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Published online before print June 11, 2007, 10.1148/radiol.2442060371
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(Radiology 2007;244:449-456.)
© RSNA, 2007

Experimental Studies

Antiangiogenic Tumor Treatment: Early Noninvasive Monitoring with USPIO-enhanced MR Imaging in Mice1

Thorsten Persigehl, MD, Ralf Bieker, MD, Lars Matuszewski, MD, Alexander Wall, MD, Torsten Kessler, MD, Hendrik Kooijman, PhD, Norbert Meier, PhD, Wolfgang Ebert, PhD, Wolfgang E. Berdel, MD, Walter Heindel, MD, Rolf M. Mesters, MD, and Christoph Bremer, MD

1 From the Departments of Clinical Radiology (T.P., L.M., A.W., N.M., W.H., C.B.) and Medicine/Hematology and Oncology (R.B., T.K., W.E.B., R.M.M.), University Hospital Muenster, Albert-Schweitzer-Str 33, D-48129 Muenster, Germany; Philips Medical Systems, Hamburg, Germany (H.K.); Bayer Schering Pharma, Berlin, Germany (W.E.); and Interdisciplinary Center for Clinical Research (IZKF Muenster, FG3), University of Muenster, Muenster, Germany (C.B.). From the 2004 RSNA Annual Meeting. Received February 27, 2006; revision requested April 27; revision received July 10; accepted August 7; final version accepted December 4. Supported in part by the German Research Foundation (BR 1653/2-1) and the Federal Ministry of Education and Research, Germany (13N8896). Address correspondence to C.B. (e-mail: bremerc{at}uni-muenster.de).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To prospectively investigate steady-state blood volume measurements for early quantitative monitoring of antiangiogenic treatment with ultrasmall superparamagnetic iron oxide (USPIO)–enhanced magnetic resonance (MR) imaging.

Materials and Methods: The institutional animal care committee approved all experiments. HT-1080 fibrosarcoma-bearing nude mice were injected with a thrombogenic vascular targeting agent (VTA) (11 nude mice, 20 tumors) or saline (12 nude mice, 20 tumors). USPIO-enhanced (SH U 555C) MR imaging was performed after the VTA was administered. USPIO-induced changes in tissue R2* ({Delta}R2*) were measured with a T2-weighted dual-echo echo-planar imaging sequence, and the vascular volume fraction (VVF) was calculated. Parametric {Delta}R2* maps were analyzed with respect to tumor perfusion patterns. Correlative histologic analysis was performed for grading of tissue thrombosis, and tissue perfusion was quantified with fluorescent microbeads. Unpaired Student t test and Spearman nonparametric correlation coefficient were used for statistical analysis.

Results: The {Delta}R2* values were significantly (P < .001) reduced shortly after treatment initiation (mean {Delta}R2*, 0.017 msec–1 ± 0.0014 [standard error] in control animals vs 0.005 msec–1 ± 0.0007 in animals that received VTA), which was also reflected by a decrease in the VVF (2.47% ± 0.18 vs 0.41% ± 0.48, P < .001). Histologic analysis revealed various degrees of tumor thrombosis after VTA treatment that correlated inversely with the {Delta}R2* values (r = –0.83). Moreover, tumor perfusion measurements corroborated the MR results, indicating a significant reduction in tissue perfusion after VTA treatment (mean tissue fluorescence, 570.4 arbitrary units [au] per gram ± 27 vs 161.7 au/g ± 17; P < .05).

Conclusion: USPIO-enhanced MR imaging enables early monitoring of antiangiogenic treatment of tumors.

© RSNA, 2007


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
More than 80 molecules that reveal antiangiogenic activity have been discovered (1,2). Results stemming from trials with antivascular endothelial growth factor antibodies used alone or in combination with classic chemotherapy enabled researchers to confirm that systemic antiangiogenic therapy affects tumor progression and thus patient survival (2,3). More recently, the first antivascular endothelial growth factor molecule was approved by the Food and Drug Administration for use in treating advanced colon cancer (4).

