Download Pericytes Promote Malignant Ovarian Cancer Progression in Mice

Published OnlineFirst November 20, 2015; DOI: 10.1158/1078-0432.CCR-15-1931
Clinical
Cancer
Research
Biology of Human Tumors
Pericytes Promote Malignant Ovarian Cancer
Progression in Mice and Predict Poor Prognosis in
Serous Ovarian Cancer Patients
€ nchgesang1,
€ ter1,4, Susann Mo
Devbarna Sinha1,2, Lynn Chong1, Joshy George3, Holger Schlu
1
2
5
3,4,6,7
Stuart Mills , Jason Li , Christopher Parish , David Bowtell
, for the
Australian Ovarian Cancer Study Group, and Pritinder Kaur1,4,8
Abstract
Purpose: The aim of this study was to investigate the role of
pericytes in regulating malignant ovarian cancer progression.
Experimental Design: The pericyte mRNA signature was used
to interrogate ovarian cancer patient datasets to determine its
prognostic value for recurrence and mortality. Xenograft models
of ovarian cancer were used to determine if co-injection with
pericytes affected tumor growth rate and metastasis, whereas coculture models were utilized to investigate the direct effect of
pericytes on ovarian cancer cells. Pericyte markers were used to
stain patient tissue samples to ascertain their use in prognosis.
Results: Interrogation of two serous ovarian cancer patient
datasets [the Australian Ovarian Cancer Study, n ¼ 215; and the
NCI TCGA (The Cancer Genome Atlas), n ¼ 408] showed that a
high pericyte score is highly predictive for poor patient prognosis.
Co-injection of ovarian cancer (OVCAR-5 & -8) cells with
pericytes in a xenograft model resulted in accelerated ovarian
tumor growth, and aggressive metastases, without altering tumor
vasculature. Pericyte co-culture in vitro promoted ovarian cancer
cell proliferation and invasion. High aSMA protein levels in
patient tissue microarrays were correlated with more aggressive
disease and earlier recurrence.
Conclusions: High pericyte score provides the best means to
date of identifying patients with ovarian cancer at high risk of
rapid relapse and mortality (mean progression-free survival time
< 9 months). The stroma contains rare yet extremely potent locally
resident mesenchymal stem cells—a subset of "cancer-associated
fibroblasts" that promote aggressive tumor growth and metastatic
dissemination, underlying the prognostic capacity of a high
pericyte score to strongly predict earlier relapse and mortality.
Introduction
tion (1). Despite aggressive surgical intervention combined with
chemotherapeutic and platinum/paclitaxel treatment to eliminate residual cancer, 60% to 70% of late-stage ovarian cancer
patients relapse with recurrence, dying within 2 years of treatment.
The predominant diagnosis of ovarian cancer at advanced stages is
attributed largely to asymptomatic spread of disease in the peritoneal cavity combined with symptoms confused with other
innocuous gastrointestinal effects (e.g., bloating, discomfort,
indigestion, and pelvic pain). Thus, an increased understanding
of the critical biological changes underlying the development and
progression of ovarian cancer, aside from notable genetic changes,
is critical to the success of earlier diagnosis and design of novel
therapeutic interventions to reduce high mortality rates associated
with ovarian cancer.
The tumor microenvironment (TME) or cancer-associated stroma, including cancer-associated fibroblasts (CAF), bone marrow–
derived mesenchymal stem cells (BM-MSC), endothelial cells,
pericytes, and immune components plus the growth factors and
extracellular matrix proteins they produce, regulates tumor cell
dissemination and metastases. Ovarian cancer is classified into
distinct histopathological subtypes, that is, high-grade and lowgrade serous, endometroid, clear cell, and mucinous and transformed cells with low malignant potential (LMP) reminiscent of
the anatomy of origin (2). High-grade serous ovarian cancer, often
diagnosed after metastatic spread into the abdominal cavity and
omentum (1), is distinguished by a highly mitotic, stratified
epithelium, with reactive stroma (2–4) making it an ideal model
to study epithelial–stromal interactions in the TME.
Ovarian cancer is the most life-threatening gynecologic cancer,
with very high rates of recurrence and mortality following diagnosis. Statistics from the United States (NCI) and United Kingdom (CRUK)—countries with some of the highest rates of ovarian
cancer—show that although diagnosis at stage 1 at a young age is
associated with approximately 90% survival, the overwhelming
majority of patients with ovarian cancer (85%) are diagnosed at
advanced stages of disease (3 and 4), after metastatic dissemina1
Epithelial Stem Cell Biology Laboratory, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia. 2Bioinformatics Core Facility,
Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.
3
Cancer Genetics & Genomics Laboratory, Peter MacCallum Cancer
Centre, Melbourne, Victoria, Australia. 4Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Victoria,
Australia. 5The John Curtin School of Medical Research, Australian
National University, Canberra, Australia. 6Department of Pathology,
University of Melbourne, Parkville, Victoria, Australia. 7Department of
Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia. 8Department of Anatomy and Neuroscience,
University of Melbourne, Parkville, Victoria, Australia.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Pritinder Kaur, Hudson Institute of Medical Research,
27-31 Wright Street, Clayton, Melbourne, Victoria 3168, Australia. Phone: 61-39594-4398; Fax: 61-3-9594-7114; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-15-1931
2015 American Association for Cancer Research.
Clin Cancer Res; 1–12. 2015 AACR.
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Sinha et al.
Translational Relevance
There is an urgent clinical need to identify those serous
ovarian cancer patients at greater risk of earlier recurrence,
relapse, and mortality at diagnosis despite similar treatment.
Although the predictive capacity of tumor-associated stroma
gene expression is well recognized in these patients, we report
that this can be highly refined into those that succumb to
relapse and mortality in less than 9 months versus those that
respond better to current treatment modalities (surviving for
up to 29 months) on the basis of stromal heterogeneity—
specifically by using the molecular signature of human stem
cell–like pericytes rather than fibroblasts. Although ovarian
cancer tends to be diagnosed at advanced stages and thus
carries inherently poor prognosis, it would be of great benefit
to select those patients at significantly higher risk for recurrence and mortality for more aggressive or alternate treatments
to increase their chances of survival.
