Antiangiogenic tumour therapy: will it work?

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Antiangiogenic tumour therapy: will it work? Hellmut G. Augustin The inhibition of angiogenesis is considered to be one of the most promising strategies that might lead to the development of novel antineoplastic therapies. This concept is supported by the dramatic results of gene inactivation experiments in mice that have identified several vascular endothelium related molecules as rate limiting for embryonic angiogenesis. Likewise, a number of recent animal studies have shown that angiogenesis inhibitors can prevent metastasis and shrink established experimental tumours to small dormant microtumours. In this review, Hellmut Augustin illustrates differences between developmental angiogenesis, physiological angiogenesis in the adult, and pathological angiogenesis in experimental animal tumours and natural human tumours. He then summarizes the experimental approaches to antiangiogenic therapies and finally discusses critical issues that need to be considered in translating these novel therapeutic strategies into clinical practice.

H. G. Augustin, Group Leader, Cell Biology Laboratory, Department of Gynaecology and Obstetrics, University of Göttingen Medical School, 37075 Göttingen, Germany.

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Growth of the vascular system is primarily a developmental process that occurs during embryogenesis and only to a limited extent in postnatal life1–3. Except for the female reproductive system4, angiogenesis in the adult is almost completely downregulated and is mostly related to pathological tissue growth, where it may be desirable (wound healing, cardiac ischaemia) or undesirable (tumour growth, diabetic retinopathy). Consequently, the goal of therapeutic manipulation could be both the stimulation and the inhibition of angiogenesis. The primary target for an antiangiogenic intervention is the inhibition of tumour growth, by attacking the tumour’s vascular supply5–11. Conceptually, therapeutic targeting of the tumour vasculature is extremely appealing for a number of reasons: (1) as an oncofoetal mechanism that is mostly downregulated in the healthy adult, targeting of angiogenesis should lead to minimal side-effects even after prolonged treatment; (2) tumour-associated angiogenesis is a physiological host mechanism and, consequently, its pharmacological inhibition should not lead to the development of resistance12; (3) potentially, each tumour capillary supplies hundreds of tumour cells, so that the targeting of the tumour vasculature should lead to a potentiation of the antitumorigenic effect; and (4) in contrast to the interstitial location of tumour cells, direct

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contact of the vasculature with the circulation allows efficient access of therapeutic agents. Antiangiogenic therapies have already proved to be very powerful in a number of experimental animal tumour models. The first substances have entered clinical trials, highlighting the fact that angiogenesis research has advanced from basic science to the bedside5. A number of excellent reviews have summarized the current concepts of the molecular mechanisms of the angiogenic cascade2–5,13–17 and highlighted the prospect of antiangiogenic tumour therapies5–11. The reader is referred to these references for a more detailed account of individual aspects of current angiogenesis research. This review will briefly conceptualize the mechanisms of angiogenesis to discuss the specific quantitative and qualitative properties of angiogenic processes in naturally occurring human tumours as opposed to experimental animal tumour models and reproductive angiogenesis.

Mechanisms of angiogenesis A plethora of angiogenic factors has been identified in the past 20 years5,16–19 (Table 1). While the angiogenesisinducing capacity of these molecules is well established, most of them are not specific angiogenesis inducers. Rather, they perform numerous other biological functions and it is little understood to what extent they contribute to the angiogenic cascade and what combination of angiogenic cytokines makes up the ‘angiogenic cocktail’ in various types of angiogenesis (e.g. developmental, reproductive, inflammatory and tumour angiogenesis). Furthermore, some of these pleiotrophic growth factors may have multiple, sometimes even antagonistic, functions in the angiogenic cascade. For example, transforming growth factor ␤ (TGF-␤) inhibits endothelial cell functions in vitro, but induces angiogenesis in vivo, supposedly by activating other proangiogenic cells20. Likewise, the angiogenesis-inducing capacity of TNF-␣ is highly dosage dependent: low concentrations are proangiogenic, whereas higher concentrations are antiangiogenic21.

