Real-Time Imaging Reveals Local, Transient

Transcription

Published OnlineFirst August 12, 2015; DOI: 10.1158/2159-8290.CD-15-0012
Research Article
Real-Time Imaging Reveals Local, Transient
Vascular Permeability, and Tumor Cell
Intravasation Stimulated by TIE2hi
Macrophage–Derived VEGFA
Allison S. Harney1,2,3,4, Esther N. Arwert1,4,5, David Entenberg1,3,4, Yarong Wang1,4, Peng Guo1,4,
Bin-Zhi Qian6,7,8, Maja H. Oktay9, Jeffrey W. Pollard6,7,8, Joan G. Jones1,3,9,10, and John S. Condeelis1,3,4
Dissemination of tumor cells is an essential step in metastasis. Direct contact
between a macrophage, mammalian-enabled (MENA)–overexpressing tumor cell,
and endothelial cell [Tumor MicroEnvironment of Metastasis (TMEM)] correlates with metastasis in
breast cancer patients. Here we show, using intravital high-resolution two-photon microscopy, that
transient vascular permeability and tumor cell intravasation occur simultaneously and exclusively at
TMEM. The hyperpermeable nature of tumor vasculature is described as spatially and temporally heterogeneous. Using real-time imaging, we observed that vascular permeability is transient, restricted
to the TMEM, and required for tumor cell dissemination. VEGFA signaling from TIE2hi TMEM macrophages causes local loss of vascular junctions, transient vascular permeability, and tumor cell intravasation, demonstrating a role for the TMEM within the primary mammary tumor. These data provide
insight into the mechanism of tumor cell intravasation and vascular permeability in breast cancer,
explaining the value of TMEM density as a predictor of distant metastatic recurrence in patients.
Abstract
SIGNIFICANCE: Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA
signaling from TIE2hi TMEM macrophages results in local, transient vascular permeability and tumor
cell intravasation. These data provide evidence for the mechanism underlying the association of TMEM
with distant metastatic recurrence, offering a rationale for therapies targeting TMEM. Cancer Discov;
5(9); 1–12. ©2015 AACR.
1
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, New York. 2Department of Radiology, Albert
Einstein College of Medicine, New York, New York. 3Integrated Imaging Program, Albert Einstein College of Medicine, New York, New York.
4
Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine,
New York, New York. 5Tumour Cell Biology Laboratory, Cancer Research
UK, London Research Institute, London, United Kingdom. 6Department of
Developmental and Molecular Biology, Albert Einstein College of Medicine, New York, New York. 7Department of Obstetrics & Gynecology and
Women’s Health, Albert Einstein College of Medicine, New York, New York.
8
MRC Center for Reproductive Health, University of Edinburgh, Edinburgh,
United Kingdom. 9Department of Pathology, Albert Einstein College of
OF1 | CANCER DISCOVERY September 2015
Medicine, New York, New York. 10Department of Epidemiology and Population Health, Albert Einstein College of Medicine, New York, New York.
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Authors: Allison S. Harney, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. Phone: 718-678-1131;
Fax: 718-678-1128; E-mail: [email protected]; and John S.
Condeelis, Phone: 718-678-1127; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-15-0012
©2015 American Association for Cancer Research.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on October 27, 2016. © 2015 American Association for
Cancer Research.
CD-15-0012_PAP.indd 1
29/07/15 7:27 AM
INTRODUCTION
For almost two decades, tumor vasculature has been
described as abnormal with increased vascular permeability
(1, 2). VEGFA is known to promote vascular permeability,
and inhibition of VEGFA results in the normalization of
tumor vasculature and a decrease in permeability (3, 4). Due
to the significant effects of VEGFA on tumor angiogenesis
and vascular permeability, inhibitors of VEGF signaling have
become an important research focus in the development of
antitumor therapies.
Tumor-associated macrophages (TAM) have been implicated in tumor progression, angiogenesis, and metastasis (5,
6). A subpopulation of perivascular TAMs that have features
of protumorigenic macrophages, promoting tumor angiogenesis and metastasis, have been identified as TIE2-expressing macrophages (TEM; ref. 7). Perivascular macrophages are
also an essential component of the microanatomical sites
termed “Tumor MicroEnvironment of Metastasis” (TMEM)
that consist of a TAM in direct contact with a mammalianenabled (MENA) overexpressing tumor cell and endothelial
cell (8). TMEMs have been associated with tumor cell intravasation (9, 10), and TMEM density predicts distant metastatic
recurrence in breast cancer patients independently of other
clinical prognostic indicators (8, 11). However, the mechanistic link between perivascular macrophages and tumor cell
intravasation has remained unclear. Further, hyperpermeability in tumor vasculature is not uniform, but rather is spatially
and temporally heterogeneous (12). In a VEGFA overexpression
model inducing vascular permeability, the presence of macrophages at vascular branch points was observed at hotspots
of vascular permeability (4). Although hyperpermeability of
tumor vasculature is widely accepted, a mechanistic understanding of the heterogeneity of vascular permeability, the
contribution of TAMs, and the link with tumor cell intravasation have not been described.
Here we show, using intravital high-resolution two-photon
microscopy, that transient vascular permeability events are
restricted to TMEM sites of TIE2hi/VEGFAhi perivascular
macrophages. Local loss of vascular junctions at the TMEM
results in transient vascular permeability and tumor cell intravasation in the spontaneous autochthonous mouse mammary cancer model where the mouse mammary tumor virus
long terminal repeat drives the polyoma middle T antigen
(MMTV-PyMT), the human patient–derived xenograft model
TN1, and human metastatic breast cancer.
RESULTS
TMEM-Associated Tumor Cells and Macrophages
Are Stationary in TMEM Structures
To examine the functional role of the TMEM in tumor
cell dissemination, we used the spontaneous autochthonous
mouse mammary cancer model where the mouse mammary
tumor virus long terminal repeat drives the polyoma middle
T antigen (MMTV-PyMT), in which tumors exhibit histo
logy similar to human luminal breast cancer and progress to
September 2015 CANCER DISCOVERY | OF2
Cancer Research.
29/07/15 7:27 AM
Harney et al.
RESEARCH ARTICLE
A
B TC/Macrophage/Dextran
TC/Macrophage/Dextran
0′
Streaming
M
B4′
B0′
B6′
TC
TC
M
TC
M
*
EC
*
*
C TC/Macrophage/Dextran
M
D TC/Macrophage/Qdots
M
TC
*
TC
4′
E
16′
20′
T2
M2
EC
EC
TC
M
M1
T2
T1
EC
TC/Macrophage/EC
Figure 1. Motile tumor cells intravasate at the TMEM. A, time 0′ in the left plot indicating the TMEM (white box) from time-lapse intravital microscopy
(IVM). Macrophages (M; cyan), tumor cells (TC; green), and blood vessels (155-kDa Dextran–TMR; red). Right plot is a single time point from time lapse of
tumor cell and macrophage streaming toward nonmigratory TMEM (asterisk, TMEM position from left plot). Streams and the TMEM are in different focal
