British Journal of Radiology (2003) 76, S3-S10
© 2003 British Institute of Radiology
doi: 10.1259/bjr/68078705
Translating angiogenesis research into the clinic: the challenges ahead
H G Augustin, DVM, PhD
Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, D-79106 Freiburg, Germany
Correspondence: Dr Hellmut G Augustin, Department of Vascular Biology and Angiogenesis Research, Tumor Biology Center, Breisacher Str. 117, 79106 Freiburg, Germany, E-mail: augustin@angiogenese.de
 |
Abstract
|
|---|
The field of angiogenesis research has evolved to become one of the most rapidly growing biomedical disciplines. The interest in basic angiogenesis research is sparked by the translational therapeutic potential aimed at developing anti-angiogenesis as a novel therapeutic modality for tumours and a number of non-oncological diseases, such as rheumatoid arthritis, psoriasis, diabetic retinopathy and age-dependent macula degeneration. The molecular determinants of the angiogenic cascade have been characterized in great detail over the last few years. Likewise, intense ongoing efforts are aimed at identifying and validating additional vascular specific determinants that may be exploited as therapeutic targets for pro-angiogenic and anti-angiogenic therapy. At the same time, a large number of angiomodulatory compounds are in various phases of clinical trials. These include the neutralizing vascular endothelial growth factor (VEGF) antibody Avastin, which has successfully passed phase III clinical trials for the combination with chemotherapy in colorectal cancers. In view of the dramatic progress in basic angiogenesis research, surprisingly little is known about the nature of the neovasculature in human tumours. The inclusion and exclusion criteria of clinical trials of anti-angiogenic compounds are devoid of angiogenesis-related parameters and reliable biomarkers to trace the efficacy of an anti-angiogenic intervention are largely missing. Based on a brief review of the biology of the angiogenic cascade, this review provides an overview of the current concepts of the angiogenic vasculature in human tumours and discusses some key unanswered questions in translating angiogenesis research into the clinic.
|
Introduction
|
|---|
Specific targeted therapies are in the process of significantly changing conventional anti-proliferative chemotherapeutic and radiotherapeutic antitumour regimens. For example, Herceptin (trastuzumab; Genentech, South San Francisco, USA; Roche, Basel, Switzerland) is an anti-HER2 monoclonal antibody used in the treatment of metastatic breast cancer. Herceptin is the first antitumour reagent that specifically targets the gene product of an oncogene, which is overexpressed or amplified in patients with breast tumours. The use of Herceptin consequently requires a specific diagnosis for the expression of HER2 to identify HER2 overexpressing breast cancer patients who will benefit from the treatment with Herceptin. Likewise, chronic myeloid leukaemia (CML) is caused by the specific gene product of the Philadelphia chromosome which results from a chromosomal translocation. The resulting oncoprotein bcr-Abl is specifically inhibited by Glivec (imatinib mesylate; STI571; Novartis, Basel, Switzerland) which has revolutionized the therapy of CML.
A large number of other therapeutic targets are being intensively explored for their suitability to selectively interfere with specific tumour pathways in order to halt tumour progression or even to actively induce tumour regression. Angiogenesis research is among the disciplines that have raised great expectations with regard to the identification and validation of novel targets that may be exploited for specific targeted tumour therapies. Before 1990, less than 100 scientific articles were published annually that contained the word "angiogenesis" in their title. Today, more than 10 times as many articles are published every year and the field of angiogenesis research has evolved to become one of the most rapidly growing biomedical disciplines (Figure 1
). A large number of anti-angiogenic compounds are in various phases of clinical trials. In fact, the neutralizing vascular endothelial growth factor (VEGF) antibody Avastin (bevacizumab; Genentech) is the first selective angiogenesis inhibitor that has successfully passed clinical trials and is currently awaiting approval for use in combination with chemotherapy in metastatic colorectal cancers (www.gene.com).
View larger version (16K):
[in this window]
[in a new window]
|
Figure 1. Number of angiogenesis-related articles published in Medline. The annual number of angiogenesis-related publications has increased by more than a factor 10 in the last 10 years (squares: number of articles with the term "angiogenesis" in the title; circles: number of articles with the term "angiogenesis" in any Medline heading).
|
|
Hand-in-hand with the implementation of specific targeted therapies, anti-angiogenic tumour therapies requires a set of complementary tools and techniques to assess the angiogenesis status of patients. These are required to mechanistically define the optimal therapeutic approach and to monitor the efficacy of anti-angiogenic interventions. It is quite sobering to conclude that inclusion and exclusion criteria of ongoing anti-angiogenic clinical trials are largely not based on angiogenesis-related parameters. Likewise, the primary readouts of clinical tumour trials, namely time to progression (TTP) and median survival, are the ultimate endpoint readouts of an antitumourigenic therapy. Yet, theses parameters do not allow a direct assessment of the efficacy of anti-angiogenic therapy. Consequently, reliable biomarkers that are indicative of the efficacy of an anti-angiogenic intervention are urgently needed.
