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This review covers progress to date in the identification of molecular targets on blood vessels in cancers, as well as agents that act on those targets, with emphasis on those currently in clinical trials. Current vascular-targeting therapies comprise two general types—antiangiogenic therapy and antivascular therapy. Advances in antiangiogenic therapies, particularly inhibitors of vascular endothelial growth factors and their receptors, have clarified the capacity of these inhibitors to change tumor-associated vessel structure to a more normal state, thereby improving the ability of chemotherapeutics to access the tumors. The responses of other antiangiogenesis target molecules in humans are more complicated; for example, αvβ3 integrins are known to stimulate as well as inhibit angiogenesis, and cleavage of various extracellular proteins/proteoglycans by matrix metalloproteinases produces potent regulators of the angiogenic process. Antivascular therapies disrupt established blood vessels in solid tumors and often involve the use of ligand-based or small-molecule agents. Ligand-based agents, irrespective of the antiangiogenic capacity of the ligand, target antivascular effectors to molecules expressed specifically on blood vessels, such as aminopeptidase N, fibronectin extra-domain B, and prostate-specific membrane antigen. Small-molecule antivascular agents, which are not targeted to molecules on blood vessels, rely on physical differences between the vasculatures in tumors and those in normal tissues.
Tumor angiogenesis has been a subject of wide interest over the past decade, following from the groundbreaking work of Folkman and colleagues. Uncontrolled angiogenesis is present in almost every tumor type and leads to immature, malformed vessels lined by endothelial cells with aberrant morphology, loosely attached pericytes, and an abnormal basal membrane. These vessels are characterized by their chaotic organization and increased permeability causing elevated interstitial fluid pressure. Vascular malformations do not provide an efficient supply of oxygen, nutrients, or therapeutic drugs to tumor tissue. This leads to significant treatment resistance.[3,4]
In this issue of ONCOLOGY, Sato et al provide a comprehensive overview of different strategies for antiangiogenic and antivascular targeted therapies, focusing on those currently in clinical trials. This commentary will focus on the need for methods to monitor changes in the microenvironment and angiogenic responses to vascular-targeted therapies, alone and in combination with cytotoxic therapies.
Considerations When Using Antiangiogenic Agents
Antiangiogenesis was first considered as a treatment option based on the hope that it would destroy tumor vasculature to the point that tumors regressed as a result of severe oxygen starvation. While this approach seems theoretically attractive, that outcome has not proven to be the case for antiangiogenic therapies.
In fact, correcting the overexpression of proangiogenic factors (such as vascular endothelial growth factor [VEGF]) by antiangiogenic compounds leads to a better balance of pro- vs antiangiogenic factors. This can lead to maturation and/or pruning of immature vessels, causing "normalization" of tumor vasculature.[5-10] Of utmost importance in "normalization" is whether oxygen and drug delivery are enhanced, and if so, the kinetics of the responses.
The concept of "vascular normalization" has been evaluated at the preclinical level in numerous studies, briefly summarized below. A murine mammary carcinoma (McaIV) and a human colon carcinoma cell line (LS174T) showed less tortuous vessels 3 days after blocking VEGF receptor (VEGFR)2 with the murine VEGF antibody DC101. Remaining vessels were more uniformly covered by pericytes and basement membrane. These normalized vessels were less leaky, as reflected by a reduction of intratumoral fluid pressure, and there was improvement in oxygenation, as assessed by hypoxia marker drug uptake. Vessel normalization was followed by vessel regression at day 5, however, demonstrating the transient nature of this response. In a neuroblastoma (NB-1691) xenograft model, VEGF inhibiton by bevacizumab (Avastin) led to better intratumoral drug delivery and efficacy. Both bevacizumab and DC101 improved tumor tissue oxygenation[9,11] in human glioma xenografts, resulting in increased efficacy of radiation treatment.