planes. Scale bar, 50 μm. B, three-dimensional (3D) reconstruction of time-lapse IVM from A of tumor cell and macrophage streaming toward the TMEM
(asterisk). Scale bar, 20 μm. C, 3D reconstruction of tumor cell intravasation (yellow arrowhead) at the TMEM (luminal surface of the endothelium dashed
white line). D, IVM time lapse of tumor cell intravasation at the TMEM [white box in 4′ plot containing stationary TMEM-Macrophage (M), TMEM-tumor
cell, and TMEM-endothelial cell boundary (EC; arrows)]. A non-TMEM tumor cell arrives at TMEM (arrowhead in plot 16′) and undergoes transendothelial
migration (arrow in plot 20′), whereas TMEM-macrophage and TMEM-tumor cell remain immobile. Scale bar, 10 μm. E, schematic summary diagram of
plots A to D where tumor cell (green; T2) and macrophage (blue; M2) stream toward nonmigratory TMEM (black box, T1 and M1), where the tumor cell (T2)
undergoes transendothelial migration.
metastasis (13). IHC revealed that TMEM structures in mouse
tumors have the same microanatomical structure as identified in humans (Supplementary Fig. S1A; ref. 11). TMEM
density and circulating tumor cells (CTC) increase with tumor
progression with elevated TMEM scores in late carcinoma as
compared with early carcinoma as seen by IHC (Supplementary Fig. S1A–S1C), though total perivascular macrophage
(including macrophages not associated with tumor cells) density is not significantly different (13). High-resolution imaging
demonstrates that in TMEM structures tumor cells and macrophages extend protrusions but are relatively nonmigratory
and stay in direct contact over time (Supplementary Fig. S1D).
Vascular Permeability and Tumor Cell
Intravasation Occur Concurrently at the TMEM
To directly observe TMEM function in vivo, we used
extended time-lapse intravital microscopy (IVM) with high
spatial and temporal resolution. To visualize blood flow, vessels were labeled with a high–molecular weight compound
(155-kDa dextran or quantum dots; refs. 1, 14; Figs. 1, 2, 3; and
Supplementary Fig. S2). In PyMT late carcinoma, migratory
tumor cells and macrophages stream toward TMEM at sites
with vascular permeability, whereupon tumor cells undergo
transendothelial migration at the TMEM (Fig. 1A–E; Supplementary Fig. S2A–S2E). In late carcinoma, transient, local
blood vessel permeability was observed at TMEM sites by
the extravasation of quantum dots (Supplementary Fig. S2A
and S2B) or 155-kDa dextran–tetramethylrhodamine (TMR;
Figs. 2A and B, 3C; Supplementary Fig. S2C–S2E; and Supplementary Movie S1). Further, tumor cell intravasation
occurs at TMEM sites concurrently with transient permeability (Fig. 2A–H and Supplementary Fig. S2C–S2E). Transient
vascular permeability at the TMEM is spatially and temporally heterogeneous (Supplementary Fig. S2F), with events
of permeability and tumor cell intravasation at the TMEM
occurring predominantly at vascular branch points (Supplementary Fig. S2G). Transendothelial crossing of tumor cells
is visualized by the hourglass shape of tumor cells, as they
are partially in the vessel lumen and partially in the tissue
(Fig. 1C, 2A, C–E; Supplementary Fig. S2E). During transendothelial migration of tumor cells, the TMEM tumor cell and
macrophage neither migrate nor intravasate, indicating that
tumor cells entering the blood vessel at the TMEM are supplied by the migratory stream of cells (Fig. 1A, B, and D). The
stationary phenotype of these cells is consistent with previous
results showing macrophage contact–initiated invadopodium
formation uniquely in the TMEM tumor cell (9) and that
perivascular invadopodium-containing tumor cells are relatively nonmotile in vivo (15).
The peak of extravascular dextran intensity and the appearance of circulating tumor cells coincide temporally and spatially (Fig. 2A–E, H; Supplementary Fig. S2; Supplementary
Movie S1), demonstrating a direct link between localized
blood vessel permeability and tumor cell intravasation at the
TMEM. The coincidence of spontaneous, transient vascular
permeability with tumor cell intravasation at the TMEM also
has been observed in a patient-derived xenograft model of
triple-negative breast cancer, TN1 (Supplementary Fig. S3).
To confirm that the TMEM is associated with transient
vascular permeability and tumor cell intravasation, a 100-μm
Cancer Research.
29/07/15 7:27 AM
Imaging the Tumor Microenvironment of Metastasis
Tumor cell/Macrophage/Dextran
F
Dextran
0.04
0.02
0.00
0.03
0.02
0.01
0.00
TM
EM
TM
EM
30′
0.06
0.04
N
N
o
9′
0.08
TM
EM
TM
EM
EC
0′
**
**
TC
B
G
30′
o
9′
M
Frequence of tumor cell
intravasation per hour
0′
Frequency of blood vessel
permeability per hour
A
RESEARCH ARTICLE
Tumor cell
0′
9′
30′
1.0
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
0
D
E
Intravasation
M
Circulating tumor cell
normalized area
C
Dextran
normalized intensity
H
10
20
30
Time (min)
Tumor cell/Dextran
J0′
J1′
J2′
J3′
TC
3
1
2
4
3
1
2
4
3
4
4
Figure 2. Transient, local blood vessel permeability events accompany intravasation at the TMEM. A, IVM time lapse of 155-kDa dextran–TMR extravasation and tumor cell intravasation. TMEM, white box. Blood vessel permeability sites (white arrowheads) and intravasating tumor cell (TC; yellow dashed
line, 9′). Clearance of dextran and decrease of CTCs at 30′. Scale bar, 50 μm. At 9′ and 30′, TMEM tumor cells and macrophages are added in false color to
increase visibility after bleaching. B, isolated 155-kDa dextran–TMR channel from A. Red arrowheads mark dextran extravasation (white). Dashed red line,
luminal side of the endothelium. C, isolated tumor cell channel from A. Yellow arrow marks site of intravasating tumor cell (yellow dashed line) at the TMEM.
White dashed line marks the luminal surface of the endothelium. Red box, region adjacent to the TMEM with elevated CTCs. D, single time point of tumor
cell intravasation (yellow dashed line) by time-lapse IVM. Scale bar, 50 μm. E, 3D reconstruction of time-lapse IVM from D of tumor cell intravasation at the
TMEM. Transmigrating tumor cells (individually numbered, dashed white lines) are isolated from other cell types for clarity with time in minutes from start
(J0′) to end of transmigration (J3′). The luminal endothelial surface is outlined in a pink dashed line. Extravascular dextran (red) at the TMEM indicated with
a yellow arrowhead and outlined in a yellow dashed line. F, frequency of blood vessel permeability events in the presence of the TMEM or away from the
TMEM in 100-μm windows (see Supplementary Fig. S4; n = 16; **, P = 0.0034). G, frequency of tumor cell intravasation events in the presence of the TMEM
or away from the TMEM in 100-μm windows (n = 16; **, P = 0.0012). H, quantification of extravascular dextran intensity and CTC area at the TMEM over
time from A. •, extravascular dextran; n, CTC.
window, the approximate width of a TMEM site, was consecutively slid along all blood vessels (window measurement)
to quantify the frequency of tumor cell intravasation and
vascular permeability events in the presence or absence of the
TMEM (Supplementary Fig. S4). Vascular permeability and
tumor cell intravasation occur exclusively within the 100-μm
