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Introduction
Cancer growth strictly depends on the expansion of the host
vasculature, which not only supplies oxygen and nutrients to the
tumor tissue, but also provides cancer cells with the metastatic
route to colonize distant organs. Therefore, angiogenesis repre-
sents a critical process during tumor initiation and malignant pro-
gression (1). Diferent strategies have been developed to reduce
angiogenesis and thus control tumor progression, as exemplifed
by the inhibition of VEGF-dependent pathways. However, while
some cancer types show a certain degree of therapeutic response,
the benefts of such antiangiogenic agents are transient and the
initial response is often followed by the establishment of resis-
tance and escape mechanisms, leading to tumor relapse (2, 3).
This highlights the need for a more comprehensive understand-
ing of the biological processes that underlie tumor vascularization,
which, in turn, would set the stage for additional angiogenesis-tar-
geted therapies.
Compared with their normal counterparts, tumor vessels are
aberrant in almost all aspects of their structure and function. They
are heterogeneous and tortuous, branch chaotically, and have an
uneven vessel lumen. In addition, they frequently lack pericyte
coverage and show an abnormal basement membrane, result-
ing in vascular instability and altered permeability. These vessel
abnormalities generate a promalignant microenvironment, char-
acterized by hypoxia, low pH, and high fuid pressure, which can
select for more malignant cancer cells and facilitate their dissemi-
nation through leaky vessels, thus causing poor response to ther-
apy (4). These fndings raise the question of whether tumor vessel
normalization provides an alternative therapeutic opportunity in
order to reduce metastatic spread and enhance tumor responses
to chemotherapy and radiotherapy (4). However, the characteris-
tics that make tumor-associated endothelial cells (ECs) diferent
from normal ECs are not yet fully identifed, and their properties
are usually extrapolated from the behavior of ECs during vascular
development. Moreover, while it is well known that diferent and
morphologically distinct EC types, such as tip, stalk, and phalanx
cells, coexist during normal vascularization, the heterogeneity of
cancer-associated ECs is still poorly understood. Hence, a deep-
er understanding of the phenotypical heterogeneity and specifc
molecular signature of tumor vasculature is essential to elucidat-
While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and
functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation,
and pathnding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we
evaluated L1 functionality in cancer vessels. Tumor-bearing mice specically lacking L1 in endothelial cells or treated with
anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth
and metastasis. In line with these dramatic efects of L1 on tumor vasculature, the ectopic expression of L1 in cultured
endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis,
enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC
transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated
pathways. In particular, L1 induced IL-6mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis
prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in
noncancerous tissue, L1 expression is specically enhanced in blood vessels of human pancreatic carcinomas and in vessels
of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and
represents a potential target for tumor vascular-specic therapies.
Endothelial deficiency of L1 reduces tumor
angiogenesis and promotes vessel normalization
Elena Magrini,
1
Alessandra Villa,
1,2
Francesca Angiolini,
1,2
Andrea Doni,
3
Giovanni Mazzarol,
2
Noemi Rudini,
4
Luigi Maddaluno,
4

Mina Komuta,
5
Baki Topal,
6
Hans Prenen,
7
Melitta Schachner,
8
Stefano Confalonieri,
2
Elisabetta Dejana,
4
Fabrizio Bianchi,
1,2

Massimiliano Mazzone,
9,10
and Ugo Cavallaro
1,2
1
Department of Experimental Oncology and
2
Molecular Medicine Program, European Institute of Oncology, Milano, Italy.
3
Department of Inammation and Immunology, Humanitas Clinical and
Research Center, Rozzano, Milano, Italy.
4
IFOM, FIRC Institute of Molecular Oncology, Milano, Italy.
5
Department of Morphology and Molecular Pathology,
6
Department of Abdominal Surgery, and
7
Department of Gastroenterology, University Hospitals Leuven, Leuven, Belgium.
8
W.M. Keck Center for Collaborative Neuroscience and Department of Cell Biology and Neuroscience,
Rutgers University, Piscataway, New Jersey, USA.
9
Laboratory of Molecular Oncology and Angiogenesis, Vesalius Research Center, Vlaams Instituut voor Biotechnologie (VIB),
Leuven, Belgium.
10
Department of Oncology, Katholieke Universiteit (KU) Leuven, Leuven, Belgium.
Authorship note: Alessandra Villa and Francesca Angiolini contributed equally to this
work.
Conict of interest: The authors have declared that no conict of interest exists.
Submitted: April 26, 2013; Accepted: July 17, 2014.
Reference information: J Clin Invest. doi:10.1172/JCI70683.
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Besides the nervous system, L1 is expressed in many human
cancers, including ovarian and endometrial carcinoma, pancreat-
ic ductal adenocarcinoma (PDAC), melanoma and glioblastoma.
L1 expression confers motile and invasive properties to tumor
cells, supporting cancer growth, metastasis, and chemoresistance
and often acting as a marker of poor prognosis (8). L1 has also been
detected in the hematopoietic system, in particular in immune
cells of myelomonocytic and lymphoid origin (9), and we have pre-
viously reported L1-dependent transmigration of dendritic cells
across the endothelium (10).
An intriguing aspect of L1 biology is its expression in the vascu-
lar system: while no or very little L1 is detectable in the vasculature
of most normal tissues, its level is markedly increased in the vas-
ing the mechanisms of pathological angiogenesis and to identify-
ing novel cancer vesselspecifc markers.
L1 (also known as L1CAM or CD171) is a transmembrane glyco-
protein belonging to the immunoglobulin superfamily and is com-
posed of an extracellular portion, containing 6 Ig-like domains and
5 fbronectin type III repeats, followed by a transmembrane region,
and a highly conserved cytoplasmic tail (5). L1 was discovered and
characterized as a cell-adhesion molecule in the nervous system
(6), where it is involved in neurite outgrowth and fasciculation
as well as cell adhesion and migration. In addition to homophilic
binding, L1 can establish cis- or trans-interactions with diferent
binding partners, such as integrins, CD24, neurocan, neuropilin-1,
and other members of the neural cell adhesion family (7).
Figure 1. Ablation of endothelial L1 in Tie2-Cre;L1
oxed
mice and related efects on tumor development and mouse survival. (A) Sections of normal pan-
creas and Panc02 tumors from L1
oxed
and Tie2-Cre;L1
oxed
mice were costained for L1 (green) and the vascular marker PECAM-1 (red), followed by confocal
analysis. Arrows indicate vessels coexpressing PECAM-1 and L1; arrowheads indicate L1-positive nerves that served as internal control. Scale bars: 10 m.
(B and C) The volume (B) and weight (C) of pancreatic tumors from L1
oxed
and Tie2-Cre;L1
oxed
mice were recorded 14 days after Panc02 injection. Data rep-
resent mean SEM from 10 mice per group. (D) Images of explanted tumors. (E) Survival rates in Panc02 tumorbearing L1
oxed
(n = 14) and Tie2-Cre;L1
oxed

mice (n = 13). **P < 0.01; ***P < 0.001.
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endothelial-specifc ablation of L1 in mice was achieved by crossing
Tie2-Cre with L1
foxed
mice (10). Indeed, L1 was readily detectable
in the tumor vessels of control, L1
foxed
mice, while no L1 immuno-
reactivity was observed in the vasculature of Tie2-Cre;L1
foxed
tumors
(Figure 1A). Interestingly, the vessels of normal pancreatic tissue in
either L1
foxed
or Tie2-Cre;L1
foxed
mice showed no L1 expression (Fig-
ure 1A). As expected, L1 expression in peripheral nerves was not
afected by Tie2-Cremediated recombination, thus acting as an
internal control (Figure 1A and Supplemental Figure 2).
Given that the Tie2 gene promoter is also active in hematopoi-
etic precursors and L1 is expressed in certain immune cell lineages
(10), we frst checked whether Tie2-Cremediated ablation of L1
afected immune cell infltration into the tumor. No diference in
the number of tumor-infltrating immune cells, assessed either as
total CD45
+
leukocytes or as individual hematopoietic cell sub-
populations identifed by lineage-specifc markers, was observed
between control and Tie2-Cre;L1
foxed
mice (Supplemental Figure
3, A and B). In agreement with our previous results (10), the abla-
tion of L1 in Tie2
+
hematopoietic progenitors did not cause major
defects in mouse hematopoiesis, as blood cell counts for the dif-
ferent leukocyte populations gave very similar values for L1
foxed

and Tie2-Cre;L1
foxed
littermates (Supplemental Figure 3C). Overall,
these data indicate that in the Panc02 tumor model, L1 defcien-
cy does not afect immune cell migration into cancer tissue and,
therefore, any efect on tumor behavior upon Tie2 promoterdriv-
en ablation of L1 in Tie2-Cre;L1
foxed
mice should be attributed to
endothelial L1 rather than to L1 expressed in immune cells.
