| Tongue cancer is the most frequently encountered malignance in oral cavity and has a high metastatic ability. The survival of cancer patients often depends primarily on whether or not the tumor metastasizes. Therefore, it is important to characterize the phenotype of metastatic tumors and to elucidate both the responsible molecules and their functions. The establishment of cell lines with various metastatic potential is necessary for the investigation of themolecular mechanisms of the metastasis process. However, there are only atfew human oral squamous cell carcinoma (SCC) cell lines suitable for investigation. By means of tail vein injection of cancer cells, cell culture and orthotopic implantation technique we established a cell line from spinal cord metastasis in nude mice. Cell counting, flow cytometry, colonogenic assay, morphological observation, immunohistochemical staining, tumor growth-6-and metastasis experiments in nude mice were used to investigate the biological properties of the cell line.1: Establishment of the cell line from spinal cord metastasisA cell line was obtained from lymph node metastasis of tonguecancer by orthotopic implantation of human tongue cancer Tb cellsin nude mice and was named TbTl .A cell line was established fromspinal cord metastasis by tail vein injection of TbTl cells in nudemice and named Ts. Ts cells have been maintained in culture for more than 1 year and transferred for more than 50 passages.2: Ts cell line is confirmed to be squamous cell carcinoma derived from human tongue cancer with cell culture, morphological observation, chromosome analysis and tumorgenesis test.2.1 Phase contrast and light microscope observation revealed similar epidermoid-cell-like morphological characteristics of Ts and Tb cells. The cells were polygonal in shape containing oval or round nuclei with 1 or 2 or more evident nucleoli and grew in a typical cobblestone pattern. Ultrastructual appearance of Ts and Tb cells were similar, There were plentiful microfibril on the cell surface, many polysomes, mitochondria, secretory granules in the cells. Some cells were giant and with multipul nuclei.2.2 The number of chromosome of Ts and Tb was 44-110 and 30-88; the mode was 60 and 50, respectively. The morphology of the chromosome of both cell lines was of human being. Both of them were heteroploidy.2.3 The tumor was proved to be squamous cell carcinoma by pathological examination: clear cell's boundary,plentiful cytoplasm,cell connection.-7-3: The growth and metastasis of Ts cells were studied by means of cell counting, flow cytometric analysis, soft agar clonogenic assay and lung colonogenic assay.The population doubling time of Ts and Tb were 26.83h and 28.81h respectively. Flow cytometric analysis showed that the population percent of Gl, S and G2 cells of Ts were 61.8, 25.5 and 12.7, that of Tb 68.4, 21.1 and 10.4 respectively. Cloning efficiency in soft agar of Ts and Tb were 62.1 and 48.0 respectively. The number of metastatic foci in the Ts and Tb on lung surface in the nude mice were 50.3 + 45.7 and 17.5 ?14.4respectivly.4: Immunohistochemical staining was carried out to detect nm23-HK PCNA, CD44Hs MMP9 expression in Tca8113,Tb, and Ts cells in culture or in sections prepared from paraffin-embedded specimens obtained from nude mice.Tca8113, Tb and Ts cells showed positive staining with the antibodies of PCNA, nm23-Hl, CD44H, MMP9.The protein expression of PCNA and MMP9 in Tca8113, Tb and Ts increased in turn, while the protein expression of nm23-Hl, CD44H decreased in Tca8113, Tb and Ts in turn.Our initial idea of the experiments was to select highly metastatic tongue cancer cells by tail vein injection and orthotopic implantation; during the study we unexpectedly found spinal cord metastasis in one nude mouse. Accordingly, we established human Ts cell line derived from spinal cord metastasis of TbTl cells. Compared with Tb, Ts cells have more S-phase in the population, and shorter doubling...
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