Stromal signatures strongly predict relapse in ovarian, colorectal, pancreatic, and breast cancers (5–9), contributing to the
prevailing view that the TME is pro-tumorigenic. Although sufficient evidence exists to support the notion that recruitment of a
cooperative stroma is essential for malignant progression, it is
equally plausible that the influx of some stromal elements is the
body's attempt to limit cancer spread through fibroblastic encapsulation typical of many tumor types. Notably, the TME has been
implicated in contributing at least partially to resistance against
cancer therapeutic reagents (10), but also in enhancing therapeutic efficacy depending on context [see ref. (11) for review].
It is, therefore, vital that the functional heterogeneity and
diverse origins of the tumor stroma are more fully mapped. It is
well known that CAFs isolated from cancers promote tumor
growth, invasiveness, and metastasis of many cancers, notably
breast, prostate, and pancreatic carcinomas (12–14) compared
with normal fibroblasts. However, the postulated origins of CAFs
include many normal stromal cells, including tissue resident
myofibroblasts, activated adipocytes, and BM-MSCs (13, 15)—
the latter representing perhaps the best defined source of CAFs.
However, BM-MSCs that are home to developing tumors inducing
increased metastases comprise approximately 20% of CAFs
(16–20), leaving some 50% to 80% of CAFs that are not BMderived and may arise from locally resident fibroblasts and
presumably MSC-like populations such as pericytes that form
the focus of this study.
Pericytes are best known for regulating endothelial cell proliferation, differentiation, and microvascular perfusion/permeability through paracrine regulators such as TGF-b and vasoactive
agents (21, 22), and are identified as a-smooth muscle actin
(a-SMA)–positive, contractile cells located abluminally in microvessels. It is increasingly evident that pericytes and BM-MSCs share
many phenotypic and functional attributes, including multilineage differentiation capacity, and may have a pro-proliferative role
in organ growth, repair, and regeneration (23–25). Notably, we
have previously demonstrated that pericytes promote normal
epithelial cell proliferation and regeneration in the absence of
angiogenesis, most likely through the secretion of the LAMA5
isoform of laminin (26). In the context of cancer, targeting both
OF2 Clin Cancer Res; 2016
endothelial cells and pericytes through kinase inhibitors of VEGF,
which promotes blood vessel growth, and PDGF-B, which promotes proliferation and survival of pericytes (27), improved the
efficacy of anti-cancer therapies in animal models (28–31), attributed to destabilizing microvascular structure (27). Although
subsequent studies claimed to show unaffected tumor growth
following pericyte ablation, complete pericyte knockdown was
not achieved by treatment with AX102—an inhibitor of PDGF-B
signaling (32), or in PDGFBret/ret mice harboring a mutation in the
PDGF-B retention motif (33) with only partial decrease in tumor
vasculature and pericyte number. A maximal 50% reduction in
pericyte number is reached in PDGFBret/ret mice (34). In contrast,
comprehensive knockdown of pericytes via NG2-promoter–driven thymidine kinase results in tumor hypoxia, leading to epithelial–mesenchymal transition (EMT) and increased metastatic
lung dissemination in mouse models of breast cancer, melanoma,
and renal cell carcinoma (35). In fact, retaining pericytes within
tumor blood vessel walls may limit metastatic spread through
leaky blood vessels (36).
Thus, arguments to support both tumor growth–limiting and
metastasis-promoting roles have been made for pericytes and
most likely depend on the nature of the experimental model used.
In this study, we show that the molecular signature of pericytes is
highly predictive for patient relapse and mortality in high-grade
serous ovarian cancer patients, demonstrating our ability to
identify with a very high degree of certainty those patients who
die in less than 9 months, despite aggressive treatment at diagnosis, suggesting a potent pro-tumorigenic/pro-metastatic role for
pericytes in ovarian cancer progression. Consistent with this
clinical correlate, we experimentally demonstrate that MSC-like
pericytes are potent stimulators of both poorly and highly tumorigenic ovarian cancer cell lines when introduced into the TME in a
xenograft model, accelerating tumor growth rates and earlier
metastasis in aggressive ovarian cancer cells, but also inducing
metastasis in nonmetastatic cell lines without affecting tumor
vasculature. These influences in malignant ovarian cancer cell
proliferation, migration, and invasion were also demonstrable in
vitro using co-culture models, further indicating a novel and as yet
unappreciated function for pericytes in malignant progression.
Materials and Methods
In silico analyses
Bioinformatics analyses for predicting prognosis based on the
pericyte signature and gene set enrichment are described in the
Supplementary data.
Cell culture
CD45VLA-1bri pericytes and CD45VLA-1dim fibroblasts were
isolated from human neonatal foreskin, as described (26) and
expanded in culture up to passage 4 (p4) (24). Fibroblasts were
maintained in DMEM with 10% FCS, and pericytes in EGM–2
(Lonza; # CC-4147); OVCAR-5 and OVCAR-8 cells obtained from
NCI were authenticated using short tandem repeat markers
to confirm cell identity against the Genome Project Database
(Wellcome Trust Sanger Institute) and were maintained in RPMI
1640 (Invitrogen; #11875) with 10% FCS, 25 mmol/L HEPES
buffer, 1% penicillin-streptomycin, and 1.5% Diflucan, and
HEK293T cells in DMEM with 10% FCS, 1% L-glutamate (2
mmol/L), and 1% sodium pyruvate (1 mmol/L) without antibiotics for lentiviral production. OVCAR-5 cells and pericytes
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Pericytes Promote Metastasis and Predict Relapse
were transduced with GFP-luciferase, as described in the Supplementary data. All human tissue experimentation was approved
by the Peter MacCallum Human Research Ethics Committee
(#03/44).
Statistical analysis
Data analyses were performed using the Prism 6.0 (Graphpad
software) or R software. Pooled data were represented as mean SD, unless otherwise indicated.
Animals
Six to eight week old female nude Balb/c mice (WEHI), housed
in a pathogen-free 12-hour light–dark environment, fed ad libitum
were used for tumorigenicity assays. All experimentation was
approved by the Peter MacCallum Animal Research Ethics
Committee (# E394).
Results
Tumorigenicity assay
OVCAR-5 cells (8 106) 10% fibroblasts or pericytes in 100
mL of 1:1 sterile PBS and standard Matrigel (BD Biosciences) were
injected subcutaneously (s.c.) into the flanks of mice. Five mice
were injected/group; all experiments performed in triplicate.
Immunostaining and morphometric analyses, invasion assays
All procedures and antibodies used are described in the
Supplementary data.
