
above-‐ligated
plasmid
was
added
to
100µl
competent
cells
prepared
in
the
laboratory.
As
negative
controls,
5ng
of
amplified
digested
human
B7-‐H4
and
5ng
of
the
double
digested
plasmid
were
added
separately
to
the
competent
cells.
The
cells
were
heat
shocked
at
42ºC
for
30
seconds.
The
samples
plated
on
LB
agar
containing
50mg/ml
kanamycin
overnight
grew
small
yellow
round
colonies
of
the
ligated
plasmid.
No
colonies
were
observed
in
plates
containing
human
B7-‐H4
or
the
double
digested
plasmid
alone
(see
Figure
4.4.1)
Figure
4.4.1
Transformation
of
ligated
pIRES-‐GFP-‐hB7-‐H4.
White
round
colonies
were
observed
in
the
plate
containing
the
ligated
pIRES-‐GFP-‐hB7-‐H4.
The
negative
control
grew
no
colonies
on
plates
containing
the
hB7-‐H4
alone
and
the
double
digested
plasmid
alone.
B7-‐H4
Competent
cells
Double
digested
plasmid
Competent
cells
Ligated
Plasmid
Competent
cells
39
Colonies
were
picked
from
the
plate
in
50µl
of
water
and
half
was
PCR
using
the
same
conditions
and
primer
sequences
that
were
used
earlier
to
amplify
the
gene
(refer
section
3.2.3).
The
PCR
samples
were
loaded
on
a
1%
agarose
gel
which
showed
clear
bands
in
lanes
2,
4,
and
5
indicating
they
were
positive
for
the
B7-‐H4
insert
(see
Figure
4.4.2).
These
samples
were
grown,
miniprepped,
and
sent
for
sequencing.
The
sequence
identity
was
screened
using
the
nBlast
tool
online
between
the
sequenced
results
and
the
known
sequence
of
human
B7-‐H4.
Figure
4.4.3
shows
that
sample
1(Lane
2)
is
identical
to
the
B7-‐H4
gene
and
was
used
for
the
further
studies
while
the
others
had
point
mutations
or
no
insert
present.
Figure
4.4.2
Colony
PCR
of
transformed
pIRES-‐GFP-‐hB7-‐H4.
Colony
PCR
was
performed
using
the
primer
sequences
with
restriction
enzyme
shown
in
Table
5.
Lanes
2,
4,and
5
showed
a
thick
band
and
these
samples
were
sent
for
sequencing
to
confirm
and
also
check
its
orientation.
Figure
4.4.3
nBlast
between
sequenced
sample
and
known
h-‐B7-‐H4
sample.
The
query
sequence
(sequence
results)
and
subject
(h-‐B7-‐H4)
were
blasted
on
nBlast.
The
two
sequences
were
identical
and
in
the
right
orientation
and
in
frame,
thus,
confirming
the
plasmid
contains
the
h-‐B7-‐H4
insert.
1
2
3
4
5
40
4.5
Stable
cell
line
Transfection
using
Lipofectamine2000:
Cells
(4T1)
were
transfected
using
different
transfection
agents
including
Lipofectamine
2000,
BP-‐Fectin,
Electroporation,
and
Genecillin,
at
different
concentrations
and
it
was
observed
that
Lipofectatime2000
to
be
the
best
for
the
4T1
cells
(see
figure
4.5).
Preliminary
screening
of
transfected
cells
was
performed,
by
detecting
green
fluorescent
protein
by
fluorescence
microscopy.
Different
concentration
of
Lipofectamine2000
(0.2,
0.3,
0.4,
0.5µl)
were
added
along
with
2.5µg
of
pB7H4IRESeGFP
plasmid
and
incubated
at
room
temperature
for
5
minutes.
This
was
added
drop-‐wise
to
2x10
5
cells
plated
a
day
before
in
a
24
well
plate.
Many
green
cells
were
observed
at
0.3µl
of
lipofectamine2000
compared
to
other
concentrations
after
48hr
incubation
Figure
4.5(B).
