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Cancer Research 68, 7042, September 1, 2008. doi: 10.1158/0008-5472.CAN-08-0285
© 2008 American Association for Cancer Research
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Experimental Therapeutics, Molecular Targets, and Chemical Biology

Vessel Fractions in Tumor Xenografts Depicted by Flow- or Contrast-Sensitive Three-Dimensional High-Frequency Doppler Ultrasound Respond Differently to Antiangiogenic Treatment

Moritz Palmowski1,2, Jochen Huppert1, Peter Hauff3, Michael Reinhardt3, Karin Schreiner1, Michaela A. Socher1, Peter Hallscheidt4, Guenter W. Kauffmann4, Wolfhard Semmler1 and Fabian Kiessling1,2

1 Medical Physics in Radiology, German Cancer Research Center; 2 Division of Experimental Molecular Imaging, RWTH-Aachen University, Aachen, Germany; 3 Global Drug Discovery, Bayer Schering Pharma AG, Berlin, Germany; and 4 Department of Diagnostic Radiology, University of Heidelberg, Heidelberg, Germany

Requests for reprints: Fabian Kiessling, Division of Experimental Molecular Imaging, University of Aachen, Pauwelsstraβe 20, D-52074 Aachen, Germany. Phone: 49-241-8036124; Fax: 49-241-8082442; E-mail: fkiessling{at}ukaachen.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
High-frequency volumetric Power Doppler ultrasound (HF-VPDU) captures flow-dependent signals in blood vessels and can be used to assess antiangiogenic therapy effects in rodent tumors. However, the sensitivity is limited to vessels larger than capillaries. Contrast-enhanced HF-VPDU reveals all perfused vessels by assessing stimulated acoustic emissions from disintegrating microbubbles. Thus, we investigated whether flow-sensitive and contrast-enhanced HF-VPDU can depict different vessel fractions and assess their early response to antiangiogenic therapy. Mice with A431 tumors were scanned before and after administration of polybutylcyanoacrylate microbubbles by HF-VPDU. Animals received either antiangiogenic treatment (SU11248) or a control substance and were imaged repeatedly over 9 days. At each time point, tumors were removed for immunohistochemical analysis. During growth of untreated tumors, vascularization decreased correspondingly on flow-sensitive and contrast-enhanced scans. Treated tumors showed a significantly (P < 0.05) stronger decline in vascularization than controls, which was more pronounced in contrast-enhanced scans. Surprisingly, whereas vascularization remained low in contrast-enhanced scans, flow-sensitive ultrasound indicated a reincrease after day 6 with a higher vascularization than the controls at day 9. Histologic evaluation indicated that immature vessels degraded markedly on therapy, whereas large mature vessels on the tumor periphery were more therapy resistant and drew closer due to tumor shrinkage. In conclusion, contrast-enhanced HF-VPDU and flow-sensitive HF-VPDU are both capable of assessing the effects of antiangiogenic therapy. Because contrast-sensitive ultrasound is more sensitive for small immature vessels and flow-sensitive ultrasound mostly captures large vessels at the tumor periphery, the combination of both methods can provide evidence of vascular maturity in tumors. [Cancer Res 2008;68(17):7042–9]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Angiogenesis is the basic prerequisite for the invasive growth and systemic spread of tumors, which makes it an important target in controlling cancer progression (1). A crucial issue for emerging approaches of antiangiogenic therapy is the ability to directly quantify their effects longitudinally in the tumor neovasculature (2). Up to now, most large animal studies related to the development of new angiogenesis inhibitors are based on caliper measurements of the tumor size or on histologic examinations (3). Functional data obtained in vivo may be superior to histologic analyses for recording therapy effects accurately because the latter do not distinguish between functional and nonfunctional blood vessels. In this respect, surrogate markers of tumor vascularization can be determined noninvasively by magnetic resonance imaging (MRI; refs. 4–6), computed tomography (7–9), positron emission tomography, and single-photon emission computed tomography (10, 11). In addition, ultrasound has been used successfully as a cheap, fast, and easily applicable method for serial imaging of animals with high throughput (12, 13).

