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British Journal of Radiology (2003) 76, S23-S35
© 2003 British Institute of Radiology
doi: 10.1259/bjr/30165281
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Measuring tumour vascular response to antivascular and antiangiogenic drugs

G M Tozer, PhD

Tumour Microcirculation Group, Gray Cancer Institute, PO Box 100, Mount Vernon Hospital, Northwood, Middlesex HA6 2JR, UK


   Abstract
Top
 Abstract
Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
 References
 
The tumour vasculature is an attractive target for therapy because of its accessibility to blood-borne anticancer agents and the reliance of most tumour cells on an intact vascular supply for their survival. For convenience, therapeutic targeting of the tumour vasculature can be divided into antiangiogenic approaches, which target the process of new blood vessel development and antivascular approaches, which target the established tumour vasculature. Many agents are now in clinical trial for the treatment of cancer by these methods. The main aim of this article is to describe the vascular effects of some of these agents and identify suitable end-points for measuring efficacy in early clinical trials. For drugs which are active below their maximum tolerated dose (MTD), measurement of vascular end-points is required to determine the most effective dosing/scheduling protocols. In addition, many of the current and developing antiangiogenic agents have additional mechanisms of action unrelated to angiogenesis per se, requiring measurement of vascular end-points to understand their mechanisms of action. Measurement of tumour microvascular density (MVD) from tumour biopsies is a common method for assessing the efficacy of antiangiogenic drugs. The limitations of this method and alternative end-points, which take into account vascular function, are discussed. Pre-clinical data regarding tumour response to the antivascular agent combretastatin A-4 3-0-phosphate (CA-4-P) are discussed in the context of guiding clinical trial planning. Finally, the accessibility of vascular end-points for clinical imaging is addressed.


   Introduction
Top
 Abstract
 Introduction
Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
 References
 
The tumour vasculature is an attractive target for therapy because of its accessibility to blood-borne anticancer agents and the reliance of most tumour cells on an intact vascular supply for their survival [1–3]. For convenience, therapeutic targeting of the tumour vasculature can be divided into antiangiogenic approaches, which aim to disrupt the processes involved in the out-growth of new blood vessels from pre-existing ones and antivascular approaches, which aim to target the established tumour vasculature. Individual agents may possess both antiangiogenic and antivascular properties. However, a practical distinction between the two approaches can be made based on the dosing strategies employed. In order to prevent angiogenesis, a chronic dosing schedule is appropriate, whereas single dose or split dose treatments are more effective for antivascular action, which aims to rapidly shut-down blood flow in established tumour blood vessels.

Many antiangiogenic and antivascular agents are now in clinical trial for the treatment of cancer. Continued research in this area is encouraged by the recent success of a Phase III clinical trial of AvastinTM (Genentech, San Francisco, CA; bevacizumab, rhuMAb-VEGF), a humanized antibody against vascular endothelial growth factor (VEGF), in combination with conventional chemotherapy, in metastatic colorectal cancer patients [4] (www.gene.com). The main aim of this article is to identify appropriate end-points for assessing antiangiogenic and antivascular efficacy of new compounds in early clinical trials. Many of these end-points are accessible by non-invasive imaging techniques. In addition, the value of analysing specific vascular markers in peripheral blood will be discussed. First, strategies for targeting tumour angiogenesis and the established tumour vasculature will be reviewed briefly.


   Antiangiogenic strategies for cancer therapy
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
 References
 
Tumour angiogenesis has been confirmed by measurement of high proliferation indices for endothelial cells, not only in rapidly growing animal tumours [5] but also in human tumours [6]. The rationale for developing antiangiogenic strategies for cancer therapy was based on the fact that physiological angiogenesis only occurs in a limited number of situations, such as wound healing and the menstrual cycle, providing the opportunity for developing highly tumour-specific drugs with little toxicity. In addition, the target endothelial cells are non-transformed and this implied that the development of acquired resistance was unlikely [7, 8]. These early expectations proved to be rather optimistic, with a number of toxicities emerging in clinical trials and the realisation that treatment resistance can develop, possibly even for angiogenesis inhibitors acting directly on endothelial cells [9]. However, antiangiogenic therapy is still an attractive option. AvastinTM, the antiangiogenic agent which is furthest along in clinical development, was well-tolerated in combination with conventional chemotherapy in the recently reported Phase III trial [4]. In addition, any acquired resistance may be more readily overcome for antiangiogenic agents than for conventional cytotoxics because of the great molecular diversity of available antiangiogenic drug targets. This should enable the effective combination of drugs to circumvent resistance [9]. Further molecular targets are also likely to emerge following the realisation that tumour recruitment of circulating endothelial progenitor cells can contribute to tumour neovascularization (vasculogenesis) [10] and that a vascular network can develop from splitting of pre-existing vessels (intussusceptive microvascular growth) [11]. These processes are distinct from angiogenesis (the outgrowth of new blood vessels from pre-existing ones), which is the classic process leading to tumour neovascularization and the target for the current agents described in this review.

