Necuparanib Inhibits Pancreatic Cancer Progression and Invasion
in a 3D Tumor and Stromal Cell Co-Culture System
Amanda MacDonald, Michelle Priess, Jennifer Curran, Savannah Moore, Guilin Wang, and Silva Krause — Momenta Pharmaceuticals, Inc., Cambridge, MA
ABSTRACT
RESULTS
BME – basement
membrane extract
m
g
N /mL
ec
u
5
Tr
ea
m
g
N /mL
ec
u
tm No
en
t
m
g
N /mL
ec
u
5
m
g
N /mL
ec
u
1
tm No
en
t
Necuparanib and gemcitabine in combination exhibit an
additive effect compared to gemcitabine alone
**
60000
Gemcitabine
30000
A
No Gemcitabine
500 μm
m
g
N /mL
ec
u
5
m
g
N /mL
ec
u
ns
100000
50000
AsPC-1 + PSC
Capan-2 + PSC
50000
0
Necuparanib
1.0
C
****
****
*
****
*
**
0.8
0.6
0.4
**** p < 0.0001; ** p < 0.01, * p < 0.05
at
m No
en
1 t
m
g
N /mL
ec
u
5
m
g
N /mL
ec
u
1
nM
ge
m
1 1
m nM
g/
m ge
L m
N +
ec
5 1
m nM u
g/
m ge
L m
N +
ec
10
u
0
nM
10
ge
1 0
m
m nM
g/
m ge
L
10 N m +
ec
5 0
m nM u
g/
m ge
L m
N +
ec
u
1
nM
ge
1 1
m
m uM
g/
m ge
L m
N +
ec
5 1
m uM u
g/
m ge
L m
N +
ec
u
0.0
p=0.8970
ea
tm No
en
t
Co-culture ratios used:
AsPC1 : PSC 1:3 (n = 8-16 per condition)
maintaining AsPC1 at 3,000 cells/well
*
Tr
e
0
C
****
0.2
M g/m
20 L
2
5 mg/mL M202
*
****
Circularity
1 mg/mL Dalt
m
B
No Treatment
****
****
****p<0.0001; *** p< 0.001
ec
N
1 mg/mL Enox
****
50%
100%
Figure 4: A) Co-cultures of AsPC-1 and PSCs treated with necuparanib and two different
low molecular weight heparins (LMWHs), enoxaparin and dalteparin. Quantitative analysis
of the cultures shows that necuparanib inhibits invasion in a similar manner to enoxaparin
and dalteparin. B) Co-cultures of AsPC-1 and PSCs with and without treatment with M202,
a structural analogue of necuparanib that has been modified to drastically reduce growth
factor and cytokine binding ability (200 – 2000 fold reduction).
B
****
****
****
60000
40000
20000
****p<0.0001
In summary, our in vitro data indicate that in this model necuparanib
affects tumor cell growth, PSC growth, and tumor cell invasion likely
by binding growth factor and cytokines that modulate tumor-stromal
cell interactions.
****p<0.0001; * p< 0.05
Tr
B
0
5
100 μm
Figure 2: A) Organoids were generated by
dissociation of an orthotopically implanted AsPC1 tumor grown in a mouse. Cultured organoids
were allowed to grow in invasion matrix for 12
days (inset, day 1), and showed invasive outgrowth
(white arrow). B) AsPC-1 cell line (red) or C)
Capan-2 cell line (red) co-cultured with pancreatic
stellate cells (PSCs, green), the most abundant
stromal cell in the pancreas. Tumor cells become
invasive in a bud-like outgrowth into the matrix.
***
5 mg/mL Necu
CONCLUSIONS
at N
m o
en
1 t
m
g
N /mL
ec
u
5
m
g
N /mL
ec
u
1
nM
ge
m
1 1
m nM
g/
m ge
L m
N +
ec
5 1
m nM u
g/
m ge
L m
N +
ec
10
u
0
nM
10
ge
1 0
m
m nM
g/
m ge
L
10 N m +
ec
5 0
m nM u
g/
m ge
L m
N +
ec
u
1
nM
ge
1 1
m
m uM
g/
m ge
L m
N +
ec
5 1
m uM u
g/
m ge
L m
N +
ec
u
All experiments were performed using 8 technical replicates per condition, with at least
3 biologic replicates each.
