点击显示 收起
1 From the Departments of Radiology (I.I., P.D., G.A.T.) and Surgery (C.B., B.Z.), Children’s Hospital and Harvard Medical School, 300 Longwood Ave, Boston, MA 02115. Received March 26, 2001; revision requested April 23; revision received June 8; accepted July 5.
| ABSTRACT |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|
MATERIALS AND METHODS: Thirty-five genetically engineered mice with spontaneously occurring prostate tumors were monitored on a weekly basis with gray-scale and color Doppler US with a 15-MHz linear transducer. Eighteen mice were treated with an adenoviral vector to deliver a soluble form of the Flk1 receptor (VEGFR-2), a vascular endothelial growth factor receptor designed to block tumor angiogenesis. The remaining 17 animals were injected with saline and used as controls. Tumor volumes were calculated on the basis of serial US measurements. Color Doppler US was performed in every tumor before and after intravenous injection of 0.1 mL per kilogram of body weight of a US contrast agent. US images were evaluated for tumor size, pattern of vascularity, and extent of vascularity (vascularity index). Findings at US were correlated with findings at autopsy in 30 animals.
RESULTS: Estimates of tumor volume at US correlated well with tumor measurements at autopsy (r = .89, P < .001). Marked differences in tumor size and slope of increasing tumor volume were evident at US between treated and control mice after treatment (P < .016, analysis of variance). The US contrast agent markedly increased color Doppler US signal intensity with an 800% (from 10% to 12,700%) change in the mean number of color pixels per imaging field, and showed vascularity in areas of tumor not identified on precontrast images in 70% (109 of 156 studies). No correlation was found between the pattern of vascularity or vascularity index before or after contrast material administration and tumor size, treatment status, or histologic assessment of tumor vascularity.
CONCLUSION: Contrast-enhanced US improves visualization of tumor vascularity. However, histologic patterns of tumor vascularity do not correlate with Doppler US depiction of blood flow in these vessels.
Index terms: Angiogenesis • Prostate neoplasms, 844.32 • Prostate neoplasms, US, 844.12983, 844.12988 • Ultrasound (US), Doppler studies, 844.12983, 844.12988
| INTRODUCTION |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|
While subcutaneously implanted tumors have been long used for screening of new compounds for potential antiangiogenic activity, there is recent evidence that suggests tumor growth is governed by interaction with the relevant organ environment. As a result, orthotopic tumor models are being used to predict the efficacy of antiangiogenic therapy for transplantable tumors (4). Unlike subcutaneous models in which tumor behavior can be directly observed and measured, tumors implanted in intraabdominal locations are difficult to monitor for therapeutic response.
Ultrasonography (US) performed with high-frequency transducers has provided accurate intermediate end points for monitoring of experimental intraabdominal tumor growth and response to therapy in the mouse model (5,6). Rooks et al (5) showed that volume estimates at US correlated well with volume measurements at autopsy and that tumor neovascularity could be identified in every mouse with color Doppler US. However, this nonenhanced color Doppler technique was not sufficiently sensitive to help evaluate moderate (20%) reductions in tumor vascularity after treatment with angiogenic inhibitors (7).
The purpose of this study was to determine the ability of contrast material–enhanced US to depict tumor growth and vascularity in a murine model of prostate carcinoma treated with an angiogenic inhibitor.
| MATERIALS AND METHODS |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|
Thirty-five mice (transgenic adenocarcinoma mouse prostate, or TRAMP) were used in this study. Among the animal models currently available, this model most closely mimics human prostate cancer progression (8). It was developed by Greenberg and colleagues (9) in 1995 by linking the prostate-specific rat probasin promoter to the SV40 large T antigen. The resulting genetically altered animals reproducibly develop various forms of dysplasia and, eventually, adenocarcinomas of the prostate.
