CANCER INVASION
DISSECTING MOLECULAR MECHANISMS BETWEEN
MACROPHAGES AND GASTRIC CANCER CELLS
ANA PATRÍCIA PEREIRA CARDOSO
TESE DE DOUTORAMENTO EM ENGENHARIA BIOMÉDICA
FACULDADE DE ENGENHARIA DA UNIVERSIDADE DO PORTO
INSTITUTO DE ENGENHARIA BIOMÉDICA
Orientadora: Professora Doutora Maria José Cardoso Oliveira
Para os meus Filhos, Clara e António
Prize L’Oreal for Women in Science (Foundation L’Óreal/FCT/UNESCO), and International Iberian Nanotechnology Laboratory (INL) - 1st PhD recruitment program.
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Tumours are considered malignant when cancer cells fail to respect boundaries and invade the surrounding tissues. Invasive cancer cells may then, through lymph and blood vessels, reach secondary organs and metastasize. During this process, several interactions supporting invasion-related activities are established between cancer cells and stromal components of the tumour microenvironment. Macrophages, in particular, constitute a large portion of the tumour mass and are frequently associated with cancer progression. In breast cancer, macrophages are found at areas of basement membrane degradation or at the invasive front of advanced tumours. A paracrine loop involving the production of colony-stimulating factor-1 (CSF-1) by breast cancer cells and of epidermal growth factor (EGF) by macrophages has been reported to modulate extracellular matrix (ECM) remodelling and intravasation of cancer cells into neighbour blood vessels. The role of macrophages in gastric and colorectal cancer progression, however, is still undetermined. Given that cancer cell invasion and macrophages are appealing and promising targets for anticancer therapies, this project aimed at i) identifying the major signalling pathways involved in the crosstalk between gastric and colon cancer cells and macrophages, ii) understanding how these molecular mechanisms contribute to cancer cell invasion and, finally, iii) designing a therapeutic strategy to target and impair these interactions.
This study demonstrated that soluble factors produced by human macrophages result in increased cancer cell invasion, motility and proteolytic activity and clarified the underlying molecular mechanisms. EGF was identified as a key pro-invasive and pro-motile factor produced by macrophages, with the ability to activate cancer cell EGFR signalling pathway, leading to increased motility and invasion. This signalling pathway involved the phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 smallGTPase activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR
Moreover, this study evidenced that the role of macrophages on gastric and colon cancer cell-related activities was dependent on macrophage phenotype. The anti-inflammatory M2-like macrophages were more efficient in stimulating cancer cell invasion, motility/migration and angiogenesis than their pro-inflammatory M1-like counterparts. Interestingly, both macrophage populations similarly induced EGFR tyrosine phosphorylation and activation of its downstream partners. Distinct abilities in promoting invasion-related activities were due to differences in matrix metalloproteinase activity. These results inspired the design of a therapeutic strategy aiming to counteract macrophage pro-tumour activities and, simultaneously, to reverse their differentiation from an M2-like anti-inflammatory into an M1-like anti-inflammatory phenotype. Therefore, interferon-γ, a pro-inflammatory cytokine known to modulate macrophage differentiation, was successfully incorporated into chitosan/poly(γ-glutamic acid)-based delivery systems. The gradual cytokine release was effective in modulating macrophage morphology, cytoskeleton organization and cytokine profile. Most interestingly, this strategy successfully reversed the stimulation of invasion provided by M2-like macrophages.
In summary, this work highlighted the role of the tumour microenvironment, and in particular of macrophages, in gastric and colon cancer cell-invasion, motility, proteolysis and angiogenesis, identifying, for the first time, the underlying molecular mechanisms. The contribution for such cellular activities of macrophage populations with distinct inflammatory profiles was also elucidated. Additionally, a strategy to modulate macrophage pro-invasive activity at the tumour microenvironment was proposed, as groundwork for a therapeutic approach targeting cancer progression.
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Os tumores são considerados malignos quando as células tumorais ultrapassam os limites do tecido a que pertencem, invadindo os tecidos adjacentes e sendo capazes de atingir e colonizar outros órgãos, através dos vasos sanguíneos e linfáticos, levando à formação metástases. Durante este processo, são estabelecidas várias interações entre as células tumorais e os elementos estromais do microambiente tumoral, que resultam no favorecimento de atividades relacionadas com a invasão. Os macrófagos, em especial, constituem grande parte da massa tumoral e são frequentemente associados à progressão da doença. No cancro da mama, os macrófagos localizam-se em áreas de degradação da membrana basal ou na frente invasiva de tumores mais avançados. Um circuito de ação parácrina entre o CSF-1 (colony-stimulating factor-1), produzido pelas células tumorais, e o EGF (epidermal growth factor), produzido pelos macrófagos, foi descrito como responsável pela remodelação da matriz extracelular e pela entrada das células tumorais nos vasos sanguíneos envolventes. Contudo, o papel dos macrófagos na progressão do cancro gástrico e coloretal permanece indefinido.
Considerando a invasão das células tumorais e os próprios macrófagos como alvos aliciantes e promissores para terapias anti-tumorais, este projeto teve como objectivos i) a identificação das principais vias de sinalização envolvidas na interação entre macrófagos e células tumorais gástricas e de cólon, ii) a compreensão de como estas vias moleculares contribuem para a invasão e, finalmente, iii) a conceção de uma estratégia terapêutica para contrariar estes processos.
Esta investigação demonstrou que fatores solúveis produzidos por macrófagos humanos aumentam a invasão, motilidade e atividade proteolítica das células tumorais, clarificando, ainda, os mecanismos moleculares subjacentes. O EGF, produzido pelos macrófagos, foi identificado como um factor crucial na estimulação da motilidade e invasão, através da ativação nas células tumorais do respetivo recetor (EGFR) e das vias moleculares associadas. Estas vias de sinalização envolvem a fosforilação do Akt, c-Src and ERK1/2 e resultam, também, num aumento da atividade das smallGTPases RhoA e
Adicionalmente, o papel dos macrófagos em atividades das células tumorais gástricas e de cólon mostrou-se dependente do fenótipo macrofágico. Os macrófagos anti-inflamatórios ou do tipo M2 estimularam mais eficientemente a invasão, motilidade/migração e angiogénese associada às células tumorais do que os macrófagos pro-inflamatórios ou do tipo M1. No entanto, níveis semelhantes de fosforilação do EGFR e das moléculas associadas a este recetor foram obtidos com ambas as populações macrofágicas. As diferentes capacidades de estimulação de invasão foram atribuídas a diferenças na atividade de metaloproteases da matriz.
Estes resultados encorajaram a criação de uma estratégia para contrariar as atividades pró-tumorais dos macrófagos e, simultaneamente, reverter a sua diferenciação de um fenótipo anti- (tipo M2) para um pró-inflamatório (tipo M1). O interferão-γ, uma citocina pró-inflamatória modeladora da diferenciação macrofágica, foi incorporado em sistemas de entrega baseados em quitosano e ácido-poly(γ-glutâmico). A libertação gradual desta citocina modelou eficientemente a morfologia, a organização do citoesqueleto e o perfil de citocinas dos macrófagos e contrariou os estímulos pró-invasivos libertados pelos macrófagos anti-inflamatórios (tipo M2).
Resumindo, este trabalho realça o papel do microambiente tumoral e dos macrófagos, em particular, na invasão, motilidade, proteólise e angiogénese associadas às células tumorais gástricas e de cólon e identifica, pela primeira vez, os mecanismos moleculares subjacentes. A contribuição para essas atividades celulares de populações de macrófagos com distintos perfis inflamatórios foi também elucidada. Adicionalmente, é proposta uma estratégia inovadora para modelar as contribuições macrofágicas no microambiente tumoral, visando uma aplicação terapêutica futura contra o cancro.
