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2006;66:11238-11246. Published OnlineFirst November 17, 2006.Cancer Res
Elaine Y. Lin, Jiu-Feng Li, Leoid Gnatovskiy, et al.
Model of Breast Cancer
Macrophages Regulate the Angiogenic Switch in a Mouse
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Macrophages Regulate the Angiogenic Switch in a Mouse
Model of Breast Cancer
Elaine Y. Lin,
1
Jiu-Feng Li,
1
Leoid Gnatovskiy,
1
Yan Deng,
2
Liyin Zhu,
1
Dustin A. Grzesik,
2
Hong Qian,
3
Xiao-nan Xue,
3
and Jeffrey W. Pollard
1
1
Department of Developmental and Molecular Biology, Center of Reproductive Biology and Women’s Health,
2
Analytical Imaging
Facility, and
3
Department of Epidemiology and Population Health, Albert Einstein Cancer Center, Albert Einstein
College of Medicine, Bronx, New York
Abstract
The development of a tumor vasculature or access to the host
vasculature is a crucial step for the survival and metastasis of
malignant tumors. Although therapeutic strategies attempting
to inhibit this step during tumor development are being
developed, the biological regulation of this process is still
largely unknown. Using a transgenic mouse susceptible to
mammary cancer, PyMT mice, we have characterized the
development of the vasculature in mammary tumors during
their progression to malignancy. We show that the onset of the
angiogenic switch, identified as the formation of a high-
density vessel network, is closely associated with the transition
to malignancy. More importantly, both the angiogenic switch
and the progression to malignancy are regulated by infiltrated
macrophages in the primary mammary tumors. Inhibition of
the macrophage infiltration into the tumor delayed the
angiogenic switch and malignant transition whereas genetic
restoration of the macrophage population specifically in these
tumors rescued the vessel phenotype. Furthermore, premature
induction of macrophage infiltration into premalignant
lesions promoted an early onset of the angiogenic switch
independent of tumor progression. Taken together, this study
shows that tumor-associated macrophages play a key role in
promoting tumor angiogenesis, an essential step in the tumor
progression to malignancy. (Cancer Res 2006; 66(23): 11238-46)
Introduction
Tumor progression is characterized by an initial ‘‘avascular
phase’’ when the tumors are small and usually dormant (1) with
diffusion being the major way to support their metabolic needs (2).
In the subsequent ‘‘vascular phase,’’ the development of a unique
tumor vasculature is required for the increased metabolic demand
of tumors that have grown beyond a certain size. The induction of
this vasculature, termed the ‘‘angiogenic switch’’ (1, 3, 4), can occur
at various stages of tumor progression, depending on the tumor
type and the environment (1). However, it is clear that malignant
tumors require its development as it has been shown that the
initiation of revascularization in dormant lesions allows them to
progress (5, 6).
The stroma of solid tumors are replete with many leukocytic
cells of which macrophages represent a major component (7).
Recent clinical and experimental studies have indicated that these
tumor-associated macrophages promote the progression to
malignancy (7, 8). In human breast cancers, macrophages cluster
in ‘‘hotspots’’ in avascular areas in human breast cancer samples
(9), which correlates with a high level of angiogenesis and with
decreased relapse-free and overall survival of the patients (10).
Macrophages play a crucial role in regulating angiogenesis in
wound healing (11). They produce many proangiogenic factors
including vascular endothelial growth factor (VEGF), tumor
necrosis factor a, granulocyte macrophage colony-stimulating
factor, interleukin (IL)-1, IL-6 (11), and other factors including
matrix metalloproteinases (MMP) and nitric oxide (12, 13) that also
have the potential to regulate angiogenesis (7, 11). Parallels have
been drawn between the microenvironment of wound-induced
inflammation and that of tumors, as proposed in the hypothesis
that tumors are ‘‘wounds that never heal’’ (14). However, whether
tumor-associated macrophages are able to promote angiogenesis is
still not clear.
We have reported in the mouse model of breast cancer caused by
the mammary epithelial cell restricted expression of the Polyoma
middle T oncoprotein (PyMT mice) that the infiltration of
macrophages in primary mammary tumors was positively asso-
ciated with tumor progression to malignancy (8). Depletion of
macrophages in this model severely delayed tumor progression and
dramatically reduced metastasis whereas an increase in macro-
phage infiltration by transgenic means remarkably accelerated
these processes (8). To identify the mechanism(s) that macro-
phages use to promote tumor progression, we have tested the
hypothesis that tumor-associated macrophages stimulate the deve-
lopment of tumor vasculature. Our results indicate that tumor-
associated macrophages were actively involved in promoting the
angiogenic switch during the malignant transition as well as in the
maintenance and/or remodeling of an established vessel network
in malignant tumors.