Cancer cells promote angiogenesis at an early stage of tumour development by the production of pro-angiogenic proteins, such as VEGF, basic fibroblast growth factor (bFGF), transforming growth factor-{beta} (TGF-{beta}) and others [2, 12]. Normal infiltrating cells such as macrophages also contribute to this process [13, 14]. In addition, down-regulation of endogenous antiangiogenic proteins such as thrombospondin-1 is critical for the shift towards a pro-angiogenic phenotype [15]. Tumour microenvironmental factors can act as "angiogenic switches", triggering these changes. The most studied of these is tumour hypoxia, which develops as tumour cells proliferate and extend beyond the diffusion distance for oxygen. Hypoxia activates the hypoxia-inducible factor-1{alpha} (HIF-1{alpha}), which leads to upregulation of several pro-angiogenic proteins including VEGF [16, 17].

Pro-angiogenic growth factors may act directly on the angiogenic process, via surface receptors on vascular endothelial cells (e.g. VEGF) or indirectly, via receptors on endothelial cells and other cell-types, to activate further growth factor production (e.g. TGF-{beta}). The early stages of angiogenesis (breakdown of basement membrane, endothelial cell migration and proliferation) have been the main targets for the development of antiangiogenic strategies and several classes of antiangiogenic agents are currently in clinical trials for cancer (Table 1Go and [18, 19] for recent reviews). It is important to note that the precise mechanisms by which these agents cause their antiangiogenic effects are often unknown (e.g. endostatin, thalidomide). Many agents were not developed specifically for their antiangiogenic effects (e.g. thalidomide) or they were developed against more than one molecular target (e.g. ZD6474). Some agents interfere directly with endothelial cell proliferation or migration pathways, whereas others act on the processes involved in matrix breakdown, which is essential for these processes, or on production or activity of pro-angiogenic growth factors, particularly VEGF.


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  		Table 1. Antiangiogenic agents in clinical trial

 
Approximately 15% of the antiangiogenic agents currently in clinical trial target VEGF. VEGF (also denoted VEGF-A) is a dimeric glycoprotein that can be expressed by virtually all cell types. It acts on two high affinity transmembrane receptor tyrosine kinases (TKs), VEGFR-1 (Flt 1) and VEGFR-2 (Flk-1 or KDR), which are expressed on vascular endothelial cells. VEGFR-2 is primarily restricted to endothelial cells and a few types of neurons, whereas VEGFR-1 can also be found on a few other cell types such as monocytes, dendritic cells and smooth muscle cells. Activation of VEGFR-2 promotes angiogenesis and vascular permeability in a variety of animal models and induces endothelial cell migration and mitosis. Less is known about the function of VEGFR-2 but it appears to play an important role in vascular development, especially in vascular maturation. A third homologous receptor, VEGFR-3 (Flt-4) is expressed on quiescent adult lymphatic but not vascular endothelial cells. The biology of VEGF and its receptors has been reviewed recently [20]. Small molecule inhibitors of VEGFR-2 kinase inhibitors have been developed as antiangiogenic drugs (Table 1Go and [21]). Achieving selectivity of these agents is difficult because of the similarity of TK enzyme active sites. Non-specific activity may result in toxicity but may also augment efficacy because inhibition of multiple angiogenic pathways are likely to be required to block the complex process of angiogenesis. A number of small molecule VEGFR-2 kinase inhibitors, with different inhibitory profiles, are now in clinical trial (Table 1Go). Alternative approaches for targeting the activity of VEGF include monoclonal antibodies to VEGF (e.g. AvastinTM), VEGF-traps consisting of fragments of the VEGF binding domains of VEGFR-1/VEGFR-2 fused to the Fc region of human IgG1 (www.regeneron.com) and ribozymes designed to cleave the mRNA for the receptors (e.g. AngiozymeTM (Sirna Therapeutics, Boulder, CO; RPI-4610)). See Table 1Go for more details.

Anticancer drugs developed for their capacity to block an oncogene product commonly have indirect antiangiogenic effects via their interference with the production of angiogenic growth factors (e.g. inhibitors of the epidermal growth factor (EGF) receptor tyrosine kinase such as ZD1839/Iressa). In addition, it has been recognised that many conventional chemotherapeutic agents that target the proliferative capacity of tumour cells, may also be active against proliferating/angiogenic endothelial cells. Exploitation of this effect is likely to require modification of conventional acute dosing regimens to continuous dosing strategies, in order to have a substantial effect on the dynamic process of new blood vessel development [22]. Availability of orally-active preparations are an obvious advantage here.


   Antivascular strategies for cancer therapy
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
 References
 
In contrast to the antiangiogenesis approach, antivascular strategies aim to cause a rapid and extensive shut-down of the established tumour vasculature, leading to secondary tumour cell death [23]. Cell death following blood flow shut-down, induced by clamping or ligation of the tumour-supplying blood vessels, is characterized by an early and extensive tumour cell necrosis [24, 25]. Therefore, this pattern of cell death following treatment is indicative of vascular-mediated cytotoxicity. There is potential for specific targeting of the tumour vasculature based on selective expression of proteins on tumour endothelial cells. Recent development of techniques for the isolation of tumour-derived endothelial cells and gene expression have led to the identification of a number of gene transcripts, which are specifically elevated in tumour-associated endothelium [26]. Although the function of the majority of these genes is unknown, their recognition holds the promise of selective tumour vascular targeting. In addition to active angiogenesis, they are likely to be associated with a range of characteristics of the established tumour vasculature, which may eventually be possible to target at the molecular level. For example, tumour blood vessels are often described as "immature", relatively lacking investment with vascular smooth muscle cells or pericytes [6] and highly permeable [27], which contributes to high interstitial fluid pressure [28]. Tumour vascular networks are complex, seemingly chaotic, leading to heterogeneous blood perfusion [29] and oxygenation [30].