Based on these introductory considerations, this review will briefly outline the key determinants of the angiogenic cascade with special emphasis on the validation of determinants of the angiogenic cascade as therapeutic targets. It will then provide an overview of the current concepts of the angiogenic vasculature in human tumours. In conclusion, key unanswered questions will be discussed that require the attention of the translational angiogenesis community in order to rationally implement anti-angiogenic tumour therapies into clinical practice.
|
The angiogenic cascade and the identification of anti-angiogenic targets
|
|---|
The concepts of the angiogenesis-dependency of tumour growth and metastasis were born through pioneering experiments by Dr Judah Folkman more than 30 years ago. As obvious as these concepts appear nowadays, they were not widely appreciated when they were first developed and the angiogenesis community was for many years a small circle of dedicated basic scientists. The search for the "tumour angiogenesis factor" (TAF) [1] led to the identification of a large number of angiogenesis-inducing molecules in the years until 1989. Among important discoveries of these early years was the heparin-affinity of many growth factors which led to the discovery of the heparin-binding growth factors, many of which are capable of inducing angiogenesis in vitro and in vivo. Angiogenesis research gained more widespread recognition when the first specific regulator of the angiogenic cascade was identified in 1989 [2]. Originally identified as vascular permeability regulating cytokine [3], VEGF was found to exert angiogenesis-controlling vascular-specific functions through the almost endothelial cell specific expression of its receptors, VEGFR-1 and VEGFR-2. Subsequent work showed that VEGF is absolutely essential for embryonic and adult angiogenesis. In fact, targeted deletion of just one allele in mice is not compatible with life and leads to early embryonic lethality around embryonic day 8.5 [4, 5]. Of the many thousands of genes that have been deleted in mice, the phenotype of heterozygously targeted VEGF-deficient mice is still the most pronounced knockout phenotype produced so far despite the increasing list of mutant mice with an overt heterozygous phenotype. The heterozygous embryonic lethal phenotype has rendered VEGF also a very attractive candidate molecule for advanced genetic studies aimed at conditionally deleting or overexpressing VEGF in a spatially and/or temporally restricted manner [6, 7].
The VEGF family of angiogenic cytokines has grown over the years and includes VEGF-B, VEGF-C, VEGF-D, the viral homologue VEGF-E, as well the related placental growth factors PlGF-1 and PlGF-2 [8]. VEGF-C and VEGF-D have been identified as key regulators of lymphatic angiogenesis by binding and activating the lymphangiogenic receptor VEGFR-3 [9]. All of these molecules have been intensely studied in terms of understanding the complexity of the angiogenic cascade and in order to validate the molecules as targets for pro-angiogenic and anti-angiogenic therapy.
Different avenues have been explored for therapeutically interfering with VEGF signalling (Figure 2). The humanized neutralizing antibody Avastin (Genentech) binds and neutralizes VEGF and thereby prevents it from binding to its receptor [10]. Similarly, the VEGF trap (Regeneron, Tarrytown, USA/Aventis, Strasburg, France) is a dimeric soluble receptor composed of parts of the extracellular domain of VEGFR-1 and VEGFR-2 [11]. In contrast to Avastin, the VEGF trap does not just bind VEGF, but also other VEGFR-1 or VEGFR-2 binding family members including PlGF. Classical large scale pharmacological screening of small molecular weight compound libraries has led to the identification of selective antagonists to the VEGF receptors. Whereas some of the early VEGF receptor inhibitors such as SU5416 or SU6668 are not pursued in clinical trials anymore, others are already in advanced clinical trials. The VEGF receptor inhibitor PTK787/ZK222584 (Novartis/Schering, Berlin, Germany) is in phase III clinical trials in combination with chemotherapy for colorectal tumours and is also in phase II clinical trials for a number of other tumours [12]. Other VEGF receptor tyrosine kinase inhibitors include ZD6474 (AstraZeneca, Macclesfield, UK) and SU11248 (Pfizer, New York, USA). More recently, screening attempts are underway, to develop small molecular weight kinase inhibitors with relevant multiple kinase inhibitory profile [13]. For example, SU11248 is an oral multitargeted tyrosine kinase inhibitor with antitumour and anti-angiogenic activity through targeting PDGFR, VEGFR, KIT and FLT3 [14].
View larger version (27K):
[in this window]
[in a new window]
|
Figure 2. Clinically pursued avenues to therapeutically interfere with vascular endothelial growth factor (VEGF)/VEGFR signalling. Production of VEGF in VEGF-producing cells can be blocked with VEGF ribozymes or VEGF siRNAs. VEGF protein can be neutralized with neutralizing antibodies (Avastin), soluble receptors (VEGF trap) or VEGF aptamers. Activation of the VEGF receptors can be effectively inhibited with specific small molecular weight tyrosine kinase inhibitors. Lastly, the synthesis of VEGF receptors can be blocked with VEGFR ribozymes or siRNAs.
|
|
Several other VEGF signalling inhibition strategies have been developed in addition to the ligand blocking agents Avastin and VEGF trap and the small molecular weight tyrosine kinase receptor inhibitors. Aptamers are DNA molecules that bind and functionally neutralize target proteins. A VEGF aptamer has been developed to trap VEGF (Eyetech, New York, USA) that is currently in advanced clinical trials for the local treatment of abnormal neovascularization associated with age-dependent macular degeneration (AMD) [15]. Lastly, a VEGFR-1 ribozyme, called Angiozyme (Sirna Therapeutics, Boulder, USA/Chiron, Emeryville, USA), has been developed to block VEGFR-1 protein synthesis [16]. A phase II clinical trial of Angiozyme in combination with standard chemotherapy for metastatic colon cancer has been completed in 2003. Presently, ribozyme therapeutic approaches are increasingly replaced by siRNA-mediated silencing of target gene production [17]. Consequently, several laboratories and companies are presently exploring the suitability of siRNAs against either VEGFR-1 and VEGF to therapeutically interfere with angiogenesis.
