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Division of Experimental Oncology

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Background information
Tumor microenvironement

Cells grow within defined environmental niches and are subject to microenvironmental control. Outside of their niche normal cells lack appropriate survival signals. During tumor development and progression, malignant cells escape the local tissue control and escape death. A bidirectional relationship is initiated between tumor cells and tumor stroma favoring tumor growth and progression.

Heterotypic cellular interactions in the tumor microenvironment. Tumor cells orchestrate directly (e.g. though the release of factors) or indirectly (though the induction of tissue hypoxia or appearance of necrosis) the modification of the microenvironment by attracting or activating many non-tumoral cells, including blood and lymphatic endothelial cells and pericytes, carcinoma associated fibroblast, bone marrow-derived cells, immune and inflammatory cells. Tumor cells can also deposit or modify the extracellular matrix. Most of these stromal modifications start early during tumor progression, often at the transition stage from premalignant to malignant lesions. In some cases they may even precede cancer formation, for example in situations of chronic inflammatory conditions. In turn, tumor microenvironmental events promote tumor progression by stimulating tumor growth and survival, and facilitating invasion and metastasis. Collectively these events will contribute to determine the outcome of tumor progression: tumor
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growth, tumor dormancy, tumor invasion and metastasis and resistance to therapy.

Abbreviations: B, B lymphocyte; BMDC, bone marrow-derived cells; BV, blood vessel; CAF, carcinoma associated fibroblast; EC, endothelial cell; ECM, extracellular matrix; EMT, epithelial to mesenchymal transition; Gr, granulocyte; LEC, lymphatic endot

Inflammation and tumor progression

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The immune system plays a dual role: the adaptive immune system tends to repress tumor growth, while the innative system stimulates it by promote tumor angiogenesis, invasion and metastasis. A causal relationship between chronic inflammation and cancer formation has been proposed over a century ago based on the observations that cancers often develop at sites of chronic inflammation. For example, chronic inflammatory bowel diseases, such a Crohn's disease or ulcerative colitis, chronic reflux esophagitis in Barrett syndrome, chronic infection, or chronic gastric ulcers (i.e. consequent of Helicobacter pylori infection) are associated with increased risk of developing colorectal, esophageal or stomach cancer, respectively. Today the functional relationship between the inflammation response and cancer is widely recognized, and some of the underlying cellular and molecular events have been uncovered. Inflammatory events can create a local microenvironment capable of promoting tumor growth progression. Tumor cells and/or cells of the tumor microenvironment, respond to tumor hypoxia, necrosis, general inflammation and release a number of growth factors and cytokines that are chemoattractive for monocytes and macrophages. In turn, infiltrating macrophages secrete growth factors that affect tumor cells or tumor endothelium and promote the recruitment of secondary inflammatory cells, such as mast cells and neutrophils. In turn these cells support tumor progression by sustaining inflammation and secreting pro-angiogenic and pro-tumorigenic cytokines and proteases. A good example to illustrate this paradigm is cyclooxygenase-2 (COX-2) expression and prostaglandin (PG) production that physiologically occurs during inflammation. Within the tumor environment COX-2/PG stimulate cell proliferation, cell survival and tumor angiogenesis, thereby promoting tumorigenesis.

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Conversely, transformed cells exploit mechanisms that occur physiologically during inflammation to invade and metastasize. A good example is the ability of tumor cells to exploit the chemokine system to invade and metastasize. Many leukocyte populations can be found at tumor sites, i.e. neutrophils, eosinophils, basophils, monocytes/macrophages, dendritic cells, natural killer cells and lymphocytes. Although many of the leukocytes are potentially capable of killing tumor cells, deficient monocyte recruitment at tumor sites in mice lacking colony stimulation factor 1 (CSF-1) expression was shown to attenuate late-stage progression and metastasis formation, suggesting that monocytes can promote tumor progression. A positive correlation between the number of tumor-associated monocytes/macrophages (TAMs), microvessel density (MVD) and poor prognosis has been reported for many cancers.

