Volume 13, Issue 1, Pages 53-61 (January 2003)

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Targeting the tumor blood vessel network to enhance the efficacy of radiation therapy☆☆☆★

Abstract

It has been well established that the vascularization of solid tumors is a prerequisite if a clinically relevant size is to be reached. For progressive tumor growth, the vessel network must continuously expand to satisfy the neoplastic cells' nutritional needs and waste product removal requirements. This utter reliance of the tumor on its vasculature provides a logical target for new approaches to cancer therapy. Indeed, there currently exists a great deal of enthusiasm for the development of interventions that compromise the growth and/or function of the tumor neovasculature. Two primary directions are being pursued. Inhibitors of angiogenesis seek to interrupt the angiogenic process to prevent new vessel formation. Antivascular approaches aim to cause direct damage to the tumor endothelium and thus lead to extensive secondary neoplastic cell death. The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies. Copyright 2003, Elsevier Science (USA). All rights reserved.

Article Outline

• Abstract

• Tumor vasculature

• Controlling blood vessel development

• Targeting the tumor vasculature

• Combinations with radiation therapy

• Antiangiogenic treatments

• Vascular targeting agents

• Combining antiangiogenic and antivascular treatment strategies

• Conclusions

• References

• Copyright

Radiotherapy is one of the most important treatment modalities for solid tumors.1 Still even though nearly 70% of cancer patients who are cured receive radiation either alone or in combination with other modalities,2 significant numbers of radiotherapy patients treated with curative intent ultimately fail.2 Major reasons for radiotherapy failures include genetic resistance, presence of abnormal tumor microenvironments, tumor progression, and metastatic spread of the disease. An important contributor underlying many of these treatment failures may be the tumor vasculature.

Tumor vasculature

Tumor growth requires the formation of new blood vessels to facilitate the delivery of nutrients such as oxygen and the removal of waste products. This process of neovascularization, also referred to as angiogenesis, is essential to all solid tumors.3, 4, 5 Tumor cells promote new vessel formation by releasing endothelial cell growth factors that support endothelial cell proliferation, migration, and survival.6 Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are 2 proangiogenic growth factors widely considered to be critically important.7, 8 The switch to an angiogenic phenotype results in enhanced tumor growth, increased endothelial cell proliferation, and extensive new vessel formation. Still, neovascularization invariably lags behind the aggressively expanding tumor mass.9 The resulting tumor vasculature is highly abnormal and chaotic, typically exhibiting torturous and dilated vessels with heterogeneous flow and permeability.10, 11, 12 It is unable to provide adequate nutritional support13 and consequently gives rise to tumors characterized by microregional variations in concentrations of oxygen, glucose, and other nutritional factors, as well as metabolic waste products.13, 14 The fact that the production of several angiogenic growth factors can be upregulated by physiologic parameters, including low oxygen or glucose and acidic pH, which are associated with these vascular insufficiencies, provides further rationale for the strong angiogenic stimulus in malignant tissue.15, 16, 17, 18, 19 Given its pivotal role in tumor development, progression, and spread, a great deal of research in recent years has focused on the development of strategies aimed at interfering with the process of neovascularization in tumors (angiogenesis inhibitors [AIs]) or destroying the existing tumor blood vessel network (vascular targeting agents [VTAs]).

Controlling blood vessel development

Although angiogenesis is a complex process with multiple, sequential, and interdependent steps, this complexity also creates many potential targets for therapeutic suppression. Many agents that are antiangiogenic have been identified and characterized20, 21, 22, 23, 24, 25; each of these affects at least one of the several stages involved in new vessel formation, (ie, tumor/endothelial cell signaling, basement membrane degradation, endothelial cell migration, endothelial cell proliferation, and tube formation). Proangiogenic growth factors are primary targets because of their significance in tumor neovascularization.26, 27, 28 Indeed, a variety of approaches targeting VEGF and bFGF, including the use of antibodies,29 the inhibition of the tyrosine kinase activity of endothelial cell receptors,30, 31, 32, 33 and the use of antisense to downregulate tumor cell expression of these angiogenic factors,34, 35 are being investigated as antiangiogenic therapeutic strategies. Another favorite target has been the matrix metalloproteinases (MMPs), a class of enzymes that are critically involved in the degradation of the underlying matrix. Inhibitors of these proteins have shown antiangiogenic effects in preclinical models36 but to date clinical trials conducted with synthetic MMP inhibitors have been disappointing. Finally, a great deal of effort has focused on the suppression of tumor-induced angiogenesis by elevating known endogenous inhibitors of angiogenesis, such as endostatin and angiostatin.37, 38, 39 Thalidomide also belongs to this class, and encouraging results have been reported from phase 2 clinical trials.40, 41 The overarching goal of antiangiogenic strategies is to interfere with the proangiogenic balance between tumor, stromal, and endothelial cells to stabilize the growth of the tumor by preventing further development of a functional vessel network.