The discovery of VEGF started a race to identify other specific regulators of the angiogenic cascade. The angiopoietins (Ang-1 and Ang-2) were identified in 1996 and 1997 as ligands of the vascular specific receptor tyrosine kinase Tie-2 [18]. Tie-2 activation transduces survival signals in endothelial cells and stabilizes a growing neovascular network. Consequently, Ang-1 or Tie-2 deletion in mice leads to embryonic lethality around embryonic day 10.5. Embryos deficient in either Ang-1 or Tie-2 can initiate the early steps of the angiogenic cascade but fail to properly assemble and mature the embryonic neovascular network. Ang-2 has been identified as functional antagonist of the Ang-1/Tie-2 axis. It binds to endothelial cell surface expressed Tie-2 without inducing Tie-2-mediated signal transduction. It thereby antagonizes the functions of Ang-1 and causes the destabilization of the vascular network. As such, Ang-2 does not directly affect endothelial cell functions. Instead, it acts contextually to facilitate the function of other regulators of the angiogenic cascade. Ang-2-mediated destabilization of the vasculature allows VEGF to exert its pro-angiogenic function. In turn, Ang-2 acts anti-angiogenic and vessel regression-inducing in the absence of pro-angiogenic activity [19]. The biology of the Angiopoietin/Tie-2 system is still poorly understood and a ligand for the related endothelial cell tyrosine kinase receptor Tie-1 has hitherto not been identified. Yet, intense attempts are presently underway to identify selective Tie-2 inhibitors or an inhibitor that targets both, VEGF receptors and Tie-2; such an inhibitor may have an attractive combinatorial targeting specificity.
The EphB receptors comprise the latest family of receptor tyrosine kinases that exert rate limiting vascular morphogenic functions [20–22]. Originally identified more than 10 years ago as axonal pathfinding molecules, EphB receptors were identified through gene targeting experiments as critical regulators of arteriovenous differentiation and vascular assembly. Mice with targeted deletion of either EphB4 or its ligand ephrinB2 are not capable of establishing arteriovenous asymmetry and die around embryonic day 10.5 as a consequence of grossly perturbed vascular differentiation and arteriovenous remodelling. Interactions of EphB receptors with their corresponding ephrinB ligands are conceptually very different from classical interactions of a secreted ligand with its corresponding transmembrane receptor. Instead, both the EphB receptors and the ephrinB ligands are transmembrane receptors and signalling occurs only if two corresponding receptor and ligand expressing cells get into juxtapositional contact [23]. Recently, evidence is emerging that EphB/ephrinB interactions do not just control neural and vascular cell positioning, but that they are also expressed by numerous other cell populations. EphB/ephrinB interactions have been shown to control positional gastrointestinal epithelial cell interactions [24]. Evidence is also emerging to suggest that ephrinB molecules are involved in lung epithelial cell morphogenic programs. EphB and ephrinB molecules have also been shown to be expressed by circulating haematopoietic cell populations and an increasing list of tumours is identified as expressing EphB and ephrinB molecules. Collectively, the emerging data indicate that EphB/ephrinB interactions may act as universal cell–cell interaction and guidance signalling system. Much needs to be learnt about the biology of the EphB/ephrinB system and the proof-of-principle experiments of their prospective therapeutic value for the treatment of tumours are just in the process of being published. Nevertheless, several pharmaceutical companies have already initiated small molecular weight screening programs to identify specific inhibitors that interfere with bi-directional EphB/ephrinB signalling.
Another vascular molecular system that holds great prospect for therapeutic targeting purposes is the platelet-derived growth factor (PDGF)/PDGFR system [25]. PDGF-B is produced by endothelial and other cells and plays a central role in the recruitment of mural cells (pericytes, smooth muscle cells). Genetic ablation of PDFG-B or PDGFR-
leads to essentially identical phenotypes characterized by perinatal lethality resulting from widespread intravascular leakage and haemorrhage. Mural cell contact is critical for the stabilization and maturation of a growing neovascular bed. Lack of mural cell recruitment leaves a vasculature in an immature and vulnerable state that may contribute to defining the therapeutic window for angioinhibitory tumour strategies (Figure 3).
View larger version (141K):
[in this window]
[in a new window]
|
Figure 3. Morphology-based histological techniques to functionally assess the vasculature of tumours. (A) Fluorescent-micrographic image of the dense vascular network of an experimental tumour using CD31 immunohistochemistry. Pan-endothelial cell markers such as vWF, CD31, or CD34 can be used to determine the overall microvessel density (MVD), which is a global angioarchitectural readout of the average intercapillary distance. (B) Double labelling of blood vessels (CD31, brown staining) and proliferating cells (Ki67, dark nuclei) in a human glioblastoma. Co-localization of CD31 and Ki67 staining allows the detection of the few proliferating endothelial cells (arrows) among the many proliferating tumour cells. (C) Fluorescent microscopic triple staining of tumour cell nuclei (Hoechst stain, blue), blood vessels (CD31, red), and proliferating endothelial cells (Ki67, green) in an experimental human tumour growing in an immunocompromised mouse. The anti-murine Ki67 stains only proliferating mouse cells and not the xenografted human tumour cells allowing the specific detection of the proliferatin, angiogenic mouse blood vessel compartment. (D) Three-dimensional co-localization of an endothelial cell marker (CD31, green) and a mural cell marker (desmin, red) demonstrating the intense mural coverage in the quiescent subcutaneous vasculature of the mouse. Correspondingly, the degree of maturation of the tumour vasculature can be traced by employing appropriate mural cell markers ( -smooth muscle actin, desmin, NG2, RGS5).