The findings of clinical cancer trials have made it clear that classic end points in oncology (eg, tumor size regression) are insufficient when monitoring treatment response to antiangiogenic treatment protocols (2,3). Thus, the availability of surrogate markers will be instrumental to reevaluate the role of tumor angiogenesis in patients with cancer, identify new molecular targets and drugs, and improve planning and monitoring of future studies and interpretation of study findings. Thus, noninvasive imaging methods that are sensitive to tumoral microvessels (<20 µm in diameter) and are capable of resolving hotbeds of angiogenesis would be useful. Dynamic contrast material–enhanced magnetic resonance (MR) imaging performed with low-molecular-weight gadolinium chelates has been applied in clinical practice to enable detection and characterization of breast tumors (5). However, dynamic contrast-enhanced MR imaging has been shown to be highly sensitive but nonspecific in the identification of malignant lesions (6–8).

High-spatial-resolution imaging capable of covering large tumor volumes requires use of a blood pool contrast agent that resides intravascularly at approximately constant concentrations during imaging. While many such agents have been used in experimental studies (9,10), only a few are currently clinically available or in phase III clinical trials (11,12). One of these nanoparticles, a dextran-coated iron oxide (monocrystalline iron oxide nanoparticle 46), has been used to extract the vascular volume fraction (VVF), which serves as a surrogate marker for tumor angiogenesis in vivo (13). A carboxydextran-coated ultrasmall superparamagnetic iron oxide (USPIO) with similar pharmacologic properties, SH U 555C (Bayer Schering Pharma, Berlin, Germany), has been used with high-spatial-resolution MR angiography of the chest and abdomen in clinical phase I trials (11,12,14). SH U 555C has a plasma half-life of approximately 6 hours in humans. High-spatial-resolution diagnostic-quality MR angiograms could be obtained up to 30 minutes after intravenous injection (11,12,14). Thus, the purpose of our study was to prospectively investigate steady-state blood volume measurements for early quantitative monitoring of antiangiogenic treatment with USPIO-enhanced MR imaging.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
An author (H.K.) who is employed by Philips Medical Systems was involved in the design of the MR sequence used in our study. Another author (W.E.) is employed by the company (ie, Bayer Schering Pharma) that provided the contrast agent used in this study. All authors who are not employed by these companies controlled data and information submitted for publication that might have presented a conflict of interest for the authors who are employed by these companies.

Cell Culture
Human fibrosarcoma cells (HT-1080) were cultured under generally established cell culture conditions in 5% CO2 at 37°C. The culture medium consisted of a minimum essential medium (Gibco, Paisley, England) supplemented by 10% fetal bovine serum (Gibco), 1% penicillin-streptomycin (Invitrogen, Paisley, England), and 1% glutamine (Invitrogen). Human breast carcinoma cells (DU4475) were cultured in Roswell Park Memorial Institute medium (RPMI 1640; Gibco) supplemented by 10% fetal bovine serum, 1% penicillin-streptomycin, and 1% glutamine. All cell lines were commercially available from the American Tissue Culture Collection (Manassas, Va).

Tumor Xenograft
All experiments were approved by the institutional animal care committee of the University of Muenster. Three authors (T.P., L.M., A.W.) subcutaneously implanted 2 x 106 human fibrosarcoma cells in the flank of athymic female nude mice (CD-1NU/NU; Charles River Laboratories, Sulzfeld, Germany). To reduce the number of animals needed, tumors were implanted bilaterally and allowed to grow for 3–8 weeks to a size of 0.5–1.0 cm (15,16). Fibrosarcoma-bearing animals (11 animals, 20 tumors) were intravenously injected with a vascular targeting agent (VTA) that induced selective tumor thrombosis, whereas control animals (12 animals, 20 tumors) received a saline solution.

MR imaging and fluorescent perfusion measurements were obtained 4–8 hours after initiation of treatment. In a separate subgroup of three animals with five tumors, the degree of revascularization was measured with contrast-enhanced MR imaging 24–36 hours after a single treatment dose was administered. At the end of the experiment, animals were sacrificed with an overdose of ketamine and xylazine. Tumors were excised and were either fixed in phosphate-buffered formalin or snap frozen.

Tumor Treatment
For induction of selective tumor thrombosis, a fusion peptide was generated by using a polymerase chain reaction assembly cloning strategy for expression in Escherichia coli, as described elsewhere (15,17,18). Briefly, the peptide contained a truncated tissue factor with an NGR (Asn-Gly-Arg) peptide linked to its C-terminus. While the NGR peptide specifically binds to CD13 (aminopeptidase N) of tumor neovasculature, the truncated tissue factor induces selective thrombosis at the binding site with a mechanism of action similar to that of the fusion protein truncated tissue factor fused to the ED-B domain of fibronectin (a sequence of 91 amino acids) described by Nilsson et al (19). In different preclinical models, the used VTA has been shown to induce selective thrombosis of tumor neovascularization immediately after treatment initiation (17). The fusion peptide was intravenously injected at a dose of 1 µg per kilogram of body weight (total volume, 300 µL). The control group received 300 µL sodium chloride (0.9%). Application of the VTA was well tolerated, without any clinically apparent symptoms of systemic thrombosis.

