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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Integr Biol (Camb). Author manuscript; available in PMC Apr 5, 2011.
Published in final edited form as:
PMCID: PMC3071431
NIHMSID: NIHMS272457
A Perspective on Vascular Disrupting Agents that Interact with Tubulin: Preclinical Tumor Imaging and Biological Assessment#
Ralph P. Mason, Dawen Zhao, Li Liu, Mary Lynn Trawick, and Kevin G. Pinney*
Department of Radiology, 5323 Harry Hines Boulevard, The University of Texas Southwestern Medical Center, Dallas, Texas, 75390-9058 USA
Department of Chemistry and Biochemistry, One Bear Place #97348, Baylor University, Waco, Texas 76798-7348, USA
* To whom Correspondence should be addressed. Tel: (254) 710-4117. Fax: (254) 710-4272. Kevin_Pinney/at/baylor.edu
The tumor microenvironment provides a rich source of potential targets for selective therapeutic intervention with properly designed anticancer agents. Significant physiological differences exist between the microvessels that nourish tumors and those that supply healthy tissue. Selective drug-mediated damage of these tortuous and chaotic microvessels starves a tumor of necessary nutrients and oxygen and eventually leads to massive tumor necrosis. Vascular targeting strategies in oncology are divided into two separate groups: angiogenesis inhibiting agents (AIAs) and vascular disrupting agents (VDAs). The mechanisms of action between these two classes of compounds are profoundly distinct. The AIAs inhibit the actual formation of new vessels, while the VDAs damage and/or destroy existing tumor vasculature. One subset of small-molecule VDAs functions by inhibiting the assembly of tubulin into microtubules, thus causing morphology changes to the endothelial cells lining the tumor vasculature, triggered by a cascade of cell signaling events. Ultimately this results in catastrophic damage to the vessels feeding the tumor. The rapid emergence and subsequent development of the VDA field over the past decade has led to the establishment of a synergistic combination of preclinical state-of-the-art tumor imaging and biological evaluation strategies that are often indicative of future clinical efficacy for a given VDA. This review focuses on an integration of the appropriate biochemical and biological tools necessary to assess (preclinically) new small-molecule, tubulin active VDAs for their potential to be clinically effective anticancer agents.
Tumor growth and metastasis require a functioning vascular network to provide oxygen and other nutrients. While the endothelium of normal, remodeled blood vessels is largely quiescent, the neovasculature of tumors is primitive, distinct in morphology, more responsive to angiogenic cell signaling, and activated in nature.1-3 Consequently, the tumor vasculature offers an excellent, potentially selective target for anticancer therapy. The term “vascular disrupting agents” (VDAs) has been coined to describe a relatively new and rapidly emerging class of anticancer agents that selectively damage established tumor vasculature.4-6 Distinct from angiogenic inhibiting agents (AIAs), such as bevacizumab (Avastin™)7 which halt the formation of new blood vessels, VDAs fall into two general classes referred to as biologics and small-molecules.4 The overarching realm of vascular targeting strategies includes both AIAs and VDAs, which are collectively described as vascular targeting agents (VTAs).4-6,8 It is important to emphasize that a very clear distinction has developed in the scientific community that defines compounds such as bevacizumab as angiogenic inhibiting agents, which represent a class of anticancer agent that is mechanistically separate and distinct from the compounds known as vascular disrupting agents that are the focus of this perspective. While bevacizumab (Avastin™) has been approved as an antiangiogenic VTA, there are no VDAs, either biologic or small-molecule, that have reached approval by the Food and Drug Administration (FDA) to date. Bevacizumab is a recombinant humanized monoclonal antibody that binds to vascular endothelial cell growth factor (VEGF) and blocks VEGF interaction with its corresponding receptors on the surface of endothelial cells. It is approved for the treatment of colon and lung cancer.9,10
The discovery and development of new small-molecule VDAs has increased significantly over the past decade and today includes approximately a dozen compounds world-wide that are in human clinical trials (Fig. 1).11-29
Fig. 1
Fig. 1
Tubulin Binding Small-Molecule Vascular Disrupting Agents (VDAs)
