Biochimica et Biophysica Acta 1865 (2016) 23–34
Contents lists available at ScienceDirect
Biochimica et Biophysica Acta
journal homepage: www.elsevier.com/locate/bbacan
Tissue-resident versus monocyte-derived macrophages in the
tumor microenvironment
Qods Lahmar a,b, Jiri Keirsse a,b,1, Damya Laoui a,b,1, Kiavash Movahedi a,b,1,
Eva Van Overmeire a,b,1, Jo A. Van Ginderachter a,b,⁎
a
b
Myeloid Cell Immunology Lab, VIB, Brussels, Belgium
Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium
a r t i c l e
i n f o
Article history:
Received 29 May 2015
Received in revised form 25 June 2015
Accepted 26 June 2015
Available online 2 July 2015
Keywords:
Tumor-associated macrophages
Tissue-resident macrophages
Monocyte-derived macrophages
Kupffer cell
Microglia
Breast cancer
Lung cancer
Pancreatic adenocarcinoma
Glioma
Hepatocellular carcinoma
a b s t r a c t
The tumor-promoting role of macrophages has been firmly established in most cancer types. However, macrophage identity has been a matter of debate, since several levels of complexity result in considerable macrophage
heterogeneity. Ontogenically, tissue-resident macrophages derive from yolk sac progenitors which either directly
or via a fetal liver monocyte intermediate differentiate into distinct macrophage types during embryogenesis and
are maintained throughout life, while a disruption of the steady state mobilizes monocytes and instructs the
formation of monocyte-derived macrophages. Histologically, the macrophage phenotype is heavily influenced
by the tissue microenvironment resulting in molecularly and functionally distinct macrophages in distinct
organs. Finally, a change in the tissue microenvironment as a result of infectious or sterile inflammation instructs
different modes of macrophage activation. These considerations are relevant in the context of tumors, which can
be considered as sites of chronic sterile inflammation encompassing subregions with distinct environmental
conditions (for example, hypoxic versus normoxic). Here, we discuss existing evidence on the role of macrophage
subpopulations in steady state tissue and primary tumors of the breast, lung, pancreas, brain and liver.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
Macrophages are among the most plastic cells of the hematopoietic
system. They are ubiquitous cells found in all tissues, in which they
display functional and anatomical diversity. Macrophages are implicated in organogenesis and are key players in tissue homeostasis maintenance, tissue repair and immune surveillance.
The traditional belief that all tissue macrophages derive from
hematopoietic stem cells (HSC) in the bone marrow via circulating
monocyte precursors [1] has been overthrown in recent years. It has
been shown that certain tissue-resident macrophage populations are
already present in the embryo before the development of HSC (Fig. 1).
Studies based on the inactivation of the transcription factor c-Myb,
crucial for HSC development, confirmed that macrophages in adulthood
can be derived from progenitor cells in the embryonal yolk sac [2]. These
Csf1r+ progenitors with erythro-myeloid potential (erythro-myeloid
progenitors or EMP) emerge at embryonal day E8.5 in the mouse yolk
sac [3] and either fully differentiate into tissue-resident macrophages
⁎ Corresponding author at: Lab of Cellular and Molecular Immunology, VIB-Vrije
Universiteit Brussel, Building E8, Pleinlaan 2, B-1050 Brussels, Belgium.
E-mail address: jvangind@vub.ac.be (J.A. Van Ginderachter).
1
The authors contributed equally.
http://dx.doi.org/10.1016/j.bbcan.2015.06.009
0304-419X/© 2015 Elsevier B.V. All rights reserved.
without the presence of a monocyte intermediate (as is the case for
microglia), or give rise to c-Myb+ liver monocytes that seem to be the
prime source of liver Kupffer cells (KC), epidermal Langerhans cells
(LC), alveolar macrophages and possibly other tissue-resident macrophages [4]. Only following these events, a second wave of HSCdependent hematopoiesis is initiated in the fetal liver [3]. Notably, full
maturation of some tissue macrophages, such as alveolar macrophages,
is only achieved early after birth [5]. Some of these tissue macrophages
are very long lived and are hardly replaced by HSC-derived cells under
steady-state (KC, microglia, LC) [2,6–9], while other macrophage populations are either rapidly replaced after birth (gut macrophages, [10]) or
gradually replaced throughout life (alveolar macrophages [3]; heart
macrophages [11–13]).
These studies raise the important question of determining the
macrophage identity and function during a situation of chronic smoldering inflammation such as cancer. Tumor-associated macrophages
(TAM) are the predominant leukocytes infiltrating solid tumors and
can represent up to 50% of the tumor mass. The clinical significance of
these cells is illustrated by the significant link between TAM number
and density and a poor prognosis in 80% of the reported studies
[14–16]. The non-redundant role of macrophages in tumor progression
flows from the fact that TAMs actively contribute to each stage of cancer.
They promote cancer cell invasion, metastasis and angiogenesis by
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Fig. 1. Ontogenic, functional and anatomical diversity of macrophages under steady state or cancer conditions. Macrophage heterogeneity in a tissue can be the result of a different origin
(yolk sac-derived/HSC-independent versus bone marrow-derived/HSC-dependent) but also the confrontation with a distinct microenvironment. Under homeostatic conditions (red),
most tissue-resident macrophages are yolk sac-derived and perform specialized functions in each organ. During cancer (green), which is a type of chronic smoldering inflammation,
the contribution of tissue-resident versus monocyte-derived macrophages to the tumor microenvironment is not always clear and might depend on the tumor type and afflicted tissue.
Most often, tumor promoting functions have been ascribed to TAM and the involvement of M2-oriented macrophages (irrespective of their origin) seems to be a recurring theme in
most organs.
releasing cytokines, growth factors, extracellular matrix-degrading
enzymes and angiogenic factors including vascular endothelial growth
factor (VEGF), Bv8 and MMP9. TAM also inhibit cytotoxic T-cell activity
by the secretion of suppressive cytokines such as IL-10 and TGF-β,
high levels of arginase activity and the production of ROS and RNI
[17–21]. Finally, TAM can contribute to tumor relapse following tumor
Q. Lahmar et al. / Biochimica et Biophysica Acta 1865 (2016) 23–34
irradiation, the administration of anti-angiogenic agents and some
forms of chemotherapy [22]. Presently, it is obvious that TAM populations are heterogeneous both inside the same tumor and among
25
tumor types [23] (Figs. 1–2). Here, we review evidence for the
ontogenic and phenotypic heterogeneity of TAM in primary tumors
from distinct organs. In this respect, we limited ourselves to tumor
Fig. 2. Tumor-associated macrophage heterogeneity. Overview of the available data describing the diversity of TAM in breast, lung, pancreas, brain and liver cancers.