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Muitas pessoas contribuíram de várias formas para este trabalho, às quais agradeço brevemente nesta secção. Naturalmente, a orientadora/amiga Maria José Oliveira. É imensa a minha admiração, respeito e confiança, não só como cientista mas também como ser humano. Obrigada pelo otimismo, paciência, compreensão, orientação e perseverança. Ao meu co-orientador, Professor Mário Barbosa, agradeço a confiança que depositou em mim para desenvolver este trabalho. Obrigada pela disponibilidade para discussão e preciosas contribuições para o trabalho.
Para além dos contributos científicos e profissionais, algumas pessoas colaboraram também com a sua amizade. Uma das melhores partes deste trabalho foi quando a Ana e a Marta integraram a equipa da Maria. Meninas, sem vocês teria sido muito mais difícil e solitário. Martinha, obrigada pelo contínuo apoio técnico, conceptual e pessoal. Raquel, admiro muito a tua serenidade e inteligência e foi com verdadeiro gosto que tive a sorte de aprender contigo na fase final do trabalho. Joana, muito obrigada pela tua dedicação e ajuda numa fase complicada para ti.
To INL, I thank the funding and also everyone for their support and assistance concerning my PhD scholarship. No INEB, encontrei pessoas incríveis que me apoiaram em momentos importantes da minha vida ;) Ana Lopes, Eliana, Dulce, Ana Paula Filipe, Sandra, Carla e, claro, Sílvia, Suse, Lili, Hugo, Rui: a vossa influência está presente neste trabalho mais do que imaginam, por isso ele é parcialmente vosso.
Mãe e Pai (e Rita): este trabalho é dedicado a vocês, que com o vosso esforço e dedicação apostaram sempre na minha formação (pessoal e não só). Ao meu Marido e Filhos agradeço a inspiração e a força para perseverar e fazer o melhor possível.
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List of abbreviations 16
Chapter I – General Introduction 22
1. CANCER 23
1.1 Hallmarks of Cancer 23
1.2 Invasion-associated cancer cell activities 29
1.2.1 Cell-cell adhesion and cell-matrix interactions 30
1.2.2 Cell migration 33
1.2.3 Proteolysis 34
2. EGFR AND CANCER 38
3. THE TUMOUR MICROENVIRONMENT 40
3.1 Fibroblasts 40
3.2 Endothelial cells 42
3.3 Immune cells 43
4. MACROPHAGES 46
4.1 Macrophage activation 47
4.2 The group of M1-like macrophages 49
4.3 The group of M2-like macrophages 50
5. MACROPHAGES AND CANCER 51
5.1 Macrophages in gastric cancer 53
5.2 Macrophages in colorectal cancer 54
6. TUMOUR-ASSOCIATED MACROPHAGES AS THERAPEUTIC TARGETS 55
7. INTERFERON-γ 57
7.1 Interferon-γ in cancer 58
7.2 Interferon-γ delivery systems 59
8. AIMS OF THE THESIS 60
REFERENCES 62
Chapter II – Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y1086, c-Src, Erk1/2 and Akt phosphorylation and small GTPase activity
Abstract Introduction Results Discussion
Materials and Methods Supplementary Material References 78 81 82 84 95 99 104 108 Chapter III – Article 2 – Matrix metalloproteases as maestros for the dual role of
Introduction Results Discussion Conclusions
Materials and Methods References 118 119 131 134 135 141 Chapter IV – Article 3 – An Interferon-γ-delivery system based on chitosan/poly(γ-glutamic acid) polyelectrolyte complexes modulates macrophage-derived stimulation of cancer cell invasion in vitro
Abstract Introduction Results Discussion Conclusions
Materials and Methods Supplementary Material References 146 149 150 152 166 171 171 180 183 Chapter V – Concluding Remarks and Future Perspectives
References
189 196
Curriculum Vitae 200
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ADAM A disintegrin and metalloproteinase
AKT v-AKT murine thymoma viral oncogene homologue
AR Amphiregulin
Bad Bcl-2-associated death promoter Bax Bcl-2-asociated X protein Bcl-2 B-cell lymphoma 2 protein Bcl-xl B-cell lymphoma extra-large
Bid BH3 interacting-domain death agonist
BTC Betacellulin
c-Src Cellular homologue of Rous sarcoma virus protein CAM Chorioallantoic membrane
Cbl Casitas B-lineage lymphoma
CD Cluster of differentiation CCL Chemokine (C-C motif) ligand Cdc42 Cell division cycle protein 42
Ch Chitosan
CM Conditioned medium
CSC Cancer stem cells
CSF-1R Colony stimulating factor receptor 1 CXCL Chemokine (C-X-C motif) ligand
DAG Diacyl glycerol
DMSA Dimercaptosuccinic acid
ECM Extracellular matrix
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
EPR Epiregulin
ErbB v-Erb avian erythroblastic leukaemia viral oncogene homologue ERK Extracellular regulated MAP kinase
FAK Focal adhesion kinase
FGF Fibroblast growth factor
Gab1 GRB2-associated-binding protein 1 GAP GTPase-activating protein
GDI GDP dissociation inhibitor
GDP Guanine diphosphate
GEF Guanine exchange factor
GM-CSF Granulocyte macrophage colony-stimulating factor Grb2 Growth factor receptor-bound protein 2
GTP Guanine triphosphate
GTPase Guanine triphosphatase
HB-EGF Heparin-binding epidermal growth factor HGF Hepatocyte growth factor
HMGB1 High mobility group box 1 hMSCs Human mesenchymal stem cells
HRG Heregulin
IC Immune complexes
IL Interleukin
IP3 inositol 1,4,5-trisphosphate IRF Interferon response factor
JAK Janus kinase
KSR1 Kinase suppressor of ras 1
LbL Layer-by-layer
Mac Macrophages
M-CSF/CSF-1 Macrophage colony-stimulating factor/ Colony stimulating factor 1 MAPK Mitogen-activated protein kinases
MHC Major histocompatibility complex
MIP-1α Macrophage inflammatory protein 1 alpha MMP Matrix metalloprotease
MR Mannose receptor
MT-MMP Membrane type-matrix metalloprotease mTOR Mammalian target of rapamycin
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural killer cells
NO Nitric oxide
p38 p38 mitogen-activated protein kinase
p53 Phosphoprotein p53
PDGF-C Platelet-derived growth factor C PEMs Polyelectrolyte multilayers
PTX3 Pentraxin
PGE Prostaglandin
PI3K Phosphoinositide 3 kinase
PIGF Phosphatidylinositol-glycan biosynthesis class F protein PIP2 Phosphatidylinositol 4,5-bisphosphate
PLC-γ Phospholipase C-gamma PLGA Poly(lactide-co-glicolide)
PU.1 PU-box binding transcription factor
RB1 Retinoblastoma 1
RGD Arginine-glycine-aspartic acid ROI Reactive oxygen intermediates SDF-1 Stromal-derived factor 1
SHC Src homology 2 domain containing
siRNA Small interfering ribonucleic acid
SLAM Signalling lymphocyte activation molecule
SOS Son of sevenless
SR Scavenger receptor
STAT Signalling transducer and activator of transcription TAMs Tumour-associated macrophages
TGF-β Transforming growth factor beta TIMP Tissue inhibitor of metalloprotease
TLR Toll-like receptor
TNF-α Tumour necrosis factor alpha VEGF Vascular endothelial growth factor
VitD3 Vitamin D3
General Introduction
1. CANCER
Gastric and colorectal cancers are the third and fourth leading causes of cancer-related deaths worldwide, respectively. In the particular case of Portugal, colorectal cancer ranks as the first most frequent and mortal cancer (Globocan, 2012). These diseases are often diagnosed in advanced stages and are associated with a poor prognosis for patients (1,2).
