Content uploaded by Dinesh Bangari
Author content
All content in this area was uploaded by Dinesh Bangari on Jun 14, 2017
Content may be subject to copyright.
Cancer Therapy: Preclinical
Placental Growth Factor Upregulation Is a Host Response
to Antiangiogenic Therapy
Rebecca G. Bagley, Yi Ren, William Weber, Min Yao, Leslie Kurtzberg, Jason Pinckney,
Dinesh Bangari, Cokey Nguyen, William Brondyk, Johanne Kaplan, and Beverly A. Teicher
Abstract
Purpose: Placental growth factor (PlGF) is an angiogenic protein. Upregulation of PlGF has been
observed in the clinic following antiangiogenic regimens targeting the VEGF pathway. PlGF has been
proposed as a therapeutic target for oncology. sFLT01 is a novel fusion protein that neutralizes mouse and
human PlGF (mPlGF, hPlGF) and mouse and human VEGF-A (mVEGF-A, hVEGF-A). It was tested in
syngeneic and xenograft tumor models to evaluate the effects of simultaneously neutralizing PlGF and
VEGF-A and to investigate changes observed in the clinic in preclinical models.
Experimental Design: Production of PlGF and VEGF-A by B16F10 and A673 cancer cells in vitro was
assessed. Mice with subcutaneous B16F10 melanoma or A673 sarcoma tumors were treated with sFLT01.
Tumor volumes and microvessel density (MVD) were measured to assess efficacy. Serum levels of hVEGF-A,
hPlGF, and mPlGF at early and late time points were determined by ELISA.
Results: Exposure of cancer cell lines to sFLT01 caused a decrease in VEGF secretion. sFLT01 inhibited
tumor growth, prolonged survival, and decreased MVD. Analysis of serum collected from treated mice
showed that sFLT01 administration caused a marked increase in circulating mPlGF but not hPlGF or
hVEGF. sFLT01 treatment also increased circulating mPlGF levels in non–tumor-bearing mice.
Conclusion: With the tumor cell lines and mouse models we used, antiangiogenic therapies that target
both PlGF and VEGF may elicit a host response rather than, or in addition to, a malignant cell response that
contribute to therapeutic resistance and tumor escape as suggested by others. Clin Cancer Res; 17(5); 976–88.
2011 AACR.
Introduction
The realization that tumors require vasculature to grow
and metastasize gave rise to the development of antiangio-
genic therapies to treat cancer. Food and Drug Adminis-
tration–approved antiangiogenic agents include the
humanized monoclonal antibody against VEGF-A bevaci-
zumab and the multitargeted small molecule tyrosine
kinase receptor inhibitors sunitinib, pazopanib, and sor-
afenib, which potently inhibit the VEGF and PDGF (plate-
let derived growth factor) pathways. Although therapies
that target vasculature are often included in clinical stan-
dard-of-care regimens, the benefit can be modest with little
improvement in overall survival (1). There is a need for the
identification of factors that enable tumor escape and
confer antiangiogenic agent resistance and the identifica-
tion of new targets for the next generation of antiangiogenic
agents.
Placental growth factor (PlGF) can form heterodimers
with VEGF and several splice variants of PlGF exist that
bind to VEGFR-1/Flt-1 and/or neuropilin-1 (2–5). The
contribution of PlGF to angiogenesis was shown in trans-
genic mice in which the overexpression of PlGF resulted in
a substantial increase in vasculature, including the number
of vessels, branching points, size of the vessels, and
increased vascular permeability (6). PlGF is expressed by
the placenta, endothelial cells, and osteogenic cells and
promotes angiogenesis during wound healing, ischemia,
and inflammation (7, 8). It is induced under hypoxic
conditions that occur in many solid tumors (9), thereby
promoting tumor neovascularization and enhancing
the survival of tumor endothelial cells and macrophages
(7, 10, 11).
PlGF is upregulated in many malignant diseases and thus
may be a useful therapeutic target. It is more highly
expressed in small and non–small cell lung cancers and
in renal cell carcinomas (RCC) than in the corresponding
normal tissues (12–14). PlGF transcripts were present in
human cervical squamous cell carcinomas and were upre-
gulated in human prostate cancer cells (15, 16). Human
melanoma cells and melanocytes also secrete and respond
to PlGF (17, 18). In addition, PlGF is of interest as a target
for breast and gastric cancers in which the expression is
higher than in other cancers (19, 20). PlGF enhanced the
Authors' Affiliation: Genzyme Corporation, Framingham, Massachusetts
Corresponding Author: Rebecca G. Bagley, Genzyme Corporation, 49
New York Ave., Framingham, MA 01701. Phone: 508-270-2455; Fax: 508-
271-4796; E-mail: rebecca.bagley@genzyme.com
doi: 10.1158/1078-0432.CCR-10-2687
2011 American Association for Cancer Research.
Clinical
Cancer
Research
Clin Cancer Res; 17(5) March 1, 2011
976
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
motility and invasion of the human breast tumor lines
MCF-7 and MDA-MB-231; a PlGF antagonist inhibited this
activity in vitro and reduced lung metastasis in vivo (21). The
therapeutic potential of neutralizing PlGF was shown with
an anti-PlGF antibody that inhibited the growth of tumors
in preclinical studies of melanoma, pancreatic cancer,
colon carcinoma, and bone metastasis (8, 22).
PlGF expression is increased following cancer therapy
and is one of several factors implicated in therapeutic
resistance, post-therapy angiogenesis, and tumor regrowth
(23). PlGF is associated with early recurrence of hepato-
cellular carcinoma following radical resection and was
upregulated in preclinical lung and colon adenocarcinoma
tumors following radioimmunotherapy (24, 25). In colo-
rectal cancer patients with metastatic disease, PlGF levels
increased following the administration of bevacizumab in
combination with chemotherapy and/or radiation (26,
27). VEGF-A and PlGF levels increased following sunitinib
treatment in patients with bevacizumab-refractory meta-
static RCC and in men with advanced prostate cancer who
were treated with sunitinib (28, 29). It has been proposed
that PlGF plays a role in resistance to antiangiogenic
therapies and that an antibody against PlGF may help
overcome resistance to VEGF receptor inhibitors (30).
Despite reports that tumors of multiple human cancers
overexpress PlGF, conflicting opinions exist on the impor-
tance of PlGF in oncology (31, 32). The therapeutic poten-
tial of an anti-PlGF antibody was shown in efficacy studies
in several preclinical tumor models (22). However, the data
presented by Bais and colleagues (31) suggest that anti-
angiogenic therapies that target PlGF will be no more
effective than those that neutralize VEGF-A. Consequently,
the significance of PlGF and value of neutralizing PlGF in
cancer have become recent subjects of controversy.
We have investigated whether a therapy that targets both
VEGF and PlGF can be efficacious with a novel fusion
protein, sFLT01 (33), which has a molecular weight of
approximately 80 kDa and consists of the VEGF/PlGF bind-
ing domain of VEGFR-1/Flt-1 fused to the Fc portion of
human IgG1 through a polyglycine linker (9Gly; ref. 34).
Intravitreal delivery of an AAV2 vector encoding sFLT01
(AAV2.sFLT01) was efficacious as a gene therapy in murine
and nonhuman primate models of retinal neovasculariza-
tion (34, 35). sFLT01 functions as a soluble VEGFR-1 decoy
receptor and neutralizes mouse VEGF-A and PlGF (mVEGF-
A, mPlGF) and human VEGF-A and PlGF (hVEGF-A, hPlGF).
The B16F10 melanoma model was selected on the basis of
reports that human melanoma cells secrete hPlGF and also
because the B16F10 model has been utilized to evaluate
antibodies against PlGF in earlier reports (8, 17, 18, 22, 31).
