| Part I Correlation of Contrast-Enhanced Ultrasound with TwoDistinct Types of Blood Vessels for Assessment of Angiogenesis in Lewis Lung CarcinomaBackgroundAngiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, is a critical factor for tumor growth and metastasis in many human tumors. Many studies have evaluated tumor angiogenesis through noninvasive imaging methods. Examples are contrast-enhanced dynamic computed tomography (CT), contrast-enhanced dynamic magnetic resonance imaging (MRI), non-enhanced magnetic resonance angiography, positron emission tomography, and Doppler ultrasonography. However, these methods are limited in the detection of low flow and flow in small vessels. Contrast-enhanced ultrasonography increases the sensitivity to slow flow, which can offer detailed information of tumor vascularity.Microvessel density is the current reference standard for characterization of tumor angiogenesis. Different blood vessel markers are used to reveal different aspects and characteristics of tumor vasculature, and in the previous studies, the blood vessel marker of CD31was used. CD31is usually expressed in both differentiated and undifferentiated endothelial cells, whereas the other blood vessel marker CD34is expressed in differentiated endothelial cells.The LLC was the mature mice model established from the lung of a C57BL mouse bearing a tumor resulting from an implantation of primary Lewis lung carcinoma. To date, studies of the different blood vessel markers have not been correlated with CEUS parameters, especially in Lewis lung carcinoma. The purpose of the present study was to assess vascularity measurements via non-invasive quantified contrast-enhanced ultrasonography and correlate CEUS parameters with different immunofluorescent analyses of MVD in LLC.Although previous studies have confirmed that the quantitative analysis of ultrasonic imaging parameters and tumor microvascular density exist certain linear relationship, so far, there is no report of relevant research on Lewis lung cancer in mice between different vascular markers of microvessel density and contrast enhanced ultrasound parameters.ObjectiveThe aim of our study was to evaluate tumor angiogenesis in Lewis Lung carcinoma (LLC) of mice by the use of a contrast-enhanced ultrasound (CEUS) examination with contrast pulse sequencing (CPS), and determine the correlation of contrast-enhanced ultrasonographic parameters with different blood vessel markers of microvessel density (MVD) in the LLC animal model,.MethodsSubcutaneous Lewis Lung carcinomas were established in25mice, which were evaluated by contrast-enhanced US using SonoVue(a second-generation US contrast agent). The results were recorded as digital video images and the time-intensity curves and hemodynamic parameters were analyzed. Pathological tumor specimens were obtained just after US examinations. Tumor specimens were stained with hematoxylin and eosin (H&E) and expression of CD31and CD34, the different endothelial cell markers, was determined by immunohistochemical staining. Then the relationship between the CEUS parameters and the level of MVD was analyzed.Results1.The model of Lewis lung carcinoma in mice was established successfully in our experiment. It is proved that the model is a practical and reliable tool that could be used for researching the tumor of soft tissue.2.Two distinct types of microvessels were identified in Lewis Lung carcinoma: differentiated (CD34+) and undifferentiated (CD31+) vessels.3.A significant correlation was found between CEUS parameters and undifferentiated MVD (CD31+vessels) in LLC(r=0.428; P<0.05).4.There was a reverse correlation between the different MVDs(r=-0.474; P<0.05).ConclusionThe study showed that the distinct types of vasculature(CD31+and CD34+) in Lewis Lung carcinoma, the former correlated with the CEUS parameters. Therefore, CEUS using a second-generation US contrast agent may be useful for the evaluation of tumor angiogenesis of LLC of mice. Part II Assessment of antiangiogenic therapeutics effect in Lewis lung carcinoma:using quantitatively contrast-enhanced ultrasound(bolus kinetics)BackgroundBecause antiangiogenic and antineovascular therapies are designed to affect the abnormal blood vessels found in tumors, changes in blood volume, blood flow, or other hemodynamic parameters may be promising biomarkers that herald a positive clinical response to therapy. Several non-invasive functional imaging methods (including dynamic contrast enhanced magnetic resonance(MR)imaging, computed tomography(CT), positron emission tomography(PET), and ultrasonography)have been used to evaluate tumour angiogenesis and antiangiogenic therapy by identification tumour perfusion. Ultrasound imaging plays an important role in this field because it is a rapid,relatively inexpensive portable and non-radiation. It has the added advantages of direct evaluation of vessel perfusion. Power Doppler sonography can detect reductions of larger tumor vessels during antivascular therapy. However,conventional and even contrast-enhanced Doppler US is not capable of visualizing capillary blood flow,an essential precondition for quantifying tissue perfusion. The rapid development of contrast enhanced gray-scale ultrasound methods has improved its ability to detect low-velocity flow and made it possible to assess capillary blood flow. On the other hand, there is a linear relationship between ultrasound signal intensity and the concentration of the contrast agent present in the vessel. With the use of time-intensity curve, functional parameters of tumour perfusion such as blood volume, blood flow and blood perfusion can be quantified.Endostar is vascular endothelial inhibition, which mechanism is to inhibit the formation of tumor angiogenesis, blocking the nutrient supply of cancer cells by inhibiting the proliferation and migration of vascular endothelial cells, so as to achieve the purpose of inhibiting tumor proliferation or metastasis.Due to the abnormal angiogenesis inhibiting drugs will not only leads to the change of the number of blood vessels within the organization, but also the tumor vessel morphology and functional changes, these changes are secondary to the abnormal tumor blood flow dynamics. So monitoring the abnormal changes can favorably evaluation of the antiangiogenic therapy.ObjectiveThe purpose of this study was to quantitative evaluate the endostar in treatment the LLC tumors,and discuss the effect of the antiangiogenic therapeutics with the CEUS(bolus kinetics).Methods30mature mice model of the LLC were divided into two groups. Mouse were treated with endostar (5mg/kg once daily)by intraperitoneal injection over14days, starting at day2post-implantation, respectively. Fifteen control mouse were treated with an equivalent volume of0.9%NS. Contrast-enhanced gray-scale ultrasound was performed on day15after bolus injection of SonoVue(0.1ml)and the imaging was recorded on cine. Regions of interest within tumour were analyzed off-line with the software of SonoLiver to determine the peak intensity (PI), time to peak(TTP) and arrival time (AT). Immediately after imaging, minces were euthanized and tumour tissue removed for fixation in a10%formalin solution. Microvascular density (MVD) was measured after anti-CD31staining.Results1.7days after implantation, the mice were all observed tumor appearancing.2.Effects on the the general state of health of LLC models.With the gradual growth of the transplanted tumor, their consumption of food, water and movement got slowly in NS group of mice. The volume of Endostar group was obviously than the NS group.3. Effect on micro vessel density in the Lewis lung carcinoma.CD31was employed as the marker of vascular endothelial cell and its cytoplasm was dyed yellow-brown. The mean values of MVD on NS group and Endostar group were98.50±13.53and56.21±5.10,respectively Compared with NS group, the mean values of MVD reduced in the intervention-drug group(P<0.05).4.Treatment with Endostar group resulted into a significant decrease in PI in comparison with control group (P<0.05).5:There-was a positive correlation between PI and CD31MVD values in Lewis Lung carcinoma, which was statistically significant (r=0.532, P<0.05).ConclusionThis study indicates that restructuring endothelial inhibition can effectively inhibit the growth of Lewis lung carcinoma in mice, and contrast-enhanced ultrasound (bolus kinetics) can be used to evaluate changes in perfusion parameters of tumour during treatment of antiangiogenic agents. PI can effectively detect the changes of tumour hemodynamic parameters caused by antiangiogenic treatment. |
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