Published OnlineFirst June 8, 2010; DOI:10.1158/0008-5472.CAN-10-0012
Angiopoietin-2 Regulates Gene Expression in TIE2-Expressing
Monocytes and Augments Their Inherent Proangiogenic
Functions
Seth B. Coffelt, Andrea O. Tal, Alexander Scholz, et al.
Cancer Res 2010;70:5270-5280. Published OnlineFirst June 8, 2010.
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Copyright © 2010 American Association for Cancer Research
Published OnlineFirst June 8, 2010; DOI:10.1158/0008-5472.CAN-10-0012
Microenvironment and Immunology
Cancer
Research
Angiopoietin-2 Regulates Gene Expression in TIE2-Expressing
Monocytes and Augments Their Inherent
Proangiogenic Functions
Seth B. Coffelt1, Andrea O. Tal3, Alexander Scholz3, Michele De Palma5, Sunil Patel1, Carmen Urbich4,
Subhra K. Biswas6, Craig Murdoch2, Karl H. Plate3, Yvonne Reiss3, and Claire E. Lewis1
Abstract
TIE2-expressing monocytes/macrophages (TEM) are a highly proangiogenic subset of myeloid cells in
tumors. Here, we show that circulating human TEMs are already preprogrammed in the circulation to
be more angiogenic and express higher levels of such proangiogenic genes as matrix metalloproteinase-9
(MMP-9), VEGFA, COX-2, and WNT5A than TIE2− monocytes. Additionally, angiopoietin-2 (ANG-2) markedly
enhanced the proangiogenic activity of TEMs and increased their expression of two proangiogenic enzymes:
thymidine phosphorylase (TP) and cathepsin B (CTSB). Three “alternatively activated” (or M2-like) macrophage markers were also upregulated by ANG-2 in TEMs: interleukin-10, mannose receptor (MRC1), and
CCL17. To investigate the effects of ANG-2 on the phenotype and function of TEMs in tumors, we used
a double-transgenic (DT) mouse model in which ANG-2 was specifically overexpressed by endothelial cells.
Syngeneic tumors grown in these ANG-2 DT mice were more vascularized and contained greater numbers
of TEMs than those in wild-type (WT) mice. In both tumor types, expression of MMP-9 and MRC1 was
mainly restricted to tumor TEMs rather than TIE2− macrophages. Furthermore, tumor TEMs expressed
higher levels of MRC1, TP, and CTSB in ANG-2 DT tumors than WT tumors. Taken together, our data show
that although circulating TEMs are innately proangiogenic, exposure to tumor-derived ANG-2 stimulates
these cells to exhibit a broader, tumor-promoting phenotype. As such, the ANG-2–TEM axis may represent
a new target for antiangiogenic cancer therapies. Cancer Res; 70(13); 5270–80. ©2010 AACR.
Introduction
TIE2-expressing monocytes/macrophages (TEMs) are a
subpopulation of circulating and tumor-infiltrating myeloid
cells with profound proangiogenic activity, found in both
humans and mice (1–4). TEM ablation studies in mice have
Authors' Affiliations: 1 Academic Unit of Inflammation and Tumour
Targeting, University of Sheffield Medical School; 2Department of Oral
and Maxillofacial Medicine and Surgery, University of Sheffield School
of Clinical Dentistry, Sheffield, United Kingdom; 3Institute of Neurology/
Edinger Institute, Frankfurt University Medical School; 4Institute for
Cardiovascular Regeneration, Centre of Molecular Medicine GoetheUniversity, Frankfurt, Germany; 5 Angiogenesis and Tumor Targeting
Research Unit, Division of Regenerative Medicine, Stem Cells and
Gene Therapy, San Raffaele Scientific Institute, Milan, Italy; and
6 Singapore Immunology Network, Biomedical Sciences Institutes,
Agency for Science, Technology and Research, Singapore, Singapore
Note: Supplementary data for this article are available at Cancer
Research Online (http://cancerres.aacrjournals.org/).
S.B. Coffelt and A.O. Tal contributed equally to this work as co-first
authors and K.H. Plate, Y. Reiss, and C.E. Lewis contributed equally to
this work as co-senior authors.
Corresponding Author: Claire E. Lewis, Academic Unit of Inflammation
and Tumour Targeting, University of Sheffield Medical School, Sheffield
S10 2RX, United Kingdom. Phone: 44-114-271-2903; Fax: 44-114-2712903; E-mail: claire.lewis@sheffield.ac.uk.
doi: 10.1158/0008-5472.CAN-10-0012
©2010 American Association for Cancer Research.
5270
shown that this subpopulation plays a greater role in regulating tumor angiogenesis than TIE2− tumor-associated macrophages (TAM; refs. 1, 5). Similarly, when circulating TEMs are
coinjected with Matrigel in mice, microvessel density (MVD) is
higher than that seen with TIE2− monocytes, suggesting that
they possess an inherent ability to stimulate angiogenesis
(1, 4). Various phenotypic differences have emerged recently
between TEMs and TIE2− TAMs in murine tumors, with TEMs
exhibiting various markers of an alternatively activated
(i.e., M2-like) phenotype (5).
