HRG inhibits tumor growth and metastasis by inducing macrophage polarization and vessel normalization through downregulation of PlGF.

Abstract

Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like) phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG) inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and vessel normalization, effects known to decrease tumor growth and metastasis and to enhance chemotherapy. Skewing of TAM polarization by HRG relies substantially on downregulation of placental growth factor (PlGF). Besides unveiling an important role for TAM polarization in tumor vessel abnormalization, and its regulation by HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.

Full text

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Cancer Cell
Article
HRG Inhibits Tumor Growth and Metastasis by
Inducing Macrophage Polarization and Vessel
Normalization through Downregulation of PlGF
Charlotte Rolny,1,2,3,10Massimiliano Mazzone,2,3,10So `nia Tugues,1Damya Laoui,4,5Irja Johansson,1Cathy Coulon,2,3
Mario Leonardo Squadrito,6Inmaculada Segura,2,3Xiujuan Li,1Ellen Knevels,2,3Sandra Costa,2,3Stefan Vinckier,2,3
Tom Dresselaer,7Peter A˚kerud,8Maria De Mol,2,3Henriikka Saloma ¨ki,1Mia Phillipson,9Sabine Wyns,2,3Erik Larsson,1
Ian Buysschaert,2,3Johan Botling,1Uwe Himmelreich,7Jo A. Van Ginderachter,4,5Michele De Palma,6
Mieke Dewerchin,2,3Lena Claesson-Welsh,1,11,* and Peter Carmeliet2,3,11
1Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory, 75185 Uppsala, Sweden
2Vesalius Research Center, VIB, Leuven, Belgium
3Vesalius Research Center, K.U.Leuven, Leuven, Belgium
4Laboratory of Cellular Molecular Immunology, Department Molecular Cellular Interactions, VIB, Brussels, Belgium
5Laboratory of Cellular Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
6Angiogenesis & Tumor Targeting Unit, HSR-TIGET, and Vita-Salute University, San Raffaele Scientific Institute, 20132 Milan, Italy
7MoSAIC, K.U. Leuven, Belgium
8Uppsala University, Department Surgical Sciences, University Hospital, 75185 Uppsala
9Uppsala University, Department Medical Cell Biology, Biomedical Center, Uppsala, Sweden
10These authors contributed equally to this work
11These authors contributed equally to this work
*Correspondence: lena.welsh@igp.uu.se
DOI 10.1016/j.ccr.2010.11.009
SUMMARY
Polarization of tumor-associated macrophages (TAMs) to a proangiogenic/immune-suppressive (M2-like)
phenotype and abnormal, hypoperfused vessels are hallmarks of malignancy, but their molecular basis and
interrelationship remains enigmatic. We report that the host-produced histidine-rich glycoprotein (HRG)
inhibits tumor growth and metastasis, while improving chemotherapy. By skewing TAM polarization away
from the M2- to a tumor-inhibiting M1-like phenotype, HRG promotes antitumor immune responses and
vesselnormalization,effectsknowntodecreasetumorgrowthandmetastasisandtoenhancechemotherapy.
SkewingofTAMpolarizationbyHRGreliessubstantiallyondownregulationofplacentalgrowthfactor(PlGF).
BesidesunveilinganimportantroleforTAMpolarizationintumorvesselabnormalization,anditsregulationby
HRG/PlGF, these findings offer therapeutic opportunities for anticancer and antiangiogenic treatment.
INTRODUCTION
Angiogenesis and inflammation are hallmarks of cancer. Tumor
vessels are irregular, disorderly structured, and inefficiently
perfused, thereby impairing perfusion and drug delivery (Jain,
2005). The resulting hypoxia creates a hostile milieu from where
cancer cells escape through a leaky endothelium (Mazzone
et al., 2009). Traditional antiangiogenic ‘‘vessel pruning’’ agents
can aggravate tumor hypoxia and worsen malignancy (Bergers
and Hanahan, 2008). Antiangiogenic ‘‘vessel normalizing’’ strat-
egies are gaining attention, as they can silence metastasis and
improve anticancer therapies. However, the basis of vessel ab-
normalization remains enigmatic.
Infiltration of tumor-associated macrophages (TAMs) is asso-
ciated with an unfavorable prognosis (Qian and Pollard, 2010).
However, not only their numbers but also their phenotype
Significance
M2-like TAMs promote malignancy by suppressing antitumor immune responses and stimulating angiogenesis. Also, since
tumor vessels function poorly, tumor perfusion, oxygenation, and chemotherapy are impaired, while enhancing metastasis.
Traditional antiangiogenic ‘‘vessel pruning’’ strategies can worsen this situation by aggravating hypoxia. Our findings are
significant as (1) they unveil an important role of TAM polarization in vessel abnormalization; (2) they imply that reeducation
of TAM polarization is a promising anticancer treatment strategy; (3) they identify HRG as an anticancer drug target, that
combats malignancy by enhancing immunity and vessel normalization; (4) they stress the potential of antiangiogenic
‘‘vessel normalizing’’ strategies in silencing metastasis; and (5) they provide support for PlGF-blockage for treatment of
cancer.
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regulates tumorigenesis. In nonprogressing or regressing
tumors, TAMs are biased to a classic macrophage activation
M1-like program, characterized by proinflammatory activity,
antigen presentation and tumor lysis. In malignant tumors,
TAMs resemblealternatively
(M2-type), that increase angiogenesis and tumor cell intra/
extravasation and growth; they suppress antitumor immunity
by preventing activation of dendritic cells (DCs), cytotoxic
T lymphocytes (CTLs), and natural killer (NK) cells (Mantovani
and Sica, 2010; Qian and Pollard, 2010). It is unknown if TAMs
regulate tumor vessel abnormalization.
TAMs consist of distinct subsets (Mantovani and Sica, 2010;
Qian and Pollard, 2010), which coexist in tumors, adapt to the
changing milieu, and can be re-educated by immunoregulatory
cues (Movahedi et al., 2010; Pucci et al., 2009). This has primed
interest in developing therapies, aimed at skewing TAMs to an
M1-like phenotype (De Palma et al., 2008). Nonetheless, only few
molecules have been identified to orchestrate this process so far.
In this study, we explored if histidine-rich glycoprotein (HRG),
a host-produced antiangiogenic and immunomodulatory factor,
regulates tumor vessel abnormalization and inflammation, for
various reasons. First, HRG is a multidomain protein that binds
thrombospondins (TSPs), heparin, FcgR receptors and other
molecules, implicated in tumorigenesis (Blank and Shoenfeld,
2008; Jones et al., 2005). Second, HRG is deposited in the tumor
stroma from plasma or platelets (Klenotic et al., 2010; Thulin
et al., 2009), but tumor HRG levels have been only analyzed in
a few human cancers (Klenotic et al., 2010; Simantov et al.,
2001). Third, binding of HRG to its ligands is facilitated by Zn2+
and low pH, conditions found in the tumor milieu. Fourth, HRG
stimulates phagocytosis of dying cells (Poon et al., 2010a), but
it is unknown if it regulates TAM polarization. Fifth, HRG inhibits
tumor growth (Dixelius et al., 2006; Karrlander et al., 2009;
Olsson et al., 2004), but its precise mechanisms remain incom-
pletely understood. Moreover, a role for HRG in metastasis has
not been documented yet.
HRG’s antitumor activity has been ascribed to effects on
tumor vessels, but these reports are not unequivocal. Indeed,
by inhibiting antiangiogenic agents, HRG may stimulate angio-
genesis(Klenoticetal.,2010;Simantovetal.,2001).Ontheother
hand, HRG inhibits endothelial cell (EC) responses. It blocks
binding of FGFs to heparan sulfate, prevents release of angio-
genic factors from the matrix (Jones et al., 2004; Poon et al.,
2010b), and inhibits growth factor-induced EC migration
(Dixelius et al., 2006). Binding of HRG to tropomyosin has also
been proposed to underly its antiangiogenic activity (Guan
et al., 2004). In tumors, HRG counteracts PDGF-driven angio-
genesis (Karrlander et al., 2009), while HRG deficiency increases
angiogenesis (Thulin et al., 2009). Nonetheless, it has not been
established if these in vitro mechanisms underlie the in vivo anti-
angiogenic effects of HRG in tumors. This is a relevant question,
as HRGaffects other cell types,suchasmacrophages, known to
regulate angiogenesis. Whether HRG regulates tumor angiogen-
esis indirectly through TAMs has not been explored.
The goal of this study was to identify mechanisms mediating
the antitumor effects of HRG, with focus on its previously docu-
mented immune modulatory function (Blank and Shoenfeld,
2008),andtorevealmolecularlinksbetweenHRGandregulators
of pathological angiogenesis.
activatedmacrophages
RESULTS
Expression of HRG in Human Cancer
We immunostained samples from 20 healthy and malignant