Vascular endothelial growth factors act as specific angiogenesis inducers In contrast to the pleiotrophic angiogenic growth factors, vascular endothelial growth factor (VEGF) has a narrow target cell specificity and acts primarily on endothelial cells16,22,23. VEGF is a member of a growing family of polypeptide growth factors consisting of VEGF-A (the original VEGF), VEGF-B, VEGF-C and VEGF-D. VEGF-A and VEGF-B appear to be involved primarily in haemangiogenesis, whereas VEGF-C and most likely VEGF-D regulate lymphangiogenesis24. VEGFs mediate their endotheliotropic activities through receptor tyrosine kinases [VEGF-R1 (flt-1), VEGF-R2 (flk-1/KDR) and VEGF-R3 (flt-4)], whose expression is upregulated on endothelial cells during angiogenesis (Fig. 1)13,23,25. The central role of VEGF-A in angiogenesis was demonstrated unambiguously through gene

Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0165 – 6147/98/$19.00

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Table 1. Positive and negative endogenous regulators of angiogenesis Stimulators

Inhibitors

Peptide growth factors Vascular endothelial growth factor A, B, C, D (VEGF-A, -B, -C, -D) Placenta growth factor (PIGF) Acidic fibroblast growth factor (aFGF) Basic fibroblast growth factor (bFGF) Platelet-derived growth factor (PDGF) Transforming growth factor ␣ (TGF-␣) Transforming growth factor ␣ (TGF-␤) Hepatocyte growth factor (HGF) Insulin-like growth factor I (IGF-I) B61/LERK-1

Proteolytic peptides Angiotensin Endostatin 16 kDa prolactin fragment Laminin peptides Fibronectin peptides

Multifunctional cytokines/immune mediators Tumour necrosis factor ␣ (low dose) Monocyte chemoattractant protein 1 (MCP-1)

Multifunctional cytokines/immune mediators Tumour necrosis factor ␣ (high dose) Interferons Interleukin 12

CXC chemokines Interleukin 8 (IL-8)

CXC chemokines Platelet factor 4 (PF-4) Interferon ␥-inducible protein 10 (IP-10) Gro-␤

Enzymes Platelet-derived endothelial cell growth factor (PD-ECGF; thymidine phosphorylase) Angiogenin (ribonuclease A homologue)

Inhibitors of enzymatic activity Tissue metalloproteinase inhibitors (TIMP 1, 2, 3, 4) Plasminogen activator inhibitors (PAI-1, -2)

Extracellular matrix molecules Thrombospondin

Hormones Oestrogens Prostaglandin E1, E2 Follistatin Proliferin

Hormones/metabolites 2-Methoxyoestradiol (2-ME) Proliferin-related protein

Oligosaccharides Hyaluronan oligosaccharides Gangliosides

Oligosaccharides Hyaluronan, high-molecular-weight species

Haematopoietic growth factors Erythropoietin Granulocyte colony-stimulating factor (G-CSF) Granulocyte–macrophage colony-stimulating factor (GM-CSF)

inactivation experiments in mice: loss of a single VEGFA allele is not compatible with life and leads to early embryonic death26,27. VEGF-A is not just central to embryonic angiogenesis, but it plays a similarly crucial role in all situations of adult angiogenesis: VEGF-A is expressed prominently during reproductive angiogenesis (ovary, uterus)18,22 and interference with VEGF-A signalling in rats (e.g. by application of soluble VEGFR1) leads to a complete block of ovarian function. Similarly, tumour angiogenesis is associated with an upregulated expression of VEGF-A and its receptors13.

a role for these receptors in blood vessel assembly and maturation. Ligands with agonistic and antagonistic activities for Tie-2 have now been identified: angiopoietin 1 (Ang-1) is an activating ligand for Tie-2 and regulates blood vessel maturation28. Angiopoietin 2 (Ang-2) is another Tie-2 ligand that binds to the receptor but does not transduce a signal29. Thus, Ang-2 acts as a functional Ang-1 antagonist. Overexpression of Ang-2 leads to destabilization of blood vessels and is involved in blood vessel regression processes as they occur in the ovarian corpus luteum29.