Materials and Methods
Mice. All procedures involving mice were conducted in accordance with
NIH regulations about the use and care of experimental animals. The study
of mice was approved by the Albert Einstein College of Medicine animal use
committee. The PyMT transgenic mice and mice carrying the CSF-1R-GFP
transgene were kindly provided by Drs. W.J. Muller (McGill University,
Montreal, Quebec, Canada) and David Hume (University of Brisbane,
Brisbane, Australia), respectively. The origin, care, and identification of
CSF-1 null mutant (Csf1
op
/Csf1
op
) mice have previously been described (8).
Because +/Csf1
op
mice have normal serum concentration of CSF-1, normal
tissue population of macrophages, and are in all aspects tested equivalent
to wild-type (+/+) mice (15), these +/Csf1
op
are used as controls. The
preparation of CSF-1-expressing transgenic mice has previously been
described (8). The mice used were in a mixed genetic background of C3H/
B6/FVB. The genotype of the CSF-1R-GFP mice was determined by directly
Requests for reprints: Jeffrey W. Pollard, Albert Einstein College of Medicine, 607
Chanin Building, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-430-2090;
Fax: 718-430-8663; E-mail: pollard@aecom.yu.edu.
I2006 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-06-1278
Cancer Res 2006; 66: (23). December 1, 2006
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visualizing the green fluorescent protein (GFP) expression in blood
monocytes.
In vivo vessel labeling methods. Texas red–conjugated dextran (mol wt
70,000; Molecular Probes, Eugene, OR) was prepared to 6.2 mg/mL in PBS;
21 Ag/g of mouse body weight was i.v. injected. The mice were killed for
tissue preparation 5 minutes after the injection. For the Lycopersicon
esculentum lectin (LEL)/dextran colabeling, 1.3 Ag FITC-conjugated LEL
(Sigma)/g body weight were mixed with dextran solution in the
concentration described above and injected into the tail vein. For analysis,
tumors were isolated and fixed with formalin, paraffin embedded, and
sectioned following standard procedures.
Macrophage labeling and quantitative analysis. Immunohistochem-
istry of macrophage with rat anti-mouse F4/80 antibody was previously
described (8). To measure the macrophage density in tumors, immuno-
histochemically stained sections were photographed to TIFF Images using
an Olympus IX70 microscope and a Sensicam QE cooled CCD camera.
Macrophages in the vicinity of a lesion were measured and normalized by
the circumference of the lesion using ImageJ program. To prepare tissue
sections from CSF-1R-GFP transgenic mice, tissues were fixed in 5%
formalin in 20% sucrose/PBS solution at 4jC for 24 hours followed by
freezing and sectioning. To label macrophages using dextran, 21 Ag/g mouse
body weight of FITC-conjugated dextran (Molecular Probes) were i.v.
injected and tumors were isolated 1 hour after the injection. Using
transgenic mice expressing GFP specifically in macrophages (CSF-1R-GFP
mice), we have observed that within the tumors, only macrophages took up
the injected dextran (data not shown). The standard procedure was used for
immunohistochemistry of anti-VEGF antibody (Santa Cruz Biotechnology,
Santa Cruz, CA) and anti–von Willebrand factor (vWF) antibody (DAKO,
Glostrup, Denmark).
Histologic analysis and vessel density measurements. For quantita-
tive analysis of vessel distribution in the tumor, the entire midline section
from a lesion was photographed into TIFF images and the area in the lesion
marked by dextran was measured using the ImageJ program. For the
comparison of vessel density at different stages of progression classified as
previously described (16), such dextran-marked areas were normalized by
the number of slides for each section and the mean vessel density at
different stages was compared. As previously described (8), a large
percentage of PyMT mice analyzed at 5 to 6, 7 to 8, 9 to 10, or >12 weeks
of age carry mammary lesions at hyperplasia, adenoma/mammary intra-
epithelial neoplasia (MIN), early carcinoma (EC), and late carcinoma (LC)
stages, respectively. To determine the density of the vessels in tumors
(Fig. 5B, iii), the dextran-marked vasculature was skeletonized using ImageJ
program. At least four mice per group were analyzed.
Results
Increased vessel density is associated with the transition to
malignancy. Mammary lesions in PyMT mice progress through
four stages from a benign hyperplasia to an adenoma/MIN and
then to the malignant EC and LC stages (Fig. 1A; ref. 8). A high
frequency of pulmonary metastasis was detected when the primary
mammary tumors progressed to the malignant stages (8, 17). This
stereotypic passage through defined stages provides an excellent
model to study the relationship of the development of the
vasculature according to stage and to test the hypothesis that
tumor-associated macrophages play a key role in angiogenesis.
To characterize the development of the vessels in tumors, PyMT
mice were i.v. injected with Texas red–conjugated, lysine-fixable
dextran 5 minutes before killing. The conjugation of the dextran
with lysine allows cross-linking of the florescent dye–labeled
dextran onto the vessel wall during fixation to permanently mark
the vessels during histologic analysis. This i.v. injection method
gives an accurate and quantitative measurement of vessel density
and architecture in tumors. It also allows to label functional blood
vessels in the tumor (18).
