Real time quantitative multiplex analysis of cell-cell interactions

描述（由申请人提供）：尽管长期以来人们都知道肿瘤细胞可以自给自足地生长和存活，但最近的研究强调了肿瘤与微环境细胞之间相互作用的重要性，这可以对肿瘤发展和持续生长起到支持作用。因此，了解癌症发展和开发更有效的治疗方法的关键是获得肿瘤细胞与其环境细胞相互作用的全貌。细胞-细胞相互作用是一个复杂的过程，可能涉及几种表面分子以及从细胞释放的分子。为了了解癌症微环境生物学，必须开发一种新技术，用于同时分析来自相互作用的不同细胞的多种分泌物和表面分子。我们的长期目标是开发一种用于细胞-细胞相互作用的通用设备，该设备可以由最终用户定制，用于分析大量的分泌物和细胞表面分子。本申请的目的是开发一种用于分析细胞间通讯的新技术，这将使我们能够测量真实的时间内相互作用的单细胞上的几种分泌物和表面分子。我们将使用这项新技术来研究慢性淋巴细胞白血病（CLL）患者的细胞与其微环境细胞之间的相互作用。我们选择使用CLL作为该应用的模型系统，因为它是一种容易获得的液体肿瘤，我们的团队成员Kipps博士在他的组织核心中有600多个表征的样品可供我们使用。此外，我们有数据表明，CLL细胞可以释放分泌物，导致细胞的环境获得的变化，这反过来又有利于白血病细胞的生存。了解CLL细胞释放的分泌物将使我们能够设计新的治疗方法。该应用程序的具体目标是：构建和验证一种新的工具，可以在两种不同的细胞类型相互作用的同时对许多分子进行定量和定性测量。使用新的装置来研究CLL细胞与其环境中的细胞之间的通信过程。研究设计和方法：将使用化学和生物工程方法来构建一系列微小的腔室，每个腔室容纳一个细胞。这些腔室将与小隧道相连，这些小隧道将允许分子通过，从而使相邻的细胞能够进行通信。小型探测器将被内置到腔室中，以报告从不同细胞释放的因子。将细胞分配到威尔斯孔中，并使用显微镜测量分泌物和表面分子。最近的证据表明，肿瘤可以操纵周围的“非肿瘤”细胞，这反过来又在肿瘤的发展和持续生长中发挥关键作用。我们将开发一种新技术，使我们能够真实的实时监测癌细胞及其环境细胞之间的复杂通信过程，目前还没有可用的工具。了解这些 相互作用将揭示治疗干预的可能新方法。

DESCRIPTION (provided by applicant): Though it has been known for long that tumor cells can be self-sufficient for growth and survival, recent studies highlight the importance of interactions between the tumor and cells of the microenvironment, which can play a supportive role for tumor development and sustained growth. Thus a key to understanding cancer development and develop more effective therapeutics is to gain a complete picture of tumor cell interactions with cells of its environment. Cell-cell interactions are a complex process that can involve several surface molecules as well as molecules that are released from cells. To develop an understanding of cancer microenvironment biology, a new technique must be developed for the simultaneous analysis of multiple secretants and surface molecules from different cells that are interacting. Our long-tern goal is to develop a generic device for cell-cell interactions that can be custom tailored by the end user for analysis of a high number of secretants and cell surface molecules of choice. The objective of this application is the development of a new technology for the analysis of cell-cell communication, which will allow us to measure several secretants and surface molecules on single cells that are interacting with one another in real time. We will use this novel technology to investigate interactions between cells from chronic lymphocytic leukemia (CLL) patients and cells of their microenvironment. We chose to use CLL as model system for this application, because it is an easily accessible liquid tumor and our team member Dr. Kipps has more than 600 characterized samples in his tissue core that are available to us. Furthermore, we have data showing that CLL cells can release secretants that cause cells of their environment to acquire changes which in turn are of benefit for leukemia cell survival. Understanding what secretants are released by CLL cells will allow us to design novel therapeutic approaches. The specific aims of this application are: Construct and validate a novel tool that can make quantitative and qualitative measurements of many molecules at the same time on two different cell types that are interacting. Use the new device to investigate the communication process between CLL cells and cells of their environment. Research Design and Methods: Chemical and bioengineering methods will be used to build a collection of tiny chambers, that each holds one cell. These chambers will be connected with small tunnels that will allow molecules to pass through and thus enable the cells that are next to each other to communicate. Small detectors will be build into the chambers that will report what factors are released from the different cells. Cells will be distributed into the wells and secretants and surface molecules will be measured using a microscope. Recent evidence demonstrates that tumors can manipulate surrounding "non-tumor" cells which in turn play a critical role in tumor development and sustained growth. We will develop a new technology that will allow us monitor the complex communication process between cancer cells and cells of their environment in real time, for which there is currently no tool available. Understanding these interactions will reveal possible new approaches for therapeutic intervention.