Angiopoietins regulate blood vessel assembly and maturation

A sequential model of the angiogenic cascade

Another family of endotheliotropic growth factors has recently been identified: two endothelial cell receptor tyrosine kinases, Tie-1 and Tie-2, have been known for a number of years, but their ligands were not known. Genetic inactivation experiments in mice have suggested

Figure 2 shows a proposed model of the angiogenic cascade that assembles the determinants of angiogenesis into a sequence of events: hypoxia leads to the expression of hypoxia-related transcription factors [hypoxiainducible factor 1 (HIF-1)30, hypoxia-inducible factor 2 (HIF-2, HIF-related factor31)] which in turn control the

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Positive and negative regulators of angiogenesis Like most biological processes, angiogenesis is regulated through a balance of stimulators and inhibitors5,15,18. A number of endogenous inhibitors of angiogenesis has been identified in the past few years. The most prominent of these are the matrix molecule thrombospondin18, the CXC-chemokine platelet factor 4 (PF-4)33, the plasminogen fragment angiostatin34, and the collagen XVIII fragment endostatin35. The list of lesswell-studied endogenous inhibitors of angiogenesis is still growing (Table 1), suggesting that the process of angiogenesis is tightly controlled by a network of inducing and inhibiting factors. Fig. 1. Molecular properties of angiogenic endothelial cells. During angiogenesis, endothelial cells display a distinct gene expression phenotype that reflects the requirements of the angiogenic cascade. The principal cellular functions involved in the regulation of the angiogenic cascade are shown adjacent to the encircled text, whereas specific molecules of angiogenic endothelial cells that have been characterized in some detail are shown in circles. The proteolytic balance of angiogenic endothelial cells is shifted towards an invasive phenotype, as evidenced by an upregulated expression of matrix metalloproteinases (MMP) and serine proteases, such as the plasminogen activator/plasmin system [tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA)]. At the same time, protease inhibitors, such as plasminogen activator inhibitor I (PAI-I), may also be upregulated, suggesting that a precise protease/antiprotease equilibrium allows localized pericellular matrix degradation during cell migration, while protecting against excessive extracellular matrix destruction. Sprouting capillaries synthesize their own extracellular matrix (ECM) and preferentially express basement membrane components such as tenascin, type IV collagen and laminin. Adhesion molecules such as the integrin heterodimers ␣v␤3 and ␣v␤5 mediate the interactions of invasive, migratory endothelial cells with the local extracellular matrix. Angiogenic endothelial cells also activates a number of autocrine growth regulating systems such as heparin-binding fibroblast growth factors (aFGF and bFGF) and the activin-binding glycoprotein follistatin (FS). A number of surface receptors are upregulated by angiogenic EC: corresponding to the angiogenic activation by endothelial cell-specific growth factors [vascular endothelial growth factors (VEGFs), angiopoietins], the expression of VEGF-R1, VEGF-R2, Tie-1 and Tie-2 is upregulated during embryonic angiogenesis and tumour angiogenesis. Tissue factor expression was also found to be upregulated by tumour angiogenic endothelial cells.

expression of hypoxia response genes, such as VEGF-A (Ref. 32). VEGF-A and related molecules act as endotheliotropic angiogenesis inducers. It might be most appropriate to conceptualize the VEGFs by designating them as ‘specificity providers’. To a varying degree and in various combinations, the pleiotrophic angiogenic growth factors might play a secondary role as locally acting angiogenesis factors, which further stimulate endothelial cells that are primed by specificity providers. Angiogenic activation through specific and pleiotrophic angiogenic growth factors induces a complex response programme in activated endothelial cells (Fig. 1). This includes the activation of autocrine growth-regulating systems; the expression of cytokines, growth factor receptors, molecules regulating the cells’ proteolytic balance and adhesion molecules that facilitate the endothelial cells’ complex functions during the angiogenic cascade; and the molecular regulation of vascular network formation and vessel maturation. Finally, in 218

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Angiogenesis in human tumours The quantitation of microvessel densities (MVDs) using endothelial cell markers such as von Willebrand Factor (vWF), CD31 or CD34 to visualize blood vessels has been used widely to assess the vascular status of a number of different human tumours36,37. In general, malignant tumours with poor prognosis were found to have high MVDs. However, a significant number of studies failed to establish a relationship between intratumoural MVD counts and poor prognosis. These discrepancies were related primarily to differences in quantitation techniques and the unavoidable subjectivity in identifying tumour vascular ‘hot spots’. It is important to note that the quantitation of MVD gives an indication of a tissue’s vascularization but does not necessarily reflect angiogenesis. This becomes most obvious when considering the lungs, which are by far the most vascularized tissue and, therefore, will give the highest MVD count, despite the fact that there is absolutely no angiogenesis in the lungs of healthy adult individuals.

