Research On Isolation And Determination Of Bladder Cancer Stem(Initiating) Cells

Objective: Bladder transitional cell carcinomas (BTCC) is the most common urologic tumor in China and it have unfavourable prognosis. Cancer stem cells hypothesis indicate that the reason for tumorigenesis, metastasis and recurrence of tumors is the existence of cancer stem cells in tumors. Cancer stem cell(CSCs) is the targets for tumor targeting therapy. Before therapy targeting to CSCs is developped,all undetermined CSCs should be isolated. CSCs have been positively identified and successfully isolated from some but not all cancers,including bladder cancer stem cells. However, the isolation and identification of undetermined CSCs has proven difficult because of the paucity of specific cell surface markers for cell sorting. This work was supported by the Doctor Foundation for Innovation, Tianjin Medical University grant yjs200604;Tian Jin Natural Science Foundation, Tianjin Municipal Science and Technology Commission grant 07JCZDJC03400.This study for isolating bladder cancer stem cells is on the basis of the previous work of Prof. Chang JiWu for bladder cancer research. Methods: 1. To confirm the existence of bladder cancer stem cells: Identifying the differentially expressed stem cells related genes, Bmi-1 and EZH2,between laser-microdissected low malignant bladder-cancer tissues and corresponding noncancerous mucosae by the combination of laser capture microdissection, oligo microarray analysis and mRNA linear amplification. Side population cells in bladder cancer were detected under fluorescence microscope. Expression of Marker of embryonic stem cells,OCT4, in bladder cancer was detected by immunohistochemistry. 2. To confirm the markers of bladder cancer stem cells : Human CD molecules were retrieved on the web. Either markers of homologous or heterologous somatic stem cells or progenitor cells or markers of tumorigenesis, progression, metastasis and recurrence in many malignant tumors, or both were retrieved on the PUBMED web.Then, confirming the potential markers of bladder cancer stem cells by immunohistochemistry. Confirming the markers of bladder cancer stem cells by colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. In the end, summarizing the methods for isolating undetermined cancer stem cells .Results: 1. Highly expressed stem cells related genes, Bmi-1 and EZH2, were found in bladder cancer cells. Side population cells in bladder cancer were confirmed under fluorescence microscope. Expression of marker of embryonic stem cells,OCT4, in bladder cancer was found 2. 227 human CD molecules were found on the web. Among the 227 human CD molecules,28 CD molecules might be the markers of cancer stem cells. They were CK34BE12,EMA ,CD9,CD10,CD14,CD19,CD20,CD24,CD33, CD34,CD38,CD44v6,CD45RO,CD49d,CD56,CD61, CD62L,CD71 ,CD72,CD81 ,CD8 2,CD90,CD95,CD117 ,CD133,CD178,MUC-1(CD227) and CD243. EMA and CD44v6 might be the markers of bladder cancer stem cells by immunohistochemistry, colony-forming assays,nude mice repopulating assay,and serial passage of nude mice repopulating assay. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers. Condusion: We conclude the persistence of BCICs. BCICs might be among EMA- CD44v6+ subset. Our strategies for isolating BCICs might be useful for isolating other undetermined epithelial CSCs, especially those in well-differentiated cancers.