Anti-vascular therapy: a new approach to cancer treatment

The understanding that the growth of tumors is dependent on angiogenesis has led to the development of new approaches to treatment and new agents directed at tumor vasculature. These have yielded striking successes in experimental models, which if translated into the clinical setting will have a substantial effect on patient survival. Such new approaches are vital because, although great strides have occurred in the treatment of certain cancers, the overall, standardized mortality from most solid tumors has altered little over the past two decades.¹

This article considers the process of tumor angiogenesis and discusses the potential of angiogenic inhibitors as therapeutic agents.

ROLE OF ANGIOGENESIS IN GROWTH OF TUMORS

The vascularity of tumors has been noted for many years.² Alguire noted that vascularization was instigated by the developing tumor: “An outstanding characteristic of the growing tumor is its capacity to elicit the production of a new capillary endothelium from the host.”³ Tannock elegantly showed that the rate of division of tumor cells decreased in proportion to their distance from the supplying blood vessel and related this to diminishing oxygen supply.⁴ Moreover, he showed that the overall rate of growth was dictated not by the proliferation of tumor cells but by the lower rate of proliferation of endothelial cells, concluding that the supply of oxygen and nutrients to the tumor limited its growth.

Tumor vascularization is a vital process for the progression of a neoplasm from a small, localized tumor to an enlarging tumor with the ability to metastasize (figure).^5,6 Anti-angiogenesis as a therapeutic concept was developed in the early 1970s based on the observation that tumors that did not vascularize failed to grow beyond a few millimeters in diameter.⁷ By comparing the growth of transplanted tumors in the avascular aqueous humor of a rabbit eye with those in the vascular iris, Folkman showed distinct avascular and vascular phases of tumor growth. The start of the vascular phase of growth coincided with tumors growing beyond 2-3 mm³ and a 20-fold increase in the rate of tumor growth. Tumors in the aqueous humor were prevented from entering the vascular phase and remained dormant.⁸ He concluded that vascularization was essential to tumor growth and inferred that preventing this process was a viable therapeutic approach.

Figure 1.

Role of angiogenesis in growth of tumors

Induction of angiogenesis by tumors

Adult endothelium is essentially quiescent, but in response to physiological or pathological stimuli (such as proliferating endometrium, injury, tumor growth or diabetic retinopathy) the endothelium can alter to a proliferating and organizing population of cells. Physiological angiogenesis can also be rapidly curtailed, indicating that the process is held in check physiologically and yet can be activated in response to the appropriate stimuli, somewhat analogous to the clotting cascade (see box).

A tumor induces this proliferative vascular response from host vessels by altering the balance of positive and negative regulators locally. This “angiogenic switch” is necessary for tumor growth and may be rate limiting.⁹ Convincing evidence exists that tumors undergo a switch to an angiogenic character as they progress. In cervical carcinoma, the development of vascularity can be associated with progression from a non-invasive premalignant stage to invasive carcinoma.¹⁰ The density of microvessels in tumors is a powerful, independent prognostic indicator of distant metastasis and survival, suggesting that tumor vascularization correlates with growth and metastatic potential.¹¹

ANTI-VASCULAR TREATMENT

There are four key approaches to anti-vascular treatment (box). All depend on targeting endothelial cells rather than tumor cells for drug action, and destruction of the tumor cells is secondary. A theoretical advantage of these approaches is that endothelial cells are not transformed and are unlikely to acquire mutations resulting in drug resistance.

Proteins that may regulate angiogenesis

• Promoters

  • Fibroblast growth factors
  • Vascular endothelial growth factor
  • Angiogenin
  • Transforming growth factor α
  • Pleiotrophin
  • Scatter factor
  • Thrombin
  • Angiopoietin 1

• Inhibitors

  • Thrombospondin
  • Angiostatin
  • Endostatin
  • Interferon α, β, γ
  • 16 kDa prolactin fragment
  • Platelet factor 4
  • Angiopoietin 2

Furthermore, treatment directed at endothelial cells is applicable to all solid tumors, irrespective of the origin of the tumor cells. Also, endothelial cells are uniquely exposed to blood-borne agents, circumventing the problem of delivering drugs to the center of a tumor, which is a major hurdle in conventional treatment.

Neutralizing angiogenic promoters

One of the favored approaches is to interfere with the balance of angiogenic proteins produced by a tumor—“turning off the switch”. This has been made possible by an understanding of the mediators of angiogenesis in normal and pathological settings. Current evidence implicates vascular endothelial growth factor (VEGF) and the family of fibroblast growth factors (FGF 1-12) as critical regulators of physiological angiogenesis. Angiopoietins 1 and 2 have been implicated as the factors controlling the recruitment of supporting cells to the developing tubule.^12,13

Both vascular endothelial growth factor and fibroblast growth factors 1 and 2 have been implicated as important promoters of tumor angiogenesis. Vascular endothelial growth factor, a mitogen and permeability factor specific for endothelial cells, is up-regulated in many different tumor types and cell lines,^{14, 15, 16} often in areas of tumor hypoxia.¹⁷

Fibroblast growth factor 2 is ubiquitous in the extracellular matrix bound in an inactive form, but in tumors the active factor is released either from the tumor or from the extracellular matrix by various mechanisms.¹⁸ Increased serum and urine concentrations of fibroblast growth factor 2 can be detected in cancer patients.¹⁹ Increasing the amounts of active fibroblast growth factors secreted by tumor cell lines, by transfecting DNA into cells, can markedly increase the ability of poorly tumorigenic cells to form tumors and to metastasize.²⁰

Because there is evidence that vascular endothelial growth factor and fibroblast growth factor 2 are promoters of tumor angiogenesis, researchers have investigated ways of interfering with their angiogenic effects and have demonstrated the ability to induce tumor regression in mice. These approaches include targeting vascular endothelial growth factor with a monoclonal antibody²¹ and preventing release of active fibroblast growth factor 2 from the extracellular matrix by eradicating a binding protein necessary for its release.²² The problem with such approaches is that they inhibit only a single positive factor. There is a range of positive angiogenic factors, and many have been shown to be up-regulated in tumors. More importantly, individual tumors can express several angiogenic factors,²³ and thus may have a number of routes around such a specific approach.

