Resistance to antiangiogenic therapy is directed by vascular phenotype, vessel stabilization, and maturation in malignant melanoma.

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J. Exp. Med. Vol. 207 No. 3 491-503
www.jem.org/cgi/doi/10.1084/jem.20091846
491
Angiogenesis is a pivotal process for growth,
invasion, and spread of tumors and is therefore
used as a therapeutic target in many types of
cancer (Hanahan and Folkman, 1996; Ferrara
and Kerbel, 2005). Sprouting of capillaries from
preexisting blood vessels is accomplished by a
hypoxia-driven mechanism, within which vas-
cular endothelial growth factor (VEGF) A has
been identified as the most potent inducer of
the angiogenic cascade (Neufeld et al., 1999).
Several strategies against VEGF-A (Siemeister
et al., 1998; W. Leenders et al., 2002; Ferrara
et al., 2004) or its receptor, VEGF receptor
(VEGFR) 1 (Flt-1), and its major signaling re-
ceptor, VEGFR-2 (KDR/Flk-1; Kunkel et al.,
2001; Sweeney et al., 2002), have been de-
veloped including neutralizing humanized an-
tibodies. Another way to efficiently perturb
VEGF-A signaling is to block the kinase activity
of VEGFRs by small-molecule inhibitors, such
as sorafenib, sunitinib, or PTK787/ZK222584
(PTK/ZK; Hess-Stumpp et al., 2005; Escudier
et al., 2007; Thomas et al., 2007). VEGF di-
rectly stimulates endothelial cell proliferation
and migration, but its role in pericyte biology
is still unclear and controversial. The interplay
of platelet-derived growth factor (PDGF) B,
CORRESPONDENCE
Dirk Schadendorf:
dirk.schadendorf@uk-essen.de
OR
Iris Helfrich:
iris.helfrich@uk-essen.de
Abbreviations used: Ang, angio-
poietin; cDNA, complementary
DNA; fl-VCT, flat-panel vol-
ume computer tomography;
MVD, microvessel density;
PDGF, platelet-derived growth
factor; PDGFR, PDGF recep-
tor; PTK/ZK, PTK787/
ZK222584; SMA, smooth mus-
cle actin; TRP, tyrosinase-
related protein; VEGF, vascular
endothelial growth factor;
VEGFR, VEGF receptor.
Resistance to antiangiogenic therapy is directed
by vascular phenotype, vessel stabilization,
and maturation in malignant melanoma
Iris Helfrich,1,3,4 Inka Scheffrahn,2 Sönke Bartling,5 Joachim Weis,6
Verena von Felbert,6 Mark Middleton,7 Masahi Kato,8 Süleyman Ergün,2
and Dirk Schadendorf 1
1Department of Dermatology and 2Institute of Anatomy, University Hospital Essen, D-45122 Essen, Germany
3Joint Research Division of Vascular Biology, Medical Faculty Mannheim, University of Heidelberg, D-68167 Mannheim, Germany
4Joint Research Division of Vascular Biology and 5Medical Physics in Radiology, German Cancer Research Center, D-69120
Heidelberg, Germany
6Institute for Neuropathology, Medical Faculty, RWTH Aachen University, 52074 Aachen, Germany
7University of Oxford, Department of Medical Oncology, Churchill Hospital, OX3 7L J Oxford, England, UK
8Unit of Environmental Health Sciences, Department of Biomedical Sciences, College of Life and Health Sciences, Chubu
University, Kasugai-shi, 487-8501 Aichi, Japan
Angiogenesis is not only dependent on endothelial cell invasion and proliferation, it also
requires pericyte coverage of vascular sprouts for stabilization of vascular walls. Clinical
efficacy of angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF)
signaling pathway is still limited to date. We hypothesized that the level of vessel matura-
tion is critically involved in the response to antiangiogenic therapies. To test this hypoth-
esis, we evaluated the vascular network in spontaneously developing melanomas of MT/ret
transgenic mice after using PTK787/ZK222584 for anti-VEGF therapy but also analyzed
human melanoma metastases taken at clinical relapse in patients undergoing adjuvant
treatment using bevacizumab. Both experimental settings showed that tumor vessels, which
are resistant to anti-VEGF therapy, are characterized by enhanced vessel diameter and
normalization of the vascular bed by coverage of mature pericytes and immunoreactivity
for desmin, NG-2, platelet-derived growth factor receptor , and the late-stage maturity
marker  smooth muscle actin. Our findings emphasize that the level of mural cell differ-
entiation and stabilization of the vascular wall significantly contribute to the response
toward antiangiogenic therapy in melanoma. This study may be useful in paving the way
toward a more rational development of second generation antiangiogenic combination
therapies and in providing, for the first time, a murine model to study this.