At clinical frequencies of 3 to 15 MHz, Power Doppler sonography can detect regression of larger tumor vessels during antivascular therapy (14). However, capillary blood flow in small nonstabilized vessels, which mostly respond to antiangiogenic treatments, cannot be assessed via conventional Doppler sonography. Even the administration of ultrasound contrast agents does not lead to reliable assessment of the relative blood volume (15–17). This constraint is very probably related to the fact that Doppler signals derived from flow (or from disintegrating microbubbles) on ultrasound images are only displayed several millimeters in size due to the limited spatial resolution of clinical ultrasound devices. This limitation holds particularly true for imaging of small animals, in which tumors are only few millimeters in diameter. As a consequence, the situation arises in which Doppler signals rapidly produce a saturation of voxels within a region of interest (18), leading to overestimation of the relative blood volume. To a certain degree, this is also an obstacle for dynamic contrast-enhanced ultrasound techniques such as harmonic or pulse inversion techniques. Another limitation is that, in most previous studies, only one or few tumor slices were analyzed. Due to the strong heterogeneity of tumor tissue, the chosen slice may not have been representative for the entire tumor (19).

Novel high-frequency Doppler ultrasound that operates beyond 20 MHz provides higher spatial resolution and showed convincing results in three-dimensional assessment of the relative blood volume in animal tumor models studied without administering contrast medium (20, 21). Although high-frequency ultrasound scanners provide a resolution of 50 to 150 µm, it is unlikely that slow flow in capillary vessels (diameter: <10 µm; flow: ~0.3 mm/s) can be detected. The use of ultrasound contrast agents in high-frequency ultrasound devices and their destruction by ultrasound pulses with a high-mechanical index may permit detection of capillaries independent of flow velocities, which would make this superior for monitoring the early effects of antiangiogenic therapy.

In this respect, we conducted a study to evaluate high-frequency volumetric Power Doppler ultrasound (HF-VPDU) for assessing early effects of the multitargeted receptor tyrosine kinase inhibitor SU11248 (3) on the vascularization of human epidermoid carcinoma xenografts (A431). We also investigated whether contrast-enhanced scans (ceHF-VPDU) could further increase the sensitivity of the method. We found that ceHF-VPDU was much more sensitive for small nonstabilized vessels, whereas HF-VPDU was more sensitive for large mature vessels. Thus, ceHF-VPDU and HF-VPDU provide different and supplementary diagnostic information. Due to the fact that nonstabilized vessels responded rapidly to the treatment, functional effects on tumor vascularization were already observed few hours after start of treatment by ceHF-VPDU, whereas it took several days until the therapy effects became significant in HF-VPDU. Nevertheless, both methods depicted response to the treatment before changes in tumor size became apparent.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Synthesis of Polymer-Stabilized Microbubbles
In this study, polymer-stabilized microbubbles with a hard polymeric shell were used as ultrasound contrast agents (22), which can be destroyed under high-powered sonication. Thereby, the disintegrating microbubbles emit a final signal that can be detected by the ultrasound device. Contrast agents were synthesized as follows: Monomeric butyl-2-cyanoacrylate (Sichel-Werke) was added to an aqueous acidic solution (pH 2.0) containing 0.02% Triton X-100 (Sigma-Aldrich). Vigorous stirring for 1 h resulted in a suspension, which was then subjected to a flotation process to separate air-filled from non–air-filled microbubbles. Purified by repeated flotation, air-filled microbubbles were partially hydrolyzed by exposure to sodium hydroxide solution. With this, ~10% of the polymer mass was hydrolyzed and the stability of the microbubbles reduced (22), enabling them to be destroyed by sonication above a mechanical index of 0.25 (23). Size distribution of the microbubbles was measured with a particle counter (Multisizer-3, Beckman Coulter). Their mean volume-weighted size was 2.61 ± 0.81 µm (mean ± SD). For in vivo use, the acidic MB suspension was diluted in PBS solution (final pH: 7.2; concentration: 5 x 109/mL microbubbles).

