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信构成它们的小生境的细胞是 对于癌症发展、进展、转移和治疗抗性的许多方面至关重要。这些 互动往往是动态的、短暂的和复杂的。此外，细胞间通讯直接发生在 通过接触依赖性细胞-细胞相互作用和间接通过可溶性因子分泌到局部 微环境设计用于识别和鉴定的邻近依赖性标记策略的当前局限性 表征体内直接和间接细胞相互作用配偶体的试剂包括毒性和/或限制性的试剂 相互作用细胞的分离和随后的表征，非特异性或大半径标记（即， 直接与间接接触），或者需要预先设计两个相互作用的细胞，这避免了 识别新的细胞相互作用伙伴的可能性。因此，更理想的体内邻近依赖 标记系统将同时但不同地标记多个细胞的直接和间接细胞接触。 使用与一系列下游分析兼容的技术， 单细胞分析。在本提案中，我们将解决当前技术中的这一缺点， 全面鉴定和表征体内直接和间接的细胞间相互作用。我们将建立 基于文献和我们开发新方法的初步工作， 或其它感兴趣的细胞同时、有差别地和暂时地直接和间接地荧光标记 癌症小生境内的细胞接触（目的1和2）。重要的是，标记的细胞将适合于阵列 下游分析，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。因此，作为概念验证，我们将联合收割机将这种小生境标记技术与单细胞 RNA-seq开始定义中枢神经系统白血病早期阶段的细胞接触 转移和生态位发育（Aim 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).