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492 Antiangiogenic therapy in melanoma | Helfrich et al.
vessel stabilization and crucial factors sensitizing blood vessels
to anti-VEGF therapy, both in melanoma patients and in the
corresponding murine tumor model.
RESULTS
Tumor growth and progression in MT/ret transgenic mice occurs
via high, but also low, angiogenic-active vascularization
For analysis of tumor angiogenesis in a physiological setting,
we used MT/ret transgenic mice, which spontaneously de-
velop multiple melanoma (Kato et al., 1998) and metastases
in lymph nodes (100%), spleen (>80%), and lung and brain
(>10%; Fig. S1 A). Tumor development started after a short
latency of 2–3 wk in the face and on the back of these mice,
with up to 100 tumors/mouse (20% ± SD) by the age of
9–10 wk (unpublished data). Immunohistological analyses re-
vealed morphological analogy to human melanoma (unpub-
lished data) and the expression of tyrosinase, tyrosinase-related
protein (TRP) 1, and gp-100, which are enzymes, regulating
the quality and quantity of pigment production in melanocytes
(Fig. S1 B). Interestingly, detailed studies on tumor vascular
beds showed two different vascular phenotypes in this tumor
model (Fig. 1, A and B). One predominant type showed a high
angiogenic-active phenotype (Fig. 1 A) with a mean microves-
sel density (MVD) of 250/mm2 (Fig. 1 C), in contrast to a sec-
ond type which is characterized by only a few intratumoral
vessels (Fig. 1 B) and a mean MVD of 40/mm2 (Fig. 1 C) and
which was subsequently described as low angiogenic tumor.
Analyzing tumor volume in relation to MVD, it became evi-
dent that the phenotype of the vascular network in MT/ret
melanoma was dependent on neither tumor volume nor
tumor location (Fig. 1 C and not depicted). Furthermore, the
intratumoral vessels of the low angiogenic tumors showed an
almost 10-fold increase in vessel perimeter in comparison with
the vessels of the high angiogenic tumor type (Fig. 1 D) and a
significant increase (P ≤ 0.001) in vessel lumina (not depicted).
In contrast to high angiogenic tumors, total coverage by endo-
thelial cells in the peritumoral tissue area of low angiogenic-
active tumor nodules was observed (Fig. 1 B, arrowhead).
In addition, lymphatic endothelial cells were detected in a
high number of tumor septa and peritumoral areas but not in
intratumoral tissue in both tumor types (Fig. S1 C). Assessment
of the relative abundance of the vascular bed phenotype per
mouse showed a mean distribution of 83% high angiogenic to
17% low angiogenic tumors (Fig. 1 E). However, vessel perfu-
sion did not differ in either tumor type (Fig. 1 F). Assessment
of vessel–vessel distance in individual nodules revealed a mean
distance of 41.8 µm (±19.1 µm) in tumors with high-angiogenic
potential in comparison with 172.2 µm (±57.7 µm) in low
angiogenic-active tumors (Fig. 1 G), which warrant the deliv-
ery of oxygen and nutrients for growth and progression.