Small-molecule inhibitors of VEGF receptor activity have also shown promise. After 4 days of treatment of a non–small-cell lung cancer (NSCLC) xenograft tumor line with imatinib (Gleevec), a platelet-derived growth factor receptor (PDGFR) inhibitor, investigators observed a significant reduction of VEGF expression and vascular normalization (defined by tight pericyte-endothelial cell coverage). In addition, they noted improved oxygenation after 1 to 2 weeks of therapy and an enhanced antitumor effect when imatinib was combined with docetaxel.[13,14]
Not all reports have been positive with respect to whether normalization occurs after initiation of VEGF inhibition, however. Fenton et al reported variable results in different tumor lines and suggested that direct sensitization of tumor endothelial cells to radiation therapy following withdrawal of VEGF signaling might be more important in explaining radiosensitization than any change in oxygenation. The mechanism of treatment failure of individual tumors is not fully understood. One possible explanation is the ability of certain tumor cells to dedifferentiate and build vessel-like-structures-the so-called vasculogenic mimicry. This process of cell plasticity occurs mainly in highly aggressive tumors and was first discovered in highly metastatic melanoma. These vessels seem not to respond to antiangiogenic treatment.
Clinically, Willett et al performed serial studies in patients with locally advanced rectal cancer to monitor the effects of bevacizumab, prior to initiation of radiotherapy. They observed reductions in perfusion (assessed by contrast computed tomography [CT]), interstitial fluid pressure, microvessel density and circulating levels of endothelial cell precursors. Another extensive study in patients with glioblastoma multiforme was reported recently by the same group. Using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) methods, they observed significant reductions in intratumoral edema and microvessel diameter response to a small-molecule VEGFR inhibitor. Although these measures strongly suggest that oxygenation of the tumors improved after antiangiogenic therapy, there have not been any clinical reports examining hypoxia as an endpoint, which is crucial for making treatment decisions that involve oxygen-dependent therapies, such as radiation and chemotherapy.
Influence of Cytotoxic Therapies on VEGF and Angiogenesis
It is important to recognize that VEGF levels in tumors change in response to various forms of cancer therapies and that these changes influence vascular responsiveness and endothelial cell survival after therapy. An important regulator of VEGF activity in tumors is the hypoxia-inducible transcription factor (HIF-1), which binds to the promoter region of VEGF to stimulate transcription. HIF-1 is active predominantly under hypoxic conditions, because its alpha subunit is targeted by the proteosome under normoxic conditions for hydroxylation reactions in its oxygen-dependent degradation domain, modifying it for recognition by the VHL complex and subsequent ubiquitinylation and proteosomal degradation.
Cytotoxic treatment of tumors often leads to transient improvement in oxygenation, in a process originally coined "reoxygenation" by Rubin and Casarett. One might think that reoxygenation would lead to reduced HIF-1 activity, but in fact, the opposite effect occurs. During the process of reoxygenation, HIF-1 levels actually increase, largely in response to production of free radicals that prevent the oxygen-dependent degradation of HIF-1.[22,23] The increase in HIF-1 activity increases VEGF levels, thereby protecting them from damage by radiation or chemotherapy. In effect, this indirectly protects the tumor, by preserving its vasculature. This provides a strong rationale for inhibiting VEGF during radiotherapy or chemotherapeutic treatment.
Teicher was the first to show that inhibition of angiogenesis led to improved chemotherapy and radiotherapy response in several different tumor lines. Gorski et al were the first to demonstrate that VEGF levels increased after radiotherapy and further demonstrated that inhibition of VEGF increased radioresponse. Our group was the first to show that elevations in HIF-1 after radiotherapy were responsible for increases in VEGF and that blockade of HIF-1 upregulation led to profound vascular sensitization to radiotherapy.
In contrast to the "normalization effects"of antiangiogenic approaches, vascular-targeting therapies more closely approximate the original goal of antiangiogenesis. They rapidly shut down tumor perfusion, leading to tumor cell starvation and death. With this form of therapy, the ability to image treatment responses is very clear. The pure vascular-targeting agents, such as combretastatin A4, cause profound reductions in tumor perfusion within minutes of treatment, yet when used alone, have virtually no effect on tumor growth. This is because the highly proliferative tumor margin is able to repopulate the tumor quite readily. The need for imaging to monitor vascular responses and demonstrate bioactivity with such agents is virtually mandatory in the absence of significant tumor volume reduction.