In vivo Experiments
Experiments were approved by the governmental review committee on animal care (Regierungspraesidium). Human epidermoid carcinoma xenografts were induced by s.c. injection of 6 x 106 A431 cells (kindly provided by Peter Huber, German Cancer Research Center, Heidelberg, Germany) in the right hind leg of female nude mice (purchased from Charles River WIGA GmbH). After 14 d of tumor growth, animals were divided randomly into different groups. Animals of the therapy groups received an antiangiogenic treatment with SU11248 (Pfizer, Inc.), a selective multitargeted receptor tyrosine kinase inhibitor that exhibits antiangiogenic activity through its potent inhibition of vascular endothelial growth factor receptor and platelet-derived growth factor receptor signaling (3). A daily dose of 80 mg/kg body weight of SU11248 (dissolved in 60 µL DMSO with a purity of 99.8% and then diluted by addition of 30 µL PBS buffer) was administered i.p. This dosage was previously reported to induce rapid tumor regression in A431 tumors (3). Control animals received DMSO (diluted by addition of PBS buffer) alone.

An additional group of animals (n = 5) was used to define the optimal scan protocol and received no medication.

Scan Protocol for Contrast-Enhanced Volumetric Imaging
Generating a volumetric data set requires assessment of multiple two-dimensional ultrasound images that can be reconstructed to the desired three-dimensional volume. A crucial precondition for performing a volumetric contrast-enhanced scan is a stable microbubble concentration in the blood during the entire scanning procedure. The required scan time thereby depends on the number of acquired slices, the frame rate, and the size of the Doppler field.

In our study, the initial tumor size was about 3 to 4 mm diameter, requiring assessment of 30 to 40 images (slice thickness: 100 µm). Ultrasound measurements were performed using the VEVO 770 Micro-Ultrasound System (VisualSonics) equipped with the RMV-704 sector ultrasound probe containing a single mechanical transducer (B-mode frequency, 40 MHz; Doppler frequency, 30 MHz). This meant that the Doppler frame rate was comparably low (frame rate, ~0.5 Hz), resulting in a prolonged scanning time. It was thus necessary to ensure a stable concentration level of the experimental contrast agent throughout the entire scan. For this purpose, contrast agent concentration and the corresponding Doppler signals within tumors were investigated over time. Examinations were performed as follows: Nude mice fixed on an examination table were anesthetized by inhalation using a mixture of isoflurane (1.5%) and O2 (98.5%). A tail vein catheter for i.v. injection of contrast medium was placed. Tumors were covered with ultrasound gel and the ultrasound transducer was fixed on a motor-driven unit above the animal. For dynamic contrast-enhanced imaging, the scan head remained in a stable position. For volumetric imaging, the ultrasound probe moved perpendicular to the beam axis, thereby acquiring consecutive images with a slice thickness of 100 µm. Subsequently, two-dimensional ultrasound images were merged to a three-dimensional data set using the implemented software of the VEVO 770 System. The following settings were applied (on n = 5 animals without any treatment):

(a) Dynamic contrast-enhanced imaging in B-mode: To investigate the principal concentration of the experimental contrast agent within a representative two-dimensional tumor slice, cine loops with a low-mechanical index (power setting, 20%) were acquired in B-mode (4 frames/s) for 180 s after bolus injection (20 µL/s) of 100 µL microbubbles. A region of interest was drawn around the tumor and time-intensity curves were created.
(b) Dynamic contrast-enhanced imaging in Power Doppler mode: To define the amount of Doppler signals after bolus injection of microbubbles, cine loops with a high-mechanical index (power setting, 100%) were acquired in Doppler mode for 180 s (1 frame/s; scan speed, 5 mm/s; wall filter, 6 mm/s). The color pixel density within the tumor was calculated, and time/color pixel density curves were created.
(c) Volumetric Power Doppler: Both previous examinations indicated a time frame with a stable amount of microbubble-derived Doppler signals within tumors. To confirm these findings, volumetric imaging of tumors was performed within and after this time interval. The following scans were used: non–contrast-enhanced volumetric Power Doppler scan set to detect slow blood flow (scan speed, 2 mm/s; wall filter, 2.5 mm/s; frame rate, ~0.5 Hz), non–contrast-enhanced volumetric Power Doppler scan with a modified setting (scan speed, 5 mm/s; wall filter, 6 mm/s) to increase the frame rate to ~1 Hz and enable completion of the scan while the blood concentration of microbubbles was stable, and 4-fold repetition of the latter scan after bolus injection of microbubbles (100 µL).

