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Advances in Brief |
Sunnybrook and Women’s College Health Sciences Centre, Department of Medical Biophysics, University of Toronto, Toronto, Ontario, M4N 3M5 Canada
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ABSTRACT |
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Introduction |
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The therapeutic agent used for our studies was ZD6126 (AstraZeneca, United Kingdom), a newly reported member5 of the tubulin-binding class of antivascular drugs (8) . These drugs destabilize microtubules in the cytoskeleton of endothelial cells, thereby inducing neovascular endothelial cell rounding that can subsequently lead to vascular shutdown and necrosis in tumors. ZD6126 has been shown in preclinical studies to rapidly disrupt tumor blood vessels and subsequently produce growth delays in a range of tumor types (7) and is, therefore, a suitable model to induce blood flow changes in the present study. In addition to ultrasound monitoring, independent validation of the effects of this therapy on tumor blood flow was conducted using Hoechst perfusion staining (9) .
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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In Vivo Studies.
The growth of tumors was monitored until an estimated volume of either 75 mm3 ("early stage") or 150 mm3 ("later stage") was reached. Volumes were determined using caliper measurements and the standard formula (
·length·width2/6). The mice then received a single tail vein injection of either ZD6126 (treated) or physiological saline (control). The treated mice received a therapeutic dose of 20 mg/kg ZD6126, a level well below the maximum tolerated dose.5
Ultrasound studies consisted of making serial measurements on the same tumor (either treated or control) at baseline, at 4 and 24 h postinjection. Perfusion staining studies were conducted on separate groups of mice at baseline, 4 and 24 h posttreatment. The acute nature of the perfusion staining experiments required that different groups of mice be used for each time point and that different mice be used than those for the serial Doppler measurements. After conducting either ultrasound or perfusion-staining experiments, mice were killed according to the Canadian Council on Animal Care guidelines.
Ultrasound Flow Imaging.
Ultrasound flow imaging provides information about the spatial distribution of blood velocities and moving blood volume. The range of blood velocities that can be assessed is limited at the low end by the clutter filter that is used to remove the signal from tissue and at the high end by the ultrasound pulsing frequency (13)
. Because of attenuation effects, which result in a reduced penetration depth at higher frequencies (14)
, it was necessary to assess the larger tumors using a lower frequency than was used for the smaller tumors. Ultrasound flow imaging of the earlier-stage smaller tumors was conducted at a center frequency of 38 MHz with system settings held constant (800-Hz pulse repetition frequency and 35-Hz wall filters) to provide sensitivity to velocities above 0.7 mm/s. The lateral and depth direction resolutions were 93 and 82 µm, respectively. Ultrasound flow imaging of later-stage tumors was conducted at a center frequency of 25 MHz with system settings held constant (800-Hz pulse repetition frequency and 25-Hz wall filters) to provide sensitivity to velocities above 0.75 mm/s. The lateral and depth direction resolutions were 113 and 122 µm, respectively.
At each time point, volumetric imaging was performed by acquiring a series of image planes, spaced at 50 or 80 µm for the 38- and 25-MHz experiments, respectively, covering the entire tumor region (Fig. 1A)
. Scan times were on the order of 30 min. Future versions of the instrumentation will be capable of reducing this time to 
10–15 min. As noted earlier, the HFD system is capable of operating in high-frequency pulsed-wave of flow-imaging modes. Before the imaging scan, a survey of flow within the tumor was performed using either 25- or 38-MHz pulsed-wave Doppler. This technique provides an audible signal reflecting the temporal variation of flow in a single sample volume and is useful for providing a qualitative real-time indication of flow levels within the tumor.
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Ultrasound Image Processing.
Region-of-interest selection was performed using custom written Matlab (Mathworks Inc., Natick, MA) software to include the entire tumor volume. Ultrasound flow imaging data consists of spatial maps of either blood velocity or Doppler power (P). In this study we consider only the Doppler power data. Using these data, a measure of the total IP (IP) flow was calculated within a tumor volume at each time point as follows:
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The test/retest variability for the measurement of IP was assessed by analyzing the two same-day (i.e., baseline and 4-h time point) IP data points for the control mice (n = 6).
Drug Preparation.
ZD6126 was dissolved at a concentration of 10% (w/v) in a solution of PBS and 0.5% sodium carbonate.
Hoechst Perfusion Staining.
Perfused vasculature in the MeWo tumors was identified by i.v. injection of 200 µl (10 mg/kg) of Hoechst 33342 dye (Sigma, St. Louis, MO) into the tail vein (11)
. Twenty min after injection with Hoechst, mice were euthanized by cervical dislocation. Tumors were quickly removed and embedded in Tissue-Tek OCT Compound (Miles Inc., Elkhart, IN), followed by rapid freezing over dry ice for subsequent sectioning. For each tumor sample, 6-µm cryosections were cut at four different levels and viewed under UV epifluorescence using an Axioskop 2 transmitted-light microscope (Zeiss, Munich, Germany). Using this method, perfused blood vessels could be visualized by the surrounding halo of fluorescent, Hoechst 33342-labeled cells. Digital images were captured using the Axiovision 3.0 image analysis system at x10. Prior to analysis, a threshold for the vascular structures was determined for the stain used, based on the background level of brightness. Image analysis was performed with Northern Eclipse Version 6.0 software to measure the percentage image area occupied by perfused vasculature. Although this analysis does not directly quantify the number of perfused vessels, it provides an estimate of the relative degree of perfused tumor vasculature. For each tumor, an average percentage image area was calculated, a minimum of four random fields were analyzed per section, and a minimum of four sections per tumor were examined. A total of four mice were imaged at each time point for the later-stage tumors.