The vast majority of these small-molecule VDAs include an interaction with the tubulin-microtubule protein system as a key component of their mechanism of action. This protein includes two small-molecule binding sites, vinca alkaloid and colchicine, located separately on the αβ-tubulin heterodimer. In addition, it features a taxoid binding domain located on the microtubule. It is instructive to note that all of the current clinically relevant small-molecule VDAs that include an interaction with tubulin involve a binding event at the colchicine site on β-tubulin.30,31 It has been previously observed that the natural product colchicine itself induces vascular damage, but only at doses that are limited by toxicity.32,33 In addition, a vascular component has been identified in the mechanism of action attributed to vinblastine and vincristine, as representative vinca alkaloids.34,35 Paclitaxel (Taxol™), however, does not induce vascular damage through its interaction at the taxoid binding domain on microtubules although it alters tubulin-microtubule dynamics through stabilization of microtubules.36 One small-molecule VDA known as DMXAA functions through a separate and distinct mechanism involving tumor necrosis factor alpha (TNF-α).37
Typically, VDAs are not administered to humans (in clinical trials) as single agents, but rather are combined with standard chemotherapy, such as carboplatin and paclitaxel. While a small-molecule, tubulin-interactive VDA is capable of selectively starving a tumor of oxygen and nutrients, this, in turn, leaves behind a “viable rim” at the periphery of the necrotic tissue that is capable of supporting tumor regrowth.38 Continued advances in understanding the subtle differences between the tumor microenvironment39,40 versus the healthy cell environment, on a molecular level, have been instrumental in providing fundamental support for the conceptualization and realization of VDAs as viable therapeutic agents for the treatment of cancer.
The VDA field has been well-reviewed both in terms of small-molecule agents,41-48 currently regarded as the “key players”, and descriptions of the biological mechanism of action2,3,49-59 that involve a complex (yet not completely understood) cell signaling pathway that is initiated by rapid microtubule depolymerization in tumor vasculature, but not in normal blood vessels, ultimately leading to selective vascular damage and collapse in the tumor microenvironment. Vascular collapse in turn can result in massive tumor necrosis. Treatment of endothelial cells in vitro with potent, tubulin-binding VDAs results, in minutes, in profound cell morphology and cytoskeletal changes that are characterized by microtubule depolymerization leading to cell retraction, rounding and detachment (Fig. 2). The cytoskeletal reorganization includes an increase in actinomyosin contractility, assembly of actin stress fibers, formation of focal adhesions and membrane blebbing in certain cell sub-populations. Cell-cell junctions and cell-extracellular matrix interactions are disrupted resulting in an increase in permeability. In some cases, apoptosis results.3 While the exact mechanism relating microtubule disassembly to vascular collapse has not been elucidated, a number of enzymes and a cell signaling pathway have been identified. An increase in myosin light chain phosphorylation is observed and the overall effects are largely abolished in the presence of Rho-kinase inhibitors indicating that in addition to RhoA kinase, the intracellular switch RhoA may be involved. RhoA, which hydrolyzes GTP (guanosine triphosphate), cycles between its active GTP binding form, and the inactive form that binds GDP.
Fig. 2
Fig. 2
Model for downstream effects of treatment with tubulin-binding VDAs in tumor endothelium. Microtubule disassembly leads to cell rounding and detachment that can result in a rapid collapse in tumor blood flow.
Guanine nucleotide exchange factors (GEFs) activate Rho-GTPases by facilitating the exchange of GDP for GTP. In a variety of cells, activated Rho GTPases regulate reorganization of the cellular cytoskeleton in response to multiple signaling pathways via GEFs.60-62 For example, in HeLa cell motility, the actin cytoskeletal rearrangements that occur as a result of microtubule depolymerization are regulated through RhoA.63 GEF-H1 (ARHGEF2) is one of the few GEFs that bind to microtubules thus inhibiting its activity. Upon microtubule depolymerization, GEF-H1 is released and activates the Rho GTPase, RhoA in a number of different cells. In lung endothelial cells, depletion of GEF-H1 attenuated the increase in cell permeability and actin stress formation that results from thrombin treatment of the microtubule depolymeriztion agent nocodazole.64
This review focuses on an integration of the appropriate biochemical and biological tools necessary to preclinically assess new small-molecule, tubulin active VDAs for their potential to be clinically effective anticancer agents.