We then analyzed Panc02 tumors 14 days after implantation.
Pancreatic tumor burden was markedly reduced in Tie2-Cre;L1
foxed

mice (Figure 1, B and C), implicating vascular L1 in cancer growth.
This efect was accompanied by an increase in TUNEL-positive
apoptotic cells in Tie2-Cre;L1
foxed
tumors (Supplemental Figure 4A),
while cancer cell proliferation, as assessed by staining for either
Ki-67 or phospho-histone H3, was not afected (Supplemental Fig-
ure 4, B and C). These data indicated that vascular L1 defciency
is associated with reduced tumor growth and increased apoptosis.
A macroscopical examination revealed that tumors of L1
foxed
mice
exhibited intensely red areas, suggesting high tumor vascularization
and/or hemorrhages. Notably, this feature was greatly reduced in
tumors from Tie2-Cre;L1
foxed
mice (Figure 1D), possibly implicating
endothelial L1 in tumor angiogenesis and/or vascular permeability.
Vascular L1 also appeared to be involved in the dissemination
of Panc02 tumors, since only 29% of Tie2-Cre;L1
foxed
mice (2/7)
showed metastatic lesions in diferent abdominal organs, while
metastases were detected in 71% of L1
foxed
mice (5/7) (Table 1).
Consistent with the reduced cancer growth and dissemination,
tumor-bearing Tie2-Cre;L1
foxed
mice also showed longer survival
times than their control littermates (Figure 1E). Overall, these
results indicated that the ablation of endothelial L1 results in
decreased tumor malignancy.
Endothelial L1 regulates tumor angiogenesis and vascular nor-
malization. To determine whether endothelial L1 plays any role
in cancer vascularization, we measured the microvessel density
in tumor tissue. Panc02 tumors in Tie2-Cre;L1
foxed
mice exhibited
a reduced number of vessels as compared with L1
foxed
mice, indi-
cating that endothelial L1 is involved in tumor angiogenesis (Fig-
ure 2, A and B). These fndings were further supported by in vivo
cular endothelium associated with pathological conditions, most
prominently cancer and infammatory diseases. Furthermore, vari-
ous cytokines and angiogenic growth factors abundantly released in
the tumor microenvironment, such as VEGF-A, TNF-, IFN-, and
TGF-1, upregulate L1 expression in ECs (10, 11). Taken together,
these observations point to L1 as a tumor vesselspecifc molecule.
However, it remains elusive whether L1 is causally involved in the
formation and/or function of cancer-associated vasculature.
In this study, we investigated the role of vascular L1 in a mouse
model of pancreatic tumor in which the endothelial-specifc abla-
tion of L1 was achieved by Cre-LoxP technology. This approach
was complemented by in vitro studies on ECs in which the expres-
sion of L1 was manipulated in order to induce overexpression or
silencing of the gene. Our data revealed that L1 plays a pivotal
role in tumor-associated vessels, thus infuencing cancer growth,
metastasis, and mouse survival. In particular, L1 was found to
modulate diferent EC functions and to promote endothelial-to-
mesenchymal transition (EndMT), a process causally linked to
neovascularization (12). We also demonstrated that antibody-
mediated targeting of L1 in tumor-bearing mice leads to decreased
neovascularization and to vessel normalization, resulting in
reduced tumor growth. Finally, we obtained mechanistic insights
into the function of L1 in ECs by showing that (a) L1 modulates
several transcriptional programs in ECs, including pathways that
govern vascular development and function; and (b) L1 promotes
the activation of the IL-6/JAK/STAT signaling pathway, and the
latter mediates L1-induced stimulation of ECs.
Results
Endothelial L1 regulates pancreatic tumor growth and metastasis in
mice. To defne the role of vascular L1 in tumor growth, we com-
bined the genetic inactivation of endothelial L1 with an orthotopic
mouse model of pancreatic carcinoma. This model is based on the
injection of mouse Panc02 cells into the head of the pancreas of
syngeneic C57BL/6 mice, resulting in the formation of highly vas-
cularized and metastatic pancreatic tumors (13). Panc02 cells do not
express L1, either in culture (Supplemental Figure 1, A and B; sup-
plemental material available online with this article; doi:10.1172/
JCI70683DS1) or upon tumor formation in mice (not shown). The
Table 1. Number of mice with metastases and number of site-
specific metastases in Panc02 tumorbearing L1
floxed
and Tie2-
Cre;L1
floxed
mice
L1
floxed
Tie2-Cre;L1
floxed
Mice with metastasis 5/7 (71%) 2/7 (29%)
Site-specific metastases (number of nodules)
Stomach 1 0
Intestine 10 0
Liver 1 1
Kidney 3 0
Urogenital tract 1 0
Diaphragm 3 3
Abdominal wall 11 8
n = 7 mice per group.
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disruption of endothelial polarity, with no or very low level of col-
lagen IV, the irregular distribution of podocalyxin within the ves-
sel wall, and a lack of VE-cadherinmarked junctions (Figure 2E).
In contrast, the vasculature of Tie2-Cre;L1
foxed
tumors displayed
abundant deposition of collagen IV in the basement membrane
(Figure 2, E and F), localization of podocalyxin at the luminal sur-
face, and a regular pattern of VE-cadherin localization, consistent
with its accumulation at cell-cell contacts (Figure 2E). These fnd-
ings implicated L1 in the maintenance of endothelial polarity.
Vascular abnormalities such as defective pericyte coverage,
altered polarity, and disorganized endothelial junctions, lead to
disruption of endothelial barrier function and, hence, to vascular
leakage, thus implying that L1 might regulate vascular permeabili-
ty. To test this hypothesis, we assessed the extravasation of intrave-
nously injected 40-kDa dextran in Panc02 tumors. Indeed, tumor
vascular permeability to dextran was remarkably higher in con-
trol, L1
foxed
mice as compared with Tie2-Cre;L1
foxed
mice (Figure 2,
G and H), thus implicating L1 in tumor vessel leakiness.
Taken together, these observations point to endothelial L1 as a
causal player in enhanced tumor angiogenesis as well as in the dis-
Matrigel plug assays, where Tie2-Cre;L1
foxed
mice showed a marked
decrease in the neovascularization induced by the angiogenic fac-
tor FGF-2 (Supplemental Figure 5).
We also tested the hypothesis that L1 regulates vascular
integrity in cancer tissue. To this goal, given that the interaction
of pericytes with the endothelium is causally linked to vessel sta-
bilization and integrity (14), we analyzed pericyte coverage in the
vasculature of Panc02 tumors. The extent of pericyte coverage,
as assessed by costaining for PECAM-1 and the pericyte marker
NG-2 (15), was signifcantly increased in Tie2-Cre;L1
foxed
mice
(Figure 2, C and D), thus implying that endothelial L1 negatively
regulates the recruitment of pericytes to the vascular wall.
Next, we asked whether endothelial polarity, another prereq-
uisite for vessel integrity, was afected by L1. Panc02 tumors were
stained for collagen IV, a major constituent of the vascular base-
ment membrane that is essential for capillary stability and organi-
zation (16), for the apical marker podocalyxin and for VE-cadherin
as a marker of endothelial junctions (17). In line with the notion
that the architecture of cancer-associated vasculature is irregular
and disorganized (4), the tumors of L1
foxed
mice showed a dramatic
Figure 2. Endothelial L1 deciency
results in reduced tumor angiogen-
esis and in vessel normalization. (A)
Representative images of Panc02 tumor
sections stained for PECAM-1 (red) to
visualize vessels. Scale bars: 50 m. (B)
Quantitation of vessel density in tumors
from L1
oxed
(n = 9) and Tie2-Cre;L1
oxed

mice (n = 5). (C) Representative images
of Panc02 tumor sections costained for
PECAM-1 (red) and the pericyte marker
NG-2 (green) to visualize pericyte cover-
age. Scale bars: 50 m. (D) Quantitation
of pericyte coverage in tumor vessels
from L1
oxed
(n = 8) and Tie2-Cre;L1
oxed

mice (n = 5). (E) Representative images
of Panc02 tumor sections costained for
the endothelial apical marker podocalyxin
(cyan), the junctional marker VE-cadherin
(yellow), and the basement membrane
marker collagen IV (red). Arrows indicate
the localization of VE-cadherin at cell-cell
contact in Tie2-Cre;L1
oxed
tumor vessels
(right), which is lost or dramatically
reduced in L1
oxed
vessels (left). Scale bars:
10 m. (F) Quantitation of collagen IV
deposition in tumor vessels from L1
oxed

(n = 3) and Tie2-Cre;L1
oxed
mice (n = 3). (G)
Representative images of Panc02 tumor
sections from mice injected with Texas
reddextran. Sections were costained
for Texas red (red) and PECAM-1 (green).
