Systems microscopy analysis of tumor cell motility in microenvironment context

描述(由申请人提供)： 候选人：格利戈里耶维奇博士在塞尔维亚贝尔格莱德主修分析化学，在乔治敦大学的研究生培训期间，她已经成为光学和活细胞显微镜方面的专家，并开发了几种基于显微镜的新技术。在博士后期间，格利戈里耶维奇博士一直专注于研究乳腺肿瘤中的肿瘤细胞在活体动物中的渗透。为了做到这一点，她引入了体内多光子显微镜的几项进展。格利戈里耶维奇博士是由美国国防部和查尔斯·雷夫森奖学金资助的，她的研究获得了几项荣誉。到目前为止，她的工作产生了一项专利，十项同行评议和十项其他出版物。最近的研究表明，肿瘤微环境的不同成分在肿瘤进展中起着至关重要的作用。为了破译塑造肿瘤环境和确定肿瘤细胞行为的相互作用网络，理想的做法是将系统生物学方法与活体显微镜相结合，因为细胞培养的复杂性有限，小鼠模型既耗时又昂贵，而计算机模型需要根据实验进行优化。在该奖项的指导阶段，Gligorijevic博士将把先进统计学和动力系统数学建模的相关原理融入到她的肿瘤生物学和显微镜背景中。她将参加爱因斯坦提供的课程，并定期与她的赞助商和共同赞助商会面。这种培训将使建立一个实验室成为可能，该实验室使用综合的系统显微镜方法并利用从分子到群体水平的信息来研究转移机制。 环境：爱因斯坦国际和平研究所的发起人约翰·康迪利斯博士是爱因斯坦大学解剖学系和生物光子学中心的联合主席。他的实验室和该中心创建了一个多学科环境，专注于通过使用显微镜回答人类疾病(如癌症)的机制。该中心以共享成像资源和创新实验室而闻名，其中新的显微镜系统是定制的，以满足不同项目的特定需求。咨询委员会咨询委员罗伯特·辛格博士是实验生物学和理论生物学相结合的专家，也是著名的导师。共同赞助人Aviv Bergman博士是爱因斯坦大学系统系的创始主席，教授的课程将成为职业发展的一部分。爱因斯坦是一所高度重视合作的机构，坚持博士后研究员、讲师和初级教员的职业发展。 研究：虽然大多数对肿瘤微环境的研究都集中在分离和理解单个参数上，但相关生物参与者之间缺乏一个完整的、系统级的相互作用网络。在原发肿瘤中，有许多生物力学信号能够引导肿瘤细胞向血管运动和进入血管。肿瘤微环境中宿主巨噬细胞、成纤维细胞和内皮细胞分泌的生长因子是主要的趋化因子，但最近的研究表明细胞外基质也起着重要作用。在松散的细胞外基质中，肿瘤细胞可以通过重组细胞骨架来迁移，产生突出力。在细胞外基质僵硬的区域，简单的运动是不可能的。在这里，肿瘤细胞变得侵袭并降解细胞外基质，主要是通过基质金属蛋白酶(MMPs)。肿瘤细胞在体内的迁移特性很好，但体内肿瘤细胞从运动状态到侵袭状态的转换以及侵袭性突起的组装机制尚不清楚。为了解决整合的微环境信号与肿瘤细胞行为之间的联系，有必要将细胞生物学、高级显微镜和系统生物学结合起来。在初步实验中，使用时间分辨3D多光子成像技术在活体小鼠体内记录了肿瘤细胞，并观察到两种不同的突起类型：a)运动性突起，它迅速导致肿瘤细胞的迁移；b)侵袭性突起，它是持久的，依赖于基质金属蛋白酶。目的1的目的是研究侵袭性突起在肿瘤细胞内渗和转移中的作用。一组荧光记者将被用于侵袭性突起组装的成像和随后的肿瘤细胞命运的调查。目的2探索驱动肿瘤细胞形成运动性或侵袭性突起或在行为之间切换的信号。在含有运动性或侵袭性突起的区域，同时记录肿瘤微环境参数(巨噬细胞数量、胶原蛋白硬度、血管大小等)。成像分析产生一个数据矩阵，该矩阵由支持向量机分类进行分析。分类表明，在非常特殊的条件下，肿瘤细胞从运动状态转换到侵袭状态。拟议的实验验证了这样一个假设，即只要稍微修改微环境参数，就可以改变肿瘤细胞的行为。在目标3中，将相关的微环境参数纳入了肿瘤细胞从运动状态到侵袭状态转换的综合数学模型中。利用该模型的预测能力，设计了抑制侵袭状态和后续转移的实验。实验结果将被用来优化和复杂化模型。这项研究的结果将有助于更好地了解肿瘤进展过程中微环境因素之间的相互作用，并将结果用于提高早期转移的诊断和治疗。

DESCRIPTION (provided by applicant): Candidate: Majoring as analytical chemist during the time in Belgrade, Serbia, Dr. Gligorijevic has become an expert in optics and live cell microscopy over the course of her graduate training at Georgetown University and developed several novel microscopy-based technologies. During the postdoctoral period, Dr. Gligorijevic has focused on studying tumor cell intravasation in breast tumors, in living animals. To do so, she introduced several advancements into in vivo multiphoton microscopy. Dr. Gligorijevic is funded by DOD and Charles Revson Fellowship and has received several honors for her research. Her work to date resulted in one patent, ten peer-reviewed and ten other publications. Recent research has shown that different components of tumor microenvironment have an essential role in tumor progression. In order to decipher the network of interactions which shape the tumor environment and determine tumor cell behavior, it is ideal to apply systems biology approaches combined with in vivo microscopy, as cell cultures are limited in their complexity, mouse models are time-consuming and expensive and in silico models need optimization based on experiments. During the mentored phase of the Award, Dr. Gligorijevic will incorporate the relevant principles of advanced statistics and mathematical modeling of dynamical systems into her background of tumor biology and microscopy. She will take coursework offered at Einstein and meet regularly with her sponsor and co-sponsor. This training will make it possible to build a laboratory which investigates mechanisms of metastasis using the integrative, systems microscopy approach and utilizing information from molecular to population levels. Environment: Sponsor of the PI, Dr. John Condeelis is a Co-Chair of Anatomy Department and Biophotonic Center at Einstein. His lab and the Center create a multidisciplinary environment focused on answering mechanisms of human diseases, such as cancer, through