In vitro assessment of tubulin-binding VDAs
There is a strong correlation between established VDAs and their ability to inhibit tubulin assembly into microtubules, and cytotoxicity against tumor cells lines. The ability of certain VDAs (tubulin-binding) to disrupt microtubule structure is presumed to be the initiating event in the profound morphological changes that occur in vascular disruption.
Inhibition of tubulin assembly into microtubules
To evaluate the effect of the compounds on tubulin assembly in vitro, varying concentrations of the compounds are preincubated with 10 μM tubulin that has been purified from calf brain65 in a solution that will promote polymerization (glutamate solution)66 at 30 °C and then cooled to 2 °C. After addition of GTP, the mixtures are warmed to 30 °C in a recording spectrophotometer and the assembly of tubulin is observed turbidimetrically at 350 nm followed by depolymerization at 2 °C to determine the baseline (Fig. 3). The IC50 value is defined as the compound concentration that inhibits the extent of tubulin assembly by 50% in a 1500 second incubation. The data obtained for a drug at different concentrations are compared to control samples.67-69 A commercially available fluorogenic variation (cytoskeleton.com) of this assay is also available using a 96-well plate format.70 Fluorescence enhancement is measured with the incorporation of a fluorescent reporter into microtubules with neuronal tubulin. Tubulin from sheep, pigs, and recombinant human tubulin isotypes have also been used for assessing inhibition of tubulin polymerization.
Fig. 3
Fig. 3
(A) Inhibition of tubulin polymerization as a function of increasing concentration of VDA (CA1) from the top two traces, 0 μM (control), to the lowest curve at 5 μM CA1. (B) IC50 determination for CA1 (1.9 μM).71
Cytotoxicity assay
The antimitotic activity of molecules that bind to tubulin and prevent microtubule assembly is well documented. Inhibition of human cancer growth can be assessed by the standard sulforhodamine B assay,72 which measures the total cellular protein as a means to determine cellular growth. Cells are distributed into 96-well plates, followed by treatment with study compounds and controls, at varying concentrations at 37 °C for 48 h. A growth inhibition of 50% in comparison to untreated controls (GI50 or the drug concentrations causing a 50% reduction in net protein increase) is calculated by nonlinear regression analysis. Alternatively, the MTT assay,73 which is based on the reduction of the yellow tetrazole, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, to a purple formazan in living cells, is used to assess the effect of the compound on cell growth.
IC50 values for the inhibition of tubulin assembly into microtubules for excellent VDAs are usually in the low micromolar range, while the GI50 values for the cytotoxicity assay are often in the nanomolar range or lower. This amplification is consistent with the involvement of the RhoA-GTP signaling system and activation of the enzyme RhoA kinase.
For the direct in vitro assessment of the VDA on endothelial cells, tube disruption, cell adherence (rounding up), and cell permeability assays are carried out.
Endothelial tube disruption and reorganization assay
Human primary umbilical vein endothelial cells (HUVECs) can be induced to form three-dimensional, capillary-like tubular structures by growing these cells on a substitute for the extracellular matrix such as Matrigel™,74 basement membrane extract (BME), or laminin-rich extracellular matrix,75 and using a growth factor rich medium, which is added to each well of a 24-well or 96-cell plate. A suspension of HUVEC cells in EGM-2 (endothelial cell growth media) without antibiotics is seeded into each well and allowed to incubate at 37 °C from 4 to 24 h to determine the amount of time required for significant tube formation.76 Tubule formation is determined by microscopy and documented by a photographic record. In preliminary experiments, the cells are treated with different concentrations of the compound to be assayed that has been dissolved in DMSO and diluted with medium, added to cells and allowed to incubate at 37 °C for 1-3 h to determine their effect. The compound is then removed and fresh media added. The disruption of tube structure is evaluated by light microscopy after a further 24 h of incubation. Cells can be conveniently stained with Calcein AM for fluorescence imaging77 including confocal microscopy. An estimated IC50 value is obtained through visual inspection of the images.
