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Cancer Letters 264 (2008) 204–215

www.elsevier.com/locate/canlet

Mini-review

Cancer related inflammation: The macrophage connection

Antonio Sica a, Paola Allavena b,*, Alberto Mantovani b,c
a
Fondazione Humanitas per la Ricerca, 20089 Rozzano, Milan, Italy
b
Istituto Clinico Humanitas, Fondazione Humanitas per la Ricerca, Via Manzoni 11a/A,
20089 Rozzano, Milan, Italy
c
University of Milan, Via Mangiagalli, 20133 Milan, Italy

Received 26 February 2008; received in revised form 26 February 2008; accepted 12 March 2008

Abstract

Tumor-associated macrophages (TAM) are key regulators of the link between inflammation and cancer. In the tumor
microenvironment neoplastic cells shape the differentiation and functional orientation of TAM which, in turn, express sev-
eral protumoral functions, including secretion of growth factors and matrix-proteases, promotion of angiogenesis and sup-
pression of adaptive immunity. This review analyzes our current knowledge of TAM and their involvement in tumor
development and progression. The interplay between TAM and neoplastic cells represents a promising target of future
therapeutic approaches.
Ó 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Tumor-associated macrophages; Macrophage polarization; Tumor promotion; Therapeutic targets; Inflammation; Cancer;
Chemokines; Cytokines; Angiogenesis

1. Origin of TAM come in some tumor types, including for instance

Solid tumors are infiltrated with leukocytes
and the cross-talk between neoplastic and
blood-borne cells have profound effects on tumor
progression. Leukocytes account for up to 50%
of the tumor mass, the most represented subsets
being lymphocytes and macrophages. The pres-
ence of immunocompetent cells within human
tumors has been considered as a proof of an
immunological anti-tumor response. Indeed, the
density of T lymphocytes has been significantly
associated with a more favourable clinical out-
*
Corresponding author. Fax: +39 0282245101.
E-mail address: paola.allavena@humanitas.it (P. Allavena).
colorectal and ovarian cancer, melanoma [1–5].
In contrast, the density of macrophages is corre-
lated in most – though not all – tumors with
increased angiogenesis, tumor invasion and poor
prognosis [6–9].
The presence of TAM at the tumor site repre-
sents one of the hallmarks of cancer-associated
inflammation. [10–13]. TAM derive from circulat-
ing monocytes which are selectively attracted
within the tumor microenvironment by locally
produced chemotactic factors. Historically, a
tumor-derived chemotactic factor (lately identified
as CCL2) was described 25 years ago [14]. Experi-
mental and human studies have confirmed that
the levels of tumor-derived CCL2 significantly

