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Integrin-Mediated Macrophage Adhesion Promotes Lymphovascular Dissemination in Breast Cancer



Lymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in β4 integrin-dependent adhesion to the lymphovasculature. β4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-β1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-β1 drives β4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-β1 signaling in this context. β4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-β signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between β4 integrin and TGF-β1.
Integrin-Mediated Macrophage Adhesion Promotes
Lymphovascular Dissemination in Breast Cancer
Graphical Abstract
db4 integrin-expressing macrophages release TGF-b1 near
breast cancer lymphovasculature
dTGF-b1 drives b4 integrin clustering on macrophages,
enhancing macrophage adhesion
dTGF-b1 signals through RhoA to drive to lymphatic
endothelial cell contraction
dLymphatic remodeling signaling cascade facilitates breast
cancer metastasis
Rachel Evans, Fabian Flores-Borja,
Sina Nassiri, ..., Frederic Festy,
Michele De Palma, Tony Ng
Correspondence (R.E.), (T.N.)
In Brief
Breast cancer metastasis through
lymphatic vessels is associated with poor
prognosis. Evans et al. describe b4
integrin-expressing macrophages that
regulate lymphatic vessel structure in
breast cancer. Macrophage-released
TGF-b1 drives lymphatic cell contraction
via RhoA activation, culminating in
lymphatic hyperpermeability. This study
defines a signaling cascade that could be
targeted therapeutically.
Evans et al., 2019, Cell Reports 27, 1967–1978
May 14, 2019 ª2019 The Authors.
Cell Reports
Integrin-Mediated Macrophage Adhesion Promotes
Lymphovascular Dissemination in Breast Cancer
Rachel Evans,
*Fabian Flores-Borja,
Sina Nassiri,
Elena Miranda,
Katherine Lawler,
Anita Grigoriadis,
James Monypenny,
Cheryl Gillet,
Julie Owen,
Peter Gordon,
Victoria Male,
Anthony Cheung,
Farzana Noor,
Paul Barber,
Rebecca Marlow,
Erika Francesch-Domenech,
Gilbert Fruhwirth,
Mario Squadrito,
Borivoj Vojnovic,
Andrew Tutt,
Frederic Festy,
Michele De Palma,
and Tony Ng
Richard Dimbleby Department of Cancer Research, Randall Division & Division of Cancer Studies, Kings College London, London, UK
Breast Cancer Now Research Unit, King’s College London, Guy’s Hospital, London, UK
Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, E
´cole Polytechnique Fe
´rale de Lausanne (EPFL),
Lausanne, Switzerland
Pathology Core Facility, University College London Cancer Institute, London, UK
Institute for Mathematical and Molecular Biomedicine, King’s College London, London, UK
King’s Health Partners Cancer Biobank, King’s College London, London, UK
Research Oncology, Division of Cancer Studies, Guy’s Hospital, King’s College London, London, UK
Division of Imaging Sciences and Biomedical Engineering, King’s College London, London, UK
Department of Oncology, Cancer Research UK and Medical Research Council, Oxford Institute for Radiation Oncology,
University of Oxford, UK
Tissue Engineering and Biophotonics, King’s College London, London, UK
UCL Cancer Institute, University College London, London, UK
Present address: Cancer Institute, University College London, London, UK
Present address: Centre for Immunobiology and Regenerative Medicine, Barts and The London School of Medicine and Dentistry, Queen
Mary University of London, London, UK
Present address: Division of Infection and Immunity, Institute of Immunity and Transplantation, University College London, London, UK
Lead Contact
*Correspondence: (R.E.), (T.N.)
Lymphatic vasculature is crucial for metastasis in tri-
ple-negative breast cancer (TNBC); however, cellular
and molecular drivers controlling lymphovascular
metastasis are poorly understood. We define a
macrophage-dependent signaling cascade that fa-
cilitates metastasis through lymphovascular remod-
eling. TNBC cells instigate mRNA changes in macro-
phages, resulting in b4 integrin-dependent adhesion
to the lymphovasculature. b4 integrin retains macro-
phages proximal to lymphatic endothelial cells
(LECs), where release of TGF-b1 drives LEC contrac-
tion via RhoA activation. Macrophages promote
gross architectural changes to lymphovasculature
by increasing dilation, hyperpermeability, and disor-
ganization. TGF-b1 drives b4 integrin clustering at
the macrophage plasma membrane, further promot-
ing macrophage adhesion and demonstrating the
dual functionality of TGF-b1 signaling in this context.
b4 integrin-expressing macrophages were identified
in human breast tumors, and a combination of
vascular-remodeling macrophage gene signature
and TGF-bsignaling scores correlates with metas-
tasis. We postulate that future clinical strategies for
patients with TNBC should target crosstalk between
b4 integrin and TGF-b1.
Tumor cells establish complex interactions with cells within their
microenvironment that determine malignancy progression (Balk-
will et al., 2012). Tumor cell dissemination can occur through
blood or lymphovasculature; however, targeting blood vascula-
ture has limited clinical efficacy when lymphatic dissemination
is prevalent (Wong and Hynes, 2006).
Breast cancer is divided into subtypes based on histopatho-
logical features and gene signatures (Gazinska et al., 2013).Tri-
ple-negative breast cancer (TNBC) is characterized by a lack
of druggable targets, is highly metastatic, and is associated
with dismal prognosis (Gazinska et al., 2013; Dent et al., 2007).
The prognostic significance of lymphangiogenesis in TNBC is
under debate. However, invasion into lymphatic vessels corre-
lates with poor prognosis, suggesting that targeting an existing
lymphatic vessel network could provide an effective treatment
strategy (Choi et al., 2005; Mohammed et al., 2007, 2011; Liu
et al., 2009).
The relationship between tumor and immune cells is often bidi-
rectional and involves both tumor-promoting and -antagonizing
mechanisms (Pollard, 2004; Quail and Joyce, 2013). Among
innate immune cells, macrophages have been implicated in the
promotion of tumor progression and, in particular, breast cancer
metastasis (Condeelis and Pollard, 2006; Kitamura et al., 2015;
Pollard, 2004; Harney et al., 2015). However, it remains unclear
how certain subsets of tumor-associated macrophages (TAMs)
influence breast cancer metastasis spatially, temporally, and at
a molecular level.
Cell Reports 27, 1967–1978, May 14, 2019 ª2019 The Authors. 1967
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1968 Cell Reports 27, 1967–1978, May 14, 2019
The integrin family are adhesion receptors of paramount impor-
tance for immunecell adhesion and migration during inflammatory
processes (Evans et al., 2009). Their ability to form adhesive con-
tacts is regulated by soluble factors, as part of the chemoattrac-
tant-adhesion crosstalk that causes a combination of changes
in integrin conformation and clustering on the plasma membrane
(PM) that regulate downstream signaling (Hynes, 2002). In malig-
nancy, many integrins common in epithelial cells are also present
in solid tumors, and some, such as avb3anda5b1, are specifically
upregulated in cancer (Desgrosellier and Cheresh, 2010). Tumor-
expressed integrins affect tumor cell migration, proliferation,
survival, and anchorage to the extracellular matrix. Endothelial-
cell-expressed integrins are implicated in angiogenesis, lymphan-
giogenesis, and vascular remodeling (Avraamides et al., 2008).
While the importance of integrins with respect to maintaining a
pro-tumoral immune microenvironment in solid tumors is not
well defined, in chronic lymphocytic leukemia, impaired integrin
signaling in non-leukemic T cells changes the immune microenvi-
ronment to be more immunosuppressive, which may facilitate
malignancy progression (Ramsay et al., 2013).
We seek to identify the role of TAMs in regulating existing lym-
phovasculature in TNBC, where lymphatic dissemination is not a
direct result of lymphangiogenesis.
We propose that macrophages have an important role in
controlling established tumoral lymphatic networks in TNBC
and that lymphatic dissemination of cancer cells is facilitated
by a cascade of signaling events initiated by integrin-mediated
adhesion of macrophages at the sites of lymphatic vessels.
Lymphovascular Macrophages in TNBC Mouse Models
Are Retained through Binding of b4 Integrin to Laminin-5
To identify endogenous macrophages with respect to lymphatic
vasculature in murine TNBC tumors, we scored F4/80+Tie2+
macrophages within podoplanin+ lymphovasculature across mul-
tiple fields of view (FOVs) from 4T1.2 and BLG-Cre;Brca1
TNBC models (Molyneux et al., 2010; Melchor et al., 2014;Fig-
ures 1A and 1B). The Tie2-expressing macrophage (TEM) subset
is associated with angiogenesis and lymphatic development
(De Palma et al., 2005, 2007; Gordon et al., 2010). Lymphovascu-
lar-associated macrophages expressing Tie2 have recently been
reported in a small breast cancer cohort (Bron et al., 2015).
In 4T1.2 tumors, we found a mean value of 6.3 F4/80+Tie2+
macrophages within podoplanin+ vasculature (versus 1.7 in
podoplaninregions) per FOV. In BLG-Cre;Brca1
mors, we observed 8.8 F4/80+Tie2+ macrophages in podopla-
nin+ vasculature (versus 2.0 in podoplaninregions) per FOV.
Therefore, F4/80+Tie2+ macrophages are enriched in lymphovas-
cular regions in murine TNBC models.
The b4 integrin subunit is a transmembrane glycoprotein asso-
ciating exclusively with the a6 integrin subunit. a6b4 integrin is
expressed predominantly on epithelial and endothelial cells
and binds to laminins to form adhesion complexes, hemidesmo-
somes (Stewart and O’Connor, 2015). Microarray analysis of
endogenous macrophages co-cultured with 4T1.2 tumor cells
showed a mean 1.8-fold upregulation of b4 integrin at the RNA
level, compared with non-educated endogenous macrophages,
and that the RAW264.7 macrophage cell line similarly exhibited a
mean 1.58-fold increase in b4 integrin levels, compared with
endogenous macrophages (Figure 1C; see also data published
in ArrayExpress: MTAB-4064).
4T1.2 tumors were excised and disaggregated at day 10.
Within 4T1.2 tumors, we defined a population of macrophages
as CD45
b4 integrin
(Figure 1D).
