The CAM As A Model For In Vivo Research Of Angiogenesis: Tumor Invation And Metastasis Of NPC

Nasopharyngeal carcinoma(NPC)is a common neoplasm in China, and the incidence of nasopharyngeal carcinoma in GuangXi province is the second high incidence in China. In the past two decades, the incidence and mortality of NPC in GuangXi show no significant change. Nasopharyngeal carcinoma specimen collection is difficult, for two reasons:on the one hand, it is due to the insidious anatomical position of NPC so that there is difficulty in obtaining clinical samples. On the other hand, NPC patients generally don't been carried out surgical treatment, resulting in a very small amount of clinical biopsy specimen. These all are bad to carry out research on the biological behavior of NPC. Therefore, the establishment of animal model of NPC in vivo is an important means of NPC investigation, and also is one of the issues we need to be resolved. The most commonly used animal model of NPC is nude mouse xenograft tumor model which requeste high experimental condition, long experimental cycle and high cost. The chick embryo chorioallantoic membrane (CAM) model with its simple, convenient, inexpensive and rapid has increasingly become a commonly used model for living by tumor researchers. Our aim is to create in vivo animal models of NPC based on the CAM which would become a technology platform for our further study on invasion and metastasis mechanism, anticancer drug screening, pre-clinical evaluation and even individual cancer treatment. By repeating exploration, we optimized the methods and conditions of establishing the xenograft model. According to the characteristics of embryo hatching we determined that the starting time of the experiment is the eighth day after hatching and the latest termination time is the fourteenth day after hatching. We established the "window method" for CAM methods and silicone ring for the inoculating carrier in CAM. We have improved and mastered the chick embryo "window" technology, so that chick "window" success rate close to 100%. We also explored and determined the best seeded number of nasopharyngeal carcinoma cells. With Optimization of the above conditions, we successfully established and perfected the chick CAM tumor model of nasopharyngeal carcinoma.Meanwhile we found that NPC cells also induce angiogenesis when they form xenograft in CAM, and the new blood vessels arrange radially toward to the center of tumor. By quantitative vessel area/chorioallantoic membrane ratio it was found that neovascularization increased with the increase in the number of inoculated cells. We constructed GFP labeled NPC cells and inoculated them in CAM. Then we observed invasion behavior of these GFP labeled NPC cells by using laser confocal microscope. With this model we can observed the tumor cells invasion, breaking through the basement membrane and early stage of blood metastasis. We also discussed the feasibility that CAM model is used to study distant metastasis of NPC cells. By detecting humanβ-globin gene with absolute quantitative real-time fluorescence quantitative PCR method, we detected small amounts of human tumor cells within the heart and lung tissue of inoculated 5-day chicken embryo. On this basis, we verified the distant metastasis difference between the two nasopharyngeal carcinoma cell lines with different metastatic potential, and confirmed that CAM model can be used for prediction of tumor metastasis.In conclusion, this study established a NPC chick embryo chorioallantoic membrane transplantation tumor model for the first time, and evaluated its application on NPC angiogenesis quantitative study in vivo, observation of early invasion behavior and anticipation of the distant metastasis potential from the morphology, molecular biology and other aspects.Our study work has laid a good working basis and provide a good platform for further work on NPC biology research.