Effect of Angiogenesis on Growth and Metastasis of Lewis Lung Carcinoma
|Keywords||Lewis lung Lung metastases Angiogenesis Vascular endothelial growth factor Microvessel density Hypoxia-inducible factor 1a|
Background and purpose of tumor growth, invasion and metastasis is more than one factor involved in the interaction between multiple genetic changes and evolution of complex multi-step process, with the tumor cells and host stromal cells, tumor angiogenesis, and host immune system reaction, tumor-induced angiogenesis in many factors much attention is currently considered a very close relationship with the biological behavior of tumor. A large number of studies have confirmed that tumor neovascularization, can only reach a size of about 1-2mm 3 sup>, and rare distant metastasis formation. However, the formation of new blood vessels, the tumor will grow exponentially, the probability of transfer will also increase. Seen tumor angiogenesis is not only critical to the growth of the primary tumor itself, but also the tumor was a prerequisite for invasive growth and distant metastases. Tumor angiogenesis is a complex process involving basement membrane degradation, endothelial cell migration, proliferation. All of these steps are subject to a variety of growth factors and their receptors, the regulation of cytokines, vasoactive adjustment factor (angiogenesis promoting factor inhibitor) plays the central role. When the angiogenesis-promoting factor inhibiting factor between the balance is broken, that will start tumor angiogenesis process. Vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) in a variety of vasoactive factors currently known strongest pro-angiogenic factor that can specifically direct or indirect role in vascular endothelial cells, thereby promoting vascular new life. The study found, have played an important role in a variety of tumor growth, metastasis. The exact mechanism of regulation of VEGF expression is still unclear, mainly that it may be related to hypoxia regulation, cytokine modulation related to cancer gene regulation. Tumor cells from the growth of the primary tumor proliferation to distant metastases formation stages of change to go through a long process and a lot of biology, often take years or even decades to the progress of the process in the human body, so the process observed in the human body can not be achieved, to complete observed that this process must establish the appropriate animal transfer model to this process, but the majority of the experimental animals in the tumor-bearing state due to its own lifetime limit our difficult dynamic, continuous observation. We found Lewis lung carcinoma (Lewis lung carcinoma, LLC) cells were seeded in C57BL / 6 inbred mice in the experimental research process, also gradually increased the probability of occurrence of lung metastases with increasing tumor passage number, On this basis, we simulated continuous passage through the Lewis lung tumor-bearing mice tumors in vivo growth and evolution process, in order to establish animal models able to simulate the tumor growth, invasion and metastasis dynamic process, looking for an observed tumor biology characteristics of the dynamic changes in the new method. To observe the animal model established on the basis of this experiment the growth of Lewis lung carcinoma, invasion and metastasis dynamic process of tumor tissue vascular endothelial growth factor (VEGF), hypoxia-inducible factor-1a (hypoxia-inducible factor 1a and HIF-1a) and microvascular density (microvesseldensity, MVD) expression changes, in order to reveal the relationship and its possible mechanism of angiogenesis and tumor growth and metastasis. LLC cells were seeded in DMEM high glucose medium containing 10% fetal bovine serum, placed at 37 ° C for 2 humidified incubator with 5% CO culture. Initial inoculation of LLC cells wind and percussion into a single cell suspension of 1 × 10 6 sup> / 0.2ml inoculated in C57BL / 6 mouse the right axilla dorsal subcutaneous, the establishment of the first generation of tumor-bearing mice model. Each subsequent passage two weeks off neck of tumor-bearing mice were sacrificed, to strip tumor tissue under sterile conditions, made the tumor cell suspension to 1 × 10 6 sup> / 0.2ml inoculated in C57BL / 6 mice right axilla dorsal subcutaneous continuous passage in mice. , Respectively, in the second generation, the 8th generation and 15 generation inoculated C57BL / 6 mice 15 as the early group, the interim group and the late group. Out tumor time for each group of mice was observed in the experimental procedure, since after inoculation from the 6th day to once every other day to measure the tumor size, tumor volume was calculated. The first 28 days after inoculation the mice were sacrificed by cervical dislocation, the tumor after peeling fixed and observe lung metastases. Using immunohistochemistry method (SP) detection MVD, VEGF and HIF-1a expression in tumor tissue, and conventional histological observation. Application SPSS13.0 statistical software for experimental data analysis of variance, x 2 sup> test, non-parametric tests, correlation analysis rank correlation analysis using binary variables (Kendall'stau-b), a = 0.05 as inspection standards. Results 1. Early, middle and late out of the mice in each group tumor time were days (4.60 ± 1.18), (3.73 ± 1.03) days (2.93 ± 0.96) days, along with the increase of the number of passages early, middle and late out the tumor of mice in each group time, the difference was significant (P <0.01). 2 by the early, middle and late tumor growth curves of mice in each group can be seen with the increase in the number of passages, the tumor growth rate gradually speed up, the difference was significant (P <0.05). Tumor gross Appearance: With the increase in the number of passages early, middle and late tumor-bearing mouse tumor tissue by pale, quality and tough to ruddy, crisp, to the infiltration of the skin to cause skin ulceration by the limitations of growth, chest wall myometrial invasion even break through the chest, by necrotic tumor tissue taken without bleeding to bleeding. 4 lung metastases: early, the late mice lung metastasis rates were 13.3% (2/15), 60.0% (9/15) and 100% (15/15), showed a trend of gradually increasing , the difference was statistically significant (P <0.01). Early group only the two microscope, is profiled by dozens of tumor cells micrometastases; the interim group visible lung metastases, the number of 1-5, more in diameter below 2mm, microscope, tumor The organization was late group, the number of lung metastases ranged from 4 to 30, the diameter of more than 1-2mm diameter greater than 2 mm metastases, some nests scattered;, the microscope See also showed nests, but multiple larger. Early, the MVD in tumor tissue of mice in each group in late (24.73 ± 10.38), (41.27 ± 13.50) and (55.80 ± 21.31), with the increase in the number of passages, increased incidence of lung metastasis early, the MVD in tumor tissue of mice in each group in late gradually increased (P <0.01). Early, the expression of VEGF in the tumor tissue of mice in each group in late gradually increased VEGF expression in metastases was significantly higher than that of lung metastasis, the difference was significant (P <0.05), and VEGF and MVD was positively correlated (r = 0.530, P <0.01). Early, HIF-1a expression in the tumor tissue of mice in each group in late gradually increased (P <0.05), and the expression of VEGF and HIF-1a into a positive correlation (r = 0.751, P <0.01). Conclusion 1.Lewis lung cancer tumor speed the continuous passaging process in C57BL / 6 mice in advance, accelerated growth, invasion ability of lung metastases increasingly high probability of occurrence, the number of metastases from less to more from small diameter to large, essentially rendering the whole dynamic process of tumor growth, invasion and metastasis of angiogenesis in tumor growth, the study of the role of the transfer process to lay the foundation. Tumor cells secrete VEGF promotes angiogenesis, and its expression with the degree of malignancy and metastatic potential. Angiogenesis play an important role in the growth and metastasis of Lewis lung dynamic process. Possible mechanism for tumor hypoxia caused by overexpression of HIF-1a, start the transcription of VEGF, and the involvement of other vasoactive factors promote angiogenesis, allowing the tumor cells to sustain growth and diffusion transfer.