The A673 Ewing’s sarcoma xenograft model was employed
since previous studies investigated antiangiogenic agents
that target VEGF-A in this model and/or the contribution of
host stroma to mVEGF production (36, 37). We exposed
mouse B16F10 melanoma and human Ewing’s sarcoma
A673 cells to sFLT01 in vitro to assess the phenotypic
changes resulting from the neutralization of both VEGF-A
and PlGF. To assess antitumor activity, sFLT01 was admi-
nistered to mice bearing syngeneic B16F10 melanoma or
human xenograft A673 sarcoma tumors. Given the clinical
observation that VEGF-A and PlGF levels in circulation
increased following antiangiogenic therapy, the serum
levels of mPlGF, hPlGF, and hVEGF were quantified in
the B16F10 and A673 tumor–bearing mice following the
administration of sFLT01. Only mPlGF levels significantly
increased at early time points when the tumors were
responding to therapy and during late disease progression.
Administration of sFLT01 to naive mice also produced
upregulation of mPlGF, indicating a systemic host response.
The increase in mPlGF in the host is consistent with reports
that other antiangiogenic therapies delivered to naive mice
elevated circulating cytokine levels (23). Thus, antiangio-
genic therapies that target both PlGF and VEGF will likely
not prevent upregulation of PlGF. Finally, our findings also
indicate that resistance to anti-VEGF therapies may be
attributed in part to a systemic host response and not
exclusively to molecular changes in the malignant cells.
Materials and Methods
sFLT01 protein
CHO DXB11 cells were transfected with an expression
vector containing the coding sequence of sFLT01 (34),
using the Lipofectamine 2000 reagent (Invitrogen). Trans-
fected cells were selected with increasing concentrations of
methotrexate in MEM alpha, with no ribonucleosides or
deoxyribonucleosides (Invitrogen), containing 10% dia-
lyzed FBS (Invitrogen). The transfected pool was grown
in 850-cm
2
roller bottles in 200 mL of selection medium.
When the cells reached confluence, the selection medium
was removed and replaced with IS CHO CD medium
(Irvine Scientific). The conditioned medium was harvested
Translational Relevance
Therapies that target the VEGF pathway offer some
benefit but often do not significantly increase overall
survival. The factors that contribute to drug resistance
and tumor escape are not clearly defined. Placental
growth factor (PlGF) has been implicated as a contri-
butor in resistance to anti-VEGF regimens. It has been
proposed as a therapeutic target for oncology. We show
that simultaneous neutralization of VEGF-A and PlGF in
both tumor-bearing and -naive mice elicits an acute
host response resulting in elevated serum PlGF levels.
The results presented here raise questions about the
specific involvement of the tumor microenvironment
in the increases of serum PlGF levels that are observed
in the clinic and the utility of targeting PlGF as a
second-line antiangiogenic therapy. Furthermore,
increased circulating PlGF may be associated with phar-
macodynamics and decreased response to VEGF-A ther-
apy but may not necessarily indicate efficacy or disease
progression.
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 977
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
3 to 4 days later, filtered using a 0.2-mm filter, and loaded
onto a Protein A column that was preequilibrated with 0.7
mol/L NaCl, 20 mmol/L Tris, pH 7.0. The column was
washed with equilibration buffer and eluted with 50
mmol/L glycine, pH 2.5. The eluted peak fraction contain-
ing sFLT01 protein was adjusted to pH 6.5 to 6.7 with 0.3
mol/L Na
3
PO
4
(dibasic). The sFLT01 was concentrated
to 10 mg/mL, using a size 4, 30-kDa cutoff cartridge
(GE Healthcare) and diafiltration with PBS (Invitrogen).
Surface plasmon resonance binding analysis
A Biacore T100 instrument was utilized to perform
affinity analysis. Biacore CM5 Series S sensor chips (GE
Healthcare) were directly immobilized with 300RUs
hVEGF, mVEGF, hPlGF, and mPlGF (R&D Systems). Bind-
ing of sFLT01 was tested at 0, 7.5, 15, 30, 60, and 120 nmol/
L concentrations in duplicate. All samples were diluted in
HBS-EP þrunning buffer (10 mmol/L HEPES, pH 7.4; 150
mmol/L NaCl; 0.05% P20 surfactant; 3 mmol/L EDTA).
The flow rate was 50 mL per minute for both association
(5 minutes per sample injection) and dissociation
(10 minutes running buffer) steps. After each cycle, the
cytokine surfaces were regenerated using 10 mmol/L gly-
cine, pH 1.5, for 40 seconds at 30 mL/min. Biacore T100
evaluation software (v1.1.1) was used to analyze binding
kinetics. Blank flow cells and 0 nmol/L samples were
double-reference subtracted from data. All samples were
fit to a 1:1 binding model.
Cell culture
Mouse B16F10 melanoma and human A673 Ewing’s
sarcoma cell lines (American Type Culture Collection) were
grown in RPMI 1640/10% FBS (Invitrogen) 150 mg/mL
sFLT01 for 9 to 28 days. At several time points, aliquots of
the cells were grown to confluency in a T25 flask and were
washed twice with serum-free RPMI 1640. The cells were
overlayed with 4 mL of serum-free medium for 24 hours in
the absence of sFLT01. The conditioned medium was
collected and centrifuged to remove any loose cells and
transferred to a new tube. The remaining cells were col-
lected by trypsin/EDTA digestions (Invitrogen), total cells
were counted, and the resulting cell pellet was lysed with
lysis buffer (Roche Diagnostics) and 2 mmol/L sodium
orthovanadate (New England Biolabs). The conditioned
medium and lysed cell pellets were analyzed for secreted
and intracellular levels of PlGF and VEGF-A by ELISA (R&D
Systems).
In vivo models
Mouse B16F10 melanoma or human A673 sarcoma cell
lines were cultured as describe earlier. For the B16F10
melanoma tumor model, C57Bl/6 mice (Charles River)
were implanted subcutaneously with 5 10
5
cells mixed
1:1 with Matrigel in RPMI in a 200 mL volume (n¼12 per
group). For the human A673 Ewing’s sarcoma tumor
model, beige nude (NIH-LystbgFoxn1nuBtkxid) mice
(Charles River) were implanted subcutaneously with 9
10
6
cells mixed 1:1 with Matrigel in RPMI in a 200 mL
volume (n¼10 per group). sFLT01 (10 or 25 mg/kg) or
vehicle was delivered by intraperitoneal injection 2 or 3
times per week. Tumors were measured with calipers, and
dosing was initiated when tumors were 100 mm
3
in
volume. Tumor volume was calculated using the following
formula: width
2
length 0.52. Mice were euthanized
when tumors measured 20 mm in diameter or became
ulcerated. Studies were blinded.
Non–tumor-bearing mice (C57Bl/6 or beige nude) were
administered an intraperitoneal injection of 0, 10, or 25
mg/kg sFLT01 in a 0.9% saline vehicle on days 0, 4, and 7.
Blood was collected from the ocular sinus at 4, 24, and 48
hours post-injection on days 0, 4, and 7. Blood was also
collected on days 14 and 21. Serum samples were assayed
using PlGF and VEGF ELISA (R&D Systems). All procedures
were carried out according to a protocol approved by the
Institutional Animal Care and Use Committee in accor-
dance with the Federal Animal Welfare Act (9 CFR, 1992)
and were conducted in an AAALAC (Association for Assess-
ment and Accreditation of Laboratory Animal Care)-accre-
dited facility.