Antivascular approaches under investigation include integrin-binding peptides conjugated to anticancer drugs [31], antibodies targeted to endothelial-specific proteins [32, 33] and gene therapy approaches [34]. Antibodies have been conjugated to a range of agents designed to shut down blood flow including radioisotopes, cytotoxic drugs and tissue factor for inducing coagulation [35]. However, closest to clinical practice are two classes of low molecular weight drugs, the colchicine-related tubulin-binding agents such as the combretastatins [36] and drugs related to flavone acetic acid (FAA) such as dimethylxanthenone-acetic acid (DMXAA) [37–43]. Although the mechanisms of action of these two classes of drug differ [44, 45], both cause a rapid and selective shut-down of tumour blood flow leading to haemorrhage and tumour necrosis. DMXAA, combretastatin A-4 3-0-phosphate (CA-4-P), the combretastatin analogue AVE8062 and the colchicine analogue ZD6126 are all currently in clinical trial as tumour antivascular agents [46, 47]. In addition, arsenic trioxide, which has been used extensively for treatment of patients with promyelocytic leukaemia, also has antivascular activity in solid tumours and is currently being tested in clinical trial [48, 49].


   Measuring the efficacy of antiangiogenic and antivascular agents in the clinic
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
Alternative methods for...
 Summary and implications for...
 References
 
Rationale
For measurement of the efficacy of both antiangiogenic and antivascular agents in clinical trials, classical assessment of tumour response is insufficient because a significant tumour growth delay may only occur in combination with conventional treatment. On the other hand, slowing of tumour growth may occur with little or no change in vascular density, if angiogenesis is prevented for extended periods without compromising blood flow in the existing vessels. Tumour regression may also occur if there is a significant antivascular action but is often slow compared with that following conventional cytotoxic chemotherapy [18]. A moderate growth effect following conventional treatment is often considered as treatment failure because the tumour cells are likely to become resistant to the drug. However, acquired resistance should be less of a problem with direct-acting vascular-targeted drugs, such that growth arrest for prolonged periods may be possible.

In order to determine the efficacy of vascular-targeted drugs in early clinical trials, it is necessary to obtain as much pre-clinical data as possible and measure both pharmacodynamic and tumour growth end-points. Pre-clinical data can provide appropriate pharmacodynamic end-points for clinical studies and these can be sub-divided into measurement of vascular response and measurement of the interaction of the drug with its molecular target or down-stream molecular events. Since the precise molecular targets of many of the current agents are unknown, this review is primarily focused on measurement of vascular response.

Cancer chemotherapeutics are traditionally administered at close to their maximum tolerated dose (MTD). However, antiangiogenic agents may be effective at drug doses below their MTD requiring measurement of vascular end-points to determine the most effective dosing/scheduling protocols. In addition, many of the current and developing antiangiogenic agents have additional mechanisms of action unrelated to angiogenesis per se, requiring measurement of vascular end-points to understand the mechanism of action of these agents.

Antivascular drugs are administered intermittently and at close to their MTD, so that the choice of dose level and scheduling resembles that for conventional cytotoxic agents. However, as for the antiangiogenic agents, measurement of vascular parameters is essential for assessing efficacy and understanding the mechanism of action of these agents, which will ultimately lead to the development of improved vascular-targeted treatments.

Measuring the tumour vascular response to antiangiogenic agents
Measuring the tumour vascular response to antiangiogenic agents is a challenge [50–52]. A direct measure of angiogenic rate would be, for example, the number of new vessels formed or the increase in vascular surface area within a tissue per day. In a non-proliferating normal tissue, this is relatively easy to measure but, in a tumour, where tumour cell proliferation accompanies angiogenesis, it is extremely difficult, even in pre-clinical models. In the clinical situation, this end-point is impossible to measure directly. Instead, the most commonly used end-point for assessing antiangiogenesis in clinical studies is tumour microvascular density (MVD), measured from biopsies taken before and at one or more times after treatment, using a variety of immunohistochemical vascular markers such as CD34, CD31, CD105 and von Willebrand factor to identify the vessels [53].

MVD measured in tumour vascular "hotspots" is a prognostic indicator in a range of human tumours. The first study was carried out in breast cancer by Weidner et al [54] and this stimulated hundreds of similar studies, most of which showed some value of MVD as a prognostic indicator in, for example, malignant melanoma, non-small-cell lung cancer, genitourinary cancers, oesophageal/gastrointestinal cancers and haematologic cancers. A possible explanation for MVD indicating poor prognosis is that high MVD reflects a high "escape rate" from tumour and/or a high angiogenic capacity of escaping cells, which can then go on to form metastases [55].