human tissues. HRG was prominent in healthy liver and less
abundant around vessels, macrophages, and other cell types
in healthy tissues (Figure 1A). Consistent with reports that HRG
binds to cell surfaces (Hulett and Parish, 2000), a HRG immuno-
reactive signal was detected on other cell types. This signal was
weaker in malignant than healthy cells (especially in hepatocel-
lular carcinoma cells) and weaker in stromal cells of tumors
compared to healthy tissues (Figure 1B; see Figures S1A–S1D
and Table S1 available online). Only in brain tumors was the
HRG signal somewhat stronger than in healthy brain, consistent
with another report (Klenotic et al., 2010), likely because plasma
HRG leaked through the disrupted blood-brain barrier (Fig-
ure S1D). Overall, HRG levels are decreased in human cancer.
Genetic Gain-of-Function Strategy to Study the Role
of HRG in Tumors
To study the role of HRG in cancer, we used a gain-of-function
approach to overcome the decreased tumor HRG levels.
Reasoning that overexpression of HRG by tumor cells would
result in stromal deposition, we transduced T241 fibrosarcoma,
Panc02 pancreatic tumor and 4T1 breast tumor lines with a lenti-
viral vector, encoding human HRG (hHRG+) and GFP; control
cells were transduced with a vector expressing only GFP (Ctrl).
GFP expression was controlled by an internal ribosomal entry
site and therefore not detected by direct fluorescence, neither
on HRG+nor on Ctrl tumor sections. None of the HRG+tumor
lines expressed mouse HRG (not shown), but produced hHRG
in vitro (HRG+T241 tumor: 991 ng/107cells/24 hr) (Figure 1C).
hHRG was deposited in HRG+but not in control T241 tumors
grown in mice (Figures 1D and 1E). When tumors grew to larger
sizes,theHRG+signalbecameweaker (Figures S1E–S1G),while
plasmalevelsofhumanHRGdecreased(from?100mg/mlatday
14 to ?5 mg/ml at day 21). Since GFP remained detectable
by anti-GFP antibodies, it raised the question whether HRG
became degraded by tumor cell-produced proteases; HRG is
indeed partially degraded to inactive fragments by tumors (not
shown). To avoid any confounding interpretation due to degra-
dationofHRG,tumor studieswereperformed duringthe window
of HRG expression. RT-PCR showed that murine HRG was
undetectable in cancer-associated fibroblasts (CAFs), TAMs
and tumor ECs (tECs), sorted from intact tumors (not shown).
HRG Inhibits Tumor Growth and Metastasis
When implanted in wild-type (WT) mice, HRG+tumors grew
slower and metastasized less. HRG reduced the growth of
subcutaneous T241 fibrosarcomas, orthotopic Panc02 pancre-
atic tumors and 4T1 breast tumors by 62%, 26%, and 36%,
respectively (Figures 1F–1I). HRG also decreased Panc02 lymph
node metastasis by 62% (Figure 1J) and 4T1 lung metastasis by
90% (Figure 1K). As HRG decreased the metastatic index
(nodules per gram tumor), the reduced tumor spread was partly
independent of tumor growth inhibition. Since metastatic nodule
formation was reduced, but lodging to the lung was unaffected
(Figure S1H), the decreased metastasis is attributable to
reduced escape from the primary tumor. However, delayed
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growth of HRG+metastases also contributed to the decreased
metastatic burden (Figure S1I). Staining for phosphohistone-
H3 showed that proliferation was reduced in HRG+T241 tumors
(Figures 1L–1N). As HRG+tumor cell proliferation was normal
in vitro (Figure S1J), stromal rather than tumor cell autonomous
mechanisms accounted for the decreased tumor cell prolifera-
tion in vivo. We thus focused on vessel normalization and
antitumor immunity, known to regulate tumor growth and metas-
tasis (Mantovani and Sica, 2010; Mazzone et al., 2009).
HRG Promotes Vessel Normalization and Improves
Tumor Perfusion
We first studied angiogenesis in HRG+tumors, not only because
the reduced growth of HRG+tumors might be caused by
blockage of angiogenesis, but also because the decreased
metastasis of HRG+tumors might be due to ‘‘normalization’’ of
tumor vessels. Staining for the EC marker CD31 showed that
vessel density and average vessel area were comparable in
control and HRG+T241 tumors (Figures 2A–2D). When analyzing
thedistribution ofvesselsize,weobservedashifttowardsmaller
vessels (Figure S2A). Since these parameters do not necessarily
correlate with vessel function, we studied perfusion by delivery
of FITC-conjugated lectin. Nearly double the number of vessels
was perfused in HRG+T241 tumors (Figures 2E–2G). HRG+
tumors were less hypoxic, as assessed by pimonidazole
(PIMO) staining at early (12 days) and late stage (19 days)
(Figures 2H–2J), and displayed smaller necrotic and hemor-
rhagic areas (Figures 2K–2N). MRI confirmed that HRG+tumors
were less necrotic (Figures S2B and S2C). HRG+tumors were
also less apoptotic (Figures 2O–2Q). Since pericyte coverage
improves vessel maturation, we double stained for CD31 and
the pericyte marker a-smooth muscle actin (aSMA) and
Ctrl
HRG
PHH3
M
L
PHH3
***
J
0
20
40
60
Metastatic index
Ctrl HRG
Panc02 lymph node metastasis
*
Metastatic index
K
Ctrl HRG
4T1 lung metastasis
0
2
4
6
*
N
Ctrl HRG
10
5
0
T241 tumor proliferation
Proliferation
index (%)
HRG DAPI
D
E
Ctrl
HRG
HRG DAPI
A
Healthy
HCC
B
Ctrl HRG
75 kDa >
C
IgG >
HRG expression
F
Days
T241 tumor volume
171512 1075
Tumor volume (mm3)
900
700
500
100
300
***
Ctrl
HRG
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0
0.5
1.0
T241 tumor burden
Tumor weight (g)
Ctrl HRG
H
Panc02 tumor burden
Tumor weight (g)
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0
0.2
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Ctrl HRG
4T1 tumor volume
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Tumor volume (mm3)
Days
Ctrl
HRG
10 121517212325
900
700
500
100
300
**
Figure 1. Reduction of Tumor Growth by HRG
(A and B) HRG immunostaining, showing stronger HRG+signal in healthy liver than in hepatocytocellular carcinoma (HCC). Bars: 20 mm.
(C) Production of HRG by HRG+but not control (Ctrl) T241 cells.
(D and E) HRG immunostaining (green) of T241 tumors, showing expression of HRG in HRG+(E) but not control (D) tumors. Bars: 50 mm.
(F and G) T241 model, showing slower growth of HRG+tumors (F; n = 13; ***p < 0.001); similar findings for end-stage tumor weight (G; n = 20; **p < 0.01).
(H and I) Panc02 (H; n = 13; **p < 0.01) and 4T1 (I; n = 8; **p < 0.01) model, showing slower growth of HRG+tumors.
(J and K) Reduced metastasis of HRG+tumors in Panc02 model (J; n = 13; ***p < 0.001; lymph node metastasis) and 4T1 model (K; n = 8; *p < 0.05; pulmonary
metastasis).
(L–N) PHH3 staining, revealing fewer proliferating tumor cells in HGR+(M) than control (L) T241 tumors; (N) proliferation index (PHH3+/total cells) (n = 5; *p < 0.05).
Bars: 20 mm.
Data represent mean ± SEM; statistical significance was assessed by t test. See also Figure S1.
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Lam
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Figure 2. HRG Improves Tumor Vessel Maturation and Normalization
(A–D) Staining for CD31 (red) in control (A) and HRG+(B) T241 tumors. (C and D) Vessel density (C) and area (D; CD31+area, %).
(E–G) Staining for FITC-conjugated lectin (green) and CD31 (red) in control (E) and HRG+(F) T241 tumors. (G) Increase in perfused lectin+CD31+vessels (% of
CD31+vessels) in HGR+tumors (n = 6; ***p < 0.001).
(H–J) Staining for pimonidazole (PIMO; brown), revealing smaller hypoxic regions in HRG+(I) than control (H) tumors. Morphometry (J) revealed reduced PIMO+
area (% of tumor area) in HRG+T241 tumors (n = 8; **p < 0.01; ***p < 0.001).
(K–N) H&E staining, showing less hemorrhaging (M; arrows) and necrosis (N) in HRG+(L) than control (K) T241 tumors. (M and N) Analysis of necrotic and hemor-
rhagic area (% of tumor area) (n = 7; *p < 0.05, **p < 0.01).
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observed an increased pericyte coverage of tumor vessels in
HRG+tumors (Figures 2R–2T); similar numbers of PDGFRa+
cancer-associatedfibroblast/myofibroblasts
present (not shown). HRG+tumor edema was also reduced
([wet-dry weight]/dry weight, 3100: 80 ± 2 for controls versus
73 ± 2 for HRG+; n = 5; p = 0.06).
Further characterization of the tumor vasculature in orthotopic
Panc02 tumors confirmed that HRG promoted vessel normaliza-
tion. HRG+tumors contained fewer ‘‘empty sleeves,’’ i.e.,
channels with a laminin+basement membrane devoid of
CD31+ECs (Figure 2U). HRG also improved the stability and
tightness of the EC layer, since the junctional molecule VE-cad-
herin was continuous over longer distances and more abundant