The values of the first unenhanced scan were set to 100%, and the values derived from the consecutive scans normalized correspondingly.

Monitoring Effects of Antiangiogenic Therapy
Treatment group. A group of n = 4 animals was used to investigate the early effects of SU11248. Ultrasound examinations were performed before, 8 h, and 24 h after treatment. Subsequently, animals were sacrificed and tumors were removed for histologic analysis (24-h values). Additional animals (which did not undergo imaging) were sacrificed before and 8 h after treatment (baseline and 8-h values).

A group of n = 7 animals was used to investigate the subsequent effects of SU11248. Animals were examined on days 0, 3, 6, and 9 of therapy. For histologic analysis, one animal from each group was killed immediately after each time point. Thus, n = 7 animals were examined on day 0, n = 6 animals on day 3, n = 5 animals on day 6, and n = 4 animals on day 9.

Control group. A group of n = 4 animals and a group of n = 7 animals were used to investigate early and subsequent time points, respectively. Animals received control substance without SU11248. The same protocol as described above was applied.

Imaging protocol. At each time point, a volumetric non–contrast-enhanced scan was applied to detect slow blood flow (scan speed, 2 mm/s; wall filter, 2.5 mm/s). After bolus injection of 100 µL microbubbles, it was followed by a scan with a modified setting to detect microbubbles within the vessels (scan speed, 5 mm/s; wall filter, 6 mm/s). During volumetric imaging, slices of 100 µm thickness were acquired and merged to a three-dimensional data set. Regions of interest were drawn manually on every two-dimensional image along the tumor margins. Tumor volumes and vascularization (determined as color pixel density) were calculated. To balance interindividual variability, values measured at day 0 were set to 100%. Consequently, intraindividual changes 8 and 24 h after start of therapy (for early effects) and intraindividual changes at days 3, 6, and 9 (for subsequent effects) are expressed as percent change. Due to the intraindividual normalization to the initial value, curve progressions of both groups are plotted in a joint graph. To investigate the influence of the heart rate on Doppler signals, electrocardiogram (ECG) measurements were performed in both groups of animals.

Immunohistochemistry
Tumors were resected, frozen in liquid nitrogen vapor, cut in slices of 10 µm thickness, and fixed with methanol/acetone. Immunostaining of endothelial cells was performed using a rat anti-mouse CD31 antibody (BD Biosciences) in combination with a Cy3-conjugated donkey anti-rat antibody (Jackson ImmunoResearch). Smooth muscle actin was stained with a rabbit anti-mouse antibody (Abcam) in combination with Cy2-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch). Cell nuclei were counterstained by 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Slices were investigated using a fluorescence microscope (DMRE), and digital images were captured by a technician who was blinded to the experimental group. For quantitative analysis of fluorescence signals, area fractions with positive fluorescence were calculated. Twenty representative area fractions were analyzed from each tumor in the control and therapy groups. Mean vessel diameter was determined by measuring the diameters of orthogonally cut vessels on CD31-stained histologic sections. Vessels, which seemed to be tangentially cut, were excluded from counting.

