免疫治疗过程中肿瘤微环境内免疫细胞可视化活体光学成像研究

发布时间：2018-07-08 13:27

本文选题：活体光学成像 + 多色标记　；参考：《华中科技大学》2016年博士论文

【摘要】：肿瘤免疫治疗的全过程类似于一个“黑匣子”,科学家们已了解的是输入信息“免疫治疗”和输出结果“肿瘤消退”。然而,人们对于整个治疗过程中在什么时间和地点发生了哪些重要的细胞与分子事件,目前仍然所知不多。活体光学成像技术具有可视化、高时空分辨率、高灵敏度、实时动态和多通道检测等优点,在肿瘤免疫学研究领域已展现了其巨大的应用潜力。活体光学成像技术可以对肿瘤免疫治疗过程中肿瘤微环境内多种免疫细胞的聚集、迁移以及细胞接触等事件进行长时程动态研究,给相关免疫学机制解析提供直观可视化动态信息。本论文中,我们使用环磷酰胺-过继细胞(CTX-ACT)联合免疫治疗方案,在活体肿瘤微环境内观察免疫细胞与黑色素瘤细胞之间的博弈过程。尽管CTX-ACT联合免疫疗法在临床已经取得了一定的抗肿瘤疗效,但是这一治疗方案是如何影响肿瘤微环境内参与的免疫细胞,进而影响免疫治疗效果,目前仍然不清楚,尤其缺乏肿瘤微环境内多种免疫细胞的运动行为特征,以及使用这些信息表征其免疫学功能这方面的研究。因此,本论文构建了多色标记的肿瘤微环境模型,通过大视野活体显微成像技术对于CTX-ACT联合免疫治疗诱发的免疫反应关键细胞事件,在肿瘤微环境内进行直观可视化观察与研究。进而,根据活体成像的结果对免疫治疗方案进行改进,获得了更加理想的肿瘤免疫治疗效果。本论文主要取得了以下研究成果:(1)建立适用于活体光学成像的多色标记肿瘤微环境的荷瘤鼠模型。本论文研究工作基于多色荧光标记技术、大视野活体光学显微成像技术以及小鼠背部皮窗模型,实现了对肿瘤微环境的蓝色荧光蛋白(CFP)标记的黑色素肿瘤细胞,近红外染料(Di R-BOA)标记的血管,以及绿色荧光蛋白(EGFP)、红色荧光蛋白(m RFP)和黄色荧光蛋白(YFP)标记的多种免疫细胞的同步动态显微成像,为活体显微光学成像系统性研究肿瘤免疫治疗过程中肿瘤微环境内多种免疫细胞的时空动态变化提供了理想的活体多色标记模型。(2)显微光学成像动态观察特异性CTL(细胞毒性T细胞)体外杀伤肿瘤细胞的全过程。激光共聚焦时间序列成像的结果显示,在体外培养的条件下,几十个CFSE(绿色荧光染料)标记的CTL围绕在一团CFP标记的B16细胞周围,相互接触30~40min后,B16细胞的形态一开始并未发生显著性变化,但是其荧光信号逐个迅速消失,细胞形态快速瓦解,出现“瞬间破裂”的快速连锁式死亡,这一成像结果提示我们,CTL在体外对肿瘤细胞的杀伤反应是快速、高效的。(3)活体光学成像研究肿瘤免疫治疗过程中的免疫细胞动态参与及其与疗效的相关性。实现了对CTX-ACT联合免疫治疗后肿瘤微环境中m RFP标记的Treg,CFSE标记的回输CTL,YFP标记的DC以及CFP标记的B16肿瘤细胞的活体长时程动态显微成像观察。活体成像结果表明,Treg在肿瘤发生发展的早期聚集到肿瘤区域,并在实体肿瘤形成后,围绕在其周围形成一个环形的“免疫耐受圈”,导致大部分回输的CTL无法迁移到肿瘤区域。在ACT治疗前对荷瘤小鼠进行CTX治疗,可以清除荷瘤鼠的大部分Treg细胞,从而无法在实体肿瘤外围形成“免疫耐受圈”,这有利于回输CTL和DC募集到肿瘤区域并浸润到肿瘤实质内部,进而有效地诱发抗肿瘤免疫反应。(4)动态研究肿瘤免疫治疗过程中多种免疫细胞的运动行为特征。活体成像结果表明,CTX-ACT联合治疗后,回输的CTL在肿瘤区域的运动可以细分为四个阶段。在进入第3阶段(CTX-ACT治疗后第5天)时,CTL可以大量募集到肿瘤区域,并且其在肿瘤边缘和肿瘤实质内的运动活跃程度都迅速增加,分别达到5.79±3.12μm/min和3.22±2.58μm/min。与此同时,对肿瘤的杀伤效应达到顶峰,肿瘤出现大面积死亡,外部开始“萎缩”,内部开始“瓦解”。在CTX-ACT联合治疗后24 h,内源的TII(肿瘤浸润性免疫细胞)被诱导活化,其平均速度迅速增至4.92±2.8μm/min,该速度是ACT治疗前的2.4倍,是联合治疗后48 h的3.26倍。与此同时,大部分TII的运动是朝向肿瘤实质内快速迁移,从而进一步激活后续的抗肿瘤免疫反应。活体显微成像对多种免疫细胞(DC、CTL以及TII)的运动行为进行观察发现,免疫细胞的活体运动具有“走走停停相互交替”以及“折线形运动”这两个显著特征,利用本实验室建立的“折线形广义利维运动”模型能够精确地描绘免疫细胞这两个活体运动特征。综上所述,本论文建立了多色标记的肿瘤微环境荷瘤鼠模型,通过活体显微成像技术,成功获取了肿瘤免疫治疗过程中肿瘤微环境多种免疫细胞的聚集、空间分布、迁移以及相互接触等动态信息,从而动态展示了CTX-ACT联合治疗抑制肿瘤生长的过程:联合治疗阻止Treg在实体肿瘤外周形成“免疫耐受圈”,诱发内源性TII短暂活化并朝向肿瘤区域快速迁移,进而促进回输CTL和DC募集到肿瘤区域并浸润到实质内。活体动态成像结果表明,外源的CTX与ACT联合治疗过程诱导内源性免疫活化,所产生的“协同免疫增强”效应是获得理想肿瘤免疫治疗效果的关键因素。
[Abstract]:The whole process of tumor immunotherapy is similar to a "black box", which scientists have known is the input information "immunotherapy" and the output "tumor regression". However, what important cell and molecular events have occurred at what time and location throughout the treatment are still unknown. Imaging technology has the advantages of visualization, high temporal spatial resolution, high sensitivity, real-time dynamic and multi-channel detection. It has shown great potential application in the field of cancer immunology. The living optical imaging technology can accumulate, migrate, and contact a variety of immune cells in tumor Microrings during the tumor immunotherapy. In this paper, we use a combined immunotherapy of cyclophosphamide adoptive cells (CTX-ACT) to observe the game between immune cells and melanoma cells in a living tumor microloop, although CTX-ACT combined immunization. The treatment has achieved a certain antitumor effect in clinical, but how this treatment affects immune cells involved in the tumor microring, and then affects the effect of immunotherapy, is still unclear, especially the lack of the movement of multiple immune cells in the tumor microring, and the use of these information to characterize its immunology. Therefore, the tumor microenvironment model of polychromatic markers is constructed in this paper. Through the large field of vision microimaging technique, the key cell events induced by CTX-ACT combined immunotherapy are visualized and studied in the tumor microloop, and then the immune system is immune to the results of living body imaging. The treatment scheme has been improved to achieve better effect on tumor immunotherapy. The main achievements of this thesis are as follows: (1) the establishment of a tumor bearing mouse model suitable for the multicolor microenvironment of vivo optical imaging. The mouse back skin window model realized the synchronous dynamic microimaging of many immune cells marked by blue fluorescent protein (CFP) labeled with the tumor microenvironment, the blood vessels labeled by the near infrared dye (Di R-BOA), and the green fluorescent protein (EGFP), the red fluorescent protein (m RFP) and the yellow fluorescent protein (YFP). Microscopical imaging systematically studies the spatiotemporal dynamic changes of multiple immune cells in tumor Microrings during tumor immunotherapy. (2) microscopic optical imaging dynamic observation of the whole process of specific CTL (cytotoxic T cells) killing tumor cells in vitro. Laser confocal time sequence imaging The results showed that, under the conditions of in vitro culture, dozens of CFSE (green fluorescent dye) labeled CTL around a group of CFP labeled B16 cells, after contact with 30~40min, the morphology of B16 cells did not change significantly at the beginning, but the fluorescence signal quickly disappeared, the cell morphology rapidly disintegrated and appeared "instantaneous rupture". Rapid linkage death, this imaging result suggests that the killing response of CTL to tumor cells in vitro is rapid and efficient. (3) living body optical imaging studies the dynamic participation of immune cells in the process of tumor immunotherapy and the correlation with the curative effect. The Tre marker of M RFP in the tumor microenvironment after CTX-ACT combined immunotherapy has been realized. G, CFSE labeled CTL, YFP labeled DC, and CFP labeled B16 tumor cells in vivo long term dynamic microimaging. The living imaging results showed that Treg was gathered into the tumor area early in the tumor development, and formed a circular "immune tolerance circle" around it, leading to large part of the tumor after the formation of solid tumors. CTL can not be migrated to the