Angiogenesis can be blocked by means of several different tactics designed to interfere with vascular endothelial growth factor (VEGF) function, including the use of antibodies to VEGF or VEGF receptors, which are peptide antagonists that block VEGF interaction with its receptors, and the use of soluble VEGF receptors to scavenge available VEGF (10). For this experiment, we chose a gene therapy approach to deliver a soluble form of the Flk1 receptor (VEGFR-2) to treat the prostate tumors. An adenoviral vector was used as a carrier for the gene construct. The continuous production of soluble receptor protein leads to elevated plasma levels of the antiangiogenic compound.
Each mouse was anesthetized with isoflurane (Baxter Healthcare, Deerfield, Ill) in an inhalation chamber and randomly assigned into a treatment or control group. Each mouse received a 100-µL injection into either the tail vein or the retro-orbital plexus. The treatment group received an intravenous injection of an adenoviral vector that delivered a soluble form of the Flk1 receptor gene (Ad Flk1-Fc) for VEGF-2. The remaining 17 control animals were injected with saline that contained control adenovirus (Ad Fc) without the VEGF receptor gene. The efficacy of this construct in controlling tumor growth and angiogenesis has been tested and confirmed (Becker CM, written communication, 2000).
US Evaluation
Lower abdominal palpation was performed weekly by one examiner (C.B.) in each animal older than 14 weeks to detect tumors of the prostate (11). Palpation findings were confirmed with transabdominal US. Tumor-bearing animals were prospectively enrolled in the study.
Transabdominal US was performed with a 15-MHz linear transducer (Sequoia System; Acuson, Mountain View, Calif). Before US, each animal was anesthetized with isoflurane, and the abdomen was shaved. Gray-scale and color Doppler US were performed every 7 days during the next 10 weeks, and each animal was examined for tumor size and presence, location, and distribution of neovascularity. In every animal, color Doppler US images were obtained before and after intravenous administration of a US contrast agent (Imavist; Alliance Pharmaceutical, San Diego, Calif). This is a microbubble-based experimental US contrast agent. The constituted preparation yields a dispersion of surfactant-coated microbubbles containing perfluorohexane nitrogen, with a volume-weighted median diameter of approximately 6 µm. Elimination from the blood is accomplished by means of evaporation through the lungs (12). The contrast material was administered in a 0.01-mL dose. Each dose was injected intravenously during 1–2 seconds by means of retro-orbital injection into the retrobulbar plexus. We were unable to reliably secure intravenous access via the tail vein despite attempts by multiple experienced investigators of mice. Use of the retrobulbar plexus allowed repeatable and nontraumatic injection of intravenous contrast material.
Imaging settings were standardized and were unchanged throughout the experiment. Visualization of small tumors was difficult with standard gray-scale US. As a result, we electronically magnified the image, and we used a wide dynamic range (90 dB) and a false color (B-mode temperature) map for anatomic depiction of the tumors. In addition, color Doppler US images were obtained in sagittal and transverse orientations by using the following settings: unidirectional color flow map, velocity range of 0.005 m/sec, color Doppler US gain of 50%, and no frequency filtering.
Imaging was performed by experienced sonologists (I.I., G.A.T.) and a sonographer (P.D.), who were blinded to treatment status throughout the study.
Image Analysis
The size and imaging features, such as echotexture and vascularity distribution, of each tumor were prospectively recorded during each imaging session. Tumor volumes were estimated by using the following formula: length x width x depth x 0.5. This measure has been shown to correlate well with actual tumor size in mice with bladder tumors (5). Color Doppler US images were analyzed for the presence and distribution of tumor vascularity. Patterns of neovascularity were described as central if color flow pixels were identified deep within the tumor, as peripheral if flow was seen surrounding the tumor, and as mixed if color Doppler US signal was present both within and surrounding the tumor.
The effect of intravenous contrast material administration was quantified by two investigators (G.A.T., I.I.) with software (PhotoShop; Adobe Systems, Mountain View, Calif). Sagittal or transverse images of the tumor obtained before and after contrast material administration were selected. The total number of color-containing pixels within and surrounding the tumor was selected by using the color range tool. The total number of color-containing pixels was counted by using the histogram function. Doppler US vascularity was normalized to tumor size and expressed as the number of color-containing pixels per cubic millimeter (C). An enhancement ratio (expressed as percentage enhancement) was calculated with the following formula: (Cpost - Cpre)/Cpre, where post is postcontrast and pre is precontrast.