One
microgram
per
milliliter
of
geneticin
was
used
to
kill
the
untransfected
cells.
A
41
B
Figure
4.5
Transfection
of
4T1
cells
by
transfection
agents.
(A)
Represents
4T1
cells
under
the
bright
field
and
green
fluorescent
cells
48hrs
post
transfection
using
Lipofectamine2000.
(B)
Transfected
4T1
cells
using
BP-‐fectin
as
a
transfection
agent.
Fewer
green
cells
are
observed
with
B
compared
to
A.
4.6
Screening
of
cells
by
Western
Blot
and
Flow
Cytometry:
4.6.1
Screening
of
transfected
4T1
cells
using
Western
Blot:
Forty
colonies
were
picked
using
3mm
discs
(Sigma),
and
thirty
colonies
grew
in
the
presence
of
1µg/ml
of
Geneticin.
These
were
screened
using
Western
blot
and
Flow
cytometry.
Total
protein
from
a
6
well
plate
containing
transfected
4T1
cells,
untransfected
4T1,
CHO,
and
SKBR3
cell
lines
were
lysed
using
300µl
of
cytobuster
protein
extraction
reagent
at
room
temperature
for
5
minutes
according
to
manufacture’s
protocol.
The
samples
were
loaded
on
a
10%
SDS
gels
along
with
4X
loading
dye
and
transferred
onto
a
nitrocellulose
membrane.
The
Western
Blot
shows
no
bands
in
lanes
1
and
2
with
untransfected
4T1
and
CHO
cell
lines
42
respectively,
as
negative
controls.
A
strong
band
at
50kDa
of
SKBR3
in
lane
3
was
seen
as
a
positive
control,
and
lighter
bands
of
the
transfected
B7-‐H4
clones
are
shown
in
lanes
4,
5,
and
6
(see
Figure
4.6(A))
In
Figure
4.6(B),
lane
1
represents
the
untransfected
4T1(negative
control),
and
lanes
2,
3,
and
5
show
a
significant
a
faint
band
or
no
band.
Lanes
4,
6,
7,
and
8
show
a
strong
band
from
the
clones
of
transfected
4T1
cells.
A
ß-‐Actin
B7-‐H4
1
2
3
4
5
6
B
SKBR3
Clone
6
Clone
7
Clone
12
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
4T1
CHO
Lane
3
Lane
4
Lane
5
Lane
6
Sample
revaltive
to
internal
contro
(ß-‐
Actin)
Samples
43
Figure
4.6.1
Screening
of
hB7-‐H4
by
Western
Blot.
(A),
(B)
Western
Blot
showing
the
B7-‐H4
binding
at
50KDa
using
a
commercially
available
antibody.
(A)
Represents
negative
control
4T1
and
CHO
cells
(lane
1
and
2)
followed
by
sample
clones.
B,
D
Plot
representing
B7-‐H4
protein
levels
were
normalized
to
the
internal
control
ß-‐Actin.
(B)
Western
Blot
of
samples
5-‐12,
demonstrating
the
expression
of
B7-‐H4
in
the
transfected
4T1
cells.
Clone
27
Clone
37
Clone
38
Clone
41
Clone
42
Clone
43
Clone
44
Clone
48
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Sample
relavtive
to
internal
control
(ß-‐
Actin)
Samples
C
D
B7-‐H4
1
2
3
4
5
6
7
8
ß-‐Actin
44
4.6.2
Screening
of
transfected
cells
by
Flow
Cytometry:
Binding
of
transfected
4T1
cells
were
screened
by
Flow
Cytometry,
using
commercially
available
antibody
specific
to
human
B7-‐H4.
Three
and
a
half
x10
5
cells
per
test
were
used
SKBR3
(positive
control),
untransfected
4T1
with
antibody
and
4T1
with
an
Isotype
(negative
control),
and
clones
of
different
transfected
4T1
cells
were
tested.
Three
microliter
of
the
commercially
available
antibody
per
test
and
20µl
of
isotype
control
were
used,
respectively.