Histologic Analysis
After MR imaging, mice were euthanized with an overdose of ketamine and xylazine, and tumors were excised and dissected. Half of the tissue was fixed in phosphate-buffered formalin, and the other half was immediately snap frozen. For hematoxylin-eosin staining, the formalin-fixed tissue slices were embedded in paraffin, cut into 4-µm-thick slices, deparaffinized in xylene, and hydrated in a serial ethanol solution. A solution of Mayer hemalaun (Merck, Whitehouse Station, NJ) and eosin (Eosin B; Sigma-Aldrich, St Louis, Mo) was used for staining. The percentage of perfused tumor tissue (ie, the percentage of tumor tissue without thrombosis or necrosis) was estimated at histologic analysis for each slice for all individual tumors by two investigators (T.P., C.B.) in consensus. Mean percentage of perfused tumor tissue was estimated for every tumor and graded with a six-point scale, as follows: grade 0, 0% thrombosis and/or necrosis; grade 1, 1%–20% thrombosis and/or necrosis; grade 2, 21%–40% thrombosis and/or necrosis; grade 3, 41%–60% thrombosis and/or necrosis; grade 4, 61%–80% thrombosis and/or necrosis; and grade 5, more than 80% thrombosis and/or necrosis.

Tumor Perfusion Studies with Fluorescent Microspheres
Correlative tumor perfusion studies were performed in a separate subset of animals (treatment group, four tumors; control group, three tumors) by using red fluorescent polystyrene microspheres (FluoSpheres; Molecular Probes, Leiden, the Netherlands) with an average particle size of 10 µm (T.P., A.W.). To fluorescently stain the tumor endothelial cells, isolectin (Isolectin GS-IB4; Molecular Probes) was coinjected into the animals. Tumor-bearing animals were anesthetized, and a midline incision was made to expose the abdominal aorta and inferior vena cava. Both vessels were ligated distal to the renal arteries, a custom 28-gauge catheter was inserted into the abdominal aorta, and 7 x 105 microspheres (200 µL) and 100 µg isolectin (100 µL) were bolus injected into the aorta. The catheter was then flushed with 1 mL of saline, and the inferior vena cava was incised to allow venous decompression. The animals were euthanized with an overdose of ketamine and xylazine, and tumors were excised, cut in half, and snap frozen.

Multiple 20-µm slices were prepared with a cryotome and visually evaluated (T.P., L.M.) with a high-power magnification (objective magnification, x40) camera (Eclipse TE 2000-U; Nikon, Tokyo, Japan). The second half of the tumor was processed to measure fluorescence after digestion by using a modified version of the protocol described by Van Oosterhout et al (20). Briefly, the tumor samples were digested in 2 mol/L of potassium hydroxide and 2% Tween 20 (Sigma-Aldrich) for 48 hours at 60°C. Homogenized samples were then centrifuged and washed repeatedly with a weaker solution of Tween 20 and distilled water. For extraction of the microspheres, 2-(2-ethoxyethoxy)ethyl acetate (Merck, Darmstadt, Germany) was added to the pellet and incubated for at least 12 hours overnight. The samples were again shaken and centrifuged. An author (T.P.) used a fluorescence spectrophotometer (F-4500; Hitachi, Tokyo, Japan) to measure the fluorescent supernatant fluid. The microspheres were excited at 580 nm and emitted fluorescence at 613 nm.

MR Contrast Agent
Schering provided USPIO nanoparticles (SH U 555C) for use in obtaining steady-state blood volume measurements. These nanoparticles consist of a carboxydextran-coated iron oxide core (ie, ferucarbotran). Electron microscopic studies, x-ray diffraction studies, and dynamic laser light scattering revealed the mean size of the iron oxide core to be about 3–5 nm and the mean hydrodynamic diameter to be about 20 nm in an aqueous environment. Relaxivity was measured (r1 = 22 L · mol–1 · sec–1, r2 = 45 L · mol–1 · sec–1) at 40°C and 20 MHz in water (11). In clinical phase I trials, the particles were found to exhibit a plasma half-life of approximately 6 hours in volunteers (11,12,14). Equilibrium-phase MR angiography was performed up to 30 minutes after injection and yielded diagnostic-quality high-spatial-resolution MR angiograms at all examined time points (12). In phase I trials, SH U 555C was intravenously injected at doses of up to 80 µmol of iron per kilogram of body weight after precontrast MR imaging (11,14,21).

