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Blood First Edition Paper, prepublished online February 13, 2013; DOI 10.1182/blood-2012-08-449819
VCAM-1 and VAP-1 recruit myeloid cells that promote pulmonary metastasis in
mice
Running title: VCAM-1 & VAP-1 recruit myeloid cells in metastasis
Špela Ferjančič1*, Ana M. Gil-Bernabé1*, Sally A. Hill1, Philip D. Allen1, Peter
Richardson2, Tim Sparey2, Edward Savory2, Jane McGuffog2 and Ruth J. Muschel1
1
Gray Institute for Radiation Oncology and Biology, Department of Oncology,
University of Oxford, Oxford, United Kingdom
2
Proximagen Group plc, London, United Kingdom
*
These authors contributed equally to this study
Corresponding author: Professor Ruth J. Muschel
Gray Institute for Radiation Oncology and Biology
ORCRB, Roosevelt Drive, Oxford
OX3 7DQ, United Kingdom
Email: [email protected]
Phone: 44-1865-225847
Fax: 44-1865-857127
Disclosure of conflict of interest: P.R., T.S., E.S. and J.M are employed by
Proximagen Group plc., which supplied the VAP-1 inhibitor PRX.A and contributed to
the consumables cost for the experiments. The rest of authors declare no competing
financial interests.
Scientific category: Vascular Biology. Thrombosis and Hemostasis.
1
Copyright © 2013 American Society of Hematology
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Key points
1. Metastatic tumor cell attachment induces endothelial VCAM-1 and VAP-1.
2. VCAM-1 and VAP-1 promote metastatic tumor cell survival by recruiting
myeloid cells, pointing to VAP-1 as a therapeutic target.
Abstract
Pulmonary metastasis is a frequent cause of poor outcome from cancer. The
formation of pulmonary metastasis is greatly facilitated by recruitment of myeloid
cells that are crucial for tumor cell survival and extravasation. During inflammation,
homing of myeloid cells is mediated by endothelial activation, raising the question of
a potential role for endothelial activation in recruitment of myeloid cells during
pulmonary metastasis. Here, we show that metastatic tumor cell attachment causes
the induction of the endothelial activation markers VCAM-1 and VAP-1. Induction of
VCAM-1 is dependent on tumor cell clot formation, decreasing upon induction of TF
pathway inhibitor expression or upon treatment with hirudin. Furthermore, inhibition
of endothelial activation, with a VCAM-1 blocking antibody or a VAP-1 small
molecule inhibitor, leads to reduced recruitment of myeloid cells, and diminished
tumor cell survival and metastasis, without affecting tumor cell adhesion.
Simultaneous inhibition of VCAM-1 and VAP-1 does not result in further reduction in
myeloid cell recruitment and tumor cell survival, suggesting that both act through
closely related mechanisms. These results establish VCAM-1 and VAP-1 as
mediators of myeloid cell recruitment in metastasis, and identify VAP-1 as a potential
target for therapeutic intervention to combat early metastasis.
2
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Introduction
Metastasis can be greatly facilitated by recruitment of myeloid cells. Since
leukocyte homing during inflammation is mediated by endothelial activation, we have
examined the role of endothelial activation in myeloid cell recruitment during early
pulmonary metastasis. Inflammation results in luminal expression of a variety of
molecules that mediate adhesion between the blood borne inflammatory cells and
the endothelium
1,2
. One class of these molecules is the selectins, lectins that bind
oligosaccharides on leukocytes with relatively low affinity. These interactions reduce
the velocity of the leukocytes in the blood stream, making them appear to roll on the
surface of the blood vessel. Tighter adhesion and arrest are then mediated by
integrin binding between the leukocyte and the endothelial activation markers VCAM1 and ICAM-1 and 2, which are transcriptionally induced and expressed on the
endothelial luminal surface.
In addition to these molecules, VAP-1, an ectoenzyme that is stored in
cytoplasmic vesicles and upon inflammatory stimulation is relocated to the luminal
surface of endothelial cells, has a dual action in endothelial adhesion
3,4
. As an
adhesion molecule, it binds different leukocyte ligands, including Siglec-9 and Siglec10, which are present on granulocytes/monocytes and B cells, respectively. As an
enzyme, it converts primary amines into aldehyde through its semicarbazidesensitive amine oxidase (SSAO) activity, releasing hydrogen peroxide and
ammonium. VAP-1 enzymatic activity enhances cellular binding, rolling and firm
adhesion during inflammation, playing mostly a supplemental role 5-7. VAP-1 deficient
mice have impaired leukocyte trafficking into mesenteric lymph nodes, spleen,
peritoneum and joints during inflammation 8; modestly diminished T and B cell
responses, and a mildly diminished response to bacterial (Staphylococcus aureus) or
viral (Coxsackie B4) infections. However, inhibition of VAP-1 activity in wild type adult
mice, with antibodies or pharmaceutical agents, did not alter the response to these
3
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infections 9. Leukocyte homing to liver sinusoids, especially of CD16+ monocytes,
might be more dependent upon VAP-1 than homing at other sites
10-12
proangiogenic monocytes may be especially reliant on VAP-1 for homing
VAP-1 gene is amplified in gastric cancer
14
. M2
13
. The
and VAP-1 is expressed in the blood
vessels of human tumors of the head and neck, liver and melanoma
15,16
.