The influence of tumor education on macrophage adhesion to
b4 integrin ligand, laminin-5, was investigated. Tumor-educated
endogenous macrophages displayed increased adhesion to lam-
inin-5 (30.7% ±7.2% to 81.7% ±13.2% adherent cells on 0.5 mM
laminin-5; Figure 1E). As laminin-5 is reportedly localized in areas
with high blood vessel density, we investigated whether laminin-
5 was also in areas of lymphovasculature. 4T1.2 tumor tissue
analysis showed laminin-5 furnished around podoplanin+ lympho-
vasculature (Figure 1F). In addition we observed macrophages
expressing a6b4integrininlymphovascularregions(Figure 1G).
To study b4 expression in vivo, we used primary 4T1.2 tumor
sections stained with Lyve1-Cy3 and b4 integrin-Cy5. Tissues
were imaged using a protocol involving laser photobleaching
to remove autofluorescence. Our methodology reveals b4 integ-
rin throughout the tumor; however, within lymphatic vessels,
there is differential distribution of b4 integrin with relative in-
creases in b4 accumulation observed in lymphovascular areas
proximal to Lyve1+ lymphatic endothelial cells (LECs) (Figure 1H,
white arrow). Additionally, there were lymphovascular areas with
an increased localized Pearson coefficient, suggesting that
LECs and b4-integrin-expressing macrophages were in close
contact (Figure 1H, blue arrow) (mean colocalization coefficient,
4.094 ±0.8146).
Figure 1. Lymphovascular Macrophages in TNBC Mouse Models Are Retained through Binding of b4 Integrin to Laminin-5
(A and B) Tumor sections from 4T1.2 (A) and BLG-Cre;Brca1
(B) were stained with F4/80-FITC, podoplanin-AF555, and Tie2 -Cy5-conjugated ant ibody.
F4/80+Tie2+ macrophages within podoplanin+ areas versus those in other areas were quantified per field of view (FOV). Vessel lumen is outlined; arrow indicates
a macrophage within a podoplanin+ area. Images were acquired with a 340 air objective. Scale bars, 100 mm (main image) and 25 mm (zoomed inset).
(C) Array-derived expression profile of b4-integrin (Itgb4) across samples. Barplot shows log
fold change of normalized expression value for b4 integrin (ratio of
the median value of probe in BMM samples).
(D) Day-12 4T1.2 tumors were disaggregated. Tie2 and b4 integrin FMO controls are indicated in 2 left panels. Right dot plot and histogram depict b4-integrin-
expressing macrophages from representative 4T1.2 tumor (n = 8).
(E) BMMs co-cultured alone or with 4T1.2-GFP cells plated on laminin-5. The percentage of adherent cells were quantifi ed in triplicate (n = 2).
(F and G) 4T1.2 tumor sections were stained with laminin-5-Dylight488 and podoplanin-AF555 (F), and Lyve1-Cy3, F4/80-FITC, and b4 integrin-Cy5 (G); inset
shows F4/80+b4 integrin+ macrophages around lymphatic endothelium.
(H) Stained sections (Lyve1-Cy3 and b4 integrin-Cy5) were imaged using a custom-built microscope (320 air objective). Area of distinct b4 integrin and Lyve1
within lymphatic vessel (white arrow) and area of close contact between b4 integrin and Lyve1 (blue arrow) are indicated. Scale bars, 50 mm (main panels) and
25 mm (inset).
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TAMs Drive Disorganized and Hyperpermeable
Lymphatic Architecture, and Contact between
Macrophages and LECs Results in RhoA-Dependent
We used a mammary image window (MIW) subcutaneously im-
planted over a 4T1.2-mCherry tumor (Kedrin et al., 2008;
Figure 2A). Injection of 76 kDa dextran-FITC (fluorescein isothio-
cyanate) allowed visualization of lymphatic vasculature. Using
multiphoton microscopy, we observed that, within the tumor,
lymphatic vessels leaked dextran dye across the FOV (Fig-
ure 2Aii, left panel), suggesting high levels of vessel permeability;
however, in more distal regions, lymphatic vessels had a distinct
structure and 4T1.2-mCherry intra-lymphatic tumor cells could
be seen within vessels, suggesting ongoing metastasis (Fig-
ure 2Aii, middle and right panels, respectively). To understand
how increasing TAMs could phenotypically influence lymphatic
vasculature, we studied the permeability of lymphatic vessels
from 4T1.2 tumor-bearing mice given an intermittent bolus of
RAW264.7 macrophages during tumor development. Both
RAW264.7 macrophages and the 4T1.2 tumor line are derived
from a BALB/c genetic background, allowing us to investigate
the effects of elevated macrophage numbers on tumor progres-
sion in vivo using a syngeneic model of TNBC.
To quantify lymphatic vessel permeability in vivo,weadapteda
protocol previously used in angiogenesis studies (Finsterbusch
et al., 2014). Using a subcutaneous injection of Evans Blue dye,
we quantified the permeability of the tumoral lymphatics. Tumors
with elevated macrophages contained hyperpermeable lymphatic
vessels with an increase in mean optical density (OD) per gram
from 0.7812 ±0.2956 to 2.290 ±0.5160 when compared with
PBS-treated control, suggesting a facilitated pathway between
the primary tumor and lymphatic vasculature (Figure 2B).
To understand the effects of elevated macrophages on tu-
moral lymphatic vessel architecture, we stained tumor sections
from mice treated with PBS or RAW264.7 macrophages with
the lymphatic vessel markers, Lyve1 and podoplanin (Figure 2C;
Figures S1A and S1B), demonstrating that both lymphatic
markers gave a similar staining distribution. Typical sections
from PBS-treated mice showed small, well-formed vessels to-
ward the tumor periphery or within the peri-tumoral areas with
a mean diameter of 13.66 mm±1.295 mm. This was in contrast
to RAW264.7-treated mice that had larger vessels with a mean
diameter of 48.00 mm±6.065 mm, indicating increased vessel
dilation (Figure S1C).
To quantify changes in lymphatic architecture in tumors with
elevated levels of macrophages, we blindly scored lymphovas-
culature for disorganization based on the following criteria.
Smaller vessels with a clear lumen were given low scores
(0 and 1) compared with larger disorganized vessels with unclear
borders (2 and 3). PBS-treated tumors had a mean disorganiza-
tion score of 0.25 ±0.16 and 1.6 ±0.33, compared with 1.8 ±
0.29 and 2.5 ±0.17 for tumors treated with RAW264.7 macro-
phages (Figure 2C).
To further investigate whether macrophages were sufficient to
induce a disorganized lymphatic phenotype, we ablated endoge-
nous macrophages using clodronate-containing liposomes post-
establishment of 4T1.2 tumors. Endogenous macrophages were
reconstituted post-clodronate treatment with non-educated
bone marrow macrophages (BMMs) or tumor-educated BMMs
for 48 h (Figure 2Di). The extent of lymphatic disorganization in
the 4T1.2 primary tumors was greater after reconstitution with
endogenous tumor-educated BMMs, compared with non-
educated BMMs (0.333 ±0.3 to 2 ±0.29; Figure 2D, ii and iii).
These results demonstrate that the presence of TAMs results in
a disorganized lymphatic vasculature around the primary tumor,
that the extent of disorganization is related to overall macrophage
levels, and that this occurs at an early time point in tumor develop-
ment (days 10–14).
To investigate how TAMs affect lymphatic endothelia, we
added endogenous macrophages to monolayers of primary
LECs isolated from BALB/c mice (Figure 2E). Primary LECs
had a mean spread area of 1,132 mm
±247.9 mm
reduced slightly to 808.6 mm
±185.9 mm
after the addition of
endogenous uneducated macrophages but dramatically
reduced to 324.1 mm
±76.43 mm
with tumor-educated
macrophages and 473.7 mm
±92.8 mm
with ex vivo TAMs
). Similar LEC contraction occurred
when the murine LEC line, SV-LEC (Ando et al., 2005), was grown
as a monolayer and endogenous macrophages (Figure S1D) or
RAW264.7 macrophages added (Figure 2Fi). SV-LEC contraction
Figure 2. TAMs Drive Dilated, Hyperpermeable, and Disorganized Lymphatic Architecture through LEC RhoA Activation
(A) (i) Mouse with mCherry-tagged 4T1.2 tumor and implanted mammary imaging window (MIW) at days 10–14. (ii) Left panel: lymphatic vessels (green) sur-
rounding tumor (red). Middle panel: lymphatic vessels (green) distal to main tumor bulk (red). Right panel: lymphatic vessel (green) with tumor cells (red) within
vessel. Scale bars, 100 mm.
(B) 4T1.2 tumor-bearing mice were treated with PBS or RAW264.7 macrophages over 3 weeks. 1% Evans Blue dye stained lymphatics in vivo. Lymphatic
permeability was calculated as optical density per gram of tumor. Data represent means ±SEM; significance was determined using unpaired t tests (**p < 0.01).
(C) (i) Lymphatic vessels within tumors from mice treated with PBS or RAW264.7 macrophages stained with Lyve1-Cy3 or podoplanin-AF555 (red) and blindly
scored for disorganization. Scale bars, 50 mm. (ii) Four FOVs in 4 PBS-treated and 4 RAW264.7 macrophage-treated tumor samples scored blindly for disor-
ganization. Data represent means ±SD; significance was determined using unpaired t tests (***p < 0.001).
(D) (i) Timeline depicting clodronate-containing liposome protocol. (ii) Tumor sections from clodronate-treated mice reconstituted with PBS, BMM, or BMM
stained with Lyve1-Cy3 or podoplanin-AF555 (red). Lymphatic disorganization within tumors from 6 mice was quantified from >3 FOVs per mouse from Lyve1-
stained sections. Data represent means ±SD; significance was determined using unpaired t tests (**p < 0.01).
(E) Primary LECs were cultured alone, with BMM, eBMM, or TAM. LECs were stained with podoplanin-AF555, and macrophages were stained with F4/80-FITC.
Confocal microscopy (x40 air objective) was used to quantify the area of LECs from 3 FOVs (n = 2). Scale bar, 10 mm.
(F) (i and ii) Monolayer of SV-LECs (CellTracker Green CMFDA) with RAW264.7 macrophages (CellTracker Orange CMTMR) after 24 h. Area of SV-LECs was
measured using ImageJ software. Data represent means ±SEM; significance was determined using unpaired t tests (**p < 0.01). Scale bars, 25 mm.
(G) (i) SV-LECs transfected with RhoA RAICHU biosensor (RAICHU R/G) or RhoA-GFP as a control. Transfected SV-LECs were cultured alone or with BMM or
eBMM for 24 h. (ii) Multiphoton microscopy was used to determine the fluorescence lifetim e decay (Tau; in nanoseconds) of SV-LECs transfected with RhoA-GFP
or RhoA RAICHU biosensor. Data represent means ±SD; significance was determined using unpaired t tests (**p < 0.01). N.S., not significant.