Statistical Analysis
Continuous variables were presented as the mean plus or minus the SD. Two-way repeated-measures analysis of variance was used to determine the effect of treatment on tumor volume and to assess changes over time in the groups on the basis of the Greenhouse-Geisser F tests for within-subjects and between-subjects effects (13). A group-time interaction was tested to compare the rate of change in tumor volume (ie, slope) between the groups. Mean tumor volume and percentage change were compared between the treatment and control groups at 3 and 6 weeks after baseline by using the two-sample Student t test, and differences with a Bonferroni-corrected P value of less than .025 (0.05 divided by two comparisons) were considered significant. The pattern of tumor vascularity was compared by using the nonparametric Fisher exact test to compare proportions between independent groups. Linear regression models were used to compare changes in pixel density at color Doppler US with tumor vascular density estimates at US and to compare volume measurements at autopsy with tumor volume estimates at US.
Histologic Analysis
Animals were euthanized just before they died, and the abdominal tumors were evaluated at histologic examination. Thirty prostate tumors removed from dead animals were measured and fixed in neutral buffered 10% formalin for 12 hours at 4°C. Afterward, the tissue samples were washed with phosphate-buffered saline and were embedded in paraffin. Sections (5 µm thick) were first stained with hematoxylin and eosin to evaluate tissue viability and quality. Microvessel density was determined with immunocytochemical staining by one investigator (C.B.), who used an avidin-biotin detection system (Vectastain; Vector Labs, Burlingame, Calif) with anti-CD31 antibody (monoclonal with dilution of 1:250; Pharmingen, San Diego, Calif), according to the manufacturers’ instructions. The slides were counterstained with hematoxylin. At low magnification (x40), regions with the highest vessel density (hot spots) were scanned as described by Weidner (14). These regions were then counted at a magnification of x100 in a blinded fashion. Three different fields were randomly chosen, and findings in each were counted twice. The mean score was determined and used as the final value.
| RESULTS |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|
|
|
|
Doppler US Findings
Effect of US contrast material on depiction of vascularity.—The number of color-containing pixels increased by approximately 800% ± 130 (standard error of the mean) after administration of US contrast material (P < .001, analysis of variance). However, there was considerable variability in the degree of enhancement among animals.
Patterns of tumor vascularity.—Before contrast material administration, three patterns of tumor vascularity were identified: central (2% [three of 156]), peripheral (52% [81 of 156), and mixed (47% [73 of 156). After contrast material enhancement, a change in the pattern of vascularity could be detected in 44 of 156 (28%) studies. In 31 of 44 (70%) studies, contrast-enhanced images showed vascularity in more central areas of tumor that were not identified on the precontrast color Doppler US images (Fig 3). Treatment status did not appear to have any effect on the postcontrast pattern of tumor vascularity (P > .1, 2).
|
|
|
|
|
|
|
|
|
| DISCUSSION |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|
Although accurate estimates of tumor size are important criteria for success with experimental therapies, they are only an indirect measure of the efficacy of experimental angiogenic inhibitors. Angiogenesis is essential for rapid tumor growth, and some tumors induce the development of an overabundance of neovascularity (15–17). As a result, an initial reduction in tumor vessel density secondary to angiogenic-inhibitor therapy may not result in tumor ischemia and an immediate reduction in tumor size (18). Therefore, the development of other techniques for direct observation of the antiangiogenic effects on tumor neovascularity is desirable.