Rapid tumor growth of high angiogenic tumors results
in increased tumor hypoxia
The observations described in the previous section strongly
suggest that both vascular beds coexist in parallel in the MT/ret
transgenic model without the need to switch from the low- to
which is secreted by endothelial cells, and pericytes, express-
ing PDGF receptor (PDGFR) , is important for mural cell
recruitment during development (Hellström et al., 1999;
Armulik et al., 2005; Betsholtz et al., 2005; Carmeliet 2005;
von Tell et al., 2006; Andrae et al., 2008). The absence of
pericytes, which play a key role in vascular development, ves-
sel stabilization, maturation, and remodeling, is thought to
be at least partially responsible for the irregular, tortuous, and
leaky blood vessels found within tumors (Morikawa et al.,
2002; Abramsson et al., 2003). These later steps of the an-
giogenic cascade are controlled by the PDGFs and angiopoi-
etins (Ang’s; Fiedler and Augustin, 2006; Andrae et al., 2008).
Ang-2, which is expressed by endothelia cells (Fiedler et al.,
2004), acts as a context-specific antagonist of Ang-1/Tie2 sig-
naling. As such, it destabilizes the quiescent endothelial cell
layer lining the vessel lumina and increases vascular leakage
(Carlson et al., 2001) but its effects appear to be contextual
and dependent on local cytokine milieu (Hanahan 1997), par-
ticularly on the presence of VEGF. The benefits of targeting
both pericytes and endothelial cells in tumor vessels have been
shown in several tumor models (Bergers et al., 2003; Erber
et al., 2004), and receptor tyrosine kinase inhibitors that block
both VEGFRs and PDGFRs have been shown to be more
efficacious in combination than in single use (Bergers et al.,
2003; Erber et al., 2004). Although systematic studies have
provided ample evidence that tumor progression correlates
with tumor-induced angiogenesis, this issue remains contro-
versial in the case of human cutaneous melanoma (Folkman
et al., 1989; Fallowfield and Cook, 1991; Ilmonen et al., 1999).
Neovascularization has been considered to be synonymous
with directed vessel ingrowth in almost all of these studies, but
alternative growth factor–independent mechanisms have been
reported, both experimentally and in human tumors (Paku
1998). It has been shown for some human cancers, including
non–small cell lung carcinomas (Pezzella et al., 1997) and
human glioma (Holash et al., 1999), that tumors in more natural
settings do not always originate avascularly, particularly when
they arise within or metastasize to vascularized tissue. In such
settings, tumor cells have the ability to incorporate (i.e., co-opt)
host vessels (W.P. Leenders et al., 2002), which has also been
shown as an important mechanism during development of
cutaneous melanoma (Döme et al., 2002) and melanoma of
the brain (Küsters et al., 2002). This leads to the speculation
that although compounds may be efficient inhibitors of angio-
genesis and tumor growth in angiogenesis-dependent tumors,
their effects may be limited in growth factor–independent
tumors using mature vessels. The present study analyzed the
vascular network and levels of pericyte-mediated vessel matu-
ration in human melanoma metastases and melanomas of a
corresponding tumor model grown during anti-VEGF ther-
apy. In this paper, first, we identify the spontaneous endog-
enously driven murine melanoma model (MT/ret) as the first
existing model where VEGF-dependent and independent
tumor growth occurs in parallel, and, second, we provide strong
evidence that the level of mural cell differentiation influenc-
ing vessel maturation and pericyte coverage is essential for

JEM VOL. 207, March 15, 2010
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493
et al., 1998). Therefore, we consequently analyzed the re-
cruitment of mural cells in both vascular phenotypes. NG-2,
Desmin, and PDGFR- have been established as markers
of early, i.e., immature, pericytes, whereas  smooth muscle
actin (SMA) has been reported as a marker of mature mural
cells including pericytes and smooth muscle cells (Nehls et al.,
1992; Morikawa et al., 2002; Gerhardt and Betsholtz, 2003).