Cancer initiating cells develop as a consequence of genetic or epigenetic occurrences that accumulate sequentially, leading to disturbance of the normal programs of cell division and cell differentiation (3). An heterogeneous population arises, and the clones that escape normal defence mechanisms are selected, eventually evolving into a population of cells able to invade the adjacent tissues and, through blood and lymph vessels, reach and colonize distant organs establishing metastasis (4).
1.1 Hallmarks of cancer
As cells progress gradually to a neoplastic state, they acquire a series of abilities that support tumour growth and progression, as proposed by Hanahan and Weinberg (5,6). These hallmark capabilities include maintenance of chronic proliferation, escape from negative regulators of cell proliferation, gain of apoptosis resistance, adjustments in metabolism to support cell growth and division, acquisition of an unlimited replicative potential, evasion of immune surveillance, stimulation of angiogenesis, and activation of invasion and metastasis (Fig.1) (5). These activities will be herein briefly addressed.
Sustained proliferation
In normal tissues, cell growth and proliferation are under careful control to maintain homeostasis. In cancer, signals that regulate cell cycle progression are modified in several ways: i) growth factors can be produced by cancer cells themselves or by instructed tumour-associated host cells, ii) cancer cell surface receptors can be upregulated, iii) structural changes in these receptors can enable ligand-independent signalling or, ultimately, iv) components from signalling pathways downstream these growth factor receptors can be mutated and constitutively activated, supporting a continuous proliferation.
Usually, deregulation of cell proliferation interferes with other cancer cell capabilities, such as unlimited replication, resistance to cell death and altered cell energy metabolism (5,7,8).
Figure 1 – Acquired abilities, referred as “hallmarks of cancer”, involved in the pathogenesis of most human cancers, as proposed by Hanahan and Weinberg in 2011. These events, although not always present simultaneously, are gradually acquired during the multistep development of a tumour and are crucial for its progression. Stromal cells at the tumour microenvironment (centre) also contribute to hallmark capabilities. Adapted from Hanahan and Weinberg, 2011 (5).
Escape from growth suppression
Besides being able to maintain their proliferative potential, cancer cells must also evade signals that negatively regulate their proliferation. This is usually accomplished by inactivation of tumour suppressor genes, such as RB1 and p53, which normally would limit inappropriate cell growth and proliferation or, eventually, activate senescence and apoptotic programs (9). By this reason, tumours harbouring p53 missense or truncating mutations, compromising protein activity, are generally associated with a worse prognosis (10).
Resistance to cell death
It is generally accepted that cancer cells are more resistant than normal cells to cell death stimuli, by the acquisition of mechanisms that counteract necrosis, apoptosis or autophagy.
Necrosis is frequently accompanied by cell membrane disruption with the release of intracellular content into the neighbour tissues. The necrotic cell burden, accompanied by the release of intracellular organelle content, recruits inflammatory cells and induces a strong inflammatory response which in turn, may promote several pro-tumour activities, as further discussed. In their revision of the new generation of cancer hallmarks, Hanahan and Weinberg (5) debated that tumours may gain advantage by tolerating a certain degree of necrosis, since by recruiting tumour-promoting inflammatory cells, they may sustain escape to cell death or promote proliferation, angiogenesis, invasion and metastasis.
In opposition to necrosis, apoptosis does not induce the burst of the dying cell, but instead results in the shrinkage and contraction of the cell body, until elimination by neighbour phagocytic cells. The apoptotic cell cytoplasm is then divided into apoptotic bodies inside of which organelles are still functional. Although apoptosis is understood as a natural barrier to cancer development, cancer cells have developed several strategies to avoid it. These include the loss of the damage sensor and tumour suppressor p53, the increased expression of anti-apoptotic or pro-survival signals (Bcl-2, Bcl-xL, IGF 1/2), the decreased expression of pro-apoptotic factors (Bax, Bid or Bad) or by circumventing the programmed death pathway triggered by specific ligands (5,11). Additionally, oncogenes may not only sustain continuous cell proliferation but may also counteract the induction of cell senescence or apoptosis. Besides the multiplicity of apoptosis-avoiding mechanisms, they generally end to an altered balance between pro- and anti-apoptotic signals, which avoids DNA damage by proteolytic caspases. Cancer cells remain alive, proliferating and perpetuating their advantageous mutations. Instead, during autophagy, cell organelles are engulfed into intracellular vesicles, termed autophagosomes, whilst the associated metabolites are being recycled and reused as an alternative energy source. Later, the autophagosomes fuse with the lysosomes, permitting organelles degradation. Recent research revealed molecular mechanisms common to autophagy and apoptosis (12). The most striking examples are i) the PI3K/Akt/mTOR pathway, which can downstream survival signals, inhibiting both apoptosis and autophagy, and ii) the Beclin-1/Bcl-2 association at the
endoplasmic reticulum, which under nutrient deprivation blocks Beclin-1-dependent autophagy (6,13). Conversely, Beclin-1 overexpression does not increase apoptosis and fails to counteract the anti-apoptotic effect of Bcl-2 (13). Exogenous stimulation may release this complex, allowing Beclin-1-dependent autophagy and the activation of pro-apoptotic signals. Autophagy may be induced during certain states of cellular stress, such as nutrient deprivation, radiotherapy or chemotherapy (12). Paradoxically, it may protect other cancer cells within a tumour from stressing conditions, by allowing them to recycle the metabolites released by the autophagic dying cells. More interesting is the ability of cancer cells to, via autophagy and under limit conditions, shrink the cell body to a state of reversible dormancy, during which minimal energy consumption is undertake (12). This adaptive response is associated with chemoresistance of several tumours and discloses the ability to persist and regrowth of dormant metastases, following treatment with chemotherapeutic agents.
Altered cell energy metabolism
The intense cell proliferation and the reduced cell death lead the tumour to reprogram cell metabolism for energetic maximization. In the presence of oxygen, normal cells convert glucose into pyruvate, releasing 2 ATP molecules, through a glycolytic pathway that occurs at the cytosol. Pyruvate is then converted in the mitochondria, through the oxidative phosphorylation, into carbon dioxide at the same time that 30 to 36 molecules of ATP are being produced. Under anaerobic conditions, glucose is frequently consumed through glycolysis, leading to the accumulation of pyruvate which is then converted into lactic acid, to minimize the consumption of oxygen by the mitochondria (5). A property of cancer cells is an altered glucose metabolism known as aerobic glycolysis, converting glucose to lactic acid, even in the presence of oxygen (14). This phenomenon, known as Warburg effect, can be considered a consequence of activated oncogenes and mutant tumour suppressors (15), but can also be induced by hypoxia itself (16), which is frequently found within tumours. Moreover, it has been proposed that glycolysis, in detriment of mitochondrial oxidative phosphorylation, favours the diversity of biosynthetic pathways, allowing the uptake and
incorporation of nutrients that are necessary for producing and sustain new cells (17). This metabolic adaptation allows tumour survival during insufficient oxygen and vascularization, providing resistance to conditions normally not tolerated by any other cell type.