Angiopoietins are a family of molecules known to bind to,
and activate, the TIE2 receptor on endothelial cells (EC; refs.
6–9). Angiopoietins play an essential role in regulating angiogenesis and vascular homeostasis. Angiopoietin-1 (ANG-1)
maintains the integrity of the endothelium, whereas angiopoietin-2 (ANG-2) was thought until recently to act as an
ANG-1 antagonist to destabilize the vasculature (10). However,
it now seems that the exact influence of ANG-2 on endothelium is highly dependent on the local cytokine milieu. Together
with other cytokines such as vascular endothelial growth
factor (VEGF), ANG-2 stimulates angiogenic responses,
but without such cofactors, it elicits vessel regression (7, 11,
12). During inflammation, ANG-2 also sensitizes the endothelium to tumor necrosis factor α (TNFα), and together, they
regulate expression of adhesion molecules and leukocyte
adherence (13).
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Influence of ANG-2 on TIE2-Expressing Monocytes
In human tumors, the ANG-1/ANG-2 balance is often
skewed in favor of ANG-2 and vascular remodeling. ANG-2
overexpression has been shown in several tumor types
(14–19), where it is produced by the endothelium and occasionally by tumor cells (14, 20). ANG-2 has a complex and
sometimes contradictory role during tumor progression.
For example, ANG-2 overexpression by tumor xenografts
leads to advanced proliferation, increased angiogenesis,
and enhanced invasiveness (11, 14–16, 21–23), but it delays
tumor growth and disrupts angiogenesis in other tumor
types (24, 25). Syngeneic tumors grown in Ang-2 knockout
mice show slower proliferation during the initial stages of
tumor progression, decreased vessel diameter, and increased
pericyte vessel coverage (26).
We recently reported that ANG-2 modulates the secretion
of certain inflammatory cytokines by human monocytes
in vitro (3, 4). However, information about the role of
ANG-2 in regulating the tumor-promoting functions
of TEMs is lacking. We therefore examined the influence
of ANG-2 on expression of various tumor-promoting genes
by human circulating TEMs in vitro and murine tumorinfiltrating TEMs in vivo.
Materials and Methods
Monocyte isolation and culture
Monocytes were isolated as previously described (3, 27).
Pacific blue–conjugated anti-CD14 (1:20 per 10 6 cells;
clone M5E2; BD Biosciences) and allophycocyanin
(APC)–conjugated anti-TIE2 (1:10 per 10 6 cells; clone
83715; R&D Systems) antibodies were used to identify
TEMs. Monocytes were sorted into cold Iscove's modified
Dulbecco's medium (IMDM; BioWhittaker) containing
2% fetal bovine serum (FBS) and 2 mmol/L L-glutamine
(Sigma-Aldrich) using a FACSAria II flow cytometer (BD
Biosciences). Typically, 2 × 106 cells were collected from
each sort with a purity of ∼90%. When required, 106 cells
were seeded onto plastic with IMDM–2% FBS and then
placed into a humidified 37°C incubator with 5% CO2.
Quantitative real-time PCR
Total RNA was extracted from monocytes using RNeasy kit
(Qiagen). RNA from 106 cells was isolated immediately following fluorescence-activated cell sorting (FACS). For experiments involving ANG-1/ANG-2 treatment, sorted populations
were cultured overnight, washed the next day, and then exposed to 300 ng/mL of recombinant angiopoietins (R&D Systems) for 6 hours. RNA (250–500 ng) was reverse transcribed
using Precision RT kit (PrimerDesign). cDNA was amplified
with Taqman master mix (Applied Biosystems) or Precision
Mastermix (PrimerDesign) containing SYBR Green. PCR cycling conditions were as described using ABI 7900HT Sequence Detection System (Applied Biosystems; ref. 27).
Samples were run on the same plate as the housekeeping
gene [tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAZ1) for SYBR Green probes,
and β2-microglobulin for Taqman probes] in triplicate. Experiments were performed four to six times. Differences in
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gene expression were determined by the quantitative comparative Ct (threshold value) method.
Soluble protein analysis
Conditioned medium was collected from sorted monocyte
subsets after 24 hours in culture. VEGF, interleukin (IL)-6,
and IL-10 were quantified by cytometric bead array using a
FACSArray bioanalyzer (BD Biosciences). Epidermal growth
factor (EGF) levels were measured by ELISA (PreproTech).
Matrix metalloproteinase-9 (MMP-9) activity was assessed
by zymography in which proteins were separated in 10%
SDS-polyacrylamide gels containing 0.1% gelatin. Following
incubation in developing buffer, the gels were washed in distilled water and then stained with SimplyBlue SafeStain (Invitrogen). Quantification of band intensity was performed by
using ImageJ software (NIH, Bethesda, MD). Human ANG-2
(hANG-2) blood serum levels in mice were detected by ELISA
(R&D Systems) after 1:2 dilution. All other soluble factors
were analyzed with Bio-Plex Cytokine Assays (Bio-Rad).