Cell Reports 27, 1967–1978, May 14, 2019 1971
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1972 Cell Reports 27, 1967–1978, May 14, 2019
occurred with areas reducing from 835.9 mm
±72.32 mm
380.5 mm
±40.82 mm
and from 632.5 mm
±83.0 mm
82.67 mm
±14.38 mm
. In addition, the area of SV-LECs was
quantified with and without contact with RAW264.7 macro-
phages. SV-LEC contraction was only observed when direct con-
tact between the 2 cell types occurred (436.4 mm
±63.3 mm
116.2 mm
±34.6 mm
)(Figure 2Fii). Collectively, our evidence sug-
gests that direct contact between TAMs and LECs is required for
contraction events to occur.
RhoA regulates many events in blood-vessel-specific endo-
thelial cells during angiogenesis, such as motility, proliferation,
and permeability (Bryan et al., 2010). We sought to test whether
RhoA regulates contraction events observed in LECs. SV-LECs
were transiently transfected with the GFP- and monomeric red
fluorescent protein (mRFP)-expressing RhoA RAICHU biosensor
(Heasman et al., 2010; Makrogianneli et al., 2009; Yoshizaki
et al., 2003), which allows measurement of the fluorescent
lifetime decay (Tau) when fluorescence resonance energy trans-
fer (FRET) occurs between the GFP and mRFP upon RhoA acti-
vation. After SV-LEC transfection, non-educated or tumor-
educated endogenous macrophages were added to SV-LECs
for 24 h. The fluorescence lifetime of the RAICHU probe
(expressed exclusively in the SV-LECs) was measured using
multiphoton microscopy. SV-LEC co-culture with tumor-
educated macrophages led to a reduction in Tau of the
biosensor from 1.797 ns ±0.0252 ns to 1.622 ns ±0.0338 ns,
indicating an increase in FRET between the GFP- and
RFP-terminal fluorophores and, consequently, an increase in
RhoA activity (Figure 2G). No change in Tau was observed
when SV-LECs were co-cultured with non-educated endoge-
nous macrophages (Figure 2Gii). These results demonstrate
that RhoA activity increases during LEC contraction and that
this only occurs in the presence of tumor-educated macro-
phages in contact with lymphatic endothelia.
LEC Contraction Is Dependent on TGF-b1 Release from
Tumor-Educated Macrophages
Transforming growth factor (TGF)-breceptor ligation in fibro-
blasts results in RhoA activation (Fleming et al., 2009). We inves-
tigated the release of active TGF-b1 and TGF-b2 isoforms from
non-educated and tumor-educated macrophages by ELISA
(Figure 3A). TGF-b1 levels increased from 2,600 pg to 4,400 pg
in tumor-educated endogenous macrophages (increase in opti-
cal absorbance at 450 nm from 1.286 ±0.07119 to 2.585 ±
0.1077). In contrast, TGF-b2 levels were not significantly
changed. While TGF-bis present throughout the tumor microen-
vironment, membrane-bound TGF-bcan have a potent effect on
downstream signaling through increasing the concentration
gradient of this molecule (Savage et al., 2008). Our data showed
that 4T1.2 education of endogenous macrophages significantly
increased the levels of plasma-membrane-bound TGF-b1(Fig-
ure S2A), allowing stringent spatial control of downstream
signaling events.
To test the hypothesis that macrophage-released TGF-b1 was
responsible for LEC contraction, we investigated the effect of a
TGF-breceptor inhibitor, SB-431542 (Inman et al., 2002;Fig-
ure S2B). As expected, RAW264.7 macrophages alone induced
LEC contraction (950.6 mm
±129.9 mm
to 335.8 mm
38.23 mm
); however, this did not occur in the presence of
SB-431542 or when TGF-b1orb4 integrin were transiently
knocked down in RAW264.7 macrophages, demonstrating that
the presence of b4 integrin and TGF-bin macrophages or
TGF-breceptor ligation on LECs was sufficient to prevent
contraction (Figures 3B, S2C, and S2D).
The role of macrophage-released TGF-b1 on lymphovascular
disorganization was investigated in vivo. A stable knockdown of
TGF-b1 was generated in RAW264.7 macrophages using lentivi-
ral short hairpin RNA (shRNA) (Figure S2E). Similar to our previ-
ous in vivo studies, macrophages were administered intrave-
nously throughout tumor development. After 2 weeks’ growth,
tissue sections were stained for Lyve1 and podoplanin. The
extent of lymphatic disorganization in tumors with RAW264.7-
TGFb1 knockdown, compared with that in RAW264.7-NTC,
was blindly scored in Lyve1-podoplanin-stained tissues as
described earlier. Our results show that absence of TGF-b1in
RAW264.7 macrophages was sufficient to significantly decrease
the extent of lymphatic disorganization observed, compared
with that in RAW264.7-NTC macrophages (1.8 ±0.16 to 1.1 ±
0.18) (Figure 3C) and that these changes were evident at an early
time point.
To functionally associate macrophage-released TGF-b1to
structural changes in the lymphatic endothelium in vivo,we
Figure 3. Macrophage-Expressed TGF-b1 Regulates b4 Integrin Clustering on the Macrophage Plasma Membrane and Is Required for LEC
(A) BMMs cultured alone (BMM) or with 4T1.2 cells (BMM coculture). Supernatants were probed for (i) TGFb1 and (ii) TGF-b2 by ELISA. Data represent
means ±SD; significance was determined using unpaired t tests (***p < 0.001). N.S., not significant.
(B) SV-LECs grown as monolayers. Tumor-educated RAW264.7 macrophages (eRAW) were added plus DMSO control or 10 mM SB-431542. After 24 h, SV-LEC
areas were quantified. Data represent means ±SD; significance was determined using unpaired t tests (****p < 0.0001). N.S., not significant.
(C) Tumor-bearing mice were injected with RAW264.7-NTC or RAW264.7-TGFb1 knockdown until day 14. Tumor sections were stained with podoplainin-AF555
or Lyve1-Cy3 and Lyve1+ vessels blindly scored for lymphatic disorganization (*p < 0.05). Scale bars, 50 mm.
(D) Tumor-bearing mice were injected with RAW264.7-NTC or RAW264.7-TGFb1 un til day 21. Tumor sections were stained with F4/80-FITC, pMLC (and Rabbit-
Cy3 secondary antibody), and podoplanin-Cy5. F4/80+ cells within podoplanin+ regions were identified, and a 65-mm
region of interest (ROI) was identified
(white circles) where the fluorescence intensity of the pMLC signal was quantified. Scale bar, 50 mm (4 FOVs from n = 2 tumors from each condition). Data
represent means ±SD; significance was determined using unpaired t tests (**p < 0.01).
(E) RAW264.7-NTC and RAW264.7-TGFb1 macrophage areas were measured by confocal microscopy. Data represent means ±SD; significance was deter-
mined using unpaired t tests (**p < 0.01).
(F) RAW264.7-NTC and RAW264.7-TGFb1 were stained with anti-b4 integrin-AF647 and imaged using structured illumination microscopy (Nikon 3100 oil
objective). Focal adhesion area was determined using ImageJ on thresholded image s. Data represent means ±SD; significance was determined using unpaired
t tests (**p < 0.01). Scale bars, 10 mm (main image) and 1 mm (insets).
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1974 Cell Reports 27, 1967–1978, May 14, 2019
quantified levels of phospho-myosin light chain (pMLC) in LECs
adjacent to macrophages. Since RhoA activity is high in con-
tracting LECs, and since active RhoA phosphorylates MLC,
pMLC can be used as a readout of LEC contractility in cells
proximal to lymphatic-associated macrophages. We observed
that, when mice were injected with RAW264.7-TGFb1 knock-
down, compared with RAW264.7-NTC, there was a significant
reduction in pMLC levels in lymphatic vasculature adjacent to
RAW264.7 macrophages when TGF-b1 was absent (1.97 3
±401,151 to 6.56 310
3187,133) (Figure 3D).
TGF-b1 Controls b4 Clustering at the Macrophage
Plasma Membrane
We studied the effect of TGF-b1 on the phenotypic functionality of
macrophages by quantifying the spreading response of macro-
phages. There was clear reduction in cell spreading when
TGF-b1 was knocked down in RAW264.7 macrophages,
compared with the non-targeted control counterpart (235.2 mm
±41.06 mm
to 91.91 mm
±11.62 mm
)(Figure 3E). To understand
how TGF-b1 could control macrophage spreading, we investi-
gated the effect of TGF-b1onb4 expression. Since integrins can
be constitutively expressedon the cell surface, we sought to study
the plasma membrane distribution of b4 integrin using structured
illumination microscopy in RAW264.7-TGFb1 shRNA versus
RAW264.7-NTC. Our results clearly show that, while there may
be small differences in the overall amountof b4 integrin expressed
on the cell surface (Figures S3A and S3B), the size of integrin clus-
ters that can form firm adhesive contact with integrin ligand are
significantly reduced when TGF-b1isabsent(1.97mm
0.12 mm
to 1.559 mm
±0.0.07 mm
;Figure 3F, i and ii). These re-
sults collectively indicate that TGF-b1 has both a paracrine role in
controlling the lymphatic endothelium and an autocrine role in
regulating b4 activity in tumor-educated macrophages.
b4 Integrin+ Macrophages and Lymphatic Remodeling
Are Associated with TGF-bSignaling and Adverse
Outcome in TNBC Patients
To establish that human macrophages express ITGB4 RNA (b4
integrin), we performed an analysis of a compendium of data
composed of macrophages from in vitro and in vivo datasets.