Scale bars: 50 m. (H) Quantitation of
vascular permeability, expressed as the
percentage of vessels showing extrava-
sated dextran in tumors from L1
oxed

(n = 3) and Tie2-Cre;L1
oxed
mice (n = 3).
*P < 0.05; ***P < 0.0005.
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notion that targeting L1 might be a suitable anti-angiogenic strat-
egy. Finally, tumor vessels from anti-L1treated mice showed a
higher rate of pericyte coverage than tumors treated with control
antibody (Figure 3E).
Thus, the treatment of tumor-bearing mice with the anti-L1
antibody, besides confrming the result of genetically ablating
endothelial L1, revealed that the pharmacological inactivation of
L1 might represent a novel strategy to interfere with tumor neo-
vascularization and to enhance vessel stabilization.
L1 orchestrates the angiogenic behavior of ECs. The reduced
tumor microvessel density upon ablation of L1 in the endothelium
prompted us to test whether L1 is involved in the critical EC pro-
cesses that underlie angiogenesis, i.e., proliferation, migration,
and tubulogenesis. Toward this goal, we employed immortalized
mouse lung ECs (luECs) because, due to the expression of moder-
ate levels of endogenous L1 (Supplemental Figure 7A), they were
amenable to both gain- and loss-of-function studies. The luECs
were stably transfected with the murine L1 cDNA (Supplemental
Figure 7, A and B). As shown in Figure 4A, forced expression of L1
resulted in increased luEC proliferation. This result was confrmed
in a classical in vivo assay for EC proliferation (18), where luECs
were injected subcutaneously into immunodefcient mice and
allowed to form hemangioma-like lesions. As shown in Figure 4B
and C, hemangiomas formed by L1-transfected luECs exhibited a
markedly higher growth rate than those formed by control luECs,
consistent with our in vitro results on L1-induced proliferation
of ECs. To further validate and extend these observations, we
employed a loss-of-function approach. The knockdown of L1 with
2 diferent siRNAs (Supplemental Figure 8A) was accompanied
by a decrease in luEC proliferation compared with control siRNA-
transfected cells (Supplemental Figure 8B). In addition, L1 silenc-
ing also reduced the proliferative response of luECs to TNF-
stimulation (Supplemental Figure 8C). Thus, L1 plays a pivotal
role in EC proliferation.
Next, a scratch-wound assay was used to evaluate the possible
efect of L1 on EC migration. L1-transfected luECs showed a high-
er migration rate than control cells (Figure 4D and Supplemental
Figure 7C). Conversely, L1 gene silencing resulted in decreased
luEC migration (Supplemental Figure 8, D and E). These data
demonstrate that L1 is causally involved in EC migration.
We also assessed the efect of L1 in in vitro angiogenesis
assays, determining the ability of ECs to form tube-like structures
in 3D reconstituted extracellular matrix. As shown in Figure 4E
and Supplemental Figure 7D, forced expression of L1 signifcantly
enhanced luEC tube formation as compared with control ECs.
Thus, L1 regulates EC proliferation, migration, and tubulo-
genesis, considered to be key cellular processes during the angio-
genic cascade.
ruption of vascular integrity and stability that is commonly associ-
ated with cancer development.
In vivo targeting of L1 reduces tumor growth and angiogenesis
and promotes vessel normalization. The results obtained with the
genetic ablation of L1 in Panc02 tumorbearing mice implied
that L1 might be a therapeutic target for preventing cancer-
associated neovascularization and/or enhancing vessel normal-
ization. To explore this possibility, we generated a polyclonal
antibody against the ectodomain of mouse L1. First, we tested
the function-blocking activity of the antibody in vitro. ECs were
transfected with L1 and then subjected to functional assays in
the presence of the antibody. As described in more detail in the
next section, L1 induced EC proliferation, migration, and tube
formation, and the anti-L1 antibody inhibited all these activities
(Supplemental Figure 6), thus confrming its neutralizing activ-
ity. The antibody was then used to target host L1 in the Panc02
mouse tumor model. As shown in Figure 3, A and B, tumor
growth was signifcantly reduced in mice treated with the anti-L1
antibody as compared with mice treated with control antibody.
Furthermore, the intensely red areas present in control tumors
were markedly reduced in anti-L1treated tumors (Figure 3C), in
agreement with our observations in Tie2-Cre;L1
foxed
mice (Figure
1D). The anti-L1 antibody caused a signifcant decrease of ves-
sel density in Panc02 tumors (Figure 3D), thus supporting the
Figure 3. Treatment with anti-L1 antibodies reduces tumor growth and
angiogenesis while increasing pericyte coverage in tumor vessels. The
volume (A) and weight (B) of pancreatic tumors from mice treated with
anti-L1 antibodies (n = 6) or control (ctrl) IgG (n = 6) were recorded 14
days after Panc02 injection. Data represent means SEM. (C) Images of
explanted tumors. (D) Quantitation of vessel density in tumors from mice
treated with anti-L1 antibodies (n = 5) or control IgG (n = 5). (E) Quantita-
tion of pericyte coverage in tumor vessels from mice treated with anti-L1
antibodies (n = 6) or control IgG (n = 5). *P < 0.05.
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Our data on Tie2-Cre;L1
foxed
mice indicated that L1 modulates
the permeability of tumor vessels. Since vascular permeability is
strictly related to the organization and function of cell-cell junc-
tions in the endothelium, we determined whether L1 afected
the expression and localization of junctional proteins in luECs.
Staining for 2 prototypical adhesion molecules of the endothe-
lium, PECAM-1 and VE-cadherin, showed that the architecture
of cell-cell boundaries was disorganized in L1-transfected luECs
(Figure 4F), despite the fact that the total level of either adhesion
molecule was unafected (Supplemental Figure 9A). This observa-
tion was consistent with the in vivo data showing that the altered
localization of VE-cadherin in the vasculature of Panc02 tumors
was reverted upon endothelial ablation of L1 in Tie2-Cre;L1
foxed

mice (Figure 2E). We found no diferences in the levels of other
components of the interendothelial adhesion complexes, such as
4 catenin family members (, , , and p120-catenin) and junc-
tional adhesion molecule-A (Supplemental Figure 9B). We then
tested whether L1 afected the expression of claudin-5, an essen-
tial component of tight junctions widely implicated in the barrier
function of endothelium (19). The level of claudin-5 mRNA was
signifcantly downregulated in L1-overexpressing luECs as com-
pared with mock-transfected cells (Figure 4G), indicating that L1
negatively regulates its expression. The immunoblotting analysis
also confrmed the decrease of claudin-5 in L1-transfected luECs
(Supplemental Figure 10A). In line with these fndings, the immu-
nofuorescence (IF) staining of Panc02 tumors revealed that no or
very little claudin-5 was present in L1
foxed
tumor vessels, while the
protein was readily detectable in the tumor vasculature of Tie2-
Cre;L1
foxed
mice (Supplemental Figure 10B), implicating L1 in the
regulation of claudin-5 expression in ECs.
Interendothelial junctions play a key role in the integrity of
vascular barrier (20). Therefore, given the junctional alterations
induced by L1, we tested whether endothelial permeability was
modifed by L1 overexpression. Indeed, the permeability of con-
fuent luEC monolayers to FITC-labeled dextran was dramatically
increased upon forced expression of L1 (Figure 4H), in agreement
with the reduced permeability observed in the vasculature of Tie2-
Cre;L1
foxed
mouse tumors (Figure 2, G and H).
Taken together with our fndings on L1-dependent alterations
in tumor vessels (Figure 2), these data indicate that endothelial L1
Figure 4. L1 confers an angiogenic phenotype to ECs and enhances endothelial permeability. (A) Proliferation curves of mock- and L1-transfected luECs.
(B) Growth curves of hemangiomas formed by mock- or L1-transfected luECs injected subcutaneously into nude mice, as determined by volume mea-
surement at the indicated time points. Representative images of hemangiomas explanted are shown (insets). (C) Weight of hemangiomas explanted 33
days after injection of mock- or L1-transfected luECs. (D) Migration assays of mock- and L1-transfected luECs were performed as described in Methods.
(E) Matrigel-based tube formation assays of mock- and L1-transfected luECs were performed as described in Methods. (F) Mock- or L1-transfected luECs
were stained for PECAM-1 (red) or VE-cadherin (green) prior to confocal analysis. Scale bars: 10 m. (G) qRT-PCR analysis of claudin-5 mRNA in mock- and
L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-
transfected cells (n = 3). (H) FITC-dextran permeability assays were performed on monolayers of mock- and L1-transfected luECs as described in Methods.
Data in A, D, E, and H represent the mean SD from a representative experiment performed at least in triplicate. Data in B and C represent mean SEM
from 10 to 12 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.
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destabilizes the vasculature not only by altering pericyte coverage
and collagen IV deposition, but also via EC-autonomous efects
on the localization and/or expression of certain junctional compo-
nents, therefore orchestrating intercellular adhesion.
