TUMOUR-ASSOCIATED LEUCOCYTES
The infiltration of leucocytes into solid tumours was remarked upon more than 100
years ago when it was suggested that they had a causal role in carcinogenesis. These
infiltrates are now known to contain myeloid cells (neutrophils, dendritic cells,
macrophages, eosinophils and mast cells) as well as lymphocytes. However, controversy
remains over the relationship between these host cells and tumour progression. In
the past, their presence has been construed as evidence for a host response against
the growing tumour. This is because such immune functions were often observed in transplantable
tumour models that by their nature represented a transplant that could elicit immune
rejection despite being placed in immunocompromised hosts. However, it is becoming
clear that tumours growing naturally are largely recognized as self and lack strong
foreign antigens. Instead, they appear to have been selected to manipulate the host
immune system to prevent rejection (Dunn et al, 2002) and use this system to facilitate
their own growth and spread (Khong and Restifo, 2002). This lack of immune response
has become particularly evident with the study of tumours induced by the restricted
expression of oncogenes in transgenic mice where it has been established that tumour-associated
leucocytes are often active participants in the neoplastic process. In addition, there
is a growing body of clinical data on a wide range of solid tumour types that has
correlated a high density of leucocytic infiltration with poor outcome (Coussens and
Werb, 2001, 2002). Furthermore, it has be recognised that cells containing DNA alterations
caused by viral or chemical carcinogens do not progress to become cancerous until
they are exposed to a second type of stimulus that often includes chronic irritants
or inflammatory agents (Coussens and Werb, 2002). For example, an inflammatory response
is required to induce cancers in chickens infected with the potent oncogenic Rous
Sarcoma virus despite it carrying the v-src oncogene that alone is competent to transform
fibroblasts in culture (Sieweke et al, 1989). This view of the role of leucocytes
in facilitating cancer progression has been further enhanced by the realisation that
many cancers are caused or promoted by infectious or other agents that induce chronic
inflammation (Coussens and Werb, 2002).
Under normal physiological circumstances, leucocytes are recruited in response to
wounding, inflammatory or pathogenic stimuli. They are attracted by the local synthesis
of chemokines (chemoattractive cytokines), cytokines and growth factors as well as
products of tissue breakdown. These are all part of a signalling system that involves
recognition of the pathological state, organisation of an appropriate cellular response
and suppression of this response once the situation is resolved. During cutaneous
wound healing, these processes require epithelial cell proliferation and migration,
angiogenesis and tissue remodelling (Nathan, 2002). In tumours, it is thought that
similar chemoattractive factors are also responsible for the recruitment of leucocytes
and that these cells play roles comparable to those observed during wound healing.
However, because of the accumulation of intrinsic mutations the epithelial cells have
lost positional identity and consequently do not stop growing and migrating on cue.
Instead, they send out continuous signals that recruit leucocytes to continue to support
the tumor's development. This concept has led to the rubric that tumours are ‘wounds
that never heal’ (Balkwill and Mantovani, 2001).
Several steps are crucial for a tumour to become metastatic. Tumour cells need to
be able to break out of their confining basement membranes in order to enter the extracellular
matrix and circulation. These processes require the proteolytic breakdown of basement
membranes, changes in epithelial cell adhesion, migration and the suppression of ankoisis.
They are matched in the surrounding stroma with angiogenesis as well as the frequent
recruitment of leucocytes. Angiogenesis, known to be the crucial process for tumour
progression by providing oxygen and nutrients and removal of waste products, as well
as, providing an expanding endothelial surface for the tumour cells to enter the circulation,
also involves degradation of basement membranes followed by migration of endothelial
cells into the tumour stroma (Folkman, 2002). Recent studies have shown that the tumour-associated
leucocytes produce factors that promote all these steps associated with malignancy
within tumours (Ribatti et al, 2001). This review will focus on the evidence that
members of the myeloid lineage, particularly macrophages, neutrophils and mast cells,
can facilitate tumour progression.