We observed that ITGB4 is expressed in both human and mouse
total macrophages (Figures 4A and S4A). From the same com-
pendium, a correlation between ITGB4 expression and signaling
downstream of TGF-b1 was established (Figure 4B). Single-cell
transcriptome analysis of non-tumor cells isolated from primary
breast tumors revealed that TAMs expressed high levels of
ITGB4, compared with other non-tumor cells within the tumor
microenvironment (Figure 4C). To identify patients who may
have enrichment of macrophages capable of lymphovascular re-
modeling, we used a gene signature containing genes enriched
in TEMs (Pucci et al., 2009) in a cohort of 122 TNBC gene expres-
sion patterns (Gazinska et al., 2013). We plotted the activation
score of the TEM gene signature against the TGF-bsignaling
pathway for each tumor and observed the enrichment of patients
with distant metastasis when both of these gene signatures were
present in the primary tumor (Figure 4D). Kaplan-Meier plots also
showed a significant reduction in distant metastasis-free survival
(DMFS) in patients classified as having a high TEM-TGF-bactiva-
tion score (Figure 4E). To investigate the presence of lymphatic-
associated macrophages in breast cancer patients, samples
from 20 patients were used. Of these patients, 10 were previ-
ously characterized as having lymphatic vessel invasion (LVI),
and the remaining 10 did not have LVI. To assess macrophage
localization with respect to lymphatic vasculature, we dual-
stained sections with an antibody against CD14 and podoplanin
(Figure 4F). The sections were scored for the presence of CD14+
macrophages within or proximal to lymphatic vasculature. In our
cohort of 20 patients, all samples exhibited some degree of
CD14 and podoplanin positivity. Six cases (30%) had macro-
phages associated with lymphatic vessels; of these, 4 were
shown to be positive for LVI. In this small study, our results sug-
gest that 67% of patients with lymphatic-associated macro-
phages also have LVI. In a separate small patient cohort
(8 patients), we demonstrated CD68+ macrophages expressing
b4 integrin (ITGB4) in close proximity to podoplanin+ vessels
using consecutive paraffin-embedded sections (Figures 4G
and 4H). We quantified CD68+ITGB4+ macrophages per square
millimeter and saw an association between CD68+ITGB4+
Figure 4. b4 Integrin-Expressing Macrophages and Lymphatic Remodeling Associated with TGF-bSignaling and Adverse Outcome in TNBC
(A) ITGB4 expression in human macrophages. The y axis indicates normalized expression on log
scale. Red line indicates median expression of all genes. Raw
gene counts were obtained from the ARCHS4 database.
(B) Correlation between ITGB4 expression and enrichment of TGF-bsignaling in human macrophages (Spearman rho = 0.26; p < 0.001. The x axis indicates
normalized expression on the log
scale. The y axis indicates single sample gene set enrichment analysis (ssGSEA) enrichment scores computed for the TGF-b
hallmark gene set obtained from the molecular signatures database (MSigDB). Red curve indicates loess fit. Associatio n strength was quantified using Spearman
correlation coefficient. Raw gene counts were obtained from the ARCHS4 database.
(C) Expression of ITGB4 in single cell RNA sequencing (scRNaseq) data of primary breast cancer (GEO: GSE75688). Data are reported as log
(TPM+1). TPM,
transcripts per million.
(D) Activation score of TEM gene signature and TGF-bsignaling. Red and green dots indicate TNBC with or without distant metastasis, respectively. Enrichment
of TNBC with distant metastasis in the top right quadrant, established by hypergeo metric testing.
(E) Kaplan-Meier survival curves showing distant metastasis-free survival in TNBC. Stratification based on samples with high TGF-bsignaling and TEM gene
signature activation score classified as ‘‘High TEM-TGFbsignature’’ versus the remainder (‘‘Low TEM-TGFbsignature’’).
(F) Representative breast cancer section (from n = 20) stained with CD14 (red) and podoplanin (brown). Scale bars, 100 mm. Zoomed inset demonstrates CD14+
macrophages associated with podoplanin+ lymphatic vasculature (black arrows). Tissues were selected from 8 patients with or without lymph node positivity.
Consecutive sections were stained singly for podoplanin lymphovasculature or doubly using pan-macrophage marker, CD68, and anti-b4 integrin antibody.
(G) Double-stained macrophages per square millimeter shown with patient clinical details (LVI and lymph node positivity).
(H) CD68+ITGB4+ macrophages are indicated in upper right panels (red arrows). CD68 and ITGB4 stainings are indicated below as 2 single panels;
CD68+ITGB4+ macrophages are indicated with red arrows. Podoplanin+ vessels shown in upper left images (black arrows). Scale bars, 20 mm.
Cell Reports 27, 1967–1978, May 14, 2019 1975
macrophage score and lymph node positivity in individual pa-
tients (Figure S4B). Future studies will endeavor to repeat this
small study in a larger patient cohort to investigate whether
this relationship is statistically significant. The combination of
our data suggests that b4-integrin-expressing lymphovascular
macrophages may be driving LVI and subsequent metastasis
to lymph nodes via the lymphatic remodeling signaling cascade.
This study demonstrates how crosstalk between a previously
unreported tumor-infiltrating myeloid subpopulation and an
existing lymphatic vasculature can promote metastasis through
quantifiable architectural changes in lymphatic vessels. We
identified a population of b4 integrin-expressing macrophages
that drive lymphatic remodeling through TGF-bsignaling and
are associated with adverse pathological response in TNBC
Our study uses both endogenous BMMs and the RAW264.7
macrophage cell line, which is strain-matched to the lympho-
tropic tumor cell line, 4T1.2. Through intravital imaging and
ex vivo tissue analysis, our TNBC model allowed us to probe
the relationship between the tumor lymphatic vasculature and
macrophages in vivo and directly translate these phenotypic
observations into in vitro assays for mechanistic studies. We
then directly assessed the prognostic significance of the key
molecules in the lymphatic signaling cascade in predicting
adverse pathological outcome for a cohort of TNBC patients.
In breast cancer samples previously characterized for LVI, we
identified lymphatic-associated macrophages in approximately
a third of the samples and show that LVI was present in the
majority of these cases. We identified b4 integrin-expressing
macrophages proximal to lymphatic endothelium in breast can-
cer samples and demonstrate that, in patients with a larger a6b4-
expressing macrophage infiltrate, there is a trend toward sentinel
lymph node metastasis. Our data suggest that b4 integrin-ex-
pressing macrophages may drive metastasis via the lymphovas-
cular route in human breast cancer.
Our study reveals that macrophages are retained in lymphatic
endothelium in a TNBC model through the upregulation of b4
integrin on tumor-educated macrophages. While the adhesion
receptor a6b4 integrin is ubiquitously expressed in early breast
cancer (Diaz et al., 2005), transcriptome analysis of breast can-
cer patient samples revealed a correlation between expression
levels and prognosis (Lu et al., 2008). Through analysis of b4
integrin at the transcriptome and protein levels, we demonstrate
a population of endogenous macrophages that express b4
integrin and are adherent to laminin-5 in lymphovascular areas.
Collectively, our data suggest that b4 integrin acts to ensure
that tumor-infiltrating macrophages are in a prime location for
sustained interaction with LECs.
We have defined dual functionality of TGF-b1 where it can
affect signaling within TAMs and LECs. First, we show that
TGF-b1 is required for b4 integrin clustering at the macrophage
plasma membrane. Integrin clustering can positively regulate
levels of cell adhesion rapidly in response to soluble stimuli (Hy-
nes, 2002). TGF-bhas previously been demonstrated to control
a6b1 and a6b4 integrin clustering in HER2-overexpressing
mammary tumor cells (Wang et al., 2009). Here, we describe
TGF-b1-dependent b4 integrin clustering in macrophages that
control the macrophage-spreading response necessary for
TAM adhesion at the site of lymphatic vasculature.
Second, TGF-b1 acts in a paracrine manner to activate RhoA
in LECs lining the lymphatic vessel, as demonstrated through
RAICHU-fluorecent lifetime imaging microscopy (FLIM) technol-
ogy (Heasman et al., 2010; Makrogianneli et al., 2009; Vega et al.,
2011). Our study shows that signaling within LECs in contact with
TAMs drives LEC contraction, which correlates to gross archi-
tectural changes and hyperpermeability of the lymphatic vessel
network that could actively facilitate metastasis. We have previ-
ously demonstrated the activation of RhoGTPases by integrin
signaling in cis (on the immune cells that are triggered by adhe-
sion processes (Makrogianneli et al., 2009; Carlin et al., 2011;
Heasman et al., 2010; Ramsay et al., 2013). Our present study
indicates that this phenomenon can also occur in trans, i.e., acti-
vation of RhoGTPases in the endothelial cells that are contacted
by the adherent macrophages, through the expression of factors
such as TGF-b1. The role of macrophage-released TGF-b1
in vivo is shown to have an effect on the RhoA pathway in prox-
imal LECs and a concomitant role in lymphovasculature
In summary, this study identifies an alternative macrophage-
mediated signaling pathway involved in the promotion of
lymphatic metastasis. Our work emphasizes the importance in
considering crosstalk between macrophages and the lymphatic
vessel network in TNBC, where aggressive tumor growth and
rapid metastasis often mean a poor outcome. We hope this
study will guide future endeavors to focus on therapeutically
targeting the lymphatic remodeling signaling cascade in TNBC
disease progression.
Detailed methods are provided in the online version of this paper
and include the following:
BTissue culture
BTumor-bearing mice
BHuman breast cancer samples
BStudy approval
BRAW264.7 macrophage treatment
BClodronate treatment
BImage acquisition and analysis for colocalization
studies in tissue
BStructured Illumination Microscopy (SIM)
BMammary imaging window implantation and intravital
BLymphatic vessel permeability
BAdhesion assay
BLymphatic endothelial cell contraction
BRhoA biosensor
1976 Cell Reports 27, 1967–1978, May 14, 2019
BTGFb1 stable knockdown in RAW264.7 macrophages
BFACS analysis
BHuman tissue staining
BGene expression microarray analysis
BAnalysis of gene signatures
Supplemental Information can be found online at
We thank Cancer Research UK King’s Health Partners Cancer Centre at King’s
College London; Nikon Imaging Centre, King’s College London; Mathew
Smalley for tumor tissue from BLG-Cre;Brca1
mice; Steven Alex-
ander for the murine SV-LEC line; UCL Pathology core facility; Kalnisha Naidoo
for advice; and James Arnold, Victoria Sanz-Moreno, Hellmut Augustin, and
Anne Ridley for reviewing the manuscript. This work was funded by the Cancer
Research UK King’s Health Partner’s Centre at KCL, KCL/UCL Comprehen-
sive Cancer Imaging Centre, and Breakthrough Breast Cancer (recently
merged with Breast Cancer Campaign, forming Breast Cancer Now).