Luciferase imaging
Metastatic spread and pericyte survival in vivo were monitored
using the Xenogen Real-Time Imaging System. D-luciferin (Gold
Biotechnology Inc.), a substrate for the luciferase enzyme, was
injected s.c. at 150 mg/g body weight in PBS. Mice were allowed free
movement for 6 to 8 minutes, anaesthetized with isofluorane, and
imaged within 10 to12 minutes of luciferin injection. Bioluminescent imaging (BLI) was repeated every 7 days to track metastatic
spread until an experimental or ethical endpoint was reached, from
d14 when primary tumors were palpable. At the endpoint, as the
luminescent signal from primary tumors was saturated potentially
masking signal from smaller metastatic nodules, primary tumors
were carefully excised after sacrifice, and the peritoneum opened
surgically and imaged with increased exposure times to improve
visualization of metastases. Metastatic burden was quantified and
organs harvested for histology/GFP staining.
Vascular permeability assay
A volume of 100 mL of 10 mg/mL FITC-dextran (2,000,000
MW, Sigma) was injected into the tail vein an hour before sacrifice.
Tumors were collected, snap-frozen in liquid nitrogen, and cryosections co-stained with the endothelial marker CD31 for fluorescence microscopy analysis.
Tissue microarrays
Patient tissue microarrays (TMA) consisting of 4-mm cores of
formalin-fixed, paraffin-embedded, high-grade serous ovarian
cancer biopsy tissues were obtained from the Australian Ovarian
Cancer Study (AOCS) approved by the AOCS review board.
In-vitro proliferation assays
Co-culture proliferation assays were performed in a 6-well
format. GFPþOVCAR-5 (2 104) cells alone or with 2 104
p4 pericytes or p4 fibroblasts were mixed and seeded in either 1%
or 10% FBS epidermalization medium. Plates were incubated at
37 C, in 5% CO2 for 24, 48, and 72 hours, fixed in 4% paraformaldehyde (w/v), and immunostained for GFP to determine the
number of OVCAR-5 cells over time.
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The transcriptional profile of pericytes predicts significantly
earlier relapse and mortality in high-grade serous ovarian
cancer patients
The AOCS Group showed that high-grade serous ovarian cancer
patients with a stromal signature had a poor clinical outcome (5),
as reported for breast cancer patients (6). Previously we demonstrated that MSC-like pericytes had high potency in increasing
epithelial proliferative capacity compared with fibroblasts (26),
so we used the molecular signature of these two distinct stromal
cell types (26), to compare their predictive capacity for clinical
outcome in the AOCS ovarian cancer patient dataset in silico, using
the AOCS ovarian cancer stromal signature as a reference (5).
Notably, the pericyte-specific signature or high pericyte score was
a potent predictor of rapid relapse and mortality (P ¼ 0.00067;
Kaplan–Meier plots Fig. 1A), identifying those patients with a
mean progression-free survival (PFS) time of 9 months or less
versus those with a low pericyte score (mean PFS time of 29
months) despite similar treatment, as compared with the AOCS
ovarian cancer stromal signature (P ¼ 0.0011; ref. 5) and the
normal fibroblast signature (P ¼ 0.01).
Analysis of genes co-expressed by laser-capture micro-dissected
ovarian CAFs in the AOCS study and normal pericytes revealed
146 genes, including well-known pericyte markers (PDGFRb,
ACTA2, RGS5, CALD1, MCAM, and ANGPT1) (26), growth
factors, adhesion ligands and receptors (FGF/FGFRs, Tenascin
C, LAMA3, LAMA5, CSPG-4, the VLA-1, VLA-3 and VLA-7 integrins), BMPs, and Notch pathway signaling genes (Supplementary
Table S1), linked to CAFs and ovarian cancer progression (4).
Importantly, minimal overlap was detected with the Gene Ontology classification angiogenesis signature (GO Angiogenesis—
GO:0001525) with our pericyte signature, given that pericytes
stabilize tumor vasculature, with only two common genes (angiopoietin 1 and 2). Moreover, no overlap was present between the
recently described angiogenic signatures (37), suggesting that the
significantly earlier relapse observed in patients with a high
pericyte score was unrelated to angiogenesis. An interrogation of
the NCI TCGA (The Cancer Genome Atlas) ovarian cancer patient
dataset further confirmed the ability of the pericyte signature
to predict decreased survival in a group of 408 patients
(Fig. 1B; P ¼ 0.008), leading us to investigate whether pericytes
could promote ovarian tumor growth experimentally without
affecting angiogenesis.
Pericytes accelerate ovarian tumor growth in vivo
OVCAR-5 cells derived from a serous ovarian cancer patient
with metastatic disease before treatment with anti-cancer agents
(38) were used as an ovarian cancer model. GFP-luciferaseþ
OVCAR-5 cells resuspended in Matrigel were injected s.c.
in nude mice, either alone (OVCAR-5) or with pericytes
(OVCAR-5þP) or fibroblasts (OVCAR-5þF), at a 10:1 tumor:
stromal cell ratio. Pericyte co-injection consistently led to
accelerated tumor growth compared with OVCAR-5 or
OVCAR-5þF (Fig. 1C; P < 0.0001; n ¼ 5 independent experiments), and increased endpoint tumor volumes (and mass) at
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day 35 (Fig. 1D; P < 0.0001); OVCAR-5þP tumors reached
200 mm3 4 to 5 days earlier. Furthermore, a dose-dependent
effect was observed when the proportion of pericytes was
increased from 10% to 50%, keeping the number of OVCAR5 cells constant, with greater endpoint tumor volumes than
OVCAR-5 controls in both the 10:1 and 1:1 pericyte co-injected
groups (P < 0.05 and P < 0.01, respectively; Fig. 1E).
Dual staining for epithelial-specific EpCam and Ki67 (with
anti-human specific antibodies) revealed a direct increase in
OVCAR-5 cell proliferation, i.e., number of EpCamþ/Ki67þ cells
compared with OVCAR-5–only controls and OVCAR-5þF tumors
(P < 0.0001 and P < 0.01 respectively; Fig. 1F). No difference in
apoptotic index was observed between experimental groups at
day 11 or day 35 by staining for cleaved caspase-3 (CC3; Supplementary Fig. S1A–S1H), excluding decreased apoptosis in
increasing tumor size. Moreover, in vitro experiments showed
that pericyte co-cultured GFPþOVCAR-5 cells displayed increased
proliferation compared with controls within 72 hours in both 1%
(P ¼ 0.0349) and 10% serum (P ¼ 0.0328), not seen in fibroblast
co-cultures (Supplementary Fig. S2A–S2C).
