| Background Bladder cancer is one of the most common urologic neoplasms. In our country, the incidence of male bladder cancer was in the eighth of all tumors, while that of women after the twelfth. In recent years, reports from different cities in China showed its incidence was on the rise, though far below that of western countries. Muscle invasive bladder cancer is the main cause of death, as it is commonly associated with local invasion and distant metastasis. Total cystectomy and pelvic lymph node dissection may not free the patient of this type of disease. Currently, there are still no effctive preventive measures or treatments against tumors with lymph node or distant metastasis. The mechanisms involved in organ-specific metastasis are still not fully characterized. Metastatic bladder cancer samples are difficult to obtain, as bladder cancer patients with distant organ metastasis are not indicated for surgery, which limits the studies on metastatic mechnisms and related treatments. Tumor cell lines are the main means for various mechanisms study, bladder cancer is no exception. At present, most of the bladder cancer lines established were derived from patients from western countries. Due to racial differences, these cell lines may not be representative of the characteristics of Chinese patients. Thus, establishment of a Chinese bladder cancer cell line with multiple organ metastasis is of great importance for the study of the metastatic mechnisms and treatments.Objective Establish a Chinese highly malignant and metastatic bladder cancer cell line and a mouse multiple organ metastatic model, probe on metastatic mechanisms of bladder cancer and lay the foundations for the further studies on metastatic bladder cancer.Materials and methods Bladder cancer fresh tissues were obtained from a female patient with muscle invasive bladder cancer(pathological stage T2). Tissues were digested with collgenase and then cultured with RPMI1640 supplemented with 10% fetal bovine serum. Differential tripsin digestion method was used to remove stromal cells. After 10 passages, 1×10(?) cells were inoculated orthotopically into 3 murines(?) bladder.5 weeks later, the mice were executed and local invasion and distant metastasis were evaluated. The tumors were then harvested for IHC study and in vitro culture. After 10 passages, the cells were reinoculated orthotopically into murine bladder. And the resulting metastatic tumors were used for further studies.Result 1.Chinese bladder cancer cell line T921 was successfully established.2.IHC confirmed its urothelial origin.3.Chromosome analysis identified abnormal karyotype.4. Nude mice multiple organ metastasis model was successfully established. IHC showed that P53, which is rarely expressed in metastatic lesions, is higher in bladder lesions, while Ki-67 is higher in metastatic lesions. EMT related gene E-cadherin andβ-catenin were lost during metastasis. Western blot showed that N-cadherin, Slug and Snail were overexpressed in lymph node metastatic lesions. Fibronectin levels were not significantly changed. P27 were downregulated in metastatic lesions. qPCR results suggested that different pathway are involved in different organ metastasis. Flow cytometry analysis indicated that CD47 levels are higher in metastatic cell lines.Conclusion T921 bladder cancer cell line and bladder cancer multiple organ metastases model were successfully established. Preliminary experiment results suggest that P53 loss may be one of the mechanisms involved in bladder cancer progression. EMT related Genes and transcription factors and oncogenes commonly observed in bladder cancer examined in this study suggest that different mechanisms may be involved in different organ metastasis. Furthermore, T921 cells may also be used for further studies on the development, progression and metastasis of bladder cancer. Taken together, these results suggest that individualized target therapy should be offered to bladder cancer patients with different organ metastasis.
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