R.E. conceptualized the study; designed, performed, and analyzed experi-
ments, and wrote the manuscript; F.F.-B. performed FACS (fluorescence-acti-
vated cell sorting) acquisition and analysis, assisted with in vivo experiments,
assisted with experiment analysis, and assisted with writing the manuscript;
S.N. performed gene analysis on macrophage populations and assisted with
writing the manuscript; E.M. stained, quantified, and analyzed CD68+ITGB4+
patient tissues; K.L. performed in vitro macrophage gene array analysis and
assisted with writing the manuscript; A.G. analyzed TNBC gene expression
data and assisted with writing the manuscript; J.M. assisted with in vivo exper-
iments and writing the manuscript; C.G. and J.O. selected, stained, and
analyzed breast cancer sections; P.G. assisted with in vivo experiments;
V.M. designed the lymphatic disorganization scoring and assisted with data
analysis; A.C. assisted with analysis; F.N. assisted with antibody optimization;
P.B. gave technical advice on analyzing FRET-FLIM data; R.M. and E.F.-D.
performed tumor transplantation; G.F. and B.V. gave technical advice on intra-
vital imaging; M.S. contributed reagents; A.T. contributed to clinical translation
and reviewed the manuscript; F.F. wrote the colocalization software and
analyzed colocalization data; M.D.P. contributed reagents and reviewed the
manuscript; and T.N. provided funding, contributed to clinical translation,
and assisted with writing the manuscript.
The authors declare no competing interests.
Received: June 12, 2018
Revised: March 14, 2019
Accepted: April 17, 2019
Published: May 14, 2019
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1978 Cell Reports 27, 1967–1978, May 14, 2019
Rat monoclonal anti-Lyve1 Novus Biologicals #NB-600-1008
Rabbit polyclonal anti-Tie2 (C-20) Santa Cruz #sc-324
Rabbit polyclonal phospho-Smad2/3 (D27F4) Cell Signaling #8828
Mouse monoclonal anti-ITGB4 Abcam #ab29042
Mouse monoclonal anti-CD68 antibody Ventana Cell Marque #168M
Mouse monoclonal anti-CD14 (EPR3653) Ventana Cell Marque #114R
Mouse monoclonal anti-podoplanin (D2-40) Ventana Cell Marque #332M
Rat monoclonal anti-CD45-APC-Cy7 Biolegend #103115
Rat monoclonal Ly6G-Biotin Biolegend #127603
Streptavidin AF488 Biolegend #405235
Rat monoclonal CD11b-eFluor450 ThermoFisher Scientific #48-0112-82
Rat monoclonal Tie-2 PE Biolegend #124007
Rat monoclonal b4 integrin-BV711 BDBiosciences #744154
CD31 PerCPCy5.5 Biolegend #102419
Rat monoclonal anti-F4/80-FITC (clone BM8) Abcam #Ab60348
Rabbit polyclonal anti-laminin-5 Abcam #Ab14509
Rabbit polycloncal Anti-Phospho myson light
chain (Ser19)
Cell Signaling #3671
Mouse monoclonal anti-podoplanin antibody Santa Cruz #sc-166906
Rabbit polyclonal anti-TGFb1 antibody Proteintech #11522-1-AP
Biological Samples
Breast cancer tumor tissues (paraffin-embedded) King’s College London breast cancer
Team lead – Dr Cheryl Gillet
4T1.2 tumor tissues (frozen) King’s College London Dr Rachel Evans
tumor tissues (frozen) King’s College London Dr Rebecca Marlow
Chemicals, Peptides, and Recombinant Proteins
Cell tracker
red (CMTMR) and Cell tracker
green (CMFDA)
Life Technologies #C34552, C2925
Murine CSF1 Sigma #M9170
Human recombinant laminin-5 Novus Biologicals #H00003911
Clodronate and PBS liposomes Liposoma Technology #CP-005-005
acetoxymethyl ester (BCECF)
Thermo Scientific #B1170
SB-431542 Sigma #S4317
Evans Blue dye Sigma #E2129
Formamide Sigma #F9037
76kDa dextran Texas Red Sigma #R05027
76kDa dextran fluorescein Santa Cruz #sc-263323
Critical Commercial Assays
Murine TGFb1 quantikine ELISA kit R&D Ltd #MB100B
Murine TGFb2 quantikine ELISA kit R&D Ltd #DB250
Deposited Data
Experiment ArrayExpress accession Array Express ArrayExpress: E-MTAB-4064.
Breast Cancer Gene Expression data Gene Expression Omnibus GEO: GSE75688
ARCHS4 database (Lachmann et al., 2018) N/A
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Cell Reports 27, 1967–1978.e1–e4, May 14, 2019 e1
Further information and requests for resources and reagents should be directed to the Lead Contact, Tony Ng (
For a detailed description of the experimental procedures please see Supplemental Information.
Tissue culture
Bone marrow macrophages
Monocytes were isolated from female BALB/c mice femurs and cultured in mCSF-1 for 5 d.
Cell lines
All cell lines were tested as mycoplasma negative and authenticated by IDEXX Laboratories Ltd, UK.
Tumor-bearing mice
BALB/c immune-competent mice were 6–8 weeks of age and maintained under pathogen-free conditions. Tumors were established
by injection of 1x10
4T1.2 (Lelekakis et al, 1999) cells into the mammary fat pad.
Mammary tumor chunks (approximately 0.2cm
) dissected from BLG-Cre;Brca1
mice (Molyneux et al., 2010) were trans-
planted orthotopically into mammary fat pads of recipient 5-week old C57BL6J mice. Tumors were grown for 4-8 weeks before
mice were culled and tumor tissues harvested.
Human breast cancer samples
Paraffin embedded samples (n = 20) (KHP Cancer Biobank Molecular Taxonomy of Breast Cancer International Consortium
(METABRIC) dataset cohort) were used. Ten patients were previously characterized as having lymphatic vessel invasion (LVI) and
the remaining 10 did not have LVI. Please see SI for details on staining.
Experimental Models: Cell Lines
4T1.2 cells derived from female BALB/C mouse (Lelekakis et al., 1999) N/A
SV-LEC (derived from male ‘‘immortomouse’’) (Ando et al., 2005) Gift from Dr Steven Alexander
Primary LEC from male BALB/C mouse Generon Ltd #BALB5064L
RAW264.7 derived from male BALB/C mouse ATCC Ltd #ATCC-TIB71
HEK293T (derived from human fetus) ATCC Ltd #ATCC-CRL-11268
Experimental Models: Organisms/Strains
Female BALB/c mice Charles River N/A
Female C57Bl6J mice Charles River N/A
RAICHU RhoA biosensor construct King’s College London (Heasman et al., 2010)
GFP-RhoA construct King’s College London (Heasman et al., 2010)
Software and Algorithms
Gray Laboratories Oxford University and
University College London
Dr Paul Barber (Barber et al., 2013)
Prism Software
ImageJ (Fiji) N/A
Colocalization plugin for ImageJ (within this manuscript) Dr Fred Festy
TGFb1 shRNA (GIPZ) Open Biosystems University College London library
ITGB4 shRNA (GIPZ) Open Biosystems University College London
RNA easy minikit Quiagen #74104
Live/Dead Yellow dye Invitrogen #L34959
Affymetrix Mouse Gene 1.0 ST arrays Thermo Scientific #901168
e2 Cell Reports 27, 1967–1978.e1–e4, May 14, 2019
Study approval
All experiments were performed in accordance with the local ethical review panel, the UK Home Office Animals Scientific Procedures
Act, 1986 and the UKCCCR guidelines.
RAW264.7 macrophage treatment
Tumor-bearing mice were injected with 100 mL PBS or 1x10
RAW264.7 macrophages starting on the second day after tumor inoc-
ulation and repeated every 2 days until the end of the experiment.
Clodronate treatment
Endogenous macrophages were ablated using clodronate-containing liposomes (Weisser et al., 2012).
Tissue sections were fixed with 4% paraformaldehyde (PFA), blocked in 5% BSA followed by staining. Hoechst-33342 (0.1 mg/ml)
was used for nuclear staining and samples mounted using Mowiol (with DABCO). Image acquisition by confocal microscopy was
performed using a Nikon Eclipse Ni-E Upright. Image acquisition was conducted using NIS Elements C software and analyzed using
ImageJ software.
Image acquisition and analysis for colocalization studies in tissue
Cy3 and AF647 dyes were imaged before and after photobleaching using (x20 0.75NA air objective, Nikon) and a cooled CCD de-
tector (Hamamatsu ORCA-03G, 1024 31024) with respective integration time of 100 ms and 1000 ms. Dyes were photobleached
using a mode-locked Titanium Sapphire Laser (Coherent, Chameleon Ultra 2) tuned at 730 nm with pulse duration of about 200
fs, a repetition rate of 80 MHz and average laser power on the sample of 30 mW. To measure the relative level of b4 integrin expres-
sion within the lymphovasculature compared with the rest of the tissue, we measured average AF647 intensity within lymphovascu-
lature areas (high Cy3 intensity) normalized by the average AF647 intensity outside lymphovasculature areas (low Cy3 intensity).
Structured Illumination Microscopy (SIM)
RAW264.7-NTC or RAW264.7-TGFb1 KD were stained with rat anti-b4 integrin antibody and anti-rat AF647 antibody. Image acqui-
sition by SIM was performed using Nikon N-SIM microscope equipped with a 640nm laser, a Andor iXon Ultra 897 EMCCD camera
and a 100x 1.49NA oil immersion objective. Images were analyzed using ImageJ software.
Mammary imaging window implantation and intravital microscopy
Mammary Imaging Window (MIW) surgery was performed 10-14 days after tumor innoculation (Kedrin et al., 2008). Images shown are
representative of a minimum of 5 independent experiments.For imaging lymphatic vasculature, mice were injected subcutaneously
at the tail base with 50 mL 76kDa dextran-fluorescein or dextran-Texas red 15 min prior to imaging. Mice were imaged for a maximum
period of 4 h per day using a x20 air objective. All post hoc image processing and image reconstructions were done using ImageJ
Lymphatic vessel permeability
Tumor-bearing mice were injected subcutaneously at the tail base with 1% Evans Blue dye. After 30 min the mice were culled and the
tumors incubated in formamide overnight at 55C. Optical density of formamide was read at 620nm and quantification of lymphatic
permeability was given as OD per g tumor.
Adhesion assay
Laminin-5 was plated onto 96 well plates overnight at 4C and non-specific interactions blocked with BSA. Macrophages (5 310
were labeled with 1 mM2
0,70-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein-acetoxymethyl ester (BCECF) for 30 min at room
temperature. 100 mL of cells (1 310
/ml) were added at 37C, plates washed, and adhering macrophages quantified using a fluo-
rescence microtiter plate reader.