Macrophages
Macrophages derived from circulating monocytes represent a major component of the
infiltrated leucocytic population in the tumour microenvironment. These cells have
a wide range of functions in immunity, during development and in tissue repair. They
can adopt a particular phenotype according to the demand and produce many factors
ranging from chemokines, cytokines, and proteases, to angiogenic and growth factors.
They therefore appear to be the ‘jack-of all trades’ of the myeloid lineage. Of all
the cells of the myeloid lineage, the evidence is strongest in support of a positive
impact of macrophages on tumour progression. For example, in greater than 80% of clinical
studies, an increase in tumour-associated macrophages (TAMs) density is correlated
with poor prognosis, with less than 10% of studies showing the converse (Bingle et
al, 2002). Similarly, overexpression of macrophage chemoattractants within tumours
has also been shown to correlate with poor prognosis (Leek and Harris, 2002). One
such example is colony stimulating factor-1 (CSF-1 or-macrophage CSF), a macrophage
growth factor as well as a potent macrophage chemoattractant (Lin et al, 2002). Overexpression
of CSF-1 correlates with poor prognosis in human breast, ovarian endometrial and prostatic
carcinomas (Kacinski, 1997). In breast cancers, this overexpression correlates with
a strong leucocytic infiltration in over 95% of cases (Scholl et al, 1994). Similarly,
the CC chemokine ligand 2 CCL2/MCP-1 (MCP=monocyte chemoattractant protein 1) has
been identified as a major chemokine for macrophages recruitment in several human
tumours, including the bladder (Amann et al, 1998), cervix (Riethdorf et al, 1996),
ovary (Negus et al, 1995), lung (Arenberg et al, 2000) and breast (Valkovic et al,
1998; Ueno et al, 2000). The level of CCL2/MCP-1 expression is correlated with the
increased infiltration of macrophage (Ueno et al, 2000) and the grade of tumour (Amann
et al, 1998; Valkovic et al, 1998). Although both CSF-1 and CCL-2 can be targeted
to the tumour cells themselves, the strong correlation of overexpression of these
macrophage chemoattractants with macrophage recruitment and poor prognosis suggests
that TAMs can play a major role in the progression of tumours to metastasis.
Several experiments have supported the role of macrophages in tumour progression.
We have observed that in a Polyoma Middle T antigen-induced mouse model of breast
cancer (PyMT), an increase of macrophage infiltration at the primary tumour site occurred
immediately before the onset of malignant transition (Figure 1
Figure 1
Leucocytic infiltration promotes tumour progression to malignancy. In a manner similar
to wounded tissues, solid tumours induce a local ‘inflammatory response’ by attracting
leucocytes into its microenvironment. Such an infiltration consists of multiple cell
types of which cells of the myeloid lineage are the major component. Leucocytes in
such ‘inflammatory sites’ produce an array of growth and angiogenic factors, proteases
and mutagenic factors that promote tumour growth, invasion and angiogenesis. However,
different from its physiological counterpart whose inflammation ceases when the wound
has healed, tumour-induced inflammation persists and eventually leads to tumour progression
and metastasis.
) (Lin et al, 2001). Using genetic approaches, we demonstrated that depletion of CSF-1
in this model markedly decreased the infiltration of macrophages at the tumor site
and this correlated with a significant delay of tumour progression to metastasis.
In contrast, overexpression of CSF-1 in the tumour dramatically increased the macrophage
density in the primary tumour and this was correlated with an accelerated malignant
switch (Lin et al, 2001). Similarly, removal of CSF-1 from transplanted tumours also
resulted in an impairment of growth with extensive necrosis and poor vascularisation,
phenotypes that could be reversed by treatment of the mice with CSF-1 (Nowicki et
al, 1996). These studies have provided strong evidence that TAMs promote the tumour
progression to malignancy. This conclusion was enhanced by the observations that treatment
of mice that had been xenotransplanted with either a human colonic or embryonic tumour
with antisense oligonucleotides directed against mouse CSF-1 reduced tumour growth
and prolonged survival. This was associated with a reduced serum concentration of
CSF-1 and a decreased TAM density. Since mouse CSF-1 does not stimulate the human
receptor, these data argue for the effects of the CSF-1 antisense molecules to be
mediated through the reduction in TAMs (Aharinejad et al 2002).