In vitro EC activation assays
Medium was conditioned by TIE2− monocytes or TEMs for
24 hours and then incubated with 20 μmol/L MMP-9 Inhibitor I (Calbiochem), which inhibits MMP-1, MMP-9, and
MMP-13, or 0.4% DMSO (vehicle control) for 1 hour at
37°C. The water-soluble thymidine phosphorylase (TP) inhibitor AEAC (6-[2-aminoethyl]amino-5-chlorouracil; 25 μmol/L;
a gift from Dr. Edward Schwartz, Albert Einstein College of
Medicine, Bronx, NY; refs. 28, 29) was incubated with sorted
monocyte populations 1 hour before exposure to 300 ng/mL
ANG-2, and then medium was collected after 24 hours. For
the tubule formation assay, serum-starved human umbilical vein ECs (HUVEC; 8,000 cells/96-well) were resuspended in conditioned medium and seeded onto growth
factor–reduced Matrigel (BD Biosciences). After 6 hours,
tubule formation was measured using ImageJ software.
For the spheroid/spouting assay, HUVEC spheroids (400
cells) were generated as described previously (30) and incubated for 24 hours with conditioned media. Images of
sprouting spheroids were taken with an Axiovert 100M microscope and Plan-NEOFLUAR 10×/0.30 objective lens.
Capillary sprouting length was quantified using AxioVision
Rel 4.4 digital imaging software (Zeiss). Every sprout from
10 spheroids per group was measured, and the mean cumulative sprout length was calculated. Both sets of in vitro assays were performed three times.
Immunoprecipitation and immunoblot analysis
Serum-starved HUVECs (2 × 106) or human monocytes (2 ×
7
10 ) were treated with 300 ng/mL ANG-2. Cells were lysed in
50 mmol/L Tris and 100 mmol/L NaCl with 1% Triton X-100
containing protease and phosphatase inhibitors. After spinning at 14,000 rpm for 10 minutes, supernatant was collected.
Protein lysates (40 μg) were precleared with protein A/G
beads (Santa Cruz Biotechnology) and then incubated with
5 μg anti-TIE2 (clone 83711; R&D Systems) overnight at
4°C. Protein A/G beads were added for 2 hours at 4°C.
IgG–protein A/G complexes were collected by centrifugation,
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Coffelt et al.
washed, and boiled in loading buffer before loading into 8%
SDS-polyacrylamide gels. Separated proteins were transferred to nitrocellulose, blocked with 5% milk in TBS–Tween
20, and probed with anti–phospho-TIE2 (0.5 μg/mL; Y992;
R&D Systems) overnight at 4°C. Membranes were washed
and incubated with biotin-conjugated anti-rabbit antibodies
(1:5,000; R&D Systems) for 1 hour at room temperature, followed by streptavidin–horseradish peroxidase (HRP; 1:200;
R&D Systems) for 1 hour at room temperature. Bands were
visualized by enhanced chemiluminescence (Amersham).
Membranes were then stripped and reprobed with antiTIE2 (1:250; clone 83711; R&D Systems) followed by
HRP-conjugated goat anti-mouse antibodies (1:5,000; Dako).
Similar experiments were repeated three times using three
different blood donors.
For detection of TP, adherent human monocytes were
treated with 300 ng/mL ANG-2, washed in cold PBS, and
lysed. Proteins were separated on 10% SDS-polyacrylamide
gels and transferred onto nitrocellulose. The remainder of
the procedure was carried out as above using anti-TP antibodies (1:1,000; clone P-GF.44C) purchased from Abcam followed by anti-mouse secondary.
Murine tumor model
TIE1-tTA–driven hAng-2 double-transgenic (DT) mice
were generated as previously described (Supplementary
Fig. S4A; refs. 31, 32). Mice were depleted of doxycycline
2 weeks before implantation of tumor cells (Supplementary
Fig. S4B). Syngeneic Lewis lung carcinoma (LLC) cells (2 ×
106 ) were inoculated s.c. and allowed to propagate for
2 weeks. Caliper measurements were taken every 3 days,
and tumor volume was calculated with the following formula:
length × width 2 /2. Each group consisted of at least five
mice and experiments were repeated four times. Animals
were cared for in accordance with German Legislation on
the Care and Use of Laboratory Animals.
Immunohistochemistry and immunofluorescence
confocal microscopy
Detection of blood vessels and pericytes, assessment of
MVD, and pericyte coverage were performed as described
previously (33) using rat anti-mouse CD31 (BD Biosciences)
and anti–α-smooth muscle actin (αSMA; Sigma). CD45,
Gr-1, CD3, and B220 antibodies were purchased from BD
Biosciences. Images were taken with a Nikon 80i microscope,
and staining was analyzed with Soft Imaging Analysis System
software at ×10 magnification. ApopTag In Situ Apoptosis
Detection kit (Chemicon) was used as before (33). Assessment of hypoxic tumor regions was conducted as described
previously (27) using Hypoxyprobe (HPI).