Injected pericytes persist but do not proliferate or contribute to
angiogenesis in OVCAR-5 tumors
BLI of xenografts generated with unlabeled OVCAR-5 cells
and GFP-luciferaseþ–tagged pericytes permitted pericyte-tracking
in developing tumors. Whilst control animals (OVCAR-5 cells
alone) gave no signal despite luciferin injection (Fig. 2A),
pericytes persisted within co-injected tumors at all time points
analyzed (Fig. 2B). Histological analyses revealed single GFPþ
pericytes in OVCAR-5þP tumors, declining in numbers over time
(Fig. 2C). Notably, GFPþ pericytes were Ki67-negative at all time
points, indicating that they did not proliferate during tumorigenesis (Fig. 2C).
We next addressed whether co-injected pericytes accelerated
tumor growth by increasing or stabilizing tumor vasculature. The
area of CD31þ blood vessels in tumors remained unaltered both
B
A
C
High-grade serous ovarian cancer samples stratified into two groups based on the expression of pericyte genes
AOCS
1.0
Tumor volume (mm3)
0.8
0.6
0.4
Probability of survival
Low pericyte score
High pericyte score
OVCAR-5
OVCAR-5+F
OVCAR-5+P
1,000
500
0.0
0.2
Probability of survival
0.2 0.4 0.6 0.8 1.0
0.0
Low pericyte score
High pericyte score
50
100
150
Time to relapse (months)
log-rank test P value: 0.00067
0
10
20
30
40
50
E
5
10 15 20 25 30
Days
35 40
(1
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1)
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+P
(1
P
200
ns
150
100
50
0
OVCAR-5+F OVCAR-5+P
A
R
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OVCAR-5
C
O
V
V
C
A
O
1)
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-5
+P
R
-5
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-5
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500
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A
1,500
0
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O
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ns
60
Time to relapse (months)
log-rank test P value: 0.00803
Day 35 tumor volume (mm3)
D
200
#Epcam+Ki67+ cells per field
(day 35)
0
Day 35 tumor volume (mm3)
1,500
TCGA
Figure 1.
The pericyte-specific gene signature predicts poor prognosis in patients with ovarian cancer, and co-injection of OVCAR-5 ovarian cancer cells with pericytes
accelerates tumor cell proliferation and tumor volume. Kaplan–Meier curves showing a significantly poorer PFS rate among 215 high-grade serous ovarian cancer
patients with a high score of pericyte-specific genes in the AOCS dataset (A) and the NCI TCGA patient dataset (B). C, nonlinear regression fit of tumor volumes
6
against time generated from the injection of 8 10 OVCAR-5 cells alone or co-injected at a ratio of 10:1 with pericytes (OVCAR-5þP) or fibroblasts (OVCAR-5þF).
Data represented as mean tumor volume SD of 26 mice per group from 5 independent experiments. Repeated measure data for each time point were
compared using two-way ANOVA. D, quantification of endpoint tumor volumes at day 35 represented as mean tumor volume SEM of 5 independent
experiments, calculated from data shown in A. E, quantification of endpoint tumor volume at day 35, demonstrating the dose effect of increasing the
6
number of pericytes on OVCAR-5 tumor growth, i.e., injection of 5 10 OVCAR-5 cells with or without pericytes at a ratio of 10:1 and 1:1. Data are shown
as mean tumor volume SEM of 10 mice per group from 2 independent experiments. Statistical analysis in E and F performed using one-way ANOVA.
F, quantification of dual immunofluorescent staining with anti-human specific antibodies to the proliferation marker Ki67 and the epithelial marker EpCam
of ovarian tumors generated by OVCAR-5 cells alone or co-injected with pericytes—OVCAR-5þP and fibroblasts, OVCAR-5þF showing a significant increase
þ
þ
in the number of Ki67 /EpCam tumor cells in the OVCAR-5þP group. Data are shown as mean SEM from 3 tumors per group from 2 independent experiments.
Statistical analyses performed by one-way ANOVA. , P < 0.05; , P < 0.01; , P < 0.0001, ns, not significant.
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Pericytes Promote Metastasis and Predict Relapse
Figure 2.
Co-injected human pericytes survive but do not proliferate in ovarian tumors in vivo. A and B, representative BLI images of mice with unlabeled OVCAR-5 tumors
or OVCAR-5þGFP-luciferase tagged pericytes imaged at days 6–35. BLI imaging conducted in 3 mice/group/time point in 2 replicate experiments. C, dual
þ
þ
immunofluorescent staining for GFP pericytes (green) and Ki67 proliferating cells (red) in days 6–35 pericyte co-injected tumors, showing decline in
þ
þ
pericyte numbers over time and absence of Ki67 GFP pericytes. Images are representative of three random fields from 3 tumors per experimental group
þ
from 2 independent experiments. D, dual immunofluorescent staining for CD34 and GFP pericytes, illustrating that injected pericytes do not incorporate into
host blood vessels. (P ¼ pericytes; BV ¼ blood vessels). Immunostaining is representative of multiple sections per mouse. Scale bar ¼ 25 mm.
in the center and in the edges of the OVCAR-5þP tumors compared with controls (Fig. 3A and B) at day 11 (Fig. 3C; P ¼ 0.1879),
confirmed further by measuring microvessel density (MVD)
(Fig. 3D; P ¼ 0.8910). The CD31þ blood vessel area (Fig. 3E;
P ¼ 0.2021) and MVD remained unaffected at day 35 (Fig. 3F;
P ¼ 0.7790).
Furthermore, we could not find any differences in the aSMAþ
pericyte coverage index (MPI) of CD34þ microvessels between
OVCAR-5þP and OVCAR-5 controls (Fig. 3G: P ¼ 0.5321),
indicating that pericyte inclusion did not alter the structural
stability of OVCAR-5 tumor vasculature.
Similar analyses of microvessels in clinical samples, i.e., TMAs
of serous ovarian cancer patients, demonstrated that CD34þ
expression in tumor microvessels did not correlate with time to
relapse or survival (Fig. 3H and I), providing independent verification that poor prognosis predicted by the pericyte signature
had minimal overlap with the angiogenic signature.