Lymphatic endothelial cell contraction
SV-LEC cells or primary lymphatic endothelial cells were grown as a monolayer. On day 3 LECs and macrophages were stained for
30min at 37C using 1 mg/ml CMTMR or CMFDA respectively. Macrophages were added to SV-LEC monolayers overnight. Confocal
images of the co-culture and the area around individual SV-LECs was calculated using ImageJ software.
RhoA biosensor
SV-LECs were transiently transfected with the RAICHU RhoA biosensor (Yoshizaki et al., 2003). The biosensor was modified to
express GFP and mRFP (Makrogianneli et al., 2009). Multiphoton time-correlated single photon counting FLIM was performed to
Cell Reports 27, 1967–1978.e1–e4, May 14, 2019 e3
quantify RhoA biosensor FRET Fluorescence excitation was provided by a Fianium laser, which generates optical pulses with a dura-
tion of 40 ps at a repetition rate of 80 MHz. For the imaging of RAICHU-transfected SV-LECs, multi-photon excitation was employed
using a solid-state pumped (8-W Verdi; Coherent), femtosecond self-mode locked Ti:Sapphire (Mira; Coherent) laser system (Peter
et al., 2005; Barber et al., 2009). Imaging data comprised of 256 3256 pixel resolution and 256 time channels. The fluorescence life-
time was calculated as described (Barber et al., 2013).
TGFb1 stable knockdown in RAW264.7 macrophages
Stable TGFb1 knockdown RAW 264.7 macrophage lines were generated by lentiviral transduction using the pGIPZ system (Open
Biosystems). Viral packaging was performed by transiently transfecting HEK293T cells with the pGIPZ shRNA transfer vector and
the accessory plasmids pCMV-dR8.91 and pMD2G. Stable cell lines were established using three different shRNA lentiviral vectors.
RAW 264.7 macrophages were cultured in puromycin (1 mg/ml) to enable the selection of successfully transduced cells and efficacy
of knockdown was assessed by western blotting.
FACS analysis
RAW264.7 cell lines (TGFb1-knockdown or NTC) were stained with a Live-Dead Yellow dye followed by staining with a primary rat
anti-b4 integrin antibody and anti-rat AF647-conjugated secondary antibody.
Tumors were disaggregated with Collagenase (Sigma UK) and DNase I (Applichem, UK) before staining with Live-Dead Yellow,
CD45-APC Cy7, Ly6G-Biotin + Streptavidin AF488, CD11b-eFluor450, Tie-2 PE b4 integrin-BV711 and CD31 PerCPCy5.5. Cells
were fixed with 1% PFA and analyzed in a FACS Canto II (BD Biosciences) cytometer. Data analyzed using FlowJo software (TreeStar
Inc., Ashland, OR, USA).
Human tissue staining
Sections were stained using anti-CD14/anti-podoplanin using Ventana Benchmark Ultra and Ultra view DAB and Alkaline Phospha-
tase detection systems. Sections were assessed independently by two histopathologists and scored for CD14+ macrophages within
or proximal to lymphatic vasculature.
Alternatively, using consecutive sections the first section was stained with anti-podoplanin and the second section stained with
anti-ITGB4 anti-CD68. All sections were stained with DAB+ substrate/chromagen. All incubations were at room temperature.
The slides were scanned in the Hamamatsu NanoZoomer S210 Digital slide scanner. The image analysis was performed on the
whole section with the color deconvolution module and the positive pixel algorithm from QuPath image analysis software.
Gene expression microarray analysis
RNA was extracted from macrophage cell cultures and profiled using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression be-
tween conditions was estimated by fitting a linear model and performing empirical Bayes moderated t tests using the package ‘limma
(v3.22.4) (Ritchie et al., 2015).The expression score for a specific gene ineach sample is defined as the weighted sum of gene-standard-
ized (Z-score) expression values, with weights +1/-1 according to relative increase or decrease in BMM + 4T1.2 compared with BMM.
Analysis of gene signatures
To establish ITGB4 expression and assess association between ITGB4 expression and activation of the TGFbsignaling in macro-
phages, processed gene counts were obtained from the ARCHS4 database (Lachmann et al., 2018) and further normalized for down-
stream analyses. Enrichment of TGFbsignaling was computed using the ssGSEA method (Barbie et al., 2009) as implemented in the
GSVA package from Bioconductor.
False zero expression due to dropout events in scRNA-seq data was corrected using the scImpute algorithm as previously
described (Li and Li, 2018). scRNaseq data is reported as log2(TPM+1).
Macrophage-mediated vascular remodeling pathway signature (Pucci et al., 2009) was converted to a human gene list using Bio-
mart ID conversion (Ensembl Genes 84// Mus musculus genes GRCm38.p4). TGFb(KEGG) gene signature was derived from
(MSigDB). Gene signature activity was calculated using a weighted average sum over all genes for each tumor. Pearson’s correlation
between the activation scores was reported. Hypergeometric testing was used to establish the significance of overlap between
TNBC with distant metastasis (DM) on those of dual high activation scores. Kaplan-Meier plots were generated for each dataset
to provide a visualization of survival stratification.
All other statistical analysis is described in the text and legends and was performed using Prism software (GraphPad). P values less
than 0.05 were considered significant. The statistical test used is indicated in the figure legends and the significance of findings is
indicated in the figures.
The accession number for the microRNA experimental data reported in this paper is ArrayExpress: E-MTAB-4064.
e4 Cell Reports 27, 1967–1978.e1–e4, May 14, 2019
... Several studies have demonstrated the important role of TAMs in tumor lymphangiogenesis (83)(84)(85)(86)(87). TAMs are positively associated with lymph node metastasis and lymphatic vessel density (LVD) in numerous cancer types, including lung cancer, tongue OSCC, pancreatic cancer, gastric cancer, cervical cancer and ovarian cancer (88)(89)(90)(91)(92)(93)(94)(95). ...
... TEMs that interact with ECs can also be enriched in lymphovascular regions (85). TEMs express β4 integrin, which interacts with LECs that express laminin-5, a β4 integrin ligand (85). ...
... TEMs that interact with ECs can also be enriched in lymphovascular regions (85). TEMs express β4 integrin, which interacts with LECs that express laminin-5, a β4 integrin ligand (85). In human BC tissues, TEMs are mainly found in the tumor center and tumor invasive edges and account for 95% of the whole population of CD14 + cells (86). ...
Full-text available
Tumor‑associated macrophages (TAMs) are crucial cells of the tumor microenvironment (TME), which belong to the innate immune system and regulate primary tumor growth, immunosuppression, angiogenesis, extracellular matrix remodeling and metastasis. The review discusses current knowledge of essential cell‑cell interactions of TAMs within the TME of solid tumors. It summarizes the mechanisms of stromal cell (including cancer‑associated fibroblasts and endothelial cells)‑mediated monocyte recruitment and regulation of differentiation, as well as pro‑tumor and antitumor polarization of TAMs. Additionally, it focuses on the perivascular TAM subpopulations that regulate angiogenesis and lymphangiogenesis. It describes the possible mechanisms of reciprocal interactions of TAMs with other immune cells responsible for immunosuppression. Finally, it highlights the perspectives for novel therapeutic approaches to use combined cellular targets that include TAMs and other stromal and immune cells in the TME. The collected data demonstrated the importance of understanding cell‑cell interactions in the TME to prevent distant metastasis and reduce the risk of tumor recurrence.
... Consistent with our findings, a decrease in the lymphatic vessel network and the possible occurrence of a Ly-EndMT process have been reported in age-related diseases [34,43,44], in experimental lymphedema [30], and in vitro in lymphatic ECs infected by Kaposi's sarcoma herpesvirus or stimulated with TGFβ [30,32,[45][46][47][48][49][50]. Besides TGFβ, which is known to be the master regulator of EndMT and other mesenchymal transitions, some microRNAs (miRNAs) have been implicated in lymphatic EC transition into myofibroblasts. ...
Full-text available
At present, only a few reports have addressed the possible contribution of the lymphatic vascular system to the pathogenesis of systemic sclerosis (SSc). Based on the evidence that blood vascular endothelial cells can undertake the endothelial-to-myofibroblast transition (EndMT) contributing to SSc-related skin fibrosis, we herein investigated whether the lymphatic endothelium might represent an additional source of profibrotic myofibroblasts through a lymphatic EndMT (Ly-EndMT) process. Skin sections from patients with SSc and healthy donors were immunostained for the lymphatic endothelial cell-specific marker lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) in combination with α-smooth muscle actin (α-SMA) as the main marker of myofibroblasts. Commercial human adult dermal lymphatic microvascular endothelial cells (HdLy-MVECs) were challenged with recombinant human transforming growth factor-β1 (TGFβ1) or serum from SSc patients and healthy donors. The expression of lymphatic endothelial cell/myofibroblast markers was measured by quantitative real-time PCR, Western blotting and immunofluorescence. Collagen gel contraction assay was performed to assess myofibroblast-like cell contractile ability. Lymphatic endothelial cells in intermediate stages of the Ly-EndMT process (i.e., coexpressing LYVE-1 and α-SMA) were found exclusively in the fibrotic skin of SSc patients. The culturing of HdLy-MVECs with SSc serum or profibrotic TGFβ1 led to the acquisition of a myofibroblast-like morphofunctional phenotype, as well as the downregulation of lymphatic endothelial cell-specific markers and the parallel upregulation of myofibroblast markers. In SSc, the Ly-EndMT might represent a previously overlooked pathogenetic process bridging peripheral microlymphatic dysfunction and skin fibrosis development.
... In addition, previous studies showed that β2 integrins on macrophages interacted with signaling lymphocytic activation molecule-7 (SLAM7) and activated immunoreceptor tyrosine-based activation motifs, which initiated actin polarization and induced phagocytosis [218]. Moreover, it has been reported that β4 integrins promoted the activation of TGF-β1 and induced the upregulation of laminin-5 and Tie2, which led to enhanced macrophage adhesion [219]. Moreover, numerous studies demonstrated that integrins can inhibit tumor growth by promoting the cytotoxic effects of NK cells. ...
Full-text available
Integrins are a group of heterodimers consisting of α and β subunits that mediate a variety of physiological activities of immune cells, including cell migration, adhesion, proliferation, survival, and immunotolerance. Multiple types of integrins act differently on the same immune cells, while the same integrin may exert various effects on different immune cells. In the development of cancer, integrins are involved in the regulation of cancer cell proliferation, invasion, migration, and angiogenesis; conversely, integrins promote immune cell aggregation to mediate the elimination of tumors. The important roles of integrins in cancer progression have provided valuable clues for the diagnosis and targeted treatment of cancer. Furthermore, many integrin inhibitors have been investigated in clinical trials to explore effective regimens and reduce side effects. Due to the complexity of the mechanism of integrin-mediated cancer progression, challenges remain in the research and development of cancer immunotherapies (CITs). This review enumerates the effects of integrins on four types of immune cells and the potential mechanisms involved in the progression of cancer, which will provide ideas for more optimal CIT in the future.