Immunohistochemistry
A673 and B16F10 subcutaneous tumors were fixed in
10% neutral buffered formalin (NBF) and embedded in
paraffin. Tumor sections (5 mm) were incubated with a rat
anti-mouse CD31 antibody (Fitzgerald Industries). A
biotinylated anti-rat antibody (Jackson Immunochem-
icals), ABC-Elite peroxidase (cat. no. PK-6100; Vector
Laboratories), and 3,30-diaminobenzidine (DAB; Dako)
chromagen were applied for detection. Tumors were
counterstained with hematoxylin, and whole image
scans were generated at 20magnification with the
Aperio SCANSCOPE XT scanning system. Microvessel
density (MVD) represents the total number of CD31
þ
vessels/mm
2
of tissue area and was quantified using
Aperio’s MVD algorithm. Additional morphometric mea-
surements such as vessel lumen size and perimeter were
also generated using the Aperio MVD algorithm and are
reported in mmandmm
2
,respectively.
A673 tumors were collected 1 day after the third (day 11)
or sixth (day 21) sFLT01 dose, were flash frozen in optimal
cutting temperature compound, and cut into 4- to 5-mm
sections. Samples were fixed in NBF for 10 minutes and
then incubated with a rat anti-mouse CD31 antibody (BD
Biosciences) or rabbit anti-mouse NG2 antibody (Chemi-
con). Secondary goat anti-rat-Cy3 or goat anti-rabbit-Cy2
antibodies (Jackson Immunochemicals) were applied for
detection.
For PlGF immunohistochemistry (IHC), heat-induced
epitope retrieval was done on deparaffinized tissue sec-
tions, using Dako citrate buffer (Dako), and then the
sections were incubated with a 1:50 dilution of a rabbit
polyclonal anti-PlGF antibody (cat. no. 250824; Abbiotec).
Dako Envision kit (cat. no. K4011; Dako) and DAB as
the chromogen were used. For VEGF-A IHC, a 1:200 dilu-
tion of a rabbit polyclonal anti-VEGFA antibody (cat. no.
sc-152; Santa Cruz Biotechnology) was used as the primary
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
978
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
antibody, a biotinylated goat anti-rabbit IgG (cat. no. 111-
065-144; Jackson Immunoresearch) was used as the sec-
ondary antibody (cat. no. 111-065-144), and ABC-Elite
peroxidase and was used for detection with DAB as the
chromogen. For IHC on negative control sections, the
primary antibody was replaced with rabbit universal nega-
tive control antibody (cat. no. N1699; Dako).
Statistical analysis
In vitro data are expressed as mean SD. Comparisons
between treatment and control groups were made using
student’s ttest. Tumor volumes are expressed as mean
SEM and were analyzed by the ANOVA test. Kaplan–Meier
survival curves were analyzed by the log-rank test. Graph-
pad Prism 5.0 (Graphpad Software, Inc.) was used for
analysis. P<0.05 was considered statistically significant.
Results
The ability of sFLT01 protein, expressed by transfected
HEK293 cells into conditioned medium, to neutralize
hVEGF-A was shown previously in vitro in binding and
human umbilical vein endothelial cell proliferation assays
(34). The binding affinity of the recombinant sFLT01
protein for mVEGF-A, mPlGF, hVEGF-A, and hPlGF was
further characterized by surface plasmon resonance tech-
nology. mVEGF-A shares an 89% amino acid homology
with hVEGF-A, and mPlGF has a 65% homology to hPlGF
(38, 39). sFLT01 bound to mVEGF in addition to hVEGF-A
(Fig. 1A and B). sFLT01 also bound to both mPlGF and
hPlGF (Fig. 1C and D). The kinetic analysis indicated that
sFLT01 has strong binding interactions with these growth
factors (Table 1). These results show that sFLT01 is cross-
species reactive and can neutralize both PlGF and VEGF-A
that are secreted by either human malignant cells or murine
cells in xenograft or syngeneic tumor models.
The expression profile of VEGF-A and PlGF was investi-
gated in mouse B16F10 melanoma cells and human A673
Ewing’s sarcoma cells in vitro. Comparisons were made
between cells exposed to 150 mg/mL sFLT01 for 9 to 28 days
and untreated (control) cells. At the end of the culture
period, cells were transferred to serum-free medium con-
taining no sFLT01, the supernatant was collected following
a 24-hour incubation, and assayed by ELISA to determine
secreted levels of VEGF-A and PlGF. Cell lysates were
prepared to quantify by ELISA intracellular VEGF-A and
PlGF levels. Higher levels of mVEGF-A were detected in the
conditioned medium of untreated B16F10 cells than in the
cell lysates (Fig. 2A). In contrast, when B16F10 cells were
exposed to sFLT01, little or no secreted mVEGF-A was
detected and the levels of mVEGF-A were higher in the
cell lysates than in the conditioned medium with a decrease
over time (Fig. 2A). Untreated B16F10 cells secreted higher
levels of mVEGF than mPlGF (Fig. 2A and B). mPlGF
secretion also decreased when B16F10 cells were exposed
to sFLT01, but there was no increase in mPlGF in the cell
lysates (Fig. 2B).
The human A673 Ewing’s sarcoma cells secreted hVEGF-
A in culture but not following exposure to sFLT01 (Fig. 2C).
Unlike the B16F10 cells, little or no VEGF-A was detected in
A
180
Response (RU)
30
80
130
C
–100 100 300 500 700 900
Time (s)
–20
400
esponse (RU)
300
200
100
–100 100 300 500 700 900
Time (s)
Re
0
B
140
180
Response (RU)
20
60
100
140
D
–100 100 300 500 700 900
Time (s)
–20
20
350
esponse (RU)
50
150
250
–100 100 300 500 700 900
Time (s)
0
Re
–50
50
Figure 1. Surface plasmon resonance (Biacore) detection of the interaction between sFLT01 and VEGF or PlGF. The binding of sFLT01 to angiogenic
growth factors was tested at 0, 7.5, 15, 30, 60, and 120 nmol/L concentrations. Sensorgrams indicate binding of sFLT01 to hVEGF-A (A) and mVEGF (B).
sFLT01 also binds to hPlGF (C) and mPlGF (D).
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 979
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
the cell lysates. Similar to the secretion profile of the
B16F10 cells, A673 cells secreted higher levels of hVEGF-
A than hPlGF (Fig. 2A–D). In contrast to the secretion
patterns of the B16F10 cells, sFLT01 exposure did not
significantly change the secreted levels of hPlGF secreted
by the A673 cells but did affect the amount of intracellular
hPlGF levels (Fig. 2B and D). Although hPlGF levels
increased over time in the control A673 cells, the hPlGF
levels decreased during the same period when cells were
exposed to sFLT01 in culture. On the basis of the expression
profile of VEGF-A and PlGF by B16F10 and A673 cells and
the resulting phenotypic changes on exposure to sFLT01 in
vitro, the syngeneic B16F10 melanoma and the A673 sar-
coma xenograft were selected as models in which to assess
the effects of sFLT01 in vivo.
In the subcutaneous B16F10 melanoma tumor model,
mice were treated with vehicle (control) or 25 mg/kg
sFLT01 twice per week by intraperitoneal injection begin-
ning when tumors were approximately 100 mm
3
in
volume. Administration of sFLT01 resulted in a 14-day
tumor growth delay determined when tumors reached
1,500 mm
3
compared with the control group (P<
0.0001; Fig. 3A). Median survival significantly increased
from 17 days in the control group to 28 days with sFLT01
treatment, an increase of approximately 60% (P¼0.0021;
Fig. 3B). B16F10 tumors from treated and control mice
were collected 1 day after the second sFLT01 dose to
evaluate the vasculature. The blood vessels in the tumors
from mice treated with sFLT01 were notably more imma-
ture and less developed than those from tumors in the
control group that presented patent lumens and greater
length (Fig. 3C). Morphometric analysis of the blood
vessels indicated that although the MVD and vessel wall
thickness were not significantly reduced at the time the
tumors were collected, the mean vessel area, perimeter,
lumen size, and vascular area were significantly reduced by
sFLT01 administration (P<0.05; Fig. 3D).