Finally, vascular permeability determined by injecting FITCconjugated dextran into tumor-bearing mice an hour before
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sacrifice followed by analysis of tumor cryosections co-stained
for CD31 and FITC-dextran showed no differences between
control and pericyte co-injected OVCAR-5 tumors with minimal
FITC-dextran signal outside the vessels at day 11 and day 35 (Fig.
3J). These data were consistent with the observation that GFPtagged pericytes did not associate with CD34þ blood vessels (Fig.
2D) in OVCAR-5 xenografts, but were "stroma-associated." These
data strongly suggest that the tumor-promoting action of pericytes
is not mediated by affecting tumor angiogenesis directly or
indirectly.
Pericytes promote aggressive invasion in OVCAR-5 cells in vitro
and in vivo
At harvest pericyte co-injected xenografts appeared macroscopically different with indistinct tumor margins indicative of
outgrowths. Histological analysis confirmed the presence of
invasive nodules of cells at the tumor edges as early as day 6
in the OVCAR-5þP group compared with controls (Fig. 4A)
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Figure 3.
Tumor-promoting activity of pericytes is independent of angiogenesis. A and B, CD31 staining of OVCAR-5 and OVCAR-5þP tumors shows no difference at the
tumor center or tumor edge at day 35. Scale bar ¼ 100 mm. C and E, quantification of combined tumor center and edge CD31 staining at day 11 or day 35; and
D and F, MVD per field at day 11 and day 35. G, quantification of MPI in day 11 tumors. Data shown as mean SEM from 3 random fields from 5 tumors/group,
from 2 independent experiments. H, CD34 staining of representative patient TMAs from early-, late-, or no-relapse groups. Scale bar ¼ 200 mm. I, quantification
of CD34 staining from patient biopsy samples versus relapse (n ¼ 7 patients/relapse group; 3 fields/patient). Statistical differences analyzed by one-way
ANOVA. J, FITC-dextran and CD31 (red) staining at day 11 and day 35 in control OVCAR-5 and OVCAR-5þP tumors. Scale bar ¼ 20 mm.
GFP-immunostaining showed clear encapsulation with GFP
stromal cells in control tumors, while invasive nodules of
GFPþOVCAR-5 cells were present at the tumor margins of
OVCAR-5þP tumors (Fig. 4B).
Moreover, in vitro Boyden chamber migration assays confirmed
that co-culture of OVCAR-5 cells with pericytes increased both
migration (2–3 fold; P < 0.05; data not shown) and invasion
through Matrigel and an 8-mm filter membrane (Fig. 4C; P < 0.05),
while fibroblasts had no significant effect (Fig. 4C).
Pericytes promote aggressive ovarian cancer metastases to
distant organs in OVCAR-5 and OVCAR-8 cells
These data led us to examine whether pericytes facilitated
metastases in xenografts—BLI analysis of GFP-luciferaseþOVCAR-5 tumors in vivo tracked at regular intervals revealed
metastatic spread of ovarian cancer cells to the peritoneal cavity as
early as day 21 in OVCAR-5þP mice (Fig. 4D). By day 28,
metastases associated with the intestine, liver, and lung were
detected in these mice (Fig. 4E), whereas control mice
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(OVCAR-5 and OVCAR-5þF injected) were completely free of
metastases (Fig. 4E and F). Moreover, a dose-dependent effect on
metastatic burden was demonstrable at day 28—increasing the
OVCAR-5 cell:pericyte ratio from 10:1 to 1:1 resulted in increased
metastases to distant organs (Fig. 4G, P < 0.05), achieving strong
statistical significance over OVCAR-5 controls (Fig. 4G; P < 0.001).
By day 42, extensive local metastases were evident throughout
the peritoneal cavity associated with the upper and lower gastrointestinal tracts in both control and OVCAR-5þP groups macroscopically (Supplementary Fig. S3A–S3D), and on GFPþ staining
of tissue sections (Supplementary Fig. S3J), confirming that they
were derived from GFPþOVCAR-5 cells. However, more extensive
metastases were evident in distant organs such as the liver, spleen,
kidney, and lung macroscopically (Supplementary Fig. S3A–S3D;
day 42) and by GFP-immunostaining (Supplementary Fig. S3E–
S3I; day 35), in the pericyte–co-injected group only.
We next tested whether pericytes could affect the less aggressive
OVCAR-8 cell line derived from an early-stage cisplatin-treated
patient reported to form noninvasive tumors with long periods of
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Pericytes Promote Metastasis and Predict Relapse
A
OVCAR-5
OVCAR-5+P
OVCAR-5
OVCAR-5+P
Day 21
D
B
GFP
OVCAR-5+P
H&E
OVCAR-5
P < 0.05
Fold invasion
4
E
3
2
1
0
OVCAR-5
10
5
O
-5
AR
VC
VC
AR
-5
O
+P
+P
(1
(1
0:
:1
)
1)
0
O
OVCAR-5+F
AR
OVCAR-5
15
VC
F
OVCAR-5+F OVCAR-5+P
-5
G
Mean number of metastastic foci
per mouse (day 28)
Day 28 - primary tumor excised before imaging
C
Figure 4.
Pericytes promote invasion and metastasis of OVCAR-5 cells. A, H&E staining of invasive nodules in OVCAR-5þP tumor edges at day 11 compared with the smooth
þ
margins of controls. B, GFP staining showing single GFP OVCAR-5 cells at the edge of OVCAR-5þP tumors (blue arrow) compared with controls encapsulated within
a GFP stromal lining (red arrows). Scale bar ¼ 100 mm. Images in A and B are representative of 12 to 15 tumors per experimental group from 3 independent
experiments. C, quantification of Transwell invasion through Matrigel towards pericytes or fibroblasts normalized to OVCAR-5 control. Mean SD from 3
þ
þ
independent experiments. D, representative BLI images of nude mice carrying control GFP-luciferase OVCAR-5 and GFP-luciferase OVCAR-5þP co-injected
tumors at day 21, indicating the position of primary tumor. E, BLI at day 28 after sacrifice and surgical excision of primary tumor and exposing organs in nude
mice, indicating metastasis on co-injection of pericytes. F, BLI at day 28 in nude mice injected with OVCARþF after sacrifice and removal of primary tumors revealing
absence of metastases. Images in D–F are representative of 10 mice per experimental group from 2 independent experiments. G, quantification of metastatic
burden calculated as the mean number of bioluminescent metastatic foci at the same exposure time after primary tumor removal in nude mice injected with
OVCAR-5 (control), 10:1, or 1:1 OVCAR-5:P cells. Data are mean SEM of 10 mice/group from 2 independent experiments. Statistical analysis performed
by one-way ANOVA. , P < 0.05; , P < 0.0001.
