Targeting tumor blood vessels: an adjuvant strategy for radiation therapy

Radiotherapy and Oncology 57 (2000) 5±12

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Review article

Targeting tumor blood vessels: an adjuvant strategy for radiation therapy Dietmar W. Siemann a*, Kenneth H. Warrington Jr. a, Michael R. Horsman b a

Department of Radiation Oncology, Shands Cancer Center, University of Florida, Box 100385, Gainesville, FL 32610, USA b Danish Cancer Society, Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark Received 20 March 2000; received in revised form 8 June 2000; accepted 21 June 2000

Abstract Background and purpose: The neovascularization of tumor cells is a prerequisite if a clinically relevant tumor size is to be reached. A continuously expanding vessel network supplying nutritional requirements and removing waste products is essential for continued tumor development, growth and survival. Results: In many tumors, the growing endothelium is unable to fully support the demands of the neoplastic cell population. As a consequence of the inadequacies of the resulting aberrant vasculature, microenvironmental conditions develop in tumors which are not only detrimental to the response of tumors to conventional anticancer treatments, but may lead to or predispose cells to genetic modi®cations resulting in more aggressive phenotypes and higher metastatic potential. Yet the utter dependence of the tumor on its induced vessel formation for growth, survival and spread has also created a great deal of enthusiasm for developing therapeutic approaches to speci®cally targeting the tumor microcirculation. Conclusions: The application of such strategies as adjuvants to conventional radiation treatments offers unique opportunities to develop more effective cancer therapies. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Tumor vasculature; Targeting; Adjuvants to radiotherapy

1. Introduction Radiotherapy is the most important non-surgical treatment for cancer [92]. Today, 45±50% of all cancer patients can be cured, and ,70% of those who are cured receive either radiation alone or in combination with other modalities [22]. Still, signi®cant numbers of radiotherapy patients treated with curative intent fail, not only because of metastatic spread of the disease, but also at the local treatment site [22]. Hence, the combination of radiotherapy with new modalities continues to hold high interest. The reasons for radiotherapy failures are varied and multiple. It is, however, becoming increasingly clear that in addition to intrinsic, genetically determined resistance, physiological properties, arising primarily from inadequate and non-uniform vascular networks, can play signi®cant roles in the lack of therapeutic responsiveness of neoplasms. The morphologically and functionally abnormal vasculature results from a disproportionate relationship between tumor tissue and its vascular supply; a situation where neovascularization invariably lags behind the expanding tumor mass [90]. As a consequence, the vascular network fails to provide adequate nutritional support [95], and leads * Corresponding author.

to heterogeneous tumor microregions varying in concentrations of oxygen, glucose and other nutritional factors, as well as metabolic waste products, both within and among tumors of the same pathological grade and stage [95,96]. The development of hypoxia, acidosis and nutrient depletion can appreciably alter the tumor response to non-surgical therapies [12,17,24,76,80]. Heterogeneity in oxygenation within tumors in particular has been implicated as a major contributing factor for failure to cure neoplastic disease by radiation [23,74]. Indeed, several recent papers have shown an excellent correlation between tumor therapy outcome and the distribution of intra-tumor oxygen concentration [7,8,41,52,67,70]. In addition, it is now becoming abundantly clear that the impact of the tumor microenvironment far exceeds its direct effects on therapeutic treatment modalities. For example, stress responses to hypoxia may involve mechanisms that favor cell survival during reoxygenation, including drug resistance and increased metastasis [89,108]. Hypoxia may also contribute to processes that directly favor malignant progression through effects on the expression and activity of tumor suppressor proteins, such as p53 [31,35]. Taken together, it is perhaps not surprising that clinical reports now suggest not only a correlation between tumor oxygenation and radiotherapy response, but also an association with poor surgical outcome and distant metastases [9].

0167-8140/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0167-814 0(00)00243-7