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types/organs (breast, lung, pancreas, brain, liver) for which an overall
tumor-promoting role of macrophages has been established, while we
will not discuss cancer types in which macrophages may be antitumoral (eg colorectal carcinoma) [15].
2. Macrophage diversity in breast and in breast tumors
Macrophages are important players in the normal physiology of the
breast and are implicated in pathological situations such as breast
cancer. During mammary gland development, macrophages are recruited to the invasive front of the duct known as the terminal end buds
(TEBs), where they are required to promote ductal outgrowth [24,25].
Indeed, macrophages accumulate around the growing TEBs and align
around the TEB shaft [24] (Fig. 1). The use of multiphoton microscopy
provided insights in the role of macrophages during collagen
fibrillogenesis and in the organization of the TEB structure [26],
as well as the phagocytosis of apoptotic epithelial cells generated
while the lumen formation occurs [24,27]. Studies in mice carrying
an inactivating mutation in the csf1 gene (csf1op/op), known as
osteopetrotic mice, have shown a severe reduction in macrophage
numbers in most tissues, including the mammary gland, leading to
defects in the development of the breast tissue [28,24,29]. The rescue
of the macrophage deficiency by the ectopic expression of the csf1
gene in the mammary epithelium resulted in the restoration of the
ductal outgrowth and branching [26,30,21,27]. Interestingly, macrophages were also involved in supporting mammary stem/progenitor
cells by potentiating the stem cell niche and enabling the engraftment
and the growth of these cells [31]. Altogether, these data indicate the
functional diversity of macrophages during mammary gland development, being involved in mammary stem cell niche potentiation, in
ductal outgrowth, in angiogenesis, in tissue remodeling and the phagocytosis of apoptotic cells. Given these important functions during breast
genesis and homeostasis, it seemed likely that macrophages also
contribute to breast cancer development and progression.
Breast cancer is the most frequent malignant tumor and the leading
cause of cancer death in women worldwide [32,33]. Besides genetic
predisposition, it is now clear that stromal cells including macrophages
play a crucial role in promoting breast cancer progression and metastasis [34–36]. Indeed, macrophages are the most abundantly recruited
cells in the microenvironment of breast tumors [37,38] [39] and there
is now clear experimental and clinical evidence of the tumor promoting
activities of these cells [14,15,17,40,41].
In breast cancer, it is not fully established whether TAM accumulation is mainly due to monocyte recruitment, to the local proliferation
of the tissue resident macrophage population or both [42]. Clinical
data provide evidence for the existence of both phenomena in breast
cancer patients. Indeed, a significant correlation between CCL2 expression and the number of CD68-positive macrophages was demonstrated,
whereby CCL2hi patients showed a significantly reduced survival,
suggestive of the importance of recruiting CCR2+ monocytes to the
tumor [43]. Another study in human breast cancer patients established
the presence of PCNA+CD68+ cells in tumors, indicative of proliferating
TAM [44] (Figs. 1–2). Proliferating TAM were significantly correlated
with high grade, hormone receptor-negative tumors, and a basal-like
subtype, and were predictors of recurrence and reduced survival. It is
therefore conceivable that TAM proliferation becomes more prominent
if tumor inflammation and the rate of monocyte influx are diminished.
Studies in transgenic mouse models of breast adenocarcinoma development confirm these data. In the MMTV-Neu model, a distinction was
made between CD11bhiF4/80loMHC-IIhi and CD11bloF4/80hiMHC-IIint
TAM subsets, both of which are derived from circulating monocytes
[45]. However, the rate of monocyte contribution to the CD11blo
population was lower, and accumulation of these cells is at least
partly mediated by their M-CSFR-mediated proliferation. Likewise,
Ly6ChiCCR2+ monocytes contribute to the formation of TAM in the
MMTV-PyMT model, but, remarkably, they require less input from
circulating monocytes than mammary tissue macrophages (MTM)
found in the surrounding non-cancerous tissue [46]. This relative
paucity of monocyte contribution to TAM is compensated for by an
increased proliferation rate of these cells. The distinct requirements
for the development of TAM versus MTM is further demonstrated in
mice lacking RBPj (recombination signal-binding protein for immunoglobulin k J region), a downstream effector of Notch, in CD11c+ cells
resulting in a specific loss of TAMs and a reduced tumor burden in the
PyMT model. These data reveal that TAMs, but not MTMs, develop in a
Notch signaling-dependent manner and are involved in promoting
breast tumor growth [46]. Remarkably, studies in the same MMTVPyMT model suggested that Ly6Clo patrolling monocytes preferentially
home to the primary tumor site, while Ly6Chi monocytes infiltrate
lung metastases in a CCL2/CCR2-dependent fashion and differentiate
into so-called metastasis-associated macrophages [17]. A subpopulation
of these Ly6Clo monocytes may express the angiopoietin-2 receptor Tie2
(so-called Tie2-expressing monocytes or TEM), as these cells were
demonstrated to infiltrate primary MMTV-PyMT tumors, to remain
associated with blood vessels in a Ang2/Tie2-dependent way and to
play a nonredundant role in tumor angiogenesis [47,48]. Also in
human breast cancer patients, TEMs were observed in blood and
tumors, where they represent the main monocyte population distinct
from TAMs [49]. Importantly, following treatment with immunogenic
cell death-inducing chemotherapeutics such as doxorubicin, stromal
CCL2 production results in a CCR2-mediated influx of monocytes
into the tumors which is responsible for tumor relapse [50]. Also the
M-CSFR ligands M-CSF (or CSF1) and IL-34 are upregulated in breast
tumors following cytotoxic therapies (eg paclitaxel, CDDP, ionizing
radiation) resulting in TAM accumulation and a reduced responsiveness
to the therapy [51]. Notably, M-CSFR signaling may mediate monocyte
recruitment, TAM differentiation and TAM proliferation, but also the
conversion of monocytes to TEM can be triggered by M-CSF, resulting
in the expansion of these cells in MMTV-PyMT tumors and increased
angiogenesis [52]. Together, these findings illustrate the ontogenic and
functional diversity of macrophages in breast tumors, which can be
influenced by the treatment regimen.
