(American Journal of Pathology. 2000;156:361-381.)
© 2000 American Society for Investigative Pathology
Vasculogenic Mimicry and Tumor Angiogenesis
Robert Folberg*
,
Mary J. C. Hendrix
§ and
Andrew J. Maniotis§
From the Departments of Pathology*
and
Ophthalmology,
the University of Illinois at
Chicago, Chicago, Illinois; and the University of Iowa Cancer
Center
and Department of Anatomy and
Cell Biology,§
The University of Iowa College
of Medicine, Iowa City, Iowa
 |
Abstract
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Tumors require a blood supply for growth and hematogenous
dissemination. Much attention has been focused on the role of
angiogenesis—the recruitment of new vessels into a tumor from
pre-existing vessels. However, angiogenesis may not be the only
mechanism by which tumors acquire a microcirculation. Highly aggressive
and metastatic melanoma cells are capable of forming highly patterned
vascular channels in vitro that are composed of a
basement membrane that stains positive with the periodic acid-Schiff
(PAS) reagent in the absence of endothelial cells and fibroblasts.
These channels formed in vitro are identical
morphologically to PAS-positive channels in histological preparations
from highly aggressive primary uveal melanomas, in the vertical
growth phase of cutaneous melanomas, and in metastatic uveal
and cutaneous melanoma. The generation of microvascular channels by
genetically deregulated, aggressive tumor cells was termed
"vasculogenic mimicry" to emphasize their de novo
generation without participation by endothelial cells and independent
of angiogenesis. Techniques designed to identify the tumor
microcirculation by the staining of endothelial cells may not be
applicable to tumors that express vasculogenic mimicry. Although it is
not known if therapeutic strategies targeting endothelial cells will be
effective in tumors whose blood supply is formed by tumor cells in the
absence of angiogenesis, the biomechanical and molecular events that
regulate vasculogenic mimicry provide opportunities for the development
of novel forms of tumor-targeted treatments. The unique patterning
characteristic of vasculogenic mimicry provides an opportunity to
design noninvasive imaging techniques to detect highly aggressive
neoplasms and their metastases.
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Introduction
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Tumors require a blood supply to sustain growth. The tumor
microcirculation plays a central role in the hematogenous dissemination
of cancers. Considerable attention has been focused on the mechanisms
by which tumors acquire their blood supply. It is a well-accepted
paradigm that tumors recruit new blood vessels from the existing
circulation1
—angiogenesis—either from factors secreted
by the tumor cells, as Folkman2,3
has emphasized, or from
surrounding stromal cells.4
There are two variations on
the theme of tumor angiogenesis: augmentation of the angiogenic
response by progenitor endothelial cells, and vessel cooption. Asahara
and associates5
described the incorporation of endothelial
cell progenitors (or angioblasts) from circulating peripheral blood
into sites of ischemic-driven angiogenesis. Holash and
associates6
described a process of "vessel cooption"
in which tumors coopt the existing vasculature, which regresses leading
to massive necrosis, and the tumor is then vascularized at the
periphery by tumor angiogenesis as described above.
We7
recently described a
novel process by which tumors develop a highly patterned
microcirculation that is independent of angiogenesis: in aggressive
primary and metastatic melanomas, the tumor cells generate acellular
microcirculatory channels composed of extracellular matrix and lined
externally by tumor cells. The de novo generation of
vascular channels by aggressive and metastatic tumor cells is not
strictly a vasculogenic event, because true vasculogenesis results in
the de novo formation of endothelial cell-lined vessels. We
therefore assigned the name "vasculogenic mimicry" to the process
by which aggressive tumor cells generate non-endothelial cell-lined
channels delimited by extracellular matrix.
The discovery of a mechanism by which an aggressive tumor generates its
own network of vascular channels challenges the prevailing assumption
that angiogenesis and related mechanisms are the only means by which a
tumor acquires a blood supply.7
Bissell8
has
further noted that vasculogenic mimicry poses challenges to the
practice of surgical pathology and provides opportunities for the
development of new imaging techniques and cancer treatment strategies.
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Background
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The patterned microcirculation characteristic of vasculogenic
mimicry was first described in uveal (intraocular)
melanoma.9,10
Although cutaneous melanoma is more
prevalent, one may exploit some unique biological properties of
uveal melanoma to study critical issues in tumor progression and
metastasis in a human model of cancer.11
Cutaneous melanomas usually originate in the epidermal compartment and
require a breach of the epidermal basement membrane for tumor cells to
interact directly with the dermal mesenchyme. Although cutaneous
melanoma may disseminate hematogenously, the first route of metastasis
is usually to regional lymph nodes. Uveal melanomas, by contrast,
develop within the mesenchyme of the choroid, ciliary body, or iris and
do not have an intraepithelial growth phase. There are no lymphatics
within the eye. Uveal melanoma, therefore, is an ideal human tumor
system in which to study the biology of hematogenous dissemination of
cancer. Moreover, uveal melanoma spreads first and preferentially to
the liver,12
making it an ideal human model to study organ
targeted metastasis.
There are some important differences in the management of cutaneous and
uveal melanoma. Cutaneous pigmented lesions are accessible to incision
and excisional biopsy without significant morbidity. Patients have a
general fear of losing vision that may be surpassed only by the threat
to life posed by cancer,11
and it is not possible to
perform incisional biopsies of intraocular tumors without interfering
with vision. Some ophthalmic oncologists perform fine-needle aspiration
biopsies (FNAB) of intraocular tumors to distinguish between melanomas
and lesions that simulate melanomas clinically such as metastases to
the eye.13-15
In so doing, they make only one pass into
the neoplasm to avoid interfering with vision. Thus, the one-pass
ophthalmic FNAB does not provide a broad sampling of the tumor (in
other tissue sites, multiple passes into the lesion from different
angles increases the likelihood of a representative
sampling16
). The ophthalmic one-pass FNAB sampling of
intraocular tumors does not yield material that is satisfactory for
prognostication in cytologically heterogeneous
neoplasms.17-19
It is possible for a patient to harbor a significant quantity of
metastatic disease to the liver and maintain normal hepatic
enzymes.20
Therefore, the application of any new therapy
likely to be effective in treating metastatic melanoma would be most
efficacious if applied before the metastatic tumor burden is great. The
identification of a patient at high risk for metastasis at the time of
diagnosis would then prompt the delivery of adjuvant
therapy.21
With the increasingly popular trend to avoid
removal of an eye containing uveal melanoma by administering
vision-sparing methods of primary tumor ablations such as radiation,
hyperthermia, and laser treatments, it is likely that pathologists will
not encounter any tissue from which to suggest a prognosis for the
medical oncologist. It would therefore be helpful for those physicians
who manage patients with uveal melanoma to be able to estimate the
clinical course of a patient with a primary uveal melanoma by a
noninvasive substitute for biopsy.