Statistical Evaluation
Data are presented as mean ± SD. Differences in heart rate, tumor size, vascularization, and immunohistologic area fractions between treated and untreated tumors were compared using the Mann-Whitney test (unpaired, two tailed). P values of <0.05 and <0.01 were considered to show significant and highly significant differences, respectively. Statistical analysis was done with GraphPad Prism (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Contrast-Enhanced Three-Dimensional Scan Protocol
By increasing the "scan speed" (motion speed of the mechanical transducer) from 2 to 5 mm/s and by adapting the "wall filter" (used to reduce artifacts induced by the motion of the transducer) from 2.5 to 6.0 mm/s, the frame rate could be doubled. It was still possible to detect the stimulated acoustic emission (SAE) signals while blood flow detection was reduced (due to the decreased sensitivity for slow blood flow; Fig. 1 ).


Figure 1
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  		Figure 1. Single-slice images (A and B) with a thickness of 100 µm and the most intensity projections (C and D, each 10 images) of contrast-enhanced and non–contrast-enhanced Power Doppler measurements. B, in the non–contrast-enhanced examination, blood flow is detectable within large tumor vessels with high flow velocities. After administration of contrast medium, disintegrating microbubbles within small vessels become visible, independent of flow velocities, enabling visualization of the capillary bed. Bar, 1 mm.

 
Time-intensity curves of tumors recorded at a fixed position by low-mechanical index imaging showed an almost stable plateau of microbubbles between 35 and 110 s after injection (Fig. 2A ). When SAE signals were measured using the destructive mode, too, the color pixel density of tumors was almost stable over this period (Fig. 2B).


Figure 2
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  		Figure 2. Results from ultrasound scans performed to define an optimal time window for ceHF-VPDU. A, dynamic contrast-enhanced imaging in B-mode indicates a stable plateau 35 to 110 s after injection of microbubbles. B, dynamic contrast-enhanced Power Doppler imaging (with a high-mechanical index) correspondingly shows a stable color pixel density during this period. Black bar, time frame (~50 s) in which the three-dimensional measurements were performed in the diagnostic trial. C, volumetric Power Doppler measurements confirmed the diagnostic window. The first scan (scan time, ~120 s) was performed with a setting to detect slow blood flow. By increasing scan speed and wall filter, the scan time decreased to ~50 s but became more insensitive to slow blood flow. After injection of microbubbles, significantly higher color pixel density was observed. Color pixel density values of entire tumor volumes were only slightly reduced in the second postcontrast volumetric scan compared with the first ceHF-VPDU scan. By contrast, lower values were observed in the subsequent third scan, and these became significant in the fourth scan (P < 0.05).

 
Volumetric scanning was then performed repeatedly. Compared with the first volumetric contrast-enhanced scan, the color pixel density values of entire tumor volumes were only slightly reduced in the second postcontrast volumetric scan (scan time, ~50 s each with ~15 s interval between each volumetric scan). By contrast, significantly lower color pixel density values were observed in the subsequent third and fourth (P < 0.05) scans (Fig. 2C). Thus, 35 s after injection of microbubbles, volumetric scans of tumors can be performed for at least 50 s.

Monitoring Effects of Antiangiogenic Therapy
Tumor size. All ultrasound examinations were successful and no animal died during the examinations. Tumor volumes steadily increased in the control group from 100% (day 0) to 99 ± 2% (8 h), to 114 ± 17% (day 1), to 129 ± 24% (day 3), to 206 ± 66% (day 6), and to 245 ± 107% (day 9). Initially, volumes of treated tumors also increased from 100% (day 0) to 105 ± 8% (8 h) and to 119 ± 12% (day 1). However, after this time point, tumor volumes decreased to 83 ± 31% (day 3), to 50 ± 21% (day 6), and to 40 ± 24% (day 9; Fig. 3A ). Differences between the two groups became significant at day 3 (P < 0.05) and highly significant at day 6 (P < 0.01).