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Neovascularization, an uncommon process in most normal adult tissues, is essential in all solid tumors. Indeed, it is widely accepted that no solid tumor can grow beyond a critical size of ,1 mm 3 without evoking a blood supply [3,28,36]. Further tumor growth depends on nutrient supply via a network of microvessels [21,95] which can be acquired, in part, by incorporation of existing host blood vessels. However, it is now well established that the majority of tumor blood vessels are newly formed as a result of angiogenesis triggered by the release of stimulators, such as vascular endothelial growth factor (VEGF) [82]. The fact that the production of several angiogenic growth factors can be upregulated by physiological parameters, including low oxygen or glucose and acidic pH, which are associated with vascular insuf®ciency, provides a logical rationale for the strong angiogenic stimulus in malignant tissue [14,60,64,83,98]. The continued proliferation of tumor cells will result in the deprivation of oxygen and glucose and the production of acidic metabolites, thus stimulating the development of additional neovasculature [82]. The new vessels facilitate the further expansion of the tumor cell mass, providing a perpetual loop. Thus, a tumor's critical need for an actively growing vasculature for its progression and survival, coupled with the established negative therapeutic consequences associated with its aberrant nature, make targeting tumor vessels an attractive strategy for cancer management. 2. Targeting the tumor vasculature Strategies for the therapeutic suppression of angiogenesis have focused extensively on the various aspects of the process of angiogenesis. Many agents that are anti-angiogenic have been identi®ed and characterized [26,27,47,62,78,79]; each of these affect at least one of the several stages involved in new vessel formation, i.e. basement membrane degradation, endothelial cell migration, endothelial cell proliferation and tube formation. Of particular interest are drugs which interfere with the delivery or export of angiogenic stimuli [78], antibodies to inhibit/inactivate angiogenic factors after their release [49], drugs which inhibit receptor action [16], invasion inhibitors [57,62,65], and inhibitors of endothelial cell proliferation [6,18,73,72]. The growth factors VEGF and basic ®broblast growth factor (bFGF) are primary targets because of their signi®cance in tumor angiogenesis [4,46,99] and because their combination gives rise to one of the most potent stimuli of angiogenesis known [75]. It is perhaps especially relevant in tumor angiogenesis that both are upregulated under hypoxic conditions [14,60,83,84,98]. Given the importance of VEGF and bFGF in the regulation of angiogenesis, various approaches targeting VEGF and bFGF are being investigated in anti-angiogenic therapeutic strategies. Such approaches have included using antibodies to the growth factors [33], the selective inhibition of the tyrosine kinase activity of ¯k-1,

the receptor for VEGF [50,87,100,102], the use of antisense phosphorothioate oligodeoxynucleotides (PS-ODNs) to these angiogenic factors [68], as well as the transfer of antisense VEGF/bFGF via plasmid or viral vectors to downregulate VEGF/bFGF in situ [66]. In addition, new and exciting agents continue to be discovered, including the peptide inhibitors of endothelial cell proliferation, angiostatin [72] and endostatin [73], and the tubulin-binding agents, combretastatin (CA4DP) and ZD6126 (a phosphate prodrug of N-acetylcolchinol), which show highly selective toxicity to proliferating endothelial cells [13,18,19,56]. The abnormal nature of the tumor microcirculation, which is quite unlike that of the well-de®ned microvascular architecture of normal tissues [53,78], also provides a critical difference between tumors and normal tissues which may be exploitable. Indeed, in addition to strategies aimed at interfering with the angiogenic process, considerable efforts have been made to identify and develop therapies that speci®cally compromise the function of the existing neovasculature in solid tumors. Such approaches aim to cause direct damage to the tumor endothelium, resulting in a rapid and catastrophic shutdown in the vascular function of the tumor, thus leading to extensive secondary ischemic tumor cell death. Since thousands of tumor cells are dependent on each tumor capillary for their metabolic requirements, an agent which induced even limited damage to these vessels could produce a cascade of tumor cell death [20,21]. Two classes of low molecular weight drugs have been shown to elicit irreversible vascular shutdown selectively within solid tumors. The ®rst are agents related to ¯avone acetic acid (FAA) [39,40,43,101,110,111], whose anti-vascular effects appear to result primarily from the induction of tumor necrosis factor alpha (TNFa) [15,54]. The second are the tubulin-binding agents, some of which, including colchicine, the vinca alkaloids and the combretastatins, were recognized to have anti-vascular action and toxic effects in the endothelial cells of growing capillaries [11,39,58]. The combretastatins are of particular interest because in preclinical investigations, the induction of tumor necrosis can be achieved with doses offering a large therapeutic window [11,13,44,56]. The preclinical success of these various approaches has led to much focus being placed on the clinical potential of therapeutic interventions which target the vascular network of tumors. Indeed, several agents which appear promising have entered patient trials, while others will soon move into the clinic. Several reviews of the various agents under consideration have been published [34,47,65,77,88,93]. Table 1 and Fig. 1 provide examples of therapeutic approaches aimed at suppressing tumor-associated blood vessel growth. Finally, it should be noted that when attacking the tumor blood supply, the distinction between approaches that suppress the angiogenic process and strategies that damage existing tumor vessels, while on the surface appears somewhat arbitrary, highlights differences that are more than semantic. Angiosuppressive approaches are likely to

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Table 1 Strategies targeting tumor neovascularization Target

Agents/approaches

References

Activators of angiogenesis Matrix/cell-matrix interactions Receptors Signal transduction pathways