Detection of F4/80 + TIE2 − TAMs and TEMs was performed using FITC-conjugated anti-F4/80 (1:25, clone CI:
A3-1; AbD Serotec) and phycoerythrin-conjugated antimurine TIE2 (1:50; eBioscience). Anti–MMP-9 (1:500; a gift
from Dr. Zena Werb, University of California, San Francisco,
San Francisco, CA), anti–cathepsin B (CTSB; 15 μg/mL;
R&D Systems), anti-TP (1:500; Abcam), and anti-MRC1
(1:25; R&D Systems) antibodies were detected by Alexa Flu-
5272
or 647–conjugated anti-goat or anti-rabbit secondary antibodies (1:500; Invitrogen). The anti–hANG-2 antibody
(clone 180102) was from R&D Systems and used at 1:500.
TP and ANG-2 antibodies were preabsorbed with secondary
antibodies before incubation with tissue. Tumor vasculature
was identified using APC-conjugated anti-CD31 (1:50;
eBioscience). Nuclei were highlighted using 30 nmol/L
4′,6-diamidino-2-phenylindole (DAPI; Invitrogen) for 2 minutes. Images were captured using a Zeiss LSM 510 laser
scanning confocal microscope. Further details about microscopy can be found in the Supplementary Data.
Statistical analysis
Student's two-tailed t test (paired or unpaired as appropriate) or one-way ANOVA was used to determine P values using
GraphPad Prism software. A P value of <0.05 was considered
statistically significant. All data shown are mean ± SE.
Results
Human TEMs are more proangiogenic than TIE2−
monocytes in vitro
Human TEMs and TIE2− monocytes were isolated from
peripheral blood by FACS (Supplementary Fig. S1A), and
increased TIE2 expression in TEMs was confirmed using
quantitative real-time PCR (qPCR; Supplementary Fig. S1B).
Interestingly, flow cytometry revealed that, unlike ECs, TIE1
expression on both monocyte subpopulations was negligible
(Supplementary Fig. S2).
In the EC spheroid/sprouting assay, we found that TEMs
induced significantly more sprouts than TIE2− monocytes
(Fig. 1A). In the tubule formation assay, TEM-conditioned
medium also significantly increased EC tubule length
(Fig. 1B) and tubule area (47.9 ± 3.7 versus 32.5 ± 2.8 μm2;
P = 0.02; data not shown) when compared with TIE2 −
monocyte-conditioned medium. Furthermore, addition of
an MMP inhibitor significantly reduced the formation of tubules by TEMs (33.8 ± 3.6% reduction for length, 23.4 ± 4.1%
reduction for area) but not TIE2 − monocytes (data not
shown).
We then used qPCR to examine mRNA expression levels of
various tumor-promoting and M2 (i.e., alternatively activated
macrophage)–associated genes, including proangiogenic factors (MMP-9, VEGF, EGF, FGF2, COX-2, CTSB, WNT5A, and
TP/ECGF1), immunomodulatory cytokines (TNFα, IL-1β, IL6, IL-8, and IL-10), cell adhesion molecule (ICAM-1), and cell
surface receptors [mannose receptor (MRC1), CXCR4, TLR-2,
and TLR-4]. TEMs expressed significantly higher levels of
MMP-9 (Fig. 1C). Densitometric analysis of gelatin zymography showed a significant difference between the amount of
pro–MMP-9 released by the cells (Fig. 1C), with TEMs producing more pro–MMP-9 than TIE2− monocytes (421.3 ±
42.1 versus 570.8 ± 57.1 relative units; P = 0.047; data not
shown). MMP-2 activity was not detected.
In addition to MMP-9, TEMs expressed higher levels of
VEGFA mRNA and released higher levels of VEGFA and
TNFα protein than TIE2 − monocytes (Fig. 1C). TEMs
expressed higher levels of COX-2, MRC1, and WNT5A
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Influence of ANG-2 on TIE2-Expressing Monocytes
Figure 1. Circulating human TEMs are
inherently more angiogenic than
TIE2− monocytes. Conditioned medium
was collected from sorted
CD14+TIE2− and CD14+TIE2+ (TEMs)
human monocyte subpopulations and
used in either a HUVEC sprouting assay
(A) or a HUVEC tubule formation
assay in vitro (B). TEMs induced
significantly more EC activation than
TIE2− monocytes in both assays.
C, RNA was isolated from monocyte
subpopulations and analyzed by qPCR.
Cells were also cultured and
conditioned medium was assayed for
pro–MMP-9 by gelatin zymography or
VEGF and TNFα by cytometric
bead array. Top, VEGF and TNFα mRNA
levels assayed by qPCR, and their
corresponding proteins; bottom,
COX-2, MRC1, and WNT5A mRNA
levels. *, P < 0.05; **, P < 0.01,
compared with TIE2− monocytes. Data
from at least three replicate assays
shown. Scale bars, 50 μm.
mRNA (Fig. 1C) but significantly lower levels of EGF mRNA
(−1.7 ± 0.1 fold difference; P = 0.007; data not shown) and
protein (13.2 ± 3.2 versus 4.6 ± 1.2 pg/mL; P = 0.035; data not
shown) than TIE2− monocytes. Expression of CTSB, CXCR4,
TP, ICAM-1, IL-1β, IL-6, IL-8, IL-10, TLR2, TLR4, or TNFα mRNA
was not significantly different between the two populations.
FGF2 mRNA was not detected in either subset.
