Effect Of Nordy-carrying Lipid Microbubble With Ultrasound Destruction On Anti-angiogenesis Of Rabbit VX2 Muscle Implanted Tumors

Background:Tumor angiogenesis plays an important role in the growth and metastasis of most solid tumors.it becomes an important target of the anti-tumor therapy. Tumor angiogenesis is a complicated pathologic process. Targeting at the angiogenesis and inhibiting tumor growth or metastasis, pecific antibodies or inhibitors of tumor angiogenesis have been developed for this purpose.With the progression of tumor, the three-dimensional distribution of the shape in tumor tissue and the structure, density of tumor angiogenesis in the tumor interstitial (ie, tumor microvascular architecture phenotype, T-MAP) showed specific tumorous microvascular architecture phenotype heterogeneity following the increase in the heterogeneity of tumor cells. This kind of heterogeneity not only affects tumorous invasiveness and prognosis,but also is a key way to lead different curative effect of anti-angiogenesis. Change in tumorous microvascular architecture phenotype heterogeneity and promotion in the normalization of tumor microvascular architecture phenotype were able to turnover the tumorous microenvironment, which could improve the therapeutic efficacy of malignant solid tumors and became the significant progress of anti-tumor angiogenesis therapy in recent years. It has been found that ultrasound contrast agent can enhance the ultrasound imaging as well as be used as a novel drug carrier to delivery drug targeted, improve the drug concentration, safety and effectiveness, as well as reduce drug consumption and side effects, which has become the main research aspect of anti-tumor therapy. Based on the construction of the new antineoplastic Nordy-carrying lipid microbubbles, this program was to study the microvascular architecture phenotype characteristics of tumor tissue and normal tissue, further to explore whether this drug ultrasound targeted microbubble destruction is a simple, safe, efficient, and new Nordy targeted delivery in vivo approach, providing a new strategy for the treatment of anti-angiogenesis. Objectives:1. To observe characteristics of microvascular architecture phenotype in rabbit VX2 muscle implanted tumor and in normal muscular tissue.2. To observe the effect of Nordy-carrying lipid microbubble with ultrasound destruction on anti-angiogenesis of Rabbit VX2 muscle implanted tumors.3. To explore the correlation between the parameters of angiogenesis perfusion using Contrast Pulse Sequencing (CPS) Technique and ACQ quantitative analysis software and Pericyte findings in immunohistochemisty stain in rabbit VX2 muscle implated tumor for evaluating anti-angiogenesis effects and estimating prognosis.Methods:1. The microvascular architecture phenotype characteristics of the rabbit VX2 muscle implanted tumor and normal muscular tissue were observed based on the VX2 muscle implanted tumor model established by canalled tissue mass embedding method. Microvascular density (MVD) and positive number (PN) of pericytes were tested by HE and immunohistochemistry staining to observe the pathological changes of the tumor tissue and the normal muscular tissue, as well as expression in the microvascular endothelial cells and pericytes. Electron microscopy was used to observe the microvascular ultramicrostructure of normal muscular and tumor. The three-dimensional microvascular features of the normal muscle and tumor tissue was displayed anatomic microvascular casting techniques.2. Thirty rabbits implanted VX2 muscle tumor were randomly divided into 6 groups (5 rabbits in each group): the control group (group A), the intravenous injection of Nordy group(group B), the intravenous injection of Nordy-carrying lipid microbubbles group(group C), the ultrasound destruction group (group D), the ultrasound destruction+intravenous injection of blank lipid microbubble group (group E), and the ultrasound destruction+intravenous injection of Nordy-carrying lipid microbubble group(group F). Tumor growth rate and tumor inhibition rate were determined by two-dimensional dynamic ultrasound monitoring the size and growth of tumors in each group. The MVD was detected by immunohistochemistry staining to observe tumor microvascular