In the passage from hyperplasia to the adenoma/MIN stage
(Fig. 1A), the tumor mass was obviously increased. However, the
density of dextran-marked vessels remained constant (Fig. 1B,
H versus A/M; P = 0.259) and was comparable to the density of the
vessels surrounded the normal ducts (Fig. 1B, duct).
A dramatic change in vessel distribution and density was
observed during the transition from the premalignant to the EC
malignant stage (Fig. 1A; ref. 16). The characteristic feature of the
EC-stage lesion is that in the center of the lesion, the acina-like
structure is replaced by a small solid nodule in which tumor cells
have an increased nuclear pleomorphism and cytologic atypia. The
lesion was classified as the transition stage of malignancy because
such solid nodule(s) with malignant features were only observed in
limited region(s) of the lesion (Fig. 1A, EC, inset), whereas the
majority of the acini were still at the premalignant stages (Fig. 1A,
EC, arrows). The high-density vessel network was only found in the
solid nodular area (Fig. 1A, EC) and not in the surrounding
premalignant acini, in which the vessel density was similar to that
found in lesions at the premalignant stages (Fig. 1A, H and A/M).
Consistent with these histologic observations, a quantitative
analysis indicated a significant increase of vessel density in the
solid nodule compared with the lesions at adenoma/MIN stage
(Fig. 1B, A/M versus EC; P = 0.006). These observations suggest
that such an increase of vessel density is closely associated with
the malignant transition in the tumor.
Further changes in vessel distribution and density were observed
in primary tumors as they progressed to the most advanced
malignant LC stage. A LC-stage lesion consists of multiple large
solid nodules that contain layers of tumor cells with little or no
remaining acina structure and stroma (Fig. 1A, LC). These solid
nodules had both high- and low-density vessel networks but the
overall density of the vessels was similar to that of the EC-stage
lesions (Fig. 1B, EC versus LC).
To confirm that the dextran injection method could precisely
mark blood vessels in tumors, we did a coinjection experiment with
the Texas red–conjugated dextran and FITC-conjugated LEL, which
specifically binds to endothelial cells (19). Using confocal
microscopy, we observed that the majority of dextran-marked
channels in the tumors were colocalized with FITC-LEL staining
(Fig. 2A and B). The representative image showed a FITC-LEL–
labeled vessel (Fig. 2B, b) in the tumor that completely overlapped
with the dextran-marked channel (Fig. 2B, c and d) and the lumen
of the channel was filled with RBC (Fig. 2B, arrows). There were
also some diffuse areas of dextran labeling in cysts or necrotic
areas in large tumors (Fig. 2A, a and c, arrows), suggesting dextran
leakage in these areas. These areas of low density and dull staining
were not included in our analysis of the vessel density.
The validity of the dextran labeling was further confirmed by
immunohistochemistry of tumor sections using antibodies recog-
nizing the endothelial cell–specific marker vWF. As shown in
Fig. 2C, the pattern of vWF staining (Fig. 2C, a) largely overlapped
with the dextran labeling in the tumor section (Fig. 2C, b and c),
indicating that dextran-marked channels are lined by vWF-
expressing endothelial cells. In fact, there are more vWF-positive
areas than dextran-labeled regions in the tumor section (Fig. 2C, c,
arrows), suggesting that injected dextran only labeled vessels with
open lumens or functional blood vessels (18). Higher magnification
confirmed that the fluorescently labeled dextran was in the lumen
formed by vWF-positive cells (Fig. 2C, d, arrow). In addition to the
tissue staining, we have also observed the flow of the RBC through
all of the dextran-filled channels in tumors of living PyMT mice
Macrophages Promote Tumor Angiogenesis
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using multiphoton microscopy (ref. 20; data not shown). This
indicates that all these marked vessels are part of the circulatory
system. We can therefore conclude that the dextran injection
marked the great majority, if not all, of the active vessels in tumors.
Macrophage infiltration precedes angiogenesis and vascular
remodeling in tumors. As a first step in testing the hypothesis
that macrophages play a causal role in angiogenesis, we examined
the distribution of macrophages at different stages of tumor
progression. Few macrophages were found in the hyperplasia and
adenoma/MIN tumors in mice between 4 and 6 weeks of age, as
determined by immunohistochemistry with antibodies to the
macrophage-specific marker F4/80 (Fig. 3A, white arrows). In
fact, the density of macrophages in these premalignant lesions
was similar to that found in the adjacent normal adipose-rich
stroma but was significantly lower than macrophages surround-
ing the developing terminal end buds of normal ducts (data not
Figure 1. Vessel development in
PyMT model correlates with the
malignant transition. A, H&E
staining (top) and dark-field
images showing the dextran
labeling in adjacent sections
corresponding to the boxed area in
the H&E sections displayed above
of normal mammary gland (NM ;
black arrows, mammary ducts)
and premalignant lesions at
hyperplasia (H), adenoma/MIN
(A/M) and malignant EC and
LC stages isolated from +/Csf1
op
PyMT mice at 8, 5, 8, 9, and 16
weeks of age, respectively. EC,
arrows, regions consisted of
premalignant acini. Bar, 500 Am
(H&E); 100 Am (dark field). White
arrows, some of the
dextran-marked vessels.
