Development of a Novel Method for the Identification and Characterization of Intercellular Communication in the Cancer Niche

摘要 癌细胞之间的细胞间相互作用和交流组成它们的利基的细胞是 对癌症的发展、进展、转移和治疗耐药的许多方面都至关重要。这些 互动往往是动态的、瞬时的和复杂的。此外，细胞间的通讯直接发生。 通过接触依赖的细胞-细胞相互作用和间接通过分泌可溶性因子进入局部 微环境。与邻近相关的标记策略目前的局限性旨在识别和 表征体内直接和间接细胞相互作用伙伴包括有毒和/或限制的试剂 相互作用细胞的分离和随后的鉴定、非特异性或大半径标记(即 直接接触与间接接触)，或者要求预先设计两个相互作用的单元，从而避免 识别新的细胞相互作用伙伴的可能性。因此，一种更理想的体内邻近依赖 标记系统将同时但不同地标记直接和间接细胞接触 时间点使用与一系列下游分析兼容的技术，从显微镜 到单细胞分析。在本提案中，我们将解决当前技术中的这一缺陷 全面识别和表征体内细胞间的直接和间接相互作用。我们将建造 根据文献和我们的初步工作，开发了一种新的方法，在这种方法中， 或其他感兴趣的细胞，同时，差分地和时间地荧光标记直接和间接 癌症缝隙内的细胞接触(目标1和2)。重要的是，标记的单元将适用于阵列 下游分析，如显微镜、流式细胞仪和细胞分选，然后是成批或单个细胞 分析。因此，作为概念验证，我们将把这种利基标记技术与单细胞相结合 RNA-SEQ开始定义中枢神经系统白血病早期的细胞接触 转移和生态位发展(目标3)。

ABSTRACT Intercellular interactions and communication between cancer cells the cells that comprise their niches are critical for many aspects of cancer development, progression, metastasis, and therapy resistance. These interactions are often dynamic, transient and complex. Moreover, intercellular communication occurs directly by contact dependent cell-cell interactions and indirectly by the secretion of soluble factors into the local microenvironment. Current limitations to proximity dependent labeling strategies designed to identify and characterize direct and indirect cellular interaction partners in vivo include reagents that are toxic and/or limit the isolation and subsequent characterization of interacting cells, non-specific or large radius labeling (i.e. direct vs. indirect contacts), or a requirement to pre-engineer both interacting cells which obviates the possibility of identifying novel cellular interaction partners. Thus, a more ideal in vivo proximity dependent labeling system would concurrently, but differentially, label both direct and indirect cellular contacts at multiple timepoints using technology that is compatible with an array of downstream analyses ranging from microscopy to single cell analyses. In this proposal we will address this shortcoming in current technologies for comprehensively identifying and characterizing direct and indirect intercellular interactions in vivo. We will build upon the literature and our preliminary work to develop a novel approach in which an engineered cancer cell, or other cell of interest, concurrently, differentially and temporally fluorescently labels both direct and indirect cellular contacts within the cancer niche (Aims 1 and 2). Importantly, labeled cells will be suitable for an array of downstream analyses such as microscopy, flow cytometry, and cell sorting followed by bulk or single cell analyses. Accordingly, as proof-of-concept we will then combine this niche labeling technology with single-cell RNA-seq to begin to define cellular contacts during early stages of central nervous system leukemia metastasis and niche development (Aim 3).