Interestingly, inhibition of VAP-1 catalytic activity (but not VAP-1 blockade with
antibodies) results in reduction in myeloid cell infiltration (CD11b+, Gr-1+), modest
tumor growth reduction and impaired neoangiogenesis
17,18
.
Lung colonization by metastatic tumor cells results in a rapid influx of myeloid
cells that are essential for survival and extravasation of the metastatic tumor cells
19-
22
. Many cancer cells express procoagulant molecules, such us tissue factor (TF),
resulting in clot formation by arrested tumor cells. These clots trigger the recruitment
of CD11b+, CD11c-, Gr1- myeloid cells
19
. At later times, after tumor cell arrest,
CCL2/MCP-1 released by the tumor cells also facilitates myeloid cell recruitment 22.
The similarity of the process of tumor cell arrest in a vascular bed, during
metastasis, to leukocyte homing has raised the possibility of the metastatic process
utilizing the same mechanisms. Stimulation of endothelial activation by inflammatory
cytokines, including TNF-α, IL-1 and CXCL12, indeed leads to enhanced metastatic
colonization mediated by VCAM-1, especially by tumor cells expressing the α4β
integrin VLA-4, the ligand of VCAM-1
23-26

. However, inhibiting VCAM-1 prior to tumor
cell introduction does not block the basal level of lung colonization (24 and
unpublished data), suggesting that the initial metastatic cell attachment can occur
independently of VCAM-1. VCAM-1 aberrantly expressed by breast cancer cells
interacts with macrophages via α4 integrins, stimulating lung colonization 27. Perhaps
this is a redundant mechanism for the recruitment of macrophages by other means.
Interaction of myeloid cells through tumor cell VCAM-1 can ablate tumor cell
dormancy in the bone marrow, enhancing bone metastasis
28
. Thus, VCAM-1
expression appears to augment metastasis especially by tumor cells that express the
4
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α4β integrin, but is not required for tumor cell adhesion. Therefore, perhaps instead

of endothelial activation driving tumor cell arrest, tumor cell arrest can drive
endothelial activation. Supporting this hypothesis, induction of VCAM-1 and ICAM-1
soon after attachment of tumor cells in the liver has been reported 29. Similarly, in the
brain, VCAM-1 was not initially evident, but was detected soon after adhesion, and
has been proposed as a surrogate marker for detection of brain metastasis
30
. In this
study, we show that endothelial activation through both VCAM-1 and VAP-1 can
contribute to the recruitment of myeloid cells that are essential for tumor cell survival.
Inhibition of VCAM-1 or VAP-1 diminished lung colony formation by decreasing the
recruitment of myeloid cells.
5
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Methods
Cell culture and cell staining
4T1-GFP murine breast cancer cells, 1205Lu-GFP human melanoma cells
31
, the
highly metastatic cell line, Met-1, derived from a PyMT mouse mammary tumor
20
and the murine B16F10, B16F10-TF pathway inhibitor (TFPI) and B16F10-vector
(pcDNA3.1/Zeo) melanoma cell lines
32
were cultured as detailed in supplemental
Methods.
Cells were stained with CMFDA or CMRA (12.5 µM, Molecular Probes), following the
manufacturer’s protocol.
Animals and drug treatments
All animal procedures were carried out in accordance with the U.K. Animals
(Scientific Procedures) Act 1986 and following local ethic review. SCID (CB17/IcrPrkdcscid/IcrCrl), BALB/c and C57BL/6J mice were purchased from Charles River
Laboratories. Cx3cr1gfp/+ mice (B6.129P-Cx3cr1tm1Litt/J) were obtained from The
Jackson Laboratory
33
. Csf1r-GFP FVB [FVB.(tg(Csf1r-EGFP)1Jwp)] and B6.Mac1-
KO mice were gifts from Professor Jeffrey W. Pollard, Albert Einstein College of
Medicine, New York, U.S.A.
