Spinal Metastatic Tumor Of Human Lung Adenocarcinoma Stem Cell In Nude Mice: Model Establishment And Experimental Study

Object:With the expectancy of lifetime, the improvement of living standards anddeterioration of environmental pollution, the incidence of tumor has been graduallyincreased.The disease of tumor has been the important threat to human life. About265million people died of tumor every year in China according to statistics,. Therefore,people started to focus on tumor and put huge manpower and financial resources tocancer research in domestic and foreign. Cancer diagnosis technology haS made greatprogress, but the prognosis of most malignant tumors is still poor.The main reason due tothe shortage of the understanding of pathogenesis and biological characteristics abouttumor,lack of ideal and effective treatments.Lung cancer is one kind of malignant tumors.In spinal metastatic tumors, theincidence of Lung cancer is only second to breast cancer. With it's high malignance, fastgrowing,early transferring, strong invasion in spine, The prognosis of patients with Lungcancer is poor in the past, the5-year survival rate is only about21%.Although thesurgery and chemotherapy has made great progress recently, but the long-term survivalrate of patients with Lung cancer still lingers at34%, most patients eventually died oflocal recurrence or distant metastasis of spine.Now, the treatment of lung cancer is stillmainly aimed at tumor whole, most lung cancer cells can be removed or killed throughthe adjuvant chemotherapy or surgical excision, but it is very difficult to preventtransferring to the spine. The theory of tumour stem cells provides a novelinterpretation and treatment direction for its happenning, transferring and attackting oflung cancer. According to theinformation, there is only a tiny number of tumor cells(tumor stem cells,TSCs) rather than all tumor cells with tumorigenicity. TSCs are noteasily killed by traditional radiotherapy or chemotherapy because it is regular that indormant period similar to normal stem cells,even99.99%tumor cells were killed bytraditional radiotherapy or chemotherapy, as long as there is the the rest0.01%cells willrecur.This theory can explain the clinical confused of high recurrence rate and poorlong-term curative effect of lung cancer and its tranferring.Aiming at high relapse and metastasis of lung cance in this study, culturing tumourstem cells of lung cancer,understanding the lung cance stem cell tumorigenicity andmetastatic, identfying specifically expressed genes and proteins of lung cance stem cells, exploring the stem cell tumor-specific markers, establishing spinal metastatic tumormodel of human lung cancer in nude mice through injection of human lung cancer stemcell by left ventricle to providing the basis for further research to pathogenesis, earlydiagnosis and targeted therapy of lung cancer and its recurring and transferring.Methods:Inducing lung cancer sphere cells by adding variety of different growth factors inserum-free medium;using immunofluorescence technique to detect the expression ofSox2,Nanog and Oct4on lung cancer sphere cells;exploring and obtaining the optimumcondition of cultivation; detecting the expression of CD133by flow cytometry; sortingCD133+cells by immunomagnetic beads,establishing spinal metastatic tumor model ofhuman lung cancer in nude mice through injection of human lung cancer stem cell by leftventricle and studing its biological characterstics.Results:1. Adding EGF,TGF-β1,bFGF and successfully induced lung cancer sphere;2. using immunofluorescence technique to test the expression of Sox2,Nanog and Oct4on lung cancer sphere cells. Using flow cytometry technique to test the expression ofCD133on lung cancer sphere cells.3. The test of cell invasion Assay, cell proliferation test, tumorigenicity experimentsterifying that CD133~+cells has strong invasion ability and proliferation.5. Sorting tumor sphere cells with CD133marker by immunomagnetic beads and obtainCD133+tumor cells.Successfully establishing spinal metastatic tumor model of humanlung cancer in nude mice through injection of human lung cancer stem cell by leftventricle.Conclusions:The cells successfully induced lung cancer sphere in serum-free medium with growthfactors of EGF,TGF-β1,bFGF.Immunofluorescence manifeted the positive expression ofSox2,Nanog and Oct4was found on the majority of the lung cancer sphere cells.CD133is the most important surface marker of tumor stem cells of lung cancer by flowcytometry.Sorting lung cancer sphere cells with CD133marker by immunomagneticbeads.The results of cell invasion assay, cell proliferation test,tumorigenicityexperiments illustrated that CD133+cells has strong invasion and proliferation, themore ability of tumor formation in nude mice. Injection of human lung cancer stem cellthrough left ventricle to establish spinal metastatic tumor model of human lung adenocarcinoma in nude mice is a feasible. This model has the similar metastaticmechanism of trans-arterial system and progression with the natural history of tumorlesions, which will be helpful for the study on various treatment protocols.