乳腺癌免疫微环境中调节性B细胞自我调控及负性协同刺激分子对肿瘤细胞浸润转移的机制研究

Despite the rapid advancement of biological therapies, breast cancer is still the leading causes of cancer mortality in women. The immunosuppression, immune escape and cancer cells invasion and metastasis are regarded as predicting factors for poor prognosis of breast cancer. The exact mechanisms of immunosuppression and metastasis remain yet to be fully understood. .Emerging data suggests that regulator B cells play important roles in inhibiting the immune response in autoimmune disorders and human tumors as well as murine tumor models. Meanwhile, PD-L1 and B7H3, well known as immune checkpoints, inhibit T-cell activation, maintain immune tolerance, and induce tumor immune evasion..In our investigation, elevated CD19+ CD24+ CD38+ regulatory B cells (Bregs) were observed in PBMCs of invasive carcinoma of breast (IBCa) patients compared with that in patients with fibroadenoma (FIBma) or healthy individuals, and a tight correlation existed between Bregs and Tregs. Furthermore, we found that PD-L1 expression was higher on Bregs in IBCa patients compared with patients with FIBma or healthy individuals, and that positive correlation exists between PD-L1+CD19+ CD24+ CD38+ Bregs and CD19+ CD24+ CD38+ Bregs. More importantly, the presence of PD-L1 on Bregs was negatively correlated with PD-1hi effector T cells or TFH. Together these observations suggested that PD-L1+Bregs may play important roles in breast cancer immunosuppression and immune escape..PD-L1 and B7H3, regarded as negative regulatory molecules in immune responses, are broadly expressed on cancer cells as well as T cells, B cells, and dendritic cells. Knocking down PD-L1 or Knocking in B7H3 in breast cancer cells, the gene expressions of Snail-1,Snail-2,Zeb-1,Zeb-2 and CXCR4 were found significantly changed. These observations suggested that PD-L1 and B7H3 in breast cancer cells maybe play important role in (Epithelial-mesenchymal transition, EMT) or cancer metastasis..So it is very important to analyze the relationship among Bregs, Tregs and TFH, and investigate the immuno-microenvironment where breast tumors evade the immune system and its molecular mechanisms as well as investigate the role of PD-L1 and B7H3 in breast cancer migration, invasion and metastasis under immune suppression. It would be helpful to understand negative costimulatory molecules and its roles in Bregs or tumor and shed lights to find new ways for tumor therapy.

肿瘤免疫微环境是肿瘤生长、浸润转移以及抵抗免疫治疗的重要内环境，与表达于免疫抑制细胞及肿瘤细胞表面的负性分子密切相关。PD-L1、B7H3等免疫系统重要负性协同刺激分子不仅表达在免疫细胞上，还广泛表达乳腺癌细胞。我们发现并证实乳腺癌患者外周血中存在高水平CD19+CD24+CD38+B细胞为Bregs，高表达PD-L1分子，且PD-L1+Bregs与Tregs成正相关，与non-Tregs成负相关，与诱导B细胞分化成熟的重要免疫细胞—TFH成负相关；表达在乳腺癌细胞表面的PD-L1分子除了与Bregs、Tregs的扩增相关，还可能介入乳腺癌上皮细胞间质化（EMT）；表达乳腺癌细胞表面的B7H3则与浸润转移相关分子CXCR4存在共表达。本课题拟通过体内和体外实验进一步明确负性协同刺激分子在对乳腺癌免疫微环境的维持、调控、分子机制及其介导肿瘤浸润转移的机制和信号通路，为乳腺癌治疗提供重要靶点。

肿瘤局部微环境是由肿瘤细胞与免疫细胞、细胞因子和基质等共同组成；肿瘤细胞所表达的负性分子通过各种方式影响着肿瘤微环境以及机体免疫大环境。.本课题系统分析了浸润性乳腺癌，尤其是三阴性乳腺癌（Triple Negative Breast Cancer，TNBC）肿瘤局部和外周血CD4+ T细胞与CD19+ B细胞及其亚群的特点、肿瘤细胞和血清中PD-L1等蛋白的表达水平以及PD-L1等负性分子与免疫细胞亚群之间的相关性。结果显示，浸润性乳腺癌患者外周血中仅Tregs、Bregs、记忆性B细胞（Memory B cell）、长寿命浆细胞（ Long-live PC）等细胞亚群的百分率显著高于健康人和乳腺纤维腺瘤患者，Th9细胞亚群则显著低于健康人和乳腺纤维腺瘤患者；同时，浸润性乳腺癌患者血清中存在高水平sPD-L1与IL-10，且成正相关性；进一步研究发现患者血清中sPD-L1、IL-10与Bregs细胞表面PD-1分子的表达也成正相关。体外实验证实肿瘤细胞微环境中存在的可溶性PD-L1蛋白，可通过扩增Bregs 细胞进而诱导Tregs细胞分化，最终促进肿瘤细胞抵抗CD4+ T细胞介导的凋亡；而这种作用能被PD-L1特异性阻断型单抗所阻断，从而减少Tregs比率，促进CD4+ T细胞对肿瘤细胞的杀伤。上述研究成果丰富了肿瘤局部微环境与患者整体免疫状态之间的相关性，丰富了负性分子PD-L1对乳腺癌免疫逃逸和拮抗凋亡中的作用，为以干预PD-L1信号为基础的肿瘤免疫治疗提供新的思路和靶点.肿瘤免疫微环境不但促进肿瘤生长、浸润与转移，也是抵抗各种治疗的重要内环境。本课题组还发现肿瘤细胞表达的IGF-1蛋白与其受体及信号参与TNBC亚型的阿霉素（DOX）耐药机制。利用研制成功的纳米载药系统，使低剂量DOX特异性富集肿瘤局部，诱导乳腺癌干细胞凋亡，减轻化疗药物对正常组织与细胞的毒副作用，并避免肿瘤细胞耐药的发生。 本研究组成功建株的一株TNBC细胞株，为针对中国人TNBC的肿瘤研究提供更精准的研究对象，也为纳米载药系统联合肿瘤免疫治疗提供物质基础。.