20
and Siamon Gordon, University of Oxford, U.K.
34
,
respectively.
Recombinant hirudin (RefludanTM, Pharmion) was administered intraperitoneally at
20 mg/Kg
35
, 5 min before and 4h after the intravenous injection of tumor cells.
Lipopolysaccharides (LPS, Sigma Aldrich) were administered intraperitoneally at 5
mg/Kg. VCAM-1 blocking antibody (clone M/K-2; Millipore) and its corresponding
IgG1-kappa isotype control (Gene Tex Inc.) were administered intravenously at 1.5
mg/Kg, 4h prior to the intravenous injection of tumor cells. VAP-1 inhibitor, PRX.A
(Proximagen) was administered intraperitoneally at 6 mg/Kg, 1h before the
intravenous injection of tumor cells, or as indicated otherwise. PRX.A causes 50%
6
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inhibition of mouse SSAO at 0.7 nM and is selective over 75 other receptors and ion
channels in vitro, except for the dopamine transporter and the batrachotoxin voltage
sensitive Na channel site (<50% inhibition of both is observed at 1 µM). The free
plasma concentrations achieved would be expected to result in greater than 90%
inhibition of the enzyme for at least 5h, without exceeding 100 nM, to ensure
selectivity.
Immunohistochemistry
Murine tissue sections (described in supplemental Methods) were stained with: rat
anti-E-selectin (ABR Affinity BioReagents), rat anti-VCAM-1 (R&D Systems), rat antiCD11b, rat anti-VAP-1 (Abcam) and rat anti-αIIb (Santa Cruz). TSA biotin
amplification system (Perkin Elmer) was performed using biotinylated secondary
antibodies (goat anti-rat IgG, Zymed Laboratories), and streptavidin-conjugated
fluorophores (streptavidin-Alexa Fluor 488 and streptavidin-Alexa Fluor 633,
Invitrogen).
Microscopy and image analysis
Images were acquired using an inverted epifluorescence microscope (DM IRBE,
Leica Microsystems) and a digital camera (Orca, Hamamatsu Photonics), and
analyzed with Simple PCI 6.5 (Hamamatsu Photonics) and ImageJ 1.46
(http://rsb.info.nih.gov/ij/) software. Confocal microscopy was performed as described
in supplemental Methods.
Percentage
of
positive
area
was
analyzed
with
ImageJ
software
(http://rsb.info.nih.gov/ij/), after setting an appropriate threshold.
Tumor cells were considered to be associated with an endothelial cell activation
antigen when expression of this antigen was observed at a distance of less than 80
µm from a tumor cell, the critical oxygen diffusion distance in respiring tissue 36.
7
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Recruitment of myeloid cells was considered positive when 5 or more cells were
clustered around the tumor cell.
Statistical analysis
Statistical analysis was performed with GraphPad Prism 5.02, as described in
supplemental Methods. The particular test performed in each experiment is indicated
in the corresponding figure legend. Differences were considered significant with P
less than 0.05. Data represent mean + or ± SD, unless specified otherwise.
8
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Results
VCAM-1 and VAP-1 are induced in experimental lung metastasis
E-selectin was not detected in the unstimulated lung endothelium, whereas
VCAM-1 and VAP-1 were only expressed at low levels (Fig. 1A, Untreated, and Fig.
1B, time zero). Lipopolysaccharides (LPS) were injected intraperitoneally as a
positive control. E-selectin and VCAM-1 were induced after LPS treatment (Fig. 1A,
LPS, Fig. 1B and Fig. S1). VAP-1 expression was further upregulated after LPS
treatment (Fig. 1A, Fig. 1B and Fig. S1). Although P-selectin and ICAM-1 were also
upregulated after inflammation, the high basal expression levels of ICAM-1 in the
resting lung and the presence of P-selectin in platelets made them unsuitable for
further evaluation in this study (data not shown).
Next, we investigated endothelial activation after tumor cell arrest in the
pulmonary bed in both immuno compromised (SCID) and immuno competent
(BALB/c, syngeneic for 4T1 tumor cells) hosts. E-selectin induction was variable,
depending on the model: not induced after 4T1 cell introduction, slightly induced by
1205Lu cells (Fig. 1A, upper panels, and Fig. 1C) and strongly, but transiently,
induced after introduction of MDA-MB-231 cells (Fig. S2A). VCAM-1 was induced in
the endothelium in proximity to the tumor cells within 2h of their intravenous
introduction (Fig. 1A, middle panels, Fig. 1D and Fig. S2A), and localized with the
endothelial marker von Willebrand factor (vWF, Fig. S2B). We did not detect VCAM-1
expression on the tumor cells. There was a reproducible, but modest, regression in
the extent of VCAM-1 staining between one and three days after tumor cell
introduction. VCAM-1 staining persisted and increased afterwards (Fig. 1D and Fig.