latency (39, 40). Co-injection of OVCAR-8 cells with pericytes
(10:1 ratio) into nude mice resulted in a 15-day decrease in
latency of tumor formation, accelerated tumor growth (Supplementary Fig. S4A), and larger tumor volumes (Supplementary
Fig. S4B: P < 0.0001). Notably, while GFP-luciferaseþOVCAR-8
cells did not yield metastases by themselves, pericyte co-injection
led to OVCAR-8 metastasis to distal organs, i.e., liver, lung,
bladder, kidney, in addition to the GI tract, peritoneum and
omentum (Supplementary Fig. S4C–S4I: GFPþ immunostaining). These data clearly demonstrate the potent ability of pericytes
to confer malignancy on nonmetastatic ovarian cancer cells.
www.aacrjournals.org
Interestingly, bioinformatic analyses of gene expression enrichment in the AOCS high-grade serous ovarian cancer patients
revealed that early-relapse patients identified by a high pericyte
score displayed upregulation of molecular pathways, involving
matrix degradation, ECM remodeling, negative regulation of
cell adhesion, invasion, and migration, compared with those
patients with late relapse, using two independent methods
(i.e., enrichment analysis of GO terms or KEGG pathways among
overexpressed genes or using Gene Set Enrichment Analysis;
Supplementary Table S2), providing a clinical correlate for our
experimental findings.
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A
OVCAR-5+P
B
C
D
E
αSMA /CD34 (day 11)
F
Late relapse
No relapse
αSMA
Early relapse
Number of stroma-associated
αSMA+ cells/mm 3
H
G
20
40
60
80
Time to relapse (months)
log-rank test P value: 0.03691
0
20
40
60
80
Time to relapse (months)
log-rank test P value: 0.006652
K
0.2
0.3
αSMA
0.1
Ratio (stained area: total area)
0.6
0.8
1.0
High αSMA expression
Low αSMA expression
0.0
Overall survival (αSMA)
0.0
0.2
0.4
0.6
0.8
Probability of survival
High αSMA expression
Low αSMA expression
0.0
0
J
0.4
1.0
Progression-free survival (αSMA)
0.2
I
Probability of survival
Number of stroma-associated
αSMA+ cells/mm 3
Number of vessel-associated
α SMA + cells/mm3
Day 35
αSMA
Day 11
OVCAR-5
–2
–1
0
1
2
3
Pericyte score
P value: 0.0078
Figure 5.
þ
þ
Pericytes increase recruitment of aSMA cells to ovarian tumors in mice; increase in aSMA cells correlates with early relapse in patients with ovarian cancer.
þ
A, aSMA staining in OVCAR-5 and OVCAR-5þP tumors at day 11 and day 35. Quantification of total aSMA cells at day 11 (B) and day 35 (C). Quantification
þ
of vessel-associated (D) or stroma-associated aSMA cells (E) in OVCAR-5 and OVCAR-5þP tumors. Mean SEM from 3 independent experiments. F, illustration
þ
of vessel-associated (arrows) and stroma-associated (arrowheads) aSMA cells by co-staining for CD34. Scale bar ¼ 100 mm. G, immunostaining of
þ
þ
representative AOCS patient TMAs for aSMA cells. Scale bar ¼ 200 mm. H, quantification of stroma-associated aSMA cells in early-, late-, and no-relapse
AOCS patient TMAs (n ¼ 7 patients/relapse group; 3 fields/patient). I and J, Kaplan–Meier curves correlating aSMA protein expression and progression-free (I) or
overall (J) survival in AOCS serous ovarian cancer patients. K, scatter plot of correlation between expression levels of aSMA and pericyte score from 105
AOCS serous ovarian cancer patients.
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Pericytes Promote Metastasis and Predict Relapse
Pericytes increase recruitment of host aSMAþ cells to the TME at
sites unrelated to angiogenesis in experimental tumors—also a
feature of early-relapse patients with ovarian cancer
An increase in the proportion of the "stromal" compartment of
tumors—cells and acellular matrix—is prognostic for poor survival in patients with advanced ovarian cancer (41). We, therefore,
immunostained OVCAR-5 xenografts for the stromal cell marker
aSMA (Fig. 5A), revealing higher numbers of aSMAþ cells in the
OVCAR-5þP group at day 11 (P < 0.001) and day 35 (P < 0.05)
compared with controls (Fig. 5B and C). Although no quantitative
difference in the percentage of vessel-associated aSMAþ cells was
observed (P ¼ 0.5762; Fig. 5D), a significant increase in stromaassociated aSMAþ cells in OVCAR-5þP tumors (P < 0.0001; Fig.
5E) was evident, evaluated by co-staining for aSMAþ cells and
CD34þ blood vessels (Fig. 5F) via immunofluorescence. Notably,
the absence of detectable aSMAþ/Ki67þ cells in xenografts even at
day 11 (Supplementary Fig. S5A) suggests that this was probably
the result of increased recruitment of host aSMAþ cells to xenografts, not proliferation.
The accumulation of aSMAþ stromal cells was then analyzed in
AOCS-patient TMAs and correlated with clinical outcome; earlyrelapse ovarian cancer patients (mean PFS time ¼ 8.98 months)
showed a significant increase in aSMAþ staining compared with
late-relapse patients (mean PFS time ¼ 28.45 months; Fig. 5G).
Closer inspection of the aSMAþ sections for blood vessels in 7
patient TMAs per early-, late-, and no-relapse group revealed
that this was attributed to increased numbers of tumor stroma–
associated aSMAþ cells in early-relapse patients (Fig. 5H;
P < 0.001), as observed for the OVCAR-5þP experimental tumors,
with no significant differences in vessel-associated aSMAþ cells
(P ¼ 0.4902; data not shown). Thus, our experimental ovarian
cancer model strongly mimics the clinical situation with common
biological features.
