in vivo live imaging and Single cell gene expression analysis of macrophages during tumour initiation

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The development and progression of cancer involve a complex interplay between pre- neoplastic cells (PNCs) and the host environment. Until recently, it was not possible to live image the earliest interactions between host cells and PNCs during tumour initiation. Earlier work in the Feng lab, using zebrafish as a model organism, has made it possible to visualize a PNC at its inception in vivo [1]. This work revealed that a PNC elicits a Trophic Inflammatory response in which recruited innate immune cells including neutrophils and macrophages promote PNC growth, at least in part, through COX-2 mediated PGE2 production [1]. This has immediately provided us with a potential target for cancer prevention.Our more recent in vivo live imaging work using several activation reporter fish (NFkB, TNFa, Irg1) and macrophage reporter fish (mpeg1, mfap4) revealed that recruited macrophages within PNC developing niche are highly heterogeneous. Evidence from the literature suggests that macrophage subpopulations can inter-convert within inflamed tissue during the course of an inflammation response. Within the PNC developing niche, the particular balance among macrophage states with different phenotypes might have a major impact on the final outcome of PNC progression. Therefore we need to characterize better the complete spectrum of macrophages and their states within the PNC developing niche and to define their roles during PNC progression.Single cell RNA-sequence analysis is a powerful technique with the potential to systematically map all macrophage phenotypes within the PNC niche in an unbiased manner. This would reveal rare populations and assign developmental relations among different cellular phenotypes. We (YF/CPP) have already applied this technique to characterize neutrophils within the PNC niche, and identified a unique but rare neutrophil population that could be key to PNC promotion.Here we propose to use a recently established inducible zebrafish PNC model [2] for single cell RNA-seq analysis of macrophages isolated from the PNC niche and in vivo live imaging studies of PNC associated macrophages. This will allow us to characterize PNC promoting macrophage and to elucidate how the PNC promoting phenotype is established. A likely outcome of this project is the identification of novel targets for cancer prevention that modulate the interplay between host immune cell and PNC.