use of microscopy. The Center is well known for its shared imaging resources and Innovation Laboratory where new microscopy systems are custom-built to accommodate specific needs of different projects. Consulting member of Advisory Committee, Dr. Robert Singer, is an expert in combining experimental and theoretical biology and a renowned mentor. The co-sponsor, Dr. Aviv Bergman, is the Founding Chair of Systems Department at Einstein and teaches coursework which will be a part of the career development. Einstein is an institution which highly values collaboration and insists on career development of postdoctoral fellows, instructors and junior faculty. Research: While most research of tumor microenvironments focuses on isolating and understanding single parameters, an integrative, systems-level network of interactions among relevant biological players is missing. In primary tumors, there are numerous biomechanical signals able to direct tumor cell movement towards and into the blood vessels. Growth factors, secreted by host macrophages, fibroblasts and endothelial cells in the tumor microenvironment are the main chemoattractants but recent studies show that the extracellular matrix also plays an important role. In loose extracellular matrix, tumor cells can migrate by reorganizing their cytoskeleton, generating a protrusive force. In regions with stiff extracellular matrix, simple locomotion is not possible. Here, tumor cells become invasive and degrade extracellular matrix, mainly by matrix metalloproteases (MMPs). Tumor cell migration was well characterized in vivo, but the mechanism of the switch from locomotory to invasive state and assembly of invasive protrusions in vivo are unknown. To address the link between integrated microenvironment signaling and the tumor cell behavior, it is necessary to combine cell biology, advanced microscopy and systems biology. In preliminary experiments, tumor cells were recorded using time-resolved 3D multiphoton imaging in living mice and two different protrusion types were observed: a) locomotory protrusions which quickly lead to migration of the tumor cell and b) invasive protrusions, which are persistent and MMP-dependent. The goal of Aim 1 is to investigate the role of invasive protrusions in the tumor cell intravasation and metastasis. A combination of fluorescent reporters will be used for imaging of invasive protrusion assembly and investigation of consequent tumor cell fate. Aim 2 explores signals which drive tumor cells to form either locomotory or invasive protrusions or to switch between behaviors. In areas which contain either locomotory or invasive protrusions, tumor microenvironment parameters are recorded simultaneously (number of macrophages, collagen stiffness, blood vessel size etc.). Imaging analysis results in a data matrix, which is analyzed by a Support Vector Machine classification. Classification shows that tumor cells switch from locomotory to invasive states under very specific conditions. Proposed experiments test the hypothesis that tumor cell behavior can be changed by slightly modifying microenvironment parameters. Relevant microenvironment parameters are incorporated into an integrative mathematical model of the tumor cell switch from locomotory to invasive state, in Aim 3. Using the predictive power of the model, experiments were designed to inhibit the invasive state and subsequent metastasis. Experimental outcomes will be used to optimize and complexify the model. Results of this study will lead to better understanding of the interplay among microenvironment components during tumor progression and the results will be used to improve diagnosis and treatment of early metastasis.