****
****
2.5 mg/mL Necu
****
1 uM
Tr
e
A
Tumor cells become invasive when cultured with pancreatic
stellate cells
B
1 mg/mL Necu
1 mg/mL Necu
Figure 7: A) Representative images of PSC spheroids treated with various doses
of necuparanib (necu). B) The corresponding quantitative analysis demonstrates
significant inhibition of PSC outgrowth in a dose dependent manner.
100000
Area Change (pixels)
No Treatment
up
ar N
an o
N 1 ib
ec
up mg
ar /m
an L
ib
En
ox
ap N
ar o
in
En 1
ox mg
ap /m
ar L
in
Da
lta
pa No
rin
Da 1 m
lta g/
pa mL
rin
m
g
N /mL
ec
u
5
m
g
N /mL
ec
u
1
Figure 1: Representative images and the quantitative analysis of three pancreatic cancer
cell lines in 3D culture with and without necuparanib. A) AsPC-1 cell line; analysis
of spheroid outgrowth demonstrates significant inhibition of tumor cell growth in a
dose dependent manner. B) Capan-2 cell line; analysis shows no significant reduction
in growth with necuparanib. C) Panc-1 cell line; analysis demonstrates significant
inhibition of tumor cell growth in a dose dependent manner.
Necuparanib has a similar effect as to other heparins in
inhibiting invasion and it is dependent on its ability to bind
growth factors and cytokines
Area Reduction (percentage)
30000
* p<0.05;****p<0.001
A
1
ea
Figure 3: A) Representative images of AsPC-1 control versus co-cultures of AsPC-1 and
PSCs treated with necuparanib (necu) at 1 mg/mL and 5 mg/mL and B) the corresponding
quantitative analysis of spheroid outgrowth demonstrates significant inhibition of tumor
cell outgrowth in a dose dependent manner.
100 nM
0.5 mg/mL Necu
0
500 μm
Tr
60000
Area Change (pixels)
Day 7
C
*
5 mg/mL Necu
Area Change (pixels)
No Treatment
tm No
en
t
****
1 nM
No Treatment
0.
5
10000
Tr
ea
90000
No Necu
B
90000
Day 0:
Day 3:
AsPC1 and/or PSC
Add BME +/- necu,
cells embedded in
M202 or gem
spheroid formation ECM
AsPc1
PSCs
100 μm
**** p<0.0001;**p<0.01
Each well of a 96 well round-bottom plate:
Legend:
5 mg/mL Necu
****
ns, p=0.1267
3D Culture Set-up
Culture for
4 additional days
at 37°C and 5% CO2
1 mg/mL Necu
20000
* * * * p < 0.0001
METHODS
&&&
No Treatment
1 mg/mL Necu
30000
ns
5 mg/mL Necu
Area Change (pixels)
60000
500 μm
ns
30000
****
1
90000
Area Change (pixels)
****
500 μm
m
g/ P
m S
L C
Ne w
cu /
2.
5
m
g/ P
m S
L C
Ne w
cu /
5
m
g/ P
m S
L C
Ne w
cu /
Day 7
500 μm
tm No
en
t
The objective of this study was to test the ability of necuparanib
to reduce proliferation and invasion of pancreatic tumor cells into
surrounding matrix in 3D cultures.
+ PSC
A
ea
Necuparanib, derived from heparin, was rationally engineered
to preserve/potentiate the anti-tumor activity while substantially
reducing anticoagulant activity, permitting administration of higher
doses (relative to heparin) in patients. Necuparanib in combination
with gemcitabine and nab-paclitaxel is currently being tested in a
Phase 2 clinical trial in patients with metastatic pancreatic cancer
(ClinicalTrials.gov Identifier: NCT01621243).
Figure 5: Representative
confocal image of an AsPC1 (red) spheroid treated with
fluorescently tagged necuparanib
(green) demonstrating that it
is located both intracellularly
(arrows) and intercellularly
(triangles).