Unfortunately, measurement of MVD is problematic for assessing the vascular efficacy of antiangiogenic agents [55]. As tumours grow, they can be accompanied by proportional blood vessel growth and conversely, blocking angiogenesis may be accompanied by a proportional reduction in tumour growth that would not result in a net change in MVD. Therefore, although a reduction in MVD following treatment is indicative of an antiangiogenic effect, it does not follow that no change in MVD is indicative of no antiangiogenic effect, as is commonly assumed.

Following effective antiangiogenic therapy, tumour blood vessel growth is inhibited, perhaps accompanied by vessel regression and subsequent tumour cell death. Since MVD reflects the ratio of tumour vasculature to tumour cells, changes within both compartments affect its measurement. Therefore, an initial vascular effect may decrease MVD but with a subsequent recovery, the exact time-course being variable for different tumours. Clearly, most information would be obtained from a comprehensive time-course of effects, which is difficult or impossible to acquire from biopsies. In a clinical study of refractory multiple myeloma patients by Singhal et al [56], bone marrow MVD from biopsies was measured before and every 50 days (up to 7 times in each patient) during treatment with thalidomide. This disease is associated with significant neovascularization of the bone marrow. Thalidomide was shown to be antiangiogenic in the rabbit cornea assay [57] but also has a number of immunomodulatory properties linked to expression of adhesion molecules, cytokines and stimulation of cytotoxic T-cells [58]. In the Singhal study, patients received thalidomide once daily in an escalating dose schedule, days of treatment ranging from 2 to 465. There was a significant response to treatment in 32% of patients, as assessed by myeloma marker proteins in serum and urine, and marked decrease in MVD in some patients with complete or nearly complete remissions. However, overall, the slopes of the MVD versus time data were not significantly different from zero for either the responding or non-responding patient groups. Unfortunately, even here, where it was possible to obtain multiple biopsies, it is difficult to draw any definitive conclusions from MVD data regarding the potential for thalidomide as an antiangiogenic agent.

Non-invasive imaging methods for measuring functional vascular volume are available and can be treated as the imaging near-equivalent of measuring MVD. Such methods allow for repeated measurements and sampling of the whole tumour mass but are problematic in that, like MVD measurements, a negative effect on vascular volume cannot be interpreted as absence of antiangiogenic effect. In addition, imaging methods for vascular volume have not been used extensively and issues such as sensitivity and validation against invasive methods need to be addressed in pre-clinical models before being widely applied in the clinic. Indeed, a recent study in a xenografted model of human breast cancer showed a poor correlation between MVD and fractional blood volume estimates as measured by functional MRI and macromolecular contrast agents [59].

Tumour blood flow rate is an accessible end-point for clinical studies. A decrease in tumour blood flow rate would be expected if MVD decreased and its measurement would provide additional functional information linked to oxygen availability and tumour growth. However, as detailed above, a significant antiangiogenic effect may occur without any change in MVD. In addition, blood flow rate and MVD are poorly coupled. Some pre-clinical studies have demonstrated an increase in tumour blood flow rate following antiangiogenic therapy. For example, Teicher et al [60] showed that tumour blood flow and oxygenation significantly increased in the first weeks of treatment with TNP-470, a synthetic analogue of fumagillon. "Normalizing tumour vasculature" following antiangiogenic therapy, whereby blood flow rate within individual vessels is improved after treatment, has also been described [61]. The mechanisms behind these effects are unclear. The most immature and inefficient tumour blood vessels may be "pruned" from the tumour vascular network by antiangiogenic therapy, leaving a more efficient system [61]. In addition, many pro-angiogenic growth factors are associated with high vascular permeability and their withdrawal can reverse this effect [62]. It is possible that a decrease in vascular permeability to macromolecules could improve blood flow rate by reducing tumour interstitial fluid pressure. In any case, measurement of vascular permeability or interstitial fluid pressure could provide alternative end-points for assessing tumour vascular effects of antiangiogenic agents. A pre-clinical method of measuring tumour vascular permeability to macromolecules is illustrated in Figure 1Go.



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  		Figure 1. Measurement of permeability of the tumour vasculature to fluoroscein isothiocyanate (FITC)-albumin using intravital microscopy of tumours growing in dorsal skin flap "window" chambers. A time sequence (0–2 h) of FITC-albumin extravasation in the P22 rat tumour was detected by multiphoton fluorescence imaging, which provides images with no out of focus fluorescence for full quantification. "Patlak" plots of four random vessels are shown. KI, the plasma clearance constant for albumin was calculated from the gradient of the straight line portion of the graph in ml blood per ml tissue per minute (ml•ml–1•min–1). The vascular permeability–surface area product (PS product) is equivalent to KI for a high molecular weight compound whose uptake into tissue is permeability (rather than flow) limited and whose backflux into the blood is low.