in HRG+tumor vessels (Figures S2D–S2G). Scanning electron
microscopy (SEM) showed that fewer vessels in HRG+Panc02
tumors contained abnormal multilayers of disconnected ECs
with multiple protrusions (Figures 2V and 2W). Administration
of the cytotoxic agent doxorubicin at a suboptimal dose
(2.5 mg/kg, 33/wk) was ineffective in reducing growth of control
T241 tumors, but decreased HRG+T241 tumor growth by 50%
(Figure 2X). Overall, HRG improved tumor vessel maturation
and perfusion, reduced hypoxia and improved chemotherapy.
Also, the tighter EC barrier is known to reduce metastasis (Maz-
zone et al., 2009). The improved tumor vessel normalization and
maturation could thus explain the antimetastatic activity of HRG.
(CAFs)were
Effects of HRG on Tumor-Associated Macrophage
Polarization
Since the vascular changes alone unlikely explained the inhibi-
tion of tumor growth by HRG, we explored if HRG affected the
immune response. We focused on TAMs, as their accumulation
correlates with tumor progression. However, F4/80+TAM accu-
mulation was only slightly increased in HRG+T241 tumors
(Figures 3A–3C), and comparable in HRG+Panc02 (see below)
and 4T1 (not shown) models. HRG+T241 tumors had compa-
rable numbers of myeloid cells (CD11b+cells, percentage of
viable cells: 15 ± 3 for control versus 20 ± 1 for HRG+; n = 3;
p = 0.18) or contained slightly more CD11b+F4/80+macro-
phages (12 ± 2 for control versus 16 ± 0.2 for HRG+; n = 3;
p = 0.01). Levels of Ccl2 (also known as Mcp1), Csf2 (Gm-csf),
and Csf1 (M-csf), i.e., chemotactic cytokines for macrophages,
were not altered in HRG+tumors (Figures S3A–S3C). Overall,
HRG only caused insignificant changes in the extent of myeloid
cell infiltration.
Because tumor growth was reduced despite persistent TAM
accumulation, we analyzed the phenotype of freshly isolated
TAMs. Protumoral and proangiogenic (i.e., M2-like) TAMs
express elevated levels of the mannosereceptor-1 (MRC1), argi-
nase-1 (Arg1), IL-10 and the chemokines CCL22 and CCL17
(Movahedi et al., 2010; Pucci et al., 2009). Conversely, antitu-
moral and proinflammatory (i.e., M1-like) TAMs express higher
levels of inflammatory and antiangiogenic cytokines such as
IL-1,IL-6,TypeIinterferons (IFNs),IL-12,andCXCL9(Mantovani
et al., 2002). TNFa marks M1- and M2-oriented TAMs (Hage-
mann et al., 2008; Movahedi et al., 2010; Qian and Pollard,
2010). We found that 2-fold fewer F4/80+TAMs expressed
MRC1 in HRG+T241 tumors (Figures 3A, 3B, and 3D). F4/80+
TAMs from HRG+tumors expressed reduced levels of other
M2-type genes (Figures 3E and 3F), as well as of TNFa (pg/ml:
141 in control versus 72 in HRG+) and IL-10 (64 pg/ml for control
versus 24 pg/ml in HRG+).
Conversely, F4/80+TAMs from HRG+T241 tumors upregu-
lated the M1-type genes Cxcl9 (Figure 3F), IFN-b (mRNA
copies/104Hprt mRNA copies: 2.7 ± 0.02 for control versus
8.7 ± 2.0 for HRG+; n = 4; p = 0.04) and IL-6 (pg/ml: 182 in control
versus 644 in HRG+). Expression of IL-12 also tended to be
upregulated (mRNA copies/103Hprt mRNA copies: 3.7 ± 0.6
for control versus 24 ± 16 for HRG+; n = 5; p = 0.10). Although
IL-1b is regarded as an M1 marker, it also has angiogenic and
metastatic activity (Arteta et al., 2010); in line with the antiab-
normalization/metastatic effects of HRG, IL-1b levels were
reducedinTAMsfromHRG+tumors(Figure3G).Similarfindings,
including downregulation of Ccl17, were obtained when
analyzing F4/80+CD11c+cells (Figures S3D–S3F), which mostly
comprise M1-type TAMs (Movahedi et al., 2010; Pucci et al.,
2009). Overall, HRG skewed TAM polarization away from the
M2-like phenotype.
Mechanistic analysis revealed that HRG skewed TAM polari-
zation via direct effects, as exposure of peritoneal macrophages
(pMØs)toHRGupregulatedCxcl9andIFN-banddownregulated
expression of Ccl22 and IL-10 (Figures S3G–S3J). However,
the M1-skewed TAM polarization in response to HRG was
not attributable to an increase in circulating inflammatory
(CD115+CD11b+Gr1high)over‘‘resident’’(CD115+CD11b+Gr1low)
monocytes(FigureS3K).Also,thelowernumberofMRC1+TAMs
inHRG+T241tumorswasnotduetoapoptosisofthispopulation,
as shown by comparable numbers of TUNEL+or 7AAD+
MRC1+F4/80+cells upon immunostaining of tumor sections or
flow cytometry of TAMs, respectively (Figure S3L; not shown).
Additional Effects of HRG on Tumor Immunity
HRG also promoted the host-antitumor immune response by
affecting dendritic cells (DCs). Indeed, HRG+tumors contained
more CD11c+cells (Figures 3H–3J; Figure S3M), the majority
(O–Q) Staining for cleaved caspase-3 (red) revealed fewer apoptotic cells in HRG+(P) than control (O) T241 tumors; (Q) apoptotic index (caspase-3+/ total cells)
(n = 6; ***p < 0.01).
(R–T) Double staining for CD31 (red) and a-SMA (green), showing more pericyte-covered tumor vessels in HRG+(S) than control (R) tumors, quantified in (T)
(a-SMA+CD31+vessels, percentage of CD31+vessels; n = 6; ***p < 0.001).
(U) Counting of vessels, stained for laminin and CD31, showing fewer ‘‘empty sleeves’’ in HRG+Panc02 tumors (n = 5; *p < 0.05).
(VandW)SEMmicrographs,showingabnormaltumorvesselcontainingmultilayersofdisconnectedECswithluminalprotrusionsincontroltumor(V)andnormal-
ized vessel, lined by monolayer of cobblestone ECs in a HRG+Panc02 tumor (W). Numbers below panels indicate fraction of abnormal vessels (% of vessels
analyzed) (n = 5; *p < 0.0.5).
(X) Doxorubicine (Doxo) treatment of T241 tumor-bearing mice, showing that a suboptimal dose did not affect growth of control tumors (Ctrl), but inhibited
growth of HRG+T241 tumor growth (n = 6; *p < 0.05). In the absence of doxorubicin, HRG+tumor growth was significantly suppressed compared to control
(n=6; **p < 0.01). Bars in all tissue section panels: 10 mm.
Data represent mean ± SEM; statistical significance was assessed by t test. See also Figure S2.
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of which coexpressed CD86 (Figures 3H, 3I, and 3K; Figure S3N)
and MHC class II (CD11c+MHC class II+, percentage of CD11b+
cells: 1.3 ± 0.5 in control versus 14.6 ± 1.7 in HRG+; n = 6;
p < 0.001), suggesting that they are activated DCs, though their
molecular signature overlaps with M1-polarized TAMs (Pucci
et al., 2009). Also, antigen-presenting cells from HRG+T241
tumors stimulated allogeneic CD8+T cell proliferation more
than corresponding cells from control tumors (Figure 3L). HRG
stimulated DC activation indirectly, since it did not affect or
only marginally influenced the expression of the DC maturation
markers MHC class II, CD83, or CD86 (not shown).
HRG+T241 tumors also contained higher numbers of CD8+
cytotoxic T lymphocytes (CTLs) (Figures 3M–3O; Figure S3O)
and NK1.1+NK cells (Figure 3P), while CD4+T cells were not
AB
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N
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JK
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Figure 3. Effects of HRG on TAM Polarization
(A–D) Double staining for F4/80 (red) and MRC1 (green), revealing slightly more F4/80+macrophages but fewer F4/80+MRC1+macrophages in HRG+(B) than
control (A) T241 tumors. (C) quantification of F4/80+area (% of tumor area) (n = 5; *p < 0.05); (D) quantification of the F4/80+MRC1+area (% of F4/80+tumor
area) (n = 6; **p < 0.01). Bars: 20 mm.
(E–G) RT-PCR, revealing that FACS-sorted macrophages from HRG+T241 tumors expressed reduced levels of Ccl22 (E), IL-10 and Arg1 (F) and IL-1b (G), while
expressing increased levels of Cxcl9 (F) (n = 5–12; *p < 0.05 and#p = 0.068).
(H–K) Double staining for CD11c (red) and CD86 (green), revealing more CD11c+cells in HRG+(I) than control (H) T241 tumors. (J) Quantification of CD11c+cells
per optical field (n = 5; ***p < 0.01). (K) Higher percentage of CD11c+CD86+cells (% of CD11c+cells; n = 5; ***p < 0.01) in HRG+tumors. Bars: 20 mm.
(L) Mixed leukocyte response (MLR) assay, showing higher induction of CD8+T cell proliferation by Cd11c+F4/80+TAMs from HRG+than Ctrl T241 tumors.
(M–O) Staining for CD8 (green), revealing more cytotoxic CD8+T cells in HRG+(N) than control (M) T241 tumors; (O) quantification of CD8+area (% of tumor area)
(n = 7; ***p < 0.001). CTL, cytotoxic T cell lymphocytes. Bars: 20 mm.
(P) Flow-cytometry, showing more NK1.1+natural killer (NK) cells in HRG+than control T241 tumors (n = 6; *p < 0.05). Cells were gated as viable (7AAD-) CD45+-
CD11b-NK1.1+cells and quantified in percent of total cells after physical gating.
Data represent mean ± SEM; statistical significance was assessed by t test. See also Figure S3.
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
36 Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc.