MR Imaging
MR imaging was performed by using a clinical 1.5-T MR system (Intera; Philips, Best, the Netherlands) with standard whole-body gradients and a microscopic surface receive-only coil with a 47-mm diameter (Microscopy Coil 47; Philips). Tumor-bearing animals were anesthetized with intraperitoneal injection of ketamine (125 mg per kilogram of body weight) and xylazine (12.5 mg/kg). To allow stable vascular access, the jugular vein was cannulated after exposure by means of a longitudinal neck incision (T.P., A.W.). The cranial part of the vein was ligated, and a 2.5-F catheter (SIMS Portex, Kent, England) was inserted with its tip placed in the superior vena cava. The position of the catheter was secured with an inferior ligation.

The animals were then transferred to the MR imager and placed in the prone position, parallel to the B0 magnetic field, in the magnet bore. To depict anatomic details, transverse high-spatial-resolution T2-weighted MR images of the tumor region were acquired by using a fast spin-echo sequence with the following parameters: repetition time msec/echo time msec, 2700/100; fast spin-echo factor, 15; 2-mm section thickness; 0.2-mm intersection gap; 384 x 384 matrix; 512 x 512 reconstruction matrix; 100 x 100-mm field of view; total acquisition time, 4 minutes 13 seconds. For measurement of the transverse relaxation rate (R2*), a T2*-weighted, fast dual-echo spoiled gradient-echo sequence with a multishot echo-planar imaging readout was performed before and after intravenous injection of SH U 555C. The sequence parameters were as follows: 605/9.5, 19; 19 echo-planar imaging readouts per echo; 2-mm section thickness; 0.2-mm intersection gap; 90° flip angle; 112 x 112 matrix; 256 x 256 reconstruction matrix; 100 x 100-mm field of view; total acquisition time, 2 minutes 14 seconds. This sequence allowed us to calculate T2* from the relative signal intensities of the two echoes. The sequence is a quick alternative to conventional gradient-echo sequences; the multishot echo-planar imaging technique allows an optimal compromise between speed of acquisition and artifact level. Choosing an even number of echo-planar imaging readouts for each echo results in chemical shift– and susceptibility-induced distortions that are identical in the images obtained with both echoes, and this enables accurate T2* maps to be constructed.

MR measurements were obtained 4–8 hours after injection of VTA or saline. In this time frame, VTA was previously shown to exhibit maximum tumor thrombosis (ie, no revascularization occurred) (17,19). To assess the degree of revascularization after one dose of VTA was administered, additional MR measurements were obtained 24–36 hours after VTA administration in a separate subgroup of three animals.

Analysis of MR Data
The fundamental assumption of steady-state blood volume measurements obtained with USPIO is that the change in R2* ({Delta}R2*) is proportional to the perfused local blood volume per unit tumor volume (V) multiplied by a function (f) of the plasma concentration of the contrast agent (P) and a contrast agent concentration–dependent constant (k), as described elsewhere (22,23). The equation is as follows: {Delta}R2* = k · f(P) · V.

Assuming a steady-state distribution of SH U 555C during MR data acquisition, the equation simplifies to a linear relationship between {Delta}R2*, the perfused tumor volume (V) at a particular time (t), and the constant (k), which includes the dose-dependent blood pool concentration of the contrast agent, as follows: {Delta}R2*(t) = k · V(t) (13). The perfused tumor volume can thus be calculated as follows: V(t) = [{Delta}R2*(t)]/k.

Assuming monoexponential signal decay, R2* can be estimated from the double-echo echo-planar imaging sequence as follows:

Formula
where TE1 and TE2 are echo times 1 and 2, respectively (measured in milliseconds), and SITE1 and SITE2 describe the signal intensity with echo times 1 and 2, respectively.

