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From the Department of Anatomy and Cardiovascular ResearchInstitute and Comprehensive Cancer Center,*
University ofCalifornia, San Francisco, California; and the Department of RadiationOncology,
Edwin L. Steele Laboratory,Massachusetts General Hospital and Harvard Medical School,Boston, Massachusetts
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionConclusionsReferences |
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-smooth
muscle actin (-SMA) and desmin-immunoreactive pericytes were present
on >97% of blood vessels viewed by confocal microscopy in
100-µm-thick sections of three different spontaneous or implanted
tumors in mice. However, the cells had multiple abnormalities.
Unlike pericytes on capillaries in normal pancreatic islets,
which had desmin but not -SMA immunoreactivity, pericytes on
capillary-size vessels in insulinomas in RIP-Tag2 transgenic mice
expressed both desmin and -SMA. Furthermore, pericytes in
RIP-Tag2 tumors, as well as those in MCa-IV breast carcinomas
and Lewis lung carcinomas, had an abnormally loose association
with endothelial cells and extended cytoplasmic processes deep into the
tumor tissue. -SMA-positive pericytes also covered 73% of
endothelial sprouts in RIP-Tag2 tumors and 92% of sprouts in the other
tumors. Indeed, pericyte sleeves were significantly longer than
the CD31-immunoreactive endothelial cell sprouts themselves in all
three types of tumors. All three tumors also contained -SMA-positive
myofibroblasts that resembled pericytes but were not associated with
blood vessels. We conclude that pericytes are present on most tumor
vessels but have multiple abnormalities, including altered
expression of marker proteins. In contrast to some previous
studies, the almost ubiquitous presence of pericytes on tumor
vessels found in the present study may be attributed to our use of both
desmin and -SMA as markers and 100-µm-thick tissue sections. The
association of pericytes with endothelial sprouts raises the
possibility of an involvement in sprout growth or retraction
in tumors.
With the increasing promise of vascular targeting in cancer, a thorough understanding of the cellular structure and function of tumor vessels becomes even more important, as this information is key to interpreting the effects of anti-angiogenic agents. The endothelial cells of tumor vessels have been studied at the tissue, cellular, and molecular level, both historically and recently,10-17 but less is known about the structure and function of pericytes on tumor vessels.18-22
Pericytes, also known as Rouget cells, periendothelial cells, or mural cells, are adventitial cells located within the basement membrane of capillaries and postcapillary venules.23 Because of their multiple cytoplasmic processes, distinctive cytoskeletal elements, and envelopment of endothelial cells, pericytes are generally considered to be contractile cells that stabilize vessel walls and participate in the regulation of blood flow in the microcirculation.24-26 Pericytes may also influence endothelial permeability, proliferation, survival, migration, and maturation.27-29 Blood vessels of mouse embryos lacking platelet-derived growth factor-B (PDGF-B) or its receptor (PDGF receptor-ß, PDGFR-ß) do not have pericytes or vascular smooth muscle cells and develop microaneurysms and vessel leakiness before the animals die in late gestation.30,31
Pericytes were initially recognized by their distinctive shape and
location, but they are now most commonly identified by molecular
markers such as -smooth muscle actin (-SMA), nonmuscle myosin,
tropomyosin, desmin, nestin, PDGFR-ß, aminopeptidase A,
aminopeptidase N (CD13), sulfatide, or high-molecular weight
melanoma-associated antigen (NG2).18,26,31-36
The
expression of these markers varies with the type of vessel.
Pericytes on normal capillaries typically express desmin but not
-SMA, whereas normal venular pericytes express both
molecules.26
Marker expression can also vary in different
organs and with pathological conditions.18,34,35,37
Because no single commonly used marker identifies all pericytes with
certainty, there may be problems with identifying pericytes in
pathological conditions such as cancer when the cells change their
expression of marker proteins.18,34,37
Little is known about the variability of marker expression by pericytes
in tumors because most studies have used a single marker, usually
-SMA or desmin, and have equated lack of immunoreactivity with lack
of pericytes.21,22,38,39
Published reports suggest that
the amount of pericyte coverage on vessels in different tumors ranges
from extensive18,20
to little or
none.21,22,40
Some of these differences may be explained
by differences in pericyte marker expression among tumors. However,
others are likely to result from differences in the markers used to
identify pericytes or differences in section thickness, where partial
pericyte coverage was missed in thin histological sections.