Quantitative aspects of angiogenesis in human tumours Surprisingly little is known about the degree of active angiogenesis in different human tumours. Active angiogenesis has been assessed in a number of studies by determining the percentage of proliferating endothelial cells38,39. Essentially, all of the early studies on the analysis of endothelial cell proliferation were performed in experimental animal tumours. However, it is important to note that the growth dynamics of experimental tumours differ greatly from human tumours: an experimental mouse tumour may grow to several grams in weight (up to 20% of body weight) within weeks. In comparison, all human tumours grow more slowly and, consequently, the intensity of active angiogenesis will be weaker in human tumours. This is also reflected by more recent experiments, quantitating the percentage of

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Fig. 2. Sequential model of the angiogenic cascade during tumour angiogenesis. (1) A conducive microenvironment (hypoxia, acidosis) and tumour-specific factors, such as the activation of oncogenes, mediates (2) the expression of tumour-derived endothelial cell-specific growth factors, such as vascular endothelial growth factor A (VEGF-A) (referred to as ‘specificity providers’). (3) Other autocrine and paracrine growth factors contribute to a specific local ‘angiogenic cocktail’. (4) Endothelial cells support the angiogenic cascade through mechanisms of autocrine growth control and the expression of molecules that mediate the interactions of angiogenic endothelial cells with their microenvironment (EC-RTK, endothelial cell receptor tyrosine kinases). (5) The recruitment of monocytes and leukocytes by endothelial cell and tumour cell-derived chemokines reflects the inflammatory contribution of tumour angiogenesis and may further enhance the angiogenic cascade. (6) The complex three-dimensional alignment of new capillaries is orchestrated by a complex network of cytokines, among which the angiopoietins play a key role. aFGF, acidic fibroblast growth factor; bFGF, basic fibroblast growth factor; PD-ECGF, platelet-derived endothelial cell growth factor; PIGF, placenta growth factor; TGF, transforming growth factor.

proliferating endothelial cells in human tumours: an intratumoural endothelial cell proliferation index as low as 0.15% was determined in prostatic carcinomas40. This value was found to be 30-fold higher than the endothelial cell proliferation index in benign prostate hyperplasia, but still lower (by a factor of 43) than the endothelial cell proliferation index in granulation tissue. Furthermore, the intratumoural endothelial cell proliferation index did not correlate with intratumoural microvessel density. Similar findings were reported for breast carcinomas41. In contrast, an endothelial cell labelling index as high as 9.9% was determined in colorectal adenocarcinomas42. In comparison, endothelial cell labelling indices in reproductive angiogenesis (e.g. growth of the corpus luteum) may be as high as 36%43. These data clearly reflect the fact that: (1) there is considerable variation in the degree of active angiogenesis in different tumour types and even within the same tumour type; and (2) the intensity of angiogenesis in human tumours is considerably lower compared with experimental animal tumours and physiological angiogenesis (granulation tissue, reproductive angiogenesis).