The following equation can consequently be used to calculate {Delta}R2*:

Formula
where SITE1post and SITE2post describe the signal intensities measured with the dual-echo echo-planar imaging sequence after contrast material administration and SITE1pre and SITE2pre describe the corresponding signal intensities measured with unenhanced sequences. The VVF of tumors (VVFT) can then be calculated by multiplying calibrated {Delta}R2* values of tumor ({Delta}R2*T) and muscle ({Delta}R2*M) tissue by 1.89%, which is the known vascular volume of muscle tissue in mice (24), as follows: VVFT = ({Delta}R2*T/{Delta}R2*M) · 1.89%.

For data quantification, one author (T.P.) placed regions of interest section by section so that they covered the whole tumor volume. The R2* signal changes were determined by automatically copying the regions of interest on corresponding unenhanced and contrast-enhanced MR images. For VVF determination, the same author placed circular regions of interest (≥20 pixels) in adjacent muscle tissue.

On the basis of the formula given previously, {Delta}R2* maps were calculated by one author (N.M.) by using a program developed to depict perfusion patterns (25). The maps were created by combining each 3 x 3 median-filtered voxel on four corresponding images to form a new image according to the formula. Neither data averaging nor smoothing were applied. The resulting real data matrix was then linearly normalized to give positive integer values to be saved and viewed in standard Digital Imaging and Communications in Medicine format.

Statistical Analysis
All data are presented as means ± standard errors of the mean. Statistical differences between the treatment and control groups were analyzed with an unpaired, two-tailed Student t test by using commercially available software (InStat; GraphPad Software, San Diego, Calif). A P value of less than .05 was considered to indicate a statistically significant difference. MR imaging data and histologic grades of tumor perfusion were correlated by using a Spearman nonparametric correlation with a confidence interval of 95% (GraphPad Software).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
At the time of imaging (ie, after injection of one VTA dose), there were no observed relevant changes in tumor size in the animals that received VTA compared with the tumor size changes observed in the control group.

Histologic Analysis
Histologic analysis in the treatment group revealed various grades of tumor thrombosis (mean grade, 3.52 ± 0.21) 4–8 hours after treatment, with a large number of erythrocytes and platelets visible in the tumor-feeding vessel (Fig 1) in animals that received VTA. Some tumors showed signs of central tumor necrosis in addition to thrombosis. Morphometric grading of the thrombosed and/or necrotic tumor areas correlated inversely with {Delta}R2* (r = –0.83) and VVF (r = –0.74) measurements (Fig 2). In contrast to animals in the treatment group, animals in the control group had little or no vascular occlusion (mean grade, 0.69 ± 0.17).


Figure 1A
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  		Figure 1a: (a, b) Histologic evaluation of the treatment model (HT-1080 fibrosarcoma cells) in the (a) control and (b) treatment groups. (Hematoxylin-eosin stain; objective magnification, x20.) Note the extensive thrombosis seen 4 hours after application of the VTA. (c, d) Corresponding perfusion assay images with fluorescence microbeads show (c) fluorescent microspheres retained in the neovasculature of the tumor in the untreated control specimens, whereas (d) essentially no microspheres can be seen after VTA treatment. (Objective magnification, x20.)

 

Figure 1B
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  		Figure 1b: (a, b) Histologic evaluation of the treatment model (HT-1080 fibrosarcoma cells) in the (a) control and (b) treatment groups. (Hematoxylin-eosin stain; objective magnification, x20.) Note the extensive thrombosis seen 4 hours after application of the VTA. (c, d) Corresponding perfusion assay images with fluorescence microbeads show (c) fluorescent microspheres retained in the neovasculature of the tumor in the untreated control specimens, whereas (d) essentially no microspheres can be seen after VTA treatment. (Objective magnification, x20.)

 

Figure 1C
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  		Figure 1c: (a, b) Histologic evaluation of the treatment model (HT-1080 fibrosarcoma cells) in the (a) control and (b) treatment groups. (Hematoxylin-eosin stain; objective magnification, x20.) Note the extensive thrombosis seen 4 hours after application of the VTA. (c, d) Corresponding perfusion assay images with fluorescence microbeads show (c) fluorescent microspheres retained in the neovasculature of the tumor in the untreated control specimens, whereas (d) essentially no microspheres can be seen after VTA treatment. (Objective magnification, x20.)