The evidence from both clinical and experimental studies supports the view that, in
most cases, TAMs facilitate tumour progression and metastasis. The mechanism(s) macrophages
used to promote tumour progression are still unknown; however, it has been proposed
that macrophages may promote tumour growth and angiogenesis through the production
of growth factors and angiogenic inducers such as Epidermal Growth Factor (EGF), vascular
endothelial growth factor (VEGF), tumour necrosis factor (TNFα) and Thymidine Phosphorylase
(TP) (Xiong et al, 1998; Leek and Harris, 2002). Macrophages also indirectly enhance
blood vessel formation by possessing a procoagulant activity through fibrin deposition
(Mantovani et al, 1992). In addition, many macrophages produced factors, proteases
and protease activators such as transforming growth factor-β (TGFβ), platelet-derived
growth factor, interleukin-6 (IL-6), urokinase plasminogen activator and Tissue-type
Plasminogen Activator (t-PA) that may cause degradation of extracellular matrix to
facilitate the tumour cell invasion and migration and induce angiogenesis (Egami et
al, 2003; Eubank et al, 2003; Hildenbrand et al, 1995; Klimetzek and Sorg, 1977).
Moreover, TAMs contribute greatly to the growth of the tumour by producing proangiogenic
and tumour-stimulating chemokines such as CCR2 ligands (Vicari and Caux, 2002).
Macrophages can display tumour cytoxicity and can potentially present tumour antigens
to induce specific immune reaction against tumours. However, these cells are believed
to have primarily a protumour function since both tumours and TAMs produce potent
immunomodulating agents that suppress macrophage tumoricidal activity. Such tumour-produced
molecules, including IL-4, IL-6, IL-10, CSF-1, TGFβ and prostaglandin E2 (PGE2), and
TAM-produced factors such as IL-10 and PGE2, contribute to the general immunosuppression
of the host as well as the antitumour activity of macrophages (Elgert et al, 1998;
Mytar et al, 2003). Although infiltration of macrophages is usually correlated with
poor outcome (Hamada et al, 2002), recent studies have also shown that infiltrated
macrophages may have an anti tumour action in colorectal cancer (Nakayama et al, 2002;
Noguchi et al, 2003). These observations indicate that the microenvironment of different
type of tumours might alter the activities of infiltrated leucocytes from tumour promotion
to tumour rejection. This has led to the idea of tumour-educated macrophages whose
functions are modified by the local cytokine/chemokine environment (Pollard, 2004).
In most cases, this enhances tumour development and directs the local immune system
away for an antitumour response (Mantovani et al, 2002).
Dendritic cells (DCs) also play an important role in both the activation of antigen-specific
immunity and the maintenance of tolerance, providing a link between innate and adaptive
immunity. Several clinical studies have reported the presence of DC within human tumours
such as the stomach, colon, prostate, kidney, thyroid, breast and melanoma (Tsujitani
et al, 1990; Enk et al, 1997; Troy et al, 1998; Bell et al, 1999; Lespagnard et al,
1999; Schwaab et al, 1999, 2001; Scarpino et al, 2000). However, the effect of such
an infiltration in tumour progression is still not clear. Some of these studies have
shown that the infiltration of DC was associated with enhanced patient survival (lung),
whereas others showed that DC present in tumour either were minimally activated (Tsujitani
et al, 1990; Troy et al, 1998), had no correlation with metastasis-free or overall
survival of the patients (Lespagnard et al, 1999) or were converted to ‘silencers’
of antitumour immune responses by tumour-produced factors (Enk et al, 1997). In addition,
studies have reported that patients with a variety of cancers have impaired function
of DCs, indicating a systemic effect of the tumours on DCs (Almand et al, 2000). Moreover,
recent studies have suggested that, instead of initiating immune responses against
tumours, DC in the tumour microenvironment may have the ability to turn off the responding
T cells and induce tolerance (Hackstein et al, 2001; Vicari and Caux, 2002). The relationship
between host DC, lymphocytes and tumour in the process of ‘tumour escape’ from the
host immune system has been reviewed in detail recently and will not be discussed
here (Khong and Restifo, 2002; Hanahan et al, 2003). Nevertheless, the evidence on
balance suggests that tumours promote the suppression of these potentially damaging
cells while enhancing the trophic nature of macrophages. The hope, however, is that
therapeutic modulation of this environment locally could result in TAMs that are tumoricidal
and that together with properly matured DCs would present antigens to infiltrating
T cells with the consequent rejection of the tumour (Dranoff, 2004).