0
0.5
1
1.5
2
2.5
4t1
4t1
ab
1
2
3
4
5
6
7
8
9
10
11
12
25
26
27
28
29
37
38
39
40
41
42
43
44
45
46
47
48
MFI
of
Sample/MFI
of
Isotype
Samples
(4T1-‐pIRES-‐eGFP-‐hB7-‐H4)
A
45
Figure
4.6.2
Screening
of
4T1
transfected
cells
by
Flow
Cytometry.
(A)
Various
clones
screened
with
B7-‐H4
APC
commercially
available
antibody
for
human
B7-‐H4.
Clones
6,37,42,43
showed
a
two-‐fold
increase
to
that
of
the
negative
control.
(B)
Demonstrates
different
clones
of
4T1-‐IRES-‐GFP-‐hB7-‐H4,
SKBR3
positive
control
(Purple),
4T1
isotype
as
negative
control
(Red),
Clone
6
(Blue),
Clone
37
(green),
Clone
43
(Pink),
Clone
44
(yellow)
shifting
towards
the
right.
(C)
Two
peaks
of
clone
28
are
observed,
representing
two
populations,
one
being
positive
by
shifting
to
the
right
(R3)
and
the
other
aligned
with
the
negative
contol
(R2).
This
sample
was
sent
for
cell
sorting
to
achieve
only
the
highest
5%
positive
cells.
4T1 Isotype
SKBR3
Clone 44
Clone 6
Clone 43
Clone 37
B
C
4T1 Isotype
Clone 28
46
4.6.3
Binding
of
in-‐house
antibodies
to
B7-‐H4-‐Fc:
Screening
for
the
binding
of
various
in-‐house
antibodies
with
the
B7-‐H4-‐Fc
and
PLGF-‐Fc
fusion
protein
was
preformed
in
the
laboratory.
All
primary
antibodies
were
incubated
overnight
at
1:1000
concentrations
with
5%
milk
(see
table
5).
A
secondary
goat
anti-‐rabbit
IgG-‐HRP
antibody
at
1:5000
was
used.
It
can
be
observed
that
5F6-‐6,
36-‐1,
4-‐5-‐10,
35-‐8
showed
specific
binding
to
B7-‐H4-‐Fc.
Antibody
B7-‐H4-‐Fc
PLGF-‐Fc
12-‐14-‐12
B7H4-‐7
#43
-‐
-‐
12-‐11-‐12
B7H4
-‐7
#48
-‐
-‐
11-‐13-‐12
B7H4-‐6
#21
-‐
-‐
11-‐3-‐12
B7H4
#33
+
-‐
12-‐11-‐12
B7H4-‐7
#19
+
-‐
10-‐17-‐12
B7H4
(5F6-‐6)
IgG
+
-‐
12-‐14-‐12
B7H4-‐7
#30
-‐
-‐
11-‐3-‐12
B7H4-‐6
#22
+
-‐
#
36-‐1
+
-‐
#
4-‐5-‐10
+
-‐
12-‐9-‐12
B7H4-‐7
#9
+
+
11-‐9-‐12
B7H4-‐6
#34
-‐
-‐
12-‐9-‐12
B7H4-‐7
#2
-‐
-‐
11-‐7-‐12
B7H4-‐6
#1
-‐
-‐
12-‐11-‐12
B7H4-‐7
#
34
-‐
-‐
12-‐22-‐12
B7H4-‐7
#63
+
-‐
12-‐14-‐12
B7H4-‐7
#
32
-‐
-‐
12-‐24-‐12
B7H4-‐7
#24
+
-‐
1-‐24-‐13
B7H4
#35
-‐
-‐
12-‐9-‐12
B7H4
#35-‐8
+
-‐
12-‐14-‐12
B7H4-‐7
#38
+
-‐
11-‐13-‐12
B7H4-‐6
#6
+
+
12-‐22-‐12
B7H4-‐7
#59
-‐
-‐
2-‐18-‐13
B7H4-‐6
#35-‐1
+
-‐
2-‐19-‐13
B7H4-‐6
#
35-‐9
+
-‐
Table
5:
Screening
of
in-‐house
antibodies:
47
Figure
4.6.3
Binding
of
in-‐house
antibodies
to
B7-‐H4-‐Fc.