One reason for determining whether pericytes are a consistent feature of tumor vessels is to explore whether they participate in angiogenesis by guiding newly formed blood vessels through their association with endothelial sprouts.41,42 Another reason is to examine the suggestion that the absence of pericytes sensitizes tumor vessels to withdrawal of vascular endothelial growth factor (VEGF).21
In the present study, we compared the amount of pericyte coverage of
tumor vessels, as determined by immunoreactivity of two markers,
-SMA and desmin, with that of normal vessels. We also questioned
whether the relationship of pericytes to endothelial cells in tumors
differs from that in normal tissues and examined the relationship of
pericytes to endothelial sprouts. We compared three tumors in mice:
spontaneous pancreatic tumors in RIP-Tag2 mice, implanted MCa-IV mouse
mammary carcinomas, and Lewis lung carcinomas. Pericytes and
endothelial cells were co-localized in immunohistochemically stained
100-µm-thick sections and examined by confocal microscopy. A
preliminary description of this work has been reported.43
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
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Spontaneous pancreatic islet cell tumors were studied in RIP-Tag2 transgenic mice with a C57BL/6 background. In these mice, expression of the SV40 virus T antigen is driven by the rat insulin promoter.44 Mice expressing the viral oncogene were identified by genotyping tail-tip DNA by the polymerase chain reaction, and tumors were studied when the mice reached 10 weeks of age.44 Implanted MCa-IV mouse mammary carcinomas9 and Lewis lung carcinomas (American Type Culture Collection, Rockville, MD) were studied in syngeneic male C3H and C57BL/6 mice, respectively (25 to 30 g body weight).10 Two-mm cubes of tumor were implanted under the dorsal skin, and the tumors were examined 10 to 20 days later when they had reached a diameter of 8 to 12 mm. Mice were housed under barrier conditions in the animal care facility at University of California at San Francisco. All of the experimental procedures were approved by the University of California at San Francisco Committee on Animal Research.
Lectin Staining and Perfusion Fixation of Vasculature
Mice were anesthetized with ketamine (87 mg/kg) plus xylazine (13 mg/kg) injected intramuscularly. In some animals, fluorescein isothiocyanate (FITC)-labeled Lycopersicon esculentum lectin (100 µg in 100 µl of 0.9% NaCl; Vector Laboratories, Burlingame, CA) was injected into the femoral vein and allowed to circulate for 3 minutes before perfusion of fixative.10 The chest was opened rapidly, and the vasculature was perfused for 3 minutes at a pressure of 120 mmHg with fixative [4% paraformaldehyde in 0.1 mol/L phosphate-buffered saline (PBS), pH 7.4] from a 18-gauge cannula inserted into the aorta via an incision in the left ventricle.10 The fixative was not preceded by a saline rinse. The right atrium was incised to create a route for the fixative to escape. After removal, tissues were stored in fixative at 4°C until they were processed for immunohistochemistry.
Immunohistochemistry and Imaging
Specimens were rinsed several times with PBS, embedded in 10%
agarose, and cut with a Vibratome or infiltrated overnight with 30%
sucrose, frozen, and cut with a cryostat. Sections 100 µm in
thickness were incubated at room temperature for 12 to 15 hours in a
mixture of anti-mouse CD31 (PECAM-1, clone MEC 13.3 rat monoclonal,
1:500; Pharmingen, San Diego, CA) antibody for endothelial cell
identification plus anti--SMA (Cy3-conjugated mouse monoclonal,
clone 1A4, 1:1000; Sigma Chemical Co., St. Louis, MO) or
anti-desmin (rabbit polyclonal 1:2000; DAKO Corp., Carpinteria, CA)
antibody for pericyte identification. Antibodies were diluted with PBS
containing 0.01% thimerosal as an anti-bacterial and 0.3% Triton
X-100 to improve penetration of 100-µm sections. After several rinses
with PBS, specimens were incubated for 6 hours at room temperature with
a goat anti-rat or goat anti-rabbit secondary antibody labeled with
FITC or Cy5 for CD31 staining or with Cy3 for desmin staining
(antibodies from Jackson ImmunoResearch, West Grove, PA). After
mounting in Vectashield (Vector Laboratories, Burlingame, CA),
specimens were examined with a Zeiss Axiophot fluorescence microscope
and a Zeiss LSM 410 laser-scanning confocal microscope.
Confocal images were stored as digital files, viewed with Photoshop
(Adobe, Mountain View, CA), and printed on a Fujix Pictography
3000 color printer (Fuji Photo Film Inc., Elmsford, NY).