0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2008.03.028
 A. Sica et al. / Cancer Letters 264 (2008) 204–215
correlate with TAM density in ovarian, breast,
pancreatic and other tumors [6,13]. Other chemo-
kines (e.g. CCL5, CCL7, CCL8, CXCL12) or
cytokines e.g.: vascular endothelial growth factor
(VEGF), platelet derived growth factor (PDGF)
and macrophage colony stimulating factor (M-CSF)
have been shown to attract blood monocytes at
the tumor site [15]. TAM accumulate into necrotic
regions of tumors, characterized by low oxygen
tension [16]. This preferential localization is regu-
lated to a large degree by tumor hypoxia which
induces the expression of hypoxia inducible factor
(HIF-1)-dependent factors, like for instance
VEGF, the chemokine CXCL12 and its receptor
CXCR4, which modulate TAM migration in avas-
cular regions [17–19].
Incoming monocytes differentiate in the tumor
microenvironment to tissue resident macrophages
mainly due to the effects of tumor-derived growth
factors, especially M-CSF. Functional plasticity is
a well known characteristic of the mononuclear
phagocyte system and the paradigm of M1 and
M2 polarization identifies the two extremes of the
whole spectrum of macrophage functional activities
(see for review 20) Briefly, while M1 macrophages
have immunostimulatory Th1-orienting properties,
M2 cells have an IL-12low, IL-10high profile, poor
antigen-presenting capacity and suppress Th1 adap-
tive immunity. In addition, M2 macrophages
actively scavenge debris, promote wound healing,
angiogenesis and tissue remodeling. Microenviron-
mental signals expressed at the tumor microenvi-
ronment play a central role in the orientation of
differentiating macrophages and recent studies have
clarified that TAM represent a distinct M2-polar-
ized macrophage population [21]. Among the fac-
tors having the potential to promote the
polarization of M2 macrophages are PGE2, TGFb,
IL-6 and IL-10 [22,23]. The immunosuppressive
cytokines IL-10 and TGFb are produced by many
types of cancer cells and by TAM themselves. IL-
10 promotes the differentiation of monocytes to
mature macrophages and blocks their differentia-
tion to DC. Thus, a gradient of tumor-derived IL-
10 may preferentially push incoming monocytes
toward macrophage differentiation, in different
micro-anatomical localizations of a tumor. Such sit-
uation was observed in breast cancer and in papil-
lary carcinoma of the thyroid, where TAM were
evenly distributed throughout the tissue, in contrast
to DC which were present only in the periphery
[24].
205
2. Protumoral role of TAM
The pro-tumorigenic activities of TAM cover
several crucial features of neoplastic cells: prolifer-
ation, migration and metastasis; survival in
hypoxic conditions due to stimulated angiogenesis;
immune evasion due to suppression of anti-tumor
immunity.
Already in the late ‘70s it was clear that in the
absence of M1-orienting signals (LPS and IFNc)
TAM rather promoted tumor cell growth in vitro
[13,25–27]. Studies in experimental murine models
showed that tumors producing CCL2 and harbor-
ing higher numbers of TAM had more spontaneous
lung metastases compared to parental non-produc-
ing tumors. In turn, genetic studies in mice have
shown decreased rates of tumor growth and metas-
tasis to be associated with decreased TAM number
[6,28].
Intercross of transgenic mice susceptible to mam-
mary cancer (PyMT), with mice containing a reces-
sive null mutation in the M-CSF gene (Csfop)
demonstrated that TAM recruitment is an absolute
requirement for productive metastatic growth [29].
Lin and colleagues [30] demonstrated a slower rate
of progression to malignancy and fewer pulmonary
metastases in macrophage-deficient CSF-1 null
mutant mice bearing spontaneous mammary carci-
noma than in CSF-1 wild-type mice. In this mouse
model, they also showed that TAM play a crucial
role in the ‘‘angiogenic switch” when hyperplastic
lesions develop into early stage mammary carcino-
mas [30]. It is interesting to note that SHIP1-defi-
cient mice, which exhibit a spontaneous drift
towards M2 macrophages polarization, experience
increased growth of transplanted tumors [31].
The protumoral role of TAM in human cancer is
supported by many clinical studies that found – in
most tumors – a correlation between the high mac-
rophage content and poor patient prognosis [6–
9,11]. Accordingly, genes associated to macrophage
infiltration (e.g. CD68) or differentiation (M-CSF)
are part of recent molecular signatures which herald
poor prognosis in lymphoma and breast cancer
[32,33].
Many macrophage products released in the
tumor stroma can directly stimulate the growth of
tumor cells and/or promote tumor cell migration
and metastasis (Fig. 1). These include epidermal
growth factor (EGF), members of the FGF family,
TGFb, VEGF, chemokines and cytokines. It is well
established that IL-1b augments metastasis [34–37].
206 A. Sica et al. / Cancer Letters 264 (2008) 204–215

Fig. 1. Protumoral functions of tumor-associated macrophages (TAM) and interplay with tumor cells. Tumor-derived chemotactic factors
(CC-chemokines, eg. CCL2), M-CSF and VEGF actively recruit blood monocytes at the tumor site, where they differentiate to resident
macrophages. Several TAM products directly stimulate the growth of tumor cells (e.g. EGF, FGF, cytokines). TAM also contribute to the
angiogenic switch by releasing angiogenic factors (VEGF, FGF, TGFb, chemokines) and to the degradation and remodeling of the matrix
with metalloproteases, MMPs. Inhibition of anti-tumor responses is achieved by the secretion of immunosuppressive cytokines, like IL-10
and TGFb, and by selective recruitment of naı¨ve T cells, via the chemokine CCL18, and of Th2 and Treg, via the chemokines CCL17 and
CCL22.