Qualitative aspects of angiogenesis in human tumours It is now well established that tumour growth is angiogenesis dependent. Although this may appear self

evident nowadays, it was established through pioneering experiments only 20 years ago. Before that time, it was widely believed that tumours grew along pre-existing blood vessels. More recent experiments on the establishment of experimental models of tumour dormancy have strengthened further the concept of the strict angiogenesis dependency of malignant tumour growth44. Considering that the crucial requirement of angiogenesis for tumour growth is almost established as a firm dogma, it may appear heretical to challenge this idea. Empirical experience, however, has taught that nature’s pathways are usually complementary and not mutually exclusive. Consequently, it is proposed that angiogenesis-dependent and angiogenesis-independent tumour growth might both be present in human tumours. Recent experiments support this concept: a very detailed analysis of angiogenesis in a large number of non-small-cell lung cancers has identified four different types of tumour growth45. Among these, as many as 16% of tumours were growing in an alveolar-like way, along pre-existing blood vessels, and showed absolutely no signs of active angiogenesis. Some of the other tumour types may represent the simultaneous presence of angiogenesis-dependent and angiogenesis-independent tumour growth. Similar studies are necessary in other tumour types to determine the extent to which angiogenesis-independent

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Table 2. Antiangiogenic therapeutic strategies Substance/approach

Comment

Synthetic/semi-synthetic inhibitors Carboxiamidotriazole (NCI) Ca2+ channel blocker, Phase 1 CM101 Analogue of group B streptococcus toxin (polysaccharide), binds to tumour endothelium, induces inflammation Marimastat (British Biotech) Metalloproteinase inhibitor, inhibits endothelial and tumour cell invasion, Phase II (pancreatic, lung, brain) Pentosan polysulphate Inhibits heparin-binding growth factors, Phase I TNP-470 (Takeda/Abbott) Analogue of antibiotic fumagillin, inhibits endothelial cell migration and proliferation, Phase III (breast, Kaposi’s sarcoma, cervical) Thalidomide (EntreMed) Polycyclic teratogen, antiangiogenic mechanism unknown, Phase II (brain, breast, prostate) Endogenous inhibitors Angiopoietin 2 (Regeneron) Angiostatin Endostatin IL-12 (Roche, Genetics Inst.) Interferon ␣ Platelet factor 4

Interferes with blood vessel maturation Plasminogen fragment, antiangiogenic mechanism unknown Collagen XVIII fragment, antiangiogenic mechanism unknown Induces IP-10, Phase I Decreases FGF production, Phase III (infant haemangiomas) Inhibits endothelial cell proliferation

Biological antagonists ␣v/␤3 integrin antagonists VEGF inhibitors VEGF receptor blockers Soluble receptors

Monoclonal antibodies LM609 and 9G2.1.3, induce endothelial cell apoptosis Humanized neutralizing antibody, antisense oligonucleotides Small receptor tyrosine kinase antagonists Angiogenesis inhibition with soluble VEGR-R1 or soluble Tie-2

Vascular targeting Regional TNF-␣ therapy Antibody targeting Vascular gene therapy

Isolated limb perfusion to target in transit metastases Use of mono- and bispecific antibodies to target angiogenic endothelial cells (e.g. VEGF receptors, endoglin) to deliver specific angio- and/or tumoricidal activity Transfer of dominant-negative receptors or suicide genes under the control of angiogenic endothelial cell-specific promoters.

The names of the company sponsors of some of the substances are given in parentheses; Phase I, II and III refer to the clinical stage of evaluation with the primary tumour targets given in parentheses. FGF, fibroblast growth factor; IL-12, interleukin 12; IP-10, interferon-␥-inducible protein 10; TNF-␣, tumour necrosis factor ␣; VEGF, vascular endothelial growth factor.

tumour growth is operative in human tumours. The recognition that both angiogenesis-dependent and angiogenesis-independent tumour growth could be present in human tumours will have major implications for tumour biology and the implementation of possible antiangiogenic therapies. Obviously, it would explain many of the discrepancies in intratumoural MVD counting studies. Indeed, high intratumoural MVD counts may reflect active angiogenesis and poor prognosis. However, low MVD counts may not necessarily be indicative of a good prognosis.

Antiangiogenic therapy A number of experimental approaches have been taken to inhibit angiogenesis5–11 (Table 2). Pharmacological inhibition of angiogenesis is aimed at interfering with the angiogenic cascade or the immature neovasculature. Pharmacological agents may be synthetic or semi-synthetic substances, endogenous inhibitors of angiogenesis or 220

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biological antagonists of the angiogenic cascade5–10. In contrast, vascular targeting is aimed at utilizing specific molecular determinants of the neovasculature for the delivery of a biological, chemical or physical activity that will then act locally in an angiocidal or a tumouricidal manner7,11.