Figure 3
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  		Figure 3. Change in size (A) and vascularization of treated and untreated tumors over time assessed by (B) contrast-enhanced and (C) non–contrast-enhanced imaging. A, until day 1, both groups increased similarly in size. After day 1, treated tumors regressed, whereas control tumors further increased. Differences between both groups became significant at day 3 and highly significant at day 6. B and C, in the control group, a similar decrease in vascularization is found on HF-VPDU and ceHF-VPDU. Both methods also indicate a significantly stronger early decrease in vascularization under therapy, which is more pronounced in the ceHF-VPDU scans. Surprisingly, in HF-VPDU scans, vascularization of treated tumors reincreases from day 6 on and reaches even higher values than control tumors on day 9. In contrast, ceHF-VPDU scans indicate that vascularization of treated tumors remains low. *, P < 0.05; **, P < 0.01, between animals of the therapy and treatment group.

 
Vascularization. The heart rate, which might have influenced the measured Doppler signals of tumors, was not significantly different in treated and untreated animals when measured by ECG. Non–contrast-enhanced flow-sensitive ultrasound predominantly visualized vessel networks on the tumor margins, some of which branched toward the tumor center. Results from contrast-enhanced ultrasound diverged as they depicted intense signals from all tumor regions including the center (Fig. 1).

Over time in control tumors, vascularization was found to decrease almost comparably on both contrast-enhanced (day 0: 100%; 8 h: 95 ± 21%; day 1: 90 ± 48%; day 3: 63 ± 29%; day 6: 47 ± 28%; day 9: 37 ± 32%) and non–contrast-enhanced scans (day 0: 100%; 8 h: 117 ± 33%; day 1: 106 ± 23%; day 3: 66 ± 25%; day 6: 47 ± 6%; day 9: 33 ± 29%; Fig. 3B and C).

In treated tumors, contrast-enhanced imaging showed a rapid decrease in vascularization, which was already visible after 8 h of treatment. At this time point, vascularization had dropped from 100% to 78 ± 41%, whereas it remained almost unchanged in untreated controls (95 ± 21%). Twenty-four hours after treatment, vascularization had dropped to 44 ± 16%, whereas control tumors remained at an almost constant level (90 ± 48%). Three days after treatment, vascularization had drastically collapsed to 11 ± 8% of its initial value and became highly significantly lower (P < 0.01) compared with control tumors (Fig. 3B). Particularly, vascularization had strongly decreased in the tumor centers, whereas minor changes were found at the outer tumor rims (Fig. 4 ). The remaining vascularization further dropped at day 6 (6 ± 4%) and at day 9 (2 ± 2%) of treatment.


Figure 4
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  		Figure 4. Contrast-enhanced and non–contrast-enhanced most intensity projections of a tumor during treatment. Already after 3 d, a rapid collapse of vascularization in the tumor center can be observed by contrast-enhanced imaging. Non–contrast-enhanced imaging depicts larger vessels predominantly on the tumor periphery, whereas the majority of microvessels in the tumor center are not displayed. Despite this limitation of non–contrast-enhanced imaging, therapy effects can also be recognized on the depicted vessels visually. Bar, 1 mm.

 
In contrast to the rapid response of the tumor as detected by ceHF-VPDU, the non–contrast-enhanced flow-sensitive measurement showed no obvious change in vascularization 8 h (95 ± 11%) or 24 h (91 ± 22%) after commence of treatment. However, significant (P < 0.05) treatment effects could be detected at day 3 (29 ± 23%) and vascularization further decreased until day 6 (19 ± 16%). Surprisingly, after day 6, color pixel density reincreased and reached higher values than in the controls at day 9 (Fig. 3C).

These results indicate that different vessel fractions are captured with each of the methods: a fraction that responds rapidly to the antiangiogenic treatment and that is only caught by ceHF-VPDU and another vessel fraction with only a moderate response and controversial change of vascularization at later therapy stages.