Statistics.
Statistical analysis of data were conducted using NCSS software for Windows (Kaysville, UT). Mann-Whitney tests were used and Ps of <0.05 were considered to be significant.
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Results |
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TopABSTRACTIntroductionMaterials and MethodsResultsDiscussionREFERENCES |
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, at baseline, as well as at 4 and 24 h postinjection. A large reduction in perfused vessels was observed 4 h after the ZD6126 injection, relative to the baseline and 24-h points. A similar pattern was observed for the 38-MHz data on early-stage tumors. This was consistent with the qualitative high-frequency pulsed-wave Doppler assessment, with which only minimal flow could be detected at the 4-h point in treated tumors. The mean IP for the control and treated tumors at different times after the ZD6126 injection are shown in Fig. 2, A and B
, for the early- and later-stage tumors, respectively. In both cases, significant drops in the treated mean IP (P < 0.05) occurred at the 4-h point and was followed by a recovery at 24 h postinjection. Furthermore, the IP for the treated tumors was significantly lower than the control data at the 4-h point (P < 0.05). No significant difference was observed between treated and control groups at 24 h.
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Hoechst Perfusion Staining Results Correspond to Ultrasound Results.
Perfused vessels were visualized in tumor cryosections by the surrounding Hoechst-labeled cells (9)
. Representative examples of baseline and 4-h and 24-h posttreatment perfusion staining are shown in Fig. 3 A–C
. A large drop in the perfused area at the 4-h point was evident, whereas perfusion at 24 h resembled that of the control tumors. The quantitative analysis of the results is shown in Fig. 4
. The data indicate a significant drop in the perfused area at 4 h (P < 0.05) and no significant difference at the 24-h point. These results indicate that at the dose and treatment schedule that was used (i.e., a single injection), ZD6126 induces a short-term reduction in tumor perfusion in the MeWo tumor xenografts.
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Discussion |
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As noted earlier, conventional-frequency Doppler ultrasound is also under investigation as a method for monitoring and for antivascular and antiangiogenic therapy in superficial tumors and has shown encouraging results (3)
. Two factors are improved by the use of high-frequency ultrasound over lower frequencies: spatial resolution and sensitivity to slow flow in small vessels (6)
. Spatial resolution scales linearly with frequency and goes from about 0.5 mm at 5 MHz to 50 µm at 50 MHz (14)
. The larger sample volume of conventional-frequency ultrasound will tend to encompass a number of microvessels, which may have a range of velocities and orientations. A velocity estimate in this situation will, therefore, represent an average velocity for the ensemble of vessels present. Improved spatial resolution permits an assessment of the spatial distribution and blood velocity of smaller vessels and may be useful in assessing differences in spatial patterns of flow that result from treatment. As with conventional frequency ultrasound, the potential exists for such measures to be complicated by heterogeneous attenuation patterns. The heightened sensitivity to small vessels at high frequencies is itself the result of two primary factors. First, the echo strength from blood increases with frequency (14)
, eventually becoming comparable with that from many tissue types. Second, Doppler shifts increase linearly with frequency, making it easier to separate blood and tissue signals (6)
. The precise detection limit depends on a number of parameters (e.g., tissue motion and tissue ultrasonic properties), but at conventional frequencies it is likely limited to vessels with diameters above 100 µm and velocities above 1 cm/s (7)
. At 50 MHz, vessels as small as 15–20 µm in diameter with velocities on the order of mm/s have been detected and imaged (6)
. The improved sensitivity to slow flow in smaller vessels may be significant in applying HFD to monitoring antivascular or antiangiogenic therapy. In particular, sensitivity to smaller vessels may be advantages in providing insight into flow conditions at a level of circulation that is closer to that associated with nutrient and waste exchange and drug delivery.
The IP measurements made in this study are related to the total volume of blood flowing within the tumor with a velocity above the clutter filter value (16) . A comparison of these data for the treated and control groups, therefore, indicate that the volume of blood flowing within the tumor was significantly reduced at the 4-h point (P < 0.05) and subsequently recovered by the 24-h point. The IP results followed a similar pattern to that of the perfusion staining data, which demonstrated an initial decrease in the density of perfused microvessels followed by a recovery. A rapid and extensive reduction in perfused vessels is characteristic of what has been observed previously with ZD61263 as well as other tubulin-binding agents (17 , 18) . The possibility of a recovery in flow after an initial drop has also been reported with another tubulin-binding agent (17, 18, 19) . The pattern of a short-term reduction in perfusion followed by recovery highlights the need to directly monitor tumor blood flow to provide an indication of drug efficacy as opposed to relying on indirect measures such as tumor volume to determine the presence of an effect.
