癌干细胞与肿瘤无限增殖、复发和转移密切相关，但化疗和放疗等常规手段并不能有效的杀灭癌干细胞。目前有关膀胱癌干细胞的研究较少。本课题组在前期工作中通过分离侧群细胞的方法成功从人类膀胱癌细胞系T24和小鼠膀胱癌细胞系MB49中分离出膀胱癌干细胞，并初步证实MAGE-A3具有特异性高表达。本项目将在此基础上：（1）通过全面的体内、外实验证实分离出的侧群细胞富集膀胱癌干细胞，并且MAGE-A3在膀胱癌干细胞中有特异性高表达；（2）利用基因转染等分子生物学技术，检测MAGE-A3对膀胱癌干细胞的调控作用，并研究相关的调控机制是否通过调控Wnt/?-Catenin信号通路完成；（3）鉴于MAGE-A3是一个肿瘤相关抗原，而且免疫治疗对膀胱癌有效，故拟制备MAGE-A3 DNA疫苗，探索针对膀胱癌干细胞的免疫治疗的有效性及相关的免疫应答机制。进而为攻克膀胱癌高复发这个临床难题提供新的治疗思路和治疗靶点。

Accumulating evidence suggests that a subgroup of cancer cells with the abilities of tumor initiation, self-renewal and differentiation, namely the cancer stem cells (CSCs), drive the formation and progression of tumors. CSCs are resistant to standard therapeutic modalities, including chemotherapy and radiotherapy in various mechanisms. Thus, CSCs are thought to cause disease recurrence post-therapy, making the disease untreatable. Therefore, efficient CSC-targeting therapy is needed to cure cancer. We have identified bladder cancer stem cells from human bladder cancer cell line T24 and murine bladder cancer cell line MB49 by flow cytometry-based side population analysis. We also detected a strong up-regulation of MAGE-A3, which is one of tumor-associated antigens, in these stem cell-like side population cells. Based on these data, we propose this project including the following aspects: (1) We plan to confirm the side population cells isolated from bladder cancer cells as cancer stem cells via in vitro assays of colony formation, sphere formation, invasion, migration and apoptosis as well as in vivo xenotransplantation of a small number of these cells in immunodeficient mice. Furthermore, we will detect the up-regulation of MAGE-A3 in these bladder cancer stem cells by RT-PCR, Western blot and immunofluorescence; (2) To address the function of MAGE-A3 in the maintenance of bladder cancer stem cells and possible mechanism, we plan to knockdown MAGE-A3 gene by RNAi and overexpress MAGE-A3 gene by transfection, then we will investigate the changes of the percentage of side population cells, their tumor-initiating ability by in vitro and in vivo assays as well as the activation status of Wnt/?-Catenin signaling pathway; (3) Given that MAGE-A3 is one of tumor-associated antigens expressed in a variety of tumors as well as bladder cancer is one of malignancies with well-documented response to immunotherapy, we plan to construct MAGE-A3 DNA vaccine and evaluate its antitumor effect as well as the immune mechanism involved in this approach in mouse bladder cancer models. Our findings will clarify the existence of bladder cancer stem cells, identify MAGE-A3 as a new marker with the capacity regulating bladder cancer stem cells via Wnt/?-Catenin signaling pathway and provide an promising therapeutic approach, CSC-targeting immunotherapy for bladder cancer.

具有肿瘤起始，自我更新和分化能力的癌症干细胞（CSCs）被认为能引起治疗后复发和促进癌症进展。然而，CSCs通常对目前的癌症治疗包括化学疗法和放射疗法具有抗性。本研究利用流式细胞术SP技术从人膀胱癌细胞系SW780中分离出癌干细胞侧群（SP）细胞。SP细胞仅约为SW780细胞的3.6％，并显示ATP结合盒亚家族G成员2（ABCG2）和CD133的更高表达。肿瘤球体生长的体外测定以及异种移植物移植的体内测定证实了分离的SP细胞具有更高致瘤性。这些数据表明SP细胞富含膀胱癌的CSC。此外，我们确定了黑素瘤抗原家族A3（MAGEA3）（这是研究最多的癌症睾丸（CT）抗原之一）在来自SW780细胞的这些SP和主要群体（MP）细胞中的表达。代表膀胱癌CSCs的SW780 SP细胞中MAGE-A3的表达表达上调，而且MAGE-A3和CD133共表达上调，表明MAGE-A3是膀胱癌CSCs中优先表达的新型CT抗原。综上所述，我们的研究结果证实膀胱癌细胞中存在癌干细胞样SP细胞，进一步表明MAGE-A3是一种新型的CSC抗原，因此可作为膀胱癌CSCs的免疫治疗靶点。