Western
Blot
represents
the
binding
of
B7-‐H4-‐Fc
with
antibodies
B7H4-‐6#1,
B7H4#35-‐8,
B7H4
#5F6-‐6.
No
bands
can
be
seen
with
the
incubation
of
the
B7H4-‐6
#1
antibody.
The
other
two
have
a
clear
banding
pattern
with
B7-‐H4-‐Fc.
4.7
Tumor
Implantation
studies,
Immunohistochemistry:
5.4x10
6
cells
were
injected
into
in
BALB/c
mice.
Three
mice
were
injected
subcutaneously
with
different
clones
of
transfected
4T1-‐B7-‐H4
cells
(Clone
43,
37,
44)
and
4T1
cells
as
a
negative
control
on
either
flank
of
the
same
mouse.
None
of
the
mice
showed
rejection
of
tumors.
Mouse
1
was
injected
with
clone
37;
mouse
2
left
side
demonstrates
a
larger
tumor
of
clone
43
compared
to
the
right
with
4T1
cells.
Mouse
3
showed
that
both
tumors
(control
and
clone
44)
were
almost
the
same
size.
The
right
tumor
representing
clone
44,
and
left
4T1
cells.
All
tumor
sizes
were
calculated
using
a
vernier
caliper
for
two
weeks
until
euthanizing
the
mice.
The
tumors
studies
showed
no
rejection
of
transfected
4T1-‐B7-‐H4
cells,
which
grew
significantly
before
euthanization
(Fig
4.7).
PLGF-‐Fc
B7-‐H4-‐Fc
PLGF-‐Fc
B7-‐H4-‐Fc
PLGF-‐Fc
B7-‐H4-‐Fc
B7H4-‐6
#1 B7H4
#35-‐8
B7H4
#5F6-‐6
48
A
B
C
Figure
4.7
Tumor
implantation
studies.
(A)
BALB/c
mice
of
tumor
clone
43.
(B)
Tumor
from
clone
37
(left
flank)
and
tumor
from
4T1
(right
flank).
(C)
Tumor
clone
44
(right
flank)
and
4T1
cells
(left
flank)
are
shown.
All
three
tumors
clones
grew
significantly
without
rejection.
(D)
Tumor
size
from
day
2
to
day
14
before
euthanizing,
all
tumor
sizes
grew
with
two-‐fold
increase
from
day
8
to
14.
0
50
100
150
200
250
300
350
Day
2
Day
5
Day
8
Day
11
Day
14
Volume
of
tumor
mm
3
Number
of
Days
4T1-‐M2
4T1-‐M3
Clone
37
Clone
43
Clone
44
49
The
tumors
were
carefully
removed
from
the
skin
of
the
mice
using
a
scalpel
and
stored
in
10%
buffered
formaldehyde
before
sending
for
tissue
sectioning
at
University
of
Southern
California.
The
sections
were
stained
using
Vectastain
kit
accordingly
to
manufactures
protocol.
Three
tissue
sections
were
treated
with
a
commercial
antibody
and
three
were
treated
with
an
in-‐house
B7-‐H4
antibody
35-‐8
(1:1
PBS-‐T)
(see
figure
4.6.3).
As
a
negative
control,
untransfected
4T1
tumor
sections
were
treated
with
both
the
antibodies
separately.
It
can
be
observed
that
4T1-‐B7-‐H4
transfected
cells
showed
sparse
positive
staining,
and
no
staining
was
seen
in
the
negative
control.
Figure
4.8
Expression
of
hB7-‐H4
by
immunohistochemistry,
(A)
Represents
10x
focus
of
untransfected
4T1
cells.