Genetic ablation of IL-1b in mice resulted in
absence of metastasis development, either with mel-
anoma cells or with mammary and prostate tumors,
suggesting the importance of IL-1b in the tumor
milieu. In co-culture experiments with tumor cells,
macrophages lead to enhanced invasiveness of the
malignant cells by a TNF-a dependent MMP induc-
tion in the macrophages [13]. In lung cancer, TAM
may favour tumor progression by contributing to
stroma formation and angiogenesis through their
release of PDGF, in conjunction with TGF-b pro-
duction by cancer cells [38]. Ahmed et al., described
a method to observe the orientation of individual
tumor cells as they enter blood vessels, in real time
and in a living animal [39]. They found that tumor
cells seem to be attracted to macrophages, which
line the outside of the vessels. Goswami et al.
described a paracrine signaling loop between tumor
cells and macrophages, in which tumor cells secrete
M-CSF which, in turn, causes macrophages to
secrete EGF, a chemoattractant for tumor cells.
Interrupting either of these signals resulted in
decreased tumor cell motility [40].
TAM contribute to tumor progression also by
producing several factors which enhance neo-angio-
genesis and the dissolution and remodeling of the
interstitial matrix (Fig. 1). Tumor angiogenesis is
often activated during the early, preneoplastic
stages of tumor development and is controlled by
a number of positive or negative regulators pro-
duced by cancer cells and tumor-associated leuko-
cytes [41,42]. Macrophages can exert a dual
influence on blood vessel formation: on the one
hand they produce molecules that are proangiogen-
ic, on the other they can express anti-angiogenic
molecules and damage the integrity of blood vessels
[43]. In general, as for interaction with neoplastic
cells, the pro-angiogenic functions of TAM prevail.
Indeed, several studies in human cancer have shown
that TAM accumulation is associated with
increased angiogenesis and with the production of
angiogenic factors such as VEGF and PDGF [44].
 A. Sica et al. / Cancer Letters 264 (2008) 204–215
TAM accumulate in hypoxic regions of tumors and
hypoxia triggers a pro-angiogenic program in these
cells. A number of molecules with possible impact
on angiogenesis have been shown to be expressed
by macrophages in low oxygen conditions, such as
VEGF, TNF-a, bFGF and CXCL8 [13,18]. There-
fore, macrophages recruited in situ represent an
indirect pathway of amplification of angiogenesis,
in concert with angiogenic molecules directly pro-
duced by tumor cells (Fig. 1).
A unique subset of tumor-promoting monocytes
has recently been identified in mouse subcutaneous
tumors, human glioblastoma xenografts and spon-
taneous pancreatic tumors [45–47]. These cells
express the angiopoietin receptor Tie-2 and prefer-
entially localize to areas of high angiogenesis where
they show marked pro-angiogenic activity [46]. Tie-
2+ monocytes have also been identified in a variety
of human tumors where they are found largely in
peri-vascular and avascular, viable areas of human
tumors, but are missing in non-neoplastic tissues
adjacent to tumors [47].
Lymphoangiogenesis is mediated by the action of
VEGF-C and VEGF-D acting on the receptor
VEGFR3. More recently VEGF-A, a chemotactic
factor for monocytes, was shown to increase lymp-
hoangiogenesis, via the recruitment of circulating
monocytes [48]. In human cervical cancer, VEGF-
C production by TAM was proposed to play a role
in peritumoral lymphoangiogenesis and subsequent
dissemination of cancer cells with formation of lym-
phatic metastasis. Additionally, TAM participate to
the pro-angiogenic process by producing the angio-
genic factor thymidine phosphorylase (TP), which
promotes endothelial cell migration in vitro and
whose levels of expression are associated with tumor
neovascularization [49].
The contribution of chemokines to angiogenesis
has been the object of intensive investigation. A
variety of CXC-chemokines have been detected in
neoplastic tissues as products of tumor cells or stro-
mal elements [6]. Several CXC- chemokines have an
important role in melanoma progression by stimu-
lating neoplastic growth, promoting inflammation
and inducing angiogenesis. Strong evidence demon-
