1. TARGETING
OF
PHOSPHATIDYLSERINE
ON
THE
SURFACE
OF
MAMMARY
TUMOR
CELLS
SLOWS
TUMOR
GROWTH
AND
LUNG
METASTASIS
Liang
Huang,
Chaobo
Yin,
Mitchel
Kent,
Joel
Shilyansky,
Dept.
of
Surgery,
University
of
Iowa
Children’s
Hospital
Purpose
Cancer
cells
employ
a
variety
of
molecular
mechanisms
in
order
to
evade
immunosurveillance.
One
of
the
major
mechanisms
by
which
tumors
subvert
immune
detecHon
and
destrucHon
is
by
suppression
of
the
host’s
immune
system1.
PhosphaHdylserine(PS)
is
phospholipid
predominantly
expressed
on
the
inner
leaflet
of
the
cell
membrane
in
living
cells.
PS
is
translocate
to
the
external
leaflet
during
apoptosis,
promoHng
rapid
uptake
and
clearance
by
phagocytes.
ApoptoHc
cells,
and
PS,
has
been
shown
to
inhibit
immune
responses2
.
When
PS
is
blocked
with
Annexin-­‐V,
a
protein
that
specifically
binds
and
blocks
PS,
immunogenicity
of
apoptoHc
cells
increased3.
PS
is
also
expressed
on
the
surface
of
tumor
cells
suggesHng
that
it
may
play
a
role
in
tumor
immune
evasion4.
The
goal
of
this
study
was
to
determine
the
effects
of
blocking
tumor
PS
on
tumor
growth.
Also,
there
are
numerous
factors
known
to
influence
the
metastaHc
potenHal
of
cancer.
Annexin
V
was
shown
the
potenHal
to
induce
tumor
immunity.
We
hypothesize
that
Annexin
V
would
inhibit
tumor
growth
and
a
reduce
lung
metastases.
Methods
Spontaneously
metastasizing
murine
mammary
4T1–luciferase
tumor
cells
(4T1Luc)
were
implanted
orthotopically
in
BALB/c
mice
and
tumors
developed
within
1
week.
Modified
Annexin
V
protein
was
then
injected
into
tumors.
Tumor
size
was
measured
every
other
day.
To
determine
the
extent
of
pulmonary
metastases,
luciferin
was
administered
and
mice
were
imaged
using
IVIS
system
on
days
15,
22,
and
29
a_er
inoculaHons.
On
day
29,
lungs
were
collected
for
pathological
examinaHon
or
placed
in
Hssue
culture
and
colony
forming
units
(CFU)
were
counted.
StaHsHcal
analysis
was
performed
using
2-­‐way
ANOVA
and
student
t-­‐test.
Results
Following
treatment
with
Annexin
V,
the
tumor
size
was
smaller
in
the
treatment
group
than
control
group(p=0.024).
Live
imaging
showed
less
luminescence
in
the
lungs
of
treated
animals
than
controls,
indicaHng
reducHon
in
lung
metastases
(p=0.037).
Pathological
examinaHon
showed
a
decrease
in
the
number
of
metastases
(p=0.0217).
Lung
cultures
demonstrated
fewer
tumor
CFUs
suggesHng
a
decrease
in
tumor
load
(p=0.008)
in
Annexin
V
treated
animals.
Conclusion
Annexin
V
has
demonstrated
efficacy
in
treatment
of
established
mammary
tumors
and
reduced
lung
metastases.
The
results
support
the
potenHal
of
targeHng
tumor
phosphaHdylserine
for
treatment
of
metastaHc
disease.
References
1. Wolchok
JD,
Chan
TA.
Cancer:
AnHtumour
immunity
gets
a
boost.
Nature.
2014
Nov
27;515(7528):496-­‐8.
2. Schujers
K,
Reutelingsperger
C.
PhosphaHdylserine
targeHng
for
diagnosis
and
treatment
of
human
diseases.Apoptosis.
2010
Sep;
15(9):1072-­‐82.
3. Yan
X,
Doffek
K,
Yin
C,
Krein
M,
Phillips
M,
Sugg
SL,
Johnson
B,
Shilyansky
J.
Annexin-­‐V
promotes
anH-­‐tumor
immunity
and
inhibits
neuroblastoma
growth
in
vivo.
Cancer
Immunol
Immunother.
2012
Nov;61(11):1917-­‐27.
4. Graham
DK,
DeRyckere
D,
Davies
KD,
Earp
HS.
The
TAM
family:
phosphaHdylserine
sensing
receptor
tyrosine
kinases
gone
awry
in
cancer.Nat
Rev
Cancer.
2014
Dec;14(12):769-­‐85.
Review.
Control
AnV
U
ntreated
A
nnexin
V
*
0
20
40
60
Groups
AverageNumberofPulmonaryMets
Pulmonary Metastases
Untreated
Annexin V *
U
ntreated
A
nnexin
V
*
0
100000
200000
300000
400000
500000
Day 22 Lung Luminescence
Groups
Photons
Untreated
Annexin V *
U
ntreated
A
nnexin
V
**
0
200
400
600
800
Culture of Lung Metastases
Groups
ColonyFormingUnits
Untreated
Annexin V **
5 10 15 20
0.0
0.2
0.4
0.6
Days After Tumor Inoculation
TumorSize(cm3
)
Effect of Annexin V on Tumor Growth
Untreated
Annexin V *

2. B
Annexin
V
A
Untreated