Unlimited replication
Unlimited replication permits the continuous proliferation of tumour cells, through several generations, without triggering responses of senescence or cell death. Due to the maintenance of telomere length, cancer cells are then able to proliferate indefinitely (18). Telomeres, located at chromosomes’ extremities, shorten progressively with cell proliferation leading to instability, which is generally culminated with cell death. Telomerase expression is frequently upregulated in these cells, which, by elongating DNA at the telomeres, reduces their progressive erosion, and, consequently, allows cells to resist proliferation barriers (6). Recently, telomerase has also been implicated in other tumour-sustaining activities, namely in the activation of proliferation pathways as the Wnt and the β-catenin/TCF/LEF, independently from its telomere maintenance (19). Their contribution to tumour progression remains, however, to be elucidated.
Circumvention of immune eradication
The immune system successfully eliminates the majority of pre-malignant and malignant cells that develop daily in the organism. However, cancer cells that thrive are able to evade this elimination or to avoid immune recognition. In fact, the immune system is now known to play a dual role in cancer: it destroys cancer cells and inhibits tumour growth but it can also promote tumour progression, either by establishing favouring conditions within the tumour microenvironment or by eliminating only highly immunogenic cells, permitting the weakly immunogenic clones to form solid tumours (20,21). During this Introduction, this dual role of the immune system and its contribution for tumour progression will be further disclosed.
Stimulation of angiogenesis
Angiogenesis is important for tumours to grow since it allows the supply of nutrients and oxygen and provides clearance of metabolic waste products and carbon dioxide, as in normal tissues. In
physiological conditions, angiogenesis is transiently activated but in tumours it is chronically stimulated to sustain continuous tumour growth (22). This process, designated angiogenic switch, is promoted by pro-angiogenic factors and supports the continuous proliferation and sprouting of quiescent endothelial cells. Due to this constant pro-angiogenic activation, a disorganized and more permeable tumour vasculature is assembled, due to the reduced presence of pericytes, presenting premature sprouting with excessive and distorted branches (23). Pro-angiogenic signals, such as vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor (b-FGF) or angiopoietin are key factors frequently upregulated by cancer cells or by other cells present at the tumour microenvironment, sustaining angiogenesis (24,25). Generally, these pro-angiogenic factors are secreted into the medium upon tyrosine kinase receptor activation or hypoxic conditions (25). Alternatively, pro-angiogenic factors arrested within ECM components may be released by the action of matrix metalloproteases (MMPs) (26). Interestingly, ECM fragments also released by proteolysis, such as angiostatin (derived from plasmin) or endostatin (derived from collagen-18), are efficient suppressors of angiogenesis (27,28). Once secreted, pro-angiogenic factors may interact with tyrosine kinase receptors at the endothelial cell surface, promoting and activating their downstream interacting partners, leading to endothelial cell migration, sprouting and proliferation (5,29,30). At the tumour site, the leaky new-formed blood vessels are also permissive to the entry of invasive tumour cells that may then, through the circulation, reach another tissues or organs, metastasizing.
Invasion and metastasis
The ability of cancer cells to cross tissue boundaries and to invade neighbouring areas is what confers them malignancy. The multistep process of cancer invasion involves the modulation of signalling pathways controlling cytoskeletal dynamics and motility, alterations in cell-matrix and cell-cell junctions, active cell migration into surrounding tissues, and extracellular proteolytic activity (31). Following invasion of tissues, cancer cells are able to reach pre-existing or newly formed lymph or blood vessels and enter into circulation (intravasation). Isolated or clustered, circulating cancer cells may then extravasate the vasculature wall (extravasation) into a new tissue or organ (32,33). This
new tumour focus may remain dormant, for several months or years. Alternatively, it can proliferate, counteract cell death mechanisms and immune system eradication, and lead to the formation of a secondary tumour, also designated metastasis (Fig. 2), which is considered the major cause of cancer-related deaths (34,35).
Figure 2 – During invasion, tumour cells gain the ability to degrade, and migrate through basement membrane and extracellular matrix (ECM) components. They reach and intravasate into blood or lymph vessels, disseminating through the bloodstream to distant sites, where they may extravasate and colonize, developing secondary tumours and establishing metastasis.
1.2 Invasion-associated cancer cell activities
Invasion is not exclusive of cancer cells, occurring also during embryogenesis, healthy physiological activities like wound healing or immune cell recruitment, and non-malignant diseases. Several molecular changes occur during invasion, namely at the genomic, transcriptional, translational and post-translational levels (33). Coordinated molecular interactions established between the invading cancer cells and the other cellular (fibroblasts, endothelial and immune cells) and non-cellular (ECM)
elements of the microenvironment regulate distinct invasion-related activities, dictating the success of the invasion process.
Some of the most pertinent current questions and challenges in the cancer research field are related to invasion and metastasis: i) understanding the molecular mechanisms that drive cancer cell invasion; ii) dissecting the contribution of other cellular and non-cellular components, present at the tumour microenvironment, in assisting cancer cell invasion and metastasis; iii) designing new therapeutic strategies which target the interactions established at the tumour microenvironment, enhancing their effectiveness; iv) understanding the mechanisms underlying tumour dormancy and subsequent tumour reactivation; and v) identifying the molecular cues that attract circulating tumour cells to the metastatic niche and consent colonization. The investigation reported throughout this thesis delivered important evidence regarding the three first questions, namely by unravelling part of the molecular crosstalk between cancer cells and macrophages (Chapter II), describing the contribution of several macrophage populations to cancer cell invasion (Chapter II and III) and, proposing a strategy to target macrophages at the tumour microenvironment to modulate their role in cancer progression (Chapter IV).
The significance of therapeutic strategies able to control cancer cell invasion arises from the possibility to prevent formation of metastasis, allowing most tumours to be completely resectable by surgery and/or targeted by radiation (36). Failure of available treatments, nonetheless, illustrates the poor understanding of the complex biological mechanisms that underlie cancer cell invasion and metastasis, both at the cellular and molecular levels.
At each step of invasion or the metastatic process, activities related to cell-cell adhesion, cell-matrix interactions, proteolysis and motility are involved and, therefore, susceptible of therapeutic intervention. These activities will be now addressed and their contribution to tumour progression briefly discussed.
1.2.1 Cell-cell adhesion and cell-matrix interactions
Invasive cancer cells frequently exhibit altered expression of cell-cell (as cadherins and immunoglobulin- like molecules) and cell-matrix adhesion molecules. Besides their crucial role on the formation of adhesion complexes, they are also involved in mechanisms of signal transduction and on the modulation of cancer cell survival, metabolism, proteolysis, differentiation and migration
(37) (Fig. 3). At the cell-cell adherens junctions, loss of E- cadherin is widely observed and known to promote cancer cell invasion, dedifferentiation and motility (38). The E-cadherin molecules of one cell form homodimers at the cell surface and establish anti-parallel interactions with other E-cadherin homodimers (homotypic adhesion) on adjacent cells (Fig. 3, left panel). Alternatively, E-cadherin homodimers may interact with other receptors such as the integrin complex αEβ7 of lymphocytes (heterotypic adhesion). When connecting adjacent cells, E-cadherin allows signal transmission to intracellular circuits via the cytoplasmic contact with β-catenin, which in turn is linked to the actin cytoskeleton via α-catenin. E-cadherin loss of function, caused by missense truncating mutations/deletion of the cdh1 gene, methylation of the E-cadherin promoter or by deregulation of its downstream interacting targets, has been widely correlated with increased invasiveness and tumour metastasis (39). The biological function of E-cadherin can also be attenuated by transcriptional repression, receptor endocytosis or even by proteolysis of its extracellular domain (40). Interestingly, p120-catenin, an interacting cytoplasmic partner of E-cadherin and responsible for E-cadherin recruitment to other receptor complexes, is a potent inhibitor of E-cadherin endocytosis, by hampering the recruitment of clathrin-coated pits and by stabilizing this cell-cell adhesion molecule at the cell surface (39). The underlying mechanisms through which loss of E-cadherin function promotes tumour progression are still poorly understood. However, three potential mechanisms have been proposed: the ability of E-cadherin to regulate β-catenin signalling of the canonical Wnt pathway; the inhibition of mitogenic signalling through growth factor receptors and, the connection with molecules that determine epithelial cell polarity (41).