IHC was done on formalin-fixed, paraffin-embedded
sections of subcutaneous B16F10 control tumors to inves-
tigate the tumor microenvironment. It revealed mPlGF and
mVEGF staining in malignant cells and in the adjacent
fibroblasts (Fig. 4A). Similarly, mPlGF and mVEGF-A stain-
ing was observed in B16F10 cells and in fibroblasts in
tumors from mice treated with sFLT01. A negative control
rabbit antibody applied to tumor sections did not show
background staining. These results indicate that the stromal
cells such as fibroblasts in addition to the B16F10 malig-
nant cells can produce and secrete mPlGF and mVEGF-A.
Blood was collected from mice at 2 time points during
the B16F10 efficacy study to quantify mPlGF in circulation.
On day 12 post–tumor cell implant, blood was collected 24
hours after the second dose of sFLT01 or vehicle when
tumors were approximately 180 or 315 mm
3
in volume,
respectively. Blood was also collected on days 18 to 25
when tumors reached the endpoint volume of more than
2,000 mm
3
and mice were removed from the study. Mice
had received a total of 4 to 5 doses and were euthanized
approximately 3 days after the last sFLT01 dose.
The circulating levels of mPlGF in the control group were
undetectable on day 12 when the average tumor volume
was 315.3 49.2 mm
3
and increased to 406 205.7 pg/
mL when tumors were 2,753.6 1,562.8 mm
3
(Fig. 4B). In
contrast, the circulating levels of mPlGF in the sFLT01
treatment group were significantly elevated on day 12
(7,038.6 1,325.6 pg/mL) when tumors were an average
volume of 177.9 24.5 mm
3
(P<0.0001). The circulating
mPlGF levels remained higher (8,584.3 1,575.2 pg/mL)
than those in the control group (406.0 205.7) through-
out the study until animals reached euthanasia criteria (P<
0.0001). The mPlGF ELISA detected only free or unbound
mPlGF. We were unable to determine the levels of mVEGF
because sFLT01 interfered with the mVEGF ELISAs from 2
vendors we tested and generated artifactual results. The
results shown in Figure 4B indicate that mPlGF was upre-
gulated not only when sFLT01 was efficacious and slowed
tumor growth but also when the tumors no longer
responded to treatment.
To further investigate the efficacy of sFLT01 treatment
and the in vivo source of circulating PlGF following sFLT01
treatment (i.e., malignant cells or host tissue), we utilized
the human A673 Ewing’s sarcoma xenograft model. Mice
were treated with vehicle (control) or 20 mg/kg sFLT01 3
times per week by intraperitoneal injection beginning
when tumors reached approximately 100 mm
3
in volume.
Administration of sFLT01 resulted in a 28-day tumor
growth delay determined when tumors reached 1,500
mm
3
compared with control group (P<0.0001;
Fig. 5A). Median survival significantly increased from
20 days in the control group to 50 days following sFLT01
treatment (P<0.0001; Fig. 5B). Double immunofluores-
cent staining for pericytes, using an anti-NG2 antibody,
and for endothelial cells, using an anti-CD31 antibody,
revealed that at 2 and 3 weeks the blood vessels in the
sFLT01-treated A673 tumors were immature and under-
developed compared with control (Fig. 5C). The images
also suggest that sFLT01 treatment may delay pericyte
coverage of blood vessels in the tumor microenvironment.
sFLT01 treatment resulted in a significant reduction in
MVD at 2 weeks post–tumor cell implantation following
3 doses of sFLT01 (P¼0.0007; Fig. 5D). However, there
Table 1. sFLT01 binding affinity
Cytokine k
a
, 1/(mol/L s) K
d
, 1/s K
D
, mol/L
hVEGF-A 2.03Eþ05 3.82E-04 1.89E-09
mVEGF-A 1.52Eþ05 4.43E-04 2.93E-09
hPlGF 6.95Eþ05 8.17E-03 1.18E-08
mPlGF 2.10Eþ05 9.63E-04 4.59E-09
NOTE: The binding affinity of sFLT01 to hVEGF-A, mVEGF-
A, hPlGF, and mPlGF was measured by surface plasmon
resonance (Biacore) binding assays. sFLT01 had compar-
able binding affinities to the mouse and human homolo-
gues.
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
980
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
Figure 2. VEGF-A and PlGF
production by B16F10 and A673
cells. Conditioned medium and
lysates from cells in culture were
assayed for VEGF-A and PlGF
levels by ELISA. Serum-free
conditioned medium was
collected over a 24-hour period
and contained no sFLT01. B16F10
melanoma cells secreted lower
levels of VEGF-A and PlGF
following exposure to sFLT01 (A)
but intracellular levels did not
change significantly (B). A673
sarcoma cells exposed to sFLT01
also decreased secretion of
VEGF-A and intracellular levels
were not significantly altered (C).
sFLT01 altered the intracellular
levels of PlGF over time in the
A673 cells but did not
substantially change the secretion
pattern (D).
VEGF-A levels - B16F10 cells
A
B
C
D
1600
1800
600
800
1000
1200
1400
Day 28
0
200
400
Control sFLT01 Control sFLT01
Control sFLT01 Control sFLT01
Control sFLT01 Control sFLT01
Control sFLT01 Control sFLT01
pg/mL per million cells
pg/mL per million cells
pg/mL per million cells
pg/mL per million cells
Conditioned medium
PlGF levels - B16F10 cells
200
Cell lysates
Conditioned medium Cell lysates
Conditioned medium Cell lysates
Conditioned medium Cell lysates
100
120
140
160
180 Day 9
Day 16
Day 28
0
20
40
60
80
VEGF-A levels - A673 cells
700
800
300
400
500
600
Day
Day 28
0
100
200
PlGF levels - A673 cells
350
200
250
300
Day 9
Day 16
Day 28
50
100
150
0
Day16
Day16
Day 9
Day 9
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 981
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
was no reduction in MVD at 3 weeks after 6 doses of sFLT01
(P¼0.9619). At 3 weeks, the MVD in the sFLT01 group was
similar to the MVD at 2 weeks (P¼0.4174) but the MVD in
the control was significantly lower because of the forma-
tion of fewer but larger bloods vessels with patent lumens
(P¼0.0012).
Human A673 Ewing’s sarcoma cells secreted 10-fold
higher levels of hVEGF than levels of hPlGF in cell culture
(Fig. 2). In contrast, in control mice bearing human A673
xenograft tumors (Fig. 6A), there were generally higher levels
of circulating hPlGF (524.7 585.9 pg/mL) than hVEGF
(85.0 36.5pg/mL), although the levels of circulating hPlGF
varied within the group (data not shown). Higher levels of
circulating hPlGF than hVEGF in mice bearing A673 tumor
model are in direct contrast to the pattern of growth factor
secretionby the same cells in vitro.Differences in the secretion
patterns of growth factors by malignant cells in vitro and in
vivo have been observed in other tumor models (40). The
serum levels of hVEGF (93.7 45.3 pg/mL) and hPlGF
(831.2 837.3 pg/mL) following sFLT01 administration
were comparable with the serum levels in the control group
(P¼0.7497 and P¼0.3912, respectively). However, the
serum levels of mPlGF (10,449.2 11,488.0 pg/mL) follow-
ing sFLT01 treatment were significantly higher than the levels
of mPlGF in the control group (16.4 49.2 pg/mL; P<
0.0001; Fig. 6A). The ability to selectively quantify hPlGF
secreted by the human malignant cells and mPlGF secreted
by the host, bothof which are neutralized by sFLT01, allowed
clear distinction between PlGF secreted by the mousenormal
tissues or tumor stroma and secretion by the human cancer
cells. These results indicate that while sFLT01 can bind and
neutralize PlGF, treatment with sFLT01 stimulates excess
production of PlGF by the host which can be detected in
the circulation.