Transmission Electron Microscopy
Tumors, fixed by vascular perfusion of 0.5% glutaraldehyde
and 1% paraformaldehyde in 0.075 mol/L sodium cacodylate buffer, pH
7.4, were removed, immersed in 2.5% glutaraldehyde in cacodylate
buffer for a minimum of 2 hours, and embedded in 10%
agarose.10
Sections 100 µm in thickness were cut with a
Vibratome. Specimens measuring 
1 x 3 mm were cut from the
sections, treated with OsO4 and uranyl acetate,
dehydrated, and embedded in epoxy resin.45
Sections 0.5
µm in thickness were stained with toluidine blue for light
microscopy, and sections 50 to 100 nm in thickness were stained with
lead citrate and examined with a Zeiss EM-10 electron microscope.
Morphometric Measurements
Morphometric measurements of blood vessels were made on images
obtained from 100-µm-thick sections of four specimens
(n = 4 mice) from each of the three tumors and
normal tissues, unless designated otherwise. Regions of necrosis were
avoided. The sections were double-stained for CD31 and -SMA
immunoreactivities. Real-time video images were viewed or digital
images were captured with a Zeiss Axiophot microscope equipped with
single and dual filters for FITC and Cy3 and a low-light, three-chip
CoolCam CCD camera (SciMeasure Analytical Systems, Atlanta, GA).
Measurements were made using image analysis software developed for this
purpose in our laboratory.45
The proportion of vessels
covered by pericytes was determined for 50 vessels in each specimen.
Pericytes were considered present if -SMA or desmin immunoreactivity
was visible anywhere around the vessel perimeter. The number, length,
and pericyte coverage of endothelial sprouts were determined in the
same specimens. Sprouts were identified as tapered CD31-immunoreactive
processes that extended away from the main axis of a vessel and ended
abruptly. For all of the sprouts identified on vessels in each specimen
(10 to 27 sprouts were analyzed per 50 vessels in each specimen), the
lengths of the CD31-immunoreactive endothelial cell strands (sprout
length) and the surrounding -SMA immunoreactive pericyte sleeve
(pericyte sleeve length) were measured. In addition, for 10
sprouts from each specimen that had FITC-lectin staining in
vivo (n = 2 mice for each type of tumor),
the lengths of the lectin-stained lumen (lumen length) and the
associated CD31-immunoreactive endothelial cell strands were measured.
The extent of pericyte coverage on vessels was determined on 15
properly cross-sectioned vessels in each specimen by measuring the
proportion of CD31-positive vessel perimeter covered by
-SMA-immunoreactive cells. Values are expressed as means ± SEM
(SEM). The significance of differences between means was assessed by
analysis of variance followed by the Bonferroni/Dunn test
(P < 0.05).
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Results |
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-SMA and Desmin Immunoreactivity of PericytesNormal Pericytes
In normal pancreatic acini and islets,
-SMA-immunoreactive cells were abundant on arterioles and venules
but not on capillaries (Figure 1A)
. By
comparison, desmin immunoreactive cells were present on all segments of
the microvasculature, as shown by the extensive co-localization of
desmin and CD31 immunoreactivities (Figure 1B)
. In normal pancreatic
islets, -SMA immunoreactivity co-localized with desmin
immunoreactivity on arterioles and venules but not on capillaries where
the adventitial cells had only desmin immunoreactivity (Figure 1
; C, D,
and E). As capillaries predominated in the pancreas, most of the
vessels were enveloped by cells that had desmin immunoreactivity but
lacked -SMA immunoreactivity (Figure 1, A and B)
.
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The distribution of -SMA-immunoreactive cells in islet cell
tumors was very different from that in normal islets. In tumors in
RIP-Tag2 mice, -SMA and desmin immunoreactivities had essentially
identical distributions (Figure 2, A and D)
. The two markers were also co-localized on most vessels in implanted
MCa-IV breast carcinomas and Lewis lung carcinomas (Figure 2
; B, E, and
F).
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-SMA immunoreactivity in RIP-Tag2 mice, which had
islet cell tumors at various stages of development, showed the
transformation in expression of this marker from the normal to
neoplastic state. The number of -SMA-immunoreactive cells increased
progressively as tumors enlarged (Figure 3A)
. Double staining for -SMA and
desmin showed that most adventitial cells on blood vessels in the
smallest RIP-Tag2 tumors (hyperplastic islets) expressed desmin but not
-SMA (Figure 3B)
, whereas those in large tumors expressed both
markers (Figure 3C)
. Most vessels in MCa-IV breast carcinomas and Lewis
lung carcinomas were enveloped by cells with both -SMA and desmin
immunoreactivities within 7 days of implantation (data not shown).