At present, there has been little quantitative work done to examine directly the effects of vascular targeting therapy on velocity and flow in tumor microvessels. Tozer et al. (19) used intravital microscopy to investigate the effects of the tubulin-binding agent combretastatin A-4 on blood velocities and flow but were restricted by the limitations of intravital microscopy to observing small microvessels. It was found that there was a dose-dependent stoppage of flow in venules, particularly in those with diameters less than 10 µm. A significant reduction in the diameter and flow in arterioles was also observed. Although in the present study, there has been no independent confirmation of the size of vessels detected, the results clearly indicate that HFD measures of IP are sufficiently sensitive to detect and monitor a level of circulation that is affected by the vascular targeting therapy.
A primary limitation of HFD is that signal attenuation increases with frequency and will limit its penetration depth to be within 5–10 mm of the tissue surface. Its ultimate depth of penetration and sensitivity to small vessel flow will be determined in large part by the specific tissue properties and tissue motion present in the region of interest. It is expected, however, that oncology application areas will likely be restricted to assessing the vascular status of superficial experimental tumors and, in clinical settings, to ocular and cutaneous tumors. Its sensitivity and utility in these circumstances remains to be investigated. Ideally it would also be desirable to avoid the use of anesthetic, particularly in clinical settings. The potential to do this will depend largely on the ability to limit tissue motion in these anatomical locations, as well as on image acquisition time. Although, at present, the acquisition is relatively slow, technical advances are being made that promise to yield frame rates similar to those at conventional frequencies (Foster et al. 2000) and may eventually make it possible to perform these measures in the absence of anesthetic. It is also important to note that, as with conventional-frequency Doppler power, HFD power cannot in general be interpreted in terms of the absolute moving blood volume. Furthermore, the correlation between HFD data and classical tissue perfusion has yet to be investigated.
This purpose of this study was to provide a demonstration of principle for HFD monitoring of antivascular effect on tumor blood flow. To evaluate the feasibility of HFD flow imaging as a robust technique in experimental and clinical settings, a number of other studies will also be necessary. An important step will be to conduct comparative studies with other established flow imaging techniques. Ideally this work should be performed in normal and tumor tissue as well as in tumors that are subjected to therapy. For such comparative imaging studies, it would be useful to examine agents or dose levels that produce a graduated response in the Doppler signal, in addition to those producing dramatic reductions such as those observed in this study. In the context of such studies it would be useful to examine the variation of absolute as well as relative Doppler parameters and to test for the potential influence of nontumor effects (e.g., cardiac rate) on these data. It will also be of interest, particularly in longer-term therapeutic studies, to investigate the relationship between Doppler parameters and the development of therapeutically induced necrosis. Experiments conducted over a longer time scale will likely involve tumor volume changes, and in these circumstances it may be beneficial to normalize Doppler power measures with respect to the tumor volume. In these cases the potential influence of attenuation variations should be investigated.
A number of other techniques, such as magnetic resonance imaging, are also under evaluation for noninvasively assessing the therapeutic modulation of blood flow in tumors (2) . It is likely that no single imaging technique will satisfy the requirements for experimental or clinical therapeutic blood flow monitoring. The combination of resolution, penetration depth, and cost, along with the sensitivity demonstrated in this study, suggest that high-frequency ultrasound flow imaging has the potential to be a viable tool for noninvasively imaging blood flow in superficial tumors, particularly in preclinical experimental therapeutic studies. In this regard, it would, therefore, be of considerable interest to study agents such as conventional chemotherapeutic drugs and antioncogenic signal transduction inhibitors for their potential ability to cause antivascular and/or antiangiogenic effects (20 , 21) using HFD.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the National Cancer Institute of Canada and the Canadian Institutes of Health Research. 
2 To whom requests for reprints should be addressed, at Room 658, Research Building, Sunnybrook and Women’s College Health Science Centre, 2075 Bayview Avenue, Toronto, Ontario, Canada, M4N 3M5. Phone: (416) 480-5716; E-mail: Stuart.Foster{at}swchsc.on.ca
3 D. E. Goertz, D. A. Christopher, J. L. Yu, R. S. Kerbel, P. N. Burns, and F. S. Foster. High frequency 3D color flow imaging of the microcirculation, submitted for publication.
4 The abbreviations used are: HFD, high-frequency Doppler (ultrasound); IP, integrated (Doppler) power.
5 D. C. Blakey, F. R. Westwood, M. Walker, M. G. Hughes, P. D. Davis, S. E. Ashton, and A. J. Ryan. Anti-tumor activity of the novel vascular targeting agent ZD6126 in a panel of tumor models, submitted for publication.
Received 6/18/02. Accepted 10/ 4/02.
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