Besides their ontogeny, macrophage heterogeneity is also instructed
by their microenvironment. Spinning disk confocal microscopy
on mouse mammary tumors revealed that non-migratory macrophages
present within the tumor mass, were mostly CD68+ CD206neg
and did not ingest intravenously injected dextran. Macrophages at
the tumor-stroma border could be distinguished as migratory
CD68+ MMR/CD206neg dextranneg myeloid cells and sessile CD68+
CD206+ dextran+ M2-type TAM, altogether clearly illustrating the
existence of distinct macrophage types in breast tumors [53]. Accordingly, in subcutaneous or orthotopically growing mouse breast cancer
models, a distinction can be made between more M2-oriented
MHC-IIloCD206hi TAM that predominantly reside in hypoxic areas
and M1-like MHC-II hi CD206 lo TAM in less hypoxic regions [54].
MHC-IIlo TAMs display a superior angiogenic, phagocytic and immunosuppressive activity. This finding can be extended to models of
spontaneous mammary carcinoma formation. Indeed, MMTV-PyMT
tumors also harbor MHC-IIhi and MHC-IIlo TAM populations, the latter
of which being more M2 oriented (including higher CD206 expression),
more immunosuppressive and mainly found in hypoxic areas [55].
Along the same line, the CD11bhiF4/80loMHC-IIhi and CD11bloF4/
80hiMHC-IIint TAM subsets from MMTV-neu tumors are located in
core regions of the tumor or scarcely vascularised regions at the
tumor periphery, respectively [45]. Interestingly, prohibiting TAM
from entering the hypoxic tumor areas in MMTV-PyMT, through a conditional deletion of the VEGF/Sema3a binding receptor Neuropilin-1 in
these cells, resulted in a more antitumoral TAM phenotype (higher
cytotoxicity, less immunosuppressive) and the initiation of an immunological cascade leading to T-cell mediated tumor attack [56]. However, it
should be noted that not all CD206hi myeloid cells are restricted to
hypoxia. TEMs in particular are CD206hi cells with an overall M2 gene
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signature and transcriptomic features of circulating patrolling Ly6Clo
monocytes [57], which are found proximal to blood vessels [48].
Earlier microscopy studies had already demonstrated that macrophages are present in large numbers at the margins of mammary
tumors and then decreasingly deeper in the tumors, where many
were found in association with blood vessels either as single cells or in
clusters [58]. Perivascular macrophages have been reported to guide
cancer cells to the vasculature and promote intravasation through a
paracrine positive feedback loop consisting of M-CSF produced by
cancer cells and EGF produced by perivascular TAM [58,59]. The tripartite interaction of macrophages, cancer cells and endothelial cells
(known as the tumor microenvironment of metastasis or TMEM) is
often detected in breast cancer patients and predicts the presence of
distant metastases [60]. Using intravital microscopy through a mammary imaging window [61] demonstrated that breast cancer cells are more
motile in a vascular environment containing perivascular macrophages,
while there was little migration in avascular regions, corroborating the
importance of TMEM for invasion and metastasis.
As a final note, it should be realized that nearly all studies on
TAM heterogeneity in breast cancer were performed in mouse and
that hardly any data are available in human. One study identified thymidine phosphorylase (TP) in TAM as an independent prognostic factor,
such that even patients with a high CD68+ TAM content in the tumor
could be categorized in two subgroups with strikingly different diagnoses: a good prognostic macrophage TPneg group and a poor prognostic
macrophage TPpos group [62]. Overall, these findings illustrate the
diversity of macrophages and TAMs in the mammary tissue and breast
cancer.
3. Macrophage diversity in lung and in lung tumors
Cells of the innate immune system, and especially myeloid cells play
an important role in lung development and physiology and can contribute to cancer [63]. Lung macrophages consist of two distinct populations, the alveolar macrophages and interstitial macrophages, which
exhibit different origins and life spans in lungs, and have been identified
as key regulators of pathological and reparative processes (Fig. 1). More
recently, a population of tissue dwelling Ly6C+ monocytes was identified in the lung, that acquired antigen for carriage to draining lymph
nodes without differentiating into macrophages or dendritic cells [64].
Alveolar macrophages populate the lung alveoli and are long-lived
tissue-resident macrophages with a peculiar phenotype in that they
are CD11chigh CD11blow SiglecFhigh [65,66]. It was recently shown that
circulating bone marrow­derived monocytes contribute only minimally
to the pool of alveolar macrophages and that alveolar macrophages constitute a population of self­maintaining, proliferating macrophages in
the lung [8,9]. As most other tissue macrophages, alveolar macrophages
originate from embryonic precursors. The lungs are colonized sequentially by yolk sac macrophages and fetal liver monocytes. The latter
are able to outcompete yolk sac macrophages and become the predominant precursors of alveolar macrophages [5]. Alveolar macrophages
develop to their mature state during the first week after birth and
require GM-CSF and M-CSF, but not IL-34 for their development [5,9,
67,68]. In contrast, the lung interstitium is populated by less studied
interstitial macrophages, which possess a CD11bint CD11clo MHC-IIhi
profile, and by bone marrow–derived monocytes with a shorter halflife [64,66,69,70]. Although the ontogeny of interstitial macrophages is
not well studied, it is hypothesized that these cells consist primarily of
embryonic macrophages with a minor contribution of adult monocytes
[71].
Lung cancer is the most common cause of cancer-related deaths
worldwide [32,72]. The clinical significance of macrophages in lung cancer is illustrated by a significant link between TAM number and density
and a poor prognosis (Figs. 1–2). In pulmonary adenocarcinoma, TAM
density is associated with angiogenesis and poor prognosis or correlated
with lymph node metastasis [73,74]. An association between TAM
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presence and survival was also reported in non-small cell lung carcinoma (NSCLC) [75], However, understanding the role of macrophages
in lung cancer is complicated by several other studies that point to a
correlation between TAM infiltration and a better prognosis in lung
cancer. For example, a high density of CD68+ macrophages in tumor
islets was reported to be a powerful favorable independent predictor
of overall survival or survival from surgically resected NSCLC [76–78].
In addition, other studies show that neither the amount of CD68+ macrophages, located either in tumor islets or tumor stroma, nor the expression of M-CSFR, correlate with survival in NSCLC patients [79–81].