Immunohistochemistry. Immunohistochemistry confirmed our findings and showed reduced area fractions of CD31-positive staining under treatment (day 0: 100%; 8 h: 97 ± 40%; day 1: 32 ± 12%; day 3: 22 ± 10%; day 6: 20 ± 15%; day 9: 9 ± 10%; Fig. 5 ). However, in untreated animals, the area fraction of CD31 remained almost constant over the entire observation time (day 0: 100%; 8 h: 94 ± 41%; day 1: 95 ± 41%; day 3: 101 ± 53%; day 6: 115 ± 35%; day 9: 93 ± 83%). Differences between both groups became significant at day 1 and highly significant at day 3. Investigation of vessel diameters indicated larger vessels on the tumor periphery (67 ± 40 µm) than in the center (17 ± 7 µm; P < 0.01). Most larger vessels were stabilized by smooth muscle actin–positive cells, indicating their maturity, whereas the smaller vessels were primarily nonstabilized (Fig. 6 ). After 3 days of treatment, predominant regression of the small, nonstabilized microvessels was observed, whereas most of the larger stabilized vessels persisted. Later at days 6 and 9, tumors had decreased in size, and the networks of larger stabilized vessels at the tumor periphery had drawn closer together.


Figure 5
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  		Figure 5. Immunofluorescence images of an untreated and a treated tumor showing endothelial cells stained with CD31 (red) and cell nuclei with DAPI (blue). During treatment, regression of small vessels and development of necrotic areas (black spots, missing DAPI staining) can be observed. Quantitative analysis confirms a decrease in area fractions positive for CD31 on therapy. In untreated control tumors, histologic vessel density remains at a stable level. Bar, 75 µm.

 

Figure 6
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  		Figure 6. Immunofluorescence images of an untreated (A) and a treated tumor after 3 d of therapy (B). Endothelial cells are detected by CD31 (red) and perivascular cells by smooth muscle actin (green). Blue, counterstaining of cell nuclei was performed with DAPI. As compared with the treated tumor (B), the control tumor (A) shows a higher vessel density with a large amount of small vessels not covered with smooth muscle actin–positive cells, indicating their immaturity. Note the necrotic area within the treated tumor (white arrow, missing DAPI staining). In both tumors, large mature vessels stabilized smooth muscle actin–positive cells, which are located at the outer rim. Bar, 75 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
In this study, we investigated the early effects of a multitargeted receptor tyrosine kinase inhibitor treatment on the vascularization of A431 xenografts in nude mice using noninvasive imaging and immunohistochemistry. Special attention was paid to differentiate nonstabilized microvessels (which respond to treatment; ref. 24) from larger, predominantly stabilized vessels (which do not respond well to antiangiogenic therapy; ref. 24). Flow-sensitive high-frequency ultrasound without contrast medium was applied to detect the "larger" vessels with higher and more laminar blood flow, as reported in a previous study (25). To assess early effects on the microvasculature, a novel imaging protocol for contrast-enhanced three-dimensional high-frequency ultrasound was established. It enables the assessment of the total functional vascularization, including both microvessels and larger vessels of the entire tumor.

After 8 h of treatment, contrast-enhanced ultrasound detected a rapid decrease of vascularization in the central parts of the tumor. Vascularization in the periphery was almost unchanged. Non–contrast-enhanced ultrasound only detected larger vessels in the periphery. Here, significant treatment effects became apparent no earlier than after 3 days. The surprisingly rapid functional response of nonstabilized microvessels to multitargeted receptor tyrosine kinase inhibitor treatment has not been reported before as previous studies mainly were based on caliper measurements of the tumor size (3).

In control tumors, vascularization also decreased over time but to a significantly lower degree. Here, the decrease in vascularization was almost identical in contrast-enhanced and non–contrast-enhanced scans. We explain these findings based on the biological and physiologic properties of tumor vessels. During tumor growth, immature and partially nonfunctional blood vessels form. Although these vessels may still be partially perfused, their flow velocities are low (26). Brown and colleagues (26) reported that the majority of blood within the microvascularization of tumors flows at 130 to 350 µm/s. This reduced level of hemodynamics severely limits detection of microvessels by high-frequency ultrasound, which visualizes flow only in the range of millimeters per second. Single microbubbles may still enter these vessels and can be visualized using the SAE signals emitted during destruction.