S2A). Although some increase in VCAM-1 staining could be due to angiogenesis, the
bulk of the staining appeared on vessels adjacent to, but not part of, the colonies
(Fig. S2C). VCAM-1 expression in the vicinity of adherent tumor cells was also found
in other models of metastasis, including spontaneous metastasis to the lung of mice
9
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bearing subcutaneous 4T1 tumors, pulmonary metastasis of LLC cells and liver
metastasis of MC38 cells (Fig. S2D). VAP-1 staining in the vicinity of adherent 4T1
and 1205Lu tumor cells increased over basal levels with time, as did the percentage
of tumor cells associated with VAP-1 expression (Fig. 1A, lower panels, and Fig. 1E).
Thus, the attachment of tumor cells consistently induced local endothelial expression
of VCAM-1 and VAP-1.
VCAM-1 expression is associated with monocyte/macrophage recruitment by
the tumor cells
We have previously shown that tumor cells induce clot formation that in turn
induces myeloid cell recruitment
19,31
. B16F10 and Met-1 tumor cells were
respectively introduced into Cx3cr1gfp/+, in a C57BL/6 background, and Csf1r-GFP, in
an FVB background, mice, in which populations of myeloid cells are fluorescently
labeled
20,33
. VCAM-1 expression was induced in the vicinity of the tumor cell-clot-
monocyte/macrophage aggregate (Fig. 2 and Fig. S3A). Clot formation around the
tumor cell and recruitment of CD11b+ cells were also observed in non-transgenic
mice (Fig. S3B). Both B16F10 and 4T1 cells express TF, leading to tumor cell clot
formation and monocyte/macrophage recruitment; in contrast to 1205Lu cells, that do
not express TF (Fig. S3C) and fail to induce extensive clot formation or
monocyte/macrophage recruitment (Fig. S3B). 4T1 tumor cells attracted a similar
population of myeloid cells as compared to B16F10 cells (CD11b+, CD11c-, F4/80+,
CD68+, CX3CR1+, CD45+, Gr-1-, CD3e-)
19,20
. 4T1 cells are known to attract high
numbers of Gr-1+ CD11b+ myeloid derived suppressor cells 37.
Clot formation contributes to endothelial cell activation in pulmonary
metastasis
To determine the relationship between monocyte/macrophage recruitment
and endothelial activation, we analyzed the induction of VCAM-1 in a mouse model
10
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with impaired function of monocytes/macrophages, the Mac1 KO mouse. This mouse
lacks the αM subunit (CD11b) of the αMβ2 (CD11b/CD18) heterodimeric integrin. As
previously reported, the recruitment of myeloid cells (CD45+ and F4/80+ cells) to the
tumor cells was decreased in the Mac1 KO mouse, although clot formation was not
affected
19
. Eight hours after the introduction of B16F10 cells, VCAM-1 was
expressed in the vicinity of tumor cells despite diminished CD11b expression (Fig.
3A). These results indicate that monocytes/macrophages are not responsible for
endothelial activation in early metastasis.
Inhibition of coagulation reduced endothelial activation induced by tumor
cells. Hirudin, a direct inhibitor of thrombin, decreased the formation of clots by 4T1
tumor cells (Fig. S4A), and the recruitment of CD11b+ (Fig. S4B), Gr-1+, and CD45+
(Fig. S4C) cells. Hirudin reduced VCAM-1 induction in this model (Fig. 3B). In
contrast, mice injected with 1205Lu cells had no alteration in the already low levels of
monocyte/macrophage recruitment or VCAM-1 induction after treatment with hirudin
(Fig. 3B and Fig. S4B). Similarly, B16F10 tumor cells expressing the endogenous
inhibitor of TF, B16F10-TFPI cells, had reduced clot formation, recruitment of
monocyte/macrophages and VCAM-1 expression in the vicinity of the tumor cells
(Fig. S5 and Fig. 3C), confirming that coagulation induced by tumor cells is a
contributor to VCAM-1 induction. Cytokine expression profiles of three of the cell
lines (4T1-GFP, 1205Lu-GFP and MDA-MB-231-GFP) were obtained (Fig. S6), but
no correlation with endothelial cell induction was apparent. In summary, abrogation
of coagulation induced by tumor cells reduced, but did not eliminate, endothelial
activation.