Agonistic effects of ANG-2 on human TEMs in vitro
To investigate the possible effect of ANG-2 on the phenotype and function of TEMs, we first exposed freshly isolated
human monocytes and HUVECs to ANG-2 and examined
phosphorylation of TIE2. Activation typically occurred within
10 minutes of ANG-2 stimulation in both cell types, as variation between monocyte donors was minimal (Fig. 2A). Den-
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sitometry revealed a 50% increase in phosphorylation at
10 minutes for monocytes.
Although expression of VEGF and MMP-9 mRNA was
not further increased by ANG-2 in TEMs (Fig. 2B), it significantly upregulated expression of two other proangiogenic enzymes, CTSB and TP (Fig. 2B and C), an effect
not seen in TIE2− cells. Increased TP protein levels were
confirmed by Western blot following exposure to ANG-2
(Fig. 2C).
The ability of ANG-2 to further enhance the inherent
proangiogenic functions of TEMs was then assessed. ANG-2
treatment of TIE2− monocytes failed to augment their relatively low capacity to induce EC sprouts or tubule formation
(data not shown), whereas ANG-2 significantly increased the
ability of TEMs to activate ECs in both assays (Fig. 2D). This
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Coffelt et al.
stimulatory effect (64% increase for cumulative sprout
length, 39% increase for tubule length, and 91% increase
for tubule area) is greater than that reported previously for
VEGF and FGF2 (34). Exposure of TEMs to a specific TP inhibitor had no effect on cell viability (data not shown) but
significantly reduced their ability to activate ECs in response
to ANG-2 (Fig. 2D).
In addition to CTSB and TP, ANG-2 also significantly upregulated the expression of the two M2-associated genes, IL10 and MRC1, by TEMs (Fig. 3A and B, left). IL-10 protein
levels were significantly higher in TEM-conditioned medium
following ANG-2 treatment (Fig. 3A, right), and flow cytometry showed an increase in MRC1 expression on TEMs following ANG-2 treatment (Fig. 3B, right). Both the percentage of
TEMs expressing MRC1 [control (11.0 ± 5.3%) versus ANG-2
(17.1 ± 5.4%)] and the median fluorescence intensity [MFI;
control (1,399 ± 58.1 arbitrary units) versus ANG-2 (1,918 ±
220.6 arbitrary units)] were significantly higher in the ANG2–treated group (P = 0.0012 and 0.045, respectively). TEM
expression of IL-6 mRNA was significantly reduced following
ANG-2 stimulation; however, the amount of IL-6 secreted was
not significantly different following ANG-2 treatment of TEMs
(Fig. 3C). CCL17 mRNA was also significantly increased in
TEMs by ANG-2 (2.7 ± 0.9 fold increase; P = 0.013; data
not shown). All the other mRNA species screened by qPCR
(EGF, COX-2, WNT5A, TNFα, IL-1β, IL-6, IL-8, ICAM-1, CXCR4,
TLR-2, and TLR-4) were not altered by ANG-2 in either
population (data not shown). Of note, only TEMs, not TIE2−
monocytes, responded to ANG-2, indicating that the TIE2
receptor is required for its effects on TEMs.
Figure 2. ANG-2 induces phosphorylation of
TIE2 and enhances the proangiogenic ability of
human TEMs in vitro. A, human monocytes
and HUVECs were exposed to 300 ng/mL
of recombinant ANG-2, TIE2 was then
immunoprecipitated from cell lysates,
and phosphorylation was analyzed by
immunoblotting. B, qPCR analysis of VEGF,
MMP-9, and CTSB mRNA. C, upregulation of
TP expression by ANG-2 as assessed by
qPCR and immunoblotting. D, HUVEC
sprouting assay (left) and tubule formation
(right) after incubation with conditioned medium
from untreated or ANG-2–treated TEMs.
Where indicated, cells were preincubated
with 25 μmol/L TP inhibitor (AEAC) before
exposure to ANG-2. *, P < 0.05; **, P < 0.01,
compared with TIE2− monocytes. ^, P < 0.05;
^^, P < 0.01, compared with untreated
TEMs; †, P < 0.05, compared with untreated
TEMs + TP inhibitor; §, P < 0.05, compared with
ANG-2–treated TEMs. Data from at least four
replicate assays shown. Sprouting assay
data are representative of three independent
experiments.
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Influence of ANG-2 on TIE2-Expressing Monocytes
Figure 3. ANG-2 upregulates
expression of two classic M2
genes by human TEMs: IL-10 and
MRC1. A to C, left, mRNA levels
as determined by qPCR; right,
cytometric bead array analysis of
soluble protein in TEM-conditioned
medium. B, right, flow cytometry
analysis of MRC1 cell surface
expression. Figures cited on the
contour maps indicate MFI of
MRC1 expression and the
proportion of MRC1+ cells in each
gate. Pooled data from at least
three replicate experiments
shown. *, P < 0.05; **, P < 0.01,
compared with untreated TEMs.
Differential effects of ANG-1 on human TEMs in vitro
Unlike ANG-2, ANG-1 had no effect on TP, MRC1, IL-10,
or CTSB mRNA levels in TEMs but significantly downregulated CCL17 mRNA and upregulated EGF mRNA levels
(Supplementary Fig. S3). Like ANG-2, only TEMs responded
to ANG-1, indicating that the TIE2 receptor is required for
its effects on TEMs.