Mast cells
An infiltration of mast cells has been found in a variety of human cancers, including
non-small-cell lung cancer (Shijubo et al, 2003), breast cancer (Kankkunen et al,
1997), colorectal cancer (Lachter et al, 1995), basal cell carcinoma (Yamamoto et
al, 1997) and pulmonary adenocarcinoma (Imada et al, 2000). The accumulation of mast
cells has been associated with enhanced growth and invasion of several human cancers
(Ribatti et al, 2001). However, there are other studies in colorectal cancer where
their presence is indicative of improved prognosis (Nielsen et al, 1999). Initial
studies using animal models have shown that increasing mast cell density in tumour
promoted tumour growth (Roche, 1985), whereas reducing their number inhibited tumour
growth and angiogenesis (Starkey et al, 1988). The role of inflammatory mast cells
in tumour progression of squamous epithelial carcinogenesis was also illustrated recently
(Coussens et al, 1999). Furthermore, Schwann cell tumours caused by inherited mutations
in the NF1 gene do not form in mouse models unless the surrounding stromal cells are
at least heterozygous for the mutation. These tumours are highly populated with mast
cells and it seems likely that a haploinsufficiency of NFI in these cells is the cause
of the tumour formation (Zhu et al, 2002).
The best known role that mast cells plays in tumour progression is their ability to
induce tumour angiogenesis (Hiromatsu and Toda, 2003). Activated mast cells produce
a variety of angiogenic growth factors, including VEGF, basic fibroblast growth factor,
IL-8 and TNFα (Meininger and Zetter, 1992; Qu et al, 1995; Hiromatsu and Toda, 2003).
In addition, they can produce specific angiogenic mediators including histamine and
heparin, which can stimulate endothelial cell proliferation and may contribute to
the hyperpermeable nature of newly formed microvessels during pathological angiogenesis
(Ribatti et al, 2001), and a variety of proteases, particularly MMP9, which are involved
in angiogenesis. How tumour cells regulate the infiltration and activation of mast
cells is still not fully understood. However, several types of tumours produce stem
cell factor that may have functions in mast cell migration, proliferation and activation
(Turner et al, 1992). In addition to the promotion of angiogenesis, the activated
mast cells are a rich source of cytokines and chemokines such as IL-1, IL-3, IL-4,
IL-8, granulocyte–macrophage colony-stimulating factor, TNFα, interferon-γ (IFNγ),
CCL-2, Macrophage Inflammatory Protein MIP-1α and β, many of which can contribute
to the tumour microenvironment by enhancing tumour cell growth and invasion either
directly or through intermediaries such as macrophages (Burd et al, 1989; Selvan et
al, 1994).
Neutrophils
The role of neutrophils in tumour progression is still controversial. During immune
responses, they are among the first cells to arrive at sites of infection where they
are highly bactericidal. They are also involved in cell killing during graft rejection
and thus they might be considered as potential antitumour cells. However, clinical
studies have been contradictory. The presence of increased numbers of tumour-infiltrating
neutrophils was linked to poorer outcome in patients with adenocarcinoma of the bronchioloalveolar
carcinoma subtype (Bellocq et al, 1998), whereas studies of gastric carcinoma suggested
that neutrophil infiltration correlated with good prognosis (Caruso et al, 2002).
