基于多光子成像的脑胶质瘤微环境演进过程三维高分辨可视化研究

The glioma microenvironment, composed of the tumor and normal cells, microvascular network, collagen, etc., plays an important role in the initiation, progression, and metastasis of glioma. Acquiring accurate morphological and functional information of each component in the glioma microenvironment dynamically is the basis for understanding the pathogenesis of glioma and the prerequisite of developing specific diagnosis and treatment strategies. In our previous studies, we have developed a multiphoton microscopy merely relying on endogenous contrast. Employing this technology, a hamster cheek model of oral squamous epithelial precancer has been successfully interrogated in vivo without applying any exogenous contrast agent. In this study, to perform a systematic investigation on glioma microenvironment development, we will further expand and optimize this technology. Due to the severe scattering property of the skull, it is usually difficult to access an intravital and orthotopic glioma by optical imaging methods. To solve this problem, we will develop novel tissue optical clearing reagents and adaptive optics methods. By integrating them into label-free multiphoton microscopy, intravital three-dimensional high-resolution imaging of glioma in its native physiological environment will be realized with a whole skull. Specifically, by exploiting the proposed technology, we will dynamically monitor the proliferation and migration of tumor cells, the progression of tumor angiogenesis, as well as the architecture and development of the collagen fiber network in the glioma microenvironment. This study may shed new light on the fundamental pathogenesis of glioma, and furtherly provide new clues for the development of novel diagnosis and treatment methods for glioma.

脑胶质瘤微环境在脑胶质瘤的发生、发展和转移过程中起着十分重要的角色。准确获取脑胶质瘤组织及周边区域的肿瘤细胞、正常细胞、微血管网络、胶原纤维等构件的形态与功能信息，并记录其随时间的变化情况，是认识脑胶质瘤发病机理的基础，也是创建抗癌策略的前提。我们前期研究开发了一整套基于内源性光学信号（荧光信号和光学谐波信号）的多光子显微成像方法，利用该方法成功实现了仓鼠口腔肿瘤模型的在体、无标记、高分辨三维显微成像。本项目将进一步拓展及优化该方法，针对脑胶质瘤有颅骨保护较难观察的特殊情况，研究结合新型组织光透明技术和自适应光学技术，实现非开颅状态下的在体三维高分辨成像。利用以上技术，我们将在小鼠脑胶质瘤真实自然环境状态下，动态监测（1）肿瘤细胞生长及迁移情况，（2）肿瘤血管新生进程，（3）肿瘤内胶原纤维网络的形态及功能变化情况。本项目的开展对了解脑胶质瘤发生发展机理、探索新型诊疗方法具有重要作用。

脑胶质瘤微环境在脑胶质瘤的发生、发展和转移过程中起着十分重要的角色。准确获取脑胶质瘤组织及周边区域的肿瘤细胞、正常细胞、微血管网络、胶原纤维等构件的形态与功能信息，并记录其随时间的变化情况，是认识脑胶质瘤发病机理的基础，也是创建抗癌策略的前提。遗憾的是，目前研究人员缺乏相关技术能同时实现：（1）高分辨三维成像，（2）非开颅、无需造影剂的微\无创成像，（3）活体连续观测的成像能力。我们前期研发了基于内源性光学信号的双光子显微成像技术。本项目在此基础上进一步研究了新型组织光透明技术和自适应光学技术。基于此系统，我们实现了活体、非开颅、高分辨、三维的脑组织和脑肿瘤成像。我们追踪了神经树突棘的生长变化，观测到神经元钙离子放电情况以及脑胶质瘤生长情况；研究了在脑血栓导致脑组织缺血状态下的神经元新陈代谢变化情况；研究了脑胶质瘤生长过程中小胶质细胞的活化情况等。本项目相关研究已发表SCI期刊论文11篇，另有2篇尚在投稿中。申请PCT专利1项、中国发明专利7项（其中授权1项）、实用新型专利2项（授权2项）。培养博士后出站2人、已毕业硕士研究生3人、在读博士研究生2人。本项目的开展为脑胶质瘤发生、发展等机理研究提供了全新的技术和方法。