Biomarkers of Activity
The discussion above illustrates the importance of biomarkers of relevant physiologic parameters for phase I/II trials of antiangiogenic and vascular-targeting agents. Markers of bioactivity of such agents can be broken down into three categories: (1) imaging-based, (2) blood-based, and (3) tissue-based markers. It is preferable to include biomarkers that reflect perfusion, permeability, and oxygenation or drug transport.
• Imaging-Based Biomarkers-The relative merits of different MR- or CT-based imaging biomarkers are outside the realm of this commentary, but we will emphasize the need to develop methods that can distinguish changes in permeability from changes in vascular surface area. Typical small-molecule contrast agents cannot do this. An alternative would be to use macromolecular contrast agents or blood pool agents that show limited transvascular transport.[27,28]
In addition, one should not ignore the importance of monitoring hypoxia. The oxygenation state of a tumor is of pivotal importance in influencing treatment outcome from either radiation or chemotherapy. Since both antiangiogenic and vascular-targeting therapies can influence oxygenation, this information is pivotal in establishing how to combine such therapies with radiation or chemotherapy. Several positron-emission tomography (PET) hypoxia-imaging reagents are under development that could be used in this context, but to our knowledge none have been. Examples include 18F misonidazole, 18F EF5,[30,31] and CuATSM.
Some investigators have advocated the use of so-called blood oxygen level detection (BOLD) imaging to monitor changes in vascular oxygenation in tumors. The method relies on changes in MRI properties of hemoglobin in different oxygenation states. The problem with this method for this particular application is that BOLD depends on the differentiation of images at baseline and after the patient breathes a hyperoxic gas. Such gases are vasoactive, leading to changes in perfusion and microvessel hematocrit, both of which independently influence the BOLD signal.
• Blood-Based Biomarkers-Recently, two plasma-based markers of hypoxia have been shown to be correlated with tumor hypoxia. Plasminogen activator inhibitor (PAI)-1 is an extracelluar protein involved in stabilizing matrix. It is regulated by HIF-1 and can be detected in plasma. Osteopontin is another secreted protein involved in stimulating cell motility and metastasis. It is regulated by hypoxia via a Ras-mediated enhancer. Both of these proteins have been strongly correlated with the presence of hypoxia in tumors, based on independent direct measurements of hypoxia, such as by Eppendorf polarographic electrodes.[34-37]
• Invasive and Tissue-Based Biomarkers-The Eppendorf electrode is a polarographic needle device that can be used to measure tumor hypoxia. Although this is a highly validated method for performing such measurements, the device is cumbersome, particularly in situations where multiple measurements are required, as would be needed in a monitoring trial.
Similarly, several endogenous protein markers, alone or in combination, have been correlated with invasive measurements of hypoxia and have been shown to be prognostically important.[34,39] However, they suffer from the same fate as the Eppendorf electrode, in that multiple biopsies would be required for monitoring. Because of the heterogeneity of tumor hypoxia, this would likely require multiple biopsies at each time point, which would not be feasible for most trials.
Both antiangiogenic and vascular-targeted therapies hold great promise. The approval of some antiangiogenic agents in combination with other therapies bode well for the future clinical use of these agents. Similarly, the increased interest in vascular-targeting approaches shows potential, particularly when combined with radiotherapy and/or chemotherapy. In both of these approaches, however, it is imperative that suitable biomarkers be employed to monitor treatment effects and ensure optimal use. To achieve the highest efficacy of these treatment modalities, it may be mandatory to determine the response of antiangiogenic and antivascular compounds to ascertain an appropriate treatment schedule on an individualized basis (see Figure 1).
-Melanie Wergin, DVM, PHD
-Christopher G. Willett, MD
-Mark W. Dewhirst, DVM, PHD
The authors have no significant financial interest or other relationship with the manufacturers of any products or providers of any service mentioned in this article.
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