Synthetic angiogenesis inhibitors The development of synthetic inhibitors of angiogenesis is most advanced and several substances have entered clinical trials; the first of these is the fumagillin derivative TNP-470 [6-O-(N-chloroacetyl-carbamoyl)fumagillol]46. Fumagillin is an antibiotic that was identified accidentally as an endothelial cell migration and proliferation inhibiting substance. The mechanism of action of AGM1470 is still poorly understood, but it was shown recently that it binds and inhibits the metalloprotease methionine aminopeptidase (MetAp-2)47. Other antibiotics with antiangiogenic activity are minocycline and herbimycin A. Carboxyamidotriazole (CAI) inhibits

R the influx of calcium into cells and suppresses the proliferation of endothelial cells. It inhibits angiogenesis and metastasis, but it is not an endothelial cell-specific substance48. Similarly, the metalloproteinase inhibitors Marimastat (BB2516) and Batimastat (BB94) are not vascular-specific substances, but are both antiangiogenic and antitumourigenic by inhibiting invasion of endothelial cells as well as tumour cells49. Thalidomide appears to be one of the most promising novel antitumourigenic and antiangiogenic substances50. Originally developed as a hypnosedative drug in the late 1950s and subsequently withdrawn from the market because of its teratogenic effects, thalidomide has been reintroduced selectively in the last few years for use in various disorders thought to have an autoimmune or inflammatory basis. The mechanism of thalidomide’s antiangiogenic activity is not known but it probably impedes cell migration by downregulating ␤-integrins50. Pentosan polysulphate51 (xylanopolyhydrogensulphate) is a semi-synthetic sulphated heparinoid polysaccharide. It has been used as an anticoagulant for many years. It exerts antiangiogenic activity by interfering with the binding of angiogenic growth factors to the cell surface. Another substance that has entered clinical trials is the analogue of a group B streptococcus toxin (GBS toxin) that has been designated CM101 (Ref. 52). The polysaccharide CM101 appears to bind preferentially to a subset of tumour endothelial cells, thereby inducing a massive local inflammatory reaction, which in turn acts in a tumouricidal manner.

Endogenous angiogenesis inhibitors The endogenous inhibitors of angiogenesis are summarized in Table 1. Of these, interleukin 12 has already entered clinical trials as an antiangiogenic and antitumourigenic substance. Some of the other endogenous inhibitors of angiogenesis hold promise for therapeutic application and are included in Table 2. Of particular interest are recently identified antiangiogenic molecules that are proteolytic fragments of larger proteins with different biological functions. Among these are angiostatin34 and endostatin35. As endogenous substances, these molecules have a long half-life in the plasma (angiostatin, 2.5 days) and, thus, are particularly attractive for long-term treatments.

Antagonists of the angiogenic cascade Inhibition of any of the key regulators of the angiogenic cascade has been effective in experimental animal models. Several experimental strategies have been taken to interfere with the interaction of VEGF with its receptors5. These include antisense and antibody approaches to inhibit VEGF, the development of small-molecularweight antagonists to the VEGF receptors and the use of soluble VEGF receptors. Likewise, antagonistic antibodies to the integrin heterodimer ␣v␤3 have been effective in interfering with the interaction of angiogenic endothelial cells with their extracellular matrix, causing the cells to detach and to die by apoptosis53.

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Vascular targeting The goal of vascular targeting is to utilize specific molecular determinants of angiogenic endothelial cells to deliver substances or activities that destroy the vasculature7,11. This concept has been used to target the cellsurface domain of human tissue factor to endothelial cells by means of a bispecific antibody to an experimentally induced marker on the tumour vasculature. This approach led to localized thrombosis and the tumour regressed as a result of the massive infarction54. Vascular targeting has also been used in the regional high dose TNF-␣ therapy of advanced extremity soft tissue sarcomas55. In these experiments, an isolated limb perfusion system was used to target TNF-␣ preferentially to tumour-associated endothelial cells, causing a rapid destruction of the sarcoma-associated microvasculature55. Similarly, genetic targeting experiments are underway to direct suicide genes, such as herpes simplex virus thymidine kinase (HSV-TK) to proliferating endothelial cells by means of endothelial cell-specific promoters11,56.