B, quantitative analysis of the
vessel density in tumors at
different stage of progression.
Columns, density of dextran-
labeled vessels. Duct, normal
mammary ducts; H, hyperplasia;
A/M, adenoma/MIN; EC and LC ,
early and late carcinoma.
Statistical analysis, Welch’s
unpaired t test.
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shown; ref. 21). However, a remarkable increase in macrophage
infiltration occurred in a large percentage of the adenoma/MIN
tumors at 7 to 8 weeks of age. Macrophages in these more advanced
lesions formed dense clusters in the stroma surrounding the
enlarged tumor acini (Fig. 3A, A/M, right, white arrows). This
infiltration preceded the formation of the dense vessel network.
A large percentage of primary tumors examined at 9 to 10 weeks
of age have progressed to the EC stage. These tumors had a further
increase in macrophage infiltration. Clusters of high-density
infiltrates were found in the vicinity of tumor acini that still were
at the adenoma/MIN stage (Fig. 3B, EC left, white arrows). These
macrophages normally had a rounded morphology. The distribu-
tion of the vessels in these areas was also similar to that found in
adenoma/MIN lesion (Fig. 3A). In contrast, fewer macrophages
were found in the center of the tumor where the dense vessel
network had developed. In these areas, macrophages were attached
to the vessel network and were stretched out along the vessels
(Fig. 3B, EC right, arrows). Thus, both the morphology and
distribution of macrophages were altered depending on the tumor
stage and context.
A further increase of macrophage infiltration was observed as
tumors progressed to the LC stage. A high density of macrophages
was found in a stroma-rich ring surrounding the solid tumor
nodules with the characteristics of LC (Fig. 3B, LC). In this ring,
which also contained a high-density vessel network, a large per-
centage of macrophages seemed to stretch out and attach to the
vessels (Fig. 3B, LC, arrows). Similar to the EC, high-density clusters
of macrophages were also found in the areas that were still at the
adenoma/MIN stage with a corresponding low density of vessels
(data not shown).
To confirm the observations of F4/80 staining in tumors, LC-
stage lesions from CSF-1R-GFP.PyMT mice were used because the
CSF-1R promoter is specifically expressed in macrophages (22).
Consistent with the F4/80 staining, a high density of GFP-positive
stromal cells was found at the vicinity of the lesion (Fig. 3C) and, as
seen from the higher power image in the area containing the vessel
network, GFP-positive cells were physically associated with the
vessel (Fig. 3C).
Macrophage depletion inhibits the angiogenic switch and
the malignant transition. The data above show a remarkable
increase in macrophage infiltration into the nonmalignant
primary tumors followed by the formation of a vessel network
and the transition to malignancy. This suggests that macro-
phages may promote these two processes. We next tested
whether macrophages play a causal role in angiogenesis by de-
pleting macrophages in tumors using mice carrying the homo-
zygous null allele (Csf1
op
) for the mononuclear phagocyte growth
factor CSF-1.
In these studies, we observed a correlation between a low density
of macrophages in the primary tumors of CSF-1 null mutant mice
Figure 2. Dextran-marked channels are lined by endothelial cells. A and B, confocal images of tumor sections from Texas red-dextran and FITC-LEL coinjected
PyMT mice at 18 weeks of age. a, 4¶,6-diamidino-2-phenylindole (DAPI) counterstaining; b, FITC-LEL; c, Texas red-dextran; d, merged image. A, arrows, cysts
and necrotic areas in the tumor. B, blow-up of a vessel in A. Arrows, RBC in the lumen of the dextran- or LEL-labeled vessel. Bar, 80 Am(A); 20 Am(B ). C, images
of tumor sections from a Texas red-dextran–injected PyMT mouse at 16 weeks of age stained with anti-vWF antibody. a, vWF staining; b, dextran labeling;
c, overlaid image of a, b, and DAPI. c, arrows, vessels that are positively stained by vWF antibody but are dextran negative. d, a higher magnification image of
the staining. Arrow, a dextran-filled vessel lumen formed by vWF-positive cells (green). Bar, 100 Am(a-c); 20 Am(d).
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and a marked delay in the angiogenic switch that was associated
with a significant delay in the malignant transition (8). In contrast
to the tumors in heterozygous controls, in which the formation of
dense vessel networks was found in the mammary lesions as early
as 8 weeks of age (Fig. 4A, a), a large percentage of CSF-1 null
mutant mice did not develop such networks in their primary
tumors even when they reached 18 weeks of age (Fig. 4A, b). This
was the case even though most of these lesions were larger than
8mm
3
in size. In addition, a ‘‘sponge’’ like structure was often seen
in these CSF-1 null tumors, indicating that these lesions were
formed mainly by densely packed hyperplastic duct-like structure
instead of the solid nodules that are the characteristic component
of malignant lesions (Fig. 4A, b).