L1 promotes EndMT. The morphological and functional
changes that L1 imparted on ECs, such as increased perme-
ability, loosening of cell-cell junctions and a migratory pheno-
type, represent typical aspects of the EndMT, a process that has
been causally linked to cancer progression (12). In the course
of EndMT, ECs gain the expression of mesenchymal markers.
Therefore, to verify whether L1 induced bona fde EndMT, we
tested whether it promoted the acquisition of mesenchymal
markers. The overexpression of L1 in luECs, indeed, resulted in
the upregulation of S100A4, N-cadherin, fbronectin, and Id1
(Figure 5A), all events associated with EndMT (21). Notably, we
also observed markedly reduced levels of collagen IV in L1-over-
expressing luECs (Figure 5A), which, besides being another
hallmark of EndMT (21), was consistent with the increased col-
lagen IV deposition in L1-defcient Panc02 tumor vessels (Fig-
ure 2, E and F). Furthermore, L1-overexpressing luECs exhib-
ited enhanced expression of the stemness-associated factors
KLF4 and CD44 (Figure 5A), in line with the notion that EndMT
is accompanied by the acquisition of a stem-like phenotype
(21). Most of the markers above were validated by quantita-
tive real-time PCR (qRT-PCR) (Figure 5B), implying a regula-
tion at the transcriptional level. In contrast, the mesenchymal
marker vimentin was not afected by L1 overexpression (Figure
5B). Besides mesenchymal markers, L1 was able to enhance
the expression of transcription factors (TFs) that are thought to
drive EndMT, such as Zeb1 and Zeb2, as well as Tbx20 (Figure
5B), which has been implicated in EndMT-like processes during
heart development (22).
These data point to L1 as a regulator of EndMT, possibly impli-
cating such an activity in L1-dependent alterations of EC behavior.
L1 regulates EC transcriptome. In an attempt to elucidate the
molecular mechanisms underlying the multiple roles of L1 in
endothelium, we checked whether manipulating its expression
afected the EC transcriptome. To this purpose, we compared the
gene expression profles of L1-overexpressing luECs with those of
control luECs by Afymetrix microarray technology, and we used
the Signifcance Analysis of Microarrays (SAM) (23) to identify
genes whose expression was altered by L1. This analysis revealed a
remarkable efect of L1 overexpression on luEC transcription (Fig-
ure 6A), with 361 genes that were upregulated and 580 that were
downregulated (q value < 5%; 1.5-fold change diference; Supple-
mental Table 1). From the list of L1-regulated genes, we selected
16 candidates for qRT-PCR validation, based on their biologi-
cal relevance. For all of them, including Cdk6, Adamts9, Hoxb9,
Stmn2, Ebf1, Dll4, Vegfa, Vegfc, Ccnb1, Il13ra2 (Figure 6B), CD44,
N-cadherin, S100A4, Tbx20, Klf4 (Figure 5B), and Cldn5 (Figure
4G), the regulation in L1-overexpressing luECs was confrmed
by qRT-PCR. Many of these genes, including Stmn2, Ebf1, Dll4,
Il13ra2, and Vegfc, exhibited a concordant L1 dependence upon
siRNA-mediated silencing of L1 (Figure 6C), indicating that L1 is
required and sufcient for their modulation.
Next, we used ingenuity pathway analysis (IPA) to analyze
the efect of L1 overexpression on genes involved in biologically
relevant functions. The bio-functions analysis revealed that
L1 afects the expression profles of several genes involved in cel-
cycle regulation, DNA replication, cellular assembly, and orga-
nization (top 5 biofunctions, P value < 10
7
, Benjamini-Hochberg
correction; Figure 6D), which is consistent with the L1-induced
proliferation of ECs (Figure 4A). Importantly, IPA also identifed
genes involved in cell migration and development (Figure 6D).
In particular, the gene expression profle of L1-transfected luECs
was consistent with the activation of pathways involved in EC
movement (Supplemental Figure 11), thus supporting our obser-
vations on L1-dependent functional changes (Figure 4, D and E).
L1 regulates EC function via the IL-6/JAK/STAT3 pathway. To
get further insights about possible efectors of L1 biological func-
tion, we performed an unsupervised ingenuity upstream regulator
analysis, which predicts the activation of specifc gene expression
modulators (i.e., TFs, microRNA, etc.). This analysis predicted the
L1-induced activation of the TFs STAT1, STAT2, STAT3, IRF7, and
ATF4. In particular, we identifed a network of 105 L1-regulated
genes downstream of the above-mentioned 5 TFs (Figure 7A).
Figure 5. L1 promotes EndMT. (A) Immunoblotting analysis of mock- and
L1-transfected luECs for S100A4/FSP1, Id1, KLF4, CD44, N-cadherin,
bronectin, and collagen IV. Actin, tubulin, and vinculin served as load-
ing controls. (B) qRT-PCR analysis of the indicated genes in mock- and
L1-transfected cells. Transcript levels were normalized as described in
Methods and are shown as fold changes in L1-transfected cells relative to
mock-transfected cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.
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Next, we checked whether STAT signaling is involved in
L1-dependent regulation of EC function. Since STAT activation
occurs through JAK-mediated phosphorylation, we treated luECs
with the JAK inhibitor I (JAKi) which, indeed, repressed L1-induced
phosphorylation of STAT3 (Figure 7F). Importantly, JAK blockade
repressed L1-dependent EC proliferation and migration (Figure 7,
G and H). Together, our transcriptomic, biochemical, and cell bio-
logical data support the notion that L1 regulates EC function via
the JAK/STAT pathway.
Endothelial L1 is upregulated in human pancreatic carcinoma
and in other tumor types. Based on our fndings on the Panc02
orthotopic mouse model, we asked whether the expression of
L1 in tumor vessels also occurs in human pancreatic carcinoma.
Immunohistochemistry for L1 was performed on 18 tissue samples
of PDAC and 11 samples of noncancerous pancreatic tissue, using
To investigate in more detail the molecular mechanisms under-
lying the efect of L1 on endothelium, we focused on STAT3, which
has been implicated in tumor angiogenesis and EC activation (24).
STAT3 activation is classically induced by IL-6 (25); therefore, we
checked whether L1 afected the expression of this cytokine in
luECs. Indeed, not only IL-6, but also its receptor IL-6R, was sig-
nifcantly upregulated in L1-overxpressing ECs, while the expres-
sion of the IL-6 coreceptor gp130 was not afected (Figure 7B).
These observations were further confrmed by the increased levels
of IL-6 in the conditioned medium (Figure 7C) and of IL-6R in the
lysate of L1-overexpressing luECs (Figure 7D). The forced expres-
sion of L1 also resulted in the strong induction of STAT3 phosphor-
ylation (Figure 7D), which was abolished by antibody-mediated
neutralization of IL-6R (Figure 7E). These results indicated that
L1 promoted STAT3 activation via the IL-6/IL-6R axis.
Figure 6. L1 regulates EC transcriptome. (A) Hierarchical clustering of genes diferentially expressed in L1-transfected versus control luECs. Three indepen-
dent experimental replicates of L1-overexpressing and control cells were screened by gene expression microarray. Data were log
2
transformed before cluster-
ing analysis. Red, upregulated genes; blue, downregulated genes. A total of 361 upregulated and 580 downregulated genes (i.e., 496 and 743 probe sets,
respectively) were identied in L1-overexpressing cells. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript levels were
normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) qRT-PCR analysis of
the indicated genes in luECs transfected either with 2 diferent L1 siRNAs (A and B) or with a control siRNA. Transcript levels are shown as fold changes in L1
siRNAtransfected cells relative to control cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (D) IPA analysis of L1-regulated genes. The enriched biofunctions
were selected based on their signicance (P < 0.05; Benjamini-Hochberg correction). Blue bars indicate the log(P value) of enrichment.
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Figure 7. L1 regulates EC function via the IL-6/JAK/STAT3 pathway. (A) Gene network of L1-regulated genes. In bold, IPA-predicted upstream modula-
tors. Lines connect modulators to direct targets, and colors indicate the consistency with the predicted activity with the expression change observed in
L1-overexpressing luECs (i.e., target expression). Orange, consistent predicted activation of TFs; blue, consistent predicted inhibition of TFs; yellow, incon-
sistent predicted activation of TFs; grey, not dened activity. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript
levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) The
amount of IL-6 released in the culture medium by mock- and L1-transfected luECs was quantied by ELISA. (D) Immunoblotting analysis of mock- and
L1-transfected luECs for IL-6R, phosphorylated STAT3, and total STAT3. (E) Immunoblotting analysis for phosphorylated and total STAT3 in mock- and
L1-transfected luECs, treated either with antiIL-6R antibody or with control IgG. (F) Immunoblotting analysis for phosphorylated and total STAT3 in
mock- and L1-transfected luECs, treated either with vehicle (DMSO) or with 20 M JAKi. Actin in DF served as loading control. (G) Proliferation curves of
mock- and L1-transfected luECs treated either with vehicle (DMSO) or with the indicated concentration of JAKi. (H) Mock- and L1-transfected luECs treated
either with vehicle (DMSO) or with 20 M JAKi were subjected to 24-hour migration assays. Data in G and H represent the mean SD from a representative
experiment performed in triplicate. **P < 0.01; ***P < 0.001.