|
|
During blood vessel formation, invasive endothelial cells have to entertain a number of distinct cell–cell and cell–matrix adhesive interactions. Among these, integrin-dependent adhesive interactions have been shown to be critically rate-limiting factors of the angiogenic cascade [26]. Integrin v-deficient mice die perinatally from brain haemorrhage and have only a mild vascular phenotype. Likewise, integrin 3- and 5-deficient mice not only support tumour growth and angiogenesis, but rather have enhanced tumour angiogenesis compared with wildtype controls. In turn, blockade of the heterodimer v3 or v5 in the adult has been shown to potently interfere with tumour growth and angiogenesis [27]. The discrepancies between the findings in genetic and in adult manipulatory systems have not fully been resolved [28, 29]. Yet, blockade of v3, v5, and other integrins is intensely explored therapeutically. The neutralizing v3 antibody Vitaxin (Applied Molecular Evolution, San Diego, USA; Medimmune, Gaithersburg, MD) is in clinical trial as are small molecular weight cyclic peptide integrin inhibitors (Cilengitide, Merck, Darmstadt, Germany).
This review is primarily focusing on angiogenic receptor tyrosine kinase signalling pathways that are being pursued for clinical applications or for which good proof-of-principle work provides a solid foundation to therapeutically explore these molecules. There are a number of other molecules in various stages of clinical development (for regularly updated summaries of anti-angiogenesis clinical trials, the reader is invited to view the following website: http://www.cancer.gov/clinicaltrials/developments/anti-angio-table). Furthermore, more than 100 angiomodulatory molecules (angiostimulatory and angioinhibitory) have been identified to date (for online review, see: http://lpgws.nci.nih.gov/html-cgap/cgl/angiogenesis.html) and intense genomic and proteomic efforts are underway to identifiy novel rate-limiting angiogenesis-regulating molecules [30]. Many of these molecules may turn out to be biologically very important, but they may only be of limited value for therapeutic applications for practical reasons (targetability, drugability) or for biological reasons (danger of uncontrollable side effects in some of the pleiotrophically acting molecules that exert other important biological functions in addition to their angiogenesis-regulating activity). A number of endogenous inhibitors of angiogenesis have been identified in recent years. Thrombospondin is the best characterized endogenous inhibitor of angiogenesis [31]. Some endogenous angiogenesis inhibitors have been identified as cryptic fragments from larger molecules that are proteolytically liberated. These include angiostatin and endostatin and a number of matrix-derived peptides (tumstatin, canstatin) [32, 33]. Several of these molecules are currently being evaluated in clinical trials and intense efforts are ongoing aimed at developing a better understanding the complex biological mechanisms of action of these molecules.
The search for angiogenesis inhibitors is primarily aimed at specifically interfering with molecularly defined steps of the angiogenic cascade. Yet, an increasing list of compounds is being characterized that exerts potent anti-angiogenic activity in a number of experimental in vitro and in vivo systems. These molecules are not specifically acting on the angiogenic vasculature, but the angiogenic vasculature appears to have a preferential susceptibility for a number of pharmacological compounds. The prime example of a non-specific anti-angiogenic therapy that may be very effective is metronomic scheduling of classical chemotherapeutics. Low dose metronomic scheduling takes advantage of the fact that endothelial cells during tumour angiogenesis are proliferating and as such these cells are much more readily accessible to chemotherapeutic agents due to their strategic location in the vascular wall [34, 35]. Similarly, a number of other mechanisms may prove useful to preferentially target the angiogenic vasculature even though the targeted mechanism may not be selectively operative in angiogenic endothelial cells. For example, increasing evidence suggests that angiogenic endothelial cells may have a preferential susceptibility towards microtubule-destabilizing agents and these may turn out to be very effective when combined with specific targeted anti-angiogenic therapies.
|
Pathology-based techniques to assess the angiogenic status of a tumour patient
|
|---|
In principle, the angiogenic status of a tumour can be assessed by three different approaches: (1) directly by applying pathology-based morphological techniques; (2) indirectly by clinical chemistry-based measurement of circulating biomarkers; and (3) through non-invasive imaging techniques. Non-invasive imaging techniques to assess angiogenesis are the focus of several articles of this special issue of the British Journal of Radiology. Undoubtedly, imaging techniques have made enormous progress over the last few years and hold great prospect to not just visualize the tumour vasculature but to assess the functional properties of the tumour vascular bed. Dynamic contrast-enhanced MRI may evolve to become a reliable biomarker for the pharmacological response to an anti-angiogenic therapy [36]. The reader is referred to the respective chapters in this special issue. This review will focus on pathology and clinical laboratory based techniques to assess the functional status of the vasculature in human tumours.
The observation that tumour growth is angiogenesis-dependent prompted Weidner and co-workers [37] to hypothesize that the number of vessels in a tumour may be reflective of the angiogenic intensity of the tumour. The intratumoural microvessel density (MVD) may therefore be prognostic for the growth of the tumour. Weidner et al [37] showed in a landmark study that the intratumoural MVD in mammary tumours with poor prognosis and metastases is twice as high as in patients with mammary tumours with good prognosis and without metastases. This study has sparked numerous investigators to pursue similar studies for other tumours. Collectively, these studies have shown that intratumoural MVDs are an independent prognostic factor that correlates with poor prognosis. A simple Medline search with the search terms "MVD" or "microvessel density" identifies more than 1800 publications which have been published in the last 12 years mostly focusing on tumours (>1300).
