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Molecular Imaging Of Mice Xenografted H22 Tumor With VEGFR2-Targeted Microbubbles And Contrast-Enhanced Ultrasound

Posted on:2010-05-29Degree:DoctorType:Dissertation
Country:ChinaCandidate:X WuFull Text:PDF
GTID:1114360278959634Subject:Biomedical engineering
Abstract/Summary:PDF Full Text Request
Malignant tumor is a serious threat to the life and health of human beings. Early diagnosis and early treatment is always the best principle to rescue patients'life.Traditional medical imaging technologies including ultrasound, CT, MRI, etc provide doctors the intuitive figures of tumor beyond certain volumes. As to the early tumorigenesis or suspicious neoplasms, blood perfusion imaging with common contrast agents was used to support diagnosis for all the various technologies.However, after decades'researches, people found that only the above methods may not provide timely and accurate information that the clinic needed. Because the common contrast agents are not specific to diseased tissue, they only bring about contrast-enhanced images during the transient arterial phase. But after the short enhancement, nothing special left in the abnormal organs. In this case, early or small tumorigenesis would be missed by the traditional means. Therefore, scientific researchers are making every effort to look for novel approaches that can detect specific molecular abnormalities of early tumorigenesis timely and sensitively.Ultrasound molecular imaging provides an encouraging tool for specific diagnosis of early disease at the molecular level. Molecular imaging with contrast-enhanced ultrasound relies on the detection of targeted microbubbles which retained in diseased tissue where they produce an acoustic signal because of their resonant properties in the ultrasound field. Targeting is accomplished either through manipulating the chemical properties of the microbubble shell or through conjugation of disease-specific ligands for the targeted molecule to the microbubble surface.In the recent years, researchers have demonstrated that angiogenesis is promoted early with cancer cells in tumorigenesis and it is a critical determinant of tumor growth, invasion and metastatic potential. One of the major regulators of tumor angiogenesis is vascular endothelial growth factor receptor 2 (VEGFR2). This molecule is overexpressed on tumor vascular endothelial cells. Upregulation of VEGF/VEGFR2 has been associated with tumor progression and poor prognosis in several tumors.Therefore, noninvasive imaging strategies for the detection and quantification of VEGFR2 may be particularly helpful for detection of early neoplasm and guidance of antiangiogenic treatments. In the current study, we hypothesized that VEGFR2-targeted microbubbles with contrast-enhanced ultrasound could particularly depict the novel vessels of mice xenografted H22 tumor, which may benefit timely detection of neoplasm as well as noninvasive assessment of angiogenesis progression.On the other side, as VEGFR2-targeted ultrasound molecular imaging is limited in tumor blood pools, a preliminary study of extravascular ultrasound molecular imaging was carried out in the aim of understanding tumors more comprehensively from the tumor cells themselves.As we have known, FR is highly overexpressed in most cancer cells of endothelial origin, especially ovarian carcinoma; while folic acid is a naturally existed substance of small molecule and no immunogenicity, we believe that folic acid and FR are potentials in tumor-targeted imaging beyond blood pools. Therefore, a preliminary exploration in this field was tried in the last part.Overall, the whole study was composed of the following four parts.PartⅠPreparation of VEGFR2-targeted Microbubbles and Characterization in VitroObjective To prepare VEGFR2-targeted ultrasound microbubbles and to characterize the agent's physical properties as well as immunocompetence. Methods Microbubbles targeted to VEGFR2 (MBv) were prepared by biotin-streptavidin interaction. The diameter and concentration of MBv were analyzed by DFY software. The immunocompetence was validated using immunofluorescence. Results The basic physical properties of MBv were similar to that of the common agents. MBv could conjugate with corresponding secondary antibody specifically in vitro, showing its active immunocompetence. Conclusion MBv were successfully synthesized and demonstrated good immunocompetence. This agent is potential for targeted imaging of tumor angiogenesis in vivo.PartⅡUltrasonic Imaging of Mice