Page 7

affected (not shown). Immunodepletion of CTLs and NK cells
showed that HRG increased their lytic activity for tumor cells.
Indeed, when administering anti-CD8 antibodies, depletion of
CTLs tended to increase the growth of control tumors by
1.6-fold (p = 0.07) as compared with CTL-complete control
tumors, treated with control IgG (Figure S3P). In contrast, CTL
depletion enhanced the growth of HRG+T241 tumors by up to
5-fold (p = 0.01; Figure S3P). Analogous studies with anti-NK1.1
antibodies revealed that NK-depleted HRG+tumors grew 7-fold
faster than NK-complete HRG+tumors treated with control IgG
(FigureS3P).AlthoughHRGitselfdidnotaffectTcellproliferation
(not shown), TAMs from HRG+tumors upregulated IL-6, IL-12,
and IFN-b, which are known activators of T cells and NK cells,
suggesting that HRG induced CTLs/immune responses indi-
rectly via skewing of TAM polarization. Overall, HRG promoted
a Th1/M1-skewed antitumor immune response, known to inhibit
tumor growth.
TAM Depletion Abrogates the Effects of HRG in Cancer
To underscore that TAM polarization inhibited tumor growth,
metastasis, and vessel abnormalization, we treated tumor-
bearing mice with clodronate liposomes to chemically deplete
TAMs (using vehicle liposomes as control), and compared its
effects in control tumors (containing more M2-skewed TAMs)
with HRG+tumors (containing more M1-skewed TAMs). Elimina-
tion of TAMs decreased the growth of control tumors, indicating
that TAMs were predominantly of the M2-like, tumor-promoting
phenotype in these conditions. Vehicle liposome-treated HRG+
T241 tumors were smaller, but depletion of TAMs by clodronate
liposomes enhanced their growth (Figure 4A), showing that HRG
skewed TAMs to a more tumor-inhibitory M1-like phenotype.
Clodronate also reduced dissemination of control tumors
(Figure S4), indicating that TAMs exerted a prometastatic
activity, in accordance with their M2-like phenotype. Metastasis
ofvehicleliposome-treated HRG+tumorswasreduced,butTAM
depletion did not further affect metastasis (Figure S4), indicating
that M1-polarized TAMs in HRG+T241 tumors did not (promi-
nently) regulate this process.
We also examined if TAMs participated in tumor vessel
abnormalization, and if such a role was related to their M1/M2-
like phenotype. In control tumors, clodronate improved tumor
perfusion (Figures 4B, 4D, and 4G) and oxygenation (Figures
4H, 4J, and 4L). Perfusion and oxygenation were improved in
vehicleliposome-treated HRG+tumors,butTAMdepletionfailed
Lectin CD31 vessels (%)
A
G
FCB
ED
HI
JK
L
Figure 4. Effects of Macrophage Depletion on the Effects of HRG
(A) Treatment with clodronate reduced control4T1 tumor growth and enhanced HRG+tumor growth(relative tovehicle liposome controls). Similar effects in T241
tumors (not shown) (n = 10; p < 0.001, 2-way ANOVA; **p < 0.01, ***p < 0.001 versus control, Bonferroni post hoc test).
(B–G) Staining for CD31 (red) and lectin (green), revealing perfused CD31+lectin+vessels and nonperfused CD31+lectin-vessels in control (B and D) and HRG+(C
and E) T241 tumors in mice, treated with vehicle (B and C) or clodronate (D and E) liposomes. (F) Quantification of CD31+vessels revealed comparable density in
control and HRG+tumors in mice receiving vehicle or clodronate liposomes. (G) Quantification of CD31+lectin+vessels (% of CD31+vessels) revealed a larger
fraction of perfused vessels in HRG+tumors in mice receiving vehicle liposomes. In clodronate-treated mice, the fraction of perfused vessels was increased
comparably in control and HRG+tumors. (F and G) n = 5; **p < 0.01 and ***p < 0.001 versus Ctrl vehicle. Bars: 50 mm.
(H–L) Immunostaining for pimonidazole (PIMO; brown), revealing PIMO+hypoxic areas in control (H and J) and HRG+(I,K) T241 tumors in mice, treated with
vehicle (H and I) or clodronate (J and K) liposomes. (L) Quantification of PIMO+area (percentage of tumor area) revealed smaller hypoxic regions in HRG+tumors
in mice receiving vehicle liposomes. In clodronate-treated mice, hypoxic tumor regions were decreased comparably in control and HRG+tumors. (L) n = 5;
*p < 0.05, **p < 0.01 and ***p < 0.001 versus Ctrl vehicle. Bars: 100 mm.
Data represent mean ± SEM; statistical significance was assessed by t test, or as indicated. See also Figure S4.
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc. 37