We next compared tumor progression and angiogenesis in the
CSF-1 null mutant mice to their heterozygous littermates at 8 to 9
weeks of age in which f60% of the +/Csf1
op
PyMT mice have
developed malignant stage tumors. As we have previously
observed, a lower percentage of CSF-1 null tumors had progressed
to the malignant stages compared with the +/Csf1
op
controls at the
same age (36% versus 60%). Notably, in both genotypes, only those
tumors that had progressed to malignant stages developed a vessel
network, although still different between genotypes (see below).
However, as previously reported (8, 23), despite the complete
lack of CSF-1, tumors in mice homozygous for the Csf1
op
mutation
are not completely depleted of macrophages. Thus, we hypothe-
sized that the reason that CSF-1 null mutant mice examined
had tumors that had progressed to malignancy at this age was due
to the partial recovery of the macrophage population in these
tumors.
To test this, we compared the macrophage density in tumors that
either progressed or failed to progress to malignancy at this age. A
quantitative analysis was done by measuring the density of
macrophages in the surrounding stroma relative to the circumfer-
ence of the tumor section. As commented on above but now
presented numerically, in +/Csf1
op
tumors, a significantly higher
density of macrophages was found in tumors as they progressed
from the hyperplastic to adenoma to the malignant stages (Fig. 4B,
Pre, 6w versus 9w). This resulted in an almost 50-fold increase in
macrophage density in malignant tumors compared with the
hyperplastic ones. Although the macrophage density in CSF-1 null
Figure 3. Macrophage infiltration in primary mammary tumors correlates with vessel network development and tumor progression. Immunohistochemistry using
anti-F4/80 antibodies of tumor sections stained with DAPI from Texas red-dextran–injected +/Csf
op
PyMT mice. The staining from immunohistochemistry was converted
to green using Photoshop. Tumors were isolated from +/Csf1
op
PyMT mice at 5 weeks (H), 6 weeks (A/M, left), 9 weeks (A/M, right), 9 weeks (EC), and
17 weeks (LC) of age, respectively. A, lesions are at the hyperplasia (H ) and adenoma/MIN (A/M). Yellow size bars in H and A/M sections, 100 Am. B, lesions are
at EC and LC stages. White arrows, some of the F4/80+ macrophages; yellow arrows, dextran-labeled vessels. White size bar in EC and LC sections, 50 Am.
C, LC-stage tumor sections from a Texas red-dextran–injected CSF1R-GFP transgenic mouse. The inset on the left (bar, 100 Am) is shown at higher magnification on
the right (bar, 20 Am). Arrows, GFP-positive macrophages.
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tumors at the premalignant stage was f25% of that found in the
+/Csf1
op
lesions at the same stage, this only bordered on
significance probably because of the wide variation in wild-type
mice as they were dynamically transitioning into the malignant
state (Fig. 4B). This similarity in density is consistent with the
similar rates of progression and growth rates of these premalignant
tumors in both genotypes. However, a significantly higher level of
macrophage density was also found in the CSF-1 null tumors as they
progressed to the malignant stages compared with the CSF-1 null
tumors at the premalignant stages (Fig. 4B ). This positive
correlation of macrophage density and tumor progression to
malignancy in both genotypes argues that tumor-associated
macrophages have a direct effect on the malignant transition and
the angiogenic switch because this is always associated with the
malignant transition. Nevertheless, when comparing the macro-
phage density in CSF-1 null tumors to that in +/Csf1
op
tumors at the
malignant stages, there was a significantly lower density of
macrophages in CSF-1 null tumors at the same stage (Fig. 4B).
This result indicates that a relatively low density of macrophage
in these CSF-1 null tumors is sufficient for the malignant transi-
tion and angiogenic switch but that it might be the cause of a
slower rate of progression and inhibition of metastasis as pre-
viously observed (8).
As shown above, even in malignant tumors, the CSF-1 null
mice had lower numbers of macrophages. Thus, we tested
whether such a low density had any effect on the development of
tumor vasculature in tumors that had progressed to late-stage
malignancy in age-matched mice. This selection of equivalently
matched tumors prevented bias that could be caused by
comparing tumors at different stages of progression. We found
that, despite the formation of a vessel network in the CSF-1 null
tumors (Fig. 5A, / ), a much lower density of such networks
was observed in these lesions compared with their +/Csf1
op
counterparts (Fig. 5A, +/ ). This was directly due to the absence
of macrophages in the tumors because restoration of macrophage
numbers in the tumors of Csf1
op
/Csf1
op
mice by the transgenic
expression of CSF-1 specifically in the mammary epithelium (8)
resulted in the vessel density returning to the +/Csf1
op
level (Fig.
5A, Trans).