 

Figure 1D
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  		Figure 1d: (a, b) Histologic evaluation of the treatment model (HT-1080 fibrosarcoma cells) in the (a) control and (b) treatment groups. (Hematoxylin-eosin stain; objective magnification, x20.) Note the extensive thrombosis seen 4 hours after application of the VTA. (c, d) Corresponding perfusion assay images with fluorescence microbeads show (c) fluorescent microspheres retained in the neovasculature of the tumor in the untreated control specimens, whereas (d) essentially no microspheres can be seen after VTA treatment. (Objective magnification, x20.)

 

Figure 2A
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  		Figure 2a: Graphs show quantitative data used to analyze the effects of antiangiogenic treatment. Note that VTA treatment resulted in a significant reduction (*) of (a) the {Delta}R2* (control group, 20 tumors; VTA group, 20 tumors) and (b) the VVF (control group, 20 tumors; VTA group, 20 tumors) values. (c) Moreover, results of a perfusion assay with fluorescent microbeads corroborated the MR results and showed a 72% reduction in tumor perfusion after VTA treatment (control group, three tumors; VTA group, four tumors). In a–c, data are mean values and error bars indicate standard error of the mean. (d) Morphometric analysis revealed an inverse correlation of the grade of tumor thrombosis and/or necrosis with the {Delta}R2* (bullet, r = –0.83) and the VVF ({circ}, r = –0.74) values.

 

Figure 2B
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  		Figure 2b: Graphs show quantitative data used to analyze the effects of antiangiogenic treatment. Note that VTA treatment resulted in a significant reduction (*) of (a) the {Delta}R2* (control group, 20 tumors; VTA group, 20 tumors) and (b) the VVF (control group, 20 tumors; VTA group, 20 tumors) values. (c) Moreover, results of a perfusion assay with fluorescent microbeads corroborated the MR results and showed a 72% reduction in tumor perfusion after VTA treatment (control group, three tumors; VTA group, four tumors). In a–c, data are mean values and error bars indicate standard error of the mean. (d) Morphometric analysis revealed an inverse correlation of the grade of tumor thrombosis and/or necrosis with the {Delta}R2* (bullet, r = –0.83) and the VVF ({circ}, r = –0.74) values.

 

Figure 2C
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  		Figure 2c: Graphs show quantitative data used to analyze the effects of antiangiogenic treatment. Note that VTA treatment resulted in a significant reduction (*) of (a) the {Delta}R2* (control group, 20 tumors; VTA group, 20 tumors) and (b) the VVF (control group, 20 tumors; VTA group, 20 tumors) values. (c) Moreover, results of a perfusion assay with fluorescent microbeads corroborated the MR results and showed a 72% reduction in tumor perfusion after VTA treatment (control group, three tumors; VTA group, four tumors). In a–c, data are mean values and error bars indicate standard error of the mean. (d) Morphometric analysis revealed an inverse correlation of the grade of tumor thrombosis and/or necrosis with the {Delta}R2* (bullet, r = –0.83) and the VVF ({circ}, r = –0.74) values.

 

Figure 2D
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  		Figure 2d: Graphs show quantitative data used to analyze the effects of antiangiogenic treatment. Note that VTA treatment resulted in a significant reduction (*) of (a) the {Delta}R2* (control group, 20 tumors; VTA group, 20 tumors) and (b) the VVF (control group, 20 tumors; VTA group, 20 tumors) values. (c) Moreover, results of a perfusion assay with fluorescent microbeads corroborated the MR results and showed a 72% reduction in tumor perfusion after VTA treatment (control group, three tumors; VTA group, four tumors). In a–c, data are mean values and error bars indicate standard error of the mean. (d) Morphometric analysis revealed an inverse correlation of the grade of tumor thrombosis and/or necrosis with the {Delta}R2* (bullet, r = –0.83) and the VVF ({circ}, r = –0.74) values.

 
Tumor Perfusion Measurements
Intravascular microspheres were evenly distributed in the tumor vasculature of untreated control animals (Fig 1). The VTA treatment substantially reduced the number of microspheres seen with high-power magnification (Fig 1). Likewise, in the treatment group, tumor endothelial cells were substantially less fluorescently stained with isolectin (Fig 1). Quantitative fluorescence measurements enabled us to confirm that there was a significant reduction in tumor perfusion after treatment with the tTF-NGR fusion peptide (control group, 570.4 arbitrary units [au] per gram ± 27; VTA group, 161.7 au/g ± 17; P < .05) (Fig 2). Thus, the reduction in perfused tumor volume was approximately 72% in this assay, which was in line with our findings obtained with MR imaging.