Page 8

to further affect these parameters (Figures 4C, 4E, 4G, 4I, 4K,
and 4L), showing that M1-polarized TAMs did not (prominently)
regulate tumor vessel abnormalization. No effects on vessel
density were observed in either condition (Figure 4F). Thus, the
antivessel abnormalization activity of HRG relied on its ability
to skew TAM polarization away from the M2-like provessel ab-
normalization phenotype.
HRG Downregulates the Expression of PlGF by TAMs
We then searched for a possible downstream target of HRG that
mediates TAM polarization, and analyzed the expression of
candidate genes by treating pMØs with HRG. HRG decreased
the expression of Plgf (Figure 5A), but not of other angiogenic
regulators (Figure S5A). This downregulation was also observed
inTAMs,freshlyisolatedfromHRG+T241tumors(Figure5B),but
it was cell type specific, as HRG failed to alter Plgf levels in ECs,
CAFs, or tumor cells (Figure 5C; not shown). Not unexpectedly,
the profile of other genes, such as Vegf and Pdgfb, in pMØs
treated with HRG differed from that in TAMs from HRG+tumors,
as the latter sensed not only the direct effect of HRG but also the
indirect environmental changes induced by HRG (Figure S5B).
Role of PlGF in the Antitumor/Antimetastatic Activity
of HRG
To investigate if downregulation of TAM-derived PlGF mediated
the antitumor activity of HRG, we compared control and HRG+
Panc02 tumor cells in WT and PlGF-deficient (PlGF?/?) mice.
In WT mice, growth of HRG+tumors was impaired (Figure 5D).
GrowthofcontroltumorswasslowerinPlGF?/?mice(Figure5D),
in accordance with findings that stromal PlGF promotes tumor
growth (Carmeliet et al., 2001). However, in PlGF?/?mice,
growth of HRG+and control Panc02 tumors was reduced to
the same extent (Figure 5D), indicating that HRG did not further
suppress tumor growth in the absence of host-derived PlGF.
Similarobservationsweremadewhenanalyzingmetastasis(Fig-
ure 5E). Use of the T241 tumor model confirmed that growth of
control and HRG+tumors was comparably reduced in PlGF?/?
mice (Figure 5F). Thus, HRG inhibited tumor growth and metas-
tasis, but only if stromal cells expressed PlGF.
To explore if a myeloid cell type in the tumor stroma was the
responsible PlGF producer, we examined if PlGF in bone
marrow-derived myeloid cells (BMCs) was required for the anti-
tumor activity of HRG and therefore transplanted PlGF?/?bone
marrow in irradiated WT hosts (KO/WT) and, as control, WT
bone marrow in WT recipients (WT/WT). Compared with
WT/WT mice, growth and metastasis of control Panc0 tumors
were reduced in KO/WT mice (Figures S5C–S5E) to the same
extent as for HRG+tumors in WT/WT mice or in KO/WT
mice. Thus, HRG overexpression phenocopied the BMC-
specific loss of PlGF, and HRG could not further affect tumor
growth and spreading in the absence of BMC-produced PlGF,
indicating that HRG blocked tumorigenesis via downregulation
of TAM-produced PlGF.
Role of PlGF in TAM Polarization by HRG
We then investigated if PlGF mediated HRG’s effects on TAM
polarization. Confirming the lack of an effect by HRG on TAM
infiltration, F4/80+TAM accumulation was comparable in control
AC
**
*
PlGF expression in tumor cells
PlGF-/- - Ctrl
PlGF-/- - HRG
B
PlGF downregulation in pMØs
***
PlGF downregulation in TAMs
***
0
0.2
0.4
0.6
NS
**
**
Ctrl HRG
Ctrl HRG
Ctrl HRG
*
Metastatic index
Tumor weight (g)
PlGF transcripts /
104 Hprt transcripts
Tumor growth in PlGF-/- miceMetastasis in PlGF-/- mice
EDF
***
Tumor volume (mm3)
Days8 101315
0
5
10
15
NS
Tumor growth in PlGF-/- mice
1
1
Ctrl HRG
WT
Ctrl HRG
PlGF-/-
1
Ctrl HRG
WT
Ctrl HRG
PlGF-/-
1
** **
WT - Ctrl
WT - HRG
**
0
5
10
0
8
12
4
500
300
100
0
0
20
40
60
PlGF transcripts /
103 Hprt transcripts
PlGF transcripts /
103 Hprt transcripts
NS NS
*
Figure 5. Role of PlGF in the Antitumor/Metastatic Activity of HRG
(A–C) RT-PCR, showing reduced Plgf transcript levels in pMØs treated with HRG (n = 4; **p < 0.01) (A), and in F4/80+TAMs from HRG+T241 tumors (n = 8–12;
*p < 0.05) (B), but not in GFP+tumor cells (n = 8–12) (C).
(D and E) Analysis of tumor growth (D) and metastatic index (E) in Panc02 tumor model, revealing reduced growth and metastasis of HRG+tumors in WT mice;
growthandmetastasisofcontrolandHRG+tumorswasreducedcomparablyinPlGF?/?mice(n=17;**p<0.01versuscontroltumorsinWTmice).Datain(D)and
(E) represent mean ± SEM; statistical significance was assessed by t test.
(F) Similar findings were observed in the T241 tumor model (n = 7, *p < 0.05, ***p < 0.001 by 2-way ANOVA). See also Figure S5.
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
38 Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc.

Page 9

and HRG+tumors in PlGF?/?mice (Figures 6A, 6C, and 6E).
In contrast, loss of stromal PlGF affected TAM polarization. In
control tumors, MRC1 was expressed in fewer F4/80+TAMs in
PlGF?/?mice (Figures 6A, 6C, and 6F), but the number of F4/
80+MRC1+TAMs was reduced to the same extent in control
and HRG+tumors in PlGF?/?mice (Figures 6B, 6D, and 6F).
F4/80+TAMs, sorted from control tumors in PlGF?/?mice or
HRG+tumors in WT mice, expressed lower levels of the M2
genes Ccl22, IL-10, and Arg1; this profile was not further altered
in HRG+Panc02 tumors in PlGF?/?mice (Figures 6G–6I). Similar
findings were obtained when analyzing F4/80+CD11c+cells (Fig-
ures S3D–S3F). In accordance with findings that the increased
infiltration of DCs and NK cells in HRG+T241 tumors in WT
mice resulted from TAM polarization, more of these cells infil-
trated control tumors in PlGF?/?mice (Figures 6J, 6L, 6N, and
6O), showing that PlGF deficiency altered tumor immunity.
However, HRG overexpression did not further affect the infiltra-
tion of these cells in PlGF?/?mice (Figures 6K, 6M–6O).
F
F4/80+MRC1+ area
*****
NSNS
Ctrl
F4/80+ area (%)
(% of F4/80+ area)
E
CtrlHRG
WT PlGF-/-
HRGCtrlHRGCtrl
WTPlGF-/-
HRGCtrl HRGCtrl
0
5
15
25
35
0
5
15
25
35
CD
TAM accumulation M2-TAM accumulation
***
F4/80
MRC1
HRG
ABF4/80
MRC1
F4/80
MRC1
F4/80
MRC1
PlGF-/-
WT
**
1
*
*
*
QRP
**
Cxcl9 transcripts /
104 Hprt transcripts
**
*
*
*
WTPlGF-/-
HRGCtrlHRGCtrl
WTPlGF-/-
HRGCtrlHRGCtrl
WTPlGF-/-
HRGCtrlHRGCtrl
NS NS
NSNS
NSNS
0
20
60
100
0
200
600
400
pMØ polarization in PlGF-/- micepMØ polarization in PlGF-/- micepMØ polarization in PlGF-/- mice
Ccl22 transcripts /
104 Hprt transcripts
IL-10 transcripts /
104 Hprt transcripts
0
20
60
100
NO
WTPlGF-/-
HRGCtrlHRGCtrl
WTPlGF-/-
HRGCtrl HRGCtrl
CD11c+ area (%)
*
*
**
NS
NS
0
4
8
12
NK1.1 cells
(% of total cells)
*
*
*
NS
NS
0
2
4
6
Dendritic cell accumulationNK cell accumulation
CD11c
HRG
CD11c
Ctrl
CD11c
HRG
CD11c
JKLM
Ctrl
PlGF-/-
WT
IGH
*
**
**
IL-10 transcripts /
103 Hprt transcripts
Arg1 transcripts /
103 Hprt transcripts
Ccl22 transcripts /
105 Hprt transcripts
#
##
TAM polarization in PlGF-/- mice
100
NSNS
0
200
400
600
WTPlGF-/-
HRGCtrlHRGCtrl
WTPlGF-/-
HRGCtrl HRGCtrl
0
NSNS
WT PlGF-/-
HRG CtrlHRG Ctrl
NS
NS
**
**
0
400
800
TAM polarization in PlGF-/- mice TAM polarization in PlGF-/- mice
20
60
#
Figure 6. HRG Attenuates the Proangiogenic M2 Gene Profile
(A-F)Staining of Panc02 tumorsfor F4/80(green)and MRC1 (red), revealing MRC1+/F4/80+(yellow)macrophagesincontrol(A and C)and HRG+(BandD) tumors
in WT (A and B) and PlGF?/?(C and D) mice. (E and F) Quantification revealed similar F4/80+TAM accumulation (E; F4/80+area, percentage of tumor) in each
condition, but fewer MRC1+/F4/80+TAMs in HRG+tumors in WT mice (F); infiltration of MRC1+/F4/80+TAMs was comparably reduced in control and HRG+
tumors in PlGF?/?mice (F; n = 5; **p < 0.01 and ***p < 0.001 versus control tumors in WT mice). Bars: 20 mm.
(G–I) RT-PCR, showing lower expression of Ccl22 (G), IL-10 (H), and Arg1 (I) in flow-sorted F4/80+TAMs from HRG+than control (Ctrl) T241 tumors in WT mice;
expression of these genes was comparably decreased in control and HRG+tumors in PlGF?/?mice (n = 5–10;#p = 0.07; *p < 0.05; **p < 0.01, versus control
tumors in WT mice).
(J–N) Staining of Panc02 tumors for CD11c (red), revealing dendritic cells in control (J and L) and HRG+(K and M) tumors in WT (J and K) and PlGF?/?(L and M)
mice.(N)QuantificationrevealedmoreCD11c+cells (CD11c+area,percentage oftumorarea)inHRG+tumorsinWTmice;infiltrationofCD11c+cellswascompa-
rably increased in control and HRG+tumors in PlGF?/?mice (N; n = 5; *p < 0.05, **p < 0.01 versus control tumors in WT mice). Bars: 20 mm.
(O) Flow sorting of NK1.1+cells, revealing larger fraction of NK cells in HRG+T241 tumors in WT mice; infiltration of NK1.1+cells was comparably increased in
control and HRG+tumors in PlGF?/?mice (n = 6; *p < 0.05).
(P and Q)RT-PCR, showingreduced expression of Ccl22(P) and IL-10 (Q) in HRG-treated WT pMØs; expressionwascomparablyreducedinCtrlor HRG-treated
PlGF?/?pMØs (n = 3–6; *p < 0.05, **p < 0.01 versus control WT).
(R) RT-PCR, showing upregulation of Cxcl9 in HRG-treated WT pMØs; expression of Cxcl9 was comparably increased in control or HRG-treated PlGF?/?pMOs
(n = 4; **p < 0.01 versus control WT).
Data represent mean ± SEM; statistical significance was assessed by t test.
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc. 39