As a result of the widespread application of MVD counting protocols, MVD counts have become the morphological gold standard to assess the neovasculature in human tumours. Yet, MVD counting protocols are not without problems. More than 100 papers on MVD counts for mammary tumours alone have been published in the last 10 years, and about a third of all investigators could not identify a strong correlation between MVD counts and prognosis. Despite repeated attempts to establish a consensus of standardized staining and counting protocols [38], there is a tremendous variation of published data. For example, MVD counts in breast tumours have been reported to range from less than 20 mm–2 to more than 200 mm–2 [39]. The mean MVD of the published mammary tumour reports is between 80 mm–2 and 90 mm–2. The use of different pan-endothelial cell markers (mostly used: vWF, CD31, CD34) may account for some of the variation. Yet, the lack of standardized protocols and variations in the manual or automated counting of immunoreactive vascular spots are the primary variables to account for the limited comparability of the published literature.
MVD counts are reflective of the angioarchitectural properties of the tumour in that they are a representative of the average intercapillary distance. This is in fact an important parameter as it is the goal of an anti-angiogenic tumour therapy to reduce the intercapillary distance to a degree that it becomes rate-limiting for the growth of the tumour. Experimental anti-angiogenic experiments in murine tumour models usually have a significant reduction of the MVD as primary experimental readout of an anti-angiogenic intervention. Human tumours have different growth kinetics compared with experimental tumours. Likewise, tumours undergoing anti-angiogenic intervention may also follow a "shrink to fit" adaptation, which as a result may not lead to reduced MVD counts. It has consequently been argued that MVD reduction may not be the appropriate and expected readout of the success of anti-angiogenic interventions in human tumours [40].
MVD counts are an important morphological readout of the tumour vasculature. However, MVD counts reflect an anatomical parameter and are not reflective of the functional properties of the tumour vasculature. A microvessel density count of histological sections of the lungs or the liver will reveal very high MDV counts and yet, the detection of blood vessels in these organs is not indicative of the process of angiogenesis per se. In fact, it is quite sobering to note that the knowledge of the angiokinetic behaviour of the human tumour vasculature is quite limited.
As early as 1972, Brem et al [41] proposed a microscopic angiogenesis grading system (MAGS) to assess the angiogenic status of the tumour vasculature. Based on the analysis of the vascular density, the number of endothelial cell nuclei, and the cytological properties of tumour-associated endothelial cells, an angiogenesis score MAGS was determined and used to establish an angiogenic ranking order of different human brain tumours [41]. Similarly, MVD counts based on marker molecules of the angiogenic endothelial cell phenotype (e.g. CD105 or VEGFR-2) are not widely used and it is not well established if MVD counts based on angiogenic endothelial cell markers have a more reliable prognostic value as compared with MVD counts based on pan-endothelial marker molecules. A number of investigators have studied endothelial cell proliferation as a parameter of the tumour neovasculature (Figure 3
). In fact, endothelial cell proliferation may hitherto be considered as the most reliable parameter to assess the degree of active angiogenesis in a tumour given that it is well established that quiescent organ vasculature in adults has very low proliferation with turnover rates of months to years. The detection of proliferating endothelial cells may therefore be considered a reliable readout of ongoing angiogenic activation. Endothelial cell proliferation indices have been determined in experimental tumours to demonstrate that endothelial cell turnover is indicative of active angiogenesis [42, 43]. Similarly, a number of investigators have studied endothelial cell proliferation in human tumours and reported turnover indices of 0.15% for prostatic carcinomas [44], around 2.5% for mammary tumours [45, 46], and as high as 9.9% for colorectal adenocarcinomas [47]. These studies are largely not comparable owing to different applied methodologies, but they provide an indication that the intensity of angiogenesis may be different in different types of human tumours. Eberhard et al [48] have performed the first systematic cross-sectional study of endothelial cell proliferation in different types of malignant human tumours under identical experimental conditions. This study reported median endothelial cell turnover indices that ranged from 1.9% (prostate tumours), 2.3% (lung tumours), 3.4% (mammary tumours), 6.6% (colon carcinomas), 8.3% (renal cell carcinomas), to 8.6% (glioblastomas). Likewise, endothelial cell proliferation indices vary strongly within one tumour type, for example for glioblastomas from 2% to as high as 25% [48]. It is too early to predict the consequences of these findings for the implementation of anti-angiogenic therapies. Yet, it appears obvious that the degree of active angiogenesis will determine the therapeutic potential of an anti-angiogenic intervention.
|
Biomarkers of the angiogenic cascade
|
|---|
Any pathology-based angiogenic diagnostic protocol requires access to the tumour. Surgical removal of the tumour makes the tumour available for functional angioarchitectural analyses to quantify MVD, endothelial cell proliferation and other functional parameters of the tumour neovasculature such as the coverage of blood vessel with pericytes. Such an analysis would facilitate the reliable determination of the tumour patient's angiogenesis status if an anti-angiogenic therapy is to be implemented following surgery of the primary tumour. Similarly, biopsy specimen may be obtained to histopathologically monitor the tumour during therapy.
An alternative to the invasive examination of the tumour is the indirect assessment of the tumour's angiogenesis status based on the measurement of circulating biomarkers ("surrogate markers") of angiogenesis [49]. A number of activation-associated endothelial-derived molecules can be detected in the circulation either as specific soluble splice forms of corresponding endothelial cell surface receptors and adhesion molecules or as a consequence of proteolytic shedding of endothelial cell surface molecules [50]. These include the soluble forms the angiogenic endothelial cell surface receptors VEGFR-1 (sFlt-1), VEGFR-2 (sKDR) and Tie-2 (sTie-2). Similarly, circulating concentrations of the angiogenic factors VEGF and bFGF are upregulated in many cancer patients [51]. Lastly, inflammatory markers, such as sE-selectin, sICAM-1, and sVCAM-1 are presently explored as circulating biomarkers of angiogenesis [52].