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Pericytes on Normal Vessels
In the normal pancreas, arterioles were completely covered by
circumferentially arranged, -SMA-immunoreactive smooth muscle cells
(Figure 4, A and B)
. Venules >50 µm in
diameter were almost completely covered by -SMA-positive cells that
had a more irregular shape and looser packing than smooth muscle cells
on arterioles (Figure 4A)
. Venules <50 µm in diameter were covered
by pericytes that had -SMA immunoreactivity,
wereirregular in shape and orientation, had multiple cytoplasmic
processes, and incompletely covered the endothelium (Figure 4B)
.
Capillaries in the pancreas had pericytes that were immunoreactive for
desmin but not -SMA (Figure 4C)
. Unlike those on venules, pericytes
on capillaries had multiple long, branched cytoplasmic processes that
projected along the longitudinal axis of the vessel and covered only a
small proportion of the endothelial surface (Figure 4C)
.
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; A, B, and C). Abnormalities of Pericytes on Tumor Vessels
Blood vessels in the three types of tumors we examined had
multiple abnormalities, and most did not have the typical morphological
features and sequential hierarchy of arterioles, capillaries, or
venules. Pericytes on tumor vessels viewed by
confocal microscopy were conspicuously abnormal in shape and had an
abnormal association with endothelial cells. These cells resembled the
pericytes on small venules more than those on capillaries, but were
irregularly scattered over the endothelium and had bizarre cytoplasmic
processes (Figure 4
; D to H). Pericyte processes
projected along the abluminal surface of the endothelium or away from
the vessel wall into the tumor parenchyma (Figure 4F)
. Some pericyte
processes contacted one another within the tumor parenchyma (Figure 4G)
. Unlike the tight association of pericytes and endothelial cells in
normal vessels (Figure 4
; A, B, and C), on tumor vessels there were
abnormal separations between the -SMA and CD31-positive cells,
indicating that pericytes were loosely associated with endothelium
(Figure 4
; D, E, and F). Some pericytes overlapped one another (Figure 4, G and H)
.
Ultrastructural observations were consistent with the findings made by
confocal microscopy (Figure 5
; A to D).
Pericytes examined by electron microscopy had cytoplasmic processes
that extended away from the vessel wall toward the tumor parenchyma
(Figure 5, A and B)
. In addition, pericytes seemed to be loosely
associated with the endothelium (Figure 5, C and D)
. Some pericytes
overlapped other pericytes (Figure 5, A and D)
.
|
The vascular architecture was strikingly different in the three
types of tumors we examined. RIP-Tag2 tumors had uniformly small,
capillary-size blood vessels (Figure 6A)
,
with a mean diameter of 8 µm (Table 1)
.
In MCa-IV tumors, capillary-size vessels intermingled with extremely
large vessels (Figure 6B)
. Vessel diameters in MCa-IV tumors ranged
from 8 to 294 µm; the mean vessel diameter of 45 µm was fivefold
that for RIP-Tag2 tumors (Table 1)
. The size of vessels in Lewis lung
carcinomas (Figure 6, C and D)
, mean diameter of 31 µm, was
intermediate between the other two tumors (Table 1)
.
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-SMA-positive
pericytes were present on at least 97% of the vessels in all three
tumors (Figure 7)
. The smallest to the
largest vessels had extensive pericyte coverage. By comparison,
-SMA-immunoreactive cells were found on only 22% of vessels,
predominately capillaries, in normal pancreatic islets (Figure 1, A and B
; Figure 7
). Approximately half of the vessel surface was covered by
-SMA-positive pericytes in RIP-Tag2 tumors and Lewis lung
carcinomas, but 80% of the vessel surface was covered by pericytes in
MCa-IV tumors (Table 1)
.
|
and MCa-IV carcinomas, the density of vessels at
the interface of the tumor and the surrounding tissue was unusually
high. Densely packed vessels at the tumor surface were accompanied by
correspondingly numerous -SMA-positive cells (Figure 6D)
. In
contrast, the density of blood vessels and -SMA-positive cells was
relatively uniform throughout RIP-Tag2 tumors (Figure 3A)
. Myofibroblasts in Tumors
All three tumors contained cells that were immunoreactive for both
-SMA and desmin but had no apparent association with blood vessels
(Figure 6
; A to D). These cells, which were continuous with and
morphologically similar to pericytes on blood vessels, fit the
characteristics of myofibroblasts (Figure 4G
and Figure 6
; A to D).