window when it contains a TMEM, but never when the 100-μm
window does not contain a TMEM in PyMT (Fig. 2F and G).
Similar results were observed in the human TN1 model (Supplementary Fig. S3C and S3D), highlighting the importance
of the TMEM in transient vascular permeability and tumor
cell intravasation.
Vascular Permeability at the TMEM Is a Highly
Localized and Transient Event
Tumor vasculature has been previously described as abnormal with increased permeability, which has been attributed
to larger vascular intercellular openings (1, 12, 16). However,
vascular permeability is not spatially or temporally uniform,
with hotspots at vascular branch points (4, 12). Here, we demons
trate that vascular permeability is transient, occurs exclusively
at TMEM sites, and is temporally heterogeneous, explaining the
previously unresolved heterogeneity in vascular permeability
(Fig. 2F and Supplementary Fig. S2E). Events of spontaneous,
Cancer Research.
29/07/15 7:27 AM
Harney et al.
RESEARCH ARTICLE
A
20′
40′
60′
0′
20′
40′
60′
0′
20′
40′
60′
Laser damage
Before
Intravenous VEGFA
B
TMEM permeability
C
D
F
0.8
Total extravascular
dextran area (µm2)
Relative intensity
extravascular dextran
1.0
0.6
0.4
0.2
80,000
60,000
40,000
20,000
0
0.0
0
10
30
40
20
Time (min)
50
60
0
E
f(t ) = Ae
–(t – µ1)
erfc
σ1
–(t – µ2)
σ2
Vessel leakiness
Spontaneous
VEGFA
Damage
10
20
30
40
Time (min)
50
60
µ1
σ1
µ2
σ2
16.80
35.55
N/A
15.21
13.92
N/A
12.44
22.07
29.69
10.15
17.02
29.17
Figure 3. TMEM-mediated vascular permeability is transient and localized. A, time-lapse imaging demonstrates that laser-induced damage to the endothe-
lium creates a hole allowing for extravasation of 155-kDa dextran–TMR. The location of the hole is marked by a white dot (2 μm) and a yellow arrowhead. 155kDa dextran–TMR extravasates and increases over time up to 60′ filling the field of view and not clearing from the tissue (n = 4). Scale bar, 50 μm. B, 155-kDa
dextran–TMR is injected by tail vein i.v. catheter followed by 8 μg of VEGFA165 at 0′. VEGFA165 induces blood vessel permeability in all of the blood vessels
in the field of view. Peak extravascular dextran is observed at 20′ followed by clearance by 60′ after resealing of vascular junctions (n = 4). Scale bar, 50 μm.
C, spontaneous vascular permeability at the TMEM is both transient and local. Local peak extravasation of 155-kDa dextran–TMR occurs after 20′ (yellow
arrowhead) and clears within 60′ (n = 11). Scale bar, 50 μm. D, quantification of average relative intensity of extravascular 155-kDa dextran–TMR after (•)
laser damage (n = 4), (n) intravenous VEGFA165 (n = 4), and (▲) spontaneous permeability (n = 11). E, table of parameters from curve fitting to an Exponentially
Modified Gaussian function using data from D. F, quantification of total extravascular 155-kDa dextran–TMR area after laser-induced damage, i.v. injection of
VEGFA165, or spontaneous permeability at the TMEM from individual animals represented in A, B, and C. Peak of 155-kDa dextran–TMR area in spontaneous
permeability at the TMEM indicated with a red arrowhead. •, laser damage; n, intravenous VEGFA165; ▲, spontaneous vascular permeability at the TMEM.
Cancer Research.
29/07/15 7:28 AM
local vascular permeability and tumor cell intravasation at
TMEM occur predominantly at vascular branch points, consistent with previous reports of vascular permeability (Supplementary Fig. S2G). If tumor blood vessels were uniformly leaky,
high–molecular weight vascular probes would extravasate immediately and continuously after injection. Although the high–
molecular weight probe, 155-kDa dextran–TMR, remains in
the vasculature in the absence of transient TMEM-associated
permeability events for the duration of the time-lapse imaging,
a low-molecular weight dextran, 10-kDa dextran–FITC, which
is below the molecular-weight cutoff size of the endothelium (1,
14), leaks from blood vessels and clears from the vascular space
(Supplementary Fig. S5).
Further, transient permeability events are distinct from
mechanical damage to the endothelium. After creating a 2-μm
hole in the endothelium with a laser, 155-kDa dextran–TMR
extravasates continuously, filling the field of view (Fig. 3A).
In contrast, VEGFA-mediated permeability is transient (12).
Intravenous injection of VEGFA165, the soluble isoform of
VEGFA with properties of native VEGF (17), results in vascular permeability with peak intensity of extravascular dextran
at 20 minutes (Fig. 3B). Spontaneous vascular permeability
at the TMEM follows similar kinetics to VEGFA165-mediated
permeability with peak intensity of extravascular dextran at
20 minutes, but is restricted to individual TMEM sites (Fig. 3C).
The curves obtained for average intensity of extravascular
155-kDa dextran–TMR after laser damage, VEGFA165, and
spontaneous permeability were fit to an exponentially modified Gaussian function (Fig. 3D and E). Although the curve
for laser damage does not have a clearance term as dextran
continues to extravasate for the entire time lapse, both the
VEGFA165 and spontaneous curves have similar extravasation
and clearance rates. A significant difference between VEGFA165
and spontaneous TMEM-mediated permeability is that permeability at the TMEM is highly local, whereas VEGFA165
results in dextran extravasation from all blood vessels within a
field of view. Thus, the area of extravascular 155-kDa dextran–
TMR from local TMEM-mediated permeability is markedly
less than permeability from VEGFA165 or laser-induced damage (Fig. 3F), further emphasizing the local nature of TMEMmediated vascular permeability.
RESEARCH ARTICLE
phages, reflecting a requirement for macrophage-dependent
signaling events to induce vascular permeability. Staining for
vascular junction proteins ZO-1 and VE-Cadherin increased
in the tumor vasculature after depletion of macrophages in
MAFIA mouse tumors (Fig. 4D, G, and H), indicating that
macrophages are involved in vascular junction disassembly
during vascular permeability events at the TMEM.
TEMs Are Localized in TMEM Structures
In PyMT mammary carcinoma, a subpopulation of TAMs
has been identified as TIE2hi perivascular macrophages (7, 20,
21). TEMs have been shown to upregulate the TIE2 tyrosine
kinase receptor by 100-fold after recruitment to the tumor
(22). TEMs have features of protumorigenic macrophages
and promote tumor angiogenesis (7). TEMs are further characterized as MRC1+/CD11b+/F4/80+/CD11c– and are associated with CD31+ tumor blood vessels (20). Thus, we sought
to determine if TEMs are located in the TMEM. Immuno
fluorescence of the TMEM markers MENA (tumor cells),
CD31 (endothelial cells), and CD68 (macrophages; Fig. 5A)
compared with TIE2, VEGFA, and CD31 in sequential tissue
sections demonstrates that TIE2hi/VEGFAhi macrophages are
enriched in TMEM structures (Fig. 5B and Supplementary
Fig. S6A–S6E). VEGFA is elevated in TIE2hi macrophages, as
compared with the adjacent endothelial cells and surrounding tumor tissue (Fig. 5C and D). Further, 100% of TIE2hi/
VEGFAhi TMEM-associated macrophages express the TEM
markers MRC1, CD11b, and F4/80 while lacking CD11c
(Fig. 5E and F and Supplementary Fig. S6).