Antibodies/antisense to angiogenic growth factors Metalloproteinase inhibitors, Marimastat, alpha(v)-integrin antagonists Antibodies to VEGF receptor, Suramin, alpha(v)-integrin binding motifs Inhibitors of tyrosine kinase activity of ¯k-1/KDR, SU5416, SU6668, ZD4190, PD 0173073 Angiostatin, endostatin, TNP-470, Tie2 antagonists FAA, DMXAA, CA4DP, ZD6126

[49], [66], [68] [57], [62], [65], [51], [94], [103] [16], [102], [2] [87], [25], [50], [100]

Endothelial cell function Existing tumor vasculature

compliment rather than duplicate strategies aimed at damaging established tumor neovasculature. Indeed, evidence is beginning to accumulate to suggest that the former may be especially well-suited for attacking micrometastatic disease or early stage cancers [57,71,106], whereas the latter may prove particularly effective against large bulky and later stage tumors [81]. 3. Combinations of anti-vascular strategies and radiation therapy As is the case for all anticancer therapies, normal tissue responses limit the total dose of radiation that can be delivered to a tumor. To improve the therapeutic ratio in radio-

[72], [73], [48], [107] [54], [56], [13], [19], [111]

therapy, efforts have focused primarily on either physical means of improving radiation dose distributions or combining radiation treatments with other therapeutic agents based on biological principles. The rationale for combined modality therapies is based predominantly on three concepts: enhanced antitumor ef®cacy, non-overlapping toxicities, or spatial cooperation [85]. Chemotherapeutic agents, radio-protectors and hypoxic cell sensitizers represent primary examples of approaches which have been employed with varying degrees of success in combination with radiotherapy [22]. Directly targeting the supportive vessel network of tumors provides an alternative strategy which could provide therapeutic bene®ts when used in fractionated radiation treatments. Preclinical investigations of strategies aimed at compro-

Fig. 1. Illustration of anti-angiogenic and anti-vascular strategies in cancer treatment. Whereas anti-angiogenic approaches predominantly interfere and/or suppress fundamental aspects of the angiogenesis process, anti-vascular agents primarily target the existing and expanding tumor vessel network. The interior of the tumor is illustrated in the cut-away section.

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mising the tumor vasculature which, to date, have demonstrated signi®cant antitumor effects in the presence of little overt normal tissue toxicity, also are less likely to generate cellular resistance than conventional anticancer approaches. Still, despite the promising results achieved, to cure with an individual modality (monotherapy) is often dif®cult, and the clinical application of approaches targeting a tumor's blood supply will undoubtedly be investigated in a neoadjuvant setting. Given the frequent use of radiotherapy, its mechanisms of action and reasons for treatment failure, investigations evaluating the therapeutic utility of combining this treatment modality with strategies aimed at disrupting the tumor vessel network are clearly warranted. Abnormal tumor microenvironments, tumor progression and metastatic spread of neoplastic cells are major factors contributing to treatment failures in radiotherapy [22]. Since all of these may be affected by angiosuppressive or vessel destructive treatments, the combination of such approaches with radiotherapy is likely to improve treatment outcomes. There already exists evidence that certain agents that induce vascular damage, for example hyperthermia [42] and photodynamic therapy [61], can successfully be combined with radiation to improve tumor cell killing, although the rationale for such combinations was based on the cytotoxic and radiosensitizing abilities of these agents, rather than their anti-vascular properties. More relevantly, recent experimental data from several laboratories have demonstrated that vascular targeting strategies can effectively enhance the antitumor effects of radiation treatment [32,33,56,59,63,81,91,101]. It is well established that the aberrant vascular morphology, spatial heterogeneity in vessels, and metabolic microenvironment associated with solid tumors, can have signi®cant adverse effects on the ef®cacy of radiation therapy [74]. Treatment with anti-vascular agents eliminates many of these problem areas by causing extensive hemorrhagic necrosis in the center of tumors [11,44,56,63]. For example, agents such as the tubulin-binding agents CA4Dp and ZD6126 as well as the FAA analog, dimethylxanthenone acetic acid (DMXAA), can produce abrupt and significant vascular effects which ultimately lead to extensive ischemic tumor cell death [18,19,54,56]. As a single tumor capillary may support the nutritional needs of as many as 10 6 tumor cells, vascular targeting could directly amplify the antitumor effects of a conventional anticancer therapy, such as radiation [38]. In addition, these agents may improve the radiation response of tumors by impacting the radiation refractory hypoxic cell subpopulation of tumors. Typically, the vascular shutdown and subsequent induction of necrosis after treatment with anti-vascular agents is not complete, thus leaving areas of viable tumor cells from which the tumor could regrow [20,21,56,101]. Interestingly, cells surviving treatment with these agents tend to be located in areas at the tumor periphery near normal tissues; most likely those areas supplied by normal tissue vessels [56,63,101]. This resi-