In 1984, we embarked on a series of studies to identify attributes of
uveal melanoma that were both strong markers of tumor progression and
that could be detected by a noninvasive imaging technique. Because the
interior of the eye and its circulation can be visualized directly by
angiography, our attention was directed first to the melanoma
microcirculation.
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The Microcirculation of Uveal Nevi
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There are no animal models that accurately reflect the histology
and behavior of primary human uveal melanoma.22
Few
animals develop these tumors spontaneously, and the transgenic models
of pigmented intraocular tumors23-26
are complicated by
histological features that indicate retinal pigment epithelial
differentiation (uveal melanomas develop from melanocytes of the iris,
ciliary body or choroid and not from the retinal pigment epithelium;
retinal pigment epithelial neoplasms are rarely encountered in
humans27
).
Following the precedent of studying the histogenesis of
primary cutaneous melanoma in animals following the application of
7,12-dimethylbenz[a]anthracene
(DMBA),28-30
Folberg et al22,31
attempted to induce primary uveal melanocytic lesions by the repeated
application of this carcinogen to the rabbit sclera. The rabbit,
although not commonly used in carcinogenesis research, provides an eye
whose interior is suitable for repeated photography, thereby affording
the opportunity to visualize the clinical emergence of pigmented
lesions from the normal tissues. It was possible to induce nevi in the
choroid of pigmented rabbits but attempts to promote these lesions to
melanomas were abandoned because the progressive corneal opacification
precluded a clinical (funduscopic) view of emerging lesions.
The nevi induced in these pigmented rabbits provided an opportunity to
study the histology of the earliest clinically detectable lesions. The
choroid of these animals became thick with pigmented, cytologically
bland melanocytes which appeared to accumulate around pre-existing
choroidal vessels. These vessels are easily identified by histological
examination because they appear to be evenly spaced throughout the
choroidal tissues. Naumann et al32
had earlier described
the histological appearance of human uveal nevi in which he indicated
that uveal nevi in humans also incorporate the pre-existing choroidal
vessels. Thompson et al33
had described the incorporation
of pre-existing vessels by a neoplasm without destruction of the
pre-existing vessels.
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The Microcirculation of Uveal Melanomas
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Most uveal melanomas (97%) contain pre-existing,
endothelial cell-lined blood vessels of the type seen in experimentally
induced choroidal nevi and in uveal nevi in humans (Figure 1)
.10
However, it is
difficult to detect smaller microvessels in many uveal melanomas,
especially in highly pigmented tumors in which the detection of
chromogens from histochemical reactions is quite challenging.
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Figure 1. Normal choroidal vessels incorporated into uveal nevi and melanomas.
A: Choroidal nevus. The nevus cells encircle four
pre-existing choroidal vessels. B: Choroidal melanoma. At
scanning magnification, few vessels are detected within the tumor,
although some vessels are identified near the tumor’s edge
(within the box).
C: Choroidal melanoma. Higher magnification of boxed zone
from B. The vessels at the tumor’s edge are lined
completely by endothelial cells and these vessels have a prominent
fibrous sheath, uncharacteristic of newly formed angiogenic vessels,
but characteristic of normal choroidal vessels. Despite the size of
this tumor, there is no evidence of necrosis. D: Normal
choroidal vessel lined by endothelial cells
(arrows) and
invested with a distinctive fibrous connective tissue sheath is
surrounded by epithelioid and spindle melanoma cells. The vessel is not
damaged, and has been incorporated into the tumor. Original
magnifications: A, scale bar, 250 µm; B, scale
bar, 2 mm; C, scale bar, 100 µm; D, scale bar,
25 µm. A–D, hematoxylin-eosin.
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Before the description of histochemical techniques to remove melanin
pigmentation by peroxide bleaching after histochemical
staining,34
Folberg et al9
explored the
microcirculation of uveal melanomas with fluorescent-labeled Ulex
europaeus agglutinin I (UEA-I) and laser scanning confocal
microscopy to visualize the Ulex signal through melanin
pigmentation. A variety of patterns of staining were identified
including long, straight, vascular structures that were frequently
arranged in parallel bundles and which occasionally cross-linked.
Ulex-positive loops surrounding circular packets of tumor
cells were also documented. These Ulex-positive channels
were presumed to be endothelial cell-lined blood vessels.9
A statistical analysis of the prognostic significance of these
interconnected patterns of Ulex-positive structures required
the study of a large series of tumors. It would have been impractical
to use laser-scanning confocal microscopy to study a large series of
tumors, and in the absence of continuous staining of these patterns by
Ulex, Folberg et al9
resorted to demonstrating
these vascular channels by staining for the basal laminar matrix
associated with these structures. Ophthalmic pathologists routinely
employ the periodic acid-Schiff stain to highlight intraocular basement
membranes of interest (such as Bruch’s membrane and Descemet’s
membrane). The PAS stain highlighted the patterns demonstrated by
Ulex in corresponding tissue sections. By omitting the
hematoxylin counterstain, the visual confusion introduced by tumor cell
nuclei was reduced and the PAS-positive patterns became more apparent.
Further, by introducing a green filter into the light path of the
microscope (or later, by selecting the green channel on digital
images), the magenta color of the PAS-positive patterns were rendered
vivid black color and easy to recognize.