Inhibition of VEGFA Signaling Reduces Vascular
Permeability and Tumor Cell Intravasation
To investigate the importance of VEGFA in TMEM function,
we blocked VEGFA binding to VEGFRs using a neutralizing
antibody (B20-4.1.1), and found a decrease in extravascular dextran and circulating tumor cells (Fig. 6A–C). Binding of VEGFA
to VEGR2 leads to junction disassembly (23). Vascular ZO-1
and VE-Cadherin immunostaining increased during VEGFA
inhibition, suggesting an increase in integrity of endothelial
adherens and tight junctions from reduced bioavailability of
VEGFA, including VEGFA from the TMEM (Fig. 6A, D, and E).
TMEM-Associated Macrophages Are Essential
for Vascular Permeability and Tumor Cell
Intravasation
VEGFA Signaling from TIE2hi/VEGFAhi TMEM
Macrophages Mediates Vascular Permeability and
Tumor Cell Intravasation
To determine if TMEM macrophages regulate vascular permeability and tumor cell intravasation, macrophages were
depleted in the mammary tumor using the previously characterized mouse model, macrophage fas-induced apoptosis (MAFIA;
refs. 18, 19), with orthotopic MMTV-PyMT tumor implants.
Depletion of macrophages is systemic, including within the
mammary tumor, thus resulting in a depletion of TAM and
TMEM by 67% and 72%, respectively (Fig. 4A–C). When macrophages are depleted, extravascular dextran decreases, as does
the number of circulating tumor cells (Fig. 4D–F). These data
demonstrate that macrophages are essential for vascular permeability and tumor cell intravasation at the TMEM.
Because blood vessel permeability observed by IVM is
restricted to TMEM, we examined whether vascular junction
protein localization was altered in the absence of macro-
To determine if the subpopulation of TIE2hi/VEGFAhi
macrophages in the TMEM is an essential source of VEGFA
in the tumor microenvironment required for transient vascular permeability at the TMEM and tumor cell intravasation, VEGFA was selectively ablated in monocytes and
macrophages using the Vegfaflox/flox;Csf1r-Mer-iCre-Mer transgenic mouse depletion model of Vegfa that targets myeloid
cells expressing CSF1R, including both Ly6Chi and Ly6Clo
populations, and the TEM population (24). Macrophagespecific depletion of VEGFA reduced transient vascular
permeability and circulating tumor cells and restored vascular junctions (Fig. 6F–J). Immunofluorescence of sequential sections demonstrates that blood vessels adjacent to
CD68+/TIE2hi/VEGFAhi TMEM macrophage have significantly
reduced vascular VE-Cadherin/CD31 relative intensity
Cancer Research.
29/07/15 7:28 AM
Harney et al.
RESEARCH ARTICLE
A
MENA/CD68/CD31
TMEM density
(per 10 40× fields)
Macrophage number
(per 10 40× fields)
B/B homodimerizer
400
300
200
100
0
le
c
hi
Ve
CD31/ZO-1/Merge
CD31/VE-Cad/Merge
E
B/B homodimerizer
Vehicle
Dextran area/CD31 area
CD31/Dextran/Merge
TMEM
****
EC
D
C
Total MΦ
Dextran
***
4
3
2
1
0
cle
hi
Ve
B
B/
***
250
200
150
100
50
0
B
B/
le
B
B/
ic
h
Ve
F
CTC
G
**
***
2.0
1.5
1.0
0.5
0.0
cle
hi
Ve
B
B/
H
ZO-1
1.0
0.8
0.6
0.4
0.2
0.0
cle
hi
Ve
B
B/
VE-Cad area/CD31 area
Vehicle
B
T M
ZO-1 area/CD31 area
CD31
Normalized CTC/mL blood
CD68
VE-Cad
*
0.8
0.6
0.4
0.2
0.0
e
icl
h
Ve
B
B/
Figure 4. Macrophage depletion reduces vascular permeability and tumor cell intravasation. A, immunofluorescence imaging of tumor sections stained
for TMEM. Vasculature (CD31; green), tumor cells (MENA; red), macrophages (CD68; gray), and DAPI (blue). TMEM is outlined in a white box. Scale bar,
20 μm. B, quantification of total CD68+ macrophages in tumor tissue (****, P < 0.0001), and (C) in the TMEM (***, P = 0.0003). D, immunofluorescence
imaging of tumor sections stained for vasculature (CD31; green), 155-kDa dextran–TMR (red) and DAPI (blue), ZO-1 (magenta), or VE-Cadherin (magenta)
as indicated, demonstrating changes in vascular permeability by extravascular dextran and vascular junction staining. Scale bar, 50 μm. E, quantification of extravascular 155-kDa dextran–TMR from D (vehicle n = 7, B/B homodimerizer n = 8; ***, P = 0.0009), (F) circulating tumor cells (***, P = 0.0007),
(G) vascular ZO-1 from D (**, P = 0.006), and (H) VE-Cadherin from D (*, P = 0.02).
compared with regions of vasculature away from TMEM
sites in Vegfaflox tumors (Fig. 7A and B). Further, when
VEGFA has been ablated in Vegfaflox;Csf1r-Cre tumors, VECadherin/CD31 relative staining intensity is the same along
the tumor vasculature as in regions away from the TMEM
(Fig. 7C and D). Therefore, vascular junction integrity, as
measured by VE-Cadherin/CD31 relative staining intensity,
is significantly reduced only in regions of vasculature adjacent
to VEGFAhi TMEM macrophages in the TMEM (Fig. 7E).
Further, pericyte coverage of the vasculature is reduced in
regions of VEGFAhi TEMs in the TMEM as compared with
regions away from VEGFAhi TMEM structures (Supplementary Fig. S6F–S6H). A decrease in pericyte coverage of
vasculature has been correlated with increased metastasis
and vascular permeability (25).
To establish the relevance of TIE2hi/VEGFAhi macrophages
in TMEM structures in mediating vascular permeability and
tumor cell dissemination in metastatic breast cancer, vascular junction staining was measured in human breast cancer
patient samples. Staining of sequential sections demonstrates
that blood vessels adjacent to TIE2hi/VEGFAhi macrophages in
the TMEM have significantly reduced vascular VE-Cadherin fluorescence intensity compared with regions of vasculature away
from the TMEM (Fig. 7F and G and Supplementary Fig. S7).
Together, these data establish that the TIE2hi/VEGFAhi
TMEM macrophages interact with endothelial cells through
VEGFA signaling to mediate local, transient blood vessel
permeability, demonstrating the mechanism underlying the
clinically demonstrated association of TMEM density with
metastatic recurrence of breast cancer.
DISCUSSION
Although the abnormality and permeability of tumor vasculature have been well characterized, the mechanism leading
to spatial and temporal heterogeneity in permeability has not
been resolved. The use of high-resolution multiphoton microscopy has allowed for the study of vascular permeability and
tumor cell dissemination in mammary carcinoma at unprecedented spatial and temporal resolution. Our data show that
in the PyMT authochthonous mouse mammary carcinoma
and human patient–derived xenograft TN1 models, vascular permeability is dynamic, localized, and restricted to the
TMEM. These data are consistent with previous findings that
hyperpermeability of tumor vasculature is heterogeneous and
often in the presence of perivascular macrophages (4), but
further explain the observed heterogeneity and that tumor cell
intravasation occurs at sites of vascular permeability.
Cancer Research.
29/07/15 7:28 AM
RESEARCH ARTICLE
A
B
CD68
CD31
MENA
CD31/MENA/CD68
CD31/VEGFA/TIE2
CD31/MENA/CD68
TC
EC
CD31/MENAhi/
CD68, VEGFAhi, TIE2hi
TC
TC
M
M
EC
TC
TIE2
VEGFA
E
E
M
TC
E
M
TC
E
M
M
TC
TC
M
EC
M
D
CD31/VEGFA/TIE2
CD31
Pixel intensity (a.u.)
C
EC
Macrophage
Endothelium
Tumor
60,000
40,000
20,000
0
0
E
50
100
Length (px)
F
F4/80
CD31
VEGFA
MRC1
F4/80/VEGFA/MRC1
TIE2
VEGFA
MRC1
TIE2/VEGFA/MRC1
CD11b
CD68