Myofibroblasts were more abundant at the periphery of Lewis lung
carcinomas (Figure 6D)
and MCa-IV carcinomas but not RIP-Tag2 tumors.
Pericytes on Endothelial Sprouts
Identification of Endothelial Sprouts in Tumors
CD31-immunoreactive sprouts were found on 24 to 33% of blood
vessel segments examined in 100-µm sections of the three types of
tumors (Table 2)
. Sprouts, which
projected away from the endothelium, were broadest at their base and
tapered progressively to blind endings (Figure 8
; A to E). Sprouts projected into the
tumor parenchyma a distance of 3 to 69 µm (Table 2)
. The sprouts in
MCa-IV carcinomas averaged approximately twice as long as those in
RIP-Tag2 tumors. Fluorescent L. esculentum lectin, injected
into the bloodstream, bound uniformly to the luminal surface of tumor
vessels but stained only the proximal portion of sprouts (Figure 8, A and B)
. On average, the lectin-stained segment was 20 to 35% of the
total length of the CD31-positive sprouts (Figure 9
, Table 2
), suggesting that the distal
two-thirds of the sprouts did not have a lumen.
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Examination of double-labeled sections revealed that
-SMA-immunoreactive pericytes were closely associated with most of
the CD31-immunoreactive sprouts in tumors. Measurements showed that
pericytes formed sleeves on 73 to 92% of the sprouts in the three
types of tumors (Table 2)
. Interestingly, the -SMA-positive pericyte
sleeves were consistently longer than the CD31-positive sprouts (Figure 8, C and E)
. The length of pericyte sleeves was from 38% (Lewis lung
tumors) to 80% (RIP-Tag2 tumors) longer than the sprouts themselves
(Figure 9
, Table 2
).
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Discussion |
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-SMA, and desmin, we
identified multiple distinctive abnormalities of pericytes. Unlike
those on corresponding normal vessels, pericytes on tumor vessels
uniformly expressed -SMA on capillary-size vessels, were loosely
associated with endothelial cells, had cytoplasmic processes that
projected into the tumor parenchyma, and formed a sleeve around
endothelial sprouts that was longer than the sprouts themselves. These
abnormalities are illustrated schematically in Figure 10
.
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-SMA and Desmin as Molecular Markers of Pericytes
We found that pericytes on normal capillaries in the pancreas had
desmin immunoreactivity but lacked -SMA, whereas smooth muscle cells
on arterioles and pericytes on venules were immunoreactive for both.
This finding matches what is known about the microvascular beds of the
mesentery.26
In contrast, >97% of blood vessels in the
three tumors we examined, including vessels the size of capillaries,
had abundant pericytes with both -SMA and desmin immunoreactivities.
Pericytes expressing -SMA as well as desmin enveloped tumor vessels
of all size, shape, and configuration. Tumor vessels were not
classified as arterioles, capillaries, or venules because they did not
have the structural characteristics and hierarchy that would make this
classification meaningful.
The uniform co-localization of -SMA and desmin indicates that both
are consistent markers of pericytes in the tumors we examined. Abundant
-SMA-positive pericytes are also present in glioblastoma
multiforme,20
but other types of tumors have been reported
to have variable proportions of -SMA-positive
pericytes.18,19,22
There are also reports that certain
tumors have few or no -SMA-positive pericytes.21
Pericytes in ovarian carcinoma uniformly express both -SMA and NG2,
but some tumors have a larger proportion of pericytes that express NG2
than -SMA,18,19
indicating that a lack of -SMA
immunoreactivity does not necessarily mean a lack of
pericytes.
Methods of tissue preparation also can influence the apparent number of pericytes on tumor vessels. Our study of three-dimensional confocal images of 100-µm sections revealed that, despite the high incidence of vessels with pericytes, on average from 30 to 50% of the endothelial surface had no pericyte coverage. Pericytes have been reported to be abundant in conventional histological sections of some types of human tumors,20,46 but uncovered regions could be misinterpreted in thin histological sections as vessels lacking pericytes.
The abundance of pericytes on tumor vessels examined in the present
study—in contrast to what has been reported in some previous
studies—can be explained in part by the use of both desmin and -SMA
as immunohistochemical markers in combination with 100-µm-thick
tissue sections. Differences among tumors may also be a factor.