... Similarly, TAMs synthesize and secrete TGF-β, proteases, and other signaling molecules that participate in the recruitment of CAFs as well as ECM degradation and remodeling. Moreover, TAMs adjacent to lymphatics promote neolymphangiogenesis and LVI, enhancing vessel hyperpermeability, dilatation, and disorganization [37,49]. ...
Full-text available
Traditionally, lymphovascular invasion (LVI) has represented one of the foremost pathological features of malignancy and has been associated with a worse prognosis in different cancers, including breast carcinoma. According to the most updated reporting protocols, the assessment of LVI is required in the pathology report of breast cancer surgical specimens. Importantly, strict histological criteria should be followed for LVI assessment, which nevertheless is encumbered by inconsistency in interpretation among pathologists, leading to significant interobserver variability and scarce reproducibility. Current guidelines for breast cancer indicate biological factors as the main determinants of oncological and radiation therapy, together with TNM staging and age. In clinical practice, the widespread use of genomic assays as a decision-making tool for hormone receptor-positive, HER2-negative breast cancer and the subsequent availability of a reliable prognostic predictor have likely scaled back interest in LVI’s predictive value. However, in selected cases, the presence of LVI impacts adjuvant therapy. This review summarizes current knowledge on LVI in breast cancer with regard to definition, histopathological assessment, its biological understanding, clinicopathological association, and therapeutic implications.
... Furthermore, macrophages have been seen to co-migrate with tumour cells while providing them with epidermal growth factor (EGF) (Wyckoff et al., 2004). M2 TAMs can promote angiogenesis and lymphangiogenesis (Bieniasz-Krzywiec et al., 2019;Ji et al., 2014) and intravasation into blood and lymphatic vessels (Bieniasz-Krzywiec et al., 2019;Evans et al., 2019;Harney et al., 2015;Linde et al., 2018;Wyckoff et al., 2007). In circulation, macrophages can associate with CTCs to promote their survival and facilitate their extravasation in a VEGF-A-dependent manner (Qian et al., 2009(Qian et al., , 2011. ...
Metastasis is the primary cause of deaths in cancer patients. Tumour dissemination can occur through the bloodstream or lymphatic system. The presence of tumour cells in the draining lymph node (lymph node involvement) represents the most important prognostic factor for breast cancer patients. Despite its clinical implications, the mechanisms governing lymphatic metastasis remain poorly understood. This thesis aims to identify novel determinants of lymphatic dissemination in breast cancer. It focuses on the influences of the tumour immune microenvironment and the cancer cell-autonomous metastatic drivers associated with lymph node involvement. Using transplantable syngeneic mammary carcinoma 4T1 model, a differential preference for using the lymphatic system as a dissemination route was observed in immunocompetent mice when compared to immunodeficient mice. Characterisation of the differences in the tumour microenvironment revealed a higher infiltration of neutrophils in the tumours with lymph node metastasis. Depletion of neutrophils reduced lymphatic dissemination in 4T1 cells. In vitro, tumour-associated neutrophils were seen to promote tumour migration. Finally, single-cell RNA-seq analysis of lymph nodes suggests that neutrophils could have an immunosuppressive role in lymph nodes. Tumour cell-intrinsic gene signatures driving lymph node organotropism were investigated in subclonal lines derived from the 4T1 model with enhanced ability to seed lymph node metastasis. Transcriptomic profiling of these lines highlighted the role of metabolic adaptation and immunosuppression as central determinants of lymphatic metastasis. Finally, other syngeneic breast cancer models were used to assess the validity of the above-mentioned findings across breast cancer subtypes. Molecular characterisation of these indicated that immunosuppression, lipid metabolism and cell plasticity are common denominators associated with lymphatic metastasis among breast cancer models. This thesis advances our understanding of the involvement of neutrophils and tumour cell-intrinsic mechanisms that contribute to lymphatic metastasis in breast cancer. This work suggests potential gene and pathway targets that could be exploited therapeutically for the management of lymph node involvement.
... In the periphery, perivascular macrophages (PVMs) chaperone anastomoses downstream of induced tip cells (Fantin et al., 2010;Cattin et al., 2015;Graney et al., 2020;Vagesjö et al., 2021), regulate permeability (Hickey and Kimura, 1988;Zhang et al., 2012;He et al., 2016), phagocytose blood-borne materials, and contribute to the proteome of the tissues that the vessels supply (Serrats et al., 2010;Pinto et al., 2012). Upon CSF-1R blockade, mice exhibit significant edema (i.e., enhanced fluid retention in tissues), associated with an increase in matrix metalloproteinases, changes to the integrin-mediated adhesion strength of vessels, and enhanced deposits of hyaluronan and proteoglycan (Evans et al., 2019;Bissinger et al., 2021), emphasizing further the role of PVMs in the preservation of vascular function. ...
The heterogeneity of tissue macrophages, in health and in disease, has become increasingly transparent over the last decade. But with the plethora of data comes a natural need for organization and the design of a conceptual framework for how we can better understand the origins and functions of different macrophages. We propose that the ontogeny of a macrophage—beyond its fundamental derivation as either embryonically or bone marrow-derived, but rather inclusive of the course of its differentiation, amidst steady-state cues, disease-associated signals, and time—constitutes a critical piece of information about its contribution to homeostasis or the progression of disease.
... Podoplanin-expressing TAMs interact with lymphatic vessels via integrin β1 and β4, thus favoring the increase in the lymphatic bed and metastatic spread. GAL8 expressed by lymphatic vessels mediates binding to integrin β1 when engaged by Podoplanin + TAMs, whereas independently of GAL8 binding, transforming growth factor-β (TGF-β) and various metalloproteinases (MMPs), including MMP2, MMP9, MMP12, and MMP13, overexpressed by TAMs, support lymphangiogenesis and lymphoinvasion via extracellular matrix remodeling [56,57]. Of note, both processes are hampered by deleting Pdpn in macrophages [57]. ...
Full-text available
The prognosis of cholangiocarcinoma remains poor in spite of the advances in immunotherapy and molecular profiling, which has led to the identification of several targetable genetic alterations. Surgical procedures, including both liver resection and liver transplantation, still represent the treatment with the best curative potential, though the outcomes are significantly compromised by the early development of lymph node metastases. Progression of lymphatic metastasis from the primary tumor to tumor-draining lymph nodes is mediated by tumor-associated lymphangiogenesis, a topic largely overlooked until recently. Recent findings highlight tumor-associated lymphangiogenesis as paradigmatic of the role played by the tumor microenvironment in sustaining cholangiocarcinoma invasiveness and progression. This study reviews the current knowledge about the intercellular signaling and molecular mechanism of tumor-associated lymphangiogenesis in cholangiocarcinoma in the hope of identifying novel therapeutic targets to halt a process that often limits the success of the few available treatments.
... Interestingly, recent reports suggest that TAMs also directly promote lymphatic invasion. In breast cancer models, TAMs adhered to tumor-associated lymphatic vessels in an integrin-dependent manner and induced lymphatic permeability and matrix remodeling, thereby facilitating tumor cell intravasation (285,286). Similarly, cancer-associated fibroblasts (CAFs) have been associated with tumor lymphangiogenesis and lymphatic invasion (287), but the molecular mechanisms have not been defined. RORct 1 innate lymphoid type 3 cells (ILC3s) have also been reported to mediate lymphatic invasion (288). ...
The lymphatic system, composed of initial and collecting lymphatic vessels as well as lymph nodes that are present in almost every tissue of the human body, acts as an essential transport system for fluids, biomolecules and cells between peripheral tissues and the central circulation. Consequently, it is required for normal body physiology but is also involved in the pathogenesis of various diseases, most notably cancer. The important role of tumor-associated lymphatic vessels and lymphangiogenesis in the formation of lymph node metastasis has been elucidated during the last two decades, whereas the underlying mechanisms and the relation between lymphatic and peripheral organ dissemination of cancer cells are incompletely understood. Lymphatic vessels are also important for tumor-host communication, relaying molecular information from a primary or metastatic tumor to regional lymph nodes and the circulatory system. Beyond antigen transport, lymphatic endothelial cells, particularly those residing in lymph node sinuses, have recently been recognized as direct regulators of tumor immunity and immunotherapy responsiveness, presenting tumor antigens and expressing several immune-modulatory signals including PD-L1. In this review, we summarize recent discoveries in this rapidly evolving field and highlight strategies and challenges of therapeutic targeting of lymphatic vessels or specific lymphatic functions in cancer patients.
... Bone marrow derived macrophages were obtained as previously described [28,29]. A bone marrow cell suspension was obtained by flushing out femurs and tibias from CD1-nude mice with RPMI (supplemented with penicillin/streptomycin, L-glutamine and 10% FCS). ...
Full-text available
Over the past decade, immunotherapy delivered novel treatments for many cancer types. However, lung cancer still leads cancer mortality, and non-small-cell lung carcinoma patients with mutant EGFR cannot benefit from checkpoint inhibitors due to toxicity, relying only on palliative chemotherapy and the third-generation tyrosine kinase inhibitor (TKI) osimertinib. This new drug extends lifespan by 9-months vs. second-generation TKIs, but unfortunately, cancers relapse due to resistance mechanisms and the lack of antitumor immune responses. Here we explored the combination of osimertinib with anti-HER3 monoclonal antibodies and observed that the immune system contributed to eliminate tumor cells in mice and co-culture experiments using bone marrow-derived macrophages and human PBMCs. Osimertinib led to apoptosis of tumors but simultaneously, it triggered inositol-requiring-enzyme (IRE1α)-dependent HER3 upregulation, increased macrophage infiltration, and activated cGAS in cancer cells to produce cGAMP (detected by a lentivirally transduced STING activity biosensor), transactivating STING in macrophages. We sought to target osimertinib-induced HER3 upregulation with monoclonal antibodies, which engaged Fc receptor-dependent tumor elimination by macrophages, and STING agonists enhanced macrophage-mediated tumor elimination further. Thus, by engaging a tumor non-autonomous mechanism involving cGAS-STING and innate immunity, the combination of osimertinib and anti-HER3 antibodies could improve the limited therapeutic and stratification options for advanced stage lung cancer patients with mutant EGFR.
