靶向并激活胶质瘤微环境中TLR9联合沉默STAT3的抗瘤作用及分子机制研究

Glioma is the most common brain tumor, although with the combined treatment strategy including surgery, radiotherapy and chemotherapy, prognosis for high grade glioma especially glioblastoma multiform (GBM) altered little over the past 10 years, with a median survival less than 15 months, thus new treatment strategy are urgently needed. Our previous study indicated that the TLR9 signaling pathway constitute an important cellular pathway in glioma. TLR9 may represent a useful biomarker in glioma. We demonstrated the relationship between TLR9 expression and glioma progression. The expression of TLR9 in glioma microenvironment is a major factor and promoting the expression and secretion of chemokines and cytokines.TLR9 has been shown to activate the NF-kB pathway, leading to Th1 immunostimulation and antitumor immune responses. TLR9 ligands released by microenvironment resulting in the activation of a myeloid differentiation primary response 88 (MYD88)- and NF-κB-dependent signaling pathway that promotes the secretion of interleukin (IL)-6 and hence the activation of JAK/STAT3 signaling. The ability of STAT3 is potently suppress Th1 immune responses and antitumor immunity. For malignant glioma, persistent activation of a STAT3-regulated gene network is critical for tumor progression, mesenchymal transition, and negatively impacts on patient survival. Because of glioma cells, microglia and tumor infiltrating lymphocytes in glioma-microenvironment are all expressing TLR9, therefore TLR9 pathway activation is a double-edged sword. In this study, we propose a strategy of linking STAT3-siRNAs to CpG-ODN, agonist for TLR9, for targeted delivery of siRNA into glioma microenvironment. The endosomal location of the TLR9 might be advantageous in facilitating the siRNA component to reach the cytosol for efficient gene silencing in cells selectively expressing the cognate TLR9. We believe that the targeted inhibition of TLR9/STAT3 signaling in the tumor microenvironment may constitute a novel approach for glioma patients. Better understanding of the function and regulation of the TLR9 signaling pathways in glioma microenvironment may shed new lights on understanding the mechanisms of glioma formation and progression, as well as provide new targets for more effective regimens to treat gliomas.

胶质瘤是最常见的恶性脑肿瘤，手术结合放化疗的疗效仍不能令人满意，迫切需要新的治疗策略。我们先前证实胶质瘤高表达TLR9，并促进肿瘤增殖和侵袭，可作为胶质瘤微环境的分子标记物。微环境中行使免疫功能的胶质细胞和淋巴细胞TLR9通路激活后，可使免疫向有利于抗瘤反应的Th1偏移。但TLR9通路激活后产生的IL-6又会激活STAT3，是肿瘤微环境中免疫抑制和免疫逃逸的主要原因。所以TLR9通路在胶质瘤微环境中具有两面性。因此我们提出“将TLR9配体CpG-ODN与STAT3-siRNA相连接，这种合成核酸片段可定向制导于表达TLR9的胶质瘤微环境中，必会产生强有力的抗瘤反应”的治疗假说。定位于胞内的TLR9将提高siRNA导入的准确度和效率，既扩大Th1样抗瘤效应，又打破STAT3促瘤的恶性循环。本课题将在体外研究其抗瘤机制，并在胶质瘤动物模型中验证其抗瘤效应。这可为胶质瘤治疗提供一种新思路。

鉴于胶质瘤较高的恶性程度以及较差的预后，我们在本项目中研发了新型的治疗方案，即对靶向胶质瘤高表达的TLR9的治疗方案进行了优化。微环境中行使免疫功能的胶质细胞和淋巴细胞TLR9通路激活后，可使免疫向有利于抗瘤反应的Th1偏移。然而，TLR9通路激活后产生的IL-6又会激活STAT3，是肿瘤微环境中免疫抑制和免疫逃逸的主要原因。所以TLR9通路在胶质瘤微环境中具有两面性。因此我们提出“将TLR9配体CpG-ODN与STAT3-siRNA相连接，这种合成核酸片段可定向制导于表达TLR9的胶质瘤微环境中，可能会产生强有力的抗瘤反应”的治疗假说。定位于胞内的TLR9将提高siRNA导入的准确度和效率，既扩大Th1样抗瘤效应，又打破STAT3促瘤的恶性循环。我们的研究数据显示，（1）内源性核酸片段通过TLR9通路增强小胶质细胞和淋巴细胞的活化。U87胶质瘤细胞系来源的核酸片段，在50 ng/ml的浓度下对小胶质细胞的免疫激活水平，甚至达到了IFN-γ激活能力的82.06%±2.56%。（2）内源性核酸片段确实可通过TLR9通路增强胶质瘤细胞的恶行生物学行为。（3）CpGODN联合STAT3-siRNA可有效的防止TLR9通路激活所造成的胶质瘤细胞恶行生物学行为增强。（4）CpGODN联合STAT3-siRNA可以更有效的抑制肿瘤的生长。联合给药方案对肿瘤生长的 抑制率可达到41.68%±4.09%，显著优于CpGODN或STAT3-siRNA的单独给药组（抑制率分别为10.68%±0.81%和12.60%±1.72%）。由此可见，CpGODN和STAT3-siRNA联合给药方案为胶质瘤治疗提供一种新思路。