MR Imaging
Between 4 and 8 hours after treatment initiation, {Delta}R2* maps revealed a clear reduction in tumor perfusion in treated animals compared with the perfusion in control animals (Fig 3). While control animals showed strong inhomogeneous tumor vascularization with hotbeds of neovasculature, VTA treatment resulted in clearly reduced tissue perfusion (Fig 3). Quantitative data analysis revealed an approximately 64% decrease in iron oxide–induced susceptibility effects after antiangiogenic tumor treatment throughout the entire tumor (mean {Delta}R2* = 0.017 msec–1 ± 0.0014 in the control group and 0.005 msec–1 ± 0.0007 in the VTA group, P < .001) (Fig 2). Likewise, VVF was significantly reduced after injection of the thrombogenic peptide (2.47% ± 0.18 in the control group; 0.41% ± 0.48 in the VTA group; P < .001) (Fig 2). Between 24 and 36 hours after one dose of VTA was administered, substantial revascularization could be observed, with {Delta}R2* values and VVF percentages returning to nearly baseline values (mean {Delta}R2*, 0.017 msec–1 ± 0.0047; mean VVF, 1.61% ± 0.51).


Figure 3A
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  		Figure 3a: (a, b) Anatomic and (c, d) parametric MR images of HT-1080 xenografts. Tumor xenografts were implanted bilaterally into flanks of nude mice. Tumors are clearly delineated in a and b high-spatial-resolution T2-weighted images. USPIO-enhanced (SH U 555C) MR imaging was performed 4 hours after injection of (a, c) saline or (b, d) VTA. (c, d) Parametric {Delta}R2* maps representing individual tumor sections of highly vascularized (c) untreated and (d) VTA-treated fibrosarcoma reveal excellent response to thrombogenic VTA.

 

Figure 3B
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  		Figure 3b: (a, b) Anatomic and (c, d) parametric MR images of HT-1080 xenografts. Tumor xenografts were implanted bilaterally into flanks of nude mice. Tumors are clearly delineated in a and b high-spatial-resolution T2-weighted images. USPIO-enhanced (SH U 555C) MR imaging was performed 4 hours after injection of (a, c) saline or (b, d) VTA. (c, d) Parametric {Delta}R2* maps representing individual tumor sections of highly vascularized (c) untreated and (d) VTA-treated fibrosarcoma reveal excellent response to thrombogenic VTA.

 

Figure 3C
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  		Figure 3c: (a, b) Anatomic and (c, d) parametric MR images of HT-1080 xenografts. Tumor xenografts were implanted bilaterally into flanks of nude mice. Tumors are clearly delineated in a and b high-spatial-resolution T2-weighted images. USPIO-enhanced (SH U 555C) MR imaging was performed 4 hours after injection of (a, c) saline or (b, d) VTA. (c, d) Parametric {Delta}R2* maps representing individual tumor sections of highly vascularized (c) untreated and (d) VTA-treated fibrosarcoma reveal excellent response to thrombogenic VTA.

 

Figure 3D
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  		Figure 3d: (a, b) Anatomic and (c, d) parametric MR images of HT-1080 xenografts. Tumor xenografts were implanted bilaterally into flanks of nude mice. Tumors are clearly delineated in a and b high-spatial-resolution T2-weighted images. USPIO-enhanced (SH U 555C) MR imaging was performed 4 hours after injection of (a, c) saline or (b, d) VTA. (c, d) Parametric {Delta}R2* maps representing individual tumor sections of highly vascularized (c) untreated and (d) VTA-treated fibrosarcoma reveal excellent response to thrombogenic VTA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
Given that antiangiogenic treatment protocols, like conventional chemotherapy regimens, are time consuming and expensive, there is an urgent need for reliable, sensitive, and quantitative in vivo measurement techniques that can be used to assess antiangiogenic therapies. Moreover, the findings of clinical trials have enabled researchers to confirm the need for surrogate markers of treatment effectiveness, since classic end points—such as tumor size regression—may not help in correctly evaluating the treatment response (2,3). For several years, parametric imaging approaches in which clinically available low-molecular-weight contrast agents, fast MR imaging techniques, and occasionally complex pharmacologic modeling would be used have been proposed (10,26–30). The specificity of this imaging approach is inherently limited since the first-pass extravasation of small molecules is high for both tumorous and nontumorous tissues (6). For example, in breast cancer detection a substantial overlap of malignant and benign tissue can be found. In this context, the use of so-called blood pool contrast agents has been shown to substantially improve capabilities to characterize benign versus malignant tissue (10,29). USPIO particles are one of many types of blood pool contrast agents.