Cell adherence (rounding up) assay
The endothelial layer of the tumor vasculature undergoes cell retraction, rounding and detachment upon treatment with VDAs. To assess cell adherence, HUVECs are cultured and seeded onto fibronectin coated 96-well plates at high density (deactivated model) and grown with serum with no added growth factors versus low density seeding and growth with VEGF containing (activated) medium.78 VDAs preferentially affect rapidly growing HUVECs.79 A comparison of IC50 values under the two conditions provides information on the vascular disrupting ability of compounds on tumor vasculature relative to normal vessels. Cell viability is assessed by the trypan blue exclusion assay.
Endothelial Cell Permeability Assay
The endothelial cell lining of vasculature defines a permeability barrier between the blood and the interstitial spaces. VDAs cause a reorganization of tumor endothelial cytoskeleton and an increase in vascular permeability.80-82 To assay endothelial cell permeability, HUVECs are seeded onto gelatin coated membrane (0.45 μm pore) inserts and incubated with growth medium in a modified Boyden chamber until confluent. Varying concentrations of VDA are incubated with the HUVECs. After treatment, FITC-Dextran is added on top of the cells. The permeation of FITC-Dextran through the cell monolayer indicates a change in the tight junctions between cells. The extent of permeability is determined by measuring the fluorescence of the plate well solution.83
Apoptosis Assay
Inhibition of tubulin assembly into microtubules and the antiproliferative effects of tubulin-binding VDAs are characterized by cell-cycle arrest in the G2/M phase, which can be followed by flow cytometry. G2/M blockade is often followed by cell apoptosis.84,85 Apoptosis can be measured by a variety of assays including activated caspases 3 and 7 to assess enzyme activity. One cell-based assay uses proluminescent caspase-3/7 DEVD-aminoluciferin substrate and luciferase after cell lysis.86 Caspase cleavage of the substrate liberates free aminoluciferin, which is consumed by the luciferase, generating a luminescent signal. The signal is proportional to caspase-3/7 activity and apoptosis. IC50 values for endothelial tube disruption are often much lower than for apoptosis, for a given VDA.
The nature and applications of VDAs have been the subject of many earlier reviews 4,38,87-89 and here we provide an update, particularly featuring the relevance of imaging techniques in assessing VDA activity. There is hope and promise that Radiology can move beyond an anatomical approach to provide effective biomarkers of potential response to a therapy and early indication of therapeutic efficacy.90
Tumor vasculature is recognized to be highly disorganized and inefficient. Many investigators have demonstrated the tortuous mesh of microvessels characterized by blind ends, constrictions and loops causing non-linear flow, as elegantly revealed in vascular casts, such as the classic studies of Konerding.91 We show a typical corrosion cast in Figure 4, achieved by infusing a liquid monomer into a rat breast tumor growing in the kidney of a rat. Once the material had polymerized, tissue was removed by caustic maceration, the cast coated with a sputtering of palladium-gold and scanning electron microscopy (SEM) performed. Many reports show such corrosion casts in some cases encompassing whole animals.92 Such casts provide an indication of vasculature in three dimensions, though quantitative analysis is not trivial potentially requiring micro CT.93-95 The polymer filaments are fragile and sometimes the finest capillaries may be lost. The cast provides no dynamic information and polymer may be forced into vessels otherwise occluded by temporary thromboses.