The discrepancies in the above mentioned studies may be explained
by the differences in stage and histological lung cancer subtypes studied
or by the fact that the TAM were considered as one uniform population
and that the possible co-existence of TAM subsets with different activation states was overlooked. It was indeed demonstrated that NSCLC
patients with more TAM in the tumor islets than in the tumor stroma
survived significantly longer, whereas increased numbers of macrophages in the stroma was associated with a worse prognosis, suggesting
that TAM subpopulations with a different intratumoral localization may
have opposing roles in lung cancer [76,82].
Lewis lung carcinoma (LLC) is one of the most used mouse models of
lung cancer and is a preclinical model for non-small cell lung cancer
(NSCLC), which represents 85% of all lung cancers [83,84]. A CCR2driven recruitment of Ly6Chi monocytes was shown to give rise to
TAM subpopulations in LLC and in the KrasLSL/G12D/+; p53fl/fl conditional
genetic mouse model of lung adenocarcinoma formation [85–87]. In
the latter model, the spleen was suggested as a reservoir for monocytes
and neutrophils that are mobilized to the tumor site and that are
intraspenically generated through an Angiotensin II-driven hematopoietic stem and progenitor cell accumulation [87,88]. Blocking this
recruitment axis strongly lowers the number of CD11b+ macrophages
in tumor-bearing lungs to levels seen in naïve lungs and reduces
tumor growth, indeed suggesting that most TAM were monocytederived and strongly protumoral. However, these data do not formally
exclude a role for tissue-resident alveolar and/or interstitial macrophages in lung cancer development and progression.
As in breast carcinoma, differentially activated macrophages are
found within the same subcutaneous LLC tumors, residing in distinctively oxygenated tumor regions, and discernable by a differential
expression of MHC-II molecules. MHC-IIhigh TAM are excluded from
hypoxic avascular areas and more M1 oriented, while hypoxic MHCIIlow TAM express higher levels of M2-associated markers, such as
CD206, and are more angiogenic [54,85,89]. Notably, orthotopically
growing LLC tumors (upon intratracheal injection of LLC cells) showed
many similarities with subcutaneous LLC tumors, as they were also
infiltrated with the same monocyte and macrophage subsets. Hence,
the infiltration and differentiation of monocytes towards differentially
polarized M2-like MHC-IIlo and M1-like MHC-IIhi TAM subpopulations
in tumors seemed to be largely maintained in the lung microenvironment (Laoui et al., unpublished results). In addition, the association of
M2-like macrophages with hypoxic areas in LLC is also confirmed via
immunohistochemistry, using CD209 as M2 marker [90]. To further
characterize TAM subsets in subcutaneous LLC tumors, Michele
De Palma's group isolated CD206hiCD11clo and CD206loCD11chi TAM
to show via quantitative RT-PCR and RNA-sequencing that the former
are more M2-like (and thus resemble the MHC-IIlo TAM subset) and
express multiple protumoral genes [57,91]. Remarkably, most of these
tumor-promoting genes are downregulated by miR-511-3p that is
encoded in the fifth intron of the Mrc1 gene (encoding for CD206)
[91]. Though these TAM subsets may reside in differentially oxygenated
tumor regions, the differentiation of Ly6Chi monocytes is not driven by
hypoxia [85]. Rather, hypoxia fine-tunes the gene expression profile of
mature MHC-IIlo TAM, increasing their angiogenic activity and altering
their metabolism. Unpublished data from our lab now demonstrate
that M-CSFR signaling is crucial for the extravasation, proliferation
and differentiation of Ly6Chi monocytes into TAM, with a particular
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importance in the formation of MHC-IIlo TAM. Conversely, GM-CSFR
signaling does not influence intratumoral monocyte differentiation
but shapes the phenotype of MHC-IIhi TAM (Van Overmeire et al., unpublished results). Notably, similar to breast cancers, the absence of
Neuropilin-1 in TAM prevents their migration to hypoxic LLC regions,
increases the TAM's inflammatory profile, and results in T-cell mediated
antitumor immunity [56].
Similar to the findings obtained in the mouse LLC model, Ohri et al.
could show that human NSCLC tumors are infiltrated with two distinct
macrophage phenotypes [92]. Macrophages expressing M1 markers —
HLA-DR, iNOS, MRP8/14 and TNF — were markedly increased in the
tumor islets of patients with extended survival. M2 TAM, defined by a
higher expression of CD163 and VEGF, were also increased in the islets
of the extended survival group but to a significantly lesser extent than
the M1 macrophages. These authors hypothesized that the survival
advantage conferred by tumor islet M1 TAM infiltration may be related
to their cytotoxic potential [92]. The presence of distinct TAM subsets in
NSCLC tumors was confirmed in another study, showing that high
CD68+ HLA-DR+ M1 macrophages are associated with a better outcome, while CD68+ CD163+ M2 macrophages have no prognostic
value [93]. However, in advanced NSCLC 95% of the TAM were located
in the tumor stroma, were CD163+ and were significantly higher in
patients with progressive disease [94]. IL-10 production is generally
associated with M2 TAM polarization. High IL-10 expression by TAM
was a significant independent predictor of advanced tumor stage, and
thus was associated with worse overall survival in NSCLC [95].
A similar trend between M2 TAM density and poor prognosis
was also found in other types of lung cancer. In lung adenocarcinoma,
a higher CD206+ M2 TAM density or high numbers of CD204+ macrophages correlated with several clinic pathological factors and poor
outcome [96,97]. The M2 polarizing cytokine IL-10 and the chemokine
CCL2 significantly correlated with the numbers of CD204+ TAM infiltrating the cancer-induced stroma [97]. As in lung adenocarcinoma,
high numbers of CD204+ TAM accompanied by high numbers of
Foxp3+ T lymphocytes (i.e. Tregs) correlates with poor clinical outcome
in squamous cell carcinoma of the lung [98]. Interestingly, the CD204+
TAM were strongly correlated with a high expression of CCL2 and a
high microvessel density, suggesting that M2 TAM may create a
tumor-promoting microenvironment by recruiting endothelial cells
and regulatory T cells [98].