strates that levels of CCL2 are associated with TAM
accumulation and that CCL2 may play an impor-
tant role in the regulation of angiogenesis [6].
Macrophages can produce enzymes and inhibi-
tors which regulate the digestion of the extracellular
matrix, thus favoring tumor invasion. TAM pro-
duce several matrix-metalloproteases (e.g. MMP2,
207
MMP9) and activators of MMPs, such as chemo-
kines. Direct evidence has been presented that
MMP-9 derived from hematopoietic cells of host
origin contributes to skin carcinogenesis [50]. Che-
mokines have been shown to induce gene expression
of various MMPs and, in particular, MMP-9 pro-
duction along with the uPA receptor [51]. Evidence
suggests that MMP-9 has complex effects beyond
matrix degradation including promotion of the
angiogenetic switch and release of growth factors
[50].
TAM contribute also to the deposition of matrix
proteins. The secreted protein acidic and rich in cys-
teine (SPARC) has gained much interest in cancer
being either up-regulated or down-regulated in pro-
gressing tumors. SPARC produced by macrophages
present in tumor stroma can modulate collagen den-
sity, leukocytes and blood vessel infiltration [52].
An additional characteristic of M2 macrophages
which is shared by TAM population is the ability to
suppress the adaptive immune response through
mechanisms including poor antigen-presenting
activity and inhibition of T cell proliferation [26].
As mentioned above, TAM produce and release sev-
eral immunosuppressive cytokines, of which IL-10
has been most studied. Moreover they produce
low levels of immunostimulatory cytokines such as
TNFa, IL-1 and IL-12 [53]. It has been reported
that IL-10, alone or in concert with IL-6 is respon-
sible for the up-regulation of macrophage B7-H4
expression, a molecule implicated in the suppression
of antigen-specific anti-tumor immunity [54]. The
defective production in TAM of the major immuno-
stimulatory cytokine IL-12 may also be due to the
activity of IL-10, produced either by TAM and by
tumor cells [53]. At least some of these immunosup-
pressive activities might be regulated by over-activa-
tion of transcription factors, such as STAT3.
STAT3 inhibition results in enhanced cytotoxicity
and antigen-presenting function in activated macro-
phages and is associated with retardation of tumor
growth [55].
Part of the immunesuppressive activity of TAM
is exerted indirectly by their release of chemokines
that preferentially attract T cell subsets devoid of
cytotoxic functions. CCL18 has been identified as
the most abundant chemokine in the ascitic fluid
of human ovarian carcinoma [56]. When the source
of CCL18 was investigated, it was tracked to TAM,
with no production by ovarian carcinoma cells. In
normal macrophages CCL18 is inducible by Th2
cytokines: IL-4, IL-13 and IL-10, and recruit naive
208 A. Sica et al. / Cancer Letters 264 (2008) 204–215
T cells by interacting with an unidentified receptor
[11]. Attraction of naive T cells in a peripheral
microenvironment dominated by M2 macrophages
and immature DC is likely to induce T cell anergy.
Two other chemokines, CCL17 and CCL22, are
abundantly expressed by TAM [12,28]. These che-
mokines interact with the CCR4 receptor, expressed
mostly by Th2 cells and by Treg [73], two T cell sub-
sets lacking anti-tumor functions (Fig. 1).
In addition to TAM, a number of reports have
identified a myeloid M2-biased cell population pres-
ent in tumors and lymphoid organs of tumor-bear-
ing hosts, referred to as myeloid-derived
suppressor cells (MDSC), which are suggested to
contribute to the immunosuppressive phenotype
[57]. These cells share properties and gene expres-
sion profile with M2 polarized TAM, yet also dis-
play distinct features, and are characterized by the
expression of the Gr-1 and CD11b markers
[57,58]. MDSC use two enzymes involved in the
arginine metabolism to control T cell response:
inducile nitric oxide synthase (NOS2) and arginase
(Arg1), which deplete the milieau of arginine, caus-
ing peroxinitrite generation, lack of CD3f chain
expression by T cells and T cell apoptosis [57,59].
3. Molecular basis of TAM polarization
As mentioned above, the tumor microenviron-
ment has the capacity to pilot recruitment, matura-