To confirm these histologic observations, a quantitative analysis
of the tumor was done to compare vessel area and density. Sections
of the entire LC-stage tumors of all three genotypes were imaged
and the area covered with vessels in each section was highlighted
and the total area occupied per section was measured as described
in Materials and Methods (Fig. 5B, ii). Afterwards, the images were
skeletonized to analyze the vessel density (Fig. 5B, iii). This method
allows vessels with either big or small lumens to be represented as
lines with similar thickness (pointed by blue or red arrows,
respectively) such that total vessel length could be measured
(Fig. 5B, arrows).
Based on the analysis of more than 300 images, the mean areas
in LC-stage tumors covered by the vessels was 40% lower in CSF-1
null tumors compared with their +/Csf1
op
counterparts (Fig. 5C, OP
versus WT; P = 0.014). Similarly, the vessel density in CSF-1 null LC-
stage tumors was significantly lower than +/Csf1
op
controls (Fig.
5D, OP versus WT; P = 0.03). Restoration of the infiltration of
macrophages by expression of CSF-1 in the CSF1 null tumors
increased this vessel area to a level similar to the +/Csf1
op
tumors
(Fig. 5C, WT versus TRANS; P = 0.35) and also restored the vessel
density to the +/Csf1
op
level (Fig. 5D, WT versus TRANS; P = 0.29). A
similar difference (f1.8-fold) was found when comparing the areas
covered by vessels and vessel densities between +/Csf1
op
and CSF-1 null tumors, suggesting that the sizes of the vessel
lumens in these two genotypes were similar. Taken together,
these data indicated that the infiltration of macrophages in tumors
regulates the density of vessels in LC-stage tumors with a high
level of macrophage infiltration correlated with a high density of
vessels.
Figure 4. The relationship between macrophage infiltration and malignant
transition. A, delayed angiogenic switch and malignant transition in macrophage
depleted tumors. Tumor sections from a +/Csf1
op
mouse at 8 weeks of age (a )
and from a Csf1
op
/Csf1
op
mouse at 18 weeks of age (b ) injected with Texas
red–conjugated dextran. The tumor sections were stained with DAPI. Bar,
100 Am. B, a quantitative analysis of macrophage density in +/Csf1
op
and CSF-1
null tumors, Csf1
op
/Csf1
op
.6wor 9w, mice were at 5 to 6 or 8 to 9 weeks of age,
respectively. Pre, tumors were at premalignant stages; Mag, tumors were at
malignant stages. The data were prepared as described in Materials and
Methods and analyzed using Welch’s unpaired t test.
Macrophages Promote Tumor Angiogenesis
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Premature macrophage infiltration enhances angiogenesis.
The results presented above suggest that macrophages induce both
the formation of the dense vessel network and the malignant
transition. However, based only on these data, it was difficult to
determine whether the vasculature development during the
malignant transition was directly regulated by macrophages or
whether it was a secondary effect induced by this malignant
transition consequent to other effects of macrophages. To
distinguish between these possibilities, we examined vasculature
development in +/Csf1
op
PyMT mice carrying the MMTV LTR-CSF-
1 transgene. Because the MMTV LTR in these transgenic PyMT
mice is active early in mammary gland development, the
expression of the transgenic CSF-1 is induced in young mice
independent of tumor progression. We have previously reported
that a striking increase in macrophage infiltration was observed in
the tumors of these transgenic mice at a young age (8), and that
this correlated with accelerated tumor progression and a doubling
of the metastatic rate compared with the nontransgenic +/Csf1
op
PyMT mice (8).
In the CSF-1-overexpressing transgenic mice, we found that a
dense vessel network developed prematurely in premalignant
tumors from mice at 5 weeks of age (Fig. 6A, top, white arrows).
This dramatic enhancement of the vessel network appeared several
weeks earlier than in the nontransgenic controls. Importantly, in
the nontransgenic mice, such dense vessel networks were found
exclusively in tumors at the malignant stages (Fig. 6A, +/op-9wk)
whereas in the CSF-1 transgenic mice, the network was already
formed in tumors still at premalignant adenoma/MIN (Fig. 6A,
top right) and even at the earliest premalignant stage, hyperplasia
(Fig. 6A, top left). No such dense vessel network was ever seen in
non-CSF-1 transgenic PyMT mice at the hyperplasia stage (Fig. 6A,
+/op-5wk). In parallel with the elevated angiogenesis, a high level of
macrophage infiltration was observed in the stroma of the CSF-1
transgenic premalignant tumors (Fig. 6A, top, yellow arrows)
compared with tumors from nontransgenic mice at similar ages
(Fig. 6A, +/op-5wk) or at the EC stage (Fig. 6A, +/op-9wk, yellow
arrows). A quantitative analysis of macrophage density in these
tumors confirmed a 6-fold increase of macrophage infiltration in
the tumors of CSF-1 transgenic +/Csf1
op
PyMT mice compared with
the nontransgenic controls (Fig. 6B; P = 0.045). This observation
shows that macrophages have a direct effect on the formation of
the vessel network independent of the transition to malignancy,
and that the formation of such a network enables tumors to
advance to malignancy. A possible mechanism for this is the
observation that the major angiogenic regulator, VEGF, was
depleted in stromal cells of tumors of CSF-1 null mutant mice
Figure 5. Vessel density in advanced carcinoma correlates with the degree of macrophage infiltration. A, vessel distribution in LC-stage tumors from a +/Csf1
op
(+/ ),
a CSF-1 null (/), or a CSF-1 transgenic Csf1
op
/Csf1
op
PyMT mouse (Trans ) at 16 weeks of age. Bar, 100 Am. B, processed images for the vessel density
comparison of a tumor section from a Texas red-dextran–injected mouse counterstained with DAPI (i). The area marked by dextran was highlighted using ImageJ
program and skeletonized to indicate the length of individual vessels (ii and iii). Blue arrows, thick vessels that were represented as thin lines after the image was
skeletonized. Red arrows, thin vessels in both images. C, a comparison of areas in tumors covered by dextran-marked vessels. D, comparison of vessel density.