 
VEGF is a survival factor for endothelial cells [63] and VEGF withdrawal can induce tumour endothelial cell death as well as prevent further angiogenesis [62, 64, 65]. Therefore, high enough doses of antiangiogenic agents, which are designed to block VEGF or its signalling pathways (see Table 1Go), may cause vascular shut-down and an initial decrease in tumour blood flow rate within tumours, effects which are reminiscent of the effects of agents defined as "antivascular". It is very likely that other agents designed to prevent tumour angiogenesis will also have direct vascular effects, which should be easier to detect than an antiangiogenic effect per se. However, care should be taken with the interpretation of results; a measured decrease in blood flow rate would be evidence for a vascular effect but does not necessarily mean that the drug in question is acting via the hypothesized mechanism. There are relatively few mechanistic studies of antiangiogenic agents in animal models, although these would be invaluable for providing essential information for choosing the most appropriate assays for testing these agents in clinical trials.

Various clinical trials have employed imaging techniques for assessing vascular effects of antiangiogenic agents. Changes in tumour blood flow, blood volume and glucose metabolism were measured in metastases of prostate cancer patients following treatment with thalidomide, using positron emission tomography (PET) [66]. Blood volume (measured using 11CO) correlated with change in serum markers for the disease. However, blood flow values (measured using 15O-water) did not change significantly with treatment. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques are being used in various early clinical trials of different antiangiogenic agents. Most use analysis of the tissue uptake curves for gadolinium-conjugated DTPA (Gd-DTPA). The small molecule inhibitor of VEGF receptors, PTK 787/ZK222584 was recently reported to decrease vascular permeability in glioblastoma multiforme at day 2 and 30 after treatment [67]. In the same meeting, the VEGFR-2 receptor inhibitor SU5416 was reported to decrease blood flow in metastatic melanoma 8 weeks after start of treatment [68]. Although an active area of research, MRI measurements remain to be fully validated. Measurement of the transfer constant Ktrans for Gd-DTPA is the most commonly used method for measuring vascular effect. In the tumour vasculature, the value for Ktrans is likely to reflect both tissue blood flow rate and the permeability of the vascular wall. However, one or other of these terms is often inappropriately applied to these measurements. Measurement of primary biochemical events with particular antiangiogenic agents such as receptor activation may be accessible with the development of specific PET tracers [69, 70] but need to be combined with methods for detecting subsequent effects on vascular morphology and function.

In summary, the tumour vascular effects of specific antiangiogenic agents are difficult to predict based on the available pre-clinical data. Vascular volume and blood flow are likely to decrease following treatment but are poorly coupled and may increase at certain times after treatment depending upon the relationship between the vascular and tumour cell compartments in individual tumours. Effects are also likely to be highly dose and time dependent. Vascular permeability, particularly to macromolecules, may represent a useful alternative measure of vascular effect. Using current imaging techniques, it is not usually possible to measure specific vascular parameters precisely. However, repeated measurements are possible, and this should provide the best opportunity for detecting any vascular effects of new anticancer agents proposed to act via inhibition of tumour angiogenesis.

Measuring the tumour vascular response to antivascular agents
Antivascular agents aim to cause an acute collapse of tumour blood flow, as opposed to the subtler effects anticipated for antiangiogenic agents. Several of these agents are currently in clinical trial, most data being available for the OxiGene, Inc. (Waltham, MA) compound, CA-4-P, which will be used here as an example for describing the process by which appropriate vascular end-points can be chosen for early clinical trials.

The Phase I/II clinical trials of CA-4-P followed from a series of pre-clinical studies in mouse and rat demonstrating significant tumour antivascular activity at well tolerated doses in ectopically and orthotopically transplanted murine tumours, xenografted human tumours, spontaneous tumours and vascularized metastases [71–78]. In the mouse, 100 mg kg–1 CA-4-P reduced the perfused vascular volume of subcutaneously transplanted CaNT tumours to less than 10%, by 6 h after treatment, and this was accompanied by massive necrosis of all but a narrow rim of tumour tissue by 24 h [71]. This peripheral sparing is a common feature of tumours following treatment with CA-4-P, as well as other antivascular agents, and accounts for the rapid re-growth of tumours when administered as single agents. A major challenge for basic research is to understand the mechanism of action of CA-4-P and related drugs and the reasons for the peripheral sparing [44].

Several pre-clinical studies investigated the dose response and time-course of vascular effects in tumours and normal tissues following CA-4-P treatment [78–80]. Pharmacokinetic studies in man and rat identified 10 mg kg–1 CA-4-P in the rat to be approximately equivalent, in terms of tissue exposures, to the maximum-tolerated dose in man [80, 81]. The time-course of absolute blood flow rate in tumour and normal tissue of the BDIX rat, following 10 mg kg–1 CA-4-P, is shown in Figure 2Go. Data such as these clearly demonstrate the potential of this agent for selective tumour blood flow shut-down and are extremely valuable for guiding the planning of clinical trials. They not only suggest appropriate times for clinical assessment of response but, via measurement of absolute blood flow rates, also provide a standard for comparison with subsequent clinical data, which is relatively easy to interpret.