In a pilot study, Folberg et al9
examined 20 pairs of
tumors matched for survival status (20 patients had died of metastatic
melanoma and 20 had survived for 15 years or more disease-free). Each
pair of tumors was also matched for size and location within the eye
(confinement to the choroid or involvement of the ciliary body). The
presence or absence of PAS-positive loops within the tumor was recorded
for each tumor. The histological detection of closed PAS-positive loops
was associated with the presence of other histological features
predictive of metastasis: the presence of epithelioid melanoma cells by
the modified Callender classification,35
and mitotic
figures.
With the histological identification of closed loops within uveal
melanomas as a prognostically strong marker of tumor progression,
attention was directed to the possibility of detecting other
PAS-positive microcirculation-associated patterns in uveal melanoma
that might be detectable by a noninvasive clinical test to serve as a
surrogate for the invasive acquisition of tissue for examination by the
pathologist. Folberg et al10
later identified seven
morphological patterns of PAS-positive channels in tissue sections of
uveal melanomas (Figure 2)
: straight
channels, arrangements of parallel straight channels, straight channels
that cross-link, arcs (incompletely closed loops), arcs with branching,
closed loops, and networks (networks were defined arbitrarily as at
least three back-to-back closed PAS-positive loops). These patterns
were later found to be organized into two hierarchical
groupings.36
Tumors that contained parallel vessels with
cross-linking also contained parallel channels and isolated straight
channels, while tumors that contained networks also contained loops,
arcs with branching and arcs without branching.
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Figure 2. Patterns in primary uveal melanoma stained by the modified PAS stain
(without hematoxylin
counterstaining), compared with sections stained
conventionally by hematoxylin-eosin. A: Straight channel.
The channel splays open and contains circulating red blood cells
(arrow).
B: Parallel straight channels cross-link
(arrow).
C: Arcs (incomplete
loops) are identified at the center of the
micrograph, and a cluster of back-to-back complete loops is identified
at the far right. D: Networks, defined as at least three
back-to-back loops. Three large back-to-back loops are evident in the
center of the micrograph above the normal choroidal vessel
(arrow), but
smaller complete loops are present throughout the upper half of the
photomicrograph. E: Three pale-staining clusters of
epithelioid melanoma cells correspond to the areas of tumor delimited
by the large-diameter PAS-positive loops in the section adjacent to
that shown in D (the arrow
indicates the normal choroidal vessel for
reference). The boxed area of the loop is
illustrated at higher magnification in F. F: Red
blood cells are identified within a space at the edge of the loop boxed
in D. Arrows point to the contour of the loop highlighted
here by hematoxylin-eosin. Original magnifications: A and
F, scale bar, 50 µm.; B–E, scale
bar, 100 µm. A–D, modified PAS without
hematoxylin counterstain;9
E and F,
hematoxylin-eosin.
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To obtain a more robust statistical analysis of the possible influence
of microcirculation-associated PAS-positive histological patterns,
Folberg et al10
studied 234 eyes that had been removed for
melanoma of the choroid or ciliary body. The prognostic significance of
each of the PAS-positive patterns was tested. Kaplan-Meier survival
curves generated from deaths secondary to metastatic melanoma indicated
that at 10-year follow-up, the survival of patients whose tumors lacked
cross-linked parallel vascular channels, loops, and networks was
significantly better (91.7%, 91.1%, and 88.3%) than for patients
whose tumor contained these patterns (56.9%, 55.4%, and 50.7%;
P = 0.0001 for all comparisons; n =
234). Multivariate Cox proportional hazards models were generated that
permitted the inclusion of conventional prognostic histological markers
such as tumor size, location within the eye, the cell type according to
the modified Callender classification, mitotic figures, and tumor
infiltrating lymphocytes, and the presence or absence of PAS-positive
vascular channel patterns, together with patient-related features
such as age and gender. The presence of PAS-positive networks entered
the model first (
2
= 40.84; P
= 0.0001). Other significant variables included (in descending order of
importance) tumor size, mitoses, cross-linking parallel vascular
channels, the presence of tumor infiltrating lymphocytes, and male
gender. Loops did not enter the model as an independent variable
because networks, the most significant variable in the model, is
composed of loops. In univariate models, the presence of arcs and arcs
with branching was each associated with a significant mortality from
metastatic melanoma.
The prognostic significance of these PAS-positive patterns, principally
loops and networks, was confirmed subsequently by a number of
independent laboratories.37-41
There is a high degree of
interobserver reliability in the histological detection of these
patterns.10,37,41
The prognostic association between any of the PAS-positive patterns
depends on the mere detection of the pattern anywhere in the tissue
section: the pattern is either present or absent. Because these
patterns tend to be continuous (eg, arcs connect to loops which form
networks), it is difficult to quantify patterns by counting discrete
structures. However, one may measure the amount of tumor remodeling by
patterns by calculating the percent of cross-sectional surface area in
a histological preparation of tumor occupied by patterns of interest.
Uveal melanoma lends itself to this technique because the entire
cross-sectional area of the tumor can almost always be included in a
standard glass microslide. Using this method, Mehaffey et
al42
associated death from metastatic melanoma with the
presence of either networks or cross-linking parallel vessels that
occupied 2% or more of cross-sectional area of tumor.
PAS-positive loops and networks were detected in hepatic metastases and
in all secondary metastatic sites.43
The ability for
aggressive melanoma to form these patterns, therefore, did not appear
to be dependent on the microenvironment of the eye, but rather
represented an intrinsic property of this aggressive tumor cell
phenotype.
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The Vascular Nature of PAS-Positive Patterns in Uveal Melanoma
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Foss et al44,45
challenged the assertion that
PAS-positive patterns in uveal melanomas were components of a
microcirculation. Unable to demonstrate PAS-positive patterns by
staining tissue sections for Factor-VIII related antigen, these
investigators demonstrated an association between the number of points
of tumor staining by Factor-VIII related antigen and survival.
Following the protocol described by Weidner et al,46
they
assumed that every discrete point stained by this putative endothelial
cell label represented a discrete blood vessel. Foss et
al44,45
discovered that PAS-positive patterns were
associated with outcome in univariate models, but dropped out of
multivariate models when "vessel counts" were allowed to enter the
model.