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tumor vascularization and enhanced vessel normal-
ization, thus delaying tumor growth. These fndings
were supported by in vitro data that revealed that L1
induces EC proliferation, migration, and tubulogen-
esis, and confers a mesenchymal phenotype to ECs.
Previous studies have shown that a soluble form
of the L1s ectodomain enhances EC proliferation
and migration (3133), implying a model whereby
exogenous (e.g., tumor cell derived) L1 stimulates
ECs. While this remains a possibility, particularly
in the case of L1-expressing tumors, our fndings on
the upregulation of L1 in cancer-associated vessels
and on the proliferative and migratory response of
L1-expressing ECs, point to a cell-autonomous efect
of L1 in pathological vasculature.
It is noteworthy that L1 promotes the expression
of various genes causally linked to tumor vascular-
ization, such as VEGF-A, VEGF-C, Dll4, and HOXB9
(3436). On the other hand, the endothelial expres-
sion of L1 itself is induced by classical angiogenic
factors, including VEGF-A, ANGPTL4, TNF-, and
IFN- (10, 11). Taken together, these observations
implicate L1 as a central hub in the transmission and amplifcation
of angiogenic stimuli within the tumor microenvironment.
Our data also indicate that L1 promotes vascular permeability.
Since VEGF-A, ANGPTL4, TNF- and IFN- (i.e., the same cytokines
that induce endothelial L1 expression) are among the most potent
inducers of vascular permeability (37, 38), it is conceivable that L1
acts as a general efector of cytokine-induced vascular permeability.
Notably, we discovered that L1 overexpression induces End-
MT, a process that may underlie, or at least contribute to, many
aspects of the EC response to L1, such as migration and tubulo-
genesis as well as increased tumor angiogenesis and vascular per-
meability (12). Various lines of evidence support the notion that
EndMT recapitulates most of the cellular and molecular events
occurring during epithelial-to-mesenchymal transition (EMT)
(12). The role of L1 in EMT has been clearly established in various
experimental models (3941), including tumor cells in which the
expression of L1 per se promoted EMT (42, 43). We now provide
evidence that L1 expression is sufcient to confer a mesenchymal
phenotype to ECs, which entails not only an increased migratory
activity but also the upregulation of several mesenchymal mark-
ers, such as N-cadherin, CD44, S100A4/FSP1, and fbronectin,
and even of TFs that are considered to be EMT/EndMT drivers,
namely KLF4, Zeb1, Zeb2, and Tbx20 (22, 44, 45). These fndings
suggest that L1 acts as a key mediator of the multiple EndMT-
inducing factors that can occur in tumor microenvironment and
as a master orchestrator of cancer-associated EndMT (12).
One of the most intriguing and unexpected results of our study
is the global efect of L1 on EC transcriptome, with the modulation
of approximately 1,000 genes. While these include individual fac-
tors that per se might account for several aspects of the endothe-
lial response evoked by L1, such as VEGF-A, VEGF-C, and Dll4 for
tumor angiogenesis (see above), or occludin and claudin-5 for vas-
cular permeability (19), our data rather implicate L1 in the control
of whole gene networks, resulting in the modulation of signaling
pathways that mediate the EC response.
an antibody against PECAM-1 to identify the vessels on consecu-
tive sections. L1 expression was markedly enhanced in PDAC
vasculature as compared with noncancerous tissue (Figure 8, A
and B), confrming and extending previous observations (11). The
presence of L1 in vascular endothelium was further validated by
confocal microscopy on PDAC tissue costained for L1 and for the
endothelial marker VE-cadherin (Supplemental Figure 12). Taken
together with our data on the Panc02 tumor model and on cultured
ECs, the expression pattern of L1 in clinical samples supports the
hypothesis that vascular L1 contributes to pancreatic malignancy.
To test whether the vascular expression of L1 also occurs in
other cancer types, we performed the immunohistochemical
staining for L1 on tissue microarrays (TMAs) containing various
tumors and their noncancerous tissue counterparts. As shown in
Figure 9 and Supplemental Figure 13, in several tumor types, the
percentage of L1-positive vessels was markedly higher as com-
pared with their corresponding nonneoplastic tissues. Thus, the
induction of L1 expression in the vasculature is common to a broad
spectrum of human tumors.
Discussion
The expression of L1 in tumor vasculature has been reported in
several cancer types, including breast, ovarian, colon, and pancre-
atic carcinoma (10, 11), neural tumors (26), smooth muscle tumors
(27), and melanoma (28). While vascular L1 has been implicated
in the adhesion and transendothelial migration of L1-expressing
cancer cells via homophilic binding (11, 29, 30), it remains elusive
whether L1 induces cell-autonomous efects in tumor endotheli-
um and whether this has an efect on cancer development.
Here, we report for what we believe is the frst time that L1
orchestrates the EC behavior in tumor vasculature. In particular,
the endothelial defciency of L1 in a mouse model of cancer led to
enhanced vessel stability and decreased tumor angiogenesis, result-
ing in reduced tumor growth and metastasis and prolonged mouse
survival. Consistently, antibody-mediated targeting of L1 reduced
Figure 8. L1 is upregulated in human PDAC-associated vessels. (A) Consecutive sections
from human noncancerous or PDAC tissue were stained for PECAM-1 (left panels) and
L1 (right panels) in order to visualize L1-positive vessels (arrows). Scale bars: 100 m. (B)
Quantitation of L1-positive vessels in noncancerous (n = 11) and tumor tissue (n = 18),
expressed as percentage of L1-positive over PECAM-1positive vessels. **P < 0.01.
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low levels in normal vasculature, L1 might show higher specifc-
ity than other molecular biomarkers that are currently in use or
under clinical testing, such as integrins and VEGFR2 (52). The
feasibility of this approach is supported both by the preclinical
imaging of cancer vessels by targeting other immunoglobulin-
like adhesion molecules such as NCAM (53) and by the suitability
of L1 antibodies for tumor-imaging purposes as shown in mouse
models (54). Second, based on the positive role of L1 in tumor
neovascularization, in EC proliferation and migration, and in
EndMT, it is reasonable to speculate that neutralizing L1 could
represent a novel antiangiogenic strategy. Indeed, we showed
that treating tumor-bearing mice with an L1-neutralizing poly-
clonal antibody delays tumor growth and reduces tumor vascu-
larization, strengthening the rationale for developing L1-target-
ing agents as therapeutic tools. In this context, promising results
have been obtained with anti-L1 monoclonal antibodies in pre-
clinical models of solid tumors and of endometriosis (55, 56).
Third, the observation that L1 destabilizes cancer-associated
vessels implies that targeting endothelial L1 and interfering with
its function might result in vessel normalization, a process that
has been proposed to improve the delivery into the tumor of sys-
temically administered chemotherapeutics (4). Thus, our results
set the stage for exploring the clinical relevance of L1 expression
and function in cancer vessels, possibly opening new avenues for
targeted treatments of malignancies.
Methods
Mice
Tie2-Cre;L1
foxed
mice were generated in the C57BL/6 genetic back-
ground as previously described (10). Since the L1 gene is located
on chromosome X (and hence, only 1 copy is present in the male
genome), Cre-mediated ablation of L1 was expected to be more ef-
cient in males. Therefore, only Tie2-Crepositive males carrying the
foxed L1 allele were used throughout the study, with L1
foxed
males
serving as controls.
In this context, it is noteworthy that L1 induces the expres-
sion of IL-6 and IL-6R. IL-6 is a potent angiogenic cytokine that
promotes neovascularization in various solid tumors, and it has
been proposed not only as a therapeutic target for antiangiogenic
therapies but also as a biomarker to predict the response to such
treatments (46). Thus, it is reasonable to speculate that the IL-6/
IL6R axis acts as a key mediator of the angiogenic response elic-
ited by vascular L1, an intriguing hypothesis that warrants further
investigation. In addition, L1-mediated regulation of IL-6 expres-
sion might also occur in diferent biological contexts where IL-6
signalling has been implicated. For example, the IL-6/IL-6R
system plays a major role in infammation and in several cancer
cell functions (47, 48), and our fndings imply that the established
function of L1 in both pathological conditions (8, 10) might be
accounted for, at least to some extent, by the induction of IL-6
expression and activity.
