Effect Of Angiogenesis On Growth And Metastasis Of Lewis Lung Carcinoma

Background & ObjectiveThe growth, invasion and metastasis of tumor is really a complex process. These features are contributed by multiple factors, multiple gene variation and multiple steps of progression, which involves the complex relationship between tumor cells and host, diverse reactions of tumor angiogenesis and host immune system. In particular, tumor-inducing angiogenesis significantly contribute to these biological behaviors. A great amount of studies demonstrate that tumor diameter could only reach 1-2mm³ and fail to form metastases before new vascularization. However, once the new vessels formation, tumor cells turn to duplicate, as a result, more opportunities for metastasis to happen. Therefore, tumor angiogenesis is not only necessary for tumor cells to proliferate and grow, but also to invasion and metastasis of tumor.Tumor angiogenesis is a complex process involving proliferation and migration of vascular endothelial cells, as well as degradation of extracellular matrix. It is clear that many kinds of cytokine, growth factor and their receptors together regulate the angiogenesis, among the total, vascular regulating factors (angiogenesis enhancing factors and inhibiting factors) play the pivotal role, when the imbalance of enhancing factors and inhibiting factors happened, angiogenesis would be induced. Vascular endothelial growth factor (VEGF) is well-known as the strongest angiogenesis enhancing factor, which is able to aim to vascular endothelial cells particularly, furthermore, it can cause endothelial cells to proliferate and also induce angiogenesis. Whereas, the indeed regulate mechanism of expression of VEGF is still unclear. At present, most people consider that it may relate to hypoxia regulation and cytokine regulation and so on.It is no doubt that tumor cells take a long time and diverse biological behaviors transformations from primary situ to distant metastasis, this process may need several years, even several decades of years in human body, so it is impossible for us to observe this process in human body, but available by animal model. During the formerly process of empirical study, we found that after inoculation of Lewis lung carcinoma (LLC) to C57BL/6 inbreeding line mice, with the numbers of passage increasing, the rate of pulmonary metastasis steps up. On this basis, we simulate the dynamic process of tumor progression and metastasis by means of continuous passage in mice, in order to establish a an animal model which can display the whole process of tumor progression, further to look for a novel method to observe the dynamic changes of biological behaviors of tumor. In the meantime, we also detected the expression variation of VEGF, HIF-1a and MVD, aiming to disclose the relationship between tumor angiogenesis and the invasion and metastasis of tumor, and it's possible mechanism.MethodsLLC was subcultured in DMEM medium which containing 10% fetal bovine serum, and kept in incubator at 37℃with 5%CO₂. Initially we inoculated the right armpit of C57BL/6 mice by subcutaneous at the density of 1×10⁶/0.2ml, then we establish the first generation bearing-tumor animal model. Then killed the bearing-tumor mice 2 weeks after inoculation, strip the tumor in sterility condition and made it into suspension, then inoculated the right armpit of C57BL/6 mice by subcutaneous at the density of 1×10⁶/0.2ml. The Lewis lung carcinoma was maintained by serial passage. Every time we chose fifteen mice to be inoculated on the second generation (the group of earlier stage), the eighth generation (the group of intermediate stage) and the fifteenth generation (the group of advanced stage). We recorded the time of oncogenesis on each group, and measured tumor size from the sixth day after inoculation every other day, and figured out tumor growth curve. The bearing-tumor animal models were killed at the twenty-eighth day; the tumor was fixed in methyl Aldehyde after removed from mice, and then observed lung metastases. Immunohistochemistry was used to detect the expression of HIF-1a, VEGF and MVD in tumor on each group. Statistical analysis was performed with SPSS13.0 statistical software, using variance analysis, x² test. Nonparametric test, correlation analysis was done by Kendall's tau-b .The test criterion was set at a=0.05.Results1. Time of oncogenesis on each group are (4.60±1.18) days, (3.73±1.03) days and (2.93±0.96) days respectively. With the numbers of passage increasing, time of oncogenesis shortened, and the difference is statistically significant (P<0.01).2. Learning from the tumor growth curve, the tumor's growth velocity speeds up with the numbers of passage increasing, and the difference is statistically significant (P<0.05).3. Gross appearance of primary tumor: With the numbers of passage increasing, tumor tissue varied from pale and tenacious quality to ruddy and crisp quality, from localized growth to skin involved, even to skin ulcer, to invade chest muscles. When tumor tissues were cut apart, they varied from non-bleeding to bleeding.4. Pulmonary metastasis: Pulmonary metastasis rate of each group are 13.3 %(2/15),60.0%(9/15)and 100%(15/15)respectively, it is steps up during the process of serial passage , and the difference is statistically significant(P<0.01).5. The expression of MVD on each group are (24.73±10.38),(41.27±13.50) and(55.80±21.31) respectively. With the numbers of passage increasing, the expression of MVD increased gradually, and the difference is statistically significant (P<0.01).6. With the numbers of passage increasing, the expression of VEGF increased gradually, and the difference is statistically significant (P<0.05). There was statistically significant correlation between the expression of VEGF and MVD.7. With the numbers of passage increasing, the expression of HIF-1a increased gradually, and the difference is statistically significant (P<0.05). There was statistically significant correlation between the expression of VEGF and HIF-1a.Conclusions1. With the numbers of passage increasing, the tumor's growth velocity speeds up, the invasive power was gradually enhanced and the rate of pulmonary metastasis steps up. All of this displayed the dynamic process of invasion and metastasis of tumor, so we can say it establish a solid basis to explore the important role of angiogenesis in tumor progression and metastasis by this model2. VEGF secreted by tumor cells contribute to tumor angiogenesis, and its expression is strongly correlated with the malignance level and metastasis potential of tumor.3. Angiogenesis play an extremely important role in dynamic process of tumor growth and metastasis. The potential mechanism is hypoxia induces up regulation of HIF-1a, and HIF-1a promotes the transcription of VEGF, thus tumor angiogenesis happens, which enable tumor cells to get used to hypoxia, even to grow, and metastasis.