3,500
AD
BC
2,500
3,000
Control
sFLT01
2,000
1,000
1,500
Tumor volume (mm3)
0
500
0 5 10 15 20 25 30
Day
80
100
Control
sFLT01
Control sFLT01
60
20
40
Percent survival
0 10203040
0
Day
300
Control
150
200
250
Control
sFLT01
0
50
100
150
essels/mm2)
el area (µm2)
erimeter (µm)
en area (µm2)
ar area (µm2)
hickness (µm)
MVD (ve
Mean vessel
Mean vessel per
Mean lumen
Mean vascular
an vessel wall thic
Mean
Figure 3. Efficacy of sFLT01 in B16F10 syngeneic melanoma tumors. A, mice bearing subcutaneous B16F10 melanoma tumors were dosed twice a
week by intraperitoneal injection with 25 mg/kg sFLT01 or vehicle control beginning on day 4 when tumors were approximately 100 mm
3
. sFLT01 inhibited
the growth of B16F10 tumors resulting in a 14-day tumor growth delay determined when tumors reached 1,500 mm
3
in volume. Error bars represent
SEM. B, sFLT01 increased the median survival of mice bearing subcutaneous B16F10 melanoma tumors from 17 days in the controls to 28 days in the
sFLT01-treated group (P<0.05). C, subcutaneous B16F10 tumors were collected on day after the mice received a second dose of sFLT01 or vehicle. The
endothelial cells in the tumor vasculature were visualized using an anti-CD31 antibody. The blood vessels from tumors treated with sFLT01 (right) were
underdeveloped and more immature than the controls (left). Scale bars, 250 mm. D, tumor vascular parameters were evaluated on tumor sections from
control and sFLT01-treated mice, using an anti-CD31 antibody immunolabeling the endothelial cells and imaging analysis software. Although there
were no significant differences in the MVD and blood vessel wall thickness between the sFLT01 and control groups, the blood vessels in the sFLT01 group
were reduced in vessel and overall vascular areas, perimeter, and lumen size (P<0.05).
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
982
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
To further investigate the role of host tissue in the
secretion of mPlGF into circulation in mice bearing
A673 Ewing’s sarcoma xenografts, sFLT01 (10 or 25 mg/
kg) was administered to naive, non–tumor-bearing mice
on days 0, 4, and 7. The circulating mPlGF levels increased
over time between 4 and 48 hours after injection of sFLT01
on days 0 and 7 (Fig. 6B). In contrast, circulating mPlGF
was not detectable in the control group. This effect was not
mouse strain specific and was also observed in non–tumor-
bearing immunocompetent C57Bl/6 mice (Fig. 6B). The
levels of mPlGF decreased from the time of sFLT01 injec-
tion and returned to baseline levels within 2 weeks after the
last injection of sFLT01 on day 21.
Discussion
PlGF contributes to angiogenesis during normal physio-
logic events and in pathologic conditions such as ischemia
and cancer (7). It may have prognostic value in several
human cancers including those of the breast (19, 41), lung
(14, 42), and colon (43). Overexpression in human tumors
and proangiogenic activity has identified PlGF as a poten-
tial target for therapeutic intervention (30). Higher circu-
lating levels of PlGF have been detected in patients
following antiangiogenic therapy with sunitinib and bev-
acizumab (26–29). The effect is not limited to PlGF, as
increased circulating levels of VEGF, sVEGFR-2, and
sVEGFR-3 were also observed in RCC patients being treated
with sunitinib (44). There are 2 protein therapeutics in
clinical trials that neutralize PlGF: aflibercept is a fusion
protein that binds to soluble VEGF-A and PlGF, and TB403
is a monoclonal antibody against PlGF (30, 45). Multi-
targeted small molecule tyrosine kinase inhibitors that
have been shown to antagonize VEGFR-1/Flt-1 in the clinic
or in preclinical development also block PlGF signaling.
sFLT01 is a novel, soluble decoy receptor which is com-
posed of the VEGF/PlGF binding domain of VEGFR-1/Flt-
1. We explored the phenotypic changes of cells exposed to
11,000
AB
4–5 doses
9,000
10,000 Tumor volume (mm3)
mPlGF (pg/mL) 2 doses
6,000
7,000
8,000
4,000
5,000
0
1,000
2,000
3,000 2 doses
Control sFLT01
3–4 doses
Figure 4. PlGF and VEGF-A production in the B16F10 melanoma model. Immunohistochemical methods were applied to detect PlGF and VEGF-A expression
in murine B16F10 melanomas (A). In a control (vehicle-treated) tumor, mild to moderate PlGF expression was observed in neoplastic cells (black arrows) and in
fibroblasts (green arrows) in the surrounding connective tissue stroma. Similar PlGF immunoreactivity was noted in a sFLT01-treated tumor. VEGFA
immunoreactivity was observed in neoplastic cells and fibroblasts of the control tumor. Neoplastic cells and fibroblasts in sFLT01-treated tumors also
showed VEGFA immunoreactivity. Control tumor immunolabeled with a negative control rabbit antibody showed no specific immunoreactivity. Similarly,
sFLT01-treated tumor showed no immunolabeling with negative control antibody. Neoplastic cells containing melanin are indicated by blue arrows.
Scale bars, 50 mm. Serum mPlGF levels in mice bearing B16F10 tumors were quantified by ELISA (B). Blood was collected from mice that received 2 to
5 doses of sFLT01 (25 mg/kg) or vehicle. Tumors were measured at the time blood was collected. Serum mPlGF levels were elevated 24 hours after the
second dose of sFLT01 when sFLT01 was effective and remained high when tumors no longer responded. mPlGF was detected in the serum of the control
mice only when tumors reached endpoint and the levels were much lower than those in the sFLT01-treated group.
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 983
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
sFLT01 in vitro and investigated PlGF secretion patterns in 2
preclinical tumors models following administration of
sFLT01.
sFLT01 neutralizes the murine and human homologues
of VEGF-A and PlGF, thereby facilitating investigations in
preclinical xenograft modelsinwhichthetumormassis
composed of human malignant cells and murine stromal
cells, vasculature, and infiltrating cells. The antitumor
efficacy of sFLT01 and host response were evaluated
andwedeterminedwhetherthesourceofVEGFandPlGF
was human cancer cells and/or host stroma in xenograft
tumor models and in syngeneic models in which the
angiogenic growth factors are solely murine-derived.
sFLT01 was efficacious in slowing tumor growth, increas-
ing survival, and reducing intratumoral MVD in the
B16F10 melanoma and in the A673 sarcoma models.
As has been observed in the clinic with VEGF inhibitors,
sFLT01 administration to tumor-bearing mice resulted in
an acute increase in the circulating levels of mPlGF when
sFLT01 was efficacious. However, high blood levels of
mPlGF persisted when tumors no longer responded to
therapy. In addition, circulating mPlGF levels were
acutely elevated in non–tumor-bearing mice on admin-
istration of sFLT01 and returned to baseline levels over
time showing dissociation between mPlGF levels and
antitumor efficacy. Changes in circulating mVEGF levels
could not be determined in the presence of sFLT01 by the
mVEGF ELISA.