Our study data show that treatment effects can be monitored early (4–8 hours) after treatment initiation before any classic signs of tumor response, such as size regression, occur. In this regard, the VVF measured with USPIO-enhanced MR imaging correlated well with the grade of tumor thrombosis, necrosis, or both. Moreover, the MR data were in line with independent perfusion measurements obtained by using fluorescent intravascular microspheres.

As opposed to conventional low-molecular-weight gadolinium chelates, USPIOs offer some important advantages for noninvasive characterization of tumor tissue. First, USPIOs, depending on the specific type, have a prolonged and almost exclusive intravascular retention and thus serve as ideal intravascular markers during MR measurement. The USPIO applied in this experiment (SH U 555C) exhibits a plasma half-life of about 6 hours in humans (12). It was shown in a previous study that this paradigm holds true for tumor neovasculature with increased vessel leakiness (13).

Second, although USPIO is useful for T1-weighted MR angiography (11), it exhibits strong effects on the T2 and T2* relaxation times. These effects are considerably stronger than the T1 effects of conventional MR contrast agents. Thus, even subtle differences in the tumoral VVF can be depicted sensitively with this approach.

Third, the results of our study show that treatment effects can be depicted and, more importantly, quantified instantly after initiation of antiangiogenic tumor treatment. This imaging approach can be transferred to a clinical setting instantaneously. It was recently shown that superparamagnetic iron oxide–enhanced imaging enabled differentiation of highly vascularized bone marrow in patients with acute myeloid leukemia from moderately vascularized bone marrow in healthy control subjects (31).

Besides imaging the VVF, USPIO can be used to investigate various aspects of oncologic diseases. The pronounced T1 effects of USPIOs can be exploited for MR angiography by using fast T1-weighted gradient-echo sequences right after contrast agent injection (12) and can therefore help to assess vascular infiltration of tumors. Moreover, nodal involvement of solid tumors can be assessed with this approach in a late postcontrast examination (eg, 24 hours after USPIO injection) (32). In this regard, a multifunctional imaging approach can be envisioned with USPIO-enhanced MR imaging.

There were limitations to this study. The histologic grade of perfused tumor tissue may not be representative of the whole tumor volume since only one 4-µm section can be evaluated, and treatment response may differ substantially in various tumor parts. Thus, we obtained additional fluorescence-based perfusion measurements that more accurately reflect the overall tumor perfusion and thus more closely resemble the effects seen with USPIO-enhanced MR imaging. Other antiangiogenic treatment protocols may yield fewer effects on the VVF compared with the VTA protocol applied in this study. Moreover, depending on the individual activation levels of the autonomous nervous system (eg, stress levels), slight variations in the vascular tone of muscle can be expected, and the VVF results could shift accordingly. Finally, for clinical applications, repetitive MR studies (ie, repetitive USPIO-enhanced MR imaging) must be performed to monitor treatment effectiveness over time. Thus, further experimental studies are warranted to determine if this imaging concept can be extended to a longitudinal imaging approach in which an individual treatment protocol is monitored over longer periods of time.

Practical application: Our study data suggest that USPIO-enhanced MR imaging may help to quantify changes in tumor perfusion in response to antiangiogenic treatment. Thus, parametric imaging of USPIO-induced changes in R2* may greatly facilitate early quantitative monitoring of novel antiangiogenic treatment protocols. With the availability of long-circulating USPIOs for clinical use, this imaging technique can instantly be applied in patients.


   ADVANCES IN KNOWLEDGE
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   FOOTNOTES
 

Abbreviations: USPIO = ultrasmall superparamagnetic iron oxide • VTA = vascular targeting agent • VVF = vascular volume fraction

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantors of integrity of entire study, T.P., T.K., W.H., C.B.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; manuscript final version approval, all authors; literature research, T.P., R.B., L.M., T.K., H.K., N.M., W.E.B., C.B.; clinical studies, T.K., W.E., W.E.B., W.H.; experimental studies, T.P., R.B., L.M., A.W., T.K., H.K., R.M.M., C.B.; statistical analysis, T.P., L.M., N.M., C.B.; and manuscript editing, T.P., R.B., A.W., W.E., W.E.B., W.H., R.M.M., C.B.


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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