Inhibition of endothelial activation results in decreased recruitment of
monocytes/macrophages and reduced metastasis
Treatment with VCAM-1 blocking antibody reduced the recruitment of CD11b+
cells to 4T1 tumor cells at 8h. The effect on recruitment to B16F10 cells was less and
11
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not statistically significant (Fig. 4A), although the number of CD11b+ cells recruited
per tumor cell was decreased after treatment with the blocking antibody (data not
shown). The more dramatic inflammatory response that 4T1 tumor cells exert in
comparison to B16F10 cells could account for this difference. The populations of Gr1+ and CD45+ cells recruited to 4T1 tumor cells were also reduced in the treated
animals (Fig. S7). Treatment with VCAM-1 blocking antibody did not significantly
affect clot formation by the tumor cells (Fig. 4B). Tumor cell survival in the lungs at
24h was reduced by 45% after treatment with VCAM-1 blocking antibody, in both
models (Fig. 4C and Fig. 4D). To confirm that the VCAM-1 blocking antibody was
functional in vivo, we noted that treatment with this antibody increased the levels of
immature neutrophils in peripheral blood by promoting their release from the bone
marrow, as previously reported
38
(Fig. S8A). Hence, inhibition of endothelial
activation results in a moderate reduction in the early recruitment of myeloid cells
and impaired tumor cell survival in the pulmonary vasculature.
PRX.A is a reversible small molecule inhibitor of the SSAO activity of VAP-1.
PRX.A administration results in free plasma concentrations that would be expected
to be sufficient to exceed 90% inhibition of the enzyme in vitro for at least 5h. Ex vivo
assay of the fat SSAO activity in these mice confirmed that the enzyme was inhibited
by a minimum of 60% (Fig. S8B). Inhibition of VAP-1 resulted in a reduced
recruitment of CD11b+ cells to 4T1 and to B16F10 tumor cells, but not to 1205Lu
tumor cells (Fig. 4A). These results reveal an important role for VAP-1 in the
recruitment of myeloid cells to tumor cells in the metastatic process. VAP-1 inhibition
also reduced the recruitment of Gr-1+ and CD45+ cells to 4T1 tumor cells (Fig. S7).
VAP-1 inhibition had a slight inhibitory effect on the formation of clots by 4T1 cells,
and no effect on clot formation around B16F10 or 1205Lu cells (Fig. 4B). VAP-1
inhibition decreased the survival of 4T1 and B16F10 tumor cells in the lungs at 24h,
but did not have an effect on the survival of 1205Lu tumor cells (Fig. 4C and Fig. 4D),
correlating with the reduction in the recruitment of monocytes/macrophages to 4T1
12
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and B16F10 tumor cells after treatment. In conclusion, inhibition of either VCAM-1 or
VAP-1 decreases the recruitment of monocytes/macrophages to the tumor cells and
tumor cell survival.
Simultaneous inhibition of VCAM-1 and VAP-1 was not greater than VAP-1
inhibition alone on myeloid cell recruitment, tumor cell survival or lung colony
formation (Fig. 4A, 4B and 4C). These results indicate that VCAM-1 and VAP-1 exert
their effects on myeloid cell recruitment and subsequent tumor cell survival through
closely related mechanisms.
Endothelial activation is essential for the early steps of metastasis, but is
dispensable afterwards
In order to investigate in which steps of the metastatic process endothelial
activation was required, we inhibited VAP-1 at different times: from the initial steps of
metastasis (1h before and 24h after introduction of the tumor cells) or after several
days, when the tumor cells began to proliferate and establish colonies. In both
models, 4T1 and B16F10, lung colony formation was decreased when the VAP-1
inhibitor was administered from the beginning of the experiment, but not when it was
given 5 days after the injection of the tumor cells (Fig. 5). This demonstrates that
endothelial activation plays a role in the early steps of metastasis, but is dispensable
afterwards.
13
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Discussion
We have focused this study in three important aspects. First, we have
identified endothelial activation molecules induced early during the process of
pulmonary metastasis. Second, we have analyzed factors regulating their
expression. Third, we have examined if inhibition of endothelial activation could
decrease metastasis. Our experiments demonstrate that VCAM-1 and VAP-1 are
induced in the endothelium in the vicinity of tumor cells after their attachment to the
vascular bed. VCAM-1 induction is enhanced by tumor cell clot formation, but is
independent of monocytes/macrophages recruitment. We further show that blocking
endothelial activation, either with an anti-VCAM-1 blocking antibody or with a VAP-1
inhibitor, leads to reduced recruitment of myeloid cells, and diminished tumor cell
survival and metastasis. These experiments suggest a means for therapeutic
intervention to combat early metastasis.