2,500
2,000
Control
sFLT01
1,000
1,500
500
Tumor volume (mm3)
0
0 10 20 30 40 50
Day
75
100 sFLT01
Control
50
25
Percent survival
020 40 60
0
Day
Control
AC
B
D
sFLT01
Week 2
Week 3
30
Vehicle
15
20
25
sFLT01
5
10
Average no. of vessels/ mm
2
0
63
Number of doses
Figure 5. Efficacy in the A673 Ewing's sarcoma xenograft. A, mice were treated 3 times per week with 20 mg/kg sFLT01 or vehicle by intraperitoneal
injection beginning on day 4 post–cell implant. The growth of A673 subcutaneous tumors was significantly inhibited (P<0.0001). sFLT01 resulted in
a 28-day tumor growth delay determined when tumors reached 1,500-mm
3
size compared with control group. B, median survival significantly (P<0.0001)
increased from 20 days in the control group to 50 days following sFLT01 treatment. C, A673 tumors were collected 1 day after the third sFLT01 dose
on day 11 or 1 day after the sixth sFLT01 dose on day 21. Tumor vasculature was stained with an anti-CD31 (red) to visualize endothelial cells and an
anti-NG2 (green) to visualize pericytes. sFLT01 treatment resulted in more immature and underdeveloped blood vessels. Scale bars, 100 mm. D, MVD was
significantly (P¼0.0012) reduced in A673 tumors from mice treated with 3 doses of sFLT01. Larger but fewer blood vessels in the control tumors
resulted in a lower MVD value at a later time point.
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
984
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
sFLT01 exposure altered the expression patterns of
m/hVEGF-A and m/hPlGF secretion by cancer cell lines
in vitro. However, mouse B16F10 melanoma cells did not
upregulate mPlGF secretion after exposure to sFLT01 in
culture, suggesting that host normal cells were likely the
primary source of circulating mPlGF in vivo, although IHC
revealed mPlGF expression in the malignant cells of
B16F10 tumors. Although this does not exclude the pos-
sibility that the malignant cells in vivo could be a source of
elevated serum mPlGF levels, PlGF production by fibro-
blasts has also been detected in the fibroblasts of the
B16F10 tumors and in fibroblasts under hypoxic condi-
tions (9). Thus, normal or tumor-associated fibroblasts
may have contributed to the increased circulating mPlGF
in mice bearing B16F10 melanoma tumors or A673
Ewing’s sarcoma tumors. Importantly, higher serum
mPlGF levels occurred in mice bearing human A673 tumor
cells and in non–tumor-bearing mice following sFLT01
administration. Our results further expand on the findings
of investigators who have observed that the induction of
angiogenic growth factors is a host response to antiangio-
genic therapy following the administration of sunitinib or
anti-VEGF-A in non–tumor-bearing mice (23, 31).
Although PlGF levels increased in some patient popu-
lations following antiangiogenic therapy, there are con-
flicting reports regarding PlGF increases in circulation and
in the tumor microenvironment in mice following treat-
ments that target the VEGF pathway (22, 31). Fischer and
colleagues observed increases in PlGF in circulation and
PlGF mRNA in B16F10, CT26, and Panc02 tumors. In
contrast, Bais and colleagues reported that anti-VEGF-A
treatment did not increase PlGF levels in several murine
Figure 6. Circulating hVEGF,
hPlGF, and mPlGF in mice bearing
A673 tumors and circulating
mPlGF in naive mice. A, in vivo,
hVEGF and hPLGF levels
were detected in serum at the time
of sacrifice. The levels of hVEGF
and hPlGF did not change
significantly after sFLT01
treatment. The serum levels of
mPlGF were significantly
(P<0.0001) higher in the
sFLT01-treated group than in the
control group. B, circulating
mPlGF levels increased in
non–tumor-bearing beige nude
mice following a single injection of
sFLT01. Error bars, SD (n¼5).
25
A
B
hVEGF
15
20 hPlGF
mPlGF
5
10
ng/mL
ng/mL
0
Control sFLT01 Control sFLT01 Control sFLT01
80
50
60
70
30
40
10
20
0
0 5 10 15 20
Day
Vehic le (nude)
sFLT01 - 10 mg/kg (nude)
sFLT01 - 25 mg/kg (nude)
Vehic le (C57Bl/ 6)
sFLT01 - 10 mg/kg (C57B l/6)
sFLT01 - 25 mg/kg (C57B l/6)
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 985
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
syngeneic models including B16F10. The anti–VEGF-A
treatment did, however, produce increased circulating
PlGF levels in non–tumor-bearing mice. Although our
findings do not distinguish whether an anti-PlGF treat-
ment will be equally or more effective than anti–VEGF-A
treatment, or whether neutralization of PlGF, VEGF-A, or
both, by sFLT01 leads to the PlGF host response, the
results reported here imply that upregulation of PlGF may
be initiated by other antiangiogenic therapies and will not
be prevented with a dual VEGF-A/PlGF antagonists using
a protein therapeutic.
The potential of PlGF as a marker can be viewed in the
context of prognosis, efficacy, or pharmacodynamics. (19,
40–42). In patients with RCC treated with sunitinib,
circulating PlGF levels were higher toward the end of
the 4 week-drug cycle and returned to baseline levels
2 weeks thereafter, suggesting that PlGF could be a puta-
tive pharmacodynamic indicator (44). PlGF has been
assessed as a marker following antiangiogenic therapy
with bevacizumab in rectal cancer patients and in RCC
patients treated with sunitinib (27, 44, 46), and it has
been suggested that serum PlGF may be a sign of tumor
escape when patients no longer respond to therapy (26).
However, our results indicate that following administra-
tion of sFLT01, circulating PlGF was elevated in A673
tumor–bearing mice when the tumor was responding and
during disease progression. The increase in circulating
mPlGF levels likely reflects a systemic host response to
sFLT01 and may limit the utility of serum PlGF as an
indicator of efficacy or loss thereof.
The increased secretion of PlGF into circulation upon
administration of antiangiogenic therapies could poten-
tially promote more aggressive disease. PlGF can recruit
VEGFR-1þprogenitor cells from the bone marrow, thereby
promoting hematopoiesis (47). Hematopoietic stem cells,
in turn, can then repopulate tumor vasculature and, in part,
support angiogenesis and tumor regrowth. Similar to
VEGF, PlGF promoted adult vasculogenesis by enhancing
endothelial precursor cell recruitment and vessel formation
at the site of neovascularization in B16F10 tumors (11).
PlGF overexpression in an engineered rat C6 brain tumor
line implanted in nude mice conferred protection against
apoptosis and induced a survival phenotype in tumor
endothelial cells and macrophages (10). PlGF enhanced
the mobilization of endothelial cells and tumor cell inva-
sion in the B16-BL6 melanoma model (48). Thus, PlGF can
exert effects on numerous cell types that comprise the
tumor microenvironment and may therefore promote
malignant disease.
Indeed, controversy exists as to whether antiangiogenic
therapies can actually accelerate metastasis. Sunitinib accel-
erated metastasis and decreased survival in mice when 231/
LM2-4
LUCþ
cancer cells were delivered intravenously either
before or after sunitinib treatment and also following the
removal of primary xenograft tumors (49). A more invasive
phenotype developed in the RIP1-Tag2 model when mice
were treated with an anti–VEGFR-2 antibody or with suni-
tinib (50). Although mice initially responded to the ther-
apy, the progression to end-stage disease was more rapid
with the tumors becoming more invasive within the tumor
microenvironment and resulting in an increase in distant
metastasis. Furthermore, preclinical studies may incorpo-
rate the antiangiogenic agent in the absence of chemother-
apy, as is the treatment regimen in the clinic. The factors
that may contribute to antiangiogenic resistance and tumor
escape are not clearly defined and have been the subject of
several review articles (51–53). In addition to PlGF, cyto-
kines such as FGF, SDF-1, IL-8, and PDGF are a few
examples of putative compensatory agents that have been
discussed. Stromal host cells and tumor-infiltrating cells
such as myeloid cells and progenitors have also been
implicated, whereas the malignant cells cannot be
excluded.