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  		Figure 2. The effects of 10 mg kg–1 CA-4-P on mean arterial blood pressure (MABP) and blood flow rate to the P22 tumour and selected normal tissues in the BDIX rat. MABP was measured via an intra-arterial cannula. Comparison of MABP and skeletal muscle blood flow results suggest that the initial hypertensive effect is due to vasoconstriction in muscle. Spleen is the most responsive normal tissue but the greatest effect is found in tumour. Blood flow rate was measured using the tissue uptake of radiolabelled iodo-antipyrine, where tracer concentration versus time curves for arterial blood and tissue are deconvolved to extract tissue blood flow rates for tumour and normal tissues. Values are the mean±1 standard error (SE). The shaded bars show the limits of the SE for the control animals. A significant difference from control at the 5% level is represented by *. Figure adapted from reference [80].

 
Measuring the spatial distribution of tumour blood flow in animal models following treatment with antivascular agents and relating the time course of blood flow changes to histological events and the extent of necrosis induction, provides further information, which helps interpretation of clinical data. This is illustrated in Figure 3Go for CA-4-P. Here it is apparent that initial blood flow collapse in the first few hours after treatment is associated with large, distended blood vessels, packed with red cells, with some showing signs of coagulation. Haemorrhage is also evident. However, sustained blood flow reduction at 24 h, which occurs in some tumours even at 10 mg kg–1 CA-4-P, is associated with extensive tumour cell necrosis. From data such as these, it is reasonable to assume that very poor uptake of a contrast agent into human tumours measured at 24 h or later after treatment with combretastatin-like drugs is indicative of tumour necrosis.



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  		Figure 3. Upper panels show pseudocolour images of tissue blood flow, in ml blood per gram tissue per minute (ml g–1 min–1), computed from autoradiograms of 20 µm thick frozen sections of ~15 mm diameter P22 rat tumours. Tumours were excised from an untreated rat (a) or at various times after treatment from rats treated with a clinically relevant dose of 10 mg kg–1 CA-4-P (b–d). Blood flow was computed pixel by pixel using the methods described in Figure 2Go. Lower panels show paraffin-embedded 3 µm thick tumour sections stained with haematoxylin and eosin from a separate group of rats treated as for the upper panel. Images in (a) show typical heterogeneity of blood flow in untreated tumours and spindle-shaped tumour cells in this sarcoma. Images in (b) show severely reduced blood flow accompanied by tumour cell shape changes and some dilated vessels and coagulation. Images in (c) show sustained blood flow reduction, more dilated, congested and coagulated vessels and extensive haemorrhage. The shaded areas in the upper panel indicate the extent of haemorrhage. Images in (d) show extensive necrosis (delineated in the upper panel) accompanied by very low blood flow, surrounded by a regrowing rim of tumour with flow values similar to controls. Figure adapted from reference [80]. Arrows indicate coagulation.

 
Figure 4Go shows additional vascular effects of CA-4-P in the P22 rat tumour, as detected by intravital microscopy. Blood vessels are very distended at early times after CA-4-P, when blood flow is severely compromised. This illustrates that measurement of MVD from tumour biopsies, as commonly used for assessing response to antiangiogenic agents (see above), would not be appropriate for antivascular agents. Clearly, measurement of blood flow rate or other related functional parameter is essential for clinical evaluation of CA-4-P and other antivascular agents.



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  		Figure 4. A P22 rat tumour (~4 mm diameter) growing in a dorsal skin flap "window" chamber surgically implanted into a BDIX rat before and 3 h after treatment with 10 mg kg–1 CA-4-P. Blood vessels are visualized by intravital microscopy under transmitted light using low power (upper panels) and high power (lower panels). After 3 h, when tumour blood flow is severely reduced (see Figures 2 and 3GoGo), blood vessels are clearly distended and there is significant haemorrhage especially around the tumour periphery.

 
In vitro studies with endothelial cells have demonstrated that tubulin-binding of CA-4 is followed by rapid cell signalling, which results in reorganization of the actin cytoskeleton and membrane blebbing, leading to increased permeability of cell monolayers to macromolecules [82]. An increase in tumour vascular permeability has also been detected in vivo [83].

The pre-clinical data described above suggest that, in addition to the rate of blood flow, the functional blood volume and the apparent tissue/plasma partition coefficient of a tracer/contrast agent are useful end-points to measure in clinical trials of antivascular agents. If there is a decrease in the perfused fraction of the tissue at the microscopic or macroscopic level (e.g. Figure 3dGo), then this would be reflected in a decrease in the apparent tissue/plasma partition coefficient (i.e. the measured concentration of tracer/contrast agent in tissue relative to that in plasma at relatively long times after administration). Measurement of vascular permeability would also be useful as an early indicator of vascular damage.

DCE-MRI has detected vascular changes within human tumours, consistent with reduced blood flow rate, for both DMXAA and CA-4-P, in recent clinical trials [46, 47]. The time-course of these vascular changes following CA-4-P were very similar to those found for DCE-MRI and blood flow changes in animal tumours [47, 80, 84]. Using positron emission tomography (PET) to measure tumour uptake of 15O-water into human tumours, a significant reduction in absolute tumour blood flow rate following CA-4-P was detected. Using radiolabelled carbon monoxide (C15O), the same authors reported a simultaneous decrease in tumour blood volume [85].


