In vivo imaging of tumor heterogeneity generation from endogenous single cells

项目摘要 从良性肿瘤到恶性肿瘤的形成与以下因素的存在密切相关： 肿瘤内的异质细胞特征。因此，阐明产生的机制， 肿瘤细胞异质性将是理解恶性肿瘤形成的重要里程碑。我们认为 我们的工作的两个应用可以是1）基于分子生物学的更早、更定量的恶性肿瘤检测， 标记物和2）可以在转移之前冻结肿瘤发展或甚至恢复到正常水平的治疗剂。 良性状态我们的方法是回答以下两个问题： 1.肿瘤起始细胞的特性对产生肿瘤细胞异质性有什么影响？ 在迄今为止的实验中，无法避免污染不同的细胞来源，包括组织干细胞， 和非干细胞进入所产生的肿瘤块， 机制等我们通过建立实验系统来解决污染问题， 精确追踪源自单细胞的肿瘤细胞谱系。我们的方法是非侵入性的，在体内成像， 内源性，单细胞衍生的肿瘤在成年透明斑马鱼的发展。本实验系统 使我们能够可视化单个肿瘤细胞，更重要的是，还可以选择性地标记和分离肿瘤细胞。 对详细细胞表征感兴趣。使用这个系统，我们已经对来自于 皮肤上皮中的单个细胞。重要的是，我们发现了肿瘤细胞增殖的一些主要差异 在肿瘤细胞簇之间和肿瘤细胞分化中，导致获得侵袭性特征。我们 鉴定显示侵袭性特征的肿瘤细胞亚群的结果很好地代表了 肿瘤内的肿瘤细胞异质性。我们将对肿瘤的发展进行长期的克隆分析， 通过对分离的个体肿瘤进行转录组分析以确定它们的细胞身份。回顾性成像 分析将用于研究肿瘤微环境对产生肿瘤细胞异质性的影响。 2.肿瘤内的异质性是如何建立的？ 肿瘤起始细胞的细胞类型鉴定之后将研究其机制 在多轮细胞分裂期间驱动单个肿瘤起始细胞变得异质。我们的能力 区分性标记和分离侵袭性肿瘤细胞与非侵袭性肿瘤细胞使我们能够研究 基因表达调控导致肿瘤细胞亚群在肿瘤发生过程中获得侵袭性特征 发展我们将进一步进行体内功能实验，以确定特定分子的作用， 肿瘤侵袭的途径。

PROJECT SUMMARY The formation of a malignant tumor from a benign tumor has been strongly linked to the existence of heterogeneous cellular features within the tumor. Thus, elucidating the mechanisms underlying generation of tumor cell heterogeneity will be an important milestone to understand malignant tumor formation. We believe two applications of our work could be 1) earlier, more quantitative detection of malignancy based on molecular markers and 2) therapeutics that can freeze tumor development before metastasis or even revert back to a benign state. Our approach is to answer the following two questions: 1. What is the impact of the tumor-initiating cell‘s identity on generating tumor cell heterogeneity? In experiments to date, the inability to avoid contamination of different cell origins, including tissue stem cells and non-stem cells into the resultant tumor mass has prevented identification of the precise molecular mechanisms. We address the contamination problem by establishing an experimental system that enables precise tracing of tumor cell lineage derived from single cells. Our method is the non-invasive, in vivo imaging of endogenous, single cell-derived tumor development in adult transparent zebrafish. This experimental system allows us to visualize individual tumor cells, and, more importantly, also selectively label and isolate the cells of interest for detailed cell characterization. Using this system, we have already imaged the tumors arising from single cells in the skin epithelium. Importantly, we have found some major differences in tumor cell proliferation between tumorous cell clusters and in tumor cell differentiation that lead to acquisition of invasive features. Our results identifying a subset of tumor cells displaying invasive features is nicely representing the generation of tumor cell heterogeneity within a tumor. We will perform long-term clonal analysis of tumor development and transcriptome analysis of isolated individual tumors to determine their cellular identity. Retrospective imaging analysis will be used to study the effect of a tumor’s microenvironment on generating tumor cell heterogeneity. 2. How is heterogeneity within a tumor established? Identification of the cell types of tumor-initiating cells will be followed by the investigation of the mechanisms driving a single tumor-initiating cell to become heterogeneous during multiple rounds of cell divisions. Our ability to distinctively label and isolate invasive tumor cells from non-invasive tumor cells enables us to investigate the gene expression modulations responsible for a subset of tumor cells acquiring invasive features during tumor development. We will further perform functional experiments in vivo to define the roles of specific molecular pathways in tumor invasion.