Effects of ANG-2 overexpression on the growth and
vasculature of LLC tumors in vivo
We used a DT mouse model in which the vasculature
specifically overexpresses hANG-2—termed ANG-2 DT
(Supplementary Fig. S4A; ref. 32). LLCs were grown s.c. in
these mice, and upregulation of hANG-2 in plasma was
confirmed using ELISA (Supplementary Fig. S4C). The ECspecific upregulation of hANG-2 was validated by the
immunofluorescent labeling of CD31, TIE2, and hANG-2 in
LLCs (Supplementary Fig. S4D). Approximately 60% to 70%
of CD31+ blood vessels expressed hANG-2 in ANG-2 DT
tumors, with negligible expression seen in LLC grown in
wild-type (WT) mice.
Although tumor volumes were not significantly different
between WT and ANG-2 DT mice (Supplementary Fig. S5A),
excised tumors from the latter group were more hemorrhagic
than those grown in WT mice (Supplementary Fig. S5B) and
contained significantly higher numbers of CD31+ microvessels
(Fig. 4A). The vessels in ANG-2 DT tumors exhibited an
immature phenotype (i.e., little or no pericyte coverage) and
displayed increased levels of EC apoptosis (Fig. 4A). This
accords well with the finding that ANG-2 DT tumors were
more hypoxic than WT tumors (Fig. 4A).
Overexpression of ANG-2 increases TEM infiltration
into tumors
As we previously showed that ANG-2 is a potent chemoattractant for human TEMs in vitro (3, 4), we investigated
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whether TEM infiltration into tumors was altered by
hANG-2 overexpression. Immunohistochemistry showed that
CD45+ leukocyte tumor infiltration (assessed as a proportion
of total tumor area) was significantly increased in ANG-2 DT
compared with WT tumors, with the majority of these cells
being F4/80+ macrophages (Supplementary Fig. S5C). Similarly, immunofluorescent analysis replicated this increase in
F4/80+ macrophages but also found a greater increase in the
proportion of F4/80+TIE2+ cells (TEMs) than F4/80+TIE2−
TAMs in ANG-2 DT tumors, suggesting selective recruitment
of TEMs (Fig. 4B). These data were confirmed by flow cytometry analysis of enzymatically dispersed tumors (data not
shown). The frequency of other leukocytes was unaffected,
including Gr-1+ cells, CD3+ T cells, and B220+ B cells (data
not shown). No differences in any leukocyte subsets were
seen between normal tissues (i.e., brain, heart, and spleen)
of WT and ANG-2 DT mice (data not shown).
TEM phenotype is affected by ANG-2
overexpression in vivo
A significantly greater number of MMP-9–expressing
TEMs but not TIE2− TAMs were present in Ang-2 DT than
WT tumors (Fig. 5A). This could be explained by the fact that
virtually all TEMs—and <10% F4/80+TIE2− TAMs—expressed
MMP-9 in both tumor types, and this difference in MMP-9
expression between these two macrophage populations was
significant (Fig. 5A). In agreement with our in vitro studies,
ANG-2 overexpression had no effect on the level of MMP-9
expression/TEM as assessed by the MFI per cell (Fig. 5A).
The frequency of MRC1+ TEMs was also greater in ANG-2
DT tumors than WT, with MRC1 expression being largely
confined to TEMs and absent in F4/80 + TIE2 − TAMs
(Fig. 5B). Figure 6 shows that the majority of both TEMs
and TIE2− TAMs expressed CTSB and TP, but both were
more abundant in ANG-2 DT tumors (due to increased
numbers of TEMs and TIE2− TAMs in ANG-2 DT tumors).
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Coffelt et al.
Moreover, the expression of MRC1 (Fig. 5B), CTSB (Fig. 6A),
and TP (Fig. 6B) per TEM was significantly increased in
ANG-2 DT tumors.
Discussion
Myeloid cells are essential for blood vessel formation,
maintenance, and function in tumors (35), and TEMs are
one of the most proangiogenic subsets of these cells (1, 4,
5). It has previously been suggested that circulating human
or mouse TEMs may be innately proangiogenic, as mice inoculated with tumor cells and TEMs form more vascularized tumors than those injected with tumor cells alone
(1, 4). In the present study, we confirm that circulating
human TEMs are indeed more proangiogenic than TIE2−
monocytes and express higher levels of such potent proangiogenic factors as MMP-9, VEGF, COX-2, and WNT5A.
Interestingly, we also found that MMP-9 was widely
expressed by the vast majority (>90%) of TEMs in tumors.
Use of an MMP-9 inhibitor in vitro suggests that this
enzyme plays an important role in mediating the innate,
proangiogenic function of circulating TEMs. However, it
should be noted that this inhibitor also reduces MMP-1
and MMP-13 activities, so the relative contribution of these
three enzymes awaits further study.
We reported previously that ANG-2 is a chemoattractant
for human TEMs in vitro, an effect mediated by TIE2 (3, 4).
