In vivo imaging of tumor heterogeneity generation from endogenous single cells

项目总结 从良性肿瘤形成恶性肿瘤与存在 肿瘤内的异质性细胞特征。因此，阐明了潜在的产生机制 肿瘤细胞的异质性将成为了解恶性肿瘤形成的重要里程碑。我们相信 我们的工作可能有两个应用：1)更早地、更定量地基于分子检测恶性肿瘤 标记物和2)可以在转移前阻止肿瘤发展的治疗方法，甚至可以恢复到 良性状态。我们的做法是回答以下两个问题： 1.肿瘤起始细胞的特性对肿瘤细胞异质性的产生有何影响？ 在迄今为止的实验中，无法避免不同细胞来源的污染，包括组织干细胞 而非干细胞进入所产生的肿瘤肿块阻碍了对精确分子的识别 机械装置。我们通过建立一个实验系统来解决污染问题， 精确追踪源自单个细胞的肿瘤细胞谱系。我们的方法是非侵入性的活体成像 成年透明斑马鱼内源性单细胞来源肿瘤的发展。这一实验系统 使我们能够可视化单个肿瘤细胞，更重要的是，还选择性地标记和分离 对详细的细胞特性感兴趣。使用这个系统，我们已经成像了从 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 在肿瘤细胞群之间和肿瘤细胞分化过程中导致获得侵袭性特征。我们的 结果鉴定具有侵袭特征的肿瘤细胞亚群能很好地代表肿瘤细胞的产生 肿瘤内肿瘤细胞的异质性。我们将对肿瘤的发展和发展进行长期的克隆分析 对孤立的单个肿瘤进行转录组分析，以确定其细胞身份。回溯成像 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起来的？ 在鉴定肿瘤启动细胞的细胞类型之后，将对其机制进行研究 使单个启动肿瘤的细胞在多轮细胞分裂中变得异质。我们的能力 区分标记和分离侵袭性肿瘤细胞和非侵袭性肿瘤细胞使我们能够研究 肿瘤细胞亚群在肿瘤过程中获得侵袭特征的基因表达调控 发展。我们将进一步在体内进行功能实验，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.