Exteaction Of Lewis Lung Cancer Stem Cells Which Express Green Fluorescence Protrin Constantly With Preliminary Identification

Objective1．The present study aims to establish an orthotopic Lewis lungcarcinoma (LLC) model on C57BL/6mice where the cancer cells couldexpress Green Fluorescence Protein (GFP) stably.2．Lewis lung carcinoma can be obtained in the serum-free conditionedmedium and is are to identify their biological properties.Methods1．To build the orthotopic mice model:103,104and105Lewis lungcancer cells which could express GFP constitutively were seeded inMatrigel, respectively. Then, these groups of Matigel were plantedorthotopically on6-8weeks female C57BL/6mice. Every3mice weresacrificed and analyzed every7days. The tumor growth and metastasis wasmonitored directly using LDP470nm bright blue flashlight and SellstromZ87fluorescence goggles. The tumorigenic rate and tumor formation timewere defined, which were further confirmed by pathological examinationand electron microscope analysis. 2.The spheres of LLC Monoclonal can be obtained by Monoclonal withserum-free conditioned medium.3.Through the clone sphere, we compare the capability of proliferativecapacity in vitro for the sphere of LLC and the original LLC cells.4.The capability of invasion is compared by the invasion experiments.5.We observe the shape difference of the two kinds of cells throughelectrical telescope.6.Tumorigenic capability are compared through tumor experiments.Results1.All animals survived after the orthotopic intrapulmonary implantationand in all three groups the Lewis lung cancer cells in the formed tumorexpress GFP stably. The tumorigenic rate was25.00%,58.33%and91.67%,respectively, which correlated with the planted dosage and reached thesignificance (P<0.01). The tumor formation time was (23.33±4.04)d,(21±5.72)d, and (18.45±7.84)d, respectively, which was independent of theplanted dosage(P>0.05).2.The sphere of the LLC cells can be produced through the monoclonalmethod in serum-free conditioned medium and they can pass downgeneration to generation stably.3.The results from the monoclonal experiments shows that the ratio offormation is （75.5±5.6）%and the ratio for the original cell is (26.4±3.1)%.They show significant statistical difference (P<0.01). the capability of proliferative capacity in vitro of the sphere is stronger the original cells.4.The results of invasion experiment shows that the number of thespheres of LLC pass polycarbonate membrane is259±26, the number ofLLC original cells is36±5. The relative invasion index for the spheres ofLLC compared the original cells is6.43±2.75. The number of the spherespass the membrane is larger the original LLC cells (t=6.812，P <0.01). Thespheres of LLC have stronger capability of invasion than that of theoriginal ones.5.The results from the electrical telescope show that the ratio of nuclearcytoplasm increases, the number of organelles is small and it has certainproperties of stem cells.6.The results obtained from the tumor experiments, the tumor can beobserved even when only100LLC cells are implanted and its tumorigeniccapability is stronger than the original ones.Conclusion1.The Lewis lung carcinoma model which constantly expresses GFPwas successfully established, and this could filter potential drug candidatesagainst lung cancer.2.The sphere of the LLC is successfully produced through serum-freeconditioned medium and it has stronger the proliferative and tumorigeniccapability.