The uniform presence of -SMA-positive pericytes on the vasculature
of RIP-Tag2 tumors reinforces the distinction between blood vessels and
the prominent collections of extravascular erythrocytes (blood lakes)
in these tumors.10
Such collections of erythrocytes have
been considered by some as examples of tumor cell-lined vascular
channels.47
However, blood vessels in RIP-Tag2 tumors are
composed of endothelial cells and pericytes, whereas blood lakes in
these tumors are lined only by tumor cells.10
Despite
their appearance, blood lakes seem not to be connected to the
bloodstream and are not channels for flowing blood.10
Thus, the erythrocytes in blood lakes are stagnant.
Changes in Pericytes during Tumor Progression
Islet cell tumors arise asynchronously in RIP-Tag2
mice.44
Multiple tumors are present in each mouse, and
these tumors are at different stages of development. This feature of
the RIP-Tag2 model led to our observation of the loss of heterogeneity
of the pericyte population during tumor progression. During
tumorigenesis, pericytes with the capillary phenotype (desmin-positive
and -SMA-negative) were replaced by or transformed into pericytes
with the venular phenotype (desmin-positive and -SMA-positive) even
though capillary-size vessels continued to predominate in RIP-Tag2
tumors. The lack of -SMA-positive pericytes in smaller tumors may
also be a reflection of an anti-angiogenic action of larger tumors in
the same mouse, representing the inhibition of tumor growth by tumor
mass.48
MCa-IV carcinomas and Lewis lung carcinomas had
mixtures of small and large vessels, but -SMA and desmin
immunoreactivities were co-localized on all vessels regardless of their
size. Thus, the change in the phenotype of pericytes was not simply a
reflection of vessel enlargement during tumor development.
Consistent with these observations, the number of -SMA-positive Ito
cells on liver sinusoids, which are considered equivalent to pericytes,
increases in metastatic liver cancer.49
During tumorigenesis, alterations in the tissue microenvironment,
including both the extracellular matrix and soluble factors, are likely
to contribute to the phenotypic transformation of pericytes in tumors.
Pericytes from human brain capillaries begin to express -SMA when
grown in culture in the presence of transforming growth factor-ß
1.50
Similarly, pericytes on retinal capillaries express
-SMA in vitro but not in vivo.26
Morphological Abnormalities of Pericytes in Tumors
Pericytes in the tumors we examined had some features in common
with pericytes on venules but were morphologically very different from
smooth muscle cells on arterioles. Nonetheless, pericytes on
tumor vessels had structural abnormalities that clearly distinguished
them from pericytes on venules and, indeed, from adventitial cells on
any part of the normal microcirculation (Figure 10)
.
One abnormality we observed was that pericytes on tumor vessels were loosely associated with endothelial cells, with wide spaces separating some regions of the two types of cells. Pericytes also had cytoplasmic processes that penetrated deep into the tumor parenchyma, a feature not found on normal vessels. Some confocal images gave the impression that pericytes are actively moving within tumors. Published ultrastructural observations provide additional clues that pericytes are activated under pathological conditions. Pericytes on inflamed vessels may become amoeboid, plump, and mitotically active.51,52
Another abnormality was the amount of pericyte coverage on
capillary-size vessels in tumors. Nearly all of the vessels in the
three tumors we examined were covered with pericytes. However, the
extent of coverage ranged from an average of 50% in RIP-Tag2 tumors
and Lewis lung carcinomas to as much as 80% in MCa-IV breast
carcinomas. Similarly, the pericyte coverage of vessels in human
cerebellar hemangioblastomas has been reported to be
69%.46
These values for tumors are higher than those for
most normal capillaries, where the amount of pericyte coverage,
expressed as a proportion of endothelial surface, has been estimated as
11% in cardiac muscle, 21% in skeletal muscle, 22 to 30% in brain,
and 41% in retina.53
Instead, the values for tumors fit
better with those for normal venules, estimated to be 81% in skeletal
muscle.53
Further, basement membrane had an abnormally loose association with pericytes and endothelial cells in the three tumors we examined, and in some regions was multilayered or extended beyond the pericytes into the tumor parenchyma (unpublished observations). Basement membrane on blood vessels in these tumors was continuous except for scattered discontinuities smaller than a few micrometers.