Integrins are adhesion molecules which mediate cell-matrix interactions, regulating cell survival, cell differentiation, ECM proteolysis, and cell migration along ECM components (32), thus playing a crucial role in cell migration, proteolysis and invasion (42). These adhesion complexes are comprised
Figure 3 – Schematic representation of: E-cadherin-mediated cell-cell interactions and, cell-extracellular matrix (ECM) integrin-mediated adhesions. IS and ES represent intracellular and extracellular space, respectively.
by heterodimeric transmembrane molecules, consisting of several α and β subunits linked by disulphide bounds, which can be arranged in several combinations allowing the interaction with multiple ECM elements. At their amino terminal, integrins act as receptors for ECM molecules such as fibronectin, fibrinogen, von Willebrand factor, vitronectin, and proteolysed forms of collagen and laminin. In addition, at their carboxy terminal, integrins are linked to the actin cytoskeleton by proteins like paxillin, vinculin, kindling, talin, which in turn, regulate focal adhesion kinase (FAK) and Src kinase family members, mediating downstream signalling (43) (Fig. 3, right panel). Integrins also regulate expression and activity of proteolytic enzymes that degrade the basement membrane, such as the matrix metalloproteases (MMPs) (42). In the course of invasion through degraded ECM
components, migrating cancer cells are able to change integrin expression to successfully adapt to molecular cues of the new local and distant sites with variable matrix components (44). Similarly to cadherins, they sustain not only adhesion but also signalling from the cell to the matrix (inside-out) and from the matrix to the cell (outside-in).
1.2.2 Cell migration
Cell migration is an important phenomenon for tissue development and homeostasis. In the case of cancer, it is also required for angiogenesis, invasion, intravasation and extravasation, and for sustaining metastasis. Activation of signalling pathways that modulate tumour cell cytoskeleton dynamics, ECM remodelling and cell migration, occur already at initial steps of invasion (45). During this process, cells reorganize their cytoskeleton, change their morphology, emit cell membrane protrusions, secrete proteolytic enzymes, redefine new ECM interactions and drive cell movement.
Cell migration can be individual, when cell-cell adhesion molecules are completely lost, or collective, when these molecules are, at least, partially maintained (46). In both types of migration, integrins or receptors for chemotactic molecules bridge environmental cues for triggering locomotion (47). This is a multistep and cyclic process that involves polymerization of actin and myosin-driven contraction of the cell cytoskeleton, leading to morphological polarization of the cell, membrane extension at the front, formation of cell-matrix attachments, forward movement of the cell body and release of attachments at the cell rear (48). Fast activation and spatiotemporal regulation of signalling networks is required to enable cellular responses to microenvironmental signals. Rho family GTPases are key players in this process and considered essential regulators of cytoskeletal dynamics (49), playing important roles also in cell cycle progression, transcriptional regulation, cell survival and vesicle trafficking (50). Typical Rho GTPases (Rho A, Rac1 and Cdc42) act as molecular switches alternating between a GTP-bound active form and a GDP-bound inactive form (51). Their activity is upregulated by guanine nucleotide exchange factors (GEFs) and downregulated by GTPase-activating proteins (GAPs). After stimulation, GEFs promote the conversion of inactive Rho-GDP to
an active Rho-GTP-bounded form. On the other hand, once activated, GAPs stimulate GTP hydrolysis and the conversion of active Rho-GTP to an inactive Rho-GDP-bounded form (52). In parallel, Rho GTPase activity can also be regulated by GDI proteins (guanine-nucleotide-dissociation inhibitors) which prevent its association with the membrane, sequestering the GTPase at the cytoplasm, thus inhibiting the access to its interacting partners and impairing the downstream signalling (53). After activation, Rho GTPases bind to effector molecules, triggering signalling cascades that will influence and direct cell cycle, migration, epithelial cell polarity, apoptosis and angiogenesis (Fig. 4).
Interplay between RhoGTPases is necessary to rearrangement of the cellular architecture during migration. Rac and Cdc42 are frequently active at the leading edge of the cell, promoting protrusion formation, while Rho is often active in the cell body and at the rear edge, providing actomyosin contraction to enable rear retraction and forward movement (36). Lamellipodia are broad, sheet-like membrane protrusions, which appear at the leading edge of moving cells regulated by Rac, whereas filopodia are thin, actin-rich extensions of the plasma membrane under the control of Cdc42. Sites of attachment to the ECM by integrins (the focal adhesion complexes) co-localize with large bundles of actin fibers termed stress fibers, which formation is induced by Rho (54). In cancer, the expression or activity of these proteins is often upregulated (55,56), being associated with increased motile behaviour of invading cells.
Figure 4 – Activation cycle of RhoGTPases. Guanine nucleotide exchange factors (GEFs) are responsible for activating the GTPase form, which interacts with effector proteins and mediate a response to a stimulus. Instead, GTPase-activating proteins (GAPs) promote the hydrolysis of GTP to GDP, leading to an inactive protein. GDIs are proteins constituting an additional step of regulation by impairment of RhoGTPase function.
1.2.3 Proteolysis
Invasion and migration depend on the activity of proteolytic enzymes which ensure i) removal of physical barriers such as basement membrane and stromal matrices; ii) activation of latent proteases, growth factors and chemotactic molecules; iii) exposure of integrin-binding sites encrypted within ECM components; and iv) production of bioactive ECM fragments (26,57). MMPs are a family of zinc-dependent endopeptidases responsible for these proteolytic activities and which play a crucial role, not only in tumour invasion and metastasis, but also in cell proliferation and angiogenesis (58). MMPs are characterized by their structure, exhibiting three common domains: the pro-peptide, the catalytic domain and the hemopexin-like C-terminal domain (Fig. 5). They are usually expressed as inactive zymogens, since the cysteine residue of the pro-domain interacts with the zinc ion of the catalytic domain. The enzymes become active upon proteolytic removal of this interaction or by chemical modification of the cysteine residue. The pro-domain requires proteolytic cleavage, which can occur intracellularly by furin, or extracellularly by other MMPs or serine proteases (59). The proteolytic activity of MMPs can be regulated at the transcription or post-transcription level, by control of their secretion, conversion from zymogen to active enzyme, or inactivation by specific inhibitors.
Figure 5 – Overview of gelatinases’ structure. The signal peptide (pre-domain) guides the MMP into maturation at the rough endoplasmic reticulum during synthesis. The propeptide domain (pro) maintains the MMPs latent, as the cysteine residue at the pro-domain interacts with the zinc ion at the catalytic domain, maintaining it inaccessible for substrate interaction. The hemopexin-like-C-terminal domain (PEX) is linked to the catalytic domain by a short hinge region. MMP-2 and MMP-9 contain collagen-binding domain (CBD), which consists in repeats of fibronectin type II-like domains that interact with collagen and gelatin.