Perspective To translate the results of experimental studies into novel, clinically applicable therapies, the qualitative and quantitative differences between angiogenesis in human tumours and in animal models, as discussed above, need to be critically assessed in order to: (1) establish the most promising antiangiogenic tumour therapies; (2) identify the types of tumours most suitable for antiangiogenic therapy; and (3) develop techniques to assess each individual tumour patient’s angiogenesis status.

Towards an individual quantitative assessment of angiogenesis The endothelial cell markers used so far in most MVD studies (vWF, CD31, CD34) are useful for assessing the vascular status of a tissue, but do not reflect active angiogenesis. Increasingly, however, marker molecules of angiogenic endothelial cells are being identified (Fig. 1). The development of MVD counting techniques that use angiogenic endothelial cell markers and, thus, quantitate angiogenesis and not just the presence of blood vessels, will further aid a more realistic assessment of angiogenesis in tumours. Corresponding concentrations of angiogenic cytokines have been measured in a number of body fluids and used as angiogenesis markers for various tumours5,57. At the same time, imaging techniques to assess tumour vascularization and perfusion applying colour Doppler ultrasound58 and dynamic contrast-enhanced magnetic resonance imaging59 have been improved. Results obtained with these imaging techniques have been correlated with histological MVD counts to determine more reliably the vascular status of individual tumours60. Imaging techniques in combination with MVD analysis should allow the reliable non-invasive and histological biopsy-based assessment of the

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angiogenesis status of an individual tumour patient. This will become a prerequisite for the identification of tumour patients who will benefit most from antiangiogenic therapies.

Likely and unlikely targets of antiangiogenic tumour therapy All antiangiogenic therapies aimed at targeting specific molecular determinants of angiogenic endothelial cells will only have a limited antitumourigenic effect because only a subfraction of tumour microvessels expresses the immature angiogenic phenotype and is accessible to this type of therapy. On the other hand, the targeting of angiogenic endothelial cells and the use of endogenous angiogenesis inhibitors will most likely allow long-term treatments without major side-effects. Furthermore, targeting of the tumour vasculature is conceptually very attractive as this type of therapy is ‘resistant to the development of resistance’12. This is in contrast to the targeting of tumour cell determinants, which may shift according to different selection pressures61. Vascular targeting techniques might be most effective for short-term applications. By combining the specific targeting of angiogenic endothelial cells with a broad effector function (‘target locally and then kill globally’), these approaches have the distinct advantage that they should be able to elicit a broad tumouricidal effect, even if only a subfraction of tumour microvessels actually expresses an immature angiogenic phenotype. Classic pharmacotherapeutical approaches to inhibit angiogenesis have advanced farthest and several of these substances have entered clinical trials (Table 2). Most likely, these substance will not prove to be magic bullets in the war against cancer, but they hold promise for adjuvant combination therapies with other chemotherapeutic agents.

Concluding remarks

Acknowledgement Work in the author’s laboratory is supported by grants from the Deutsche Forschungsgemeinschaft and the Deutsche Krebshilfe.

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Antiangiogenic tumour therapies are on the verge of entering the clinic. They will undoubtedly have the potential to change standard chemotherapeutic tumour therapies. However, it will be necessary to develop techniques for the assessment of the angiogenesis status of individual tumour patients to identify those patients for whom antiangiogenic therapies will be most effective. Furthermore, additional molecular determinants of the angiogenic cascade have been identified: the first molecules regulating blood vessel maturation, the angiopoietins, have just been identified28,29. It could well be that the vascular bed in human tumours is not so much characterized by active angiogenesis but rather by an inability to mature fully. Casting experiments of human tumours support this possibility62. Consequently, therapies aimed at molecular determinants of incompletely mature tumour blood vessels could add yet another approach to vascular targeting therapies of tumours.

TiPS – June 1998 (Vol. 19)

Selected references

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