Targeting the tumor vasculature

As an alternative to interfering with the angiogenic process per se, significant strides have been made in identifying and developing agents that specifically compromise the function of the existing vasculature in solid tumors. These strategies have focused primarily on agents that cause direct damage to the tumor endothelium.42, 43

Two classes of agents that can induce extensive hemorrhagic necrosis in tumors as a result of vascular collapse have been identified. The first includes flavone acetic acid (FAA) and its analogs.44, 45, 46, 47 The mechanism of action of this class appears to be largely indirect, through the induction of cytokines, particularly tumor necrosis factor alpha (TNF-α).48, 49 One of the most promising flavone derivatives is 5,6-dimethyl-xanthenone-4 acetic acid (DMXAA). This agent has shown significant potency in preclinical in vivo studies evaluating the induction of tumor necrosis and treatment response.45, 46, 47, 50, 51, 52

A select group of tubulin-binding agents, most notably combretastatin A4 disodium phosphate (CA4DP) and the phosphate prodrug of N-acetycolchinol (ZD6126), are the lead compounds in a second class of interesting agents. Both agents have shown potent antivascular and antitumor efficacy in a wide variety of preclinical tumor models at well-tolerated doses.53, 54, 55, 56, 57, 58, 59, 60, 61 The principal mechanism of action responsible for the tumor-specific antivascular effects is believed to be the selective disruption of the cytoskeleton of proliferating endothelial cells that results in endothelial cell shape changes54, 58, 62, 63, 64 and leads to thrombus formation and a consequent secondary cascade of ischemic tumor cell death.62, 65

Combinations with radiation therapy

There are 2 major reasons for examining the combination of radiation treatments with strategies aimed at compromising the tumor vasculature. The first is that the inclusion of such strategies may improve radiotherapy outcomes by capitalizing on principles of enhanced antitumor efficacy, nonoverlapping toxicities, or spatial cooperation.66 Abnormal tumor microenvironments, tumor progression, and metastatic spread of neoplastic cells are major factors contributing to treatment failures in radiotherapy.2 Because all of these resistance factors may be affected by angiosuppressive or vascular damaging treatments, the combinations of such approaches with radiotherapy are likely to improve treatment outcomes. Alternatively, the inclusion of such agents in radiotherapy protocols may allow for reductions in the total radiation dose or the delivery of reduced radiation treatments to larger field sizes. The second reason that such combined modality approaches may have merit is that, although strategies aimed at compromising the tumor vasculature have shown significant antitumor effect in preclinical investigations, achieving tumor cures in patients with either AIs or VTAs alone is likely to be extremely difficult. In the case of the former, the complexity of pathways available for neovascularization means that disrupting only a single aspect of angiogenesis probably will not be enough. It further is likely that the greatest benefit from Als may lie, not in their ability to cure, but rather in their capacity to control tumor growth. VTAs also may be best used in combination with conventional anticancer approaches because by the nature of their highly selective mechanism of damaging tumor blood vessels, they are not able to eliminate those pockets of tumor cells whose nutritional supply is derived from blood vessels in the surrounding normal tissues.

These considerations, along with the extensive clinical use of radiotherapy, make the thorough investigation of strategies combining this conventional treatment modality with therapies aimed at disrupting the tumor vessel network imperative.

Antiangiogenic treatments

Tumor progression during the course of treatment is a major reason for radiotherapy failures. The ability of a tumor to progress is dependent on the formation of new blood vessels. Consequently, the application of antiangiogenic strategies that disrupt the proangiogenic balance between neoplastic, stromal, and endothelial cells may result in growth stabilization of the tumor by preventing further development of a functional vessel network.

A number of angiogenesis suppressive agents or approaches have been evaluated in conjunction with radiation in a variety of preclinical tumor models. One of the earliest agents investigated was TNP470, a synthetic analog of fumagillin that displayed an antiangiogenic effect in vivo and in vitro.67 When given during fractionated radiotherapy, this agent led to increased tumor growth delays in murine and human tumor models.67, 68 Given the importance of proangiogenic growth factors, particular emphasis has been placed on evaluating in the combined modality setting strategies that disrupt the VEGF signaling pathways. Indeed, numerous reports are now available to indicate that VEGF inhibition achieved by protein or receptor targeted antibodies or receptor signaling inhibitors, when combined with radiation, could significantly improve treatment outcomes.29, 69, 70, 71, 72, 73 An example of this is shown in Figure 1, which shows that the administration of bFGF antisense significantly increases the tumor growth delay in a human renal cell carcinoma xenograft treated with a single 10-Gy dose of radiation.