It has been reported that tumours prolong alveolar neutrophil survival through the
production of soluble factors (Wislez et al, 2001). Using transplantable tumour models,
studies have shown that tumour-associated neutrophils were involved in tumour angiogenesis
by the production of proangiogenic factors such as VEGF and IL-8 (Schaider et al,
2003), proteases such as matrix metalloproteinases (Shamamian et al, 2001) and elastases
(Iwatsuki et al, 2000; Scapini et al, 2002). In addition, studies using animal models
have also shown that neutrophils may contribute to genetic instability in tumours
(Haqqani et al, 2000).
Furthermore, neutrophil-recruiting cytokines such as GRO (IL-8 homologues) may also
directly stimulate tumour proliferation in melanoma (Haghnegahdar et al, 2000). Taken
together, an environment that recruits neutrophils might enhance angiogenesis, promote
tumour invasion and stimulate growth.
CONCLUSION
Solid tumours are not just composed of malignant cells, but are complex microcosms
of many cell types including a wide range of haematopoietic cells. The evidence described
above suggests that cells of the myeloid lineages, particularly macrophages, mast
cells and neutrophils, on balance play an active role in enhancing tumour progression
and metastatic capacity. This is through their ability to promote angiogenesis and
tissue remodelling as well as direct effects on epithelial cell viability, growth
and migration. In wound healing or in response to an inflammatory stimulus, a similar
panoply of cells is recruited. Sentinel cells, particularly macrophages and mast cells
that send out chemotactic signals that in the first wave, bringing in neutrophils
and monocytes, initiate this recruitment. These not only eliminate pathogens but also
effect tissue repair, a process that involves angiogenesis and an induction of vascular
permeability, tissue remodelling and the migration and proliferation of epithelial
cells. These events are coordinated by a sophisticated and as yet, not understood
language of soluble mediators involving cytokines, chemokines and growth factors.
Similarly, during development, myeloid cells, particularly macrophages, play an important
role in tissue formation and their absence often results in attenuated poorly formed
structures in tissues as wide ranging as bone, skin and mammary gland (Pollard, 2004).
It would seem highly likely that tumours send out signals similar to those found in
normal physiology to recruit myeloid cells and instruct them to perform similar tasks
in tissue remodelling. However, unlike their normal counterparts, the epithelial tumour
cells do not stop growing in response to positional cues and continuously send out
signals to demand help from the invading myeloid cells.
The tumour microenvironment also educates those invading cells to promote epithelial
growth, viability, motility and invasion. Thus, it is noticeable in many tumour types
that there are dense lymphocytic infiltration sites adjacent to areas of basement
membrane breakdown and tumour invasion (Figure 1). In our studies of the PyMT oncoprotein-induced
mammary cancers, these sites marked the transition from nonmalignant to malignant
tumours, suggesting that they had a causal role in this process. Furthermore, it appears
in this model that macrophages are the sentinel cells that recruit other myeloid cells.
This was confirmed by the ablation of macrophages that stopped the infiltration sites
from occurring and which resulted in an inhibition of metastatic capacity (Lin et
al, 2001, 2003). The challenge, therefore, will be to define whether there are unique
phenotypes of these tumour-associated myeloid cells that can distinguish them from
those involved in immune or tissue repair responses that could be a target for therapeutic
agents. In addition, it will be important to understand the cytokine network in the
tumour microenvironment that promotes tumour progression so that it can be tilted
away from eliciting tropic activities to one that enhances the detection of the tumours
as an aberrant state with the resultant suppression of its development and perhaps
immunological rejection of the tumour.
Note added in proof
Ablation of macrophage recruitment in transplantable breast cancers by a chemokine
receptor antagonist significantly inhibited tumour development (Robinson et al, 2003).
Similarly, inhibition of MMP9 production in tumour-associated macrophages by a hypomorphic
Ets-2 mutation also inhibited tumour development in the PyMT mouse model of breast
cancer (Man et al, 2003). These data confirms the involvement of macrophages in tumour
development in mouse models.