Fig. 4
Fig. 4
Vascular heterogeneity in tumors identified ex vivo
More commonly, vascular extent is assessed using histological specimens with immunohistochemistry, e.g., Figure 4b reveals blood vessels based on anti-CD31 monoclonal antibody binding. Vascular perfusion may also be observed by infusion of a vascular reporter prior to sacrifice. Figure 4c shows distribution of Hoechst 33342 dye, which had been infused intravenously 60 seconds prior to sacrifice. Overlay on the CD31 image reveals the fraction of vessels that were perfused (Figure 4d). We have used this approach to show the change in vascular extent and perfusion in tumors with respect to administration of typical VDAs.96,97 Two hours after administration of combretastatin A-4P vasculature was detected based on CD31 (similar to baseline), but essentially all perfusion had ceased. Of course, such measurements generally require separate specimens (tumors) for each time point relying on the similarity of matched tumor-pairs. Hoechst dye extravagates from the vasculature, whereas other markers may reveal perfusion based on endothelial binding (e.g., tomato lectins) or trapping of fluorescent or radioactive microspheres.98 Indeed, sequential administration of stains of different colors before and after an intervention in a pulse chase fashion can reveal dynamic changes post facto 99 as demonstrated by Chaplin et al. with respect to vascular collapse following administration of the vasodilator hydralazine.100
Superficial vasculature may also be assessed in vivo using intra vital microscopy particularly as applied to window chamber models.101 Vascular development may be examined repeatedly over a period of days and, with respect to drug interventions and video loops, has revealed passage of individual erythrocytes sometimes revealing fluctuations and reversal of flow within individual vessels.102,103 Addition of fluorescent markers of pH and hypoxia has allowed correlation of multiple physiological parameters in small regions at microscopic resolution.104 While microscopy is often limited to a surface depth of about 100 um, multi-photon approaches have revealed three-dimensional structure and multimodality interactions as represented by photoacoustic tomography allow deeper penetration.105 Laser Doppler flowmetry has provided non-invasive evidence for acute response to CA4P.106
Pre-clinical studies allow sacrifice and high resolution post-mortem analysis of tissues, but a requirement for biopsies to define vascular extent is less satisfactory in patients. It is particularly unsatisfactory for evaluating vascular dynamics since samples may not be representative of the whole tumor and the same tissue cannot be examined twice precluding dynamic studies. Thus, there is a need for non-invasive approaches to reliably, reproducibly, and simply examine changes in tumor vasculature at depth. Today, this is most commonly achieved using dynamic contrast enhanced (DCE) proton MRI (magnetic resonance imaging).107 MRI is widely available, measurements are non-invasive and semi-quantitative measurement is facile based simply on changes in tissue water signal in response to distribution of paramagnetic contrast agent. Indeed, DCE MRI is now included in the clinical development of many VDAs.108-111 Dynamic computer tomography (CT) has also been used,112 but it is often considered to be less attractive due to the radiation dose of repeated CT scans and potential for anaphylactic response to iodinated contrast agents or other adverse side effects.112,113 Paramagnetic gadolinium based contrast agents are recognized to be much safer. Recently, a few instances of NSF (nephrogenic systemic fibrosis) have been reported, but these appear to be associated with poor renal function.114 DCE does require a bolus IV (intravenous) infusion of contrast agent and there is extensive discussion of the optimal procedures regarding data acquisition (temporal and spatial resolution) and interpretation (analytical approaches).115-120 Quantitative analysis is generally more relevant to subtle investigations of angiogenesis and vascular leakiness, while the beauty of many VDAs is the massive acute vascular response, which is readily detected even with simple semi-quantitative analyses.
Pre-clinical studies allow much greater versatility in imaging methodology and we show techniques in our laboratory both emulating the successes of other investigators and introducing novel paradigms.