In reviewing the work by Foss et al,44,45
Folberg47
and Rummelt et al48
pointed out
that by connecting discrete points labeled by Factor VIII-related
antigen in the photomicrographs published by Foss et al,45
one could demonstrate looping patterns in tissue sections of uveal
melanoma. Parenthetically, even the initial studies from Folberg et
al9
using fluorescein-tagged Ulex and
laser-scanning confocal microscopy suggested discontinuous labeling of
loops, networks, and cross-linked parallel vascular channels.
Folberg et al36
suggested that PAS-positive patterns in
uveal melanoma were indeed a form of a tumor microcirculation for the
following reasons. First, they36
and
others41,44,49
labeled these PAS-positive patterns (albeit
in a discontinuous fashion) with putative markers for vessels with
Ulex, CD31, and CD34. Second, they traced these patterns
directly to the vortex vein (Figure 3)
36
(the major venous
drainage of the choroid) and to pre-existing vessels within the choroid
(Figure 3)
.50
Third, they36,51
performed
three-dimensional reconstructions of Ulex-labeled
PAS-positive patterns in uveal melanoma and demonstrated relatively
flattened channels that branched and formed looping patterns.
At least three other observations argue for these patterns representing
a functional microcirculation: 1) the absence of necrosis in uveal
melanomas that measure 1 cm or more in diameter (Figure 1)
suggests
that these tumors are well-perfused: these tumors may lack histological
evidence of internal angiogenesis but contain large areas of
interconnected PAS-positive patterned channels7
; 2) red
blood cells, often in a single-file (rouleaux) formation are frequently
detected within these patterns (Figure 4)
7
; and 3) ophthalmologists
have detected looping patterns in uveal melanomas in patients using
confocal imaging systems within seconds after injection of indocyanine
green into the antecubital vein.52-54
Additionally, the
angiographic detection of looping patterns before removal of the eye
has been correlated with the detection of PAS-positive looping patterns
in histological sections of the corresponding tumors.7,53
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Figure 4. Perfusion in vasculogenic mimicry patterns. A: Column of red
blood cells in an arc without branching. Endothelial cell nuclei are
not identified lining this channel. A thin layer of extracellular
matrix (arrow)
extends from this channel. B: Thin parallel channels that do
not appear to be perfused with blood
(arrows) splay
open focally to reveal red blood cells in the lumen. None of these
channels is lined by endothelium. Original magnifications:
A, scale bar, 10 µm; B, scale bar, 50 µm;
A and B, hematoxylin-eosin.
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The Patterned Microcirculation of Uveal Melanoma: Is It
Angiogenesis?
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Foss et al44,45,55
also argued that PAS-positive
looping patterns identified by Folberg et al10
could not
have been vascular because of the topological arrangement of these
patterns: vascular structures would not be expected to form looping
patterns in two-dimensional histological sections.
What is the histological appearance of angiogenesis in intraocular
tumors? Retinoblastoma, the most common intraocular tumor of children,
is highly angiogenic and is characterized typically by a large number
of vessels within tumors which are clearly lined by endothelium.
Characteristically, zones of necrosis are present distal to the cuff of
viable tumor cells surrounding the intratumoral blood vessels in
retinoblastoma (Figure 5)
.56
Up-regulation of vascular endothelial growth factor has been
demonstrated within these highly angiogenic tumors.57,58
Curiously, despite evidence of florid intratumoral angiogenesis in
retinoblastoma, deaths from metastatic retinoblastoma are vanishingly
rare as long as the tumor is confined to the eye; the risk of mortality
increases only when the tumor invades into the optic nerve, the uveal
tract, or extends outside the eye.59
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Figure 5. Angiogenesis in retinoblastoma. A: Numerous discrete vessels
are identified. Note the zones of necrosis
(left and upper
right). B: Higher magnification.
Viable tumor surrounds vessels in a cuff. Necrosis is identified
farther away from the angiogenic vessel. C: With additional
magnification, endothelial cell sprouting is identified histologically
within the retinoblastoma tumor. Endothelial cells
(arrows) are
identified by light microscopy in every vessel. Original
magnifications: A, scale bar, 500 µm; B, scale
bar, 100 µm; C, scale bar, 50 µm.
A–C, hematoxylin-eosin.
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By contrast, one seldom sees the pattern of angiogenesis characteristic
of retinoblastoma (with perivascular tumor cell cuffing around vessels
that are clearly lined by endothelial cells interspersed with zones of
necrosis, Figure 5
) in tissue samples of primary human uveal melanoma.
Significantly large zones of necrosis are seldom encountered within
uveal melanomas.41
Also, although Kvanta et
al57
detected VEGF mRNA by in situ
hybridization within retinoblastoma, they were unable to do so in
posterior uveal melanoma. Peer et al60,61
have used uveal
melanoma as negative controls for mRNA VEGF expression when studying
classic examples of intraocular angiogenesis such as ischemic central
retinal vein occlusion.
The interconnected channels characteristic of PAS-positive vascular
channels are clearly different from the expected histological profile
expected of tumor angiogenesis. Moreover, the incorporation of normal
pre-existing choroidal vessels into uveal nevi and
melanomas33
(Figure 1)
is clearly different from the
mechanism of vascular cooption described by Holash et al6
in which the inclusion of normal pre-existing vessels in the tumor
results in destruction of the vessels, significant necrosis, and
angiogenesis at the tumor periphery.
In light of these observations, the histology and ultrastructure of the
PAS-positive channels in uveal melanoma was re-examined. The
PAS-positive patterned channels were discovered to be lined externally
by melanoma cells (Figure 4)
but lacked an internal lining of
endothelial cells by light and transmission electron
microscopy.7
Endothelial cells were detected lining the
interior of pre-existing uveal vessel lumens incorporated into these
tumors of the same type found in nevi50
present within the
same sections that contained PAS-positive patterned channels.
It is important to emphasize that a layer of extracellular matrix
(corresponding to the PAS-positive channel lining) separated the blood
column from the tumor cells. Thus, the red cells appeared to be
contained within a tube of extracellular matrix. Tumor cells were
apposed to the external surface of the tube. In this regard, the
PAS-positive vascular channels in uveal melanoma are therefore
different from the "angio-tumor complex" described by Lugassy et
al62
in which endothelial cells lining the interior
of vessels are separated by laminin from melanoma cells (Lugassy et
al63
further proposed that melanoma cells migrate along
the abluminal surface of endothelial cell-lined vessels, a process they
term "extravascular migratory metastasis").