Given that BM-MSCs can be recruited to the TME, we co-stained
for aSMA and the murine BM-MSC markers CD73 or Sca-1 in all
xenografts. Interestingly, only the pericyte co-injected OVCAR-5
tumors contained CD73þ and Sca-1þ populations with only a
small proportion of aSMAþ cells co-expressing these markers
(Supplementary Fig. S5B and S5C). Since CAF-derived CXCL12
has been strongly implicated in recruiting BM-MSCs to tumors
and driving metastatic spread (42), we immunostained for this
chemokine (Supplementary Fig. S5D), not detecting it in the
stroma of any experimental tumors, despite abundant CXCL12
expression in OVCAR-5 cells, as reported previously for other
ovarian cancer cell lines (43) in all xenografts not correlated with
metastasis, suggesting a role for alternate signaling pathways in
inducing metastasis, while not excluding a role for CXCL12 in
promoting ovarian cancer tumor growth by increasing angiogenesis, as reported previously (43).
Greater aSMA levels predict earlier relapse in serous ovarian
cancer patients
We next sought to determine if a single pericyte marker at the
protein level could be prognostic at diagnosis. TMAs from AOCS
serous ovarian cancer patients were immunostained for aSMA
and PDGFRb (and CD34 control), and their expression levels
quantitated for individual patients morphometrically and correlated with time to relapse. The levels of CD34 or PDGFRb
expression were not predictive for early-relapse (P ¼ 0.1342,
n ¼ 112 patients; and P ¼ 0.1861, n ¼ 102 patients, respectively;
Supplementary Fig. S6A and S6B); however, higher levels of aSMA
www.aacrjournals.org
correlated significantly with early relapse for both PFS
(P ¼ 0.03691, n ¼ 105 patients; Fig. 5I) and overall survival
(P ¼ 0.006652; Fig. 5J). Consistent with this, a significant correlation was obtained between aSMA expression levels and pericyte
score (Fig. 5K; P ¼ 0.0078), but not CD34 (P ¼ 0.8412) or
PDGFRb (P ¼ 0.3761; Supplementary Fig. S6C and S6D).
Discussion
Pericytes are widely known to regulate microvascular function,
including structural stability, limiting hypoxia, and blood–brain
barrier permeability. In the context of cancers, killing pericytes
destabilizes tumor vasculature, resulting in tumor regression (27),
or causes hypoxia, inducing EMT and increased metastatic dissemination in various cancers (35). Our data demonstrate that
placing pericytes in the tumor stroma of OVCAR-5 and -8 ovarian
cancer cells while leaving the tumor vasculature intact results in
accelerated tumor expansion via increased cell proliferation,
shortening the latency of OVCAR-8 tumors by 15 days. Moreover,
pericytes induced invasion and metastatic spread in nonmetastatic OVCAR-8 cells—a core clinical feature of aggressive serous
ovarian cancer (1, 44, 45), and faster, distal spread of OVCAR-5
cells compared with controls that metastasized only locally within
the peritoneal cavity to the gastrointestinal tract. These data
demonstrate that normal MSC-like pericytes placed in close proximity to ovarian cancer cells drive malignant conversion, while
normal fibroblasts do not affect tumor growth or metastasis, as
reported previously. Notably, this was observed despite the use of
heterologous, that is, non-ovarian stromal cells (primarily due to
the difficulties in obtaining human ovarian tissue in sufficient
quantities and at regular frequencies to undertake adequate
experimentation), indicating sufficient conservation of function
exists in MSC-like pericytes, despite being tissue of origin, consistent with published data (24). Indeed, current transcriptional
profiling work in our laboratory comparing adult and neonatal
pericytes from male and female donors and from different anatomical sites reveals minimal differences in mRNA expression
profiles. We speculate that pericytes are a more potent stromal
stem-cell–like population than fibroblasts, whose involvement is
a harbinger for poor clinical outcome in patients.
Consistent with this notion, we demonstrated that the pericyte
signature had strong clinical relevance for high-grade serous
ovarian cancer patients—outperforming the stromal signature
derived from ovarian cancer patient stroma (5) in predicting
significantly earlier patient relapse, despite similar treatment in
both the AOCS (n ¼ 215) and the NCI TCGA patient datasets (n ¼
408). The early-relapse patient group expressed gene sets enriched
for biological processes clearly increased experimentally by pericytes such as invasion and migration that are key features of
aggressive metastatic disease, that is, cell motility, negative regulation of cell adhesion, and EMT. In contrast, the inability of
normal fibroblasts to promote malignant ovarian cancer progression was correlated well with their signature performing relatively
poorly as a predictor of early patient relapse. These data illustrate
the need to understand the nature of stromal heterogeneity in
both normal and cancerous tissues. The ability of tumor cells to
attract specific subtypes of stromal cells may facilitate tumor
progression to a malignant state. Presumably, the process of
pericyte association and dissociation from blood vessels during
tissue remodeling in wound healing and cancer requires tight
molecular regulation. The contribution of pericytes to malignant
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progression has remained unappreciated, masked by the fact that
the markers used to detect "CAFs" or BM-MSCs are also coexpressed by pericytes, e.g., aSMA, MCAM/CD146, and CD73.
A further potentially confounding factor is the low incidence at
which these cells may exist in the TME—like most stem cell
populations, a large number is not required to effect significant
change.
Apart from their ability to affect tumor cell proliferation and
invasive capacity directly in co-culture Transwell assays in vitro,
also mirrored in xenografts in vivo, the most striking feature of the
pericyte co-injected tumors was the recruitment of host aSMAþ
yet Ki67 cells that formed a nonvascular network between the
OVCAR-5 tumor cells in early day 11 xenografts. The correlation
with increased aSMAþ cell numbers in TMAs from early-relapse
patients suggested that this was a critical functional component of
the TME, indicative of tumor progression, leading to the finding
that aSMA protein levels yield prognostic significance in a large
sample of patient TMAs. However, the combined pericyte signature at the mRNA level was much more effective at predicting
relapse (P ¼ 0.00067) than the level of aSMA staining (P ¼
0.03691). High aSMA protein has also been reported to be of
prognostic value in colorectal cancer (7) and at the mRNA level in
pancreatic adenocarcinoma (8, 46). In contrast, recent studies in
murine models of pancreatic adenocarcinoma provide evidence
in favor of aSMAþ cells having a role in limiting tumor growth and
metastasis by either suppressing immune surveillance (47) or
perhaps decreasing tumor angiogenesis (48). Notably, in pancreatic adenocarcinoma patients low aSMA levels were associated
with poorer survival (47). These studies further substantiate the
need to examine the intratumoral heterogeneity of aSMAþ stromal subsets and examine their role in epithelial cancers of
different tissue origins (e.g., ovarian vs. pancreas).