Among the signalling cascades that are elicited by IL-6, the
JAK/STAT pathway appears particularly important in the context
of L1-dependent regulation of EC function. Indeed, L1 induces
high phosphorylation of STAT3, a TF that plays a key role in EC
activation and pathological angiogenesis (49, 50), consistent with
the hypothesis that L1-dependent regulation of tumor vasculature
is mediated by the IL-6/JAK/STAT pathway. Our data on the inhi-
bition of L1-induced EC proliferation and migration upon block-
ade of JAK/STAT signaling not only support this hypothesis, but
provide mechanistic insights into the role of L1 in pathological
vessels and shed light on a signaling axis that links L1 to the JAK/
STAT pathway. Given the broad spectrum of functions that have
been ascribed to L1 in diferent cellular contexts, including cell-
cell adhesion, axon guidance, tumor cell invasion, and stem cell
self renewal (7, 8, 51), we propose that at least some of these activi-
ties are mediated by JAK/STAT signaling.
The expression and function of L1 in cancer vessels has rel-
evant translational implications. First, it provides the rationale
to test L1 as an imaging biomarker suitable for visualizing path-
ological angiogenesis. In this application, due to its absence or
Figure 9. Vascular L1 is upregulated in various human tumor types. TMAs containing diferent tumor types and their normal tissue counterparts were
stained for PECAM-1 (A and C) and L1 (B and D). Examples of thyroid carcinoma (A and B) and gastric carcinoma (C and D) are shown. Scale bars: 200 m.
(E) Quantitation of L1-positive vessels in noncancerous and tumor tissue expressed as percentage of L1-positive over PECAM-1positive vessels.
**P < 0.002 (n = 5 for each tissue type).
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antiKi-67 (1:100 in IF; Abcam); anti-KLF4 (1:1000 in WB; R&D Sys-
tems); antimouse L1 (obtained by rabbit immunization with mouse
L1-Fc; 1 g/ml in IF and WB); antiPECAM-1 (M-20; 1:500 in WB;
Santa Cruz Biotechnology Inc.); antiNG-2 (1:200 in IF; Millipore);
antiphospho-histone H3 (1:250 in IF; Millipore); anti-podocalixin
(1:400 in IF; R&D Systems); antiVE-cadherin (C-19; 1:500 in WB
and 1:200 in IF on human tissues; Santa Cruz Biotechnology Inc.);
anti-tubulin III (1:500 in IF; Covance). Antibodies used for FACS
analysis are listed in the corresponding section.
Cells
Mouse luECs were immortalized with polyoma middle T antigen as
previously described (18) and cultured in MCDB131 medium (Gibco;
Life Technologies) supplemented with 20% FBS (Invitrogen), 2 mM
l-glutamine (Lonza), 1 mM Na-pyruvate (Gibco; Life Technologies),
100 g/ml heparin (Sigma-Aldrich), and 50 g/ml EC growth supple-
ment (ECGS) obtained from calf brain. ECs were seeded on 0.1% gel-
atin (Sigma-Aldrich), except as otherwise indicated. To enhance EC
adhesion, plates were coated with glutaraldehydecrosslinked gelatin
as follows. Plates were incubated overnight with 1% gelatin at 37C
followed by a crosslinking with 2% glutaraldehyde for 15 minutes at
room temperature (RT). Glutaraldehyde was replaced with 70% etha-
nol for 1 hour at RT. After 5 washes with PBS, plates were incubated for
2 hours at 37C with 2 mM glycine in PBS. Prior to cell seeding, plates
were washed 5 times with PBS.
Murine Panc02 (pancreatic carcinoma) and MOVCAR7 (ovarian
carcinoma) cell lines were provided by S. Sebens (Institute for Experi-
mental Medicine, Kiel, Germany) and D. Connolly (Fox Chase Cancer
Center, Philadelphia, Pennsylvania, USA), respectively, and cultured
in RPMI 1640 medium (Lonza) supplemented with 10% FBS, 2 mM
l-Glutamine, and 1 mM Na-pyruvate. All cells were cultured in a
humidifed incubator with 5% CO
2
.
Cell transfection and RNA interference
Mouse cDNA encoding full-length L1 was cloned into the pcDNA3.1/
Hygro(-) expression vector (Invitrogen) using standard DNA cloning
procedures. The parental pcDNA3.1/Hygro vector and pcDNA3.1/
Hygro-L1 were amplifed in competent bacteria cells (TOP10) and
purifed using Maxi-Prep kits (QIAGEN) according to the manufac-
turers instructions. Transfection with either pcDNA3.1/Hygro or pcD-
NA3.1/Hygro-L1 was performed with LipofectAMINE 2000 (Invitro-
gen) according to the manufacturers instructions. Transfected cells
were selected with 300 g/ml of Hygromycin-B (Roche) and main-
tained under selection conditions as a bulk cell population.
Stealth RNAi Duplexes and the corresponding Medium GC Stealth
RNAi Control Duplexes (Invitrogen) were used to knock down L1 in
murine ECs. The following L1 target sequences were used: 5-CCUG-
GUACCCGGACCAUCAUUCAAA-3 (siRNA A) and 5-UGCAC-
CUUUCUUCUCAAUUGCGCUC-3 (siRNA B). ECs were subjected
to 2 rounds of transfection with 40 nM siRNA the 2 days prior to the
experiment. Transfection was performed with LipofectAMINE 2000
(Invitrogen) according to the manufacturers instructions.
Cell proliferation assay
ECs were seeded at a density of 2 10
3
cells/well in 96-well plates
coated with glutaraldehydecrosslinked gelatin. After overnight
incubation in medium containing 5% FBS, cells were stimulated with
In vivo models
Pancreatic carcinoma model. The syngeneic mouse model of pancreatic
cancer has been described previously (13). Briefy, 10- to 12-week-old
C57BL/6 mouse males were anesthetized by intraperitoneal injection
of 500 mg/kg avertin (Sigma-Aldrich), the stomach was exterior-
ized via abdominal midline incision, and Panc02 tumor cells (1 10
6

cells in 30 l PBS) were injected into the head of the pancreas using
a 29-gauge needle. The intrapancreatic injection was considered suc-
cessful with the appearance of a fuid bleb without intraperitoneal leak-
age. Peritoneum and abdominal wall were closed with individual sur-
gical sutures. Where indicated, mice were treated every 48 hours with
7 mg/kg of afnity-purifed anti-L1 polyclonal antibodies (obtained
from rabbits immunized with mouse L1-Fc) or control, nonimmune
rabbit IgG (Sigma-Aldrich), starting from the day after Panc02 injec-
tion. At day 14, primary tumors were removed, and tumor weight and
volume were recorded. Tumor volume was calculated using the for-
mula V = (d1 d2 d3)/6, where d1, d2, and d3 are the 3 tumor axes.
To assess tumor invasion to adjacent organs and metastasis, mice
were sacrifced at day 26 and subjected to whole-mount body fxation
with 4% paraformaldehyde. Necropsy was performed by the Mouse
and Animal Pathology Laboratory, Fondazione Filarete (Milan, Italy).
Hemangioma model. For EC transplantation, 1 10
5
mock- or L1-
transfected mouse luECs in 200 l of PBS was injected subcutaneous-
ly into the right fank of CD-1 nude (nu/nu) mice (9 weeks old, female;
Charles River Laboratories) as previously described (18). Hemangio-
ma volumes were recorded at the indicated time points and calculated
using the formula V = (d
2
D)/6, where d and D are the minor and
the major hemangioma axes, respectively.
Chemicals and antibodies
The JAK inhibitor 1 was provided by Calbiochem. The following
monoclonal antibodies were used as indicated: anti-mouse L1 (clone
S10.33 [ref. 10, 5 g/ml in IF]; clone 555 [1 g/ml in IF]; clone 324
[ref. 6] hybridoma supernatant [1:2 in IF]; and clone I4.2 [ref. 10]
hybridoma supernatant [1:2 in Western blotting (WB)]); anti-human
L1 (clone UJ127; 1:30 in IHC on human tissues; Thermo Scientif-
ic); antiPECAM-1 (clone 2H8; 1:500 in IF on cells and mouse tis-
sues, Millipore; clone JC70A; 1:30 in IHC on human tissues, Dako);
anticlaudin-5 (clone 4C3C2; 1:200 in IF, 1:250 in WB, Life Tech-
nologies); anti-actin (clone AC-40; 1:1000 in WB; Sigma-Aldrich);
anti-catenin (clone 5/-catenin; 1:1000 in WB; BD Biosciences);
anti-catenin (clone 14/-catenin; 1:200 in IF and 1:500 in WB;
BD Biosciences); anti-catenin (clone 15/-catenin; 1:1000 in WB;
BD Biosciences); anti-CD44 (clone IM7; 1:100 in WB; BD Biosci-
ences); antiJAM-A (clone BV19/BV20) (57); antiN-cadherin (clone
32/N-cadherin; 1:2000 in WB; BD Biosciences), antip120-catenin
(clone 98/pp120; 1:1000 in WB; BD Biosciences); antiphospho-
Stat3 (Cell Signaling, clone D3A7; 1:1000 in WB); anti-Stat3 (clone
124H6; 1:1000 in WB; Cell Signaling); antiVE-cadherin (clone
11D1.4; 1:200 in IF on cells and mouse tissues; BD Biosciences); anti-
vinculin (clone hVIN-1; 1:2000 in WB; Sigma-Aldrich); anti-tubulin
(clone DM1A; 1:2000 in WB; Sigma-Aldrich). The following poly-
clonal antibodies were used as indicated: anti-collagen IV (1:900 in
IF and 1:400 in WB; Serotec); anti-fbrin/fbrinogen (1:1000 in IF;
Dako); anti-fbronectin (1:4000 in WB; Abcam); anti-S100A4/FSP1
(1:1000 in WB; Millipore); anti-Id1 (C-20; 1:400 in WB; Santa Cruz
Biotechnology Inc.); antiIL-6R (1:1000 in WB; R&D Systems);
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formaldehyde and embedded in parafn. Sections were then subjected
to IF costaining with rat anti-CD34 (BD; clone RAM34; 1:100) and rabbit
antiTexas red antibodies (1:100; Life Technologies). Vascular permea-
bility was assessed as the ratio between the number of leaky vessels (i.e.,
vessels showing perivascular, extravasated Texas red staining) and the
total number of CD34-positive vessels in 5 randomly selected felds (13).