The PAS-positive patterns of uveal melanoma were studied with
conventional markers for endothelial cells including Factor
VIII-related antigen, Ulex, CD31, CD34, and KDR (the flk
receptor of vascular endothelial growth factor).7
Although
the endothelium in the vascular rich choroid adjacent to the tumor
stained brilliantly with these markers, there was limited staining
within tumors that contained the interconnected PAS-positive patterns
such as loops, networks, and cross-linking parallel vascular channels
(Figure 6)
. At higher magnification,
there was discrete labeling of these patterns with putative endothelial
cell markers, but a careful examination of these labeling points
indicated that the lumen contents (rather than the vessel walls) were
stained, often discontinuously.7
In some areas, the
labeling was interrupted by the presence of red blood cells within the
channel lumen (Figure 6)
.
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Figure 6. Immunohistochemical staining of primary uveal melanoma with putative
endothelial cell markers. A: Factor VIII-related antigen
stains normal choroidal vessels (left of the
tumor mass), but does not stain the interior of
the tumor. B: Same tumor in A stained by the
modified PAS stain without hematoxylin counterstain. Vasculogenic
mimicry patterns are identified within the tumor. C: Primary
uveal melanoma stained with Ulex
europaeus agglutinin I. Note the intermittent staining
of this vascular channel that contains red blood cells. The material
between the red blood cells
(plasma) stains with the
Ulex lectin. The section is counterstained with
hematoxylin, and despite the long segment of the channel illustrated,
no endothelial cell nuclei are identified. There are no difficulties in
identifying endothelial cell nuclei lining normal vessels incorporated
into primary uveal melanomas (Figure 1)
. D: CD31 staining tumor cells in
the vicinity of vasculogenic mimicry patterns
(not illustrated).
Original magnifications: A and B, scale bar, 200
µm; C and D, scale bar, 10 µm. A:
Factor VIII related antigen-hematoxylin; B: PAS without
hematoxylin counterstain; C: Ulex
europaeus agglutinin I with hematoxylin counterstain;
D: CD31 counterstained with hematoxylin.
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What explains the observation that endothelial cells are clearly
present in normal vessels within the tumor but not within the
anastomosing vascular channels outlined by PAS? One might argue
that the interconnected PAS-positive patterns represent regressed
angiogenesis: the endothelial cells are attacked, are destroyed, and
leave behind their basal lamina. If this were correct, then the basal
lamina of the PAS-positive patterns should resemble the basal laminar
profiles of angiogenic blood vessels such as those demonstrated in
retinoblastoma. However, the PAS-positive patterns do not appear to be
vascular from the vantage point of topology. In fact, one might ask the
following question: if highly invasive tumors are generally destructive
of host tissues, what mechanisms permit a tropic growth of fragile
endothelial cell sprouts to penetrate and survive within these
tumors?33
These issues prompted us to explore the hypothesis that the patterned
PAS-positive vascular changes in uveal melanoma developed through
mechanisms other than angiogenesis.
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In Vitro Observations: Aggressive Uveal
Melanoma Cells Are Capable of Generating Patterned Vascular Channels in
the Absence of Endothelial Cells through Vasculogenic Mimicry
|
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Daniels et al64
and Hendrix et al65,66
developed primary and metastatic uveal melanoma cell lines to explore
the relationship between the aggressive tumor cell phenotype, and the
generation of prognostically significant patterning in uveal melanomas.
Aggressive uveal melanoma cells (but not non-aggressive cells) were
found to produce type VI collagen which was thought to contribute to
the histogenesis of these patterns.64
Hendrix et
al65
described the relationship between the co-expression
of mesenchymal (vimentin) and epithelial (keratin 8,18) intermediate
filaments with respect to the invasive behavior of uveal melanoma
cells, and found that this interconverted phenotype specifically
expressed the c-met proto-oncogene which permitted the
aggressive melanoma cells to respond to its ligand, hepatocyte growth
factor/scatter factor (HGF/SF).66
The relationship between
aggressive melanoma cells that co-expressed vimentin and keratin 8,18
intermediate filaments was particularly interesting because these cells
often aligned along the external walls of microvascular channels that
conducted red blood cells, which did not appear to be lined by
endothelial cells (Figure 7A)
. This
observation suggested further that the interconverted aggressive
melanoma cell phenotype had a role in at least maintaining the
patterned PAS-positive microcirculation of uveal melanomas.
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Figure 7. Cytokeratin expression in primary uveal melanoma and vasculogenic
mimicry. A: Histological section of primary uveal melanoma.
A channel containing red blood cells is lined externally by spindle
melanoma cells that stain positive for pan-cytokeratin. Note the lack
of endothelial cells along the inner channel wall.
B–E:, Tissue cultures of metastatic uveal
melanoma cell line MUM2B. B: Phase contrast showing loop
encircling a small cluster of epithelioid melanoma cells. C:
The same field illustrated in B photographed with
fluorescence. The culture has been stained with antibody to keratins
8,18. Note the alignment of keratin-positive tumor cells alongside the
looping pattern formed in vitro. D: Phase
contrast of another aggressive melanoma culture showing two parallel
straight channels. E: The same field illustrated in
D photographed with fluorescence. The culture has been
stained with antibody to keratins 8,18. Note the alignment of
keratin-positive tumor cells alongside the straight channels, similar
to that seen in tissue section
(A). Original
magnifications: A, scale bar, 10 µm;
B–E, scale bar, 5 µm; A:
pan-cytokeratin counterstained with hematoxylin; B and
D: phase contrast; C and E:
fluorescence (cultures labeled with antibody to
keratins 8,18).