Recruitment of myeloid cells is well known to be required for early metastasis
in many experimental systems 21,39. Our previous work demonstrated that coagulation
is required for the recruitment of myeloid cells to arrested tumor cells 19. In our model,
coagulation was triggered by expression of TF by tumor cells, and this clot then
recruited myeloid cells. MCP-1/CCL2 production by tumor cells has also been found
to recruit myeloid cells
22
. Whether coagulation and MCP-1 are redundant or both
required is not known currently. In both of these models, ablation of the myeloid cells
or inhibition of their recruitment led to failure of metastasis. Thus, the recruitment of
myeloid cells can be a critical component of the metastatic process.
Homing of myeloid cells to inflamed tissues involves endothelial activation,
leading to the question of whether myeloid recruitment by tumor cell induced clots
also utilizes this pathway. We found evidence for aspects of endothelial activation,
such as expression of VCAM-1 and VAP-1, but only inconsistently increased
expression of E-selectin. VCAM-1 was induced by syngeneic cell lines: 4T1 in
14
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immuno competent BALB/c mice, B16F10 in C57BL/6 mice and Met-1 in FVB mice;
or by human cell lines, 1205Lu and MDA-MB-231, in immuno compromised (SCID)
mice. There was little evidence of VCAM-1 expression prior to introduction of tumor
cells. Moreover, inhibition of VCAM-1 with a blocking antibody introduced prior to the
tumor cells did not reduce the number of tumor cells attached to the pulmonary
vasculature, in agreement with previous reports (24 and data not shown). However,
VCAM-1 was clearly upregulated in the endothelium adjacent to the tumor cells after
attachment, and persisted as microscopic colonies formed. These data suggest that
VCAM-1 has functions in the process of metastasis other than initial adhesion of
tumor cells to the endothelium.
The inconsistent involvement of E-selectin is reflected in the literature, where
Laubli et. al. found that E-selectin was upregulated by MC38 murine colon
adenocarcinoma cells, although E-selectin deficiency did not block metastasis.
However, E-selectin inhibition attenuates liver metastasis in other models
40-42
. We
did not evaluate the potential roles of the endogenously expressed ICAM-1 and Pselectin.
We investigated factors regulating the expression of endothelial activation
molecules in this system. Mac1 KO mice have diminished recruitment of
monocytes/macrophages, yet the expression of VCAM-1 was not altered in this
model. Hence, recruited macrophages do not appear to amplify endothelial
activation. We asked whether tumor cell-induced coagulation was a regulator of
endothelial activation. We used two different strategies to decrease clot formation
directed by tumor cells, and they both decreased expression of VCAM-1 in the
proximity of the tumor cells, implicating clot formation in the induction of VCAM-1.
The tumor cell line 1205Lu, which does not express TF, did not exhibit extensive clot
formation or induction of VCAM-1 expression. TF expressed by leukocytes promotes
systemic and local inflammation in mouse models of sickle cell disease. Inhibition of
TF in sickle cell disease models reduced the plasma levels of soluble VCAM-1, and
15
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not those of soluble ICAM-1 or soluble E-selectin. This process was dependent on
MCP-1 and KC
43
. Administration of low molecular weight heparins reduces the
plasma levels of soluble VCAM-1 to a similar extent
44
. Continued VCAM-1 induction,
on the other hand, appears coagulation independent and may be due to cytokine
production from the tumor cells themselves, although no single cytokine is apparent
from the profiling. Thus, clot formation appears to be a significant contributor to the
induction of endothelial VCAM-1 by tumor cells.
We then asked whether blockade of endothelial activation reduced the
recruitment of myeloid cells to attached metastatic tumor cells, and hence reduced
the establishment of metastasis. To test the involvement of VCAM-1, we
administered a blocking antibody. We observed a basal level of expression of VAP-1
in the quiescent murine lung, consistent with previous reports
45
. The induction of
VAP-1 is based upon both altered localization to the endothelial luminal surface and
increased amounts at the transcriptional and translational levels. Thus, activation
may occur without increased staining of VAP-1 by immunohistochemistry
3
.
Nevertheless, VAP-1 staining increased after tumor cell adhesion, peaking at 8h,
coincident with the maxima for monocyte/macrophage recruitment. To test the
involvement of VAP-1 in metastasis, we resorted to the use of a small molecule
inhibitor. Neither inhibition of VCAM-1 nor VAP-1 affected the extent of coagulation
around the tumor cells, but both decreased the recruitment of myeloid cells and the
survival of tumor cells. In Gil-Bernabe et. al.