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Fig. 1. The effect of bFGF antisense treatment on the radiation response of Caki-1 xenografts. Mice were treated with two 10 mg/kg doses of bFGF antisense either alone or in combination with a 10-Gy dose of radiation delivered locally. Antisense bFGF was prepared in liposomes and administered in 2 increments, the first given immediately after irradiation and the second 3 days postradiotherapy.

Another approach, the elevation of endogenous Als, such as endostatin and angiostatin, also enhances the radiation response of tumors.74, 75 In addition, a variety of other agents expressing some antiangiogenic effects, such as the cyclooxygenase 2 inhibitor rofecoxib, the tetracycline derivative minocycline, and epidermal growth factor receptor antibodies, have been shown to improve the antitumor effects of radiation.76, 77, 78, 79, 80, 81

The mechanisms underlying the enhancement of radiation response by antiangiogenic therapies likely include an increase in tumor oxygenation,69, 76, 82 a decrease in vascular density,69 and possibly the radiosensitization of endothelial cells.29, 75 Although there has been some concern that the inhibition of tumor angiogenesis might increase the fraction of hypoxic tumor cells and thereby induce radiation resistance, this view is not supported by the aforementioned reports of improved tumor oxygenation after treatment,69, 76, 82 as well as recent studies showing that blocking the VEGF signaling pathway improves radiotherapy response under fully oxygenated and hypoxic conditions.69, 70, 71 Still, this clearly is an important issue that is strongly dependent on the agent in question and one that will warrant rigorous scrutiny before advancing an agent to the clinic.

Another setting in which Als might play a significant role in cancer management is in the metastatic spread of the disease. The recruitment of new blood vessels is an essential component in the metastatic process because these vessels provide not only the principal route by which tumor cells enter the circulation but also are critical for the subsequent establishment and progression of the peripheral disease.5, 25, 83, 84 Indeed, in some tumor types, vessel density can serve as a prognostic indicator of the overall survival rate of patients and incidence of metastases.83, 84, 85, 86, 87, 88, 89 Because many angiosuppressive strategies have been reported to inhibit the metastatic spread of cancer cells,90, 91, 92, 93, 94 it would appear logical to postulate that the combination of antiangiogenic strategies with localized radiation therapy might improve patient survival by affecting the development of distant metastases. However, the impact of antiangiogenic approaches when used in combination with radiation therapy on the development of peripheral disease and overall tumor response has yet to be delineated.

Vascular targeting agents

It is well established that the aberrant vascular morphology, spatial heterogeneity in vessels, and metabolic microenvironment associated with solid tumors can have significant adverse effects on the efficacy of radiation therapy.95 Although the classical example of this is the well-known oxygen effect during treatment with ionizing radiation, more recently it has become clear that metabolic characteristics of tumors, such as lactate accumulation and perhaps glucose levels, also may serve as prognosticators of outcomes in clinical oncology.96 By causing extensive hemorrhagic necrosis in the centers of tumors, treatment with VTAs may reduce or eliminate many of these potential problem areas. For example, agents such as DMXAA, CA4DP, and ZD6126 have been shown to produce abrupt and significant vascular effects in a variety of preclinical tumor models including transplanted and spontaneous rodent tumors and human tumor xenografts.53, 54, 55, 56, 57, 60, 61, 97 Once initiated, these disruptions of the integrity of the tumor vasculature lead, in a matter of hours or days, to widespread and treatment dose-dependent tumor necrosis. Although the consequential secondary ischemic tumor cell death can be extensive, the presence of a viable rim of tumor cells at the tumor periphery is a fairly characteristic feature of tumors treated with VTAs.51, 55, 56, 60, 61 Although direct evidence is lacking, these cells are believed to be able to survive because they receive their nutritional support from vessels in the normal tissues adjacent to the tumor that are unaffected by treatment with such agents. This residual tumor tissue is likely to be well oxygenated, a view supported by studies showing a reduction in the fraction of hypoxic cells in tumors treated with VTAs.51, 55, 61 Thus, a logical rationale for combining a VTA with radiation might be that the 2 treatments interact in a complimentary fashion at the tumor microregional level (ie, the former reducing or eliminating the poorly oxygenated and hence radioresistant tumor cell subpopulations, the latter destroying cells not affected by the VTA).

A number of preclinical studies have shown that agents that induce vascular damage can successfully be combined with radiation to improve tumor cell killing.51, 55, 61, 98, 99, 100, 101, 102 An important issue in combining such therapies is the question of sequence and timing. Results indicate that administering VTAs after irradiating the tumors yields maximally enhanced tumor responses, whereas sequences in which the VTAs are given shortly before radiotherapy should be avoided (Fig 2).51, 55, 61

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Fig. 2. Impact on KHT sarcoma tumor cell survival of administering a 17.5 mg/kg dose of DMXAA at various times before or after a 10-Gy dose of radiation. Data shown are the mean ± standard error of 3 to 6 experiments. The hatched area shows the survival range for radiation alone.