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Maniotis et al7
reported the unexpected finding that
highly invasive and interconverted primary and metastatic uveal
melanoma cell lines generated acellular channels in vitro
(in three-dimensional cultures of Matrigel or Type I collagen) in the
absence of endothelial cells or fibroblasts and without the addition of
soluble growth factors such as bFGF, TGF-ß, VEGF, PDGF. Thus, highly
aggressive primary and metastatic tumor cells in vitro
reconstituted channels that were interconnected into the
patterns seen histologically in tissue samples of patients at high risk
of dying from metastatic melanoma. By contrast, poorly aggressive uveal
melanoma cells that were not interconverted (cells expressing vimentin
but not co-expressing keratins 8,18) were incapable of generating
channels under identical culture conditions as the aggressive cell
lines, even after the induction of hypoxia, and the addition of
conditioned media from the aggressive uveal melanoma cell lines and
soluble growth factors. Furthermore, highly aggressive and metastatic
melanoma cells expressing keratins 8,18 along with vimentin were
frequently observed aligned outside the vascular channel wall (Figure 7, B–E)
.
Maniotis et al7
also demonstrated that the patterned
channels generated by aggressive uveal melanoma cells in
vitro were capable of conducting dye over short distances. They
further demonstrated a striking comparison between the appearance of
dye contained in the in vitro looping channels and those
visualized angiographically in the tumors of patients after systemic
injection of the dye, indocyanine green.52,53
The differential ability of highly invasive and metastatic melanoma
cell lines to generate patterned vascular channels (in comparison with
poorly invasive melanoma cell lines) provided a biological basis for
the use of these patterns in histological sections of human melanomas
as a marker of tumor progression (Table 1)
. Additionally, the generation
of these patterned acellular channels by melanoma cells in the absence
of endothelial cells provided an explanation for the histological
appearance of patterned, matrix-lined vascular channels in melanomas
that are not lined by endothelial cells.
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Table 1. Comparison between Patterned Circulatory Channels in Human Uveal
Melanoma and in Vitro Melanoma-Generated Vascular Channels
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Cutaneous Melanoma
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Although uveal melanoma provides an interesting human model of
cancer in which to study pure hematogenous dissemination of cancer,
organ targeted metastasis, and the host response to a tumor that
develops in an immunologically privileged site, uveal melanomas are
considered rare tumors. The incidence of cutaneous melanoma is
increasing and by contrast, cutaneous melanoma is a significant public
health problem.
Busam et al67
stained histological sections of primary
cutaneous melanoma with UEA-1, CD34, and CD31 and failed to demonstrate
any association between microvascular counts and outcome. In the course
of this study, these investigators looked for the microcirculation
patterns of uveal melanoma,10
but did not identify them
using these markers. In retrospect, Busam et al67
stained
for the presence of endothelial cells, whereas the patterned
microcirculation of uveal melanoma was demonstrated in histological
sections by staining for the matrix associated with the vascular
channels using the PAS stain. If vasculogenic mimicry develops in
cutaneous as well as uveal melanoma, then conventional markers for
endothelial cells may not identify vasculogenic mimicry patterns.
Loops and networks can be demonstrated in the vertical growth phase of
primary cutaneous melanoma (Figure 8)
and
in metastases from cutaneous melanoma7,68
using the PAS
stain without hematoxylin counterstaining. As in uveal melanoma,
columns of red blood cells can be identified in these channels which
are not lined by endothelial cells.
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Figure 8. Primary cutaneous melanoma, vertical growth phase. Networks are
abundant. Original magnification: scale bar, 200 µm. PAS without
hematoxylin counterstaining (tissue section
courtesy of Prof. T. K. Das Gupta).
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The identification of PAS patterns is not limited to histological
observation. Maniotis et al7
also demonstrated the
generation of patterned vascular channels by a cell line of metastatic
cutaneous melanoma under the same conditions for which the generation
of patterned vascular channels was identified in cell lines derived
from highly invasive primary and metastatic uveal melanoma. Therefore,
the phenomenon of vasculogenic mimicry is not confined to the rare
uveal melanoma, but appears both in vitro and in
vivo in cutaneous melanoma. The prognostic significance of
detecting looping vasculogenic mimicry patterns in histological
sections of cutaneous melanoma is presently under investigation.
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Counting Microvessels and Vasculogenic Mimicry
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As mentioned above, some investigators67,69-72
have
not been able to establish a relationship between tumor vascularity as
it is defined conventionally (the demonstration of microvessels by
histochemical markers for endothelial cells) and outcome in cutaneous
melanoma. On the other hand, some investigators73,74
report an association between high "vascularity" and outcome in
cutaneous melanoma while Ilmonen et al75,76
recently
reported that high vascularity was associated with a favorable outcome.
Likewise, there has been a significant difference of opinion in the
value of counting microvessels in uveal melanoma. Lane et
al77
were unable to demonstrate a relationship between
microvascular density and outcome from uveal melanoma, whereas Foss et
al44,55
and Makitie et al78
demonstrated a
relationship. The latter group cautioned that microvascular density may
only be a "rough measure" of the relative vascularization rather
than an exact number of vessels and cited several reasons for this
insight: 1) some microvessels did not stain for endothelial cell
markers (thus under-representing the number of vessels present); and 2)
cell types other than endothelial cells might be labeled with these
markers.
The discovery of vasculogenic mimicry in melanoma not only confirms the
concerns of Makitie et al78
but raises additional
questions about the validity of counting structures that stain with
putative endothelial cell markers as a measure of vascularization. When
vasculogenic mimicry is present in a tumor (such as a melanoma), then
pre-existing normal vessels10,51
which contain endothelial
cells will be labeled by the endothelial cell marker, making it
difficult to equate "vascular density" with "angiogenesis" (the
production of new vessels from pre-existing vessels). Additionally, the
channels generated by tumor cells in vasculogenic mimicry may not stain
with a variety of endothelial cell markers (because endothelial cells
are not present in these channels) or the channels may stain in a
discontinuous fashion because the contents of the lumen stain with
these markers, assumed to be endothelial cell specific;7
discontinuous staining of a vasculogenic mimicry channel may lead to
the over-counting of one vascular structure (Figures 6 and 9)
. Finally, tumor cells may themselves
stain for endothelial cell markers (Figure 6)
.7
If the
investigator uses slides that are stained to develop the chromagen for
putative endothelial cell markers without counterstaining to identify
the structure that is being labeled, then counting every labeling point
may not accurately reflect the number of endothelial cell-lined vessels
in the tumor.