dual tissue is likely to be well-oxygenated, and hence, responsive to radiation. The theory that this may indeed be the case is supported by preclinical evidence indicating that when such agents are used in conjunction with radiotherapy, the tumor's hypoxic cell population can be dramatically reduced or eliminated [56,63,101]. These ®ndings are consistent with the notion that the two treatments (radiation and vascular targeting agent) are acting in a complimentary fashion at the microregional level, i.e. the vascular targeting agent is preferentially eliminating the poorly oxygenated, and hence, radioresistant tumor cell subpopulations [56,101]. Tumor progression during the course of radiation therapy is another reason for the failure of this conventional treatment modality to completely eradicate tumors. The ability of a tumor to progress is dependent on the formation of new blood vessels. Proliferation of the tumor endothelium can be induced by the production of soluble endothelial cell growth factors by the endothelial cells themselves (autocrine), or by neighboring tumor and/or stromal cells (paracrine). These new vessels have been observed to preferentially express speci®c molecules on their surface, such as VEGF and bFGF receptors. Furthermore, hypoxic cells, commonly associated with solid tumors, may play a role in tumor progression as a result of the increased production of endothelial growth factors in response to this stress [60,64,83,84,98]. Likewise, the stress response to radiation involves the induced expression of angiogenic growth factors, such as VEGF [1,55]. Consequently, the tumor vasculature may be induced to proliferate as a result of the aberrant tumor microenvironment and/or radiation therapy per se. Since tumor angiogenesis is a regulated balance between angiogenic and anti-angiogenic factors [29,30,36], the application of anti-angiogenic strategies that disrupt the `proangiogenic' balance between the tumor, stromal and endothelial cell populations may result in growth stabilization of the tumor by preventing further development of a functional vessel network. Angiogenic inhibitors, such as angiostatin and endostatin, have been reported to inhibit angiogenesis, and overexpression of these peptides has been observed to lead to primary tumor regression and growth inhibition [72,73,105]. Similarly, a variety of angiosuppressive approaches, particularly ones employing recombinant DNA technologies to disrupt angiogenic signaling, are under active investigation to determine their effects on tumor growth. Amongst others, successful approaches include the use of oligodeoxynucleotides against VEGF and bFGF mRNA [68], and plasmid and recombinant viral gene delivery systems expressing antisense and ribozyme mRNA targeting VEGF and bFGF message [45,66] or endostatin cDNA [5]. The inclusions of such treatments to inhibit or stabilize tumor growth during the course of radiotherapy could be of signi®cant therapeutic value. Preliminary preclinical investigations examining angiostatin use in conjunction with radiation treatments indicate that such approaches can lead to

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improved tumor responses [32,33,56,59] and clearly warrant further evaluation. Another signi®cant factor involved in the failure of radiation therapy delivered with curative intent is the development of distant metastases. The recruitment of new blood vessels is an essential component in the metastatic process as these vessels provide not only the principal route by which tumor cells enter the circulation, but are also critical for the subsequent establishment and progression of the peripheral disease [36,38,47,88]. The importance of angiogenesis in the metastatic process may well be re¯ected in the observation that in some tumor types, vessel density can serve as a prognostic factor for the overall survival rate of patients and incidence of metastasis [37,38,69,86,88,97,104]. Several angiosuppressive strategies have been reported to inhibit the development of metastatic disease [5,10,103,106,109]. However, the impact of anti-angiogenic or vascular targeting approaches when used in combination with radiation therapy on the development of peripheral disease and overall tumor response has yet to be delineated.

4. Conclusions In the treatment of cancer, effective antitumor therapy must be delivered with manageable normal tissue side effects. Yet despite signi®cant efforts, the development of highly selective non-toxic therapies for the treatment of cancer remains an elusive goal. 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 and early patient trials suggest that such approaches can have a favorable impact on disease at low toxicity. There are several reasons why the combination of vascular targeting strategies and radiation therapy warrants further investigation. In general, it could be argued that a greater antitumor effect might be achieved when combining agents have fundamentally different mechanisms of action, different cellular targets and non-overlapping toxicities. Speci®cally, the application of angiosuppressive or vascular targeting strategies might overcome factors known to adversely affect the ef®cacy 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, two processes dependent on new blood vessel formation. Consequently, the utilization of therapies which target the tumor vessel network in conjunction with radiation therapy could lead to signi®cant improvements in the overall treatment outcome. Recent research advances, better understanding of the pathways and mechanisms of the angiogenic process, and the promise of more potent and effective agents in the future, clearly indicate that the role of vascular targeting strategies as adjuvants to radiotherapy needs to be thoroughly explored.

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