CD31/CD11b/CD68
CD31
CD11c
CD68
CD31/CD11c/CD68
Figure 5. TIE2hi/VEGFAhi TEMs are present in the TMEM. A, immunofluorescence imaging of the TMEM. Macrophages (CD68; gray), blood vessels
(CD31; green), tumor cells (MENA; red), and DAPI (blue). TMEM in white box (right). Scale bar, 15 μm. B, immunofluorescence imaging of VEGFAhi macrophages in the TMEM in sequential sections. Scale bar, 10 μm. Tumor cell, spotted line; macrophages, solid line; and blood vessels, dashed line. Left, macrophages (CD68; gray), tumor cells (MENA; red), blood vessels (CD31; green), and DAPI (blue). Sequential section (center), VEGFA (red), TIE2 (gray), blood
vessels (CD31; green), and DAPI (blue). Schematic representation (right) of protein expression in the TMEM; tumor cells with MENAhi (red); endothelial
cells CD31 (green); and macrophages CD68, VEGFAhi, and TIE2hi (gray). M, macrophage; TC, tumor cell; and EC, endothelial cell. C, immunofluorescence
images of TIE2, VEGFA, and CD31. Yellow lines, regions of intensity profiling of VEGFA intensity for CD31 (EC), macrophage (M), and tumor cells (TC).
Scale bar, 25 μm. D, fluorescence intensity profile of VEGFA from C of macrophage, endothelial cell, and tumor cells. E, immunofluorescence imaging of
sequential PyMT tumor sections for TEM markers. VEGFAhi TMEM macrophages express F4/80, MRC1, CD11b, and CD68 as indicated by a yellow arrowhead in sequential sections. CD31+ endothelium is indicated by a white arrowhead. F, VEGFAhi TMEM macrophages express TIE2, MRC1, and CD68 but not
CD11c as indicated by a yellow arrowhead. CD31+/TIE2+ endothelium is indicated by a white arrowhead. Scale bar, 25 μm.
The sites of dynamic tumor vascular permeability have
been identified at sites of VEGFAhi perivascular macrophages
at the TMEM. The clinical significance of TMEM density in
predicting metastatic risk has been recently expanded to a
large cohort of patients, further emphasizing the importance
of the TMEM in breast cancer metastasis (11). These data
demonstrate that TIE2hi/VEGFAhi perivascular macrophages
in TMEM share the characteristics of the proangiogenic and
prometastatic TEMs (7). Thus, we have been able to expand
our understanding of the function of this subset of TAMs in
the tumor microenvironment in promoting metastasis.
Mechanistically, macrophage/tumor cell streams migrate
to TMEM sites through the EGFR/CSF1R paracrine loop
(26). Elevated expression of VEGFA in the TIE2hi TMEM
macrophage results in transient permeability of tumor blood
vessels proximal to the TMEM that occurs by disassembling
endothelial cell junctions. The simultaneous attraction of
migratory tumor cells and transient blood vessel permeability
results in a concurrent spike in tumor cell intravasation with
vascular permeability at TMEM sites (Fig. 7H). These data,
together with the clinical association of the TMEM with
distant metastatic tumor recurrence in human breast cancer
patients, explain why TMEM density can predict metastasis
and argue for the development of therapeutic approaches
targeted against both TMEM formation and function.
METHODS
Mice
All studies involving mice were carried out in accordance with the
NIH regulation concerning the care and use of experimental animals
and approved by the Albert Einstein College of Medicine Animal Care
and Use Committee. PyMT (MMTV-PyMT) transgenic mice were bred
in house. MAFIA mice [C57BL/6-Tg(CSF1R-EGFP-NGFR/FKBP1A/
TNFRSF6)2Bck/J] were obtained from The Jackson Laboratory and
were implanted with tumor pieces (2 mm × 2 mm) into the fourth mammary fat pad on the left side. For multiphoton microscopy, transgenic
mice were generated to label the myeloid lineage and mammary tumor
cells by crossing MacBlue mice [Csf1r–GAL4-VP16/UAS-enhanced
cyan fluorescent protein (ECFP); ref. 27] in a C57BL/6 background
with Tg(MMTV-iCre)-Tg(loxP-stop-loxP-PDendra2)jwp (28) mice of FVB
Cancer Research.
29/07/15 7:28 AM
Harney et al.
RESEARCH ARTICLE
3
2
Vegfaflox;Csf1r-Cre
1
Ve
g
fa
flo
x
0
1.5
1.0
0.5
0.0
ZO-1 area/CD31 area
0.4
0.2
0.0
b
C
I
ZO-1
**
0.8
0.6
0.4
0.2
0.0
lA
ro
t
on
C
J
1
1.
4.
20
B
VE-Cad
*
0.5
0.4
0.3
0.2
0.1
0.0
Ve
g
;C fa flox
sf
1r
-C
re
4
CTC
*
0.0
b
.1
l A .1
tro 0-4
n
o
B2
C
H
0.2
0.6
flo
x
Dextran
**
0.4
0.8
fa
G
b
.1
l A .1
tro 0-4
n
o
B2
0.6
CD31/VE-Cad/Merge
0
0.8
VE-Cad
*
Ve
g
CD31/ZO-1/Merge
Ve
g
;C fa fl
o
sf
1r x
-C
re
CD31/Dextran/Merge
1
ZO-1 area/CD31 area
b .1
l A .1
tro 0-4
n
o B2
2
E
ZO-1
**
fa fl Ve
ox g
;C fa flox
sf
1r
-C
re
0
3
CTC
*
Ve
g
2
4
D
Control Ab
B20-4.1.1
6
Dextran
**
C
Vegfaflox
F
C
CD31/VE-Cad/Merge
CD31/ZO-1/Merge
fa fl Ve
ox g
;C fa flox
sf
1r
-C
re
B
CD31/Dextran/Merge
Ve
g
A
Figure 6. Inhibition of VEGFA or macrophage-specific ablation of Vegfa from TIE2hi/VEGFAhi TMEM macrophages reduces vascular permeability
and tumor cell intravasation. A, immunofluorescence imaging of tumor sections after blocking VEGFA with anti-VEGFA blocking antibody (B20-4.1.1).
Tumors are stained for vasculature (CD31; green), 155-kDa dextran–TMR (red) and DAPI (blue), ZO-1 (magenta), or VE-Cadherin (magenta) as indicated,
demonstrating changes in vascular permeability by extravascular dextran and vascular junction staining. Scale bar, 50 μm. B, quantification of extravascular 155-kDa dextran–TMR from A (n = 10; **, P = 0.0015); (C) circulating tumor cells (*, P = 0.0497); (D) vascular ZO-1 from A (**, P = 0.005); and (E)
vascular VE-Cadherin from A (*, P = 0.0463). F, immunofluorescence of tumor sections stained for vasculature (CD31; green), 155-kDa dextran–TMR
(red) and DAPI (blue), ZO-1 (magenta), or VE-Cadherin (magenta) as indicated, demonstrating changes in vascular permeability after ablation of Vegfa by
extravascular dextran and vascular junction staining. Scale bar, 50 μm. G, quantification of extravascular 155-kDa dextran–TMR from F and (Vegfaflox n =
5, Vegfaflox;Csf1r-Cre n = 3; **, P = 0.0029); (H) circulating tumor cells (*, P = 0.0177); (I) vascular ZO-1 from F (**, P = 0.0054); and (J) vascular VE-Cadherin
from F (*, P = 0.0457).
background. The Friend Leukemia Virus, strain B (FVB) macrophagespecific (Csf1r promoter), tamoxifen-inducible Cre-expressing Tg(Csf1rMer-iCre)1jwp transgenic mice were crossed with Vegfaflox/flox mice. Depletion of Vegfa in myeloid cells was induced by daily subcutaneous injection
of 3 mg tamoxifen in corn oil per mouse for 2 days before injection of
155-kDa TMR–dextran (24). TN1 cells were isolated from a pleural effusion sample from a patient with triple-negative breast cancer (ER−/PR−/
HER2−) and transduced to express GFP (29). TN1-GFP patient-derived
cells were only passaged in vivo in NOD.SCID mice (The Jackson Laboratory) by orthotopic injection of 1 × 106 dissociated tumor cells mixed
with 50% Matrigel (BD Biosciences) into the fourth mammary fat pad
on the left side of the mouse. No cell lines were used in this study.
Intravital Imaging
Multiphoton IVM was performed using a skin flap procedure as
previously described (30) with a custom-built 2-laser multiphoton
microscope (28). The animal was placed in a heated chamber maintained at physiologic temperature during the course of imaging and
monitored using MouseOx (Starr Life Sciences). Three milligrams of