DCE MRI has been most widely applied in the development of the VDAs. Measurements are non-invasive though they do require the IV infusion of a contrast agent. Essentially all imaging approaches require that animals be anaesthetized, but modern fluorinated gaseous anesthetics, such as isoflurane and sevoflurane, appear to be much less vasoactive, toxic and perturbing than earlier agents such as halothane or pentobarbital or ketamine.121 MRI can provide high temporal resolution and may generate 3D data sets for whole tumor coverage. More typically, a single slice through the center of a tumor is examined, since this reveals heterogeneity (e.g., differential response of tumor center and periphery) with high temporal resolution. Assessment of vascular dynamics requires administration of sequential doses of contrast agent and measurements could be perturbed by wash-out of residual material from prior measurements. This can be overcome by increasing the dose of successive injections or simply allowing a sufficient interval for wash-out (generally, greater than 30 mins.) and most reports have used intervals of two hours or more between examinations. Since effective VDAs generally cause massive acute effects, experimental protocols and interpretation are quite facile. Even if an animal is removed from the magnet, precluding precise correlation of individual voxels, large regions tend to behave similarly and data are readily compared based on histograms or spatial consideration of regions of interest. Animals may be allowed to wake up between scans, but it may generally be assumed that subtle physiological changes attributable to tumor development are minimal over a few hours. Thus, observed changes due to VDAs are readily identified. This is very different from antiangiogenesis agents, which normally act over days, and thus any changes in vasculature must be separated between “normal” tumor progression and response to drug.122
There are extensive reports of DCE MRI applied to many VDAs including combretastatin A-4P (Zybrestat™)96,97,106,123-126 and combretastatin A-1P (Oxi4503),127 5,6-dimethylxanthenone 4-acetic acid (DMXAA, also called ASA404 (vadimezan),128,129 ZD6126 (N-acetylcoichinol-O-phosphate),130-132 ABT-751,133 protamine134 and CYT997.135 In many cases simple DCE used small paramagnetic contrast agents, but in other cases larger materials designed to be retained in the vasculature such as macromolecular contrast agent albumin-gadolinium diethylenetriaminepentaacetate (albumin-GdDTPA)136 or SPIOS were used. Diverse tumors have been examined for research in animals (mice and rats) and as part of clinical trials in patients.135 Several investigators have taken the opportunity to use MRI to compare the efficacy of different VDAs.124,137,138 Such measurements have accelerated development of agents providing insight into efficiency, dosing, timing and heterogeneity of activity. An example from our laboratory is in Figure 5 for a 13762NF rat breast tumor with respect to a single dose of combretastatin A-4P administered intraperitoneally (IP). At 2 h, vascular perfusion was severely reduced and delayed, but substantial recovery was observed at 24 h, notably in the tumor periphery. We have presented more extensive data in this tumor system and in a mouse tumor previously.96,97 Analyses of DCE MRI with respect to VDAs have used various levels of complexity ranging from changes in relative si gnal intensity following infusion of contrast agent (semi-quantitative) to rigorous calculation of perfusion fraction, vascular leakage and transit times. Ultimately, parameters are required to reflect efficiency and data reduction may provide averaged values such as mean and median or perfused fractions.
Fig. 5
Fig. 5
Evaluating VDAs non-invasively by MRI
Alternate vascular dependent contrast mechanisms might be exploited including vascular spin labeling, though often tumors have such small blood vessels with sluggish flow that measurements are impractical. Oxygen may be considered as a contrast agent with changes in BOLD (Blood Oxygen Level Dependent) or TOLD (Tissue Oxygen Level Dependent) contrast response accompanying oxygen breathing challenge before and after the VDA administration. Certainly, tumor vascular extent has been correlated with BOLD response139,140 and flow must be considered, as noted in the FLOOD concept.141 Dynamic response to a hyperoxic gas challenge may reveal vascular shut down, but direct response to drug alone may be confused by coincidental changes in local hematocrit (blood volume), fraction of deoxyhemoglobin, and flow and Thomas et al. reported a complex pattern in response to carbogen challenge following CA4P treatment in rat bladder tumors growing in nude mice.142 Indeed, Howe et al.,143 have observed apparently contradictory results whereby BOLD signal increased following death, attributable to vascular collapse and deoxyhemoglobin clearance rather than improved oxygenation.
In this regard, vascular volume and oxygenation may be monitored directly using near infrared spectroscopy (NIRS) noting the differential absorption coefficients of oxy- and deoxyhemoglobin.144-146 To date, NIRS has generally lacked spatial resolution, but multi-exponential behavior implies heterogeneity.
