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

癌症的发生和发展涉及到肿瘤前细胞(PNC)和宿主环境之间复杂的相互作用。直到最近，还不可能实时成像肿瘤起始过程中宿主细胞和PNC之间最早的相互作用。冯实验室的早期工作，使用斑马鱼作为模式生物，使人们有可能在活体内一开始就看到PNC[1]。这项工作揭示了PNC引起的营养炎症反应，在这种反应中，包括中性粒细胞和巨噬细胞在内的招募的先天免疫细胞至少部分地通过COX-2介导的PGE2产生促进了PNC的生长[1]。这立即为我们提供了一个潜在的癌症预防靶点。我们最近使用几种激活报告FISH(NFkB，TNFa，IRG1)和巨噬细胞报告FISH(mpeg1，mfap4)进行的活体成像工作显示，在PNC发育的生态位内招募的巨噬细胞是高度异质性的。来自文献的证据表明，在炎症反应过程中，巨噬细胞亚群可以在炎症组织内相互转换。在PNC发育的生态位中，不同表型的巨噬细胞状态之间的特殊平衡可能对PNC进展的最终结果产生重大影响。因此，我们需要更好地表征PNC发育生态位中巨噬细胞的全谱及其状态，并确定它们在PNC进展中的作用。单细胞RNA序列分析是一种强大的技术，有可能以一种公正的方式系统地定位PNC生态位中的所有巨噬细胞表型。这将揭示稀有种群，并分配不同细胞表型之间的发育关系。我们(YF/CPP)已经应用这项技术来表征PNC生态位内的中性粒细胞，并确定了一种独特但罕见的中性粒细胞群，这可能是PNC促进作用的关键。在此，我们建议使用最近建立的可诱导斑马鱼PNC模型[2]来分析从PNC生态位分离的巨噬细胞的单细胞RNA-SEQ分析以及PNC相关巨噬细胞的活体成像研究。这将使我们能够确定PNC促进巨噬细胞的特征，并阐明PNC促进表型是如何建立的。该项目的一个可能的结果是确定新的癌症预防靶点，调节宿主免疫细胞和PNC之间的相互作用。

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.