Early tumorigenesis in vivo imaging, scRNA-seq to functional assays

项目总结/摘要 肿瘤在细胞行为、细胞微环境和基因表达方面的异质性已成为肿瘤发生的标志。 肿瘤进展，并提供了一个重要的挑战，在了解癌症。例如，通过 一些肿瘤细胞的侵袭性表型是癌从良性肿瘤状态发展的关键步骤。 肿瘤细胞从表皮的上皮层向真皮的迁移提供了肿瘤细胞进入表皮的通道。 血管系统，为远端转移提供了直接途径。了解一些细胞如何以及为什么能够 侵袭周围组织的能力是理解肿瘤侵袭的关键，但目前的体内侵袭模型 不容易回收和研究特定的侵袭性细胞以阐明肿瘤侵袭的驱动因素， 进展 我们的目标是建立一个跟踪肿瘤发生的新管道，允许直接研究肿瘤细胞， 多样化和入侵。该实验管道旨在提供以下功能： (i)纵向跟踪肿瘤发生，从肿瘤的第一个起源到关键事件，如侵袭; (ii)标记和选择性纯化在这些时程研究中鉴定的肿瘤细胞亚群; (iii)以无偏的方式表征分离群体中的单个肿瘤细胞。 这种方法将允许识别伴随的细胞事件和发育轨迹 肿瘤生长和侵袭性。在肿瘤的关键亚组分中表征的分子相关物可以 与细胞亚组分的事件历史直接相关，这些细胞和分子的重要性 可以通过扰动、消融和功能阻断实验来测试事件。这样的功能和 纵向实验管道是创建更具针对性和信息量的药物筛选系统的关键。 我们已经开发了一套转基因斑马鱼品系和成像工具， 成年动物内源性肿瘤发展的可视化。这使得肿瘤可以从它们的 起源于皮肤上皮细胞中癌基因表达后的长时间。我们的初步结果显示， 从肿瘤形成的最早阶段就存在异质性，因为只有一小部分细胞被诱导为 表达的癌基因继续形成侵袭性肿瘤;这些结果为 我们对肿瘤异质性的出现和发展的分析。 在这里，我们建议采用成像和标记选定的细胞群，结合单细胞 RNA测序分析，以研究细胞行为和分化途径对侵袭性肿瘤的关键 发展我们将建立一个实验管道，通过调查：细胞类型的具体侵袭性 肿瘤（目标1）;导致侵袭性表型的关键分化途径（目标2）;以及利用这些 通过功能测定的见解（目标3）。这条管道将为侵入性肿瘤的发展提供新的见解， 支持更大规模的研究和设计针对关键细胞亚群的抗肿瘤疗法。

PROJECT SUMMARY/ABSTRACT Tumor heterogeneity, in cell behavior, cell microenvironment and gene expression, has become a hallmark of tumor progression, and provides an important challenge in understanding cancer. For example, the adoption of an invasive phenotype by some tumor cells is a key step as carcinomas advance from the benign tumor state. Migration of tumor cells from the epithelial layers of the epidermis to the dermis provides tumor cell access to the vasculature, offering a direct path for distal metastasis. Understanding how and why some cells are capable of invading surrounding tissues is key to understanding tumor invasion, but current in vivo models of invasion do not readily allow specific invasive cells to be recovered and studied to elucidate drivers of tumor invasion and progression. Our goal is to create a new pipeline for following tumorigenesis, permitting direct studies of tumor cell diversification and invasion. This experimental pipeline is designed to offer capabilities to: (i) track tumorigenesis longitudinally over time from the first origins of a tumor to key events such as invasion; (ii) label and selectively purify tumor cell subsets identified in these time-course studies; (iii) characterize individual tumor cells in isolated populations in unbiased way. This approach would allow identification of the cellular events and developmental trajectories that accompany tumor growth and invasiveness. Molecular correlates characterized in the key sub-fractions of the tumors can be directly related to the event history of the cell sub-fractions, and the importance of these cellular and molecular events can be tested through perturbation, ablation and function blocking experiments. Such a functional and longitudinal experimental pipeline is key to creating more targeted and informative drug screening systems. We have developed a set of transgenic zebrafish lines and imaging tools that allow repeated, non-invasive visualization of endogenous tumor development in adult animals. This permits tumors to be tracked from their origins, over long periods, after oncogene expression in skin epithelial cells. Our preliminary results show that heterogeneity is present from the earliest stages of tumor formation, as only a small fraction of cells induced to express oncogenes go on to form invasive tumors; such results provide the motivation for and the foundation for our analyses of the emergence and elaboration of tumor heterogeneities. Here, we propose to employ imaging and labeling of selected cell populations, in combination with single-cell RNA-sequencing analyses, to study the cell behaviors and differentiation routes key to invasive tumor development. We will establish an experimental pipeline by investigating: the cell-types specific to invasive tumors (Aim 1); the key differentiation pathways leading to an invasive phenotype (Aim 2); and leveraging these insights by functional assays (Aim 3). This pipeline will offer new insights into invasive tumor development, supporting larger studies and the design of anti-tumor therapeutics targeting key cell subsets.