Interestingly, fibroblast co-injected OVCAR-5 tumors did not
show a sustained increase in the number of aSMAþ cells (data not
shown), correlating with unchanged tumor growth, absence of
invasive cells at tumor margins, and absence of metastasis.
Whereas the influx of higher aSMAþ cell numbers in both experimental tumors and early-relapse patient TMAs may be an attempt
by the host to limit tumor growth and therefore a red herring, it
remains possible that their recruitment or perhaps a subtype
therein is required for metastatic spread. Another major difference
in pericyte co-injected OVCAR-5 tumors was the recruitment of
host Sca-1þ/CD73þ BM-MSCs not observed in OVCAR-5 controls
or fibroblast co-injected tumors—given their widely reported role
in cancer cell dissemination, their recruitment by pericytes may
well contribute to metastasis.
The inability of the normal pericyte marker PDGFRb to subset
serous ovarian cancer patients for the probability of relapse
suggests that aSMA and PDGFRb do not identify pericytes exclusively in cancer and are expressed by other stromal cells in the
TME. Consistent with this, PDGFRb expression was observed in
the tumor stroma in addition to its classic perivascular localization in patient TMAs. Attempts to define a single pericyte marker
to predict poor prognosis in patients with ovarian cancer at
diagnosis were only partially successful. Poor correlation between
high pericyte score and PDGFRb expression levels in TMAs belied
its inability to predict relapse with a high degree of certainty.
CD34 served as a negative control, given poor correlation between
MVD and early versus late relapse. Thus, although angiogenesis is
obviously critical for tumor development, it is not relevant to
malignant progression at advanced stages of malignancy predom-
OF10 Clin Cancer Res; 2016
inant in the patients analyzed here. Although aSMA protein levels
achieved reasonable significance levels for predicting relapse
(P ¼ 0.03691), it is likely that a number of pericyte markers
might be required to identify patients at greater risk of relapse.
An obvious target of further work is to understand the process
by which pericytes become dissociated from blood vessels
during physiological tissue remodeling. We speculate that cytokines used by endothelial cells to attract pericytes to newly
forming blood vessels such as PDGF-B may also be synthesized
by tumor cells—indeed, overexpression of PDGF-B in squamous
carcinoma models promotes tumor cell proliferation and acts as
a chemoattractant and activator for mesenchymal cells (49).
However, metastases were not observed in this model, suggesting that this single factor is unlikely to cause malignant progression. Certainly the mRNAs co-expressed by pericytes and
early-relapse ovarian cancer patients point to a coordinate
regulation of genes enriched in processes essential for tissue
remodeling.
These data represent a paradigm shift in the current thinking
about the contribution of pericytes to the TME while providing
an effective means of identifying those patients that are at
significantly greater risk of earlier relapse and mortality.
Undoubtedly, this brings a further level of complexity to
therapeutic approaches aimed at inhibiting angiogenesis, but
provides new opportunities to develop effective strategies
against stem-cell–like pericytes in the TME, given that antipericyte reagents not only exist, but are in clinical use in the
guise of anti-angiogenic therapies. The in vitro invasion data
suggest that pericytes secrete soluble factors that induce tumor
cell dissemination forming the basis for identifying specific
proteins that promote malignant progression that could also
serve as biomarkers for ovarian cancer, particularly early-stage
disease, given that experimentally, pericyte involvement in the
TME results in the induction of metastases in the poorly
tumorigenic and nonmetastatic OVCAR-8 cells. Perhaps the
greatest barrier to translating the significance of our findings
to early diagnosis and thereby increasing the chances of overall
patient survival is the lack of early-stage ovarian cancer patient
databases combining transcriptional and proteomic profiling
with clinical outcome following diagnosis. The collation of
patient cancer proteomic analysis being undertaken by the NCI
CPTAC initiative is eagerly anticipated, given the corroboration
of our findings between the AOCS and TCGA patient datasets.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: D. Sinha, L. Chong, H. Schl€
uter, D. Bowtell, P. Kaur
Development of methodology: D. Sinha, L. Chong, H. Schl€
uter, S. Mills, J. Li,
C. Parish, P. Kaur
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): D. Sinha, L. Chong, H. Schl€
uter, S. Mills, D. Bowtell,
P. Kaur
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): D. Sinha, L. Chong, J. George, H. Schl€
uter,
S. M€
onchgesang, J. Li, P. Kaur
Writing, review, and/or revision of the manuscript: D. Sinha, S. M€
onchgesang,
J. Li, C. Parish, D. Bowtell, P. Kaur
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): D. Sinha, L. Chong, H. Schl€
uter, P. Kaur
Study supervision: H. Schl€
uter, S. Mills, C. Parish, P. Kaur
Clinical Cancer Research
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Published OnlineFirst November 20, 2015; DOI: 10.1158/1078-0432.CCR-15-1931
Pericytes Promote Metastasis and Predict Relapse
Other (obtained funding for the project and oversaw its execution and
publication): P. Kaur
Acknowledgments
The authors thank Prof. Robin Anderson, Drs. Nick Clemons, Clare Slaney,
and Izhak Haviv for valuable discussions and technical advice, and Prof. Steven
Stacker and Prof. Ruth Ganss for critical reading of the manuscript.
Medical Research and Materiel Command Grant DAMD17-01-1-0729, the
Cancer Council Tasmania, the 618 Cancer Foundation of Western Australia,
NHMRC # 400413 and Cancer Australia # 1004673 to D. Bowtell. D. Sinha
was supported by an International HDR PhD scholarship from ANU,
Canberra.
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.
Grant Support
This work was supported by the CASS Foundation, Cancer Council of
Victoria # 807184 and NHMRC # 1025874 grants to P. Kaur and US Army
Received August 13, 2015; revised October 28, 2015; accepted October 30,
2015; published OnlineFirst November 20, 2015.
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Pericytes Promote Malignant Ovarian Cancer Progression in
Mice and Predict Poor Prognosis in Serous Ovarian Cancer
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Devbarna Sinha, Lynn Chong, Joshy George, et al.
Clin Cancer Res Published OnlineFirst November 20, 2015.
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