19
, we have shown that coagulation
facilitates cell survival indirectly through the recruitment of myeloid cells. Here, we
show that the recruitment and retention of myeloid cells is reduced by inhibition of the
function of the endothelial activation molecules VCAM-1 and VAP-1, which at least in
part is regulated by coagulation. In addition to adherence to endothelial activation
markers, myeloid cells also have receptors that could allow them to bind to clots,
both binding to fibrin and to urokinase
46,47
. Inhibition of VCAM-1 and VAP-1 led to
decreased myeloid cell retention, suggesting that endothelial activation plays a role
16
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in recruitment and leaving the significance of the potential homing to the clot
components to be determined. It should be noted that CD11b+ cell recruitment and
tumor cell survival by the TF-deficient cell line 1205Lu was not affected by the VAP-1
inhibitor. Inhibition of both VCAM-1 and VAP-1 together had no greater effect than
either alone, suggesting that they act on a single pathway. Neither affected clot
formation. VAP-1 inhibition leads to reduced hydrogen peroxide production, a
compound that has been reported to induce E- and P-selectins and VCAM-1
6,7
. This
suggests a possible mechanism explaining the non-additive effect of VAP-1 and
VCAM-1 inhibition. Although VAP-1 inhibition appeared more effective than VCAM-1
blocking antibody, it is hard to determine the efficacy of either, making this
comparison uncertain. Inhibition of VAP-1 reduces angiogenesis, tumor growth and
infiltration of CD11b+Gr-1+ myeloid cells into the tumors, being inhibition of its
enzymatic activity sufficient for these effects
17,48
. In our experiments with VAP-1
inhibitor, we find reduced metastatic cell survival and reduced myeloid cell
recruitment. Since some metastatic cancer cells may utilize alternative mechanisms,
stratification of patients would be essential for clinical application of VAP-1 inhibition.
Since VAP-1 inhibition, at least in mice, has only a slight decreased effect on
immunity or defense against infection, administration of VAP-1 inhibitor might be
clinically feasible. The early phases of metastasis and not the later phases were
affected, indicating that this target would be expected to be useful only in cases
where metastasis was being established, such as the recurrence of cancer after
treatment.
17
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Acknowledgments
The authors thank Karla Watson and Magdalena Flieger (Gray Institute for
Radiation Oncology and Biology, University of Oxford) for their assistance with in
vivo experiments; Lei Zhao and Thomas Tapmeier (Gray Institute for Radiation
Oncology and Biology, University of Oxford) for their help with flow cytometry; and
Michael Ho and Leon Widdowson (Proximagen Group plc.) for performing VAP-1
activity assays on mouse fat tissue. We also thank Vittorio Katis for his help in the
preparation of the manuscript.
This study was supported by Cancer Research U.K. and in part by
Proximagen Group plc. R.J.M. is supported by Cancer Research U.K. and Medical
Research Council.
18
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Authorship contributions
S.F. designed and performed research, and analyzed and interpreted data.
A.M.G.-B. designed and performed research, analyzed and interpreted data,
performed statistical analysis, and wrote the manuscript. S.A.H. provided expert
assistance. P.D.A. analyzed data. P.R. and T.S developed the VAP-1 inhibitor
PRX.A, and P.R. edited the manuscript. E.S. synthesized the VAP-1 inhibitor PRX.A.
J.M. analyzed the pharmacokinetics and dosage of the VAP-1 inhibitor PRX.A.
R.J.M. designed research, analyzed and interpreted data, and wrote the manuscript.
Conflict-of-interest disclosure: P.R., T.S., E.S. and J.M are employed by
Proximagen Group plc., which supplied the VAP-1 inhibitor PRX.A and contributed to
the consumables cost for the experiments. The rest of authors declare no competing
financial interests.
Correspondence: Ruth J. Muschel, Gray Institute for Radiation Oncology and
Biology, ORCRB, Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom; e-mail:
[email protected].
19
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Figure legends
Figure 1. Endothelial activation in response to LPS treatment and to tumor cell
challenge. BALB/c mice were treated with LPS or injected with 5x105 4T1-GFP
tumor cells (green) intravenously. SCID mice were injected with 5x105 1205Lu-GFP
tumor cells (green) intravenously. Lungs were harvested at the indicated times and
analyzed by immunohistochemistry for the presence of different antigens of
endothelial activation. (A) Representative images, acquired with a confocal
microscope, of stainings for E-selectin, VCAM-1 and VAP-1 are shown (Alexa Fluor
633, red). (B) Dynamics of endothelial activation upon LPS treatment. Time zero
represents untreated mice and indicates the basal expression levels of the adhesion
molecules. Data represent mean ±SD, n=3 mice. Representative images are shown
in Fig. S1. E-selectin (C), VCAM-1 (D) and VAP-1 (E) induction upon 4T1-GFP tumor
cell challenge was evaluated as described in Methods. In (C-E) n≥3 mice and data
represent mean ±SD (C) or ±SEM (D) and (E). In (C) values corresponding to the
time points 15 minutes, 24h and 72h are zero. Scale bars represent 50 µm.