This lack of improvement in the treatment response under the latter conditions may indicate that, with such sequences, parts of the tumor may experience transient reductions in blood flow sufficient to render cells hypoxic at the time of irradiation but not for a time long enough to lead to ischemic tumor cell death. Taken together, these findings suggest that administering the VTAs after radiotherapy may provide the most appropriate scheme for combining these therapies. When this has been done in studies using endpoints of clonogenic cell survival, tumor growth delay, and tumor control, all have shown improvements in tumor response in the combination treatment51, 55, 61, 98, 99, 100, 101, 102 (see Fig 3).

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Fig. 3. Tumor cell survival in KHT sarcomas treated with a 17.5 mg/kg dose of DMXAA 1 hour after a range of single doses of radiation. Tumors were evaluated 24 hours after completing the treatment. Results are the mean ± standard error of 3 to 6 experiments.

Although most have used single-dose radiation exposures, evidence exists to indicate that the inclusion of a VTA also can enhance the tumor response to fractionated dose radiotherapy.61

Combining antiangiogenic and antivascular treatment strategies

It should be abundantly clear from the preceding sections that, when attacking the tumor blood supply, a distinction needs to be made between approaches that suppress the angiogenic process and strategies that damage the existing tumor blood vessels. Angiosuppressive approaches are likely to compliment rather than to duplicate strategies aimed at damaging established tumor neovasculature. Indeed, evidence is beginning to accumulate to suggest that Als may be especially well suited for attacking micrometastatic disease or early stage cancers,90, 103, 104 whereas VTAs may prove particularly effective against large bulky and late stage tumors.102 Figure 4 shows that the effectiveness of the VTAs CA4DP and ZD6126 is strongly dependent on the tumor size at the time of treatment.

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Fig. 4. Tumor surviving fractions in KHT sarcomas of varying sizes treated with a single dose of either CA4DP or ZD6126 and assayed 24 hours later. Bars are the mean ± standard error of 3 to 6 tumors except those without errors that represent individual tumors. Tumor surviving fractions were determined by multiplying the calculated fraction of surviving cells by the ratio of cells recovered in treated versus untreated tumors.

Finally, it should be noted that data are beginning to emerge to indicate that the combination of antiangiogenic and vascular targeting approaches may provide particularly beneficial therapeutic effects.105 Taken together, these findings provide impetus to future investigations of the efficacy of radiotherapy in the presence of strategies aiming to optimally compromise the tumor vasculature through combinations of Als and VTAs.

Conclusions

The possibility of targeting a tumor's blood vessel support network as a cancer treatment strategy has recently received a great deal of attention. Preclinical investigations of approaches directed at disrupting a tumor's vital support network have shown favorable responses at low toxicity. Currently, more than 80 clinical trials using such strategies are underway (http://cancertrials.nci.nih.gov/), reflecting the high pace of developments. Early returns from initial clinical trials with angiosuppressive approaches are somewhat disappointing but to a large degree probably reflect symptoms of a young field of new therapeutics rather than a condemnation of the general approach. Indeed, these trials have raised important questions regarding clinical trial design and endpoint assessment when using such agents. Still, it also is becoming increasingly clear that strategies targeting the tumor blood vessel network will ultimately be most effective if used in conjunction with or as adjuvants to conventional anticancer therapies. Preclinical evaluations have clearly shown that agents that induce vascular damage or approaches that impair the angiogenesis process can be combined successfully with radiation to improve tumor response. There are several reasons why this should be the case. In general, it could be argued that a greater antitumor effect might be achieved when combining agents having fundamentally different mechanisms of action, different cellular targets, and nonoverlapping toxicities. Specifically, the application of angiosuppressive or vascular targeting strategies might overcome factors known to adversely affect the efficacy of radiation therapy. These include the metabolic microenvironments associated with the aberrant vascular morphology of solid tumors as well as tumor progression and metastatic spread, 2 processes dependent on new blood vessel formation. Conversely, radiotherapy serves to eliminate neoplastic cell populations surviving vessel targeting treatment strategies. Consequently, the combination of therapies that target the tumor vessel network in conjunction with radiation therapy holds the promise of providing significant improvements in treatment outcomes and hence should be thoroughly explored.

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Department of Radiation Oncology, University of Florida, Gainesville, FL.

☆ Supported by the U.S. National Cancer Institute (PHS grants CA84408 and CA89655).

☆☆ Dietmar W. Siemann serves as a consultant for AstraZeneca Pharmaceuticals and Oxigene Inc.

★ Address reprint requests to Dietmar W. Siemann, PhD, Department of Radiation Oncology, University of Florida Shands Cancer Center, Box 100385, Gainesville, FL 32610.

PII: S1053-4296(03)50008-X

doi:10.1053/srao.2003.50005

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