155-kDa TMR–dextran or 100 μL of 8 μmol/L Qdots 705 [obtained
from A. Smith, University of Illinois Urbana-Champaign (UIUC) or
Life Technologies Qdots ITK 705; refs. 31–33] was administered via
a tail vein catheter.
Inhibitory Antibodies
Animals were administered 5 mg/kg B20-4.1.1 VEGFA-neutralizing antibody (Genentech) or antibody isotype control by intravenous
injection 6 hours before termination of the experiment.
Macrophage Depletion with B/B Homodimerizer in
MAFIA Mice
Animals were administered 10 mg/kg B/B homodimerizer
(AP20187; Clontech) diluted in 4% ethanol, 10% PEG-400, and 1.7%
Tween-20 or vehicle control by intraperitoneal injection daily for
5 days. Twenty-four hours after the last injection of B/B homodimerizer, animals were administered 3 mg of 155-kDa dextran–TMR for
1 hour and 100 μL of anti-CD31 antibody for 10 minutes.
Labeling of Tumor Vasculature and Extravasation
of 155-kDa Dextran–TMR
One hour before the termination of the experiments with inhibitors
in MAFIA mice, 3 mg of 155-kDa TMR–dextran was administered by
i.v. tail vein to label sites of vascular permeability. In tumor tissue,
several transient permeability events may occur at any given time
due to the spatial and temporal heterogeneity of vascular permeability, thus quantitation of extravascular dextran over the course of
Cancer Research.
29/07/15 7:28 AM
RESEARCH ARTICLE
A
C
At TMEM
1.0
0.5
1.5
1.0
0.5
0.0
5 10 15 20 25
Distance (microns)
Relative intensity
1.5
1.0
0.5
1.0
0.5
5 10 15 20 25
1.5
EM EM EM EM
TM TM TM TM
o
o
N
N
Vegfaflox Vegfaflox;
Csf1r-Cre
1.0
0.5
0.0
5 10 15 20 25
0
0
Distance (microns)
5 10 15 20 25
Distance (microns)
G
VEGFA
TIE2
M
M
EC
M
VE-Cad
M
EC
EC
EC
TC
EC
EC
EC
***
1.5
1.0
0.5
0.0
N
o
EC
VE-Cad
2.0
EM
VE-Cad/VEGFA/TIE2
EM
MENA/CD68/CD31
TM
At TMEM
Away from TMEM
1.5
Distance (microns)
TC
H
Away from TMEM
2.0
0.0
0
**
**
*
TM
0
At TMEM
2.0
Relative intensity
Relative intensity
1.5
2.0
0.0
D
Away from TMEM
2.0
E
Normalized vascular VE-Cadherin
fluorescence intensity
2.0
0.0
F
CD31/VE-Cad/VEGFA
Relative intensity
Vegfa flox
Relative intensity
B
CD31/TIE2/CD68
CD31/VE-Cad/VEGFA
Vegfa flox;Csf1r-Cre
CD31/TIE2/CD68
T3
VEGF
EGF/CSF-1
M2
T3
T2
T2
T1
M1
T1
T1 M1
M1
T2
Figure 7. Macrophage-specific ablation of Vegfa in PyMT implant tumors blocks blood vessel permeability and tumor cell intravasation at the TMEM.
A and C, immunofluorescence of tumor sections stained for the presence of vascular junction proteins at TMEM macrophages. Tumor sections are stained
for VE-cadherin (red), CD31 (green), and VEGFA (gray). Sequential sections are stained for CD31 (green), TIE2 (red), and CD68 (gray). A, control tumors
(Vegfaflox) or (C) after ablation of Vegfa (Vegfaflox;Csf1r-Cre). CD68+ macrophage in the TMEM outlined in white box, and adjacent endothelium in the TMEM
in pink box. Yellow indicates merged signal of CD31 and TIE2 (left) or CD31 and VE-Cadherin (right). Decreased VE-Cadherin at the TMEM (F, right) seen as
decreased VE-Cadherin, resulting in green (CD31) at vascular junction. Scale bar, 15 μm. B and D, quantification of the relative intensity of VEGFA or vascular junction proteins (ratio of VE-cadherin to CD31 in blood vessels) in A and C at TMEM or away from the TMEM in (B) control tumors (Vegfaflox) or (D)
after ablation of Vegfa in Vegfaflox;Csf1r-Cre tumors along 25 μm lengths of blood vessel (n = 3). •, relative fluorescence intensity of VE-Cadherin/CD31;
n, relative VEGFA intensity. Red dashed line, presence of a CD68+ macrophage. E, quantification of average pixel intensity of VE-Cadherin/CD31immuno
fluorescence staining in 25-μm lengths of blood vessel at TMEM or away from the TMEM in the presence of VEGFAhi macrophages (Vegfaflox, n = 3) or after
macrophage-specific ablation of Vegfa (Vegfaflox;Csf1r-Cre, n = 3) from data in B and D. Asterisks indicate post-ANOVA comparisons with significant difference. F, human breast cancer tumor sections stained for the presence of vascular junction proteins at TMEM macrophages. Tumor sections are stained for
TMEM; MENA (red), CD68 (brown), and CD31 (blue), (RBC in aqua) by IHC. Sequential sections are stained for VE-cadherin (green), TIE2 (gray), and VEGFA (red)
by immunofluorescence. TMEM outlined in black box in IHC and white box in immunofluorescence. Scale bar, 15 μm. G, quantification of normalized average
pixel intensity of VE-Cadherin staining in vasculature at TIE2hi/VEGFAhi macrophages of the TMEM or away from the TMEM (n = 23 at the TMEM, n = 24 away
from the TMEM in 5 individual patient samples; ***, P = 0.0001). H, diagram summarizing TMEM macrophage-mediated induction of blood vessel permeability
promotes tumor cell intravasation. TMEM assembly with close association between the nonmigratory TMEM tumor cell (TC; green, T1) and TIE2hi/VEGFAhi
macrophage (blue, M1) on blood vessels. VEGFA destabilizes vascular junctions, resulting in vascular permeability and tumor cell (T2) intravasation.
1 hour will capture these dynamic events. Anti-mouse CD31-biotin
was administered by i.v. tail vein for 10 minutes to label flowing
blood vessels. Tumors were fixed in 4% paraformaldehyde and cryoprotected in 30% sucrose in PBS before freezing in optimal cutting
temperature. Sections (5 μm) of tumors were cut, and immunofluorescence was performed.
CTCs for PyMT
CTCs were isolated from anesthetized mice from blood drawn
from the right ventricle of the heart. Blood burden experiments
obtained by cardiac puncture were used for endpoint experiments to
capture all of the CTCs in the animal blood at the experiment endpoint. Red blood cells were lysed using RBC lysis buffer (multispecies;
Cancer Research.
29/07/15 7:28 AM
Harney et al.
RESEARCH ARTICLE
eBioscience) before the cells were placed in culture with Dulbecco’s
Modified Eagle Medium:Nutrient Mixture F-12 (DMEM/F-12) with
20% FBS. Adherent tumor cells were counted at a time of no tumor
cell growth, which is a count of CTCs. Tumor cells were identified by
fluorescence microscopy as Dendra2- or CFP-expressing tumor cells
as previously described (34, 35). CTCs were also scored by quantifying
tumor cells in blood vessels adjacent to the TMEM as seen in Fig. 2 and
Supplementary Fig. S4 in live animals. This method is described under
IVM image analysis in Supplementary Methods.
Immunofluorescence
Tumor sections were fixed and permeabilized with cold acetone,
washed with PBS, and blocked with block solution (1% BSA, 10% FBS,
and 0.0025% fish skin gelatin in PBS/Tween). The following primary
antibodies were used: rat anti-mouse CD68 (1:200, clone FA-11; Serotec), AlexaFluor647-conjugated CD68 (1:100, eBioscience), mouse antiMENA (1:400, from F. Gertler), rat anti-ZO-1 (1:100, clone R40.76;
Millipore), goat anti–VE-Cadherin (1:200, clone C-19), rabbit anti-VEGFA
(1:200, clone A-20; Santa Cruz Biotechnology), hamster anti-CD11c
(1:100, clone HL-3; BD Bioscience), rat anti-CD11b (1:200, clone ICRF4;
BD Biosystems), rat anti-F4/80 (1:50, clone BM8; eBioscience), goat antiMRC1/CD206 (1:200, R&D Systems), rat anti-TIE2 (1:50, clone TEK4;
eBioscience), and rabbit anti-NG2 (Millipore). Sections were washed with