From the existing clinical studies, it is not clear whether the tumorinfiltrating macrophages comprise resident interstitial macrophages or
are strictly monocyte-derived. In addition, the role of alveolar macrophages in lung cancer is still elusive. It was reported that their numbers
decreased in cancer patients [99] and that their phenotype was altered,
resulting in their inability to stimulate anti-tumor immunity [100], but
such data are rather sporadic. Altogether, a high prevalence of TAM
with M2 characteristics generally correlates with poor prognosis,
while high M1 TAM number were associated with extended survival
in lung cancer patients. However, a more comprehensive analysis of
the clinical impact of tumor-infiltrating versus lung resident macrophage subsets is still needed.
4. Macrophage diversity in pancreas and in pancreatic tumors
Under steady-state conditions, the tissue macrophages of the pancreas are almost all derived from the yolk sac; only about 10% have a
c-Myb-dependent HSC origin (Fig. 1). Interestingly, these F4/80bright
yolk sac-derived macrophages were found in proximity to insulinpositive beta cells, suggesting a potential crosstalk between these cell
types [2]. Macrophages have indeed been shown to play an important
role during pancreas development, similar to their reported role in the
mammary gland. Comparing op/+ and op/op mice, which lack M-CSF,
it was demonstrated that M-CSF-dependent macrophages are essential
for full insulin-producing cell mass development and postnatal islet
morphogenesis [101,102]. Recently, steady-state tissue resident
pancreatic macrophages were reported to consist of two main subsets
which can be discerned based on a differential MHC class II expression
level — the MHC-IIlo and the more prominent MHC-IIhi macrophages —
and which are more M2- or M1-like polarized, respectively [103,104].
Notably, these pancreatic macrophage populations show several
similarities to the MHC-IIlo and MHC-IIhi TAM subsets that were originally identified in breast and lung cancer models [54,85,89]. Importantly, upon severe injury to the pancreas and eradication of the tissue
resident macrophage populations (eg following partial duct ligation or
PDL), an intricate dynamics of CCR2- and M-CSFR-dependent Ly6Chi
inflammatory monocyte recruitment and M-CSFR-dependent monocyte/macrophage proliferation lead to a restoration of the macrophage
subpopulations [103]. In the PDL model, M2-like macrophages were
shown to contribute to beta cell proliferation [105], and, employing
CCR2-deficient mice, evidence was provided that the tissue-resident
macrophages may be sufficient for this phenomenon [103]. However,
to what extent monocyte-derived MHC-IIlo and MHC-IIhi pancreatic
macrophages upon sterile inflammation resemble their steady-state
embryonically-derived counterparts remains unknown, a question
that is also relevant in the context of a chronic sterile inflammation
such as cancer.
Pancreatic cancer is one of the most aggressive and lethal malignancies. It has a very high mortality rate and is worldwide the eighth and
ninth leading cause of death from cancer in men and women, respectively [106], with a 5-year survival rate of only 5%. Of all pancreatic
cancers, 85% are classified as pancreatic ductal adenocarcinoma
(PDAC), which finds its origin in the exocrine cells of the pancreas.
Cancers of the endocrine cells of the pancreas or pancreatic neuroendocrine tumors (PanNET) are less common and will not be discussed in
this review [107,108]. Macrophages play an important role in pancreatic
cancer as well as in other diseases of the pancreas [104]. Indeed, TAM
have been shown to be present in pancreatic cancer in all of its stages,
starting from the preinvasive stage and persisting throughout progression [109]. Macrophage-derived RANTES and TNF drive acinar-toductal metaplasia (ADM) by inducing NF-κB activation in acinar cells.
This may lead to pancreatic intraepithelial neoplasia (PanIN) which
can progress to pancreatic ductal adenocarcinoma [110]. Especially
M1 macrophages appear to be attracted to KrasG12D transformed acinar
cells through soluble ICAM-1, and depletion of these macrophages
diminishes oncogenesis [111]. In addition, TAMs were shown to promote epithelial-to-mesenchymal transition and metastasis of pancreatic cancer cells through TLR4-dependent IL-10 secretion [112]. Finally,
TAMs also play a decisive role during treatment of pancreatic cancer
by stimulating chemoresistance to gematicibine of the cancer cells.
Mechanistically, TAM induce the expression in cancer cells of the enzyme cytidine deaminase, which catalyzes gemacitibine to its inactive
form [113]. However, in all of these scenarios it is unclear whether
tissue-resident or monocyte-derived macrophages play a dominant role.
Distinct TAM subsets were identified in an orthotopic Kras-Muc1
PDAC tumor model. Four subsets were discerned on the basis of
differential MHC-II and CD11c expression, with the MHC-IIhi subsets
(MHCIIhiCD11chi and MHCIIhiCD11clo) being the most abundant. Interestingly, these subsets also showed heterogeneity for expression of
the M2 marker CD206 [114]. Importantly, these data can be extended
to other PDAC models, such as the Kras-INC orthotopic model [115].
Although a thorough examination of the phenotype and function of
these cells is lacking, they are strikingly reminiscent of the macrophage
subsets present in the naive pancreas and in the tumor microenvironment of various other tumor models of distinct histological origins.
A specific macrophage subset, termed tumor-activated endoneurial
macrophages (EMΦ), was shown to be involved in perineural invasion
of cancer cells (CPNI) during pancreatic cancer. These EMΦ accumulated around nerves invaded by cancer cells and were attracted by secreted
M-CSF. In their turn, EMΦ produce GDNF (glial-derived neurotrophic
factor), which leads to increased migration of cancer cells, establishing
a paracrine response. Interestingly, EMΦ numbers were strongly
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reduced in Ccr2−/− mice, resulting in diminished CPNI, illustrating that
these macrophages are monocyte-derived [116].
In human PDAC a high presence of M2 macrophages (CD204+/
CD163+) is associated with lymph node metastasis and worse survival
[117,118]. In contrast, total CD68+ TAM numbers did not show a link
with survival, which is indicative of TAM heterogeneity in human
pancreatic cancer and a protumoral role for the M2-like subset [117].
Indeed, the co-existence of HLA-DR+CD68+ M1-like macrophages and
CD163+/CD204+CD68+ M2-like macrophages was shown in PDAC
samples by immunohistochemistry. A high ratio of M1/M2 TAM significantly correlated with prolonged overall survival [119]. Interestingly,
this TAM heterogeneity was confirmed on ex vivo TAMs from freshly
resected PDAC tissue based on a differential HLA-DR expression.