The initial effects of antiangiogenic therapies are most pronounced on these newly developed, immature vessels (24, 27). Because their major fraction is not recognized by high-frequency ultrasound scans, the higher sensitivity of the contrast-enhanced method for the effects of antiangiogenic therapy is plausible.

The finding that the decrease in vascularization of the control tumors is almost equal in non–contrast-enhanced and contrast-enhanced scans can also be explained by the different vessel fractions recorded by each of the methods. Here, the decrease in vascularization is not related to selective destruction of immature angiogenic vessels but to the loss of vessel functionality due to increasing interstitial pressure and subsequent necrotic degeneration of tumor tissue from insufficient oxygen and energy supplies. The fact that the continuous decrease in vascularization during control tumor growth did not correspond to the almost stable vessel density determined by CD31 staining further supports the idea of a decrease in the fraction of functional vessels (28), perhaps due to increasing interstitial pressure.

Another interesting observation is the slight reincrease in vascularization in the treated tumors seen on the non–contrast-enhanced scans. A comparable result was obtained by Kiessling and colleagues (29) treating squamous cell carcinoma xenografts with a "vascular endothelial growth factor receptor 2" blocking antibody and imaging vascularization by dynamic contrast-enhanced MRI. In this article, the reincrease during therapy was interpreted as the result of tumor shrinkage and the fact that large mature vessels from the tumor periphery draw closer. These results are highly concordant with the assumption that HF-VPDU does indeed principally detect the more mature vessel fraction.

Immunohistochemistry confirmed these findings, showing a rapid decrease in microvessel density in treated tumors, which is in line with previous reports (3). Immunostaining of stabilized mature vessels with "smooth muscle actin" as well as measurement of the mean vessel diameter confirmed a relative increase in mature vessel fraction with larger diameters during therapy but an almost identical vascular architecture in control tumors. However, we cannot exclude that some of the vessels collapsed after removal and fixation of the tumor, resulting in a systemic error. Thus, one has to admit that the results may not resemble the exact diameter of blood-filled vessels in vivo.

A crucial precondition for performing a three-dimensional contrast-enhanced scan is a stable microbubble concentration in the blood during the entire scanning procedure. An almost stable plateau of microbubbles could be observed between 35 and 110 s after injection. This plateau can be explained by the heterogeneous in vivo stability of the microbubbles within one charge (e.g., due to differences in size, wall thickness, and wall homogeneity; ref. 30). The sum of injected particles causes the initial peak of the enhancement curve. Then, the more unstable particles rapidly disintegrate, whereas the more stable particles circulate for a longer period, thus being responsible for the observed plateau. Nevertheless, because the microbubble concentration also slowly declines during the plateau phase, a certain systematic error might be present, which increases with longer scan time (respectively with an increased scan field). However, as the scan distance was comparably short, we do not assume that this error influenced our data significantly.

In a clinical scenario, this limitation might be solved technically by a defined acoustical focusing of the ultrasound beam and by the use of a multiarray scan head for a rapid three-dimensional scan.

In conclusion, although HF-VPDU can assess early changes in the vascular profile of tumors, the sensitivity of the method is limited to a certain vessel fraction with higher blood velocity and higher maturity. Administration of contrast medium permits assessment of the functional vascular volume down to the capillary bed and increases the sensitivity of the method to very early antiangiogenic therapy effects. Therefore, the combination of contrast-enhanced and non–contrast-enhanced imaging could be useful in monitoring of early antiangiogenic therapy effects and in describing the degree of tumor vessel maturation.


   Disclosure of Potential Conflicts of Interest
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: German Research Foundation (DFG Project: SFB-TR23 Vascular Remodeling and Differentiation) and German Federal Ministry for Education and Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Jennifer Hermes and Sarah Floesser for their technical assistance during animal experiments and immunohistochemical analyses. We also thank Pfizer, Inc., which kindly provided pure substance of SU11248.

Received 1/24/08. Revised 5/23/08. Accepted 6/12/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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