The abnormal relationship of pericytes with endothelial cells may alter the influence of pericytes on the endothelium and contribute to the leakiness of tumor vessels9,10 and explain the sensitivity of tumor vessels to VEGF withdrawal.21
An important next step will be to address the question of whether pericytes on blood vessels in human tumors have abnormalities similar to those observed in mouse tumors. However, answering this seemingly straightforward question is likely to be a challenge. Our study of three mouse tumors took advantage of the many attributes of experimental tumors, including well-defined growth conditions, uniform age and genetic background of the host, multiple stages of tumorigenesis, and no previous treatment, along with the technical advantages of using vascular perfusion and other conditions of optimal tissue preservation. Because corresponding studies of pericytes in human tumors cannot readily be performed under such idealized conditions, the immunohistochemical and morphological properties of pericytes in human tumors are likely to depend on many variables, including the tumor’s histological type, grade, stage, anatomical location, age, treatment history, and fixation conditions. The heterogeneity of pericyte coverage of vessels in different types of human cancer is one manifestation of these variables.22 Future studies that examine pericyte abnormalities in human tumors should take these issues into account.
Pericytes Associated with Endothelial Sprouts in Tumors
CD31-positive endothelial sprouts arising from the wall of blood vessels were found in all three tumors we examined. The CD31 immunoreactivity, origin from endothelial cells, incomplete lumen, and blind termination fit the identification of the structures as sprouts. Because the L. esculentum lectin circulating in the bloodstream stained only the proximal third of the sprouts, it is likely that the distal portion did not have a lumen.
Nearly all of the sprouts in the tumors we examined were covered by
sleeves of pericytes. The sleeve extended well beyond the end of most
endothelial sprouts. The leading position of the pericyte sleeve may
reflect a role of these cells in sprout growth and
retraction.54,55
Pericytes regularly accompany endothelial
sprouts on growing blood vessels, seem to be involved in the earliest
stages of sprout formation, may determine the location of sprout
formation, and guide the outgrowth of sprouts.41,42
In the
ovary, pericytes are among the first cells to invade newly vascularized
corpora lutea,56
and pericytes in tumors proliferate early
in angiogenesis.26
Observations made in the present study
are consistent with reports that pericytes are located on blood vessels
at the growing front of tumors where angiogenesis is most
active.18-20,57
Pericytes are found on newly formed
vessels in other conditions as well.19,41
As to the source
of the pericytes, although we did not assess pericyte migration, the
cells accompanying sprouts could have come from pericytes on the parent
vessels or have been -SMA-positive stromal cells recruited to the
sprouts.
If pericytes play a significant role in blood vessel growth in tumors, these cells would be a potential target in anti-angiogenic therapy. In this regard, the apparent dependence of pericytes on PDGFR-ß signaling30,31 raises the likelihood that inhibitors of these tyrosine kinase receptors may disrupt angiogenesis in tumors.58
Pericytes may promote angiogenesis by secreting basic fibroblast growth factor59,60 or VEGF.61 Some VEGF-expressing stromal cells near endothelial sprouts in tumors62 may indeed be pericytes. Pericytes may also be associated with regressing sprouts, and may signal blood vessel degeneration and undergo apoptosis before endothelial cells.63,64
Origin and Function of Pericytes in Tumors
Pericytes or their precursors migrate into tumors with the
developing vasculature. Studies of implanted tumors have shown that
desmin-positive cells accumulate initially at the interface of the
tumor and host tissue and later around new blood
vessels.65
A large proportion of stromal cells in some
tumors express smooth muscle proteins and are potential pericyte
precursors.66
We found that cells expressing -SMA and
desmin were abundant at the tumor-host tissue interface of MCa-IV
carcinomas and Lewis lung carcinomas. Some of these cells were closely
associated with blood vessels, but others were not.
Stromal cells that express -SMA and desmin but are not associated
with blood vessels are usually designated myofibroblasts. These cells
are abundant in many tumors.67
Fibroblasts co-cultured
with tumor cells can differentiate into myofibroblasts.67
The stromal (mesenchymal) origin of pericytes is well
documented,26
and myofibroblasts are likely to contribute.