Gene expression proling
ECs were seeded on plates coated with glutaraldehydecrosslinked
gelatin and cultured in complete medium for 4 days to reach confu-
ence. Total RNA was extracted with RNeasy Mini Kit (QIAGEN).
Quality control of the RNA samples was performed using Agilent Bio-
analyzer 2100 (Agilent Technologies). Three diferent RNA extrac-
tions were processed for each of the cell lines under analysis. Each
sample was labeled and hybridized to a Mouse Gene 1.0 ST Genechip
array according to the manufacturers instructions (Afymetrix). Data
were normalized using the Robust Multi-array Average (RMA) (ref. 58;
raw and normalized data were deposited in the NCBIs Gene Expres-
sion Omnibus [GEO GSE45859]).
All analyses were performed on log
2
data using parametric tests.
BRB ArrayTools (http://linus.nci.nih.gov/BRB-ArrayTools.html) was
used to run SAM analysis (http://www-stat.stanford.edu/~tibs/SAM/).
A false discovery rate of less then 5% (i.e., the q value) was used to
select statistically signifcant diferentially expressed genes. Cluster
3.0 for Mac OS X (http://bonsai.hgc.jp/~mdehoon/software/cluster/)
and Java Treview (http://jtreeview.sourceforge.net) were used for
hierarchical clustering analysis using the uncentered correlation met-
ric and centroid clustering method.
Ingenuity Pathway Analysis and Upstream Regulator Analysis
were performed using the online available web tool (http://www.inge-
nuity.com/). For gene network analysis, only direct relationships in
mammals (i.e., human, mouse, and rat) were considered. P values for
biofunction enrichment were corrected for multiple testing using the
Benjamini-Hochberg correction.
Immunoblotting
ECs were cultured on petri dishes coated with glutaraldehydecross-
linked gelatin for 4 days. Where indicated, cells were incubated for 2 or
24 hours with 2 g/ml goat polyclonal antiIL-6 R (R&D Systems) or
control goat IgG. Total proteins were extracted by solubilizing cells in
boiling Laemmli bufer (4% SDS, 16% glycerol, 40 mM Tris-HCl [pH
6.8]). Lysates were incubated for 15 minutes at 90C to allow protein
denaturation and then centrifuged for 5 minutes at 14,000 g to discard
cell debris. The supernatants were collected, and the concentration of
protein was determined using a BCA Protein Assay Kit (Pierce) accord-
ing to the manufacturers instructions. Equal amounts of protein were
separated on SDS polyacrylamide gel (SDS-PAGE), transferred to a
Protran Nitrocellulose Transfer Membrane (Whatman), and blocked
for 1 hour at RT in TBS, 0.1% Tween 20 containing 5% nonfat milk
or BSA (blocking solution). The membranes were incubated overnight
at 4C or for 1 hour at RT with primary antibodies diluted in block-
ing solution. Membranes were then incubated with horseradish per-
oxidaseconjugated secondary antibodies (Bio-Rad). The signal was
detected by the ECL system (GE Healthcare) using Hyperflm (Amer-
sham Biosciences). The molecular masses of proteins were estimated
relative to the electrophoretic mobility of contransferred prestained
protein marker Precision Plus Protein Standards (Bio-Rad).
medium containing 20% FBS, 100 g/ml heparin, and 50 g/ml
ECGS. Where indicated, cells were treated with JAK inhibitor 1 or vehi-
cle (DMSO) or with 3 g/ml of polyclonal anti-L1 antibodies or control,
nonimmune rabbit IgG throughout the assay. Cells were fxed at 0, 24,
48, and 72 hours, followed by staining with 0.1% crystal violet in 20%
methanol. Bound dye was solubilized with 10% acetic acid, and the
absorbance at 590 nm was measured. Cell growth was normalized on
absorbance measured at 0 hours. The experiments were performed in
quintuplicate and repeated at least 3 times.
Cell migration assay
To assess cell migration, we employed the wound-healing assay.
Briefy, confuent monolayers of luECs seeded on fbronectin-coated
(1 g/cm
2
) 24-well plates were starved for 24 hours in medium con-
taining 0.5% FBS medium. Monolayers were wounded with a plastic
pipette tip to induce EC migration into the wound, and images were
acquired at 0 and 24 hours. Where indicated, cells were pretreated for
1 hour with 20 M JAK inhibitor 1 or vehicle (DMSO), or with 3 g/ml
anti-L1 polyclonal antibodies or control, nonimmune rabbit IgG; such
treatments were maintained throughout the assay. Optical images of
the wounds were acquired at 0 and 24 hours. The width of the wounds
was measured with ImageJ (http://imagej.nih.gov/ij/) software, and
the distance covered luECs, expressed as m in 24 hours, was calcu-
lated according to this formula: (wound width at 0 wound width at
24 hours)/2. The experiments were repeated 3 times, each time with
triplicate wells.
Tube formation assay
A Matrigel-based tubulogenesis assay was performed to assess the
ability of ECs to form an organized capillary-like network. Confu-
ent ECs were starved overnight with medium containing 1% FBS.
Growth factorreduced Matrigel (BD Biosciences) was thawed over-
night at 4C on ice and, the day of the assay, plated on the bottom of
a 96-well plate and left at 37C for 1 hour for gelifcation. Thereafter,
1 10
4
cells/well were seeded on Matrigel and incubated at 37C.
Where indicated, cells were treated with 3 g/ml anti-L1 polyclonal
antibodies or control, nonimmune rabbit IgG. Optical images of the
wells were acquired after 8 hours at 4 magnifcation. The tubes in
each well were manually counted. The experiments were repeated 3
times, each time with triplicate wells.
Vascular permeability assays
In vitro. The luECs were cultured on Transwell inserts (clear, 24-well
plate, 0.4-m pores; 6.5 mm diameter; tissue culturetreated; Costar),
coated with glutaraldehydecrosslinked gelatin, and allowed to form
confuent monolayers (typically after 4 days). The day of the experiment,
the medium in the upper chamber was replaced with complete medium
containing 1 mg/ml FITC-labeled 40-kDa dextran (Sigma-Aldrich). At
each time point, 50 l of medium in the bottom chamber were taken,
and fuorescence was measured at 488 nm. The medium taken from the
bottom chamber was replaced each time with fresh complete medium in
order to maintain a constant volume in the bottom chamber. The experi-
ments were performed in quintuplicate and repeated 3 times.
In vivo. Texas redlabeled 40-kDa dextran (Life Technologies) was
administered intravenously by retroorbital injection into anesthetized
Panc02 tumorbearing mice (0.25 mg/mouse). Thirty minutes after
injection, mice were sacrifced and their tumors were fxed in 2% para-
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Immunohistochemistry
The clinicopathological data of PDAC patients are described in Sup-
plemental Table 2. Formalin-fxed, parafn-embedded specimens
were prepared from both cancerous (n = 18) and noncancerous pan-
creatic tissue samples (n = 11). Sections were rehydrated through
xylene and graded alcohols. Antigen retrieval was accomplished
using 1 mM EDTA and 0.05% Tween. Samples were incubated with
3% H
2
O
2
for 5 minutes,followed by blocking in 2% goat serum in PBS
for 1 hour. Primary antibodies were incubated for 2 hours at RT in 2%
goat serum. Horseradish peroxidaseconjugated secondary antibod-
ies were used. Samples were developed with DAB and counterstained
with hematoxylin.
The vascular expression of L1 on tissue sections was measured as
the ratio between L1-positive vessels and the total number of vessels
identifed by PECAM-1 staining on consecutive sections. The analysis
was performed on 10 felds per sample at 40 magnifcation.