An emerging novel biomarker of angiogenesis is quantification of circulating endothelial progenitor cells (EPCs). EPCs are recruited from the bone marrow and can contribute to the vascularization of tumours. Their precise contribution to tumour angiogenesis is presently subject of intense study. Correspondingly, the quantitative analysis of circulating EPCs may prove to be a useful systemic biomarker of the intensity of local angiogenesis [53]. Lastly, gene array analyses of peripheral blood cells have been proposed as surrogate biomarkers in clinical oncology studies, including angiogenesis targeting therapies [54].
|
Perspective and key unanswered questions
|
|---|
Anti-angiogenic compounds are being tested clinically. It is likely that the first inhibitor for tumour angiogenesis may receive FDA approval in 2004. Given the advances in basic angiogenesis research and the speed with which anti-angiogenic therapies are moving into the clinic, it is increasingly recognized that reliable tumour angiodiagnostic techniques and methods to monitor the success of an anti-angiogenic intervention may become rate-limiting in rationally translating angiogenesis research into the clinic (Figure 4). These techniques, which may arise from the combined effort of pathology-based morphological techniques, clinical chemistry-based assays and non-invasive imaging approaches, will allow the monitoring of patients undergoing anti-angiogenic therapy to eventually develop reliable diagnostic tools and methods to predict which patients will benefit most from an anti-angiogenic intervention. Such knowledge is also urgently needed to rationally develop schemes for combination therapies of anti-angiogenesis with chemotherapy or radiotherapy. It is presently not completely understood how different therapeutic strategies can best be combined. Classical chemotherapy is dependent on the tumour's perfusion for drug delivery. In fact, poor tumour perfusion is one of the primary reasons for the overall poor bioavailability of most chemotherapeutic agents at the tumour site. An anti-angiogenic intervention may therefore not be very compatible with classical chemotherapy. Yet, the clinical evidence has shown that the neutralizing VEGF antibody Avastin, which was not effective as a monotherapy, had good efficacy in combination therapy with classical chemotherapy. Likewise, there is good evidence to suggest that antiproliferative chemotherapies are also acting on the angiogenic tumour neovasculature to a significant degree by targeting proliferating endothelial cells. This observation is being exploited with the development of low dose metronomic chemotherapies which are aimed at preferentially targeting a chemotherapeutic agent to the angiogenic tumour vessel compartment [55]. Such anti-angiogenic metronomic chemotherapy may act synergistically with specific anti-angiogenic therapies.
View larger version (51K):
[in this window]
[in a new window]
|
Figure 4. Research hierarchy from basic research-driven target identification and validation to clinical exploitation. The arrow denotes the relative knowledge gradient of the discipline. Much has been learnt about the basic mechanisms of angiogenesis within the last 20 years and solid proof-of-principle and proof-of-concept experiments have been performed in laboratory animals to demonstrate that anti-angiogenic intervention can not just halt tumour progression but actually induce active tumour regression. More than 50 compounds with anti-angiogenic activity are presently in various phases of clinical development. Yet, the inclusion and exclusion criteria of these clinical trials are largely not angiogenesis-based. Likewise, monitoring techniques to trace the efficacy of an anti-angiogenic intervention are urgently required. The necessary intermediate step, a better knowledge of the functional properties of the vasculature in human tumours, is emerging as the rate-limiting step in translating basic angiogenesis knowledge rationally into the clinic. Work aimed at better analysing the human tumour vasculature will also lead to the development of useful techniques for individualized functional angiodiagnostic procedures.
|
|
Similar to the difficulties of rationally combining anti-angiogenic therapies with classical or metronomic chemotherapies, the combination of anti-angiogenesis and radiotherapy is difficult to conceptualize. Anti-angiogenic therapy is by definition a pro-hypoxic therapy. Hypoxia is one of the primary reasons for radioresistence. Yet, there is good experimental and clinical evidence that the combination of anti-angiogenesis and radiotherapy may act synergistically rather than antagonistically [56].
In conclusion, the implementation of reliable angiodiagnostic techniques and a better functional understanding of the neovasculature in human tumours beyond the detection of tumour blood vessels (MVD counts) will be key to rationally implementing anti-angiogenic therapies in the clinic. Future work will have to develop new imaging methods that can bridge the resolution gap between pathology-based morphological analyses and non-invasive imaging techniques to specifically identify angiogenic vessels [57]. Given that specific targeted therapies require a specific and individualized set of diagnostic procedures, such angiodiagnostic techniques will not just contribute to the identification of those patients that will benefit most from an anti-angiogenic intervention. Angiogenic monitoring of anti-angiogenic therapy will also be useful to determine the efficacy of such an intervention and will provide a better rationale for the development and tracing of powerful combination therapies.
|
Note added in proof
|
|---|
Since submission of this review, the United States Food and Drug Administration (FDA) has given clinical approval for the first anti-angiogenic tumour drug. Approval was granted on 26 February 2004 for the use of the VEGF-neutralizing antibody AvastinTM (bevacizumab) in combination with any chemotherapy that involves 5-FU in patients with advanced colorectal cancers. The Phase III clinical trial on which the approval of AvastinTM is based included 813 patients with metastatic carcinoma of the colon or the rectum. Average survival time in patients treated with the combination of chemotherapy and anti-VEGF therapy was 20.3 months, which was approximately 25% longer compared with the patient group treated with chemotherapy alone. The overall response rate of AvastinTM when used in advanced colorectal tumours in combination with chemotherapy is 45%. This stresses the importance of individualized angiodiagnostic procedures as discussed in detail in this review.
|
Footnotes
|
|---|
Research in the author's laboratory is supported by grants from the Deutsche Forschungsgemeinschaft, the Deutsche Krebshilfe and the European Union. 
|
References
|
|---|
- Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med 1971;285:1182–6.