Because pericytes may migrate away from the basement membrane and
become extramural pericytes under some conditions,26
pericytes and myofibroblasts may be interconvertible.
|
Conclusions |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
-SMA and desmin even on
capillary-size vessels. This abnormal expression pattern appears as the
tumors enlarge. Pericytes in tumors also have multiple structural
abnormalities, including a loose association with endothelial cells and
cytoplasmic processes that invade the tumor parenchyma, which may make
the vessels sensitive to VEGF inhibitors. Similarities of pericytes in
spontaneous tumors and implanted tumors suggest that the abnormalities
are common in neoplasms. Pericytes form sleeves around endothelial
sprouts that arise from tumor vessels, may participate in blood vessel
growth, and are a potential target in anti-angiogenic therapy.
|
Acknowledgements |
|---|
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Footnotes |
|---|
Supported in part by National Institutes of Health grants HL-24136 and HL-59157 from the National Heart, Lung, and Blood Institute and a grant from MBT Munich Biotechnology GmbH (to D. M.).
Present address of S.M.: Department of Anatomy and Developmental Biology, Tokyo Women’s Medical University, 8-1 Kawada-Cho, Shinjyuku, Tokyo, 162-8666, Japan.
Present address of T. K.: Department of Anatomy, Faculty of Medicine, Tottori University, Yonago, 683-8504, Japan.
Accepted for publication December 7, 2001.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionConclusionsReferences |
|---|
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P. Baluk, S. Morikawa, A. Haskell, M. Mancuso, and D. M. McDonald Abnormalities of Basement Membrane on Blood Vessels and Endothelial Sprouts in Tumors Am. J. Pathol., November 1, 2003; 163(5): 1801 - 1815. [Abstract] [Full Text] [PDF]
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J. D. Hood and D. A. Cheresh Building a better Trap PNAS, July 22, 2003; 100(15): 8624 - 8625. [Full Text] [PDF]
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J. Huang, J. S. Frischer, A. Serur, A. Kadenhe, A. Yokoi, K. W. McCrudden, T. New, K. O'Toole, S. Zabski, J. S. Rudge, et al. Regression of established tumors and metastases by potent vascular endothelial growth factor blockade PNAS, June 24, 2003; 100(13): 7785 - 7790. [Abstract] [Full Text] [PDF]
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L. Zhang, N. Yang, J.-W. Park, D. Katsaros, S. Fracchioli, G. Cao, A. O'Brien-Jenkins, T. C. Randall, S. C. Rubin, and G. Coukos Tumor-derived Vascular Endothelial Growth Factor Up-Regulates Angiopoietin-2 in Host Endothelium and Destabilizes Host Vasculature, Supporting Angiogenesis in Ovarian Cancer Cancer Res., June 15, 2003; 63(12): 3403 - 3412. [Abstract] [Full Text] [PDF]
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 L. Vallieres and P. E. Sawchenko Bone Marrow-Derived Cells that Populate the Adult Mouse Brain Preserve Their Hematopoietic Identity J. Neurosci., June 15, 2003; 23(12): 5197 - 5207. [Abstract] [Full Text] [PDF]
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R. Giavazzi, B. Sennino, D. Coltrini, A. Garofalo, R. Dossi, R. Ronca, M. P. M. Tosatti, and M. Presta Distinct Role of Fibroblast Growth Factor-2 and Vascular Endothelial Growth Factor on Tumor Growth and Angiogenesis Am. J. Pathol., June 1, 2003; 162(6): 1913 - 1926. [Abstract] [Full Text] [PDF]
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 H. Amano, I. Hayashi, H. Endo, H. Kitasato, S. Yamashina, T. Maruyama, M. Kobayashi, K. Satoh, M. Narita, Y. Sugimoto, et al. Host Prostaglandin E2-EP3 Signaling Regulates Tumor-Associated Angiogenesis and Tumor Growth J. Exp. Med., January 20, 2003; 197(2): 221 - 232. [Abstract] [Full Text] [PDF]
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M. S. Gee, W. N. Procopio, S. Makonnen, M. D. Feldman, N. M. Yeilding, and W. M. F. Lee Tumor Vessel Development and Maturation Impose Limits on the Effectiveness of Anti-Vascular Therapy Am. J. Pathol., January 1, 2003; 162(1): 183 - 193. [Abstract] [Full Text] [PDF]
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D. M. McDonald and P. Baluk Significance of Blood Vessel Leakiness in Cancer Cancer Res., September 15, 2002; 62(18): 5381 - 5385. [Abstract] [Full Text] [PDF]
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E. S. Kim, A. Serur, J. Huang, C. A. Manley, K. W. McCrudden, J. S. Frischer, S. Z. Soffer, L. Ring, T. New, S. Zabski, et al. Potent VEGF blockade causes regression of coopted vessels in a model of neuroblastoma PNAS, August 20, 2002; 99(17): 11399 - 11404. [Abstract] [Full Text] [PDF]
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