Within the tumour microenvironment, MMPs and their respective inhibitors are expressed not only by cancer cells but especially by tumour-recruited stromal cells such as fibroblasts, lymphocytes, adipocytes, endothelial cells and macrophages (57). The molecular crosstalk established at the tumour microenvironment between cancer and stromal cells modulates the expression and activity of such MMPs. Cancer cells might stimulate tumour stromal cells to synthesize MMPs in a paracrine manner through secretion of interleukins, interferons, extracellular MMPs inducers and growth factors (26). In the majority of tumours, the expression and activity of MMPs is increased and correlated with advanced stage, increased invasion and metastasis, reduced overall survival and poor prognosis (58,60,61). In fact, MMPs modulate several processes with important consequences at the tumour microenvironment. Concerning cancer cell proliferation, MMPs are able to regulate the function and the availability of important growth factors, such as transforming growth factor-β (TGF-β) and epidermal growth factor (EGF) (26). Activation of epidermal growth factor receptor (EGFR) results in MMP-9 upregulation, which in turn, cleaves E-cadherin extracellular domain, affecting cell-cell adhesion interactions (62). MMPs also allow cancer cells to evade apoptosis, by cleaving ligands or receptors that transduce proapoptotic signals. As an example, MMP-7 is able to cleave Fas receptor from the surface of cancer cells, avoiding apoptosis initiation (63). Angiogenesis is another process in which MMPs play important roles, specifically MMP-2, -9, -14, -1 and -7 (64). MMP-9 is responsible for making sequestered VEGF bioavailable and to promote endothelial cell migration and branching (64). Furthermore, neutrophil-derived MMP-9 is able to activate FGF-2, another crucial pro-angiogenic factor (65).
Although MMPs are reported to participate in the previously mentioned cancer-related activities, they assume a central role in modulating cancer cell invasion and metastasis, by structurally remodelling the surrounding ECM through proteolysis of collagens, fibronectin, laminins and ECM-bound proteins, such as tenascin and glypican (45). They are also responsible for the unbalanced proteolytic processing of receptors, growth factors and chemokines, available at the tumour microenvironment, and which impact cancer cell invasion (66). Among the diverse MMPs involved in cancer, MMP-2
(gelatinase A) and MMP-9 (gelatinase B) are emphasized due to high enzymatic activity against collagen type IV, which is the major component of the basement membrane (Fig. 5). MMP-2 and MMP-9 are overexpressed in many types of malignant tumours (breast, brain, ovarian, pancreas, colorectal, bladder, prostate and lung cancers and melanoma) and their expression and activity are frequently correlated with tumour aggressiveness and poor prognosis (66). These MMPs are secreted as inactive zymogens (pro-MMP-2 with 72 kDa; pro-MMP-9 with 92 kDa) and become active upon cleavage of the prodomain (MMP-2 with 65 kDa; MMP-9 with 82 kDa). Activation of pro- MMP-2 on the cell surface requires the formation of a molecular complex constituted by pro-MMP-2, MT1-MMP and TIMP-2 (67) but can also occur by action of MT1-MMP-1, MT1-MMP-7, thrombin and activated protein C (66). Regarding MMP-9, activation can be accomplished by the action of plasmin, trypsin-2, MMP-trypsin-2, MMP-13 (which is activated by MMP-2) and MMP-3 (68). Still, other activation mechanisms possibly exist. Namely, pro-MMP-9 binding to collagen type IV or gelatin, in vitro, can result in the disengagement of the propeptide and consequent reversible activation (69). Autocatalytic activation has also been described, both for pro-MMP-9 and pro-MMP-2, upon interaction with hemin or collagen α VI chain, respectively (66).
Interestingly, the coordination of the previously highlighted invasion-associated activities, namely the reorganization of cell-cell and cell-matrix adhesions, the increase of cell motility and enhancement of proteolysis, are crucial for the establishment of invadopodia and for the success of the invasion process. Invadopodia are specialized proteolytic structures, recently found at the basal membrane of invading cancer cells, which consist of tubular-like basal protrusions responsible for the degradation and invasion of the underlying extracellular matrix (70) (Fig. 6).
Figure 6 – Invadopodia are actin-rich structures formed on the basal membrane of the cell, which protrude into the extracellular matrix (ECM). Generally, the F-actin filaments in invadopodia colocalize with sites of proteolytic degradation of the matrix.
Invadopodia are rich in actin filaments and integrins, tyrosine kinase signalling components, soluble and membrane-bound proteases, like MT1-MMP, MMP-2 and MMP-9, and actin-binding regulating proteins, such as cortactin (71). The formation of invadopodia occurs spontaneously when cancer cells are grown in substrates like collagen or gelatin, suggesting a signalling mechanism through the ECM, perhaps involving mechanical stimulus and mechanical signal transduction (72,73). Through these tubular-like ECM adhesion structures, cancer cells release MMPs-containing vesicles from the cytosol to the ECM matrix (70). These delivery and motility structures enable cancer cells to combine focal matrix degradation with directional movement, improving their invasive ability.
Among all these functions, MMPs also play non-enzymatic, signal transduction roles, with consequences on cell survival, migration and angiogenesis. Particularly, the heterodimerization of the hemopexin-like C-terminal domain of pro-MMP9 with CD44 at epithelial cell’s membrane leads to the activation of epidermal growth factor receptor (EGFR) and subsequent phosphorylation of its downstream kinase effectors as the extracellular regulated MAPK (ERK), the cellular homologue of v-AKT murine thymoma viral oncogene (AKT) and the focal adhesion kinase (FAK) (74). The EGFR and its family members are major contributors of a complex signalling pathway that modulates cell migration, invasion, growth, differentiation and survival (75).
2. EGFR AND CANCER
mutated or upregulated in several cancer cells. Also designated as ErB1, it belongs the ErbB family of cell surface receptors, together with ErbB2, ErbB3 and ErbB4. ErbBs are transmembrane glycoproteins, structurally composed by an extracellular N-terminal ligand binding domain and a dimerization arm, a hydrophobic transmembrane domain, and an intracellular cytoplasmic C-terminal tyrosine kinase domain with several phosphorylation sites. EGFR signalling cascades can be activated through homodimerization or heterodimerization, through transactivation with other ErbB family members or with other receptor families. Upon activation by EGF or related ligands, such as transforming growth factor alpha (TGF-α), amphiregulin (AR), betacellulin (BTC), epiregulin (EPR), and heparin-binding epidermal growth factor (HB-EGF), occurs receptor dimerization. Consequently, the intrinsic kinase domain of one receptor phosphorylates specific aminoacid residues in the kinase domain of the other receptor of the dimer and vice versa (76). These phosphorylated residues act as docking sites, leading to the recruitment of downstream adaptor and effector molecules, triggering several downstream signalling events. These include mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/AKT, signal transducers and activators of transcription (STAT) and Src kinase signalling pathways, which are involved in the modulation of growth, survival, cell motility, adhesion, and differentiation (77) (Fig. 7).
Figure 7 – Signalling network of the epidermal growth factor receptor (EGFR). Upon ligand binding EGFR autophosphorylates tyrosine residues in its cytoplasmic domain. This creates docking sites for protein complexes, which transduce EGF signals to generate specific biological responses. This schematic representation includes only the better characterized downstream pathways.Kinases are light blue, scaffolds are dark blue, adaptor proteins are yellow, G proteins are green and transcription factors are orange. AP1, activator protein 1; CAMK, calcium/calmodulin-dependent protein kinase; CREB, cyclic AMP-responsive element-binding protein; DAG, diacylglycerol; EGR1, early growth response 1; FAK, focal adhesion kinase; GRB2, growth factor receptor-bound protein 2; IP3, inositol-1,4,5-triphosphate; KSR1, kinase suppressor of ras 1; MEF2, myocyte enhancer factor 2; PIP2, phosphatidylinositol-4,5-bisphosphate; PKC, protein kinase C; PLCγ, phospholipase C gamma; SHC, Src homology 2 domain-containing; SOS, son of sevenless homologue; STAT, signal transducer and activator of transcription. From Kolch, 2010 (78).