- Ferrara N, Henzel WJ. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun 1989;161:851–8.[CrossRef][Medline]
- Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science 1983;219:983–5.[Abstract/Free Full Text]
- Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 1996;380:435–9.[CrossRef][Medline]
- Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 1996;380:439–42.[CrossRef][Medline]
- Dor Y, Djonov V, Keshet E. Induction of vascular networks in adult organs: implications to proangiogenic therapy. Ann N Y Acad Sci 2003;995:208–16.[Abstract/Free Full Text]
- Haigh JJ, Morelli PI, Gerhardt H, Haigh K, Tsien J, Damert A, et al. Cortical and retinal defects caused by dosage-dependent reductions in VEGF-A paracrine signaling. Dev Biol 2003;262:225–41.[CrossRef][Medline]
- Carmeliet P. Angiogenesis in health and disease. Nat Med 2003;9:653–60.[CrossRef][Medline]
- Alitalo K, Carmeliet P. Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 2002;1:219–27.[CrossRef][Medline]
- Jain RK. Tumor angiogenesis and accessibility: role of vascular endothelial growth factor. Semin Oncol 2002;29:3–9.
- Huang J, Frischer JS, Serur A, Kadenhe A, Yokoi A, McCrudden KW, et al. Regression of established tumors and metastases by potent vascular endothelial growth factor blockade. Proc Natl Acad Sci USA 2003;100:7785–90.[Abstract/Free Full Text]
- Thomas AL, Morgan B, Drevs J, Unger C, Wiedenmann B, Vanhoefer U, et al. Vascular endothelial growth factor receptor tyrosine kinase inhibitors: PTK787/ZK 222584. Semin Oncol 2003;30:32–8.[Medline]
- Adams J, Huang P, Patrick D. A strategy for the design of multiplex inhibitors for kinase-mediated signalling in angiogenesis. Curr Opin Chem Biol 2002;6:486–92.[CrossRef][Medline]
- Abrams TJ, Murray LJ, Pesenti E, Holway VW, Colombo T, Lee LB, et al. Preclinical evaluation of the tyrosine kinase inhibitor SU11248 as a single agent and in combination with "standard of care" therapeutic agents for the treatment of breast cancer. Mol Cancer Ther 2003;2:1011–21.[Abstract/Free Full Text]
- Eyetech Study Group. Anti-vascular endothelial growth factor therapy for subfoveal choroidal neovascularization secondary to age-related macular degeneration: phase II study results. Ophthalmology 2003;110:979–86.[CrossRef][Medline]
- Weng DE, Usman N. Angiozyme: a novel angiogenesis inhibitor. Curr Oncol Rep 2001;3:141–6.[Medline]
- Shuey DJ, McCallus DE, Giordano T. RNAi: gene-silencing in therapeutic intervention. Drug Discov Today 2002;7:1040–6.[CrossRef][Medline]
- Thurston G. Role of angiopoietins and Tie receptor tyrosine kinases in angiogenesis and lymphangiogenesis. Cell Tissue Res 2003;314:61–8.[CrossRef][Medline]
- Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997;277:48–50.[Free Full Text]
- Augustin HG, Reiss Y. EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis. Cell Tissue Res 2003;314:25–31.[CrossRef][Medline]
- Adams RH. Vascular patterning by Eph receptor tyrosine kinases and ephrins. Semin Cell Dev Biol 2002;13:55–60.[CrossRef][Medline]
- Palmer A, Klein R. Multiple roles of ephrins in morphogenesis, neuronal networking, and brain function. Genes Dev 2003;17:1429–50.[Free Full Text]
- Kullander K, Klein R. Mechanisms and functions of Eph and ephrin signalling. Nat Rev Mol Cell Biol 2002;3:475–86.[CrossRef][Medline]
- Batlle E, Henderson JT, Beghtel H, van den Born MM, Sancho E, Huls G, et al. Beta-catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 2002;111:251–63.[CrossRef][Medline]
- Gerhardt H, Betsholtz C. Endothelial-pericyte interactions in angiogenesis. Cell Tissue Res 2003;314:15–23.[CrossRef][Medline]
- Hodivala-Dilke KM, Reynolds AR, Reynolds LE. Integrins in angiogenesis: multitalented molecules in a balancing act. Cell Tissue Res 2003;314:131–44.[CrossRef][Medline]
- Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets 2003;4:123–31.[CrossRef][Medline]
- Cheresh DA, Stupack DG. Integrin-mediated death: an explanation of the integrin-knockout phenotype? Nat Med 2002;8:193–4.[CrossRef][Medline]
- Hynes RO. A reevaluation of integrins as regulators of angiogenesis. Nat Med 2002;8:918–21.[CrossRef][Medline]
- Sullivan DC, Bicknell R. New molecular pathways in angiogenesis. Br J Cancer 2003;89:228–31.[CrossRef][Medline]
- Lawler J. Thrombospondin-1 as an endogenous inhibitor of angiogenesis and tumor growth. J Cell Mol Med 2002;6:1–12.[Medline]
- Folkman J. Role of angiogenesis in tumor growth and metastasis. Semin Oncol 2002;29:15–8.[Medline]
- Cao Y. Endogenous angiogenesis inhibitors and their therapeutic implications. Int J Biochem Cell Biol 2001;33:357–69.[CrossRef][Medline]
- Browder T, Butterfield CE, Kraling BM, Shi B, Marshall B, O'Reilly MS, et al. Antiangiogenic scheduling of chemotherapy improves efficacy against experimental drug-resistant cancer. Cancer Res 2000;60:1878–86.[Abstract/Free Full Text]
- Klement G, Baruchel S, Rak J, Man S, Clark K, Hicklin DJ, et al. Continuous low-dose therapy with vinblastine and VEGF receptor-2 antibody induces sustained tumor regression without overt toxicity. J Clin Invest 2000;105:R15–24.