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Tumor-host interactions and inflammatory cells in tumor progression. Growing tumors attract new blood vessels (a process termed tumor angiogenesis) and lymphatic vessels (i.e. lymphangiogenesis), which, in turn, promote local tumor growth, regional lymph node and distant metastasis formation. Tumors are often infiltrated by bone-marrow derived inflammatory cells. Some of these cells, in particular monocytes / macrophages, are 'educated' by the tumor cells to promote angiogenesis, lymphangiogenesis and tumor cell invasion.

Bone marrow-derived cells and circulating endothelial cells

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Clinical, pathological, and experimental evidence indicates that tumor progression can be stimulated by bone marrow-derived precursor cells that are recruited to tumor sites. At least three different bonemarrow-derived cell populations may be involved in this sequence of events: (1) endothelial cell progenitors incorporated into the vasculature at low frequency; (2) monocyte-like cells (i.e., CD11b/VEGFR-1/Tie-2 positive), which infiltrate the tumor stroma and stimulate angiogenesis by producing vascular growth factors; and (3) bone marrow-derived pericyte precursors, differentiating into mature pericytes and stabilizing angiogenic tumor vessels, thereby contributing to increased resistance of angiogenic endothelial cells against anti-angiogenic drugs. Circulating endothelial cells (CEC) of putative vessel wall origin have been detected in the peripheral blood of healthy individuals and, at higher frequencies, in breast cancer and lymphoma patients [Mancuso, 2001]. The frequency of CEC or CECP correlates with the degree of growth factor induced angiogenesis, while suppression of angiogenesis caused a dose-dependent reduction in CECPs paralleling anti-tumor activity. These data suggest that CECP may represent a diagnostic tool for monitoring the angiogenic status of patients before and during anti-angiogenic therapy.

Tumor angiogenesis and metastasis

Blood vessel formation and tumor angiogenesis. The formation of a 'tumor-associated vasculature', a process referred to as tumor angiogenesis, is essential for tumor progression. Tumor-associated vessels promote tumor growth by providing oxygen and nutrients and favor tumor metastasis by facilitating tumor cell entry into the circulation. During development, VEGF induces differentiation and proliferation of endothelial cells from its progenitors (the hemangioblast and angioblast) to form a poorly differentiated primitive vascular plexus (vasculogenesis). Angiopoietin-1 (Ang1) and other morphogens (e.g. Ephrins-Eph) induce remodeling of the vascular plexus into a hierarchically structured mature vascular system through endothelial cell sprouting, trimming differentiation and pericyte recruitment (angiogenesis). During tumor angiogenesis, angiopoietin-2 (Ang-2) destabilizes the vessel wall of mature vessels. Quiescent endothelial cells become sensitive to VEGF (or other angiogenic factors), proliferate and migrate to form new vessels. Bone marrow-derived endothelial cell progenitors are found in
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the peripheral blood and can recruit at sites of angiogenesis.

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Classes of molecules mediating and regulating angiogenesis. There are today hundreds of molecules known to mediate or regulate angiogenesis. They can be grouped in the seven classes a depicted here. Many molecules have been considered as potential therapeutic targets or tools to inhibit pathological angiogenesis, in particular tumor angiogenesis.

Integrins in angiogenesis Preclinical and clinical evidence indicate that vascular integrins may be valid therapeutic targets. In preclinical studies, pharmacological inhibition of integrin function efficiently suppressed angiogenesis and
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inhibited tumor progression. alphaVbeta3 and alphaVbeta5 were the first vascular integrins targeted to suppress tumour angiogenesis. Subsequent experiments revealed that at least four additional integrins (i.e. alpha1beta1, alpha2beta1, alpha5beta1 and alpha6beta4) might be potential therapeutic targets. In clinical studies low molecular weight integrin inhibitors and anti-integrin function-blocking antibodies demonstrated low toxicity and good tolerability and are now being tested in combination with radiotherapy and chemotherapy for anticancer activity in patients.