Angiogenic inhibitors as therapeutic agents

An alternative approach is to use an angiogenesis inhibitor to counter the effects of all angiogenic factors produced by a tumor. These agents can be synthetic inhibitors of endothelial cell proliferation, such as synthetic derivatives of fumagillin, or endogenous inhibitors of angiogenesis, often fragments of larger inactive circulatory proteins, that function physiologically in maintaining vascular quiescence or curtailing physiological angiogenesis. These endogenous agents can be highly potent inhibitors of endothelial proliferation and may be more effective than synthetic agents because they may also inhibit the capillary remodeling involved in expansion of tumor vessels. Angiostatin and endostatin are currently the most potent agents, both having striking anti-tumor activity. Others include a 140 kDa fragment of thrombospondin, a protein normally involved in platelet aggregation under the regulation of the tumor suppressor gene p53.^{24, 25}

Angiostatin and endostatin

The discovery of angiostatin and endostatin, two potent endogenous inhibitors of angiogenesis with powerful anti-tumor activity in mice, has produced great interest in the clinical use of angiogenesis inhibitors.^{26, 27, 28} Both were discovered as a result of the observation that the presence of a primary tumor can occasionally inhibit the development of metastases: when the primary tumor is removed, metastases develop rapidly.

Folkman hypothesized that the primary tumor produced angiogenesis inhibitors, perhaps incidentally as a result of proteolytic degradation. These inhibitors persist in the circulation while local angiogenic promoters are degraded and exert no systemic effect. Angiostatin and endostatin were identified from experimental tumors that demonstrated this phenomenon.

Both are fragments of larger circulatory proteins with no angiogenic activity. Angiostatin is a 38 kDa fragment of plasminogen, and endostatin is a fragment of collagen XVIII, a type of collagen found exclusively in blood vessels. Their anti-tumor activity in mice is impressive, being able to cause regression in various solid tumors of up to 1% of body mass. There is no evidence of drug resistance, even after multiple treatment cycles.²⁹ Research efforts are currently directed at elucidating the precise mechanism of action of these agents and at their large scale purification before they are tested in clinical trials.

Targeting endothelial cells of tumors

Another experimental anti-vascular approach to treating tumors is to target tumor endothelial cells with a toxin directed at a cell marker specific for tumor endothelium and to cause infarction of the tumor by inducing coagulation within the tumor vessels. This has been shown to be effective experimentally in a neuroblastoma that had been genetically engineered to express class II histocompatibility antigens on tumor endothelial cells. These antigens are normally absent from endothelial cells, so they served as specific markers for tumor vessels. A toxin was constructed consisting of an antibody to class II antigens linked to a truncated form of tissue factor that would cause coagulation only when bound to an endothelial cell by the antibody. This toxin induced complete regression of the experimental tumor by thrombosis of the tumor vessels while leaving the host vasculature intact.³⁰

Potential endogenous target molecules include integrin α_vβ₃, which is expressed only on proliferating vessels in healing wounds and in tumors. Antibodies to integrin α_vβ₃ promote tumor regression by inducing endothelial cell apoptosis.³¹

FUTURE DEVELOPMENTS

Pre-clinical research into tumor angiogenesis has led to the identification of several anti-vascular treatments with impressive efficacy in animal models of human cancer. Currently, 19 anti-vascular agents are being assessed in clinical trials, mostly in phase I and II trials that involve treating patients with advanced metastatic disease resistant to other treatments. (Information about current trials can be obtained from the National Cancer Institute's website at cancertrials.nci.nih.gov.)

There are occasional reports of striking clinical remissions,³² but the real efficacy of these agents will only become apparent over the next decade as they are fully evaluated in extensive clinical studies either alone or with standard treatments.

As inhibition of angiogenesis may induce dormancy of a tumor rather than killing it, there is growing appreciation that the administration of these agents, and their assessment in clinical trials, may need to be different from that used for cytotoxic drugs. Indeed, it is possible that these agents could be effective in maintaining long-term remission, an approach not currently used for solid tumors.

Approaches to anti-vascular treatment

• Neutralizing angiogenic promoters

  • Interfere with the positive effect of angiogenic factors produced by a tumor
  • Examples include
  • Antibodies to vascular endothelial growth factor
  • Viral delivery of dominant negative receptors to vascular endothelial growth factor
  • Prevent release and activation of fibroblast growth factor 2

• Endogenous angiogenic inhibitors

  • Use endogenous inhibitory proteins to counter the angiogenic stimulus produced by tumors
  • Examples include
  • Supply of angiogenic inhibitors directly—such as angiostatin, endostatin
  • Gene transfer of DNA to angiogenesis inhibitors—angiostatin, platelet factor 4

• Endothelial cell targets

  • Use specific markers for tumor endothelial cells to direct a toxin or antibody to tumor vasculature to cause tumor infarction
  • Targets include
  • Integrin α_vβ₃
  • Vascular endothelial growth factor-receptor complexes
  • Endoglin

• Synthetic angiogenic inhibitors

  • Inhibit tumor angiogenesis with drugs that specifically prevent endothelial cell division
  • Can act synergistically with conventional cytotoxic treatment and with “hypoxia-activated” cytotoxic drugs