   Alternative methods for measuring efficacy of antiangiogenic agents
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
Summary and implications for...
 References
 
Biopsy techniques
As we have seen, measurement of angiogenesis and its disruption is very difficult in tumours, especially in clinical studies. Where it is possible to obtain biopsies, measurements of vascular parameters in addition to MVD are feasible. It has also been proposed to monitor angiogenesis in accessible wound tissue of cancer patients, as a surrogate for effects in the tumour [86]. Both these approaches were used in a clinical study by Mundhenke et al [87]. Here, 21 patients with a range of solid tumours (8 fully analysed) were treated with daily infusions of endostatin, a proteolytic fragment of the basement membrane component collagen XVIII, which has profound antitumour effects in animal models [88]. Tumour biopsies were taken prior to and 8 weeks into treatment. In addition, a punch biopsy device was used to create a skin wound site in each patient from which skin biopsies were taken prior to and 3 weeks into treatment. In addition to measurement of MVD, tumour and wound tissues were also assessed for endothelial cell proliferation (Ki67 staining), endothelial cell apoptosis (TUNEL staining) and vascular maturity (muscle-specific actin staining). These end-points may provide more appropriate methods for measuring antiangiogenic effect than the MVD method. That is, the balance of endothelial cell proliferation and death would be expected to reflect blood vessel growth. In addition, newly formed vessels are immature, lacking actin-positive pericytes, and the proportion of immature blood vessels has been postulated to be a measure of angiogenic activity within a given tissue [6, 64]. Other workers have consistently identified pericytes in a range of different tumour types, using alternative markers for pericyte identification [52], and the relationship between pericytes and angiogenesis remains to be fully established.

Unfortunately, none of these end-points changed significantly in either tumour or wound tissue samples following endostatin treatment. This may have been due to the very small sample size, very advanced disease, insufficient levels of endostatin, insensitivity of the assays, as well as a true lack of effect. It is clear that more studies of this sort need to be carried out, with larger patient groups. Pre-clinical studies are also crucial in order to evaluate these end-points in systems where antiangiogenic effects can be measured directly. Effects of antiangiogenic agents on wound healing need to be further assessed to determine their relationship to effects on tumour angiogenesis. Ultimately, development of non-invasive imaging methods for measuring these end-points is required to acquire a fuller time-course of events.

Analysis of peripheral blood
Haematopoietic cells, endothelial cells and angiogenic growth factors circulate in the blood and their levels tend to increase with tumour growth. Tumours may also recruit circulating progenitor endothelial cells for blood vessel formation [10]. A simple blood test would be ideal for monitoring efficacy of antiangiogenic agents. Although it is not clear how these levels relate to tumour angiogenesis and the effects of antiangiogenic agents, many studies have investigated the effect of antiangiogenic agents on circulating haematopoietic cells, endothelial cells and angiogenic growth factors. For example, Bertolini et al [89] measured the levels of VEGF and bFGF in the circulation of 17 patients with multiple myeloma before and during twice daily treatment with thalidomide. There were some significant reductions in growth factor levels at the time of maximum response to treatment. However, interpreting this type of data is problematic. First, measurement of one or two growth factors is unlikely to truly reflect angiogenic capacity and takes no account of potentially circulating inhibitory factors. Second, levels may represent tumour burden rather than angiogenesis per se. Third, there is high VEGF production by platelets for instance, which can lead to great variability in results depending on whether VEGF levels are measured in plasma, serum or whole blood [90]. Fourth, the source and mechanism of externalization of relevant growth factors are unknown. Finally, growth factors are usually cleared from plasma within minutes so that levels only rise for a large tumour burden.

The utility of measuring circulating angiogenesis regulators (growth factors and inhibitors) extends far beyond monitoring the efficacy of antiangiogenic agents. For instance, there is potential for early detection of cancer and for distinguishing between benign and malignant disease. In patients with established disease, there is potential for determining prognosis, predicting response to therapy and monitoring tumour burden following treatment. These areas have been reviewed recently [91].

An interesting variant on measurement of angiogenic growth factors in blood was described by Gradishar et al [92]. In this study, nine breast cancer patients were treated with suramin for 5 days and then every few days for 11 more weeks. Suramin is a polysulfonated napthylurea with direct activity against tumour cells as well as an antiangiogenic effect, which is likely to relate to its ability to bind/antagonize various peptide growth factors such as aFGF, bFGF, EGF and VEGF. In this study, instead of measuring circulating levels of specific growth factors, the angiogenic capacity of the platelet-poor plasma or serum samples from patients was measured by testing its activity in an in vitro endothelial cell migration assay. Although time-consuming, this biological assay circumvents the first of the problems associated with peripheral blood analysis described above.

An increase in circulating endothelial cells in cancer patients could be explained by shedding from the lining of tumour blood vessels, adjacent normal vessels or distant normal vessels, activated by tumour-derived cytokines. Endothelial cells are assayed by immunostaining for specific endothelial cell markers followed by sorting by flow cytometry. Interestingly, in an animal model of lymphoma, most circulating endothelial cells in control mice with no tumours were annexin V positive, suggesting initiation of an apoptotic process in these cells. However, the proportion of annexin-V positive endothelial cells circulating in tumour-bearing mice was significantly reduced, possibly because of high VEGF levels generated by tumour cells [93]. There was a strong correlation with numbers of circulating endothelial cells and both tumour volume and tumour-derived VEGF. In cyclophosphamide-treated mice, most of the circulating apoptotic cells were haematopoietic, whereas following endostatin treatment, most were endothelial [93]. More studies need to be carried out to determine whether the effect of antiangiogenic agents on circulating endothelial cells is a good surrogate for the effect on endothelial cells within tumour blood vessels.