This is again reminiscent of the situation in naïve and tumor-bearing
mouse pancreas. Interestingly, some TAM co-expressed HLA-DR and
CD163, showing that a more mixed phenotype can also exist in vivo
[120]. This finding was confirmed in vitro, whereby conditioned
medium from pancreatic cancer cells induced a mixed M1/M2
CD11c+CD204+ TAM phenotype [121]. Finally, another subset of
human TAM was identified that expressed high levels of folate receptor
beta (FR-β) and VEGF and that mainly resided perivascularly and in the
invasive front of the tumor [122].
Interestingly, repolarization of TAM seems a promising avenue
for the treatment of pancreatic cancer (Fig. 2). Intratumoral expression
of histidine rich glycoprotein (HRG) led to TAM repolarization from
M2 to antitumoral M1, which led to vessel normalization and decreased
tumor growth and metastasis [123]. Similarly, inhibition of Reg3β,
which is overexpressed in PDAC, led to repolarization of TAM to an
M1 phenotype, increased T cell infiltration and impaired tumor growth
[124]. The absence of SPARC (secreted protein acidic and rich in cysteine), a glycoprotein involved in the regulation of ECM deposition, led
to more metastasis in the Panc02 model. Remarkably, the Sparc−/− tumors were less hypoxic, but had a higher infiltration of CD206+CD163+
macrophages. These cells were suggested to be immunosuppressive
and contribute to the observed metastatic phenotype of these tumors
[125]. The enzyme heparanase is also thought to play a role in the
protumoral polarization of TAM in PDAC. Human and murine tumors
that overexpress this enzyme have increased TAM infiltration, with
TAM expressing CCL2, macrophage scavenger receptor 2 (MSR-2), IL10 and VEGF [126]. Modulating TAM by M-CSFR blockade in combination with chemotherapy overcame TAM-induced CTL suppression and
inhibited tumor growth as well as metastasis in a murine orthoptopic
PDAC model [114]. The TAM subset targeted by anti-M-CSFR treatment
were the CD206hi TAM, which were reprogrammed from an immunosuppressive phenotype to a more antitumor phenotype. Anti-M-CSFR
also enhanced immune checkpoint therapy with CTLA4 and PD1 antagonists, which led to tumor regression [115]. Additionally, treatment
of PDAC with anti-CD40 mAb was shown to increase the amount of
tumoricidal TAM that expressed CD86 and MHC-II, with significant
antitumor effects in mice and human [127,128]. Despite these efforts,
pancreatic cancer remains a very difficult disease to treat and new treatment avenues are being sought after.
5. Macrophage diversity in brain and in brain tumors
The potentially differential contribution of resident versus peripheral myeloid cells during tumor growth is also particularly relevant in the
brain. Microglia, the macrophages of the central nervous system, are
critical regulators of neuroinflammation and accumulating evidence
suggests that they are important actors during tumor progression. The
ontogeny of microglia makes them somewhat unique in the spectrum
of resident tissue macrophages (Fig. 1). Microglia are the only resident
macrophages that are known to be exclusively derived from yolk sac
macrophages without monocyte intermediate [4]. Yolk sac macrophages derive from erythromyeloid precursors that arise during the
primitive hematopoiesis in the yolk sac as early as E8.5 and infiltrate
29
the brain between E9.5 and E10.5 before the emergence of HSC [2,
129–131]. After embryonic colonization, the early postnatal days are
accompanied by a massive microglial expansion, which relies on in
situ proliferation [131]. Besides microglia, which reside in the brain
parenchyma, there are at least three other resident macrophage populations in the healthy brain: the perivascular, meningeal and choroid plexus macrophages [132]. While all these populations share typical
macrophage markers (e.g. CD11b, Iba-1, F4/80 and CX3CR1), they can
be distinguished from microglia by their higher levels of CD45 expression [132]. The ontogeny of the non-parenchymal CNS macrophages is
not completely clear, but they are likely to be HSC-derived [133,134].
In addition, while being absent in the healthy brain, peripheral myeloid
cells, such as Ly6Chi monocytes infiltrate the CNS following neuroinflammation. It is becoming increasingly clear that microglia and
monocyte-derived myeloid cells play distinct, in some cases perhaps
even opposing, roles during neuro-inflammatory conditions [129,135].
Understanding the differential roles of the resident brain vs. infiltrating
bone-marrow derived myeloid cells is therefore important in the
context of brain tumors.
Notably, the term “resting” to denote microglia under steady state is
misleading. In vivo imaging experiments have shown that under
homeostatic conditions, microglia are highly dynamic cells that are
continuously surveying the microenvironment by extending and
retracting motile processes at high speed [136]. Besides their important
role as scavengers, new discoveries are suggesting the potential involvement of microglia in refining neural circuits, in neural plasticity and in
learning and memory [137,138]. It is therefore not surprising that
microglial dysfunction is gradually being linked to a wide variety of
neurological diseases, with brain tumors not forming an exception.
The large majority of malignant brain tumors are gliomas, which are
graded I–IV, with grade IV gliomas also termed glioblastoma multiform
(GBM) [139,140]. GBM, which has an invariably terminal prognosis, is a
highly invasive tumor characterized by hypoxic and necrotic centers
and shows clear signs of inflammation and neoangiogenesis. A large
accumulation of myeloid cells in GBM is a common feature, with
TAMs often constituting up to 30% of the tumor mass [141]. The number
of TAMs also positively correlates with the histological grade of human
gliomas [142]. Whether the TAM compartment is mainly composed of
resident microglia or infiltrating monocyte-derived macrophages is
unclear and still debated (Figs. 1–2). It is not straightforward to distinguish microglia from infiltrating monocyte-derived macrophages, especially since inflammation and/or glioma-derived factors may alter both
the microglial and peripheral macrophage phenotype. For example, it
has been suggested that in gliomas, microglia increase their expression
of CD45, a marker that is often used to distinguish resident from bloodderived myeloid cells [143]. In mice, bone-marrow chimeras may provide insights, with the confounding factor that the irradiation process
can alter blood–brain barrier integrity and induce significant levels of
inflammatory cytokines [144]. More recently, a set of microgliaspecific markers have been identified, which are not expressed in
monocytes or other tissue macrophages [145,146]. Such markers may
more easily allow the discrimination of resident vs. blood-derived
macrophages, provided that the glioma microenvironment does
not induce their expression in the latter subset. For example, the
microglial-expressed protein F11R is also acquired by monocytes upon
their differentiation in the glioma microenvironment [147].