(B)
5%
of
B7-‐H4
staining
was
observed,
which
is
very
sparse
stained
using
the
in-‐house
antibody.
(C)
7%
Tumor
clone
37-‐showed
maximum
staining
yet
sparse
by
using
the
commercial
antibody.
There
was
no
uniform
staining
pattern
observed.
All
positive
staining
is
shown
in
dark
brown,
compared
to
the
unstained
section.
A
B
C
50
CHAPTER
5
DISCUSSION
Breast
cancer
is
widely
studied
carcinoma,
yet
no
cure
has
been
developed.
To
grow
in
patients,
cancer
cells
avoid
recognition
by
evade
the
immune
system
25
.
To
destroy
the
tumor
cells
with
the
help
of
the
immune
system,
cancer
immunotherapy
using
monoclonal
antibodies
can
be
employed.
Therapeutic
antibodies
bind
to
specific
targets
such
as
CD19
on
B-‐cell
leukemia,
CTLA4
on
metastatic
melanoma
or
Her2
on
breast
cancers
as
examples
and
have
shown
great
promise
as
therapeutics
26
.
Nevertheless,
over
200,000
woman
in
the
US
still
die
of
breast
cancer
every
year
27
and
new
and
more
efficient
therapeutics
or
combinations
of
therapeutics
are
needed.
Here
we
investigated
the
potential
of
anti-‐B7-‐H4
antibodies
to
target
and
destroy
breast
cancer
cells.
Though
the
B7-‐H4
receptor
is
unknown,
B7-‐H4
deficient
mice
have
only
minor
immune
irregularities
28,29
,
help
in
tumor
progression
and
blockade
of
B7-‐H4
by
antisense
oligonucleotides
restored
the
function
of
macrophages
and
led
to
tumor
regression
in-‐vivio
30
making
it
an
ideal
target
for
cancer
therapy.
In
this
study,
human
B7-‐H4
derived
from
the
SKBR3
cell
line
31,16
was
cloned
into
pIRES-‐EGFP,
transfected
into
4T1
cells,
and
in-‐vivo
tumor
growth
studies
in
BALB/c
mice
were
performed.
Flow
cytometry
and
Western
Blot
confirmed
the
presence
of
hB7-‐H4
in
the
transfected
4T1
cells.
Expression
levels,
however
of
the
gene
were
extremely
low
in
tumor
sections
stained
by
immunohistochemistry.
51
Reasons
for
Low
expression
by
Immunostaining:
This
study
was
performed
before
cell
sorting.
Clones
were
picked,
grown
up
for
approximately
3-‐4
weeks
and
tested
for
B7-‐H4
expression
before
injections.
Nevertheless,
as
Genetecin
is
unstable
at
37ºC
some
untransfected
4T1
cells
could
have
survived.
In
the
mouse
system,
it
may
have
been
that
the
untransfected
cells
had
a
growth
advantage
and
outgrew
B7-‐H4
expressing
cells.
Furthermore,
the
in-‐vivo
selection
pressure
using
antibiotics
(Geneticin)
was
withdrawn
unlike
in
the
in-‐vitro
system,
where
the
cells
are
forced
to
produce
the
target
gene.
Also,
there
could
have
been
some
regulatory
mechanisms
that
down-‐
regulates/inhibits
B7-‐H4
expression
in
mice,
which
could
not
be
observed
in
the
cell
lines
14
.
Finally,
unpurified
antibody
was
used
to
test
and
may
have
caused
non-‐specific
binding.
The
B7-‐H4
contains
a
large
hydrophobic
domain
and
may
have
undergone
conformational
change
and
disorientation
of
the
binding
pocket.
These
structural
changes
could
have
affected
the
binding
of
antibodies
to
the
target.
B7-H4 is extensively and variably N-glycosylated, which may also serve
as a “barrier” mechanism to evade immunosurveillance
28
Transfection
and
screening
of
cells
by
Flow
Cytometry
and
Western
Blot:
Integration
sites
of
plasmids
into
the
host
genome
via
homologues
recombination
are
more
or
less
random.
