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

摘要

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).