Establishment of Lingual Squamous Cell Carcinoma Cell Line, Cancer Stem Cell Line and the Grafted Tumor Cell Line Derived From Same Cancer Tissue
Oral squamous cell carcinoma is characterized by a high degree of local invasiveness to surrounding tissue and a high incidence of metastasis to cervical lymph nodes, which are the most common causes of treatment failure without operation. Therefore, we reported the establishment of new cell lines, such as, Nialym cell line and cancer stem cell (CSC) line derived from metastatic foci of lymph node of squamous carcinoma of the tongue, Nialymx cell line derived from the grafted tumor of Nialym cells in scid mice.
[Materials and Methods]
The tumor tissue was obtained from the metastatic foci of lymph node of 50 years-old Japanese male patient who debt squamous cell carcinoma of tongue. The tumor tissue extracted by operation was cut into small pieces by razor blades and dissociated with 0.1% trypsin-0.02% EDTA/PBS (-). The fibroblastic cells were decreased gradually during course of culture. The growth medium was used in this study as follows; DMEM/F12 supplemented 15% FBS, 100µM of glutaMAX, 0.1% of MEM-NEAA, 50U/ml of penicillin, 50µg/ml of streptomycin, and 0.25µg/ml of Fungizone.
The Nialym cell line and CSC cell line were established from the metastatic foci of lymph nodes, and Nialymx cell line was established from grafted tumor of Nialym cells into subcutis of scid mice. The CSC cell line was established by colonial cloning of pilling up colony composed of small round cells in the long-term primary cultures of metastatic foci of lymph nodes.
[Results]
Nialym cells had epithelial like cell shape, and composed of two kinds of cells, one was clear cell and the other was dark were observed by electron microscope. The dark cells had many waving tonofilaments which runs much in all directions in the cytoplasm. Nialym cells and expressed CK-17, p53 and Ki67, while the cells had no CK-13 expression by immunocytochemical. These results well assumed that the Nialym cells had a feature of oral squamous cell carcinoma. The grafts of Nialym cells into the subcutis of scid mice were dark red color, and hardness was intermediary degree. Histopathological findings of transplanted tumor was very similar to the original tumor. Nialymx was established from this grafted tumor tissue showed the similar of immunocytochemical findings to the Nialym cells. And, we recognized both of Nialym and Nialymx secreted vascular endothelial growth factor (VGEF) and hepatocyte growth factor (HGF). Furthermore, the CSC cell line composed of small round structured cells established from primary cultures of metastatic foci of lymph nodes had exiguous cytoplasm and the organelles which poorly developed. This cells expressed Nanog, Oct3/4 and Sox2 by RT-PCR analysis, showed the feature of cancer stem cell.
[Conclusion]
We successfully established lingual squamous cell carcinoma from the metastatic foci of lymph nodes cell line (Nialym), CSC cell line and the grafted tumor cell line (Nialymx) derived from squamous cell carcinoma of tongue of same patient. We suspect cell lines of three types of squamous cell carcinoma of tongue from the same patient is a valuable elucidation of the mechanism of metastasis of cancer, the development of immunotherapy and chemotherapy.
[Reference]
・Aidan G. Major, Luke P. Pitty, and Camile S. Farah, Cancer Stem Cell Markers in Head and Neck Squamous Cell Carcinoma, Stem Cells International, Volume 2013:1-13, 2013
・Yasuo Okada and Michiko Moride, Immunohistochemical Study of Differential Expressions of Cytokeratin-13, -14, - 17 and p53 in Epithelial Dysplasia and Carcinoma of the Tongue, Journal of Hard Tissue Biology, 19[2]:123-130, 2010