Increasingly, it is recognized that combined therapy approaches are needed to successfully treat patients, particularly in the case of VDAs, which often leave a surviving peripheral tumor rim causing rapid tumor reoccurrence. Vascular shut down has implications for concomitant chemotherapy based on effective drug delivery and retention. It is also crucial for combination with radiotherapy, where vascular occlusion is expected to cause regional hypoxia, and hence, radio resistance. Indeed, several studies have shown that the combination of irradiation and VDA is crucially dependent on timing.147-148 We recently examined tumor oxygen dynamics directly based on 19F MRI oximetry with respect to VDA. Using FREDOM (Fluorocarbon Relaxometry Using Echo Planar Imaging for Dynamic Oxygen Mapping)140 we found significant acute hypoxiation in the 13762NF rat breast tumor within 30 min of administering combretastatin A-4P.96 Heterogeneous regional re-oxygenation was observed 24 h later. An example of such a measurement is shown in Figure 6, although here the hypoxiation was a little slower. Crucially, sequential pO2 measurements are non-invasive and can be repeated every 6½ min. In comparison, DCE approaches require repeated administration of the contrast agent requiring a priori choice of measurement times. Such pO2 measurements may be accelerated further by using a Look-Locker approach (90 s) as presented recently by Gallez, et al. 149 or based on a partial saturation measurement (1 s in a perfused heart 150). Most significantly, such measurements allowed us to optimize timing of combined irradiation and combretastatin to enhance tumor growth delay.151
Fig. 6
Fig. 6
Assessment of hypoxia accompanying vascular disruption
Vascular imaging may also be achieved using ultrasound,152 notably, with the availability of the new small animal VisualSonics systems, which can provide microscopic resolution or exploit micro bubble contrast agents. Doppler approaches are attractive since they require no contrast agent, hence avoiding the associated costs and technical challenge of IV administration. However, sluggish perfusion of small vessels may handicap observations in some tumors. In Figure 7, we show vascular changes based on Power Doppler in a rat breast tumor, but the effect is quite subtle. In other tumors, we have seen much more extensive vasculature (manuscript in preparation). Vascular shutdown was readily apparent in this tumor based on infusion of contrast micro bubbles (Figure 7b and d). More extensive ultrasound studies have been reported by others, notably with respect to vascular disrupting agents or vascular flare following irradiation.153-159 As with MRI, such measurements may be applied clinically.
Fig. 7
Fig. 7
Assessment of acute changes in tumor vasculature using ultrasound
We recently introduced a novel approach exploiting dynamic bioluminescent imaging (dBLI) to investigate the acute effects of vascular disrupting agents.97 Various reports have considered the dynamics of light emission for luciferase expressing cells growing in tumors in animals following the administration of luciferin substrates.160-162 Most reports have focused on magnitude and duration of light emission together with reproducibility, e.g., intravenous administration gives most rapid and intense, yet highly transient, light emission kinetics, while intraperitoneal administration is technically easier and gives a longer signal plateau, so that the timing of imaging acquisition is less critical.163 However, we and others have noted a substantial failure rate with no (or minimal) light emission being observed. On the other hand, we find that subcutaneous (SC) administration of luciferin in the back/neck region provides highly reproducible light emission kinetics.164 Noting that light emission requires delivery of luciferin substrate to the tumors by the vasculature this provides an effective assay of vascular patency. We have shown that following administration of CA4P to nude mice with human breast tumor xenografts consistent results were achieved using dynamic BLI or dynamic contrast enhanced MRI.97 MRI does of course provide spatial information including potentially 3D representations. An example of dBLI is shown in Figure 8 for human prostate PC3-Luc tumors growing in two nude mice. Each mouse shows intense BLI signal prior to CA4P with diminished signal at 2 h and significant recovery at 24 h. Kinetic curves of light emission for one of the tumors are shown in the graph. We have now applied this procedure to several VDA drugs, disease sites and tumor types. The method is particularly simple to implement, cheap and offers high throughput. The primary drawback of this approach is the need for luciferase expressing cells.