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Figure 9. Perfusion of vasculogenic mimicry patterns may simulate the appearance
of angiogenesis histologically. A: Primary uveal melanoma:
perfusion in parallel vasculogenic mimicry channels. This is the same
field illustrated in Figure 4B
. Here, the areas in which the channels
splay open and contain red blood cells are highlighted with
arrowheads. Because the blood column itself stains with
multiple putative endothelial cells markers such as Factor VIII-related
antigen (Figure 6A)
and
Ulex (Figure 6)
, as well as CD31, CD34, and
KDR,7
it is possible to count each focus of blood as a
separate vessel if the tissue section is not counterstained with
hematoxylin and one is not attuned to the existence of continuous
vasculogenic mimicry patterns in the section. B and
C: Co-localization of CD34 to PAS-positive loops and
networks. A histological section of primary uveal melanoma containing
multiple loops and networks was stained with CD34
(Texas Red chromagen) and
counterstained subsequently by PAS without hematoxylin counterstaining.
The tissue section was photographed Bio-Rad MRC-600 laser scanning
confocal microscope (Bio-Rad, Cambridge,
MA) by capturing both the direct illumination
channel (for the PAS-positive
patterns) and the rhodamine channel
(for Texas Red).
B: Back-to-back loops form networks. C: The same
field illustrated in B, showing CD34
(in red) co-localizing to
the loops by staining the lumen contents rather than endothelial cells.
A pathologist looking only at the CD34 stain
(C) might
conclude erroneously that this is an angiogenic hot-spot. Original
magnifications: A–C, scale bar, 50 µm;
A, hematoxylin-eosin; B, CD34 and periodic
acid-Schiff without hematoxylin counterstaining
(direct illumination);
C, CD34 and periodic acid-Schiff, rhodamine channel.
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Many technical factors contribute to the accuracy of applying
microvessel counts to prognosis by pathologists including the method of
sampling and the selection of the marker for demonstrating
microvessels.79-81,81
Despite the popularity of using
microvascular counts as a marker of tumor progression in many types of
cancer,81
there are a considerable number of reports that
show no relationship between vascular counts and
prognosis67,69-72,77,82-107
and even a study that
associates an increased vascular count with a longer rather than a
shorter survival time.96
It is possible that the presence
of vasculogenic mimicry in tumor types other than melanoma may
contribute to reports in which the association between vascular
counting and outcome is not established.
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Non-Endothelial Cell-Lined Vascular Channels in Animal and Human
Tumors
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Others108-110
have hinted at the possibility of
non-endothelial cell-lined channels in melanomas and other tumors.
Jensen108
described large sinusoids that were lined by
tumor cells and not endothelial cells in the portion of a melanoma
superficial to a break in Bruch’s membrane. Radnót and
Antal109
identified small vessels lined by endothelium in
uveal melanoma (as did Folberg et al,9
in an early study),
but they also described vascular channels not lined by endothelium.
Hammersen et al111
identified cells lining the interior of
vascular channels in animal models of melanoma and suggested that it
would be difficult to identify these cells accurately by either
transmission electron microscopy or with immunohistochemical stains.
These authors suggested that mesenchymal cells and tumor cells may be
incorporated into tumor blood vessels.
Konerding et al110
described tumor-cell lined
sinusoids in xenografts of melanomas and sarcomas that were clearly
different from normal vessels. Konerding et al110
also
observed more of these abnormal tumor cell-lined vessels in
the interior of tumors than normal endothelial cell-lined vessels. The
scanning electron micrograph provided by Konerding et
al110
of flat, noodle-shaped vascular channels in a
sarcoma that was not lined by endothelium is strikingly similar
to three-dimensional reconstructions of the microcirculation of human
uveal melanoma by Rummelt et al51
Warren and Shubik112
suggest from their in vivo
observations of the vascularization of tumor implants in animals that
an endothelial tropism develops adjacent to the tumor, and blood
travels between loose cords of tumor cells as identified by
transmission electron microscopy. The phenomenon described by Warren
and Shubik112
differs from vasculogenic mimicry in three
key aspects: 1) capillary sprouts were observed to enter the tumor
(angiogenesis) with leakage of red blood cells between tumor cells (one
seldom sees leakage of red blood cells from the matrix-lined tubes
characteristic of vasculogenic mimicry); 2) microthrombi were observed
in the microcirculation (curiously, microthrombi are seldom observed in
the patterned channels of vasculogenic mimicry, leading to speculation
that either the tumor cell or the matrix outlining the vascular channel
interferes with hemostasis); and 3) central necrosis was a feature of
established animal tumors: central necrosis is not a feature typical of
tumors containing vasculogenic mimicry patterns.7
Nasu et al113
implanted 0.1 mm3
chunks of rat mammary carcinoma 13762 into transparent quartz chambers
in female Fisher rats. They then recorded blood flow within the tumor
26 days after transplantation when the tumor measured 6 mm in mean
diameter. By video microscopy, they observed back-to-back loops forming
networks within these implants. Histological examination of these
implants revealed little evidence of fibrous tissue adjacent to
vascular structures within the tumor in contrast with fibrous
connective tissue associated within the tissue surrounding the tumor.
Moreover, these investigators demonstrated a uniform staining of Factor
VIII related antigen in endothelial cells in the interstitium
surrounding the tumor, but a non-uniform distribution within the tumor.
They further observed that vessels within the tumor contained
"extremely rare endothelial cells." Of interest is the fact that
they did not observe sprouting of new blood vessels and instead
described the "primitive" flow of blood between tumor cells forming
loops. This interesting observation differs from vasculogenic mimicry
in two key aspects: 1) in vasculogenic mimicry, a layer of PAS-positive
material of variable thickness separates the blood column from tumor
cells (the red blood cells flow within channels formed by PAS-positive
material as suggested by the in vitro reconstitution of
these acellular channels by tumor cells), instead of blood coming
directly in contact with tumor cells; and 2) vasculogenic mimicry
requires the active participation of deregulated, highly aggressive
tumor cells in the formation of a patterned non-endothelial cell-lined
vascular channel, whereas Nasu et al113
attribute the
formation of non-endothelial cell-lined vascular channels to the
passive sculpting of tumor by hemodynamic forces. Vasculogenic mimicry,
therefore, is linked to the aggressive tumor cell phenotype; in the
system described by Nasu et al,113
it is not clear that
the tumor plays any active role in establishing its circulation.