Intratumoral microvessels of MT/ret transgenic melanoma
were covered by Desmin-positive mural cells without signifi-
cant difference in both vascularization phenotypes (80% in
high angiogenic vs. 92% in low angiogenic tumors; Fig. 3,
A and B). A comparable percentage of vessels also expressed
the early markers NG-2 and PDGFR- (Fig. 3 B). In con-
trast, coverage of intratumoral microvessels by -SMA–positive
mural cells was significantly higher (P ≤ 0.001) in tumors of
low vessel density (98%) compared with high angiogenic-active
tumors (2%; Fig. 3, B and C). In accordance with the matura-
tion defect and partial lack of pericyte coverage, excessive vessel
leakiness was observed in tumors of high vessel density using
FITC-conjugated dextran (unpublished data). Alongside the
the high angiogenic vascular phenotype to grow. To address
potential differences in the tumor growth kinetic, we mea-
sured tumor volume of individual nodules starting early in life
with tumor-free mice at weekly intervals over a period of 4 wk
using flat-panel volume computer tomography (fl-VCT).
Over the first 3 wk, tumor growth kinetics of high angiogenic-
active tumors were significantly increased (P ≤ 0.001) com-
pared with tumors with low vessel density (Fig. 2 A). This was
paralleled by increased expression of the proliferation marker
Ki-67 (Fig. 2 B) and apoptosis indices (not depicted) in high
angiogenic tumors, associated with increased intratumoral
hypoxic regions in those tumors (Fig. 2, C and D). The distri-
bution of hypoxic areas within both vascular beds was paral-
leled by detection of glutase-1 (unpublished data).
Lack of pericyte coverage and defects in vessel maturation
promote neovascularization in angiogenic tumors of MT/ret
transgenic mice
It has been shown that a plasticity window for remodeling
neovasculature is defined by pericyte coverage (Benjamin
Figure 1. Immunohistological and morphometric analyses of the vascular network in melanoma of MT/ret transgenic mice. (A and B) Repre-
sentative images for immunoperoxidase detection of blood vessels using the endothelial marker CD31 in melanoma of high angiogenic (A) and low
angiogenic (B) potential (n = 478 tumors of 63 mice, independently performed). Arrowheads indicate peritumoral coverage of endothelial cells in low
angiogenic tumors. (C) Scatter blot for MVD (in millimeters squared) versus tumor volume (in millimeters cubed) in high and low angiogenic tumors
(n = 20 tumors/vascular bed of four mice). (D) Quantification of vessel perimeter (in millimeters) for both vascular beds of MT/ret transgenic melanoma
(n = 500 intratumoral vessels [100 vessels/tumor] of five high angiogenic and 100 intratumoral vessels of nine low angiogenic tumors isolated from two
mice). (E) Immunohistochemically based distribution analyses for the incidence of high and low angiogenic-active tumors per mouse (in percentage) cal-
culated after isolation of all tumors (n = 478 tumors of five mice). (F) Perfusion analysis (in percentage) of intratumoral vessels was performed after injec-
tion of FITC-conjugated lectin into tumor-bearing mice. Analyzing the number of double-positive lectin- and CD31-positive tumor vessels in comparison
with CD31 single-stained vessels resulted in calculation of vessel perfusion (n = 100 vessels/vascular phenotype in 10 tumors each of four mice). Injection
experiments were independently performed in each mouse. (G) Analysis of vessel–vessel distances (in micrometers) in both vascular beds of MT/ret-transgenic
melanoma (n = 500 intratumoral vessels [100 vessels/tumor] of five high angiogenic and 100 intratumoral vessels of nine low angiogenic tumors of two
mice). Median values of the experimental groups are indicated by the horizontal lines (D and G). All morphometric analyzes were microscopically quantified
using CD31-stained tissue sections. Error bars, mean ± SD. Bars, 50 µm.