Page 10

We also assessed if autocrine production of PlGF by TAMs
controlled their polarization. PlGF?/?pMØs expressed lower
levels of Ccl22 and IL-10 (Figures 6P and 6Q; Figures S3I and
S3J), but higher levels of Cxcl9 and IFN-b (Figure 6R; Figures
S3G and S3H). Moreover, WT pMØs treated with anti-PlGF anti-
bodies displayed a similar M1-skewed profile as PlGF?/?pMØs
(Figures S3G–S3J). HRG did not further affect the expression of
M2- or M1-specific genes in PlGF?/?pMØs or in WT pMØs
treated with anti-PlGF (Figures 6P–6R; Figures S3G–S3J). Over-
A
0
100
200
300
E
α-SMACD31 F
αSMACD31
C
D
J
H
G
PL
LECTINCD31
Ctrl
M
LECTIN CD31
HRG
N.
LECTINCD31O
LECTIN CD31
Lectin+ CD31+
vessels (%)
Lam+ CD31-
vessels (%)
WTPlGF-/-
HRGCtrlHRG Ctrl
Vessel density in PlGF-/- mice
Pericyte-covered vessels in PlGF-/- mice
Empty sleeves in PlGF-/- mice
α-SMA+ vessels (%)
*
**
NS
WTPlGF-/-
HRGCtrl HRGCtrl
40
30
0
NS
WTPlGF-/-
HRG
Ctrl
HRG
Ctrl
40
10
20
30
0
*
*
*
NS
NS
Vessel perfusion in PlGF-/- mice
WTPlGF-/-
HRGCtrlHRG Ctrl
0
20
80
60
40
*
*
*
NS
NS
K
Abnormal vessels (%)
Vessel abnormalization in PlGF-/- mice
*
*
**
NS
NS
WTPlGF-/-
HRGCtrlHRGCtrl
0
100
50
I
Vessels / mm2
20
10
B
Vessel area (%)
Vessel area in PlGF-/- mice
8
0
2
4
6
WTPlGF-/-
HRGCtrlHRGCtrl
Ctrl
HRG
Ctrl
HRG
WT
PlGF-/-
CtrlHRG
CtrlHRG
PlGF-/-
WT
PlGF-/-
PlGF-/-
*
αSMACD31 αSMACD31
αSMACD31
Figure 7. Improved Tumor Vessel Maturation and
Normalization in PlGF–/–Mice
(A and B) Immunostaining for CD31, revealing similar
vessel density (A) and area (B; CD31+area, percentage
of tumor area) in control and HRG+Panc02 tumors in WT
or PlGF?/?mice (n = 5).
(C–G) Double staining for CD31 (red) and a-SMA (green),
revealing pericyte-coated CD31+aSMA+vessels in control
(C and E) and HRG+(D and F) tumors in WT (C and D) and
PlGF?/?(E and F) mice. (G) Quantification revealed more
CD31+aSMA+vessels (percentage of CD31+vessels) in
HRG+tumors in WT mice; the fraction of CD31+aSMA+
vessels was comparably increased in control and HRG+
Panc02 tumors in PlGF?/?mice (n = 7; *p < 0.05,
**p < 0.01 versus control tumor in WT).
(H) Counting of vessels, double stained for laminin and
CD31 showed fewer ‘‘empty sleeves’’ (laminin+structures
devoid of a CD31+EC lining) in HRG+tumors in WT mice;
the fraction of empty sleeves was comparably decreased
in control and HRG+Panc02 tumors in PlGF?/?mice
(laminin+CD31–vessels, percentage of laminin+vessels;
n = 5; *p < 0.05 versus control tumor in WT).
(I–K) SEM micrographs, showing normalized vessel, lined
by monolayer of cobblestone-like ECs, in a control (I)
and HRG+(J) Panc02 tumor in PlGF?/?mice. (K) Quantifi-
cation revealed fewer abnormalized vessels (percentage
of vessels analyzed) in HRG+than control tumors in WT
mice; the fraction of abnormalized vessels was compa-
rably decreased in control and HRG+tumors in PlGF?/?
mice (n = 5; *p < 0.05 **p < 0.01 versus control tumors in
WT mice).
(L–P) Staining for CD31 (red) and perfusion dye FITC-
conjugated lectin (green), revealing perfusedCD31+lectin+
vessels and nonperfused CD31+lectin-vessels in control
(L and N) and HRG+(M and O) Panc02 tumors in WT
(L and M) and PlGF?/?(N and O) mice. (P) Quantification
showed more CD31+lectin+
CD31+vessels) in HRG+tumors in WT mice; the fraction
of CD31+lectin+vessels was comparably increased in
control and HRG+tumors in PlGF?/?mice (n = 5;
*p < 0.05 versus control tumors in WT mice).
Data represent mean ± SEM; statistical significance was
assessed by t test. Bars: 10 mm in C–F, I, J; 50 mm in
L–O. See also Figure S6.
vessels (percentage of
all, genetic loss or pharmacologic blockage of
PlGF as well as downregulation of PlGF by
HRGskewed polarization
away from the M2-like phenotype.
of pMØs/TAMs
Role of PlGF in HRG-Mediated Vessel
Normalization
We also explored if PlGF downregulation acted
downstreamof HRG in regulating
normalization.Vesseldensityandaveragesizewerecomparable
in WT and PlGF?/?mice, both for control and HRG+Panc02
tumors (Figures 7A and 7B). Also, the same changes in the distri-
bution of vessel area detected in HRG+tumors in WT mice were
observed both in control and HRG+tumors in PlGF?/?mice (Fig-
ure S2A). Blocking stromal- or tumor-produced PlGF inhibits
tumor angiogenesis and TAM accumulation (Fischer et al.,
2007; Van de Veire et al., 2010); however, the normal vessel
density and infiltration of TAMs in the absence of stromal PlGF
vessel
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
40 Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc.