Figure 2. VCAM-1 expression is associated with myeloid cell recruitment.
Cx3cr1gfp/+ (left panels; green, myeloid cells) or Csf1r-GFP mice (right panels; green,
myeloid cells) were injected intravenously with 5x105 CMRA-stained B16F10 or Met1 cells, respectively (magenta). Eight hours later, lungs were harvested and
assessed by immunohistochemistry for VCAM-1 expression (red, Alexa Fluor 633).
Representative images, acquired
with a confocal microscope, from
three
independent experiments performed, are shown. Scale bars represent 50 µm.
Figure 3. Clot formation contributes to endothelial activation in pulmonary
metastasis. (A) C57BL/6-wt or Mac1 KO mice were intravenously injected with
5x105 CMFDA-stained B16F10 tumor cells (green). Lungs were harvested eight
26
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hours later and assessed for VCAM-1 expression (red, Alexa Fluor 633; imaged with
a confocal microscope). Percentage of tumor cells associated with VCAM-1
expression is shown; n=3 mice (Mann-Whitney) (B) BALB/c or SCID mice were
treated with hirudin and intravenously injected 5x105 4T1-GFP or 1205Lu-GFP tumor
cells, respectively. Eight hours after the introduction of tumor cells, lungs were
harvested and assessed for VCAM-1 expression (red, Alexa Fluor 633).
Representative images, acquired with a confocal microscope, are shown.
Percentage of tumor cells associated with VCAM-1 expression was analyzed; n≥4
mice (Mann-Whitney for each cell line). (C) C57BL/6 mice were intravenously
injected 5x105 CMFDA-stained-B16F10-wt, -B16F10-TFPI or -B16F10-vector tumor
cells (green). Lungs were harvested eight hours after and assessed for VCAM-1
expression (red, Alexa Fluor 633). Representative images, acquired with a confocal
microscope, are shown. Percentage of tumor cells associated with VCAM-1
expression was analyzed; n=3 mice (one-way ANOVA and Tukey’s test). In (A)-(C)
data represent mean +SD and *P < 0.05, **P < 0.01. Scale bars represent 50 µm.
Figure 4. Inhibition of endothelial activation decreases pulmonary metastasis.
BALB/c, C57BL/6 or SCID mice were treated with VCAM-1 blocking antibody, with
VAP-1 inhibitor or with both. Mice were injected intravenously with 5x105 4T1-GFP,
CMFDA-stained B16F10 or 1205Lu-GFP tumor cells, respectively. Lungs were
harvested eight hours later to assess for CD11b+ cell recruitment (A) and for clot
formation (B), analyzed by immunohistochemistry against CD11b and the platelet
specific integrin αIIb, respectively. Lungs were harvested 24h after the introduction of
tumor cells to analyze tumor cell survival, scored in lung sections (C; ≥10 sections
analyzed per mouse) or by whole lung imaging, (D, as described in supplemental
Methods). Data represent mean +SD; n≥3 mice (one-way ANOVA and Tukey’s test
or Mann-Whitney test when only two groups were compared, for each cell line) and
*P<0.05, **P<0.01, ***P<0.001.
27
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Figure 5. VAP-1 activity is essential at the early steps of metastasis, but
dispensable afterwards. BALB/c or C57BL/6 mice were treated with VAP-1 inhibitor
either 1h before and 24h after the intravenous injection of 5x105 4T1-GFP (left) or
B16F10 (right) tumor cells, respectively, (VAP-1 inhib. early), or on days 5, 7, 9 and
11 after the introduction of the tumor cells (VAP-1 inhib. late). Lungs were isolated on
day 14 and the number of metastatic lung nodules was analyzed. Data represent
mean +SD; n≥10 mice (one-way ANOVA and Tukey’s test) and **P<0.01,
***P<0.001.
28
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Prepublished online February 13, 2013;
doi:10.1182/blood-2012-08-449819
VCAM-1 and VAP-1 recruit myeloid cells that promote pulmonary
metastasis in mice
Spela Ferjancic, Ana M. Gil-Bernabé, Sally A. Hill, Philip D. Allen, Peter Richardson, Tim Sparey, Edward
Savory, Jane McGuffog and Ruth J. Muschel
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