Xenografted H22 Tumor with Microbubbles Targeted to VEGFR2Objective To investigate the efficiency of contrast-enhanced ultrasound with microbubbles targeted to VEGFR2 (MBv) for imaging tumor angiogenesis in vivo. Methods Contrast-enhanced ultrasound imaging with MBv or control microbubbles (MBc) was performed in 20 mice bearing xenografted H22 tumors. Sonograms were analyzed by video intensity (VI) by DFY software, and time-intensity curve (TIC) was depicted according to VI change. Internal tracer experiment with DiI-labled microbubbles was carried out to show the distribution difference between MBv and MBc when they circulated for 7 min in vivo. Finally, tumor samples were harvested for analysis of VEGFR2 expressions by immunohistochemistry. Results TIC showed that the time to peak was earlier, peak intensity was higher, and the time to wash out was prolonged in the MBv contrast group compared to that of the MBc control. Internal tracer experiment demonstrated that several MBv were still shown attaching to the tumor vascular endothelium after circulating for 7 min, whereas few or no MBc left in the tumor vessels. Immunohistochemistry demonstrated that VEGFR2 was highly expressed inside the tumor tissues. Conclusion Contrast-enhanced ultrasound with MBv could enhance mice xenografted H22 tumor much more significant than the control. MBv would be a promising molecular probe for imaging tumor angiogenesis in the near future.PartⅢAssessment of VEGFR2 Dynamic Change in Mice Xenografted H22 Tumor Neovessels by Ultrasound Molecular ImagingObjective To explore the feasibility of ultrasound molecular imaging for assessment of VEGFR2 dynamic change in mice xenografted H22 tumor neovessels. Methods Tumor-bearing mice were allowed to live for different stages of 1 week, 2 weeks, 3 weeks and 4 weeks respectively. Contrast-enhanced ultrasound imaging with VEGFR2-targeted microbubbles (MBv) or control microbubbles (MBc) was performed in each mouse in a"perfusion-distruction–reperfusion"mode. Sonograms were analyzed by video intensity (VI) using DFY software, and the mean VI difference (mean VId) before and after microbubble destruction was calculated and regarded as signals from adherent microbubbles inside tumor neovessels. VEGFR2 expression in the tumor tissues were quasi-quantitatively determined by Western Blotting. The correlation between mean VId and VEGFR2 expression was analyzed by SPSS software. Results In the MBv group, mean VId was the highest in 2-week tumor, but lowest in 4-week tumor (P<0.01). There was a strong correlation between mean VId and tumor VEGFR2 expressions in the MBv group (r=0.866, P<0.01). In the control group, there was a narrower fluctuation of mean VId among the 4-stage tumors (P<0.01). However, no correlation was found between mean VId and tumor VEGFR2 expressions in the control group (P>0.05). Conclusion Ultrasound molecular imaging could assess VEGFR2 dynamic change in mice xenografted H22 tumor neovessels accurately. It may serve as a novel guidance for antiangiogenesis therapy.PartⅣPreliminary study of extravascular ultrasound molecular imaging targeted to folate receptorObjective To prepare folate-targeted microbubbles (MBF) as ultrasound contrast agent for potential molecular imaging beyond blood pools. The physical properties of the agent were characterized, and its affinity to folate receptor (FR) was investigated on ovarian cancer cells in vitro. Methods MBF was synthesized by incorporating DSPE-PEG(2000)Folate into the lipid membrane of microbubbles. The diameter and concentration of MBF were determined by DFY software. Ovarian cancer SKOV3 cells of FR overexpression was cultured in vitro. The affinity of MBF to FR was validated by targeting-conjugation test on SKOV3 cells with the observation of light microscope and confocal imaging microscope respectively. Results The mean diameter and distribution of MBF were similar to that of the common microbubbles. In the targeting-conjugation test in vitro, it was shown that a number of MBF conjugated with SKOV3 cells tightly; but after free folic acid influence, the conjugation would be inhibited. There was always no conjugation in the control group. Conclusion MBF were prepared successfully and demonstrated highly affinitive to ovarian cancer SKOV3 cells. MBF is potential to be an ideal ultrasound contrast agent for extravacular molecular imaging of cancers overexpressin FR.
Keywords/Search Tags:Ultrasound, Contrast agents, Tumor, Vascular endothelial growth factor 2, Folic acid
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