Page 11

were likely rescued by production of PlGF (or other factors) by
tumor cells (Coenegrachts et al., 2010); also, angiogenesis and
TAM infiltration in certain tumor models are PlGF independent
(Van de Veire et al., 2010).
However, other parameters of vessel function in control
tumors were affected by stromal loss of PlGF, similar as in
HRG+Panc02 tumors in WT mice. Indeed, in control tumors in
PlGF?/?mice, vessel maturation was improved, while vessel re-
modeling was reduced (Figures 7C, 7E, 7G, and 7H). Similar
changes were observed in HRG+Panc02 tumors in PlGF?/?
mice, indicating that HRG was unable to further regulate these
processes in the absence of stromal PlGF (Figures 7C–7H).
Also, SEM revealed that the fraction of vessels, lined by an
abnormal EC layer, was comparably decreased in control and
HRG+Panc02 tumors in PlGF?/?mice, to the same extent as
in HRG+tumors in WT mice (Figures 7I–7K; see Figures 2V and
2W for comparison). Similar findings were made when analyzing
the number of perfused vessels (Figures 7L–7P) and the tight-
ness of the EC barrier (Figure S6).
Bone marrow transplantation studies revealed that PlGF in
BMCs was the target of HRG’s activity to improve vessel perfu-
sion. Indeed, the decrease in hypoxia in HRG+tumors in WT/
WT mice also occurred in control tumors in KO/WT mice, while
hypoxia was not further decreased in HRG+tumors in KO/WT
mice (Figure S7). Thus, loss of stromal BMC-derived PlGF phe-
nocopied the effects of HRG overexpression in WT mice, but
HRG did not further affect these processes in PlGF?/?or KO/
WT mice, suggesting that PlGF acted downstream of HRG in
regulating vessel abnormalization.
Finally, in vitro experiments established that HRG affected
ECsindirectlyviaTAMpolarizationinaPlGF-dependentmanner.
Indeed, HRG did not affect EC proliferation or migration, while
these responses were reduced by conditioned medium from
HRG-treated WT pMØs and from PlGF?/?pMØs (Figures 8A
and 8B) or from anti-PlGF-treated WT pMØs (all M1 skewed;
not shown). EC migration and proliferation were not further
reduced by HRG treatment of PlGF?/?pMØs (Figures 8A and
8B).Overall,HRGinducedvesselnormalizationprimarilyviaindi-
rect effects through TAM polarization, rather than via direct
effects on ECs, and PlGF acted downstream of HRG in this
process.
DISCUSSION
Here, we report that HRG, a host-produced protein deposited in
the tumor stroma, combats tumor progression and dissemina-
tion by enforcing the anticancer immune response and
promoting tumor vessel normalization, respectively. Critically
underlying these activities is the ability of HRG to skew TAM
polarization away from their proangiogenic /immune-suppres-
sive M2-like phenotype.
HRG Induces Changes in TAM Phenotypes
Macrophage depletion showed that TAMs mediated the anti-
tumor effects of HRG. While not affecting infiltration, HRG
skewed TAM phenotypes. Indeed, depletion of TAMs decreased
progression of control tumors, while increasing growth of HRG+
tumors, implying that TAMs acquired a tumor-suppressive M1-
like phenotype when exposed to HRG. Expression profiling
andimmunophenotypingconfirmedTAMskewingbyHRG,while
in vitro experiments showed that HRG affected polarization via
direct effects. In the tumor milieu, HRG might also influence
TAM polarization indirectly via effects on oxygenation. Since
hypoxia stimulates M2-like polarization (Lewis et al., 2007),
improved oxygenation in HRG+tumors can provide a self-rein-
forcing stimulus for further polarizing TAMs away from an M2-
like phenotype.
Rather than reprogramming TAMs to an ‘‘M1-only’’ profile in
an all-or-none fashion, HRG induced a ‘‘HRG-specific’’ polariza-
tion signature. Indeed, HRG downregulated established M2
markers (Arg1, IL-10, MCR1, CCL17, CCL22), but also induced
changes in gene expression, that at first sight may appear
more atypical. For instance, HRG reduced the expression of
TNFa and IL-1b, cytokines with a proinflammatory activity.
However, M1-TAMs may express reduced levels of TNFa(Hage-
mann et al., 2008; Movahedi et al., 2010) and IL-1b (Mantovani
and Sica, 2010), in line with our findings. Moreover, since IL-1b
promotes angiogenesis and metastasis (Arteta et al., 2010), its
reduced levels in HRG+tumors could contribute to the
decreased metastasis and enhanced vessel normalization.
Overall, the HRG-induced polarization signature endowed
TAMs with an ability to inhibit tumor (vessel) growth and
metastasis.
***
**
**
***
*
+
+
EC Proliferation (% of pMO-WT)
NS
NS
***
NS
NS
+
-
-
A
Migrated ECs / optical field
B
Endothelial cell migrationEndothelial cell proliferation
+
-
-
-
+
-
-
+
-
-
--
-
+
-
-
-
-
--
-
+
-
-
-
-
pMØ - WT
pMØ - WT + HRG
pMØ - PlGF-/-
pMØ - PlGF-/- + HRG -
0
20
80
40
60
100
80
40
0
60
20
pMØ - WT
pMØ - WT + HRG
pMØ - PlGF-/-
pMØ - PlGF-/- + HRG
Figure
Inhibits EC Responses
(A) ECs were stimulated in vitro with medium, conditioned
by vehicle- or HRG-treated WT pMØs (pMØ-WT and
pMØ-WT+HRG, respectively),
HRG-treated PlGF?/?pMØ (pMØ-PlGF?/?
PlGF?/?+HRG, respectively). EC proliferation was only
minimally affected by HRG directly (not shown), but in-
hibited by medium conditioned by pMØ-WT+HRG,
pMØ-PlGF?/?, and pMØ-PlGF?/?+HRG. y axis: number
of ECs, percentage of ECs stimulated with medium condi-
tioned by pMO-WT (n = 4; *p < 0.05, **p < 0.01,
***p < 0.001).
(B) Similar results were obtained when analyzing direct
and indirect effects of HRG on EC migration. y axis:
number of ECs per optical field (OF) (n = 3; **p < 0.01
***p < 0.001).
Data represent mean ± SEM; statistical significance was
assessed by t test. See also Figure S7.
8. MacrophagePolarizationbyHRG
orbyvehicle-
or pMØ-
or
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc. 41