Several authors have reported mixed results in the evaluation of neovascularity in murine tumors with Doppler US techniques. Lassau et al (19) identified vessels as small as 100 µm in diameter in subcutaneously implanted tumors by using color Doppler US techniques. The presence of intratumoral vessels at Color Doppler US examination correlated with high vessel density (more than 30 vessels in each high-power field) at histologic analysis. At color Doppler US, Asselin-Paturel and colleagues (20) were able to detect a diminution in the number of vessels detected in subcutaneously implanted melanoma treated with antiangiogenic gene therapy compared with the number of vessels detected in untreated tumors. Drevs and colleagues (21) evaluated orthotopically implanted tumors with pulsed-wave Doppler US. They examined the main renal artery to assess changes in tumor hemodynamics in a murine renal carcinoma model and found a diminution in both the systolic and diastolic blood flow velocities in tumors treated with a VEGF inhibitor. In addition, they found a weak correlation between blood flow velocity changes and vascular density. Rooks et al (5), with color Doppler US equipment and techniques similar to those used in the current study, were not able to identify any significant differences in detection of neovascularity in orthotopic bladder tumors between mice treated with an angiogenic inhibitor (TNP-470) and control mice.
In the current study, we used mean pixel density at color Doppler US as a measure of patent tumor vascularity. This measure showed an inverse correlation with tumor size, such that smaller tumors had much higher identifiable tumor vascularity than did large tumors, irrespective of treatment status. However, intratumoral vascularity at Doppler US did not correlate with microvessel density at histologic examination. One likely explanation is that Doppler US techniques depict only the proportion of tumor vessels that are patent to flow and not the absolute number of vessels present, as measured on the basis of the number of antibody-labeled endothelial cells. It is known that tumor vessels may be patent to flow in only the periphery of larger tumors. During periods of rapid growth, tumor neovessels exhibit a high degree of leakage of transudate. The absence of intratumoral lymphatic vessels results in accumulation of fluid and markedly increased interstitial pressure in central parts of the tumor (22). While more central parts of a tumor are also vascularized, intratumoral pressure is often high enough to cause compression and cessation of flow in existing vessels. We postulate that flow in intratumoral vessels is more readily detectable at Doppler US in small lesions because of their relatively low intratumoral pressure. Intratumoral pressures rise with increasing size, which results in relatively decreased vascular patency depicted at Doppler US.
While unenhanced color Doppler US was able to depict the presence of peripheral tumor vascularity, flow in central portions of the lesion was identified infrequently. The use of an intravenous contrast agent increased the conspicuity of flow in areas of vascularity seen on unenhanced images and improved the detection of flow in deeper vessels that were not visualized prior to contrast material administration. Contrast-enhanced images showed a pattern similar to that on unenhanced images of decreasing vascular density with increasing tumor size. However, contrast material did not improve our ability to distinguish between treated and untreated tumors. A major limitation of our study was the large amount of variability in contrast enhancement. Since the contrast material was injected without direct visualization of the retrobulbar plexus, it is likely that a variable amount of contrast material reached the intravascular space with each injection. Nonetheless, this limitation would not have affected the results of the unenhanced Doppler US examination.
We conclude that transabdominal US in murine tumors is an effective method of monitoring tumor response to experimental therapies and that contrast material enhancement shows promise for the improved detection of deep intratumoral vascularity. Additional studies with newer contrast material–specific imaging techniques, such as gray-scale harmonic or subharmonic imaging, may result in better maps of tumor vascularity.
Practical application: Before we began using US in our laboratory, we evaluated drug response in experimental mice by performing laparotomies repeatedly to determine the size and vascularity of tumors treated with angiogenic inhibitors. Experimental cycles were prolonged, and many mice were required because of animal loss, the need for larger tumors, and the use of multiple observation times. Transabdominal US has allowed us to abandon laparotomy as a mode of assessing tumor response. Now, one tumor can be followed up over time in one mouse, which obviates killing many mice at different time points to achieve the same information. We believe that US will become increasingly important in the reduction of the number of animals and time needed to complete initial trials of new antitumor therapies in small-animal models, particularly with orthotopically placed intraabdominal tumors.
|
| ACKNOWLEDGMENTS |
|---|
| REFERENCES |
|---|
| Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES |
|---|