PBST, and the primary antibodies were detected with AlexaFluor488,
555, or 647 secondary antibody conjugates (Molecular Probes/Invitrogen) and nuclei stained with 4,6-diamidino-2-phenylindole (DAPI). All
fluorescently labeled samples were mounted with Prolong Gold antifade
reagent (Molecular Probes/Invitrogen) and analyzed with a compound
fluorescent microscope (Zeiss Axio Observer; 40× objective with numerical aperture 1.3). Images of tumor sections were acquired using mosaic
tiling of 40× images in AxioRel version 4.8. All images were acquired as
16-bit TIFF images, and all quantitative analyses were performed on the
raw 16-bit TIFF images in ImageJ.
TMEM IHC
Mouse tumor sections were fixed overnight in 10% neutral buffered saline prior to embedding in paraffin. Tumor sections (5 μm)
were deparaffinized and stained for hematoxylin and eosin (H&E)
or TMEM using anti–IBA-1 (macrophages), anti-endomucin (blood
vessels), and anti-MENA (tumor cells; ref. 36), and the TMEM was
quantified as previously described (8).
Human IHC and Immunofluorescence
Formalin-fixed paraffin-embedded patient tissue from 5 invasive ductal carcinomas was collected under the Montefiore-Einstein Institutional
Review Board approval. Paraffin-embedded human breast cancer tumors
were cut into 5-μm sections, deparaffinized, and stained for H&E or
TMEM. The sequence was anti-CD31 (clone JC70A; DAKO) and Vector
Blue chromogen (for endothelial cells); anti CD-68 (clone PG-M1; DAKO)
with and 3′,3′-diaminobenzidine (DAB) chromogen (for macrophages);
and anti–pan-MENA with Fast Red chromogen (for carcinoma cells;
refs. 8, 11). Sequential sections were cut for tyramide signal amplification
(TSA) for quantitative immunofluorescence using the Opal 3-plex Kit
(Perkin Elmer) according to the manufacturer’s directions. The sequence
was rabbit anti-VEGF (1:2,000; Rb 9031-P0-A; Thermo) with TSA Plus
Cy3; rabbit anti-TIE2 (1:3,000; clone C-20; Santa Cruz Biotechnology)
with TSA Plus Cy5; goat anti–VE-cadherin (1:200; clone C-19; Santa Cruz
Biotechnology) with TSA Plus fluorescein and nuclei stained with DAPI.
All quantitative analyses were performed on the raw 16-bit TIFF images,
and images of TMEM were validated independently by a pathologist.
Exponentially Modified Gaussian Function Fitting
Vascular leakage is composed of two competing processes: an
increase in extravascular signal due to leakage from the vasculature
and a diffusive clearance. The Exponentially Modified Gaussian
asymetic function (37) is composed of the product of a sigmoidal
error function with an exponential decay. Least squares curve fitting
was performed utilizing a nonlinear Generalized Reduced Gradient
(GRG2) solver and values were directly compared (38).
Statistical Analysis
Individual animals are presented as individual points, a horizontal
line indicates the mean, and error bars represent the SEM. One-way
or two-way ANOVA analysis was performed for data sets with more
than two groups to determine significance. Statistical significance
was determined by the comparison of the means of two groups using
an unpaired, two-sided t test using Prism (Graph Pad Inc.). Data sets
were checked for normality (D’Agostino & Pearson omnibus normality test or Shapiro–Wilk normality test) and unequal variance using
Prism (Graph Pad Inc.). Welch’s correction was applied to t tests as
needed. P values of less than 0.05 were deemed significant.
Disclosure of Potential Conflicts of Interest
A.S. Harney reports receiving commercial research support from Deciphera Pharmaceuticals, LLC. J.G. Jones has ownership interest (including patents) in MetaStat and is a consultant/advisory board member for
the same. J.S. Condeelis reports receiving a commercial research grant
from Deciphera; has ownership interest (including patents) in MetaStat;
and is a consultant/advisory board member for Deciphera and MetaStat.
No potential conflicts of interest were disclosed by the other authors.
Disclaimer
Views and opinions of, and endorsements by, the authors do not
reflect those of the U.S. Army or the Department of Defense.
Authors’ Contributions
Conception and design: A.S. Harney, D. Entenberg, Y. Wang,
J.W. Pollard, J.S. Condeelis
Development of methodology: A.S. Harney, D. Entenberg, Y. Wang,
J.W. Pollard, J.S. Condeelis
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.S. Harney, E.N. Arwert,
D. Entenberg, Y. Wang, B.-Z. Qian, M.H. Oktay, J.W. Pollard, J.G. Jones,
J.S. Condeelis
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): A.S. Harney, D. Entenberg,
P. Guo, M.H. Oktay, J.W. Pollard, J.G. Jones, J.S. Condeelis
Writing, review, and/or revision of the manuscript: A.S. Harney,
E.N. Arwert, D. Entenberg, J.W. Pollard, J.G. Jones, J.S. Condeelis
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A.S. Harney, D. Entenberg, Y. Wang
Study supervision: J.S. Condeelis
Other (performed experiments and analyzed data): J.G. Jones
Acknowledgments
The authors thank H. Guziak at the Analytical Imaging Facility of
the Albert Einstein College of Medicine for technical assistance. They
also thank A. Smith (UIUC) for Quantum dots and F. Gerlter (MIT)
for the gift of the pan-MENA antibody. Finally, they thank Jiufeng Li
for assistance in generating the MMTV-Dendra mouse.
Grant Support
This research was supported by the Department of Defense Breast
Cancer Research Program under award number W81XWH-13-1-0010
(to A.S. Harney), the Wellcome Trust (WT096084MA, to E.N. Arwert),
NIH CA100324, NIH CA179507, the Integrated Imaging Program, and
the NCI cancer center support grant (P30CA013330).
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
Cancer Research.
29/07/15 7:28 AM
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received January 5, 2015; revised June 8, 2015; accepted June 9,
2015; published OnlineFirst August 12, 2015.
References
1. Yuan F, Dellian M, Fukumura D, Leunig M, Berk DA, Torchilin VP,
et al. Vascular permeability in a human tumor xenograft: molecular
size dependence and cutoff size. Cancer Res 1995;55:3752–6.
2. Gerlowski LE, Jain RK. Microvascular permeability of normal and
neoplastic tissues. Microvasc Res 1986;31:288–305.
3. Huang Y, Goel S, Duda DG, Fukumura D, Jain RK. Vascular normalization as an emerging strategy to enhance cancer immunotherapy.
Cancer Research 2013;73:2943–8.
4. Dvorak HF, Dvorak AM, Manseau EJ, Wiberg L, Churchill WH. Fibrin
gel investment associated with line 1 and line 10 solid tumor growth,
angiogenesis, and fibroplasia in guinea pigs. Role of cellular immunity, myofibroblasts, microvascular damage, and infarction in line 1
tumor regression. J Natl Cancer Inst 1979;62:1459–72.
5. Lin EY, Nguyen AV, Russell RG, Pollard JW. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp
Med 2001;193:727–40.
6. Lin EY, Li JF, Gnatovskiy L, Deng Y, Zhu L, Grzesik DA, et al. Macrophages regulate the angiogenic switch in a mouse model of breast
cancer. Cancer Res 2006;66:11238–46.
7. De Palma M, Venneri MA, Galli R, Sergi LS, Politi LS, Sampaolesi
M, et al. Tie2 identifies a hematopoietic lineage of proangiogenic
monocytes required for tumor vessel formation and a mesenchymal
population of pericyte progenitors. Cancer Cell 2005;8:211–26.