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Vasculogenic Mimicry and Angiogenesis: The Issue of
Compartmentalization
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Before the discovery by Maniotis et al7
that the
PAS-positive acellular vascular channels were formed by tumor cells,
some pathologists considered the patterns described by Folberg et
al10
to be a stromal response to the presence of the tumor
as implied by terms used by some to describe these patterns:
"fibrovascular loops."38,44,45
Foss et
al45
concluded that the PAS-positive patterns described by
Folberg et al10
were "mostly formed from connective
tissue, including perivascular connective tissue." The in
vitro studies by Maniotis et al7
do not support these
conclusions: not only is the patterned tumor microcirculation of uveal
melanoma generated by aggressive tumor cells themselves, these patterns
form in the absence of fibroblasts, other stromal cells, and
endothelial cells.7
Uveal melanoma is distinctive because there is usually no induction of
a stromal host response at the interface between the tumor and the
surrounding host stroma (Figure 6)
. Moreover, stromal ingrowth (a
fibrovascular connective tissue stroma) is seldom seen within the
expanding cellular compartment of most uveal melanomas unless the tumor
has been treated previously by irradiation or necrosis is evident.
However, uveal melanoma may not be unique: Birck et al114
failed to demonstrate CD31-positive vessels within the expanding tumor
mass in most cases of primary cutaneous melanoma.
In studying transplantable mouse mammary adenocarcinoma, Thompson et
al33
noted a greater number of vessels in the adjacent
connective tissue than in the tumor itself. In studying human breast
cancer, de Jong et al115
noted a lower density of vessels
within the tumor than in the stroma. These authors used this
observation to caution pathologists who relate microvascular density to
note whether their observations are taken from the cellular part of the
tumor or the stroma. In prostatic carcinoma, Bigler et
al116
also demonstrated an increase in vascularity within
the tumors, but the increased vascularity was confined to the stroma.
These observations suggest that the angiogenic response to tumors may
be a component of the stromal compartment of tumors, rather than the
tumor cell compartment. Indeed, in the initial description of tumor
vessel counts as a marker of prognosis in breast cancer, Weidner et
al46
illustrated vascularity in the stromal compartment of
the tumor adjacent to masses of tumor cells. Brown et
al,117
who likened the tumor stroma to a healing wound,
recently observed118
that the formation of a vascular rich
stroma in breast cancer precedes invasion and suggested that breast
cancer invades into a richly vascular stroma induced by the tumor. It
is interesting to speculate that more aggressive tumors induce a more
robust stromal response and that the intensity of the stromal response
is the basis for relating counts of microvessels from histological
sections to outcome. Along similar lines, one might argue that therapy
targeted against angiogenesis would be expected to interfere with or
reduce the stromal response to the cellular component of the tumor
rather than affect the cellular compartment of the tumor directly.
The identification of vasculogenic mimicry in melanoma as an event
separate from angiogenesis suggests that different tumor types may
acquire their blood supply by different mechanisms. Uveal melanoma
perhaps sits at one end of the spectrum. In the earliest phases of
tumor growth, the uveal melanocytic neoplasm incorporates pre-existing
vessels without destroying them, without provoking central necrosis,
and without inducing angiogenesis at the tumor periphery (unlike
cooption as described by Holash et al6
). Relatively
indolent uveal melanomas do not show evidence of vasculogenic mimicry,
but aggressive tumors develop a perfused microcirculation comprised of
acellular vascular channels generated by the tumor cells themselves
that generally precludes necrosis, even in relatively large tumors. The
vascular channels generated by aggressive tumor cells hook up to either
pre-existing vessels incorporated into the tumor50
or to
the venous drainage of the eye at the vortex vein (Figure 10)
.36
Angiogenesis may
accompany focal zones of necrosis and may be seen after radiation
treatment but does not play a significant role in supplying the tumor
with a microcirculation.
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Figure 10. Diagrammatic scheme of vasculogenic mimicry. Uveal melanomas develop in
an environment devoid of lymphatics. Aggressive tumors
(but not non-aggressive
tumors) form looping non-endothelial
cell-lined channels that are delimited by PAS-positive material. These
vasculogenic mimicry channels link directly to normal vessels in the
choriocapillaris, the vortex vein, or normal vessels incorporated into
the tumor without evidence of angiogenesis. Channels generated by tumor
cells (vasculogenic
mimicry) are lined externally by tumor cells, in
contrast to blood vessels which are lined internally by endothelial
cells. Diagram courtesy of Dr. Dawn Kirschmann.
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Vasculogenic mimicry is also known to develop in cutaneous melanoma,
and angiogenesis is seldom seen within the expanding cellular
compartment of these tumors except adjacent to zones of ulceration or
necrosis. However, vascularization in the dermis is a known component
of the regression response to cutaneous melanoma.119
The
compartmentalization of vasculogenic mimicry to the cellular
compartment and angiogenesis typically to the stromal response may
account for the variability of associating vascular counts with
prognosis in cutaneous melanoma.
Maniotis et al7
showed that vasculogenic mimicry patterns
form in vitro in the absence of hemodynamic forces,
suggesting that the formation of a patterned non-endothelial cell-lined
microcirculation may be an attribute of the tumor cell. The phenotypic
properties of the tumor implants used in experiments by Warren and
Shubik112
and Nasu et al113
were not
described. If the animal tumor fragments used in these experiments
contained vasculogenic mimicry patterns, then it is possible that the
flow of blood from capillary sprouts that penetrated the tumor either
dissected around tissue planes generated by tumor cell remodeling or
that the flow of blood into the tumor hooked up with vasculogenic
mimicry channels.
At the other end of the spectrum, there may be tumors that develop a
prominent stromal vascular (angiogenic) response exclusive of
vasculogenic mimicry. Until the various contributions of angiogenic
stromal responses and non-angiogenic mechanisms are identified for
different types of tumors, pathologists may wish to exercise caution in
establishing and relying on conventional
Top
Abstract
Introduction