WT, +/Csf1
op
; OP, CSF-1 null or Csf1
op
/Csf1
op
; TRANS, CSF-1 transgenic Csf1
op
/Csf1
op
mice. Significance following analysis by the Mann-Whitney test.
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compared with +/Csf1
op
mice, suggesting that this may be a
significant part of the reason for this reduced angiogenesis
(Fig. 6C). Thus, overexpression of macrophage VEGF may have
caused the premature vessel formation.
Discussion
Studies in dormant and malignant tumors have suggested that
malignant tumors require the development of a unique vasculature
(5, 6). In this article, using a mouse model where the mammary
tumors undergo a natural progression through premalignant to
malignant stages, we show that the angiogenic switch is required
for the malignant transition.
At the malignant transition, although the lesions mostly
consist of premalignant acini, small solid nodules form in the
center that are the site of the initial malignant transition (16).
The angiogenic switch, identified by the formation of a dense
vascular network, occurred exclusively in this malignant transition
region. This is consistent with other studies using transgenic mouse
models of cancers in which a transition stage, termed angiogenic
dysplasia, occured before the malignant carcinoma was identified
(24–28). However, the close association between the angiogenic
switch and malignant transition was not reported in these studies.
Furthermore, by manipulating the formation of the vasculature, we
were able to show that this angiogenic switch was required for the
malignant transition. In support of this conclusion was the delay in
the malignant transition following the inhibition of the formation of
such vascular networks in CSF-1 null mutant mice. Even more
decisive was the accelerated progression of the tumor to malig-
nancy following premature induction of the angiogenic switch in
premalignant lesions in the CSF-1 transgenic +/Csf1
op
mice.
Tumors are highly populated with macrophages, and in
clinical studies of breast cancer their density correlates with
areas of angiogenesis and with poor prognosis (9). Despite these
clinical correlations, the role(s) of macrophages in promoting
tumor angiogenesis is largely unexplored. In this study, we show
that macrophages promote the development of the tumor
vasculature. We observed an increase of macrophage infiltration
in the stroma of primary tumors shortly before the development
of a dense vessel network in the area. Depletion of this
macrophage infiltration inhibited the angiogenic switch. Further-
more, increased macrophage infiltration in premalignant lesions
through the transgenic expression of CSF-1, of which the only
target is CSF-1 receptor–bearing macrophages (8, 29), induced
premature formation of the vessel network and accelerated the
malignant transition (8). These data indicate that macrophages
have a direct effect on angiogenesis and regulate the angiogenic
switch.
This study identified a unique feature of tumor vasculature in
the PyMT model that reflects the evolution of the tumor through
benign to malignant stages. We found that a dense vessel network
is initiated in the center of the PyMT lesion only where the
malignant transition occurs. This contrasts with that observed in
transplanted tumors that are uniform in malignant progression
and that need to gain vascular support from the host for the
survival of the graft. Therefore the vasculature development that
occurs at the periphery of the tumor (30) does not reflect the
angiogenic switch in naturally evolving cancers. This is different
from PyMT tumors that develop spontaneously and establish a
vascular system in premalignant stages that is similar to that
observed in the surrounding normal tissues. Therefore, the
angiogenic switch identified in this model more closely reflects
that seen during human cancer progression.