A
1
No PSC
Tr
BACKGROUND
5 mg/mL Necu
Necuparanib inhibits pancreatic stellate cell growth
m
g/ P
m S
L C
Ne w
cu /
No Treatment
B
Day 7
A
5 mg/mL Necu
Necuparanib locates both intracellularly and intercellularly
no PSC
Ne w
cu /
No Treatment
Necuparanib inhibits tumor cell invasion into the
surrounding matrix
Area Change (pixels)
Necuparanib inhibits tumor cell growth
Area Change (pixels)
Pancreatic cancer is one of the most aggressive types of cancer,
with only about 5% of patients surviving 5 years past the initial
diagnosis. Despite advances with new chemotherapy combinations,
overall survival outcomes are still dismal and novel therapeutic agents
or combinations are needed. Therapies able to both disrupt the
aggressive fibrotic microenvironment and restrict tumor proliferation
are a current focus. Necuparanib (formerly M402) is a heparan sulfate
mimetic that binds and inhibits multiple heparin-binding growth
factors, chemokines, and adhesion molecules. Here we explore how
necuparanib affects the tumor and its microenvironment using a
3-dimensional (3D) culture system mimicking pancreatic cancer. The
system contains a co-culture of human pancreatic tumor cells and
pancreatic stellate cells (PSCs), the most abundant stromal cell in the
pancreas. We found that necuparanib inhibits a) pancreatic tumor cell
growth, b) pancreatic tumor cell invasion, and c) PSC growth in a dosedependent manner in vitro. We also established that necuparanib’s
activity is growth factor binding dependent. Necuparanib treatment
in combination with gemcitabine, a chemotherapeutic agent, was
shown to have an additive effect. The ability to inhibit proliferation
in both tumor and stromal cells and enhance the therapeutic effect
of gemcitabine demonstrates that necuparanib is a promising multitargeting agent for difficult-to-treat cancers such as desmoplastic
pancreatic tumors. These findings support the current Phase 1/2
safety and efficacy study being performed in patients with metastatic
pancreatic cancer.
Figure 6: A) Representative images of AsPC-1 and PSC co-cultures treated with
necuparanib (necu) and/or gemcitabine (gem) in various combinations. B) Corresponding
quantitative analysis of the treatment groups shows that the addition of necuparanib to
gemcitabine treatment significantly decreases invasion in a dose dependent manner. C)
Corresponding quantitative analysis of the treatment groups shows that the addition of
necuparanib to gemcitabine treatment significantly increases circularity, a measure of the
ratio of the circumference to the area of the spheroid in the image, in a dose dependent
manner. As the number of buds or invasive area decreases, circularity will increase.
• Necuparanib inhibited the
growth of pancreatic tumor
cells in 3D culture.
• Pancreatic tumor cells such as
AsPC-1 and Capan-2 are only
found to be invasive when cocultured with pancreatic stellate
cells (PSCs) in 3D culture.
• Necuparanib inhibited invasion
of AsPC-1 and stellate cell
cultures in a dose-dependent
manner.
• Necuparanib, enoxaparin and
dalteparin inhibited invasion
in a dose-dependent manner.
Although necuparanib is
engineered for low anti-
•
•
•
•
coagulant activity, anti-tumor
activity was maintained at a
level equivalent to enoxaparin
and dalteparin.
Low anti-coagulant activity
in necuparanib allows for
higher concentrations to be
administered to patients.
Necuparanib’s efficacy is, at
least in part, dependent on its
ability to bind chemokines,
cytokines and growth factors.
Necuparanib was found to
localize inter- and intracellularly
in 3D tumor spheroids.
Necuparanib inhibits stellate
cell outgrowth.
Acknowledgements: Sucharita Roy for the synthesis of M202 and fluorescently tagged
necuparanib, Tom Ferrante (Wyss) for his assistance with the confocal microscope, and the Wyss
Institute of Biologically Inspired Engineering for allowing us to use their confocal microscope.
Presented at the AACR Special Conference on The
Function of Tumor Microenvironment in Cancer Progression
January 7 –10, 2016
San Diego, California, USA
© 2015 Momenta Pharmaceuticals, Inc. All rights reserved.