Our data here show that ANG-2 overexpression by the tumor
vasculature results in greater infiltration of murine TEMs into tumors. This observation could be due to the direct chemotactic effect of ANG-2 on TEMs and/or the increased
number of blood vessels present in ANG-2 DT tumors, allowing TEMs and other leukocytes greater access into tumors.
At first glance, the latter possibility seems to be supported
Figure 4. Tumors grown in
ANG-2–overexpressing mice
contain more immature
microvessels and increased
numbers of F4/80+ macrophages
and TEMs. hANG-2 expression in
ECs was achieved using a DT
Tet-Off expression system. LLC
cells were allowed to propagate for
2 wk. A, the vascular phenotype of
ANG-2 DT and WT LLC tumors was
assessed by CD31/αSMA double
immunofluorescence. αSMA+
pericytes are denoted by white
arrows. For pericyte quantification,
103 CD31+ vessels per tumor were
analyzed and percentage of αSMA+
pericyte coverage was determined.
MVD was determined by CD31
immunohistochemistry and
quantified by Soft Imaging
Analysis System software.
EC apoptosis was determined by
the terminal deoxynucleotidyl
transferase–mediated dUTP nick
end labeling assay coupled
with CD31 immunohistochemistry.
Regions of hypoxia were identified
using anti-PIMO antibody,
analyzed by NIS Elements
software, and represented as a
percentage of the entire tumor
area. B, LLC tumor sections
were immunofluorescently
double labeled with F4/80- and
TIE2-specific antibodies. Scale bar,
50 μm. Arrows denote TEMs in
representative images. The total
F4/80+ (including both TIE2− and
TIE2+) macrophage population
and TEM subpopulation were
quantified from six high-powered
fields (HPF) for each tumor section.
Data are pooled from five tumors
per group at minimum. *, P < 0.05;
**, P < 0.01; ***, P < 0.005,
compared with WT tumors.
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Influence of ANG-2 on TIE2-Expressing Monocytes
Figure 5. TEMs are the main
source of MMP-9 and
MRC1 in both WT and
ANG-2–overexpressing tumors.
Frozen sections of LLC
tumors grown in WT or ANG-2 DT
mice were stained with
F4/80 (green), TIE2 (red), and
either (A) MMP-9 or (B) MRC1
antibodies (both white) and then
analyzed by fluorescence confocal
microscopy. Representative
images are shown for each antigen
alone as well as merged images
(DAPI, blue). MMP-9 or MRC1–
expressing TEMs are denoted by
arrows. The number of MMP-9–
expressing or MRC1-expressing
TEMs per HPF, the percentage of
F4/80+TIE2− and F4/80+TIE2+
TAM populations expressing
MMP-9 or MRC1, as well as the
MFI of MMP-9/MRC1 expression
by TEMs are represented
graphically. TIE2+F4/80− structures
are blood vessels. *, P < 0.05,
compared with F4/80+TIE2+ cells in
WT tumors; ^, P < 0.05;
†
, P < 0.05, compared with
F4/80+TIE2− cells in WT and
ANG-2 DT tumors, respectively.
Scale bar, 50 μm.
by our finding that the frequency of all CD45+ leukocytes was
increased in ANG-2 DT tumors. However, the majority of
CD45 + cells were F4/80 + TAMs and TEMs, whereas the
number of Gr-1+ granulocytes, T cells, and B cells was not
increased in ANG-2 DT tumors. The enhanced recruitment
of TEMs may also be due to the increased level of tumor hyp-
www.aacrjournals.org
oxia seen in ANG-2 DT tumors, as hypoxia-induced CXCL12
attracts increased numbers of TEMs into tumors (36).
Here, we confirm that ANG-2 stimulates TIE2 receptor
phosphorylation in human TEMs and upregulates their expression of several tumor-promoting factors. This result
may seem unexpected given that some in vitro studies report
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Coffelt et al.
that ANG-2 simply inhibits or dampens TIE2 signaling
in response to ANG-1 in ECs (7). However, as mentioned
previously, tumors often produce higher levels of ANG-2 than
ANG-1 (10, 14–20), so the antagonistic role of ANG-2 on
ANG-1 signaling is likely minimal in such tissues. Moreover,
agonistic effects of ANG-2 on ECs and its ability to phosphorylate TIE2—even in the absence of ANG-1—have been reported (37, 38). TIE1 receptors on ECs can also modulate
the effect of angiopoietins on TIE2 (39–41), although the agonistic effects of ANG-2 do not involve TIE1 (39, 41). As TIE1
is absent on TEMs, the agonistic effects of ANG-2 reported
here also seem to be independent of TIE1.
We show that exposure to ANG-2 enhances the angiogenic
potential of human TEMs in vitro by augmenting expression
of two proangiogenic genes: TP and CTSB. TP catalyzes the
breakdown of thymidine that forms a proangiogenic sugar,
Figure 6. TEMs upregulate
both CTSB and TP in
ANG-2–overexpressing tumors.
LLC tumor sections from WT or
ANG-2 DT mice were stained with
F4/80 (green), TIE2 (red), and
CTSB (white, A) or TP (white, B)
antibodies and then analyzed by
fluorescence confocal microscopy.