tumor area. Before ACT treatment, CTX treatment of tumor bearing mice can remove most of the Treg cells of the tumor bearing mice, which can not form the "immune tolerance circle" on the periphery of the solid tumor, which is beneficial to the return of CTL and DC to collect the tumor area and soak into the tumor essence, and then effectively induce the anti swelling. The tumor immune response. (4) dynamic study of the movement behavior of various immune cells in the process of tumor immunotherapy. In vivo imaging results showed that after CTX-ACT combined treatment, the movement of the returned CTL in the tumor area could be subdivided into four stages. In the third stage (fifth days after CTX-ACT treatment), the tumor area could be raised in a large amount. At the same time, the activity activity in the tumor edge and tumor substance increased rapidly, reaching 5.79 + 3.12 m/min and 3.22 + 2.58 mu m/min. respectively, at the same time, the tumor killing effect reached the peak, the tumor occurred large area death, the outside began to "atrophy", and the internal "disintegration". After the combined treatment of CTX-ACT, 24 h, endogenous TII ( The tumor infiltrating immune cells were induced to be activated, and the average speed increased rapidly to 4.92 + 2.8 m/min, 2.4 times as much as before ACT treatment and 3.26 times that of 48 h after combined treatment. At the same time, most of the TII movement was moved towards the tumor substance rapidly, thus further activating the subsequent anti-tumor immune response. The exercise behavior of multiple immune cells (DC, CTL and TII) was observed. The activity of the living body of the immune cells was characterized by the two distinct characteristics of "walking and stopping each other" and "folded line movement". The model of "folded line generalized Levi movement" established in our laboratory could accurately describe the two living bodies of immune cells. In summary, a tumor microenvironment tumor mouse model with multicolor markers was established in this paper. Through the microimaging technique of living body, the dynamic information on the aggregation, spatial distribution, migration and contact of various immune cells in the tumor microenvironment was successfully obtained by living microscopic imaging technique, which dynamically demonstrated the combined treatment of CTX-ACT. The process of tumor growth: combined therapy prevents Treg from forming a "immune tolerance circle" in the periphery of the solid tumor, inducing transient activation of endogenous TII and moving rapidly towards the tumor area, and then promoting the retransmission of CTL and DC to collect the tumor area and infiltrate into the substance. The synergistic immune enhancement effect induced by endogenous immune activation is the key factor to achieve the desired effect of tumor immunotherapy.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.51

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