共刺激分子OX40联合STAT3/Akt信号轴介导乳腺肿瘤干细胞浸润、转移及免疫抑制的分子机制

Despite the rapid advancement of biological therapies, the mortality rate of metastatic breast cancer remains high. The exact mechanisms of tumor progression and metastasis remains yet to be fully understood. Emerging data has shown that malignant tumors are composed of a small population of distinct cancer cells that are responsible for tumor initiation, termed cancer stem cells (CSCs). CSCs possess the ability to self-renew, have high proliferative potential and produce differentiated progeny, and postulated to be responsible for tumor initiation, metastasis and relapse after conventional therapies. OX40, a membrane-bound member of the tumor necrosis factor receptor (TNFR) superfamily, is an important costimulatory molecule in immune response. We first found and reported that OX40 was expressed on breast cancer cell lines as well as invasive breast carcinomas. We revealed that the level of OX40 was positively correlated TNM stages or lymph node status of breast cancer. We also examined using immunohistochemistry method to detect the expression and significance of OX40, CXCR4, MMP-9, and tumor cancer stem cell markers CD44, CD24 and their relationship with the histopathological parameters of breast cancer. We showed that OX40 were closely related with CXCR4, MMP-9, CD44, CD24 or cancer stem cells. More importantly, our cell cultrue study demonstrated that OX40 only expressed on surface of CD44+CD24-/lowCSCs in MCF-7 cells. Meanwhile, we demonstrated that OX40 significantly enhanced breast cancer cell migration. Together these observations suggested that OX40 on CSCs may play important roles in breast cancer aggressiveness and metastasis. As an important costimulatory molecule in immune response, OX40 plays an important role in proliferation, survival and infiltration of activated T cells via binding to its ligand OX40L. More recently, a novel role for OX40 was uncovered showing that OX40 promotes host immune tolerance by expanding functional Regulatory T cells. Therefore, administration of OX40 agonists was considered a useful method to treat cancer. So it is very important to investigate the role of OX40 in CSCs migration, invasion and metastasis as well as immune suppression. It would be helpful to understand OX40 and its roles in tumor and shed lights to find new ways for tumor therapy.

肿瘤转移是导致乳腺癌患者死亡的最直接原因，但其转移途径和确切机制尚不明确。现认为具有自我更新、多向分化潜能的肿瘤干细胞是肿瘤浸润、转移、治疗失败及复发的源泉；本课题组首次发现OX40分子在乳腺癌肿瘤干细胞CD44+CD24-/lowCSC中高表达，乳腺癌细胞株MCF-7的体外实验也证实该分子仅表达在CD44+CD24-/lowCSC表面。OX40分子与乳腺癌的恶性演进、淋巴结及远处转移成正相关，与肿瘤黏附、浸润及转移相关分子均存在密切联系。推测OX40信号的活化可能介导乳腺癌CD44+CD24-/lowCSC的黏附、浸润及转移；新近移植免疫的研究成果提示OX40信号的激活可扩增调节性T细胞导致免疫耐受。为证实上述观点，我们采用分子生物学等技术，从体外到体内乳腺癌转移模型，对OX40信号与乳腺癌CSC浸润、转移、免疫抑制及其信号传导通路进行研究，明确其分子机制，为转移性乳腺癌治疗提供新靶点。

OX40、PD-L1分属正性、负性共刺激分子，在平衡免疫应答中起着极为重要的作用，也与肿瘤免疫及肿瘤免疫逃逸息息相关。本课题系统分析了乳腺癌细胞表面、肿瘤浸润淋巴细胞以外周血中不同免疫细胞上OX40、PD-L1的表达变化，结果提示乳腺癌细胞均高表达OX40、PD-L1分子，其中OX40分子与乳腺癌淋巴结转移密切相关，进一步研究表明OX40表达于CD44+CD24-/low CSCs表面，其信号的活化可上调CXCR4的蛋白及mRNA表达从而介导肿瘤细胞浸润及转移；本课题组首先发现并报道了乳腺癌细胞所表达的PD-L1则与CD19+CD24+CD38+ Bregs扩增密切相关，Bregs为新近证实的一群重要的B细胞亚群，可通过诱导Tregs扩增来抑制免疫应答，参与肿瘤免疫逃逸；对不同免疫细胞亚群进行检测结果显示，乳腺癌患者外周血中存在高水平Bregs、Tregs，且Bregs高表达PD-L1分子并与Tregs成正相关，与效应性T细胞成负相关。相反，乳腺癌患者外周血中T细胞及Tregs表面则低表达OX40分子，体外实验也证实缺乏OX40信号活化可阻碍T细胞向效应性T细胞转化。上述研究丰富了共刺激分子在肿瘤微环境中的作用理论，丰富了共刺激分子对乳腺癌等肿瘤生长、浸润转移及免疫逃逸的认识，为以干预OX40、PD-L1信号为基础的肿瘤免疫治疗提供新的思路和靶点。.肿瘤免疫微环境是肿瘤生长、浸润转移以及抵抗免疫治疗的重要内环境，我们的发现CD44+CD24-/low CSCs与肿瘤免疫微环境存在密切关系，基于此，利用纳米载药系统借助丰富的肿瘤微血管特异靶向CD44+乳腺癌细胞，并在荷瘤小鼠模型中获得良好的治疗效果，为纳米载药系统走向肿瘤临床治疗奠定理论基础，也为干预OX40、PD-L1信号为基础的免疫治疗提供新的手段。.