Fig. 8
Fig. 8
Assessment of acute changes in tumor vasculature using dynamic bioluminescent imaging (dBLI)
In essence, any technology providing signal sensitive to vascular extent and flow may be applied to investigate VDA activity. In other cases, radionuclides have been used in conjunction with autoradiography, biodistribution, PET and SPECT.165-167
In summary, new, innovative biological assays, modifications of existing techniques, and tumor imaging strategies are combined to assess the preclinical effectiveness of promising tubulin-binding VDAs as single agents and in combination therapy. In regard to cell-based technologies, the application of three-dimensional cell culture using components of the extracellular matrix provides a more physiologically relevant model for following the disruption of tubular networks. In the realm of tumor imaging, there now exists a diverse portfolio of imaging methods to evaluate VDAs. The ultimate choice of a particular approach may depend on the imaging needs for spatial and temporal resolution, costs, study size and nature of investigation, e.g., high throughput preliminary survey studies versus detailed mechanisms and translatability to patients. dBLI is an economical and rapid technique to screen new VDAs. Collectively, these preclinical assessment tools should prove valuable in the identification of new drug candidates for human clinical trials.
Insight, innovation, integration
New small-molecule therapeutic intervention in the treatment of cancer relies heavily on insightful and innovative strategies that integrate molecular design and chemical synthesis with clearly defined biological evaluation techniques. The discovery and development of vascular disrupting agents (VDAs) provides such an example. Recent advances in preclinical tumor imaging such as bioluminescence imaging (BLI) coupled with DCE MRI (dynamic contrast enhanced magnetic resonance imaging) rapidly assess the selectivity of tumor blood-flow shutdown. Advanced biological assessment expands the standard inhibition of tubulin assembly and cytotoxicity assays to include endothelial tube disruption and reorganization, cell adherence, and endothelial cell permeability studies. In a highly integrative sense, these assays and tumor imaging strategies collectively provide information that is potentially predictive of clinical efficacy for VDAs.
Acknowledgments
Studies reported here were facilitated by grants from the NIH NCI (R01 CA139043-01A1 (to R.P.M.); 1R01 CA140674-01A1 (to K.G.P and M.L.T)), the Cancer Prevention and Research Institute of Texas (CPRIT (RP100406), to K.G.P. and M.L.T.), Oxigene Inc. (to K.G. P. and M.L.T), and Department of Defense Congressionally Directed Breast Cancer Research Program, Department of the Army (DAMD17-03-1-0363 and DAMD#17-00-1-0437 (to R.P.M)), together with Shared Instrumentation Grants from NIH (1S10RR024757-01 and 1S10RR025648-01) and the infrastructure provided by the Southwestern Small Animal Imaging Research Program (SW-SAIRP) supported in part by 1U24 CA126608 and Simmons Cancer Center (P30 CA142543-01) as well as AIRC (P41 RR02584). The authors are appreciative to Dr. James Karban and Dr. Michelle Nemec (Director) for use of the shared Molecular Biosciences Center at Baylor University. In addition, we are indebted to Dr. Peter Peschke of the German Cancer Center Heidelberg for advice on tumor pathophysiology and Dr. David J. Chaplin of Oxigene Inc. for helpful discussions and for providing CA1P and CA4P. Finally, the authors are grateful to Dr. Tracy Strecker and Ms. Amanda K. Charlton-Sevcik for assistance with biochemical and biological assay descriptions, and Ms. Cristin McAnear and Ms. Tracy A. Pinney for expert assistance with the final manuscript drafts. As a potential conflict of interest statement, two of the authors (KGP and MLT) have current funding support from Oxigene Inc., and one author (KGP) wishes to disclose a consulting relationship with Oxigene Inc.
Footnotes
#Dedicated to Professor Mina J. Bissell of Lawrence Berkeley National Laboratory for her lifetime contributions to the overall field of cancer research and specifically the extracellular matrix and the tumor microenvironment.
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