EGFR signalling is involved in physiological processes like organ morphogenesis, maintenance and repair. However, EGFR signalling is deregulated in several cancers as a consequence of gene amplification, mutation (leading to constitutively activated receptor) or increased ligand production at
the tumour microenvironment (79), eliciting enhanced tumour growth, invasion and metastasis (80). In this dissertation, along Chapter II and III, the relevance of EGFR signalling for gastric and colorectal cancer cell invasion and invasion-associated activities such as motility/migration and proteolysis, will be extensively discussed. We report that human macrophages, frequently present at the tumour microenvironment release an EGF-like factor, which stimulates cancer cell EGFR tyrosine phosphorylation. Subsequently, interacting downstream partners were identified and the underlying signalling pathway dissected.
3. THE TUMOUR MICROENVIRONMENT
Solid tumors are surrounded by a complex microenvironment composed not only by cancer cells but also by several recruited host cells and extracellular matrix
components.
(Fig. 8). Consequently, the current understanding of tumour biology includes the components of tumour-associated stroma as active participants in tumourigenesis and cancer progression (81).Through the secretion of cytokines, chemokines and growth factors, cancer cells influence resident and recruited endothelial cells, pericytes, smooth muscle cells, fibroblasts, myofibroblasts, neutrophils, eosinophils, basophils, mast cells, T and B lymphocytes, natural killer (NK) cells, dendritic cells and macrophages. In turn, the activated microenvironment, with both cellular and ECM components, contribute to the diverse hallmarks of cancer (82). These participations will be here briefly discussed.
3.1 Fibroblasts
Fibroblasts are important cells in this context since they are responsible for the synthesis, deposition, and remodelling of the ECM, as well as for the production of many soluble paracrine growth factors, such as EGF and TGF-β that regulate cell proliferation, morphology, migration, survival, death and invasion (83). Cancer-associated fibroblasts constitute a heterogeneous population awaiting a subtyping classification as the one established for macrophages, leukocytes and even dendritic cells.
Figure 8 – Solid tumours are surrounded by a complex microenvironment composed not only by cancer cells but also by several recruited host cells and extracellular matrix components. Some of the most common and relevant cellular counterparts are herein illustrated.
Their effect on tumour cells includes promotion of tumour growth and invasion, stimulation of angiogenesis by production of VEGF, FGF-2 and platelet-derived growth factor C (PDGF-C), and modulation of the cancer stem cell (CSC) phenotype (84). In breast cancer, tumour-associated fibroblasts were described to promote cancer cell invasion, release of proteases such as MMPs, and assist cancer cell motility (8,85-87). In colorectal cancer, fibroblasts are described to create a favourable microenvironment (88), playing important roles in nearly all cancer hallmarks and promoting metastasis (89). In particular, myofibroblasts isolated from colon cancer surgical resections or obtained through transdifferentiation of colon fibroblasts by transforming growth factor-β (TGF-factor-β) were described to stimulate colon cancer invasion. The associated mechanisms implicate a
complex molecular network established between cancer cells and cancer-associated fibroblasts. Cancer cell secreted factors as TGF-β and EGF were described to promote fibroblasts into myofibroblasts differentiation, which in turn produced and released into the tumour stroma chemokines and growth factors, MMPs and other proteases with the ability to promote cancer cell invasion-associated activities. Some of these factors include hepatocyte growth factor (HGF), Interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), MMP-2 and MMP-9 and Tenascin-C (89).
3.2 Endothelial cells
Tumour-derived factors, released under hypoxic or stress conditions, recruit endothelial cells from progenitor cells or from resident vessels to form a new vasculature network. The released p ro-angiogenic factors act on endothelial cell surface receptors of nearby blood vessels, leading to the production of molecules, such as proteolytic enzymes. The degradation of basement membrane components allows endothelial cells to proliferate, migrate and branch towards the tumour cells producing stimuli. Through the involvement of adhesion molecules, secretion of proteolytic enzymes and remodelling of ECM components, endothelial cells organize into tubular structures connected with the pre-existing vessels, recruit supporting pericytes, and form a new blood vessel, leading to angiogenesis and tumour revascularization (90,91). The newly formed vasculature promotes tumour growth and tissue repair, by delivering nutrients and oxygen, recruits endothelial progenitor cells, fostering angiogenesis, orchestrates immune cell recruitment and, as recently described, releases pro-tumorigenic factors that act on cancer cells on a paracrine manner (92). Additionally, such vascular niche was appointed to sustain tumour- initiating cells, colonizing new tissues, but also to maintain and reactivate dormant cancer cell populations (92).
Angiogenesis might be genetically induced by inactivation of the tumour suppressor genes p53 and VHL or activation of the tumour promoters Ras, EGFR and ErbB2. Epigenetic inducers of angiogenesis include hypoxia, cytokines such as IL-6, growth factors as VEGF and basic-fibroblast growth factor (b-FGF) or chemokines as SDF-1. In summary, vascularization of tumours serves to
counteract cancer cell death ensuing hypoxia and lack of nutrients, sustaining cancer cell metabolic activity and promoting the delivery of pro-survival, proliferation, pro-invasive and metastatic factors (81).
3.3 Immune cells
An association between cancer and inflammation is often considered (93-97). The presence of immune cells within the tumour microenvironment can be considered a double-edged sword (98) and could be either due to a previous chronic inflammation condition, which triggered tumourigenesis, or caused in response to tumour growth and subsequent tissue disruption (99). Tumour-associated immune cells can produce anti-angiogenic cytokines and anti-tumour immune responses involving antigen-presenting cells (APCs) and B or T lymphocytes. Although immune cells can still be found in expanding tumors, most of them appear to be modulated by cancer cells, becoming tolerant or, even worse, participate in dampening of the immune system (93) Commonly, however, inflammatory cells are instructed by cancer cells to promote tumour angiogenesis, growth, cancer cell proliferation, tissue breakdown and remodelling, contributing to tumour progression (93). Immune cells may then contribute to cancer development mainly due to their release of strong soluble mediators that promote cancer cell activities (97). For the development of more efficient anti-cancer therapies it is crucial to understand how cancer cells escape immunesurveillance and modulate immune cell behaviour, in benefit of tumour progression. The role of distinct immune cell populations on cancer promotion and progression will be here briefly addressed.
Natural killer cells
NK cell infiltration, due to the generation of a pro-inflammatory microenvironment, has been correlated with good prognosis in colorectal, gastric, lung, renal and liver cancers (100). In colorectal cancer, the high incidence of NK cells has been associated with enhanced cancer cell death and improved prognosis (101). In a syngeneic rat model of colorectal cancer, with liver and lung metastasis, NK cells were selectively recruited to the tumours and stroma surrounding tumour cell clusters. Elimination of cancer cells was then initiated directly by NK cells or by their activation of
other immune effectors, suggesting that NK cells can act directly as APCs or as inducers of a secondary Tcell response (102). Nevertheless, the anti-tumour function of these cells is considerably suppressed by tumour cells and the tumour microenvironment (103). In fact, recent studies in lung cancer revealed that infiltrated NK cells are unable to produce IFN-γ and to kill cancer cells, exhibiting several phenotypic and functional alterations (104). The maintenance of NK cells in such an anergic state is attributed to the release of TGF-β by cancer cells or by other stromal components present at the tumour microenvironment. Additionally, NK cells may display pro-angiogenic activity in several physiological processes, such as pregnancy and tissue repair (105). Nevertheless, stimulation of angiogenesis within a tumour context can have detrimental consequences. Overall, NK cells are proposed to have a protective role in early stages of cancer progression, but little influence once it reaches the state of clinical detection.