tion and differentiation of infiltrating leukocytes
and play a central role in the activation of specific
transcriptional programmes expressed by TAM.
Evidence shows that polarized M2 macrophage
functions expressed during tumor development
have an important impact on tumor progression
[26]. We and others have analyzed the molecular
basis of the TAM phenotype and identified the
transcriptional factors NF-jB, STAT3 and HIF-1
as master regulators of their transcriptional
programmes.
3.1. NF-jB and STAT3 in inflammation-associated
cancer
NF-jB induces several cellular alterations associ-
ated with tumorigenesis and more aggressive pheno-
types, including: self-sufficiency in growth signals;
insensitivity to growth inhibition; resistance to
apoptotic signals; immortalization; angiogenesis;
tissue invasion and metastasis [60]. Constitutive
NF-jB activation often observed in cancer cells
may be promoted by either microenvironmental sig-
nals, including cytokines, hypoxia and reactive oxy-
gen intermediates (ROI), or by genetic alterations
[60]. In particular, pro-inflammatory cytokines
(e.g. IL-1 and TNF), expressed by infiltrating leuko-
cytes, can activate NF-jB in cancer cells and con-
tribute to their proliferation and survival. These
findings propose NF-jB as a possible target for
development of anticancer treatments and clinical
trials with drugs that block NF-jB are currently
in progress with promising results. In contrast with
cancer cells, TAM from advanced established neo-
plasia display defective NF-jB activation in
response to various pro-inflammatory signals [53].
Defective NF-jB activation in TAM correlates with
impaired expression of NF-jB-dependent
inflammatory functions (e.g. expression of cytotoxic
mediators, such as NO, cytokines, TNFa, IL-1 and
IL-12) observed in these cells [53,61,62]. We have
recently identified the molecular mechanisms driv-
ing TAM unresponsiveness toward pro-inflamma-
tory signals (e.g. IL-1, TNF) and demonstrated
that these cells are characterized by a massive
nuclear localization of the p50 NF-jB inhibitory
homodimer, associated with lack of M1-functions
and tumor progression [61].
Two recent papers [63,64] outlined the impor-
tance of autocrine IL-6 as an important activator
of oncogenic STAT3 in lung adenocarcinomas and
in breast tumor mammospheres [65]. Interestingly,
STAT3, which is a point of convergence for numer-
ous oncogenic signaling pathways, is constitutively
activated both in neoplastic and in immune cells in
the tumor microenvironment and plays a role in
oncogenesis by both promoting cell transformation
and inhibition of apoptosis [66]. Its role in cancer
development was further proved in various human
malignancies, including prostate, lung, brain,
breast, and squamous cell carcinomas [67]. Evidence
has been provided showing that STAT3 activation
in cancer promotes tumor immune evasion by inhib-
iting the expression of Th1-type immunostimulating
molecules and promoting the expression of immu-
nosuppressive factors, leading to the inhibition of
DC maturation [55,68]. In agreement, inhibition of
STAT3 activity, which is required for both the bio-
logical functions and gene transcription of IL-10,
resulted in both restored expression of pro-inflam-
matory mediators (eg. IL-12 and TNF-a) by infil-
trating leukocytes, as well as tumor inhibition [55].
The role of p50 NF-jB and STAT3 in establish-
ing protumoral functions of infiltrating leukocytes
 A. Sica et al. / Cancer Letters 264 (2008) 204–215
relies on their role in transmitting M2 polarizing sig-
nals to the nucleus (e.g., IL-10, IL-4, and IL-13)
[58]. This scenario is in agreement with accumulat-
ing evidence suggesting that high expression of
genes belonging to a type 2 immunological profile
correlates with bad prognosis in colorectal cancer
(eg. high IL-10, TGFb1 and VEGF) [1] and hepato-
cellular carcinoma (eg. high IL-10, IL-4, IL-5 and
M-CSF) [69]. Thus, overexpression of master regu-
lators of M2 polarized functions (eg. STAT3 and
p50 NF-jB) during tumor growth concur to estab-
lish permissive conditions for tumor progression
[58].
These observations were obtained in TAM iso-
lated from tumors characterized by advanced stages
[53,61] and are in apparent contrast with the inflam-