Integrin Signaling Signaling pathways initiated by integrins at focal contacts. The four major signaling pathways activated by integrin engagement in adhesion complexes are shown. The key element in all these pathways is FAK, which becomes activated through autophosphorylation at Y397 and thereby allow binding of Src and Fyn for further phosphorylation and full activation. Phosphorylation of FAK at specific sites dictates its subsequent
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interactions with other proteins (i.e. Grb2, p130Cas, PI3K, Graf) which in turn elicit a cascade of events that lead to cell proliferation, migration or survival. FAK can also be activated by cell surface receptors for growth factors, hormones and chemokines. Plain lines, direct activation or inhibition; dashed line, indirect functional interaction; red lines, FAK-mediated events mediated by specific phosphorylation events.

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Anti-angiogenesis as anticancer therapy Inhibition of tumor angiogenesis suppresses tumor growth in many experimental models. In 2004 an antibody (Avastin) directed against vascular endothelial growth factor (VEGF) was reported to provide a survival benefit to patients with advanced colorectal cancer, in combination with chemotherapy, and was approved as the first anti-angiogenic drug for use in human cancer therapy. This result validates the concept that inhibition of tumor angiogenesis suppresses tumor growth. Additional antiangiogenic drugs were approved thereafter. Attempts are under way to target lymphatic vessels to inhibit lymphgangiogenesis for therapeutic purposes.

Monitoring angiogenesis Quantification of tumor angiogenesis and measuring antiangiogenic drugs activities in patients remain unresolved issues. Many approaches have been tested in experimental models and clinical studies, but to date none has been validated for routine use in patients. It is not clear which biomarker best represents angiogenesis and, as a matter of fact, whether there is one at all. The intrinsic complexity of tumor angiogenesis, its multiple regulatory mechanisms and adaptation during therapy, suggest in fact hat different biomarkers will be necessary to give a comprehensive representation of angiogenesis and its therapeutic modulation, depending on the tumor of interest, its stage, the tested drug, the question asked and the clinical stage of development (phase I/II/III). One can distinguish three kinds of biomarkers: I) Molecular biomarkers. They consist of individual molecules such as growth factors (eg VEGF, FGF), cell surface receptors (eg VEGF-R2, integrins), downstream signaling molecules (eg ERK, AKT) and their modifications (eg activation, phosphorylation), or transcripts for single or multiple genes (eg signatures) and their modifications. Molecular biomarkers are indicative of the molecular events associated with angiogenesis or drug activity. II) Biological biomarkers. In order to obtain initial information on the effects of these molecular events and their modification on endothelial cell biology, it will be necessary to monitor basic characteristics of endothelial cell functions, such as cell proliferation or death. Blood circulating angiogenesis-associated cells (eg CEC, CECP) may reflect these changes and serve as easily accessible surrogate markers of biological effects. Molecular and cellular biomarkers are important in phase I/II studies, where demonstration of target modification and drug activity is the main goal.
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III) Functional biomarkers. Tumor perfusion is the ultimate function of angiogenic vessels, and its modification is likely to reflect significant changes in their physical or functional state. However, acquisition and meaningful interpretation of perfusion and permeability data may be challenging, since changes may be only transient, occur late after drug administration, or not necessarily be representative of all drug effects (eg some TKI concomitantly target tumor and stromal cells). Measurement of perfusion-related parameters (rBV, MTT, rBF, Ktrans) by imaging technique is probably more useful at late phases of development (III) when evidence of activity is already available. Also, imaging techniques can be used to monitor the effects on tumor biology (tumor regression, metabolism). Clinical endpoints (OS, PFS) will be eventually used to validate the impact of the tested drug on disease progression and patient survival. Data generated by different classes of biomarkers can be compared to extract valuable information on their relative value and suitability for monitoring, but also to dissect mechanisms of action of the tested drug.

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