Importantly, accumulating evidence suggests that the myeloid
compartment is essential for glioma progression. An important first
question is whether TAMs are involved in the transition of low- to
high-grade tumors. Interestingly, inhibiting M-CSFR signaling via the
chemical inhibitor BLZ945 significantly blocks tumor formation and
malignant progression in a mouse model of platelet-derived growth
factor B-driven glioma initiation [148]. BLZ945 treatment could also
induce regression in large established tumors, indicating that TAMs
can play a prominent role both in the early and late phases of glioma
progression. Depleting myeloid cells in the brain via ganciclovir
30
Q. Lahmar et al. / Biochimica et Biophysica Acta 1865 (2016) 23–34
treatment in CD11b-HSVTK mice also dramatically inhibits tumor progression [149,150]. An important glioma-promoting function of TAMs
may be their assistance in tumor invasion, for example via the production of membrane type-1 metalloprotease [149]. Conversely, myeloid
cells in gliomas may also display anti-tumor responses. Tumor necrosis
factor produced by TAMs has been suggested to inhibit glioma progression [151]. Furthermore, i.p. injections of ganciclovir in CD11b-HSVTK
mice, results in a moderate reduction of tumor-infiltrating myeloid
cells, which, surprisingly, was reported to accelerate glioma growth
[152]. Since a full myeloid depletion — by continuous local ganciclovir
delivery via osmotic pumps — strongly suppresses glioma growth in
CD11b-HSVTK mice [149,150], this may suggest that the myeloid
compartment consists of both pro- and antitumoral subsets and that
perhaps mainly the latter was targeted in the study by Galarneau et al.
[152]. As a matter of fact, elimination of bone marrow-derived Tie2expressing monocytes/macrophages in human glioma xenografts growing in the brain of nude mice seems sufficient to strongly diminish
angiogenesis and cause tumor regression [47].
To assess the activation state of myeloid cells in gliomas, a recent
study performed microarray analysis on purified CD11b+ cells from
two different mouse glioma models and compared them to CD11b+
cells from naive brains [153]. This showed an increase in both M1 and
M2 genes, which most likely reflects the highly heterogeneous myeloid
compartment in gliomas. In this regard, it is important to consider
whether microglia, who clearly have a different ontogeny and perform
unique functions in the CNS, can adopt M2 phenotypes similar to
monocyte-derived macrophages. It seems that at least in vitro, M2 stimuli less efficiently induce the expression of some typical M2 markers in
microglia [154]. However, glioma progression does seem to be coupled
to an M2-like phenotype in the myeloid compartment. In human
gliomas, the number of macrophages that express the M2 markers
CD163 and CD204 positively correlates with tumor grade [142]. In addition, M-CSFR blockade, which significantly inhibits glioma progression,
does not deplete TAMs, but reduces the expression of several M2
markers, suggesting a tumor-detrimental switch in macrophage polarization [148]. Finally, periostin, a major factor secreted by GBM stem
cells, was shown to be instrumental for attracting monocytes to the
tumor site and driving their differentiation into M2-like cells [155].
Silencing of periostin specifically reduced M2-type TAM and potently
inhibited tumor growth of the GBM stem cell-derived xenografts.
Other approaches that have suggested to curb glioma growth by altering the macrophage activation state rely on the use of cyclosporin A
and amphotericin B [156–158] or on TEM that have been engineered
to express IFNα specifically at the tumor site [159]. A more thorough
understanding of the molecular and functional heterogeneity of
myeloid cells in gliomas will undoubtedly provide even more treatment
opportunities.
6. Macrophage diversity in liver and in liver tumors
The liver is an intriguing organ to study the role of macrophages
in cancer, since Kupffer cells (KC) represent the largest pool of tissueresident macrophages in the body, making up to 35% of the liver nonparenchymal cells, which equals on average 80% of the whole body
tissue macrophage population at steady state [160]. KC appear to be
entirely derived from Csf1r+ progenitors with erythro-myeloid potential (erythro-myeloid progenitors or EMP) that emerge in the yolk sac
at embryonal day E8.5 in mouse [2,161] and fully differentiate into
tissue-resident macrophages without the presence of a monocyte intermediate stadium (Fig. 1). KC are long-lived and are hardly replaced by
HSC-derived cells under steady-state conditions.
KC reside on the luminal side of hepatic sinusoids, playing a role of
scavengers and guardians of immunological tolerance at steady state,
but also contributing to the pathogenesis of various acute and chronic
liver diseases leading to liver injury and fibrosis [162]. In these pathological conditions, bone marrow-derived monocytes are recruited to
the liver, locally differentiating into populations of monocyte-derived
macrophages. Studying the relative contribution of these ontogenically
distinct macrophage populations to functions as diverse as initiating
and perpetuating inflammation and promoting fibrosis, but also restoring homeostasis and abrogating fibrogenesis, is an active field
of research [163]. Likewise, the relative contribution of KC versus
monocyte-derived macrophages during different stages of hepatocellular carcinoma development is an outstanding question.
Hepatocellular carcinoma (HCC), the most abundant type of primary
liver cancer and the third leading cause of cancer-related death worldwide [164], is a prototypical example of an inflammation-associated
cancer (Figs. 1–2), as the presence of a chronic inflammatory state in
the liver appears instrumental for its initiation and development.
Hence, a better insight in the relative importance of KC and monocytederived macrophages during this process is mandatory. In the case
of HCC, multiple mouse models have been developed to unravel the
link between inflammation and liver cancer formation. For instance,
Mdr2-knockout mice, liver-specific lymphotoxin LTα and β overexpressing mice, hepatocyte specific NEMO-deleted mice and hepatocyte
specific TAK1-deleted mice all represent inflammation-based HCC
models, where activated inflammatory signaling pathways lead to
chronic hepatitis, which ultimately results in hepatocarcinogenesis
[165]. In contrast, chemical induction of HCC (eg via diethylnitrosamine
or DEN administration) provides a model whereby initial DNA damage
and the resulting genetic mutations in an otherwise healthy liver, only
secondarily lead to inflammation. Alternatively, mutated oncogenes
(eg NrasG12V) can be directly inserted in hepatocytes via hydrodynamic
injection of transposable elements [166]. In all these scenarios, and also
in livers of HCV-infected patients, pre-malignant senescent hepatocytes
are suggested as the initiators of HCC [166]. Senescence surveillance,
i.e. the elimination of senescent hepatocytes, depends on the helper
function of CD4+ T cells and monocytes/macrophages as effector cells
[166]. Strikingly, administration of the RB6-8C5 (anti-Ly6G/Ly6C)
antibody, which depletes neutrophils and monocytes but not tissueresident macrophages, completely disrupted the clearance of premalignant hepatocytes. Hence, monocyte-derived macrophages, but
not liver-resident KC, were suggested to be the effector cells.