Full-text available
The pathophysiology of autoimmune disorders is multifactorial, where immune cell migration, adhesion, and lymphocyte activation play crucial roles in its progression. These immune processes are majorly regulated by adhesion molecules at cell–extracellular matrix (ECM) and cell–cell junctions. Integrin, a transmembrane focal adhesion protein, plays an indispensable role in these immune cell mechanisms. Notably, integrin is regulated by mechanical force and exhibit bidirectional force transmission from both the ECM and cytosol, regulating the immune processes. Recently, integrin mechanosensitivity has been reported in different immune cell processes; however, the underlying mechanics of these integrin-mediated mechanical processes in autoimmunity still remains elusive. In this review, we have discussed how integrin-mediated mechanotransduction could be a linchpin factor in the causation and progression of autoimmune disorders. We have provided an insight into how tissue stiffness exhibits a positive correlation with the autoimmune diseases’ prevalence. This provides a plausible connection between mechanical load and autoimmunity. Overall, gaining insight into the role of mechanical force in diverse immune cell processes and their dysregulation during autoimmune disorders will open a new horizon to understand this physiological anomaly.
Full-text available
RNA sequencing (RNA-seq) is the leading technology for genome-wide transcript quantification. However, publicly available RNA-seq data is currently provided mostly in raw form, a significant barrier for global and integrative retrospective analyses. ARCHS4 is a web resource that makes the majority of published RNA-seq data from human and mouse available at the gene and transcript levels. For developing ARCHS4, available FASTQ files from RNA-seq experiments from the Gene Expression Omnibus (GEO) were aligned using a cloud-based infrastructure. In total 187,946 samples are accessible through ARCHS4 with 103,083 mouse and 84,863 human. Additionally, the ARCHS4 web interface provides intuitive exploration of the processed data through querying tools, interactive visualization, and gene pages that provide average expression across cell lines and tissues, top co-expressed genes for each gene, and predicted biological functions and protein-protein interactions for each gene based on prior knowledge combined with co-expression.
Full-text available
The emerging single-cell RNA sequencing (scRNA-seq) technologies enable the investigation of transcriptomic landscapes at the single-cell resolution. ScRNA-seq data analysis is complicated by excess zero counts, the so-called dropouts due to low amounts of mRNA sequenced within individual cells. We introduce scImpute, a statistical method to accurately and robustly impute the dropouts in scRNA-seq data. scImpute automatically identifies likely dropouts, and only perform imputation on these values without introducing new biases to the rest data. scImpute also detects outlier cells and excludes them from imputation. Evaluation based on both simulated and real human and mouse scRNA-seq data suggests that scImpute is an effective tool to recover transcriptome dynamics masked by dropouts. scImpute is shown to identify likely dropouts, enhance the clustering of cell subpopulations, improve the accuracy of differential expression analysis, and aid the study of gene expression dynamics.
Full-text available
In experimental mouse models of cancer, increasingly compelling evidence point toward a contribution of tumor associated macrophages (TAM) to tumor lymphangiogenesis. Corresponding experimental observations in human cancer remain scarce although lymphatic metastasis is widely recognized as a predominant route for tumor spread. We previously showed that, in malignant tumors of untreated breast cancer (BC) patients, TIE-2-expressing monocytes (TEM) are highly proangiogenic immunosuppressive cells and that TIE-2 and VEGFR signaling pathways drive TEM immunosuppressive function. We report here that, in human BC, TEM express the canonical lymphatic markers LYVE-1, Podoplanin, VEGFR-3 and PROX-1. Critically, both TEM acquisition of lymphatic markers and insertion into lymphatic vessels were observed in tumors but not in adjacent non-neoplastic tissues, suggesting that the tumor microenvironment shapes both TEM phenotype and spatial distribution. We assessed the lymphangiogenic activity of TEM isolated from dissociated primary breast tumors in vitro and in vivo using endothelial cells (EC) sprouting assay and corneal vascularization assay, respectively. We show that, in addition to their known hemangiogenic function, TEM isolated from breast tumor display a lymphangiogenic activity. Importantly, TIE-2 and VEGFR pathways display variable contributions to TEM angiogenic and lymphangiogenic activities across BC patients; however, combination of TIE-2 and VEGFR kinase inhibitors abrogated these activities and overcame inter-patient variability. These results highlight the direct contribution of tumor TEM to the breast tumor lymphatic network and suggest a combined use of TIE-2 and VEGFR kinase inhibitors as a therapeutic approach to block hem- and lymphangiogenesis in BC.
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Integrin α6β4 is a cellular adhesion molecule that binds to laminins in the extracellular matrix and nucleates the formation of hemidesmosomes. During carcinoma progression, integrin α6β4 is released from hemidesmosomes, where it can then signal to facilitate multiple aspects of tumor progression including sustaining proliferative signaling, tumor invasion and metastasis, evasion of apoptosis, and stimulation of angiogenesis. The integrin achieves these ends by cooperating with growth factor receptors including EGFR, ErbB-2, and c-Met to amplify downstream pathways such as PI3K, AKT, MAPK, and the Rho family small GTPases. Furthermore, it dramatically alters the transcriptome toward a more invasive phenotype by controlling promoter DNA demethylation of invasion and metastasis-associated proteins, such as S100A4 and autotaxin, and upregulates and activates key tumor-promoting transcription factors such as the NFATs and NF-κB. Expression of integrin α6β4 has been studied in many human malignancies where its overexpression is associated with aggressive behavior and a poor prognosis. This review provides an assessment of integrin α6β4 expression patterns and their prognostic significance in human malignancies, and describes key signaling functions of integrin α6β4 that contribute to tumor progression.Laboratory Investigation advance online publication, 29 June 2015; doi:10.1038/labinvest.2015.82.
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Pulmonary metastasis of breast cancer cells is promoted by a distinct population of macrophages, metastasis-associated macrophages (MAMs), which originate from inflammatory monocytes (IMs) recruited by the CC-chemokine ligand 2 (CCL2). We demonstrate here that, through activation of the CCL2 receptor CCR2, the recruited MAMs secrete another chemokine ligand CCL3. Genetic deletion of CCL3 or its receptor CCR1 in macrophages reduces the number of lung metastasis foci, as well as the number of MAMs accumulated in tumor-challenged lung in mice. Adoptive transfer of WT IMs increases the reduced number of lung metastasis foci in Ccl3 deficient mice. Mechanistically, Ccr1 deficiency prevents MAM retention in the lung by reducing MAM-cancer cell interactions. These findings collectively indicate that the CCL2-triggered chemokine cascade in macrophages promotes metastatic seeding of breast cancer cells thereby amplifying the pathology already extant in the system. These data suggest that inhibition of CCR1, the distal part of this signaling relay, may have a therapeutic impact in metastatic disease with lower toxicity than blocking upstream targets. © 2015 Kitamura et al.
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limma is an R/Bioconductor software package that provides an integrated solution for analysing data from gene expression experiments. It contains rich features for handling complex experimental designs and for information borrowing to overcome the problem of small sample sizes. Over the past decade, limma has been a popular choice for gene discovery through differential expression analyses of microarray and high-throughput PCR data. The package contains particularly strong facilities for reading, normalizing and exploring such data. Recently, the capabilities of limma have been significantly expanded in two important directions. First, the package can now perform both differential expression and differential splicing analyses of RNA sequencing (RNA-seq) data. All the downstream analysis tools previously restricted to microarray data are now available for RNA-seq as well. These capabilities allow users to analyse both RNA-seq and microarray data with very similar pipelines. Second, the package is now able to go past the traditional gene-wise expression analyses in a variety of ways, analysing expression profiles in terms of co-regulated sets of genes or in terms of higher-order expression signatures. This provides enhanced possibilities for biological interpretation of gene expression differences. This article reviews the philosophy and design of the limma package, summarizing both new and historical features, with an emphasis on recent enhancements and features that have not been previously described. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
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Microvascular plasma protein leakage is an essential component of the inflammatory response and serves an important function in local host defense and tissue repair. Mediators such as histamine and bradykinin act directly on venules to increase the permeability of endothelial cell (EC) junctions. Neutrophil chemoattractants also induce leakage, a response that is dependent on neutrophil adhesion to ECs, but the underlying mechanism has proved elusive. Through application of confocal intravital microscopy to the mouse cremaster muscle, we show that neutrophils responding to chemoattractants release TNF when in close proximity of EC junctions. In vitro, neutrophils adherent to ICAM-1 or ICAM-2 rapidly released TNF in response to LTB4, C5a, and KC. Further, in TNFR(-/-) mice, neutrophils accumulated normally in response to chemoattractants administered to the cremaster muscle or dorsal skin, but neutrophil-dependent plasma protein leakage was abolished. Similar results were obtained in chimeric mice deficient in leukocyte TNF. A locally injected TNF blocking antibody was also able to inhibit neutrophil-dependent plasma leakage, but had no effect on the response induced by bradykinin. The results suggest that TNF mediates neutrophil-dependent microvascular leakage. This mechanism may contribute to the effects of TNF inhibitors in inflammatory diseases and indicates possible applications in life-threatening acute edema.
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The heterogeneous nature of mammary tumours may arise from different initiating genetic lesions occurring in distinct cells of origin. Here, we generated mice in which Brca2, Pten and p53 were depleted in either basal mammary epithelial cells or luminal oestrogen receptor (ER) negative cells. Basal cell-origin tumors displayed similar histological phenotypes regardless of the depleted gene. In contrast, luminal ER negative cells gave rise to diverse phenotypes, depending on the initiating lesions, including both ER negative and, strikingly, ER positive Invasive Ductal Carcinomas. Molecular profiling demonstrated that luminal ER negative cell-origin tumours resembled a range of the molecular subtypes of human breast cancer, including basal-like, luminal B and ‘normal-like’. Furthermore, a subset of these tumours resembled the ‘claudin-low’ tumour subtype. These findings demonstrate that not only do mammary tumour phenotypes depend on the interactions between cell-of-origin and driver genetic aberrations, but also that multiple mammary tumour subtypes, including both ER positive and negative disease, can originate from a single epithelial cell type. This is a fundamental advance in our understanding of tumour etiology.
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 TIE2(hi) 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. Tumor vasculature is abnormal with increased permeability. Here, we show that VEGFA signaling from TIE2(hi) 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. ©2015 American Association for Cancer Research.