The data we have generated using a novel fusion protein,
sFLT01, that binds with great affinity to mouse and human
PlGF and VEGF show that simultaneously neutralizing
these 2 angiogenic growth factors in preclinical tumor
models is effective and similar therapeutic protein strate-
gies may offer some clinical benefit in treating cancer. The
administration of sFLT01 resulted in elevated serum mPlGF
levels both in tumor-bearing and non–tumor-bearing mice
indicating a systemic host response. These results hold
implications for therapies that simultaneously target PlGF
and VEGF pathways.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received October 5, 2010; revised November 15, 2010; accepted
December 1, 2010; published OnlineFirst February 22, 2011.
References
1. Staton CA, Brown NJ, Reed MWR. Current status and future pro-
spects for anti-angiogenic therapies in cancer. Exp Opin Drug Discov
2009;4:961–79.
2. Cao Y, Chen H, Zhou L, Chiang MK, Anand-Apte B, Weatherbee JA,
et al. Heterodimers of placenta growth factor/vascular endothelial
growth factor. J Biol Chem 1996;271:3154–62.
3. Maglione D, Guierriero V, Viglietto G, Delli-Bovi P, Persico M. Isola tion
of a human placenta cDNA coding for a protein relation to the vascular
permeability factor. Proc Natl Aacd Sci U S A 1991;88:9267–71.
4. Migdal M, Huppertz B, Tessler S, Comforti A, Shibuya M, Reich R,
et al. Neuropilin-1 is a placenta growth factor-2 receptor. J Biol Chem
1998;273:22272–8.
5. Park JE, Chen HH, Winder J, Houck KA, Ferrara N. Placenta growth
factor. Potentiation of vascular endothelial growth factor bioactivity, in
vitro and in vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR.
J Biol Chem 1994;269:25646–54.
6. Odorisio T, Schietroma C, Zaccaria ML, Cianfarani F, Tiveron C,
Tatangelo L, et al. Mice overexpressing placenta growth factor exhibit
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
986
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
increased vascularization and vessel permeability. J Cell Sci
2002;115:2559–67.
7. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M,
et al. Synergism between vascular endothelial growth factor and
placental growth factor contributes to angiogenesis and plasma
extravasation in pathological conditions. Nat Med 2001;7:575–83.
8. Coenegrachts L, Maes C, Torrekens S, Van Looveren R, Mazzone M,
Guise TA, et al. Anti-placental growth factor reduces bone metastasis
by blocking tumor cell engraftment and osteoclast differentiation.
Cancer Res 2010;70:6537–47.
9. Green CJ, Lichtlen P, Huynh NT, Yanovsky M, Laderoute KR, Schaff-
ner W, et al. Placenta growth factor gene expression is induced by
hypoxia in fibroblasts: a central role for metal transcription factor-1.
Cancer Res 2001;61:2696–703.
10. Adini A, Kornaga T, Firoozbakht F, Benjamin LE. Placental growth
factor is a survival factor for tumor endothelial cells and macrophages.
Cancer Res 2002;62:2749–52.
11. Li B, Sharpe EE, Maupin AB, Teleron AA, Pyle AL, Carmeliet P, et al.
VEGF and PlGF promote adult vasculogenesis by enhancing EPC
recruitment and vessel formation at the site of tumor neovasculariza-
tion. FASEB J 2006;20:E664–73.
12. Takahashi A, Sasaki H, Kim SJ, Tobisu K, Kakizoe T, Tsukamoto T,
et al. Markedly increased amounts of messenger RNAs for vascular
endothelial growth factor and placenta growth factor in renal cell
carcinoma associated with angiogenesis. Cancer Res 1994;54:
4233–7.
13. Woo IS, Park MJ, Byun JH, Hong YS, Lee KS, Park YS, et al.
Expression of placental growth factor gene in lung cancer. Tumor
Biol 2004;25:1–6.
14. Zhang L, Chen J, Ke Y, Mansel RE, Jiang WG. Expression of placental
growth factor (PlGF) in non-small cell lung cancer (NSCLC) and the
clinical and prognostic significance. W J Surg Oncol 2005;3:69.
15. Kodama J, Seki N, Tokumo K, Nakanishi Y, Yoshinouchi M, Kudo T.
Placenta growth factor is abundantly expressed in human cervical
squamous cell carcinoma. Eur J Gynaecol Oncol 1997;18:508–10.
16. Matsumoto K, Suzuki K, Koike H, Hasumi M, Matsui H, Okugi H, et al.
Placental growth factor gene expression in human prostate cancer
and benign prostate hyperplasia. Anticancer Res 2003;23:3767–73.
17. Graeven U, Rodeck U, Karpinski S, Jost M, Andre N, Shmiegel W.
Expression patterns of placenta growth factor in human melanocytic
cell lines. J Invest Dermatol 2000;115:118–23.
18. Lacal PM, Failla CM, Pagani E, Odorisio T, Schietroma C, Falcinelli S,
et al. Human melanoma cells secrete and respond to placenta growth
factor and vascular endothelial growth factor. J Invest Dermatol
2000;115:1000–7.
19. Escudero-Esparza A, Martin TA, Douglas-Jones A, Mansel RE, Jiang
WG. PGF isoforms, PLGF-1 and PGF-2 and the PGF receptor,
neuropilin, in human breast cancer: prognostic significance. Oncol
Rep 2010;23:537–44.
20. Heath VL, Bicknell R. Anticancer strategies involving the vasculature.
Nature 2009;6:395–404.
21. Taylor AP, Goldenberg DM. Role of placenta growth factor in malig-
nancy and evidence that an antagonistic PlGF/Flt-1 peptide inhibits
the growth and metastasis of human breast cancer xenografts. Mol
Cancer Ther 2007;6:524–31.
22. Fischer C, Jonckx B, Mazzone M, Zacchigna S, Loges S, Pattarini L,
et al. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors
without affecting healthy vessels. Cell 2007;131:463–75.
23. Ebos JML, Lee CR, Christensen JG, Mutsaers AJ, Kerbel RS. Multiple
circulating proangiogenic factors induced by sunitinib malate are
tumor-independent and correlate with antitumor efficacy. Proc Natl
Assoc Sci U S A 2007;104:17069–74.
24. Ho MC, Chen CN, Lee H, Hsieh FJ, Shun CT, Chang CL, et al. Placenta
growth factor not vascular endothelial growth factor A or C can predict
the early recurrence after radical resection of hepatocellular carci-
noma. Cancer Lett 2007;250:237–49.
25. Taylor AP, Rodriguez M, Adams K, Goldenberg DM, Blumenthal RD.
Altered tumor vessel maturation and proliferation in placenta growth
factor-producing tumors: potential relationship to post-therapy tumor
angiogenesis and recurrence. Int J Cancer 2003;105:158–64.
26. Kopetz S, Hoff PM, Morris JS, Wolff RA, Eng C, Glover KY, et al. Phase
II trial of infusional fluorouracil, irinotecan, and bevacizumab for
metastatic colorectal cancer: efficacy and circulating angiogenic
biomarkers associated with therapeutic resistance. J Clin Oncol
2010;28:453–9.
27. Willet CG, Duda DG, di Tomaso E, Boucher Y, Ancukiewicz M, Sahani
DV, et al. Efficacy, safety, and biomarkers of neoadjuvant bevacizu-
mab, radiation therapy, and fluorouracil in rectal cancer: a multi-
disciplinary phase II study. J Clin Oncol 2009;27:3020–6.