angiogenesis in each groups.3. The parameters of blood perfusion and RPI were measured by Contrast-enhanced ultrasound with contrast pulse sequence technique and ACQ (AxiusTM auto tracking contrast quantification) in 10 rabbits implanted VX2 muscle tumor. Correlation was analyzed with MVD and PN labelled immunohistochemically.Results1. The animal model of VX2 implanted tumor was successfully established, with a success rate of 100%.2. There are two results of the immunohistochemistry staining of the rabbit VX2 muscle implanted tumor and normal muscular tissue. Firstly, in the anti-CD34-antibody stained sections, the endothelial cells were stained brown and distributed unevenly in tumors, which mostly clustered at the edge of the tumor; while those of normal muscular tissue distributed evenly. The expression of MVD in tumor was (18.10±6.10), which is much higher than the expression of MVD in normal muscle tissue (8.60±2.55) (P<0.01). Secondly in the anti-α-SMA-antibody stained sections, brown granules were observed in pericytes plasma of both the tumor tissue and normal muscular tissue. Pericytes in the edge of the tumor tissue showed patterns of cluster, tube or stripe. The expression of PN in tumor was (27.20±8.01), which is much higher than the expression of MVD in normal muscle tissue (14.10±3.84) (P<0.01).3. Microvascular structures of tumor tissue and normal muscular tissue were observed by the electron microscopy. In normal muscle, the structure of microvessels is normal,the distribution of microvascular diameter is uniform, basement membrane thickness is of uniformity and integrity, endothelial cells and pericytes are attached tightly with the basement membrane. However, the structure of tumorous microvessels in tumor tissure display the obvious abnormality including uneven distribution of microvascular diameter, expansion of cystic lumen, thinning and uneven basement membrane, loose attachment between endothelial cells and basement membrane and parts of the separation.4. The microvascular casting directly showed the three-dimensional microvascular features of the normal muscle and tumor tissue. The former was relatively sparse winding smoothly like branches and distributed evenly; the latter was condensed, winding chaotically like snake and distributed unevenly, and with vascular network and vascular mass with complex anastomotic vessels formed by co-existence of dilated or sac-like vessels and twisted, narrowed, even occluded vessels. 5. Both the tumor growth velocity and microvascular density in the ultrasound destruction+intravenous injection of Nordy-carrying lipid microbubble group was the lowest compared to other groups ( P < 0.05).6. The quantitative analysis of the ultrasound contrast perfusion parameters showed that the PI and RPI of the tumor tissue were (15.29±4.14) dB and (2.95±1.26 ) dB/s respectively, which were significantly higher than those of the surrounding muscular tissue〔PI: (11.19±3.52) dB; RPI: (2.16±1.26 ) dB/s (P<0.01)〕.However, there were not obvious different in either AT or TTP between the tumor tissue and surrounding muscular tissue(P>0.05).7. The mean PI and RPI of the tumors were (15.29±4.14)dB and (2.95±1.26)dB/s, respectively. The mean MVD and PN of the tumors were(17.75±7.60) and (26.10±10.59), respectively. The PI has significant positive relationship with MVD and PN(r =0.866 and r=0.857respectively,P<0.01) and the RPI has significant positive relationship with MVD and PN(r=0.647 and r=0.648respectively,P<0.01).Conclusion1. The microvascular architecture phenotype characteristics of both tumor and normal tissues can be observed by immunohistochemistry staining of the endothelial cells and pericytes, electron microscopy and microvascular casting, which lay the foundation for observation of anti-angiogenesis effects.2. The Nordy-carrying lipid microbubbles and ultrasound destruction, with its significant anti-angiogenesis effects against rabbit VX2 muscle implanted tumor, is a safe, efficient, and target-oriented in vivo Nordy-delivering system, providing a new strategy for the treatment of anti-angiogenesis.3. The PI and RPI are reliable parameters to reflect the information of pericyte in tumor angiogenesis for evaluating anti-angiogenesis effects and estimating prognosis.