Our studies here indicated that the angiogenic switch was not
simply induced by the expansion of tumor size. We found that
the formation of the dense vessel network was initiated in the
malignant transition region of EC-stage lesion that was often
<1 mm
3
in sizes. This indicates that the event is not induced by
the overall growth of the lesion but is stimulated by a regional
factor(s) associated with the progression of the tumor cells to
malignancy. Furthermore, the angiogenic switch was not detected
in more than 40% of CSF-1 null mutant mice that reached 16
weeks of age whereas all of the wild-type mice at this age had
tumors developed to the most advanced malignant stage with
vessel network formation. Interestingly, although the angiogenic
switch was not detected in these CSF-1 null tumors, most of the
Figure 6. Premature expression of CSF-1 induces vessel network formation
in premalignant lesions. A, +/opT sections from a hyperplasia (left ) and adenoma
(right) from CSF-1 transgenic +/Csf1
op
PyMT mouse at 5 weeks of age. Blood
vessels in the tumor were labeled by i.v. injection of Texas red-dextran
(white arrows ) and macrophages were labeled by FITC-conjugated dextran
(yellow arrows ). Bar, 100 Am. +/op-5wk, representative image of F4/80
immunohistochemistry of a tumor lesion at hyperplasia stage isolated from
a+/Csf1
op
PyMT mouse at 5 weeks of age. Note that the densities of both
vessels (white arrows ) and macrophages (yellow arrows ) are much lower than
in the +/opT sections. Bar, 100 Am. +/op-9wk, representative F4/80
immunohistochemistry image of a lesion at the EC stage isolated from a +/Csf1
op
PyMT mouse at 9 weeks of age. Note that macrophages (yellow arrow) seem
to attach to the vessels network. Bar, 50 Am. B, quantitative analysis of
macrophage infiltration in CSF-1 transgenic (Trans ) and nontransgenic (Control )
+/Csf1
op
PyMT mice at 5 to 6 weeks of age. Columns, mean; bars, SD.
C, immunohistochemistry for VEGF in LC-stage tumor sections from a +/Csf1
op
or a Csf1
op
/Csf1
op
PyMT mouse at 18 weeks of age. VEGF-positive cells
were mainly in stroma as indicated by arrows. Representative of those
obtained from six +/Csf1
op
and five Csf1
op
/Csf1
op
PyMT mice with tumors at
LC stage.
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tumors were often >8 mm
3
, further confirming this lack of
association with size.
This study has shown that macrophages play a causal role in
promoting tumor angiogenesis and the induction of macrophage
infiltration in tumors of PyMT model occurred over a narrow time
frame when lesions are still at premalignant stage and small in size.
These observations suggest that the tumor microenvironment at
the premalignant stage produces local factors that attract the
infiltration of macrophages that promote tumor angiogenesis.
Studies have shown that tumor cells produce various chemo-
attractants for leukocytes including CSF-1, CCL2, CCL5, VEGF, and
IL-8 (11), suggesting that such infiltration might be manipulated by
tumor cells. Clearly, overexpression of CSF-1 is sufficient to recruit
abundant macrophages. This is consistent with the clinical
observation that overexpression of CSF-1 correlates with leukocytic
infiltration, which in turn correlates with poor prognosis (31, 32).
This raises the question of what tumor and/or environmental
factor(s) triggers the release of these leukocytic chemoattractants.
Numerous studies suggest that hypoxia might be the factor that
induces the up-regulation of chemoattractants in tumor cells (33).
However, we did not see any obvious colocalization of macrophage
infiltration and hypoxia in tumors when Hypoxyprobe-1 (pimoni-
dazole hydrochloride) was used to map the hypoxia regions in the
PyMT model (data not shown).
The mechanisms macrophages use to promote angiogenesis in
tumor are unclear. Macrophages can produce angiogenesis
regulators (7, 11) and may also induce tissue remodeling by
producing various proteinase activators and inhibitors that may
destroy the integrity of the basement membrane and extracellular
matrix, liberating matrix-bound factors. Indeed, a recent study
using the human papillomavirus-16–induced model of cervical
carcinoma showed that macrophages produce MMP-9 that seems
to be required for release of VEGF locally in the tumor, and that
inhibition of this by bisphosphonate inhibited angiogenesis and
reduced tumor incidence and growth (25). Furthermore, inhibition
of CSF-1 function in human tumors xenografted into immuno-
compromized mice reduced their growth and this was correlated
with poor macrophage recruitment and reduced angiogenesis due
to a depletion of VEGF (34). Indeed, we showed that most of the
VEGF-expressing cells in the PyMT model were in stroma and that
that they were reduced in the CSF-1 null tumors and presumably,
therefore, are macrophages.
In summary, this study uses genetic means to show that
macrophages regulate the angiogenic switch, an essential step for
the progression of mammary tumors to malignancy. Such
information will be very important for the development of novel
and effective therapeutic strategies against tumors.
Acknowledgments
Received 4/7/2006; revised 8/2/2006; accepted 9/1/2006.
Grant support: Albert Einstein Cancer Center Analytical Imaging Facility and
histopathology facilities, National Cancer Institute grants CA 94173 and CA 100324,
and Albert Einstein Cancer Center Core grant P30 CA 13330.
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.
We thank Dr. J.E. Segall for critical comments on the manuscript; Drs. S. Patan,
A.W. Ashton, R.N. Kitsis, B. Terman, P. Davies, A. Orlofsky, and V. Gouon-Evans for
helpful suggestions; and Jim Lee for technical support.
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11246
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