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通讯（组成其生态位的细胞是） 对于癌症发生、进展、转移和治疗耐药性的许多方面都至关重要。这些 相互作用通常是动态的、短暂的和复杂的。此外，细胞间通讯直接发生 通过接触依赖性细胞间相互作用以及间接通过可溶性因子分泌到局部 微环境。目前设计用于识别和识别邻近依赖标记策略的局限性 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后续表征、非特异性或大半径标记（即 直接接触还是间接接触），或者需要预先设计两个相互作用的细胞，从而避免 识别新的细胞相互作用伙伴的可能性。因此，更理想的体内接近依赖 标记系统将同时但有区别地标记多个细胞的直接和间接接触 使用与显微镜等一系列下游分析兼容的技术来确定时间点 单细胞分析。在本提案中，我们将解决当前技术的这一缺陷 全面识别和表征体内直接和间接细胞间相互作用。我们将建设 根据文献和我们的初步工作，开发一种新方法，其中工程化癌细胞， 或其他感兴趣的细胞，同时、差异和时间荧光标记直接和间接 癌症生态位内的细胞接触（目标 1 和 2）。重要的是，标记的细胞将适用于阵列 下游分析，例如显微镜、流式细胞术和细胞分选，然后进行批量或单细胞分析 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结合起来 RNA-seq 开始定义中枢神经系统白血病早期阶段的细胞接触 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).