Representative images are shown
for each antigen alone as well
as merged images (DAPI, blue).
CTSB- or TP-expressing TEMs are
denoted by arrows. The number
of CTSB+ and TP+ macrophage
subpopulations per HPF is
represented graphically. The
percentage of F4/80+TIE2− and
F4/80+TIE2+ TAM populations
expressing each protein and the
MFI per TEM are also shown.
TIE2+F4/80− structures are blood
vessels. *, P < 0.05, compared with
F4/80+TIE2+ cells in WT tumors;
†
, P < 0.05, compared with
F4/80+TIE2− cells in WT tumors.
Scale bar, 50 μm.
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Influence of ANG-2 on TIE2-Expressing Monocytes
2-deoxy-D-ribose (42). Previously, we and others reported
that human TAMs express TP and that this correlates
positively with tumor angiogenesis (43, 44). CTSB expression
in human cancers has also been implicated in tumor progression and poor prognosis (reviewed in ref. 45). Recently, TAMs
have been shown to be the major source of CTSB in various
tumor models, and the proteolytic activity of this enzyme is
critical for tumor growth, angiogenesis, and invasion (46, 47).
Consistent with our in vitro results using human TEMs, murine TEMs in ANG-2 DT tumors exhibited greater expression
of TP and CTSB than in WT tumors. ANG-2 upregulation of
both TEM infiltration into tumors and their expression of
such proangiogenic enzymes could explain the increased
number of microvessels in ANG-2 DT compared with WT
tumors. However, the tumor-promoting effect of these
activated TEMs may have been countered by the direct
deleterious effect of ANG-2 overexpression on the maturity
and viability of ECs in ANG-2 DT mice, resulting in similar
growth between tumor types.
We previously showed that human TEMs respond to
ANG-2 by decreasing their expression of TNFα and IL-12
(3). We report here that ANG-2 further stimulates their high
basal level of MRC1 in vitro. Although care needs to be taken
when comparing the responses to ANG-2 of human blood
TEMs in vitro with those of murine tumor TEMs in vivo, it
is interesting to note that MRC1 expression by murine TEMs
was higher in ANG-2 DT than WT tumors. We also found
that ANG-2 upregulated the expression of the potent immunosuppressive factor IL-10 and the regulatory T-cell (Treg)
chemokine CCL17. Interestingly, high levels of IL-10,
CCL17, and MRC1 coupled with low levels of TNFα, IL-12,
and IL-6 expression are indicative of an alternative (“M2”)
activation status in macrophages, a phenotype commonly
associated with TAMs (48, 49). Furthermore, Pucci and
colleagues (5) recently reported that murine tumor TEMs
express a pronounced M2 phenotype. Our data indicate that
ANG-2 in tumors may drive this M2-like polarization of
TEMs. By contrast, ANG-1 induced a completely different
gene expression profile from that seen with ANG-2, suggesting that TEMs can be skewed away from an M2-like phenotype in tissues where ANG-1 is more abundant.
In our study, high levels of ANG-2 expressed by the tumor
vasculature seem to act in an autocrine manner, causing
vascular disruption, as shown by increased EC apoptosis
and consequent tumor hypoxia. Such antivascular effects of
high-dose ANG-2 may override the enhanced, proangiogenic
activity of ANG-2–stimulated TEMs. However, such effects of
ANG-2 may be dose dependent and it is possible that lower
levels of ANG-2 in tumors might have less pronounced antivascular effects so that ANG-2–stimulated TEMs would then
induce further tumor angiogenesis and progression. On the
other hand, the genetic knockout of Ang-2 in tumor models
increases vascular maturation and pericyte coverage, resulting in an “angiostatic” phenotype, possibly from the enhanced availability of TIE2 to bind ANG-1 in the absence
of ANG-2 (26). Our data (Supplementary Fig. S3) also suggest
that, in the absence of ANG-2, ANG-1 stimulation of TEMs
would not induce a proangiogenic or tumor-promoting phenotype. It will be interesting to investigate the effects of
depleting ANG-2 in tumors on these two cell types using
new-generation anti–ANG-2 inhibitors (50). Such studies
are now warranted to see whether the ANG-2/TEM axis is a
suitable target for anticancer therapy, as drugs that selectively
target the ANG-2–TIE2 interaction may impair the biological
activity of both angiogenic ECs and proangiogenic TEMs in
the tumor microenvironment. However, it also remains to
be seen whether other tumor-derived signals also contribute
to shaping the proangiogenic activity and the insidious
functions of TEMs in tumors.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank Dr. Dan Dumont for the ANG-2 DT mice, Dr. Zena Werb for the
MMP-9 antibody, Dr. Edward Schwartz for the TP inhibitor, and Susan Newton
and Kay Hopkinson (University of Sheffield Flow Cytometry Core Facility).
Grant Support
Breast Cancer Campaign, UK (C.E. Lewis, C. Murdoch, and M. De Palma)
and German Research Foundation grant SFB/TR23 [Y. Reiss, K.H. Plate (Project C1), and C. Urbich (Project B5)].
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 01/05/2010; revised 04/09/2010; accepted 04/30/2010; published
OnlineFirst 06/08/2010.
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