TMAs were prepared with formalin-fxed, parafn-embedded
samples from diferent tumor types (thyroid, gastric, colon, lung,
and prostate carcinoma) and from their normal tissue counterparts.
Representative areas were frst selected on H&E-stained sections by
a trained pathologist; 2 representative core biopsies for every tissue
block were included in the TMAs. TMAs were assembled on a custom-
built tissue arrayer (Beecher Instruments) as previously described (61).
TMAs were stained for PECAM-1 and L1, and the vascular expression
of L1 was measured on serial sections as described above.
FACS analysis
Tumors were collected 14 days after Panc02 injection, washed in PBS,
and digested in RPMI medium containing 0.1% collagenase type I
(Gibco; Life Technologies) and 10 g/ml DNase (Roche) for 45 min-
utes at 37C. Digested tissue was passed 10 times through a 20-gauge
needle and fltered through a 40-m poresized mesh. After red blood
cell lysis, cells were resuspended in FACS bufer (PBS, 1% FBS, 0.05%
NaN
3
) and subjected to FACS staining. After a blocking incubation in
FACS bufer containing 1% normal mouse serum (Sigma-Aldrich) for
45 minutes on ice, cells were stained for 45 minutes on ice with the
indicated antibodies against diferent immune cell markers. Finally,
cells were washed 3 times and fxed with 1% PFA. Percentage of posi-
tive cells was measured by FACSCalibur fow cytometer and analyzed
with CellQuest software.
The following monoclonal antibodies, all purchased by BD Bio-
sciences except otherwise indicated, were used for FACS staining: PE
anti-CD4 (clone RM4-5; 1:200), PE anti-CD8 (clone 53-6.7; 1:50), PE
anti-CD11b (clone M1/70; 1:100), PE anti-CD11c (clone HL3; 1:50),
FITC anti-CD14 (clone rmC5-3; 1:50), FITC anti-CD19 (clone 1D3;
1:200), FITC anti-CD45 (clone 104; 1:100), PerCP anti-CD45 (clone
30-F11; 1:100), FITC anti-F4/80 (Caltag Laboratories, clone CI:A3-
1; 1:100); PE antiGr-1 (clone RB6-8C5; 1:50); and PE antiI-A/I-E
(clone M5/114.15.2; 1:200).
Statistics
Data are expressed as mean SEM, except as otherwise indicated.
Students 2-tailed nonpaired t test or ANOVA and Bonferronis mul-
tiple comparison tests were used to determine statistical signifcance
(GraphPad Prism 4). Tumor-free survival was drawn using the Kaplan-
Meier method and compared by the log-rank test. Diferences were
considered signifcant at P < 0.05.
qRT-PCR analysis
ECs were cultured on petri dishes coated with glutaraldehydecross-
linked gelatin for 4 days. Total RNA was isolated by extraction with
RNeasy Mini Kit (QIAGEN), and 1 g was reverse-transcribed with
random hexamers (SuperScript Vilo cDNA Synthesis Kit; Invitrogen)
according to the manufacturers instructions. cDNA (5 ng) was ampli-
fed in triplicate in a reaction volume of 15 l with the TaqMan Gene
Expression Assay (Applied Biosystems) and an ABI/Prism 7900 HT
thermocycler (Applied Biosystems) using a pre-PCR step of 10 minutes
at 95C, followed by 40 cycles of 15 seconds at 5C and 60 seconds
at 60C. Preparations of RNA template without reverse transcriptase
were used as negative controls. For each sample, the expression level
was normalized against the geometric mean of the housekeeping genes
encoding GAPDH and 18S. Normalized expression changes were deter-
mined with the comparative threshold cycle (Ct) method (59).
IF
ECs were cultured on 35-mm petri dishes coated with glutaraldehyde
crosslinked gelatin for 4 days and then fxed with 4% PFA for 10 minutes
at room temperature. Panc02 tumors were fxed with 2% PFA overnight
at +4C and embedded in OCT after a graded sucrose series. Fixed cells
and tumor sections (5 m) were permeabilized with PBS and 0.5% Triton
X-100 and incubated for 1 hour at RT with a blocking solution of PBS, 2%
BSA, 5% donkey serum, and 0.05% Triton X-100. Samples were then
incubated overnight at +4C with primary antibodies diluted in blocking
bufer, followed by secondary antibodies (2 hours at room temperature).
Samples were then washed and mounted with Vectashield mounting
medium with DAPI (Vector Laboratories). For staining of L1 on cells or
on tumor sections, the permeabilization step was omitted and Tween
20 was used instead of Triton X-100 in all the bufers. TUNEL staining
(Roche) was performed according to the manufacturers instructions.
Confocal microscopy was performed with a Leica SP2 confocal
microscope equipped with a motorized stage and violet (405 nm laser
diode), blue (488 nm Argon), yellow (561 nm laser diode), and red
(633 nm HeNe laser) excitation laser lines. In some cases, tile scans
of the tumor sections were acquired using 20 magnifcation. Mosaic
images were created using the Leica LCS software.
The number of Ki-67
+
, PHH3
+
, or TUNEL
+
cells/mm
2
were calcu-
lated by counting the number of positive cells in 10 diferent felds at
63 magnifcation for each section. Density of PECAM-1positive blood
vessels was measured on images of tumor sections acquired as reported
above and quantifed with a custom-written plug-in of ImageJ software.
Vessel pericyte coverage was calculated on the images of tumor
sections and quantifed with a custom-written plug-in, as previously
described (60). Collagen IV and fbrin(ogen) deposition was mea-
sured on images of tumor sections and quantifed with ImageJ as the
mean of the fuorescence intensity of the pixels in the tumor area
analyzed. Fluorescence microscopy was performed with a Olympus
BX61 automated upright wide-feld microscope equipped with a Pho-
tometrics Coolsnap Camera.
IL-6 detection
ECs were seeded on plates coated with 0.1% gelatin and cultured in
complete medium for 4 days to reach confuence. At day 4, medium was
replaced by fresh complete medium, and supernatants were collected
after 24 hours. Mouse IL-6 was quantitated using the Quantikine ELISA
Kit (R&D Systems) according to the manufacturers instructions.
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The Journal of Clinical Investigation R E S E A RC H A RT I C L E
1 5 jci.org
Tania Roskams for providing tissue samples; Giovanni Bertalot for
helping with vessel counting; and Pier Paolo Di Fiore for his sup-
port. This work was supported by research grants from the Associa-
tion for International Cancer Research (AICR grant 10-0091), the
Associazione Italiana Ricerca sul Cancro (AIRC grants 2214, 1378,
and 5815), the Fondazione Cariplo (grant 2004-1587 and 2008-
2375, the Fondazione Telethon (grant GGP04078), and the Italian
Ministry of Health (Ricerca Finalizzata), and by a travel grant to E.
Magrini from the European Society of Cardiology.
Address correspondence to: Ugo Cavallaro, Molecular Medi-
cine Program, European Institute of Oncology, Via G. Ripamonti
435, I-20141 Milano, Italy. Phone: 39.02.9437.5165; E-mail: ugo.
cavallaro@ieo.eu.
Study approval
Mouse housing and all experimental animal procedures were performed
in accordance with the guidelines established in the Principles of Labo-
ratory Animal Care (directive 86/609/EEC) and approved by the Italian
Ministry of Health. Specimens of PDAC and noncancerous pancreatic
tissue were obtained upon informed consent from patients operated
at University Hospitals Leuven. All tissue samples used for the TMAs
were collected via standardized operative procedures approved by the
Institutional Ethical Board of the European Institute of Oncology, and
informed consent was obtained for all samples linked with clinical data.
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Supplemental Table 1. Genes regulated by L1 in lECs.
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Supplementary Table S2. Clinico-pathological data of pancreatic carcinoma patients.
Patient
number
Gender Age
(years)
Tumor
diameter
pG pT pN pM
85 F 61 >4 cm 3 3 0 0
221 F 53 >4 cm 3 4 1 0
76 M 60 <2 cm 2 3 1 0
61 F 59 2-4 cm 2 3 1 0
59 F 63 2-4 cm 3 3 0 0
247 F 70 <2 cm 2 3 0 0
3 M 53 2-4 cm 3 3 1 0
301 M 33 <2 cm 3 3 1 0
66 F 55 2-4 cm 2 2 0 0
214 M 46 >4 cm 2 3 1 0
46 M 68 2-4 cm 3 3 0 0
216 F 56 2-4 cm 3 3 1 0
47 M 59 >4 cm 3 3 1 0
73 F 75 <2 cm 2 3 0 0
209 M 73 2-4 cm 3 2 1 0
228 F 75 2-4 cm 2 3 1 0
10 M 52 2-4 cm 3 3 0 0
220 M 58 2-4 cm 3 3 0 0

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