matory functions expressed by TAM during can-
cerogenesis [70,71]. It is tempting to speculate that
inflammatory cells and circuits supporting tumor
growth undergo to functional evolution during dis-
ease progression and that key therapeutic molecules
(e.g. NF-jB) can be transiently expressed and tar-
getable at different stages of tumor progression
[58]. Thus, although full activation of NF-jB in
inflammatory cells resident in preneoplastic sites,
macrophages in particular, might exacerbate local
M1 inflammation and favor tumorigenesis, tumor
evolution would result in progressive inhibition of
NF-jB, as well as enhanced STAT3 activity in infil-
trating leukocytes, as observed in tumor bearers
[58]. In line with this hypothesis, restoration of
NF-jB and inhibition of STAT3 activity in tumor-
associated myeloid cells were shown to restore
anti-tumor functions in vivo [58]. These observa-
tions are in agreement with a Toll-like receptor 4-
dependent contribution of the immune system to
anticancer chemotherapy and radiotherapy. In par-
ticular, tumor antigen-specific T-cell immunity
involved the secretion of the high-mobility-group
box 1 (HMGB1) alarmin protein by dying breast
tumor cells and the action of HMGB1 on TLR4
expressed by DC. Cancer patients who carry a
TLR4 loss-of-function allele relapsed more quickly
after radiotherapy and chemotherapy than those
carrying the normal TLR4 allele [72].
3.2. Intratumoral hypoxia and HIF-1
Intratumoral hypoxia and genetic alterations can
lead to HIF-1a overexpression, which activates the
transcription of genes that are involved in crucial
aspects of cancer biology, including angiogenesis,
209
cell survival, glucose metabolism and invasion. Acti-
vation of HIF-1a-dependent genes has been associ-
ated with increased patient mortality in several
cancer types [73]. In pre-clinical studies, inhibition
of HIF-1 activity has marked effects on tumor
growth and efforts are underway to identify inhibi-
tors of HIF-1 and to test their efficacy as anticancer
therapeutics [73]. Under conditions of normal oxy-
gen tension, the HIF-a subunit is hydroxylated by
specific prolyl-hydroxylases and targeted for rapid
proteasomal degradation by the von Hippel-Lindau
(VHL) tumor suppressor, which is the substrate rec-
ognition component of an E3-ubiquitin ligase
[74,75].
TAM accumulate preferentially in the poorly vas-
cularized regions of tumors which are characterized
by low oxygen tension [17]. Such environment pro-
motes TAM adaptation to hypoxia, which is
achieved by the increased expression of hypoxia
inducible and pro-angiogenic genes such as VEGF,
bFGF and CXCL8, as well as glycolytic enzymes,
whose transcription is controlled by HIF-1 and
HIF-2 [17,76]. The in vivo relevance of this meta-
bolic adaptation of macrophages was recently dem-
onstrated by Cramer et al. [77]. Ablation of the
hypoxia responsive transcription factor HIF-1a
resulted in impaired macrophage motility and inva-
siveness of extracellular matrix. This evidence high-
lights the relevance that the hypoxia-HIF-1
pathway may play in the recruitment and activation
of TAM into solid tumors and may be instrumental
for TAM-mediated angiogenesis and tumor metas-
tasis. We have recently shown that HIF-1a activated
in TAM by hypoxia influences the positioning and
function of tumor cells, stromal cells and TAM, by
selectively upregulating their expression of chemo-
kine receptor CXCR4 [19]. Moreover, HIF-1 activa-
tion can have a role in the induction of the CXCR4
ligand, CXCL12 [78], a chemokine involved in can-
cer metastasis [79]. Hypoxia may also have impor-
tant consequences on L-Arg metabolism in TAM,
and thereby on the suppression of adaptive immu-
nity, since it can induce NOS2 and ARG expression
(in this case with a certain variability in terms of
ARG1 and ARG2) in various cell types [58].
Accumulating evidence suggests that intersec-
tions and compensatory pathways may exist
between the NF-jB and HIF-1 systems. Colon can-
cer cells knockdown for HIF-1a preserved their
capability to promote strong angiogenic response
through the production of hydrogen peroxide and
subsequent activation of NF-jB-mediated and
210 A. Sica et al. / Cancer Letters 264 (2008) 204–215