Dendritic cells
Dendritic cells (DCs) are professional APCs extremely effective in inducing naïve T cell response but also capable of promoting immunological tolerance and regulation of T cell-mediated immune responses. The heterogeneous DC family comprises two main subsets with specific phenotypic and functional properties: the plasmocytoid DCs (pDCs) and the myeloid DCs (mDCs). Overall, DCs are seen as the first to infiltrate a tumour and to recognize tumour cells, inducing a specific immune response by presenting tumour antigens to T cells (106). In fact, Dcs express Toll-like receptors (TLRs), participating actively in the innate immune response against viruses and other microbial stimuli. DCs are also able of activating other immune cells, as NK cells, and to elicit potent cytotoxic immune responses towards tumour cells. High infiltration of DCs is generally associated with good prognosis in several primary tumours. In colorectal cancer, DCs were found to be more frequent in normal colon mucosa than within the tumour microenvironment, and nearly absent at metastatic tumours (107). In another study, lower infiltration of DCs, within the tumour stroma or at the tumour invasive margins, were associated with higher metastatic frequency and reduced patient overall survival (108). Despite the distinct clinical trials using DC vaccination as an anti-cancer therapeutic
strategy, the results were disappointing since only a minority of the treated cancer patients exhibited sustained clinical responses (109). There is still uncertainty concerning the role of DCs in generating successful anti-tumour immune responses in human cancers, as it is dependent on their maturation status and on other pro- and anti- inflammatory factors present at the surrounding milieu (110). Moreover, cancer cells are able to alter the tumour microenvironment to induce tolerance and inhibition, triggering the recruitment of immature and immunosuppressive DCs, which causes Treg activation and T cell deletion and anergy (111,112).
Lymphocytes
The role of T cells in anti-tumour immune responses is widely accepted. In few cancers, such as melanoma and virus-associated tumours, stimulation of infiltrated T cells mediates cancer regression (113). At the core and at invasive margins of colorectal cancers, distinct T cell subpopulations may be found, and their association with tumour progression and disease outcome has been recently well-established (101). A significant association between high densities of tumour-infiltrating T lymphocytes (TILs) and improved patient overall survival and prognosis was described. Approximately 80% of the tumour infiltrating lymphocytes were described as CD2+, 42% as CD4+,
and 27% as CD8+, similarly to lamina propria lymphocytes isolated from adjacent colon mucosa.
Pioneer studies performed by Galon and collaborators demonstrated that an increase in intra-tumour expression of markers for cytotoxic effector T cells associated with absence of early metastasis and a decrease in tumour recurrence (114). In general, the high density of CD4+ and CD8+ memory T cells
diminishes with local tumour invasion and metastasis (115). Consistently, the proportion of primary tumours with high infiltrates of CD4+ and CD8+ memory T cells, particularly in the centre of the
tumour, was lower in patients with recurrent tumours. One of the factors that may explain this anti-tumour effect is the expression of IFN-γ, a pro-inflammatory cytokine, by CD4+ T cells. In the
majority of cancers, however, T cells are ineffective in detecting tumour cells, due to the reduction of antigen presenting cells and to immunosuppressive factors produced by other immune cells populations. A specific subset of T cells seems to favour tumour progression: Treg cells are able to
limit the action of effector T cells and constitute part of the immune infiltrate of tumours, being locally activated by exposure to antigen in the presence of immunosuppressive conditions (116).
Myeloid-derived suppressor cells
Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells with diverse phenotypes, which are recruited to the tumour microenvironment where they are transformed into potent immunosuppressive cells. They inhibit T lymphocytes, NK cells and dendritic cell differentiation (117), and also up-regulate reactive oxygen intermediates (ROI), nitric oxide (NO), L-arginine metabolism and immunosuppressive cytokines, hampering immune cell responses (110). Furthermore, MDSCs also modulate the activation and proliferation of immunosuppressive Treg cells (116). Factors produced by tumour cells, such as CCL2, are responsible for recruiting MDSCs to the tumour site. Recent studies have demonstrated that targeting of MDSCs and of their immunosuppressive functions guides to recovery of CD8+ T cell anti-tumor activity, assisting tumor suppression.
Although all the cell types present within the tumour microenvironment can influence tumour progression to some extent, depending on tumour type, evidences have been demonstrating that macrophages have a central role as they appear to be directly involved in tumour angiogenesis, invasion and metastasis (6,118).
4. MACROPHAGES
Macrophages are encountered in all mammalian tissues, displaying pronounced anatomical and functional diversity. The most successful classification of macrophages is the mononuclear phagocytic system (MPS), which encloses bone marrow-derived precursors (monoblasts and pro-monocytes), bone marrow-associated monocytes and macrophages, peripheral blood monocytes, and tissue-associated macrophages (119). According to this classification, bone marrow pluripotent stem cells originate a common colony-forming unit granulocyte and macrophage (CFU-GM) progenitor cell which is able to differentiate into neutrophils or monocytes (120). Within the monocytic lineage,
the monoblast is the least mature cell and, under the influence of colony-stimulating factor 1 (CSF-1 or M-CSF) can divide and originate pro-monocytes. Still in the bone marrow, pro-monocytes can differentiate into osteoclast progenitors or into monocytes. The differentiation from progenitor cells into monocytes can be regulated by other environmental factors such as the granulocyte-monocyte colony stimulating factor (GM-CSF), the interleukin 3 (IL-3) and Kit and PU.1 transcription factors. The MPS definition proposes that recently formed monocytes enter the peripheral blood, where they are distributed between circulating and marginating groups. Prior to differentiation into macrophages in the target organ, peripheral blood monocytes migrate to extravascular tissues engaging in processes of adherence to the endothelium, diapedesis between endothelial cells and migration through in subendothelial areas (Fig. 9). During this process many adhesion molecules, such as β1/β2-integrins, selectins and immunoglobulin family-members, relevant to cell-cell or cell-ECM adhesion, are expressed. The migration of monocytes into tissues may be elicited by chemoattractant signals originating from sites of infection, injury or tumour growth leading to a vast variety of recruited macrophages, or it may follow intrinsic programs of differentiation and result in organ-specific populations of resident macrophages (121).
The MPS system describing a strict chronological succession of macrophage progenitors has, nonetheless, been questioned. Experimental studies in mice revealed that macrophages originate from several sources during development, remaining through lifetime (122). In brief, the first origin is described to be the yolk sac from which derive progenitors of resident macrophages (123). Then, adult Langerhans cells present a mixed origin from yolk sac and fetal liver (124). Finally, circulating monocytes and their progeny of macrophages and dendritic cells originate from the bone marrow (123). These macrophage populations derived from embryonic progenitors were, in addition, found to renew independently of hematopoietic precursors and to proliferate in response to specific stimuli, resembling the self-renewal potential of stem cells (125).
Regardless of their origin, a large number of cell surface molecules are present on monocytes and macrophages that are fundamental for the interaction with the environment. These include growth
