The Cultivation Of The Primary Lesion Cells Of The Mouse Oral Squamous Cell Carcinoma Modeland The Preliminary Exploration On The Influence Of Different Microenvironments On Cell Growth

Background: In previous studies,we constructed a mouse model of oral squamous cell carcinoma formation and lymphatic metastasis,and found that there were single-form disseminated tumor cell(DTC)scattered into the bone marrow and submandibular lymph node,when primary lesion of the oral mucosa was still in precancerous stage(moderate or severe dysplasia).The DTC grdually increased along with the development of cancer,and finally formatted metastases mass in the lymph nodes,but sustained dormancy in the bone marrow.This is similar to the phenomenon of oral cancer metastasis target to lymph node in clinical.We hypothesize that in early precancerous period,the DTC were scattered into the bone marrow and submandibular lymph node randomly,and which proliferation or dormancy were affected by the different microenvironments,and result in the later targeting lymph nodemetastasis.Objective:(1)The primary oral lesion cells of moderate,severe dysplasia and high differentiated squamous cell carcinoma were collected from the mouse model we constructed in previous studies,and cultured in vitro to provide the cytological basis of studying DTC.(2)Four different microenvironments of bone marrow and lymph nodes in normal Balb/c mice and immunodeficient Balb/c nude mice were used to observe the influence of different microenvironments on cell growth and explore the mechanism preliminarily.Method:(1)Enzyme digestion and tissue pieces method were used to culture the tongue epithelial cells of the mouse model in the stage of moderate,severe dysplasia and high differentiated squamous cell carcinoma.Fibroblasts were removed by digested with trypsin.After isolation and purification,the epithelial cells were used for further study.(2)Phase contrast microscope and transmission electron microscope were used to observe the morphology and ultrastructure of the primary oral lesion cells.Immunohistochemical method was used to detect the expression of Pan-CK,Vimentin,CD44 and ESA in primary oral lesion cells.(3)Transwell insert was used to establish the microenvironments and primary oral lesion cells co-cultured model in vitro.The experiment was divided into 5 groups: primary oral lesion cells cultured alone group(control group),primary oral lesion cells and mice bone marrowco-cultured group,primary oral lesion cells and mice lymph nodes co-cultured group,primary oral lesion cells and nude mice bone marrow co-cultured group,primary oral lesion cells and nude mice lymph nodes co-cultured group.Direct counting method was used to count the number of primary oral lesion cells at 24 h,48h,72 h,96h,120 h,144h.Result:(1)The success rate of the cultivation of primary oral lesion cells was as high as 100%.The longest passages of dysplasia and high differentiated squamous carcinoma cells were up to 40 passages and 80 passages,respectively.The longest survival days of dysplasia and high differentiated squamous carcinoma cells were up to 103 days and 212 days,respectively.(2)Under phase contrast microscope,the morphology of moderate,severe dysplasia cells were similar to normal epithelial cells,and the high differentiated squamous cancer cells were more heterogenetic.Under transmission electron microscopy,the cell membrane of high differentiated squamous cancer cells had morphological changes.And the changing degree of moderately,severe dysplasia cells was between the normal epithelial and high differentiated squamous carcinoma cells.(3)The positive rates of moderate,severe dysplasia and high differentiated squamous carcinoma cells had no difference with normal epithelial cells in Pan-CK,Vimentin,CD44 and ESA immunocytochemistry staining(P>0.05).(4)Different microenvironments influenced the growth of moderate,severe dysplasiaand high differentiated squamous carcinoma cells similarly.The counting numbers of primary oral lesion cells in nude mice lymph node microenvironment were all higher than the control group(P<0.05).The counting numbers of primary oral lesion cells in mice bone marrow,nude mice bone marrow and nude mice lymph nodes microenvironments were all lower than the control group(P<0.05).Conclusion:(1)The moderate,severe dysplasia and high differentiated squamous carcinoma cells can be successfully cultured through enzyme digestion or tissue pieces method and can be purified by digested with trypsin.(2)The purified primary oral lesion cells derive from epithelial cells and have no cancer stem cells characteristic.(3)Different microenvironments may affect the proliferation or dormancy of DTC by the difference of immune factor such as T cells deficient and cell factors such as TGF β1 and TGF β2,and finally cause the passive targeted lymphatic metastasis.