CXCL8-dependent angiogenesis [80]. In contrast, clodronate-encapsulated liposomes [82] or amino-

the small ubiquitin-related modifier (SUMO), which
was found to be expressed in the necrotic zone of
gliomas, inhibits NF-jB activity through the stabil-
ization of IjB, which leads to the inhibition of its
target genes (eg. Cox-2 and CXCL8) and activates
the HIF-1a-dependent VEGF expression [81].
3.3. TAM as therapeutic target
TAM express a multifaceted protumoral pheno-
type which may offer therapeutic targets, including
their recruitment, survival, mechanisms of activa-
tion and polarization, immunosuppression, angio-
genic activity and matrix remodeling (Fig. 2).
3.4. TAM recruitment and survival
Depletion of macrophages using transgenic
mouse approaches or the use of pharmacological
drugs that knock down macrophages in vivo, like
bisphosphonate (Zometa; Novartis) has been shown
to reduce tumor progression in several experimental
tumor models. Clodronate-mediated depletion of
TAM in mice bearing F9 teratocarcinoma or human
A673 rhabdomyosarcoma xenografts resulted in
reduced tumor angiogenesis [83]. Anti-tumor agents
with selective cytotoxic activity on monocyte-mac-
rophages would be ideal therapeutic tools for their
combined action on tumor cells and TAM. We
recently reported that Trabectedin, a natural prod-
uct derived from the marine organism Ecteinascidia
turbinata, with potent anti-tumor activity [84] is spe-
cifically cytotoxic to macrophages and TAM, while
sparing the lymphocyte subset. This compound
affects the transcription of selected genes, including
NF-Y, a transcription factor of major importance
for mononuclear phagocyte differentiation. In addi-
tion Trabectedin inhibits the production of CCL2
and may therefore interfere with monocyte recruit-
ment at the tumor site. Inhibition of IL-6 both by