28. Michaelson MD, Regan MM, Oh WK, Kaufman DS, Olivier K, Michael-
son SZ, et al. Phase II study of sunitinib in men with advanced prostate
cancer. Ann Oncol 2009;20:913–20.
29. Rini BI, Michaelson MD, Rosenberg JE, Bukowski RM, Sosman JA,
Stadler WM, et al. Antitumor activity and biomarker analysis of
sunitinib in patients with bevacizumab-refractory metastatic renal cell
carcinoma. J Clin Oncol 2008;26:3743–8.
30. Loges S, Schmidt T, Carmeliet P. "Antimyeloangiogenic" therapy for
cancer by inhibiting PlGF. Clin Cancer Res 2009;15:3648–53.
31. Bais C, Wu X, Yao J, Yang S, Crawford Y, McCutcheon K, et al. PlGF
blockade does not inhibit angiogenesis during primary tumor growth.
Cell 2010;141:166–77.
32. Van de Veire S, Stalmans I, Heindryckx F, Oura H, Tijeras-Raballand A,
Schmidt T, et al. Further pharmacological and genetic evidence for the
efficacy of PlGF inhibition in cancer and eye disease. Cell 2010;
141:178–90.
33. Bagley RG, Kurtzberg L, Weber W, Nguyen T-H, Lovewell G, Nguyen
C, et al. sFLT01: a novel fusion protein with anti-angiogenic activity.
Mol Cancer Ther 2010 Jan 20. [Epub ahead of print].
34. Pechan P, Rubin H, Lukason M, Ardinger J, DuFresne E, Hauswirth
WW, et al. Novel anti-VEGF chimeric molecules delivered by AAV
vectors of inhibition of retinal neovascularization. Gene Ther
2009;16:10–6.
35. Lukason M, DuFresne E, Rubin H, Pechan P, Li Q, Kim I, et al.
Inhibition of choroidal neovascularization in a non-human primate
model by intravitreal administration of an AAV2 vector expressing a
novel anti-VEGF molecule. Mol Ther 2010 Oct 26. [Epub ahead of
print].
36. Dalal S, Berry AM, Cullinane CJ, Mangham DC, Grimer R, Lewis IJ,
et al. Vascular endothelial growth factor: a therapeutic target for
tumors of the Ewing's sarcoma family. Clin Cancer Res 2005;
11:2364–78.
37. Liang W-C, Wu X, Peale FV, Lee CV, Meng YG, Gutierrez J, et al.
Cross-species vascular endothelial growth factor (VEGF)-blocking
antibodies completely inhibit the growth of human tumor xenografts
and measure the contribution of stromal VEGF. J Biol Cehm
2006;28:951–61.
38. Keck PJ, Hauser S, Krivi G, Sanzo K, Warren T, Feder J, et al. Vascular
permeability factor, an endothelial cell mitogen related to PDGF.
Science 1989:1309–12.
39. Persico MG, Vincenti V, DiPalma T. Structure, expression and recep-
tor-binding properties of placental growth factor (PlGF). Curr Top
Microbiol Immunol 1999;237:31–40.
40. Keyes KA, Mann L, Cox K, Treadway P, Iversen P, Chen YF, et al.
Circulating angiogenic growth factor levels in mice bearing human
tumors using Luminex multiplex technology. Cancer Chemother
Pharmacol 2003;51:321–7.
41. Parr C, Watkins G, Boulton M, Cai J, Jiang WG. Placenta growth factor
is over-expressed and has prognostic value in human breast cancer.
Eur J Cancer 2005;41:2819–27.
42. Pompeo E, Albonici L, Doldo E, Orlandi A, Manzari V, Modesti A, et al.
Placenta growth factor expression has prognostic value in malignant
pleural mesothelioma. Ann Thorac Surg 2009;88:426–31.
43. Wei SC, Liang JT, Tsao PN, Hsieh FJ, Yu SC, Wong JM. Preoperative
serum placenta growth factor level is a prognostic biomarker in
colorectal cancer. Dis Colon Rectum 2009;52:1630–6.
44. DePrimo SE, Bello CL, Smeraglia J, Baum CM, Spinella D, Rini BI,
et al. Circulating protein biomarkers of pharmacodynamic activity
of sunitinib in patients with metastatic renal cell carcinoma: modula-
tion of VEGF and VEGF-related proteins. J Transl Med 2007;5:32–
43.
PlGF Upregulation Is a Host Response
www.aacrjournals.org Clin Cancer Res; 17(5) March 1, 2011 987
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
45. Tew WP, Gordon M, Murren J, Dupont J, Pezzulli S, Aghajanian C, et al.
Phase I study of aflibercept administered subcutaneously to patients
with advanced solid tumors. Clin Cancer Res 2010;16:358–66.
46. Motzer R, Michaelson M, Redman B, Hudes GR, Wilding G, Figlin RA,
et al. Activity of SU11248, a multitargeted inhibitor of vascular
endothelial growth factor receptor and platelet-derived growth factor
receptor, in patients with metastatic renal cell carcinoma. J Clin Oncol
2006;24:16–24.
47. Hattori K, Heissig B, Wu Y, Dias S, Tejada R, Ferris B, et al. Placental
growth factor reconstitutes hematopoiesis by recruiting VEGFR1þ
stem cells from bone-marrow microenvironment. Nat Med
2002;8:841–49.
48. MarcelliniM, De Luca N, Riccioni T, CiucciA, Orecchia A, Lacal PM,et al.
Increased melanoma growth and metastasis spreading in mice over-
expressing placental growth factor. Am J Pathol 2006;169:643–54.
49. Ebos JML, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG,
Kerbel RS. Accelerated metastasis after short-term treatment with a
potent inhibitor of tumor angiogenesis. Cancer Cell 2009;15:232–39.
50. Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals F,
et al. Antiangiogenic therapy elicits malignant progression of tumors
to increased local invasion and distant metastasis. Cancer Cell
2009;15:220–31.
51. Ellis LM, Hicklin DJ. Resistance to targeted therapies: refining antic-
ancer therapy in the era of molecular oncology. Clin Cancer Res
2009;15:7471–8.
52. Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature
2005;438:967–74.
53. Shojaei F, Ferrara N. Role of the microenvironment in tumor growth
and in refractoriness/resistance to anti-angiogenic therapies. Drug
Resist Updat 2008;11:219–30.
Bagley et al.
Clin Cancer Res; 17(5) March 1, 2011 Clinical Cancer Research
988
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
2011;17:976-988. Published OnlineFirst February 22, 2011.Clin Cancer Res
Rebecca G. Bagley, Yi Ren, William Weber, et al.
Antiangiogenic Therapy
Placental Growth Factor Upregulation Is a Host Response to
Updated version
10.1158/1078-0432.CCR-10-2687doi:
Access the most recent version of this article at:
Cited articles
http://clincancerres.aacrjournals.org/content/17/5/976.full.html#ref-list-1
This article cites 50 articles, 19 of which you can access for free at:
Citing articles
/content/17/5/976.full.html#related-urls
This article has been cited by 5 HighWire-hosted articles. Access the articles at:
E-mail alerts related to this article or journal.Sign up to receive free email-alerts
Subscriptions
Reprints and
.pubs@aacr.orgDepartment at
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Permissions
.permissions@aacr.orgDepartment at
To request permission to re-use all or part of this article, contact the AACR Publications
Research.
on June 14, 2017. © 2011 American Association for Cancerclincancerres.aacrjournals.org Downloaded from
Published OnlineFirst February 22, 2011; DOI: 10.1158/1078-0432.CCR-10-2687