32,33
Integrations
can
hence
cause
the
loss
of
gene
function
or
activate
an
oncogene
which
can
have
dramatic
alterations
in
the
cell
line
morphology,
its
ability
to
migrate
and
its
survival
and
proliferation
which
might
not
be
due
to
the
expression
of
the
target
gene.
The
integration
site
could
52
also
have
an
impact
on
the
strength
of
expression
if
inserted
into
a
less
active
site.
To
investigate
whether
the
integration
site
might
have
caused
changes,
deep
sequencing
could
be
performed
to
identify
the
integration
sites.
Also,
an
inducible
promoter
could
have
been
used
to
better
compare
the
impact
of
B7-‐H4
on
the
cell
line
by
comparing
the
same
clones
with
and
without
B7-‐H4
expression.
Previously,
Tetracycline-‐
inducible
promoters
have
been
employed
in
making
stable
cell
lines.
33
Pipetting
errors
and
screening
so
many
colonies
was
very
tedious
and
a
better
approach
would
have
been
easier
though
a
good
unique
restriction
enzyme
is
hard
to
find.
Alternative
studies
to
come
over
these
problems
include
using
viruses
to
create
a
stable
cell
line
instead
of
a
plasmid.
Lentiviruses
or
retrovirus
may
provide
a
more
stable
integration,
“sleeping
beauty
transposons
is
another
method
wherein
the
use
of
Sleeping
beauty
transposase
enzyme
and
a
transposon
allows
the
DNA
to
translocate
from
one
site
to
another.
53
FUTURE
DIRECTIONS:
Using
lentivirus
to
integrate
hB7-‐H4
into
the
4T1
cell
line
genome
in
a
stable
manner
would
be
useful
approach
to
test.
In
addition,
the
biodistribution
and
other
target
specificity
studies
of
chosen
in-‐house
antibodies
might
shed
light
on
the
antigen
epitopes
being
bound.
Finally,
antibody
drug
conjugates
can
be
prepared
to
enhance
the
killing
activity
of
newly
generated
anti-‐B7-‐H4
antibodies.
From
our
studies,
B7-‐H4
looks
like
a
promising
target
for
breast
cancer
immunotherapy.
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Abstract (if available)
Abstract
B7‐H4 is a member of the B7 family, which is a costimulatory molecule and negatively regulates the immune response of T cells. Its mechanism of tumoral immune escape and therapeutic targeting is still unknown. B7‐H4 is overly expressed in breast cancer, ovarian cancer, and many solid tumors and is being investigated as a good immunotherapy target for antibodies or antibody drug conjugates. To elucidate its immune function as well as investigate newly developed anti‐B7‐H4 antibodies, the murine mammary tumor cell line, 4T1, was transfected with the human B7‐H4 gene to generate a syngeneic tumor model in immuno‐competent BALB/c mice. To facilitate the generation of a stabile cell line for in vivo studies, 4T1 a mouse breast cancer cell line with metastatic potential similar to that seen in human breast cancer, was transfected with the pIRES‐eGFP plasmid containing an internal ribosomal entry site of encephalomyocarditis virus (CMV), the gene for green fluorescent protein (GFP), and the Genetecin resistant gene for selection. The human B7‐H4 gene was first amplified from the SKBR3 human breast cancer cell line known to be a high expressor of B7‐H4 and cloned into pIRES‐eGFP via the EcoRI and BamHI restriction sites. Tumors grown on BALB/c mice with transfected 4T1 cells were not rejected demonstrating the utility of this approach.
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Asset Metadata
Creator
Sankaranarayanan, Ishwarya
(author)
Core Title
Stable expression of human B7-H4 in a mouse mammary tumor model as a target for cancer immunotherapy
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Publication Date
07/17/2014
Defense Date
06/05/2014
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cancer therapy,OAI-PMH Harvest
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Advisor
McMillan, Minnie (
committee chair
), Akbari, Omid (
committee member
), Epstein, Alan L. (
committee member
)
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isankara@usc.edu
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