Fig. 2. Therapeutic strategies to prevent TAM protumoral functions. TAM accumulate in the hypoxic areas of tumor, favouring
angiogenesis, suppression of adaptive immunity, matrix remodeling, tumor progression and metastasis. The figure summarizes strategies
impairing selective TAM protumoral functions ( ) or restoring anti-tumor activities (+). Cytotoxic drugs (eg. Trabectedin and
Clodronate) may decrease TAM number and prevent protumoral functions. A similar result may be obtained by limiting TAM
recruitment (Linomide). Restoration of M1 immunity (STAT-3 and p50 NF-jB inhibitors; anti-IL-10 plus CpG; Arginase I and IDO
inhibitors) would provide cytotoxic activity and re-activation of Th1 specific anti-tumor immunity. Inhibition of both pro-inflammatory
cytokines and growth factors expression (NF-jB inhibitors) may disrupt inflammatory circuits supporting tumor growth and progression.
MMPs inhibitors would prevent cancer cells spread and metastasis. Finally, inhibitors of TAM-mediated angiogenesis (HIF-1 inhibitors)
would restrain blood supply and inhibit tumor growth.
 A. Sica et al. / Cancer Letters 264 (2008) 204–215
TAM and tumor cells was also observed [85]. These
anti-inflammatory properties of Trabectedin may
add on and extend its anti-tumor activity.
3.5. Targeting TAM activation and polarization
Restoration of NF-jB activity in TAM is there-
fore a potential strategy to restore M1 inflammation
and intratumoral cytotoxicity [58]. In agreement,
recent evidence indicates that restoration of an M1
phenotype in TAM may provide therapeutic benefit
in tumor-bearing mice. In particular, combination
of CpG plus an anti-IL-10 receptor antibody
switched infiltrating macrophages from M2 to M1
and triggered innate response debulking large
tumors within 16 h [86]. It is likely that this treat-
ment may restore NF-jB activation and inflamma-
tory functions by TAM. Moreover, TAM from
STAT6 -/- tumor-bearing mice display an M1 phe-
notype, with low level of arginase and high level
of NO. As a result, these mice immunologically
rejected spontaneous mammary carcinoma [87].
These data suggest that switching the TAM pheno-
type from M2 to M1 during tumor progression may
promote anti-tumor activities. In this regard, the
SHIP1 phosphatase was shown to play a critical role
in programming macrophage M1 versus M2 func-
tions. Mice deficient for SHIP1 display a skewed
development away from M1 macrophages (which
have high inducible nitric oxide synthase levels
and produce NO), towards M2 macrophages (which
have high arginase levels and produce ornithine)
[31].
MDSC are a myeloid M2-biased cell population
present in lymphoid organs and peripheral tissues
of tumor-bearing hosts and potently contributing
to T cell immunosuppressionvia the release of
NOS2 and Arg1[59]. In prostate cancer, selective
antagonists of these two enzymes were proved ben-
eficial in restoring T cell-mediated cytotoxicity [59].
Finally, the IFN-c-inducible enzyme indoleamine
2,3-dioxygenase (IDO) is a well known suppressor
of T cell activation. It catalyzes the initial rate-lim-
iting step in tryptophan catabolism, which leads to
the biosynthesis of nicotinamide adenine dinucleo-
tide. By depleting tryptophan from local microenvi-
ronment, IDO blocks activation of T lymphocytes.
Recently it was shown that inhibition of IDO, which
is highly expressed by macrophages, may cooperate
with cytotoxic agents to elicit regression of estab-
lished tumors [88] and may increase the efficacy of
cancer immunotherapy [89].
211
3.6. Angiogenesis
The potent angiogenic factor VEGF is com-
monly produced by tumors and there is evidence
that it significantly contributes to macrophage
recruitment in the tumor site. TAM themselves pro-
mote angiogenesis and there is evidence that inhibi-
tion of TAM recruitment plays an important role in
anti-angiogenic strategies. In a murine prostate can-
cer model, the anti-angiogenic agent Linomide
caused significant reduction of tumor volume by
inhibiting the stimulatory effects of TAM on tumor
angiogenesis [90]. Based on this, the activity of Lin-
omide, or of other anti-angiogenic drugs, on the
expression of pro-angiogenic molecules by TAM
may be considered valuable targets for anticancer
therapy. Due to the localization of TAM into the
hypoxic regions of tumors, viral vectors were used
to transduce macrophages with therapeutic genes,
such as IFNc, that were activated only in low oxy-
gen conditions [91]. These works present promising
approaches which use macrophages as vehicles to
deliver gene therapy in regions of tumor hypoxia.
3.7. Matrix remodeling
TAM produce several matrix-proteases (MMPs)
and also release MMP activators, such as chemo-
kines [92]. Inhibition of this pathway may prevent
matrix degradation as well as tumor cell invasion
and migration. The biphosphonate zoledronic acid
is a prototipical MMP inhibitor. In cervical cancer
this compound suppressed MMP-9 expression by
infiltrating macrophages and inhibited metallopro-
tease activity, reducing angiogenesis and cervical
carcinogenesis [93].
3.8. Effector molecules
Cyclooxygenase-2 (COX-2) is a key enzyme in
the prostanoid biosynthetic pathway. COX-2 is
up-regulated by activated oncogenes (i.e., ß-catenin,
MET, RET/PTC) but is also produced by TAM in
response to tumor-derived factors like mucin in the
case of colon cancer. The usage of COX-2 inhibitors
in the form of nonsteroidal anti-inflammatory drugs
is associated with reduced risk of diverse tumors
(colorectal, esophagus, lung, stomach and ovary).
Selective COX-2 inhibitors are now thought as part
of combination therapy [12]. Finally, pro-inflamma-
tory cytokines (e.g. IL-1 and TNF), expressed by
infiltrating leukocytes, can activate NF-jB in cancer
212 A. Sica et al. / Cancer Letters 264 (2008) 204–215
cells and contribute to their proliferation, survival
and metastasis [11,35], thus representing potential
anticancer targets.
4. Conclusions
TAM are key orchestrators of the smouldering
inflammation present in the tumor microenviron-
ment. In the majority of experimental and clinical
studies TAM have been associated with cancer pro-
gression. TAM produce a host of growth factors for
epithelial and endothelial cells, as well as inflamma-
tory cytokines and chemokines that contribute to
tumor survival, proliferation and invasion. In addi-
tion, immunosuppressive mediators released by
local inflammatory or tumor cells extinguish host-
mediated anti-tumor responses and facilitate tumor
progression. Thus TAM appear as attractive candi-
date of novel therapeutic strategies. Three major
aspects of TAM, potentially amenable of therapeu-
tic interventions are:
(i) inhibition of their recruitment and/or of their
survival at the tumor site; (ii) inhibition of their
positive effects on angiogenesis and tissue remodel-
ing; (iii) reversal of their immune-suppression and
restoration of anti-tumor cytotoxicity.
An increasing amount of pre-clinical studies spe-
cifically targeting TAM have yielded encouraging
results. Although these results have not yet been suc-
cessfully translated into the clinic, therapeutic target-
ing of macrophages represents a valuable strategy to
complement established anticancer treatments.
Acknowledgments
This work was supported by Associazione Itali-
ana Ricerca sul Cancro (AIRC), Italy; European
Community and Ministero Istruzione Università
Ricerca (MIUR), Italy; Istituto Superiore Sanita’
(ISS).
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