   Summary and implications for clinical imaging
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
References
 
Investigations of the tumour vasculature in pre-clinical models can be made at the microscopic level using intravital microscopy or radiotracer uptake methods. These studies are invaluable for planning and interpreting results of clinical trials of antiangiogenic and antivascular agents for cancer therapy. There are relatively few antivascular agents currently available and all are designed to cause a rapid and catastrophic decrease in tumour blood flow. However, the antiangiogenic agents are many and varied and their vascular effects are difficult to infer from in vitro studies. More studies in pre-clinical tumour models are therefore warranted.

Vascular end-points, which will inform on the efficacy of both antiangiogenic and antivascular agents, have been described in this review and are summarized in Table 2Go. Some of these end-points are accessible by modern clinical imaging techniques, which are described in the accompanying papers. Imaging techniques usually rely on measurement of the tissue uptake of a contrast agent, followed by mathematical curve fitting of the time-activity data to extract specific vascular parameters. Concurrent measurement of the contrast agent in arterial blood (the arterial input function) allows full quantification but this is not always possible to obtain. These techniques have spatial resolutions ranging from approximately 0.5 mm to 5.0 mm, which means that there is some degree of averaging of results within an individual voxel. Therefore direct imaging of vascular parameters is not usually possible in clinical imaging. However, parameters such as the apparent tissue/blood partition coefficient of a contrast agent (often referred to as the volume of distribution in the positron emission tomography (PET) literature) are possible to measure (see Figure 5Go) and have functional significance. For instance, a decrease in the apparent tissue/blood partition coefficient following treatment is indicative of an increase in the average distance between perfused vessels or the presence of defined regions of unperfused tissue caused by vascular shutdown.


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  		Table 2. Vascular end-points for measuring response to antiangiogenic and antivascular agents

 


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  		Figure 5. Vascular end-points, which will inform on the efficacy of antiangiogenic and antivascular drugs and which are accessible by clinically-applicable imaging techniques, are shown. Analysis of the initial tissue uptake kinetics of an intravenously administered contrast agent/radiotracer allows calculation of blood flow rate or the vascular permeability–surface area product, depending on whether the tissue uptake of the marker is flow or permeability limited. This will depend on characteristics of both the contrast agent itself and the vascular wall. Full quantification requires an arterial input function. The tissue/blood partition coefficient is the relative concentration of the agent in tissue and blood at equilibrium. The apparent tissue/blood partition coefficient can be accurately determined given relatively long imaging times and is equal to the true tissue/blood partition coefficient in well-perfused tissues. In poorly perfused tissue, it is reduced and reflects the perfused fraction of the tissue (see text). Measurement of the fraction of the tissue occupied by functional blood vessels (blood volume) is made using agents which are essentially confined to the vasculature. Analysis of venous blood samples allows for full quantification.

 
Choice of contrast agent dictates the measurable parameters. For instance, low molecular weight readily diffusible tracers, such as 15O-labelled water for PET, can be used to measure blood flow rate. PET is also the only currently available technique for monitoring the molecular events following treatment, using highly specific PET radiotracers. However, all available clinical methods have their advantages and disadvantages and short-lived isotopes such as 15O are expensive to produce and only available in a few centres. DCE-MRI is more readily available and has already been used in several clinical trials of vascular targeting agents. However, tumour uptake of the low molecular weight gadolinium-based contrast agent Gd-DTPA is dependent on both blood flow rate and the vascular permeability–surface area (PS) product, making results difficult to interpret. Larger molecules, such as gadolinium-bound proteins and ultrasmall particles of iron oxide (USPIO) for DCE-MRI have potential for measuring vascular volumes and vascular permeability to macromolecules. CT and ultrasound techniques, described in the subsequent papers, are also valuable for monitoring tumour vascular effects.

As new clinically-applicable techniques for imaging tumour vascular morphology and function are developed, pre-clinical tumour models will be invaluable for assessing their potential for monitoring the effects of vascular-targeted treatment. It will be equally important to determine the predictive power of these techniques for treatment outcome and this may be best achieved by combining imaging techniques with information gained from blood and biopsy samples, as briefly reviewed in this paper.


   Acknowledgments
 
Many thanks to Professor Vincent Cunningham for helpful advice and reading this manuscript. I would also like to thank my colleagues at the Gray Cancer Institute who collaborated on producing the data presented in this paper, especially Vivien Prise and Ian Wilson, and Boris Vojnovic and his group for developing the intravital microscopy technology.


   References
Top
 Abstract
 Introduction
 Antiangiogenic strategies for...
 Antivascular strategies for...
 Measuring the efficacy of...
 Alternative methods for...
 Summary and implications for...
 References
 

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