8. Robinson BD, Sica GL, Liu YF, Rohan TE, Gertler FB, Condeelis
JS, et al. Tumor microenvironment of metastasis in human breast
carcinoma: a potential prognostic marker linked to hematogenous
dissemination. Clin Cancer Res 2009;15:2433–41.
9. Roh-Johnson M, Bravo-Cordero JJ, Patsialou A, Sharma VP, Guo P,
Liu H, et al. Macrophage contact induces RhoA GTPase signaling to
trigger tumor cell intravasation. Oncogene 2014;33:4203–12.
10. Wyckoff JB, Wang Y, Lin EY, Li JF, Goswami S, Stanley ER, et al.
Direct visualization of macrophage-assisted tumor cell intravasation
in mammary tumors. Cancer Res 2007;67:2649–56.
11. Rohan TE, Xue X, Lin H-M, D’Alfonso TM, Ginter PS, Oktay MH,
et al. Tumor microenvironment of metastasis and risk of distant
metastasis of breast cancer. J Natl Cancer Inst 2014;106.
12. Monsky WL, Fukumura D, Gohongi T, Ancukiewcz M, Weich HA,
Torchilin VP, et al. Augmentation of transvascular transport of macromolecules and nanoparticles in tumors using vascular endothelial
growth factor. Cancer Res 1999;59:4129–35.
13. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression to malignancy in the polyoma middle T oncoprotein mouse
breast cancer model provides a reliable model for human diseases. Am
J Pathol 2003;163:2113–26.
14. Dreher MR, Liu W, Michelich CR, Dewhirst MW, Yuan F, Chilkoti
A. Tumor vascular permeability, accumulation, and penetration
of macromolecular drug carriers. J Natl Cancer Inst 2006;98:
335–44.
15. Gligorijevic B, Bergman A, Condeelis J. Multiparametric classification
links tumor microenvironments with tumor cell phenotype. PLoS
Biol 2014;12:e1001995.
16. Hashizume H, Baluk P, Morikawa S, McLean JW, Thurston G, Roberge S, et al. Openings between defective endothelial cells explain
tumor vessel leakiness. Am J Pathol 2000;156:1363–80.
17. Leung DW, Cachianes G, Kuang WJ, Goeddel DV, Ferrara N. Vascular
endothelial growth factor is a secreted angiogenic mitogen. Science
1989;246:1306–9.
18. Burnett SH, Kershen EJ, Zhang J, Zeng L, Straley SC, Kaplan AM,
et al. Conditional macrophage ablation in transgenic mice expressing
a Fas-based suicide gene. J Leukoc Biol 2004;75:612–23.
RESEARCH ARTICLE
19. Priceman SJ, Sung JL, Shaposhnik Z, Burton JB, Torres-Collado AX,
Moughon DL, et al. Targeting distinct tumor-infiltrating myeloid
cells by inhibiting CSF-1 receptor: combating tumor evasion of
antiangiogenic therapy. Blood 2010;115:1461–71.
20. Pucci F, Venneri MA, Biziato D, Nonis A, Moi D, Sica A, et al. A
distinguishing gene signature shared by tumor-infiltrating Tie2expressing monocytes, blood “resident” monocytes, and embryonic
macrophages suggests common functions and developmental relationships. Blood 2009;114:901–14.
21. Mazzieri R, Pucci F, Moi D, Zonari E, Ranghetti A, Berti A, et al.
Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis
by impairing angiogenesis and disabling rebounds of proangiogenic
myeloid cells. Cancer Cell 2011;19:512–26.
22. De Palma M, Mazzieri R, Politi LS, Pucci F, Zonari E, Sitia G, et al.
Tumor-targeted interferon-alpha delivery by Tie2-expressing monocytes inhibits tumor growth and metastasis. Cancer Cell 2008;14:
299–311.
23. Nakayama M, Berger P. Coordination of VEGF receptor trafficking
and signaling by coreceptors. Exp Cell Res 2013;319:1340–7.
24. Qian BZ, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, et al.
CCL2 recruits inflammatory monocytes to facilitate breast-tumour
metastasis. Nature 2011;475:222–5.
25. Cooke VG, LeBleu VS, Keskin D, Khan Z, O’Connell JT, Teng Y, et al.
Pericyte depletion results in hypoxia-associated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway.
Cancer Cell 2012;21:66–81.
26. Wyckoff J, Wang W, Lin EY, Wang Y, Pixley F, Stanley ER, et al. A paracrine loop between tumor cells and macrophages is required for tumor
cell migration in mammary tumors. Cancer Res 2004;64:7022–9.
27. Ovchinnikov DA, van Zuylen WJM, DeBats CEE, Alexander KA, Kellie S, Hume DA. Expression of Gal4-dependent transgenes in cells of
the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic
mice. J Leuk Biol 2008;83:430–3.
28. Entenberg D, Wyckoff J, Gligorijevic B, Roussos ET, Verkhusha VV,
Pollard JW, et al. Setup and use of a two-laser multiphoton microscope for multichannel intravital fluorescence imaging. Nat Protoc
2011;6:1500–20.
29. Liu H, Patel MR, Prescher JA, Patsialou A, Qian D, Lin J, et al. Cancer
stem cells from human breast tumors are involved in spontaneous
metastases in orthotopic mouse models. Proc Natl Acad Sci U S A
2010;107:18115–20.
30. Wyckoff J, Gligorijevic B, Entenberg D, Segall J, Condeelis J. Highresolution multiphoton imaging of tumors in vivo. Cold Spring Harb
Protoc 2011;2011:pdb.top065904.
31. Smith AM, Nie S. Compact quantum dots for single-molecule imaging. J Vis Exp 2012:e4236.
32. Smith AM, Nie S. Bright and compact alloyed quantum dots with
broadly tunable near-infrared absorption and fluorescence spectra
through mercury cation exchange. J Am Chem Soc 2011;133:24–6.
33. Smith AM, Nie S. Minimizing the hydrodynamic size of quantum
dots with multifunctional multidentate polymer ligands. J Am Chem
Soc 2008;130:11278–9.
34. Wyckoff JB, Jones JG, Condeelis JS, Segall JE. A critical step in metastasis: in vivo analysis of intravasation at the primary tumor. Cancer
Res 2000;60:2504–11.
35. Roussos ET, Wang Y, Wyckoff JB, Sellers RS, Wang W, Li J, et al.
Mena deficiency delays tumor progression and decreases metastasis
in polyoma middle-T transgenic mouse mammary tumors. Breast
Cancer Res 2010;12:R101.
36. Lebrand C, Dent EW, Strasser GA, Lanier LM, Krause M, Svitkina
TM, et al. Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron
2004;42:37–49.
37. Foley JP, Dorsey JG. A review of the Exponentially Modified Gaussian
(EMG) function: evaluation and subsequent calculation of universal
data. J Chromatogr Sci 1984;22:40–6.
38. Fylstra D, Lasdon L, Watson J, Waren A. Design and use of the Microsoft Excel Solver. Interfaces 1998;28:29–55.
Cancer Research.
29/07/15 7:28 AM
Real-Time Imaging Reveals Local, Transient Vascular
Permeability, and Tumor Cell Intravasation Stimulated by
TIE2hi Macrophage−Derived VEGFA
Allison S. Harney, Esther N. Arwert, David Entenberg, et al.
Cancer Discovery Published OnlineFirst August 12, 2015.
Updated version
Supplementary
Material
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
doi:10.1158/2159-8290.CD-15-0012
Access the most recent supplemental material at:
http://cancerdiscovery.aacrjournals.org/content/suppl/2015/07/24/2159-8290.CD-15-0012.DC
1.html
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Cancer Research.

Real-Time Imaging Reveals Local, Transient

Transcription

Similar documents

we removed a tumor from her heart. her kids filled it

Don Listwin Canary Foundation don@canaryfoundation

Exsanguination - Northern Health

AMPULLA OF VATER STAGING FORM

Cervix Uteri Cancer Staging - American Joint Committee on Cancer

/ Print JoH_Poster_2016 - Fox Valley Brain Tumor Coalition

Men`s VasoPlex-PE - biometrixlife.com

angionesesis emt metastasis

Lung Cancer Staging - American Joint Committee on Cancer

Innovative Strategy in Nanomedicine for Targeted Cancer Therapy