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HRG Improves Tumor Vessel Normalization
Untreatable metastasis is often the cause of mortality in cancer
patients. A prominent environmental stimulus of tumor dissemi-
nationishypoxia,resultingfrompoorlyfunctioningabnormalized
tumor vessels. Our findings suggest that HRG inhibited metas-
tasis in part by altering vessel morphology. These changes,
ranging from an increased pericyte coverage, tightened EC
barrier and smoother EC layer, promoted vessel normalization,
perfusion, and oxygenation. Hence, by creating a less hypoxic
milieu, HRG diminished the need for tumor cells to escape. In
addition, by tightening the EC layer, HRG likely created a more
impenetrable barrier for tumor cells to intravasate and spread
to distant tissues.
A noteworthy finding was that the changes in vessel function
were accompanied by subtle changes in vessel numbers,
possibly because vessel branching is more sensitive to changes
in TAM accumulation, which was not affected by HRG. Nonethe-
less, the clodronate studies indicate that depletion of the
predominantly M2-skewed TAMs from control tumors normal-
ized the tumor vasculature, while elimination of the mainly M1-
skewed TAMs from HRG+tumors was ineffective. This suggests
that M2-like TAMs induce vessel abnormalization, while M1-like
TAMs are not/less involved. Although TAMs promote tumor
angiogenesis (De Palma et al., 2007; Qian and Pollard, 2010),
we here link TAM polarization to vessel abnormalization. We
speculate that M2-polarized TAMs render vessels abnormal by
expressing increased amounts or different sets of angiogenic
factors. Also, upregulation of angiogenic M2-cytokines (IL-10,
CCL22, IL-1b, TNFa) or downregulation of angiostatic M1-cyto-
kines (IFN-b, CXCL10, IL-12) could contribute to vessel abnorm-
alization. Though HRG can counteract vessel abnormalization
via direct effects on ECs as well as indirectly via effects on
TAM polarization, the finding that depletion of TAMs abrogated
the vascular effects of HRG suggest that the indirect effects of
HRG via TAM polarization are likely predominant in the tumor
microenvironment in situ.
HRG Promotes Tumor Immunity
HRG also increased the host-antitumor immune response.
Indeed, HRG not only increased tumor infiltration by antigen pre-
sentingDCs,cytolyticNKcellsandcytotoxicT-lymphocytes,but
also enhanced their antigen presentation and tumor cell lysis
potential, immune changes known to inhibit tumor growth
(Mantovani and Sica, 2010). Even though HRG binds to T cells
and stimulates their adhesion in vitro, it is unknown if these
changes activate T cells. Since HRG did not affect T cell prolifer-
ation in vitro (not shown), HRG likely promoted immunity indi-
rectly via effects on TAM polarization. The observed shift in cyto-
kine/chemokine profile is consistent herewith. Indeed, TAMs
from HRG+tumors expressed lower levels of IL-10, known to
inhibit the potential of DCs and macrophages to activate
T cells (Koppelman et al., 1997), while producing higher levels
of IL-6, IL-12, and IFN-b, known to stimulate T cell proliferation
and activation of DCs and NK cells (DeNardo et al., 2010;
Mantovani and Sica, 2010). HRG may further enforce the anti-
tumor response by improving perfusion and thereby tumor influx
of immune effector cells (Hamzah et al., 2008). Moreover, by
facilitating clearance of dying tumor cells (Blank and Shoenfeld,
2008),HRGcouldfurthercontributetotumorshrinkage.Thus,by
enhancingnotonlytheeffectorperformancebutalsotheinfluxof
immune cells, HRG fuels the host-antitumor response.
HRG and PlGF: A Molecular Link
That PlGF downregulation by HRG is relevant was evidenced by
in vitro and in vivo experiments. First, loss of PlGF phenocopied
the inhibitory effects of HRG on tumor growth, metastasis, and
vesselabnormalization.Second,
PlGF?/?macrophages or anti-PlGF-treated WT macrophages
phenocopied HRG’s effects on EC responses. Third, loss or inhi-
bitionofPlGFinmacrophagesphenocopiedtheeffectofHRGon
TAM polarization. Fourth, HRG was ineffective in the absence of
PlGFintheinvitro andinvivoexperiments.However,while these
studies identified PlGF downregulation as a downstream mech-
anism of HRG, HRG might also engage additional pathways
given its multidomain structure and binding characteristics.
Genetic and pharmacological studies implicated PlGF in angio-
genesis and inflammation in pathological conditions (Fischer
et al., 2008; Van de Veire et al., 2010). In contrast, Hedlund
and coworkers reported that overexpression of PlGF in tumor
cells promotes tumor vessel normalization (Hedlund et al.,
2009). This is, however, only an apparent paradox, as overex-
pression of PlGF leads to formation of VEGF/PlGF heterodimers
and therefore a reduction in proangiogenic VEGF homodimers.
The present findings underscore the dual activity of PlGF on
vessels and myeloid cells, but also unveil unknown roles of
PlGF on TAM polarization.
conditionedmediumof
Possible Implications
Our findings have a number of implications: (1) it is tempting to
speculate that deposition of an antiangiogenic/immunomodula-
tory molecule like HRG in the tumor stroma is a host defense
mechanism against the growing cancer and that HRG partici-
pates in the recognition of ‘‘malignant danger,’’ in line with its
presumed role as a ‘‘pattern recognition molecule’’ (Poon
et al., 2010a); the tumor, in turn, may try to escape from this
host-attack by downregulating or degrading HRG; (2) our
findings unveil a role of M2-TAM polarization in vessel abnormal-
ization and imply that re-education of TAM polarization is
a promising anticancer strategy; (3) they further highlight the
potential of antiangiogenic ‘‘vessel normalizing’’ strategies in
silencing metastasis and stress the importance of analyzing
vessel/TAM function rather than their numbers alone; how anti-
abnormalization strategies best fit in current antiangiogenic
therapywarrantsfurtheranalysis;finally,(4)thedataalsoprovide
further support for PlGF-blockage strategies for the treatment of
cancer.
EXPERIMENTAL PROCEDURES
More detailed methods can be found in the Supplemental Experimental
Procedures.
Animals
C57BL/6 mice and Balb/c (8–12 weeks old) were obtained from VIB mouse
facility or from Mollegard/Bomhultgard, Denmark. PlGF-deficient (PlGF?/?)
mice were described previously (Carmeliet et al., 2001). Housing and experi-
mental animal procedures were approved by the K.U. Leuven Animal Care
and Research Advisory Committee and Uppsala University board of animal
experimentation.
Cancer Cell
PlGF in Antitumor Skewing of TAMs by HRG
42 Cancer Cell 19, 31–44, January 18, 2011 ª2011 Elsevier Inc.

Page 13

Tumor Models, Cell Depletion, and BM Transplantation
T241 fibrosarcomaand Panc02 tumor models were described (Mazzone et al.,
2009). Tumor volumes were measured with a caliper (length 3 width23 p /6).
TAM depletion was achieved by using clodronate as described (Mazzone
et al., 2009). CD8+lymphocytes were depleted by the rat anti-CD8 antibody
53.6.72 (25 mg/kg; Bio X Cell) and NK cells by the mouse anti-NK1.1 antibody
PK136 (25 mg/kg; Bio X Cell). T cells purified from Balb/c splenocytes were
cultured with C57BL/6 TAMs, and proliferation was measured. Bone marrow
from PlGF?/?mice was infused in the tail vein of lethally irradiated WT mice,
and 6 weeks later, Panc02 tumor cells were injected orthotopically in the
pancreas.
Histology of Human and Mouse Tissues
Tissue microarrays (TMAs) of healthy and malignant tissues, containing
multiple samples from different humans with the same diagnosis (432 tumor
samples, 20 different cancers) produced by the Human Proteome Atlas
(HPA) facility (http://www.proteinatlas.org) were stained using anti-HRG anti-
serum (#0119) (Dixelius et al., 2006; Kampf and Ostermeyer, 2004; Olsson
et al., 2004), and stained TMA sections were scanned by high-resolution
scanners (ScanScope XT, Aperio Technologies), separated in individual spot
images, and evaluated by experienced pathologists. Ethical permit to use
anonymized,decoded (i.e., nontraceable)human fresh-frozen normal or tumor
tissue for generation of tissue slides or TMAs was granteed by the Uppsala
ethical review board in full agreement with the Swedish Ethical Review Act.
Staining, SEM, and analysis of mouse tissues were as described (Mazzone
et al., 2009).
Lentiviral Vectors
Full-length human HRG cDNA was cloned in the FUGIE lentiviral backbone,
carrying an internal ribosomal entry site; 105tumor cells were transduced
with 106U/ml of the specific lentivirus.
Flow Sorting
Tumors were minced in RPMI medium + 0.1% collagenase I (1 hr; 37?C),
passed through a 19 G needle, and filtered and cells were centrifuged (5
min; 1000 rpm). After RBC lysis, cells were centrifuged and washed with
PBS, incubated with 10% FCS and with anti-F4/80, CD31, CD45, PDGFRa
antibody for 30 min. Tumor cells were sorted as F4/80-/GFP+cells, TAMs as
F4/80+/CD45+cells, tECs as CD31+/CD45-cells, CAFs as PDGFRa+cells.
For FACS analysis, collagenase-digested tumor cells were incubated with
rat anti-mouse FcgIII/II receptor (CD16/CD32) blocking antibodies (4 mg/ml)
to block unspecific binding, labeled with 7-amino-actinomycin D (7-AAD) to
stain nonviable cells and then with the proper antibodies.
pMØS
pMØS were extracted by peritoneal lavage, counted, and plated overnight and
stimulated with appropriate reagents for 4 or 16 hr.
Protein and mRNA Assays
Protein extraction and immunoblot analysis were described (Olsson et al.,
2004); RT-PCR was described (Fischer et al., 2007). For cytokine measure-
ments, ELISAs were performed according to the manufacturer’s instructions.
Tumor Hypoxia, Perfusion, Edema, and Necrosis
Tumor hypoxia (pimonidazole staining) and perfusion (FITC-labeled lectin)
were analyzed as described (Mazzone et al., 2009). Tumor edema was
measured as the wet and dry tumor weight. Tumor necrosis was scored on
H&E-stained sections. We also used MRI to evaluate tumor edema and
necrosis on viable animals at different time points.
Statistics
Data represent mean ± SEM of representative experiments unless otherwise
stated. Statistical significance wascalculatedby t test unless otherwise stated
(Prism v4.0b), considering p < 0.05 as statistically significant.
SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
seven figures, and one table and can be found with this article online at
doi:10.1016/j.ccr.2010.11.009.
ACKNOWLEDGMENTS
This study was supported by the Swedish Cancer Society (3820-B04-09XAC),
Swedish Research Council (K2005-32X-12552-08A), and by a Wallenberg
Scholar grant to L.C.W.; the Swedish Cancer Society (CAN 2008/980) to
C.R.; a European Research Council starting grant to M.D.P.; the Belgian
State–Federal Science Policy Office (IUAP06/30); a Fund for Scientific
Research Flanders (F.W.O.) grant (G.0651.08); and a Susan G. Komen grant
(KG080498), Long-term structural funding–Methusalem funding from the
Flemish Government to P.C.
Received: February 13, 2010
Revised: August 12, 2010
Accepted: October 25, 2010
Published online: January 6, 2011
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