- Morgan B, Thomas AL, Drevs J, Hennig J, Buchert M, Jivan A, et al. Dynamic contrast-enhanced magnetic resonance imaging as a biomarker for the pharmacological response of PTK787/ZK 222584, an inhibitor of the vascular endothelial growth factor receptor tyrosine kinases, in patients with advanced colorectal cancer and liver metastases: results from two phase I studies. J Clin Oncol 2003;21:3955–64.[Abstract/Free Full Text]
- Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. N Engl J Med 1991;324:1–8.[Abstract]
- Vermeulen PB, Gasparini G, Fox SB, Colpaert C, Marson LP, Gion M, et al. Second international consensus on the methodology and criteria of evaluation of angiogenesis quantification in solid human tumours. Eur J Cancer 2002;38:1564–79.
- Fox SB. Tumour angiogenesis and prognosis. Histopathology 1997;30:294–301.[CrossRef][Medline]
- Hlatky L, Hahnfeldt P, Folkman J. Clinical application of antiangiogenic therapy: microvessel density, what it does and doesn't tell us. J Natl Cancer Inst 2002;94:883–93.[Free Full Text]
- Brem S, Cotran R, Folkman J. Tumor angiogenesis: a quantitative method for histologic grading. J Natl Cancer Inst 1972;48:347–56.
- Hobson B, Denekamp J. Endothelial proliferation in tumours and normal tissues: continuous labelling studies. Br J Cancer 1984;49:405–13.[Medline]
- Denekamp J, Hobson B. Endothelial-cell proliferation in experimental tumours. Br J Cancer 1982;46:711–20.[Medline]
- Vartanian RK, Weidner N. Endothelial cell proliferation in prostatic carcinoma and prostatic hyperplasia: correlation with Gleason's score, microvessel density, and epithelial cell proliferation. Lab Invest 1995;73:844–50.[Medline]
- Fox SB, Gatter KC, Bicknell R, Going JJ, Stanton P, Cooke TG, et al. Relationship of endothelial cell proliferation to tumor vascularity in human breast cancer. Cancer Res 1993;53:4161–3.[Abstract/Free Full Text]
- Vartanian RK, Weidner N. Correlation of intratumoral endothelial cell proliferation with microvessel density (tumor angiogenesis) and tumor cell proliferation in breast carcinoma. Am J Pathol 1994;144:1188–94.[Abstract]
- Vermeulen PB, Verhoeven D, Fierens H, Hubens G, Goovaerts G, Van Marck E, et al. Microvessel quantification in primary colorectal carcinoma: an immunohistochemical study. Br J Cancer 1995;71:340–3.[Medline]
- Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res 2000;60:1388–93.[Abstract/Free Full Text]
- Davis DW, McConkey DJ, Abbruzzese JL, Herbst RS. Surrogate markers in antiangiogenesis clinical trials. Br J Cancer 2003;89:8–14.[CrossRef][Medline]
- Drevs J. Soluble markers for the detection of hypoxia under antiangiogenic treatment. Anticancer Res 2003;23:1159–61.[Medline]
- Gasparini G. Clinical significance of determination of surrogate markers of angiogenesis in breast cancer. Crit Rev Oncol Hematol 2001;37:97–114.[Medline]
- Byrne GJ, Ghellal A, Iddon J, Blann AD, Venizelos V, Kumar S, et al. Serum soluble vascular cell adhesion molecule-1: role as a surrogate marker of angiogenesis. J Natl Cancer Inst 2000;92:1329–36.[Abstract/Free Full Text]
- Monestiroli S, Mancuso P, Burlini A, Pruneri G, Dell'Agnola C, Gobbi A, et al. Kinetics and viability of circulating endothelial cells as surrogate angiogenesis marker in an animal model of human lymphoma. Cancer Res 2001;61:4341–4.[Abstract/Free Full Text]
- DePrimo SE, Wong LM, Khatry DB, Nicholas SL, Manning WC, Smolich BD, et al. Expression profiling of blood samples from an SU5416 Phase III metastatic colorectal cancer clinical trial: a novel strategy for biomarker identification. BMC Cancer 2003;3:3.[CrossRef][Medline]
- Hanahan D, Bergers G, Bergsland E. Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest 2000;105:1045–7.[Medline]
- Garcia-Barros M, Paris F, Cordon-Cardo C, Lyden D, Rafii S, Haimovitz-Friedman A, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis. Science 2003;300:1155–9.[Abstract/Free Full Text]
- McDonald DM, Choyke PL. Imaging of angiogenesis: from microscope to clinic. Nat Med 2003;9:713–25.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
L. A. Kunz-Schughart, J. A. Schroeder, M. Wondrak, F. van Rey, K. Lehle, F. Hofstaedter, and D. N. Wheatley
Potential of fibroblasts to regulate the formation of three-dimensional vessel-like structures from endothelial cells in vitro
Am J Physiol Cell Physiol,
May 1, 2006;
290(5):
C1385 - C1398.
[Abstract]
[Full Text]
[PDF]
|
|
|
Top
Abstract
Introduction