494 Antiangiogenic therapy in melanoma | Helfrich et al.
Reduced levels of proangiogenic factors and their receptors
in endothelial cells of low-vascularized tumors are
associated with resistance to anti-VEGF therapy
We and others were able to show that the angiogenic cascade
of the Ang–Tie system is important for controlling vessel as-
sembly, maturation, and quiescence (Maisonpierre et al., 1997;
Nasarre et al., 2009). Thus, enhanced expression of Ang-2,
which is responsible for vessel destabilization and immaturity
(Carlson et al., 2001), may explain the defective integration
and the partial loss of pericytes, vessel instability, and leakiness
immunohistological findings, endothelial cells without or with
partly developed basal lamina were assessed in high angiogenic
tumors by electron microscopy (Fig. 3 D). Partly reduced peri-
cyte coverage was observed, and integration of pericytes into
the basal lamina could not be detected (Fig. 3 D). Based on
pericyte loss, direct connection of tumor cells to endothelial
cells was observed (Fig. 3 D). In contrast, nearly all blood ves-
sels of low-vascularized tumors exhibited a well constructed
basal lamina underlining the endothelial cell layer and intact
integration of pericytes into the vascular wall (Fig. 3 E).
Figure 2. Comparative analysis of tumor growth rate, tumor cell proliferation, and induction of hypoxia in MT/ret transgenic mice. (A) Tumor
growth curve of high and low angiogenic-active melanoma. Tumor volume (in millimeters cubed) of individual nodules was measured weekly over a
period of 4 wk in mice of concordant sex and age using fl-VCT (n = 10 tumors/mouse). The experiment was independently performed three times using
five mice (***, P ≤ 0.001). (B) Immunofluorescence labeling of tumor cell proliferation using double staining of the proliferation marker Ki-67 (red) and the
endothelial marker CD31 (green) in tumors of high and low angiogenic potential (n = 15 tumors/vascular phenotype of five mice, analyzed in five sepa-
rate experiments). (C) Immunohistochemical assessment of hypoxic areas in high and low angiogenic-active tumors using pimonidazole injection (n = 10
tumors/vascular bed of three mice). Filled arrowheads indicate selection of hypoxic tumor cells, empty arrowheads show tumor vessels, the double-
headed arrow indicates the hypoxic-free tumor margin, and the star indicates tumor septa. Injection experiments were independently performed three
times with the corresponding outcome. (D) Quantification of hypoxic area per tumor (in percentage) in high and low angiogenic-active tumors (n = 10
tumors/vascular bed of three mice) of three independent experiments; ***, P ≤ 0.001. Representative images are presented (B and C). Error bars, mean ±
SD. Bars, 100 µm.

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Figure 3. Quantitative assessment of mural cell maturation and stabilization in MT/ret melanoma. (A and C) Immunohistochemical double
staining for the endothelial marker CD31 (green) and the early pericytic marker Desmin (red) in low-vascularized (I) and high-vascularized (II) tumors
(A), as well as for the late differentiation marker -SMA (C; red). Representative images of >10 independently performed experiments are presented (n =
43 high angiogenic and 27 low angiogenic tumors of four mice). (B) Quantification of vessel coverage, calculated as the percentage of NG-2–, Desmin-,
PDGFR-–, or -SMA–positive cells compared with the number of CD31-positive vessels (n = 1,087 high angiogenic and 352 low angiogenic tumor ves-
sels of 10 tumors of four mice; ***, P ≤ 0.001). Data are collected from >10 independent experiments. (D and E) Electron microscopic evaluation of the
vascular wall structure in sections of tumor tissues with high vascular density (two to three blood vessels per microscopic field; D) and low vascular den-
sity (one to two blood vessels in three to four microscopic fields; E), analyzed for their construction of a basal laminar, availability of pericytes, and peri-
cyte integration (n = 9 tumors/vascular bed of three mice). Data are representative of three independent experiments. EC, endothelial cell; Ery,
erythrocytes within the vessel lumen; TC, tumor cells; star, pericyte; arrowheads, basal lamina. Representative images are presented (A and C–E). Error
bars, mean ± SD. Bars: (A and C) 100 µm; (D and E) 2 µm.