Conversely, macrophages were also suggested to contribute to
DEN-induced HCC formation. For instance, specific loss of IKKβ in
hepatocytes, thereby impairing NF-κB activation, augments DENinduced hepatocyte death and carcinogenic compensatory proliferation [167]. Interestingly, loss of IKKβ in both hepatocytes and Kupffer
cells (but also B cells and other macrophages through inducible Mx1Cre mediated excision) had the opposite effect, reducing compensatory proliferation and carcinogenesis. Hepatocyte cell death was
found to drive the NF-κB-dependent hepatomitogen (mainly IL-6)
production by KC, thus explaining the loss of hepatocarcinogenesis in
Mx1-Cre x IKKb-f/f mice. Notably, a higher IL-6 production through
MyD88 signaling in KC of male mice is responsible for the gender disparity in HCC formation [168]. Along the same line, Trem1 (triggering
receptor expressed on myeloid cells-1) deficiency in mice attenuated
hepatocellular carcinogenesis triggered by DEN, a phenotype reverted
by the administration of WT CD11b+F4/80+ macrophages/KC [169].
Trem1 is needed for KC/macrophage activation by inducing transcription and protein expression of IL-6, IL-1β, TNF, CCL2 and CXCL10.
Combined, the above results illustrate the necessity of the presence of
pro-inflammatory Kupffer cells in the liver at the early stages of chemically induced hepatocarcinogenesis. Especially tissue-resident KC,
but not recruited monocyte-derived cells, might be needed in the DEN
model, since tumor incidence and growth are unaltered in livers
of DEN-treated D6-deficient mice, which are heavily infiltrated by
monocyte-derived macrophages [170].
Once a primary tumor is established, macrophages can further
contribute to HCC progression, although the distinction between KC
and monocyte-derived macrophages has not been made in most studies. One study reports a reduced presence of KC — defined as CD68+
Q. Lahmar et al. / Biochimica et Biophysica Acta 1865 (2016) 23–34
cells present in the blood space of cancerous tissue or in the sinusoids
of noncancerous tissues — in HCC tissue compared to noncancerous
tissue from the same livers and a further decrease of intratumoral KC
presence as tumor size increases, suggesting that monocyte-derived
macrophages may play a more prominent role [169]. TAM presence
in cancerous tissue of HCC patients is most often defined via immunohistochemistry as CD68+ or CD14+ cells, and their numbers correlate
with HCC stage, with markers related to tumor progression and stemness
and with reduced overall and disease-free survival [171–173]. Notably,
also the presence of CD14+CD16+Tie2+ cells (i.e. TEM) in the blood or
tumors of HCC patients significantly correlates with HCC angiogenesis
[174]. Conversely, CD68+ cell density in nontumor areas does not correlate with survival, suggesting a different phenotype of macrophages
at the tumor site [173]. Indeed, macrophages infiltrating the tumor
mass in patients and orthotopic mouse models were shown to be more
M2-oriented, with high expression of CD206, CD163, SR-A and CCL22,
the latter of which contributes to venous infiltration of cancer cells
and metastasis [175,176]. Tim3, a well described immunosuppressive
molecule for T cells, is highly expressed on patient HCC-associated
TAM and facilitates M2-like macrophage activation [177]. One of the
key molecules secreted by these TAM is IL-6, that stimulates HCC
growth at least in part by driving CD44+ HCC cancer stem cell expansion [171]. Moreover, the presence of these TAM correlates with
intratumoral Treg numbers and their depletion (via GdCl3) reduces
Treg accumulation [178].
Interestingly, in line with the concept of TAM heterogeneity, macrophages located in the HCC peritumoral stroma may be somewhat more
M1-like, expressing higher levels of HLA-DR and inflammatory cytokines such as IL-1β, and IL-23 allowing them to induce protumoral
Th17 and Tc17 responses [179,180]. In addition, these cells express
high levels of PD-L1, under the influence of autocrine IL-10 and TNF,
resulting in the suppression of anti-tumor T-cell activity [172,173].
The ontogeny of these cells is not entirely clear, but one paper describes
their presence in the peritumoral sinusoids and in close proximity of
M-CSF production, which may classify them as bona fide KC [181].
7. Concluding remark
Macrophages have been proposed as novel therapeutic targets and
several strategies to eradicate or modulate these cells are being evaluated. However, a major gap in our current understanding of TAM biology
is the existence of ontogenically, molecularly and functionally distinct
TAM subsets in tumors. It is therefore conceivable that particular TAM
populations are strongly promoting tumor progression and metastases
and contribute to therapy resistance, while other populations are rather
anti-tumoral. For example, CD206+ TAM are typically strongly
proangiogenic and immunosuppressive and mediate tumor relapse following anti-angiogenic, radiation and chemotherapy [22,54], suggesting that the specific targeting of this TAM population would be
beneficial. Developing such approaches requires a more detailed insight
in the environmental stimuli that instruct TAM heterogeneity within a
tumor, in the signaling pathways and effector molecules that contribute
to TAM functionality and in the appreciation that macrophages residing
in different tissues display different characteristics. Though more and
more studies are appearing describing the presence, regulation and
function of tumor-associated macrophages, Tie2-expressing monocytes
and other myeloid cells in human tumors (eg [174,182–184]), the available evidence for TAM heterogeneity in patients is scarce. Future work
will need to identify useful markers to discriminate between human
TAM subsets and to firmly establish correlations between particular
TAM populations and outcome for the patient.
Acknowledgments
QL is supported by a grant from Stichting tegen Kanker. JK is
supported by a doctoral grant of Vrije Universiteit Brussel. DL is
31
supported by a postdoctoral Emmanuel van der Schueren grant from
the Vlaamse Liga tegen Kanker. KM is supported by an Attract Brains
to Brussels grant from Innoviris Brussels, EVO is supported by a doctoral
grant from FWO-Vlaanderen, JAVG is supported by several research
grants from Vlaamse Liga tegen Kanker, Stichting tegen Kanker, FWOVlaanderen, IWT-Vlaanderen and Vrije Universiteit Brussel.
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