Analysis To The Effect Of Different Microenvironment On Metastatic Tumor Cells In Lung Adenocarcinoma

Objective: Lung adenocarcinoma cells proliferate in primary sites, and always transfer to distant sites. In this process, microenvironment is changeable, to adapt to new microenvironment, tumor cells constantly change their characters. Therefore,the lung adenocarcinoma cells in metastatic sites not only inherit the characters of the tumor cells in primary sites, but also acquire some new characters in accordance with changeable environment. However, histological analyses to the morphology of tumor cells were mostly performed with conventional tissues preparation methods,which always cause some inevitable problems, for instances, artificial operations or chemical reagents could constrict blood vessels, loose blood supply, which contribute to the hypoxia of tissues. These problems may influence the judgment and analysis of results, which consequently could affect the validity of results. The“in vivo cryotechnique”(IVCT) is a method in which isopentane-propane cryogen is poured over the living animal organs to directly cryofix them, avoiding the problems which could be caused by artificial operations or chemical reagents.To get better results and avoid the problems which could be caused by conventional methods, IVCT is performed in this study to analyze the behavior of metastatic tumor cells in lung adenocarcinoma.Methods: the culture of Lewis lung adenocarcinoma cells; establishment of animal model with Lewis lung adenocarcinoma cells; IVCT is performed in lung tissues of animal model, which then is embedded in paraffin wax; The samples are used for section-staining, and record the experimental results.Results:1. The samples of tumor cells made with immersion-fixation followed by dehydration(IMDH) show that blood vessels were collapsed, cells shrinked, the distance between tumor cells was larger, the density of cytoplasm and nuclei increased; The morphology and inner structure such as cytoplasm and nuclei of tumor cells were clear and complete in The samples of tumor cells made with IVCT,2. The tumor cells around blood vessels in primary sites and metastatic sites all express HIF-1α. However, necrotic regions show negative, the further from blood vessels, the stronger the signal of HIF-α was.3. In the metastatic mediastinal lymph nodes, flowing tumor cells could be detected which show negative to HIF-1α in cortical sinus and marginal sinus.There was no tumor formation, but only few nomadic tumor cells existed in follicles, which also show negative to HIF-1α.4. In primary tumor, tumor tissues severely compressed normal tissues, and the structure of normal tissues was damaged. In metastatic tumor, tumor tissues also compressed normal tissues, but not as severely as primary tumor, the structure of adipose cells was clear without damage.5. In metastatic tumor, plasma cells mostly assembled around the blood vessels.The number of Ig M-positive cells was the most, by comparison, the number of Ig A-positive cells was least, the number of Ig G-Fc-positive cells between them.Conclusion:1. By comparison with conventional methods, IVCT could not only make the morphology of structure of tumor cells show more clear and complete, but also prevent cells shrinkage which affect the analysis of results. IVCT is an useful method for tissues fixation.2. The same to the tumor cells in primary sites, metastatic tumor cells around blood vessels are hypoxic. Because of fast growth of tumor cells, the tumor cells which are far from blood vessels could not get ample oxygen, and the classical structure “blood-hypoxic regions-necrotic regions” forms. Hypoxia is closely related to the occurrence and development of tumor.3. Because of alteration of microenvironment or the need for the proliferation and metastasis of tumor cells, tumor cells do not express HIF-1α to adapt to microenvironment or satisfy their own need in mediastinal lymph nodes.Mediastinal lymph nodes is an important way for tumor cells to metastasis.4. The invasion of tumor cells is different in different microenvironment. Primary tumor cells show stronger ablitiy of proliferation and invasion. However, the invasion of metastatic tumor cells is weaker or do not exhibit completely.5. Lymphocytes play an